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Catecholamines bind to enkephalins, morphiceptin, and morphine
Brain Reseurch
Bulkfin,
Vol. 18, pp. 5-532.
0361-9230/87 $3.00 + .@I
B Pergamon Journals Ltd., 1987.Printed in the U.S.A.
Catecholamines Bind to Enkephalins, Morphiceptin, and Morphine ROBERT
SCOTT
ROOT-BERNSTEIN’
Neurobiochemistry, T-85, Veterans Administration Hospital, Brentwood 11301 Wilshire Boulevard, Los Angeles, CA 90073 Received
17 September
1986
ROOT-BERNSTEIN, R. S. Catecholamines bind tu enkephalins, morphiceptin, and morphine. BRAIN RES BULL lS(4) 509-532, 1987.-Nuclear magnetic resonance spectroscopy, pH titration, and color reactions demonstrate that the catecholamines dopamine, epinephrine, and norepinephrine bind to the enkephalins. Binding constants are c. 6~ 103,per mole. Catecholamines also bound to the mu opiate receptor agonist morphiceptin (Tyr-Pro-Phe-Pro-NH,). Very little binding was found to enkephalin and morphiceptin fragments and analogues, indicating that the entire molecules are necessary. Serotonin binding peptides do not bind the catecholamines. Morphine and apomorphine, however, do bind these catecholamines (with a binding constant for morphine of c. 4x lo* per mole). The opiate antagonist naloxone and a number of other drugs do not bind catecholamines. Morphine, morphiceptin, and the enkephalins also retard the formation of colored reaction products by catecholamines in vitro. These results may help to explain observations that the enkephalins are co-stored and co-transmitted with dopamine and norepinephrine, and may provide a basis for the elucidation of other known cases of peptide-monoamine co-transmission. Possible implications for understanding opiate effects on catecholamines during addiction and withdrawal are discussed, and suggestions concerning drug design are made. Co-transmission
Opiate addiction
NMR
Drug design
binding sites
specifically to the serotonin and SHIAA precursor, 5-hydroxytryptophan [65]. The enkephalins, which were used as controls in previous experiments, did not appreciably bind serotonin or related indoles [66]. Preliminary experiments using nuclear magnetic resonance spectroscopy, however, yielded evidence that both Met-enkephalin and Leu-enkephalin bind dopamine (DA), epinephrine (EPI), and norepinephrine (NE) [@I. Further experiments demonstrated that these catecholamines also bound to morphiceptin (a mu opiate receptor agonist, Tyr-Pro-Phe-Pro-NH* [ 13,30]), and morphine [69,71]. Since the enkephalins are often co-stored and co-released with DA and NE, and since both peptide and alkaloid opiates are known to affect catecholamine metabolism (see the Discussion section), I have investigated these chemical interactions in detail with the goal of possibly elucidating a simple, underlying chemical basis for the observed correlations of anatomical localization and physiological interaction. The purpose of this paper is to report the results of some 120 combinations of peptides and drugs with catecholamines demonstrating the specificity of the binding of NE and DA to the enkephalins, morphiceptin and morphine.
WHY are some peptides co-stored or co-released with particular monoamines? Why do such co-transmitters behave as
modulators for each other? Must neurotransmitter activity be expressed solely through complex cellular receptor systems? If so, how did such complex systems evolve? How do exogenous drugs affect endogenous mechanisms of neurotransmitter regulation? Questions such as these have led me to consider the possibility that a simple chemical basis might exist for the anatomical distribution and interactive functions of many neurotransmitters and hormones, as well as for the mechanisms by which some drugs affect neurotransmitter systems. My initial thoughts on this subject were formed by the elucidation of serotonin (S-hydroxytryptamine [SHT]) binding sites on the tryptophan peptide of myelin basic protein, luteinizing hormone-releasing hormone (LHRH), and a sequence common to alpha melanocyte stimulating hormone (MSH) and adrenocorticotropic hormone (ACTH) [63, 64, 661. The activities of each of these peptides are known to be modulated by serotonin. Similar binding sites were located by sequence analysis of serum albumins, which are known to transport tryptophan and serotonin, and the red pigment stimulating hormone of crustaceans, which is also modulated by serotonin [64,66]. Furthermore, it is thought that several drugs may produce their effects by direct binding to a metabolite involved in the pathway disrupted by the drug 1731. An example is the anorexigenic agent fenfluramine, which lowers blood serotonin and S-hydroxyindoleacetic acid (SHIAA) levels in man and animals, and which binds ‘Requests for reprints should be addressed
Catecholamine
METHOD
Seventeen peptides (Table 1) were used to investigate the specificity of binding of catecholamines to peptides. These peptides were chosen for their structural similarities to enkephalins, to fragments of enkephalins, or to peptides previously shown to bind serotonin [66]. All peptides were
to Robert Scott Root-Bernstein,
509
3514 Veteran Avenue, Los Angeles, CA 90034.
ROoT-BERNSTf:IN
obtained from Vega Biochemicals (Tucson, AZ) or BAChem (Torrance, CA), or both. Nine drugs having varied physiological effects were tested for binding to neurotransmitters, Drugs were obtained from the following manufacturers: morphine sulphate and apomorphine hydrochloride (Merck, Sharpe and Dohme, Rahway, NJ); naloxone hydrochloride (Endo, Garden City, NY); desiprimine (USV Pharmaceuticals, Tackahoe, NY); trifluoperazine dihydrochioride (Smith. Kline and French, Philadelphia, PA); pargyline (Regis. Morton Grove, IL); amphetamine D,L sulphate and 3-methoxy-4-hydroxyphenethanol (MOPET) (Sigma. St. Louis, MO). All other chemicals were from either Sigma or Calbiochem (San Diego, CA), or both. All peptides were dissolved in distilled water, 5.0 mg per ml, and then adjusted to pH 7.0 using minute amounts ofO.01 N NaOH and 0.01 N HCI. All other chemicals were dissolved in distilled water, 2.0 mg per ml, and similarly adjusted to pH 7.0. All peptide and other chemical soiutions were immediately frozen at -40 degrees C and kept in the dark until immediately prior to use (usually within a few hours). Nuclear magnetic resonance (NMR) spectroscopy was zarried out using a Brucker WM~~ very high field multinuclear spectrometer at the Southern California Regional NMR Facility, California Institute of Technology. To facilitate ease in preparation of the samples, and so as to have the ability to examine the amide region of the spectra, IH spectra were obtained utilizing the Redfield solvent suppression proce;lure 1611. Usually, spectra for both the region O-4.5 ppm and :he region 5.5-10 ppm were obtained in separate runs using newly mixed samples each time. This procedure assured that most results were verified at least once. All positive results have been obtained at least four times using freshly prepared samples, often from different suppliers as an added control. No internal reference was used in any sample so as to obviate the possibility of unexpected interaction with peptides, drugs, or catecholamines. (This is also the reason that a standard buffer was not employed.) In all cases, the middle af the water peak, which was never completely damped by the Redfield procedure, was used as a reference set at 4.65 ppm. The resulting ppm assignments are not, therefore, exact, but the results were consistent within the study, and previous utilization of this method yielded results in good accord with published spectra [66]. One drop 10.5 ml} of D?O ;99.98% pure, Aldrich, St. Louis, MO) was added to each 0.35 ml of sample to permit spectrometer lock. Standard Five-mm NMR tubes (Gold Label, Aldrich) were employed. At the concentrations utilized here, 500 MHz spectra typically required 256 to 400 scans at 32K data points to achieve the signal-to-noise ratio displayed in the accompanying figures. All spectra obtained were of this quality. or were rerun until they were. Spectra of various concentrations of the enkephalins were obtained, and within the limits of the dilutions employed in these experiments, no peak shifts due to dilution were observed. Further controls were obtained for this particular variable as the various catecholamines and related compounds were added to the peptides. Thus, there is no doubt that the peak shifts that result from the addition of DA, EPI, and NE are not due to dilution effects or to non-specific interactions with sulphate, chloride or other ions. Since these catechols had no effect upon the spectra of most of the ather peptides tested, it is very unlikely that the peak shifts observed in the enkephalins are due to unexpected contaminants such as paramagnetic ions.
Initially, 0.15 ml DA, NE, and the other catechoiamines and neurotransmitters were added to 0.15 ml of peptidc (making in most cases about a one-to-one combination by molarity). If no shifts in any peaks were observed. then the interaction was reported to be negative. If peak shil’[\ did appear, then the interaction was measured a4 ii fraction of the amount of peak shift that was caused by NE. which caused the greatest peak-shift-per-amount-of-chemicaladded (Table 2). The binding constants of the various chemi.. cals to the peptides and morphine was determined by adding 0.01. 0.02, 0.04, 0.08, 0.16. etc. ml aliquots to 0.30 ml of peptide or morphine until saturation was achieved. The percent of the shift compared to saturation at each concentrntion was then plotted in a Scatchard plot (Figs. 3, 7. 17). Peak assignments for the enkephalins were made with regard to 270 MHz NMR spectra previously assigned by Jones, Gibbons and Garsky [4lj, 300 MHz spectra of Rocques c,t crl. 1701, NMR studies of Gly-Gly-X-Ala peptides by Bundi and Wuthrich [IO]. and the spectra of enkephalin fragments such as Gly-Gly-Phe-Leu, Tyr-Gly-Gly. and Phe-Met, which were obtained during the present study. Peak assignments for morphiceptin were made with regard to the enkephalin spectra and the spectrum of Gly-Pro-Arg-Pro 1671. pH titration studies were also performed for a number of the catecholamine-peptide and catecholamine-opiate combinations. Ten mgiml solutions of peptides, opiates, and other drugs were made in distilled water; and approximately equimolar solutions of catecholamines or other neurotransmitters. Aliquots (0.20 m1) of each compound were titrated using 0.04 ml aliquots of 0.0025 N NaOH additively. The pH was measured after each addition of NaOH. Predicted values for the combinations of peptides or drugs with catecholamines were then calculated by averaging the pH values of the individual compounds. Combinations of the original solutions of the compounds were then made by adding 0.20 ml of each. The combinations were titrated using 0.04 ml aliquots of 0.0050 V NaOH. The experimental values were then compared with the predicted values. Theoretically, if the two compounds do not interact in any way, then the experimental values should match the predicted values, since the titration will proceed in the same manner whether the two compounds are in solution together or not. However. if the compounds interact so that hydrophilic residues or side chains are involved, then the bonding of these residues will change their susceptibility to titration and the experimental value will differ significantly from the predicted value. The one difficulty with this technique is that the titration values cannot differentiate between interactions between the peptide-monoamine or drug pairs and the ion pairs if the peptides and monoamines were salted. Thus. all experiments were run with NE or DA free acid. A variety of experiments were also made utilizing the fact that DA, EPI, and NE will oxidize into colored products and polymerize into very dark melanins. Molar excesses of morphine, morphiceptin, Met-enkephalin, Met-enkephalin fragments, and naioxone were added to 1.0 mgiml solutions of DA, EPI, and NE and allowed to stand open to the air for several days at room temperature. (Both test tubes and wells were employed with no difference in results.) A set of controls was also prepared of the individual solutions. The combinations were then compared in color with the individual solutions as a general, qualitative measure of the ability of the various opiates and peptides to prevent and/or catalyze oxidation and melanin formation.
CATECHOLAMINE
BINDING
TABLE 1 SUMMARYOFNMRRESULTSOFBINDINGOF DOPAMINE (DA), NOREPINEPHRINE (NE), AND SEROTONIN TO A NUMBER OF PEnIDES AND DRUGS Norepinephrine Binding + + -
Dopamine Binding
Serotonin Binding
+ + _ _
_ _
_ _ _ _ 2 +
_ _ _ -
_ -
Sequence
Peptide or Drug Met-enkephalin Leu-enkephalin Enkephalin (l-3) Met-enkephalin (2-5) Leu-enkephalin (2-5) Met-enkephalin (4-5) Morphiceptin Substance P Arg
Tyr Tyr Tyr
Pro
Lys
-
+
_ _
_
+
_
-
+
_
+
+
5
+ _ _ _ _
+ _ _ _
_ _
+
_ & -
Proctolin Gas&in tetrapeptide MSH-ACTH tetrapeptide LHRH Trp peptide Morphine Apomorphine Naloxone Desiprimine Trifluoperazine Pargyline Amphetamine Fenfluramine MOPET
Phe Phe Tyr Tyr Pro
Gly Gly Gly Gly
Gly Gly Gly Gly
Phe Phe
Met Leu
Phe
Met
Gly
Gly
Phe Phe
Leu Met
Gly Gly Tyr Pro Gln
Phe Gly Tyr Phe Gln
Gly Phe
Pro
Pro
Gly Pro Thr Phe
Arg
Gly Tyr Trp
Pro Leu Met
Arg Pro Asp
His
Phe
Arg
Trp
His
Trp Phe
Ser Ser
Tyr Trp
Pro Phe
Gly Gly
Phe
Gly
Leu Met NH,
Leu
Arg
Ala
Glh
Pro Gly Gly Gln
NH,
Arg
The results of serotonin binding are from Root-Bernstein and Westall [65,66], in which many other controls may be found. Plus signs indicate large shifts in peaks occur in an equimolar combination. Minus signs indicate that no or only very small shifts were observed. Plus-minus signs indicate that significant shifts occurred, but were not as large as those produced by one of the other neurotransmitters
tested. The major conclusion to be drawn from this table is that catecholamine binding is highly correlated with opiate or opioid peptides, whereas serotonin binding is not. With the exception of morphine, catecholamine and serotonin binding peptides and compounds appear to be distinct.
RESULTS
The results of the NMR study are summarized in Tables 1 and 2. Significant shifts appeared in the spectra of only a limited number of peptides and drugs upon addition of NE and DA (Table 1). These were Met- and Leu-enkephalins (Figs. 1, 2, 4, 5, 6), morphiceptin (Figs. 9 and lo), possibly substance P (although there was some difficulty in obtaining consistent results), gastrin tetrapeptide (which is a sequence common to cholecystokinin as well), morphine, and apomorphine. No significant shifts occurred in the spectra of the enkephalin fragments tested (e.g., Figs. 6 and 8), several enkephalin-like peptides, proctolin, a sequence common to melanocyte stimulating hormone and adrenocorticotropic hormone, luteinizing hormone-releasing hormone, or the tryptophan peptide of myelin basic protein, naloxone, des-
ipramine, tritluoperazine, pargyline, amphetamine, or MOPET. Other controls for the interactions of these hormones and drugs and their analogues with monoamines may be found in previous publications [65,66]. Further research demonstrated that binding constants for NE binding to Met- and Leu-enkephalin were virtually identical: 5.3 and 5.8~ 103/mole, respectively (Figs. 3 and 7). The binding constant for NE binding to morphine was determined to be 4x104/mole (Fig. 12). Using these values, the peak shifts caused by NE at a given concentration of peptide or drug were arbitrarily assigned a unitary value, and the peak shift caused by other catecholamines, indoleamines, and drugs was determined under the same conditions. These peak shifts were assigned a value between zero and one according to the percent of peak shift caused by NE at the same concentration (Table 2). Thus, EPI was found to bind
ROO’I -HERNSTElN
TABLE 2 SUMMARY OF NMR RESULTS OF BINDING OF A LARGE NUMBER OF CATECHOLAMINES, CATECHOLAMINE PRECURSORS AND METABOLITES, OTHER NEUROTRANSMIlTERS, AND VARIOUS DRUGS TO MET-ENKEPHALIN, LEU-ENKEPHALIN. AND MORPHINE
MetEnk*
Phenylalanine Tyrosine L-DOPA D-DOPA Dopamine Epinephrine Norepinephrine 3-OH-4-OCH,,-phenethylamine 3,4-diOH-phenethylamine 3-OH-tyramine 3,4-diOH-mandellic acid Octopamine Homovanillic acid 3-OH-4-OCH,-phenylacetic acid S-OH-indoleacetic acid Serotonin Histamine Acetylcholine Amphetamine Fenfluramine
0 0 0.3 I.0 0.6 0.8 I.0 0.6 0.2 0.4 0 0.2 0.2 0 0.2 0 0 0.4 0.2 0.5
LetIEnkt
Morphine*
0 0 0.4
0 0 0
0.8 0.9
0.8 0.9
I .o
I .o
0 0.1 0.2 0 0 0 0 0.2
0 0.2 0 0 0.7 0 0 0.5
*Binding constant, K=5.3x lW/mole= 1.0. tBinding constant, K=5.8x lOVmole= I .O. *Binding constant, K=4x lOYmole= 1.0. Binding constants were determined by Scatchard plots from shifts produced by varied concentrations of NE with these opioids (see Figs. 3. 7. 12). NE binding was arbitrarily assigned a value of one. and the binding of the other compounds was measured as a percent of the shift in a given peak (see Figs. 3, 7. I2 for identification) caused by NE at the same concentration and pH. Related controls for binding (or lack thereof) of many of the same compounds to serotonin binding peptides (LHRH, MSH tetrapeptide, and the tryptophan peptide of myelin basic protein) may be found in RootBernstein
and Westall
1661.
to the peptides and morphine whereas other neurotransmitters
very
nearly
as well as NE,
such as histamine and acetylcholine had very little affinity. One interesting difference between the affinities of the enkephalins and morphine is the latter’s afftnity for serotonin (SHT) and amphetamine. Examination of the NMR spectra yields specific information about the nature of the observed binding beginning with the Met-enkephalin spectra (Figs. 1 and 2). Peak shifts occur in several regions of the spectrum with addition of NE. The Tyr alpha hydrogen peaks shift, possibly indicating a change in the position of the Tyr residue relative to the rest of the molecule. The Tyr beta hydrogen peaks also shift and split, indicating at the least a significant change of conformation, and possibly direct interaction with NE. Small changes occur in the Gly peaks, although it was not possible to determine whether these changes occurred in the Gly* peaks, the Gly3 peaks, or both. No shifts or other changes occurred in any of the Phe peaks at any concentration of NE except for splitting of the beta hydrogens, suggesting that a conformational change involving the angle of the Phe residue
relative to its alpha carbon may occur. There I> no evtdence interaction between the NE hydrogens and any 01 the Phe hydrogens. Large changes did OCCUI. however, ic the Met CH,, peak, which alters its width considerably, eventually diminishing in height, and splitting. These changes probably indicate direct interaction with NE. None of the other Met peaks displayed any significant changes, suggesting that the interaction with NE is limited to the CH:, portion of the side chain. No significant changes were observed in the amide or aromatic portion of the spectra tnot shown). The spectral changes in the Tyr beta H\ and Met CH,,s were used to determine the binding constant. Both \ets of peak shifts yielded the same number: 5.3 y IO:’per mole ( Fig. of direct
3).
The only peak changes observed in the NE spectra m the presence of either Met- or Leu-enkephalin were a splitting of the beta hydrogen peaks (Figs. I and 4). There were no noticeable changes in the aromatic region of the NE spectrum. It is unlikely therefore that van der Waals forces play a significant role in the binding. The results of the NMR analysis of NE binding to Leuenkephalin are broadly similar to those just described for Met-enkephalin (Figs. 4 and 5). Shifts and splitting of Tyt alpha and beta hydrogen peaks, splitting of Phe beta hydrogen peaks, splitting of the Leu CH,, peak. Also shown (Fig. 6) is the spectrum of DA binding to Leu-enkephalin, which clearly shows a shift in one of the Gly amide peaks. The most reasonable interpretation of this shift is a conformational change involving the twisting of the peptide backbone near a Gly amide. No such conformational change was apparent when DA was added to Gly-Gly-Phe-Leu (Des-Tyr-Leuenkephalin) at the same concentrations (Fig. 6). The Tyr beta Hs were used to calculate the binding constant to NE, and yielded the number: 5.8~ IO: per mole (Fig. 7). Also shown are a pair of the dozens of control spectra obtained (Fig. 8). An equimolar combination of NE with Phe-Met produced no spectral changes whatsoever in either molecule. Very slight (c. one tenth of the corresponding changes in Met-enkephalin) shifts occurred when an equimolar combination of NE was made with Tyr-Gly-Gly (enkephalin l-3). Such spectra clearly indicate that NE binding to enkephalins requires the complete pentapeptide. In comparison with the enkephalins, the spectral changes induced in morphiceptin by NE were more limited (Figs. 9 and 10). The NE hydroxyl peak completely disappeared, indicating probable hydrogen or ionic bonding. Peak broadening occurred in most of the aromatic peaks of both morphiceptin and NE. And only one hydrophobic interaction was apparent: a shift in one of the two Pro gamma CH peaks of morphiceptin. No shifts or other changes were observed in the morphiceptin amide peaks, nor in any of the alpha, beta, gamma, or delta hydrogens, with the single exception already noted. Thus, it is unlikely that van der Waals forces are important to the binding. No significant changes were observed in the spectra of a number of peptides similar in sequence to morphiceptin, including Gly-Pro-Arg-Pro, ProPro-Gly, and Tyr-Tyr-Tyr. The type of spectral changes observed m the morphiceptin-NE interaction were not appropriate for calculating a binding constant, as the changes were not easily measurable. Evidence to be presented below suggests, however, that the binding constant for morphiceptin for NE may be of the same order of magnitude as that of morphine for NE, which is discussed next.
CATECHOLAMINE
513
BINDING
aH
aH
aHs
.
.
.
.
410
.
.
.
.
. 3Io
PPM
.
.
.
FIG. 1. 1H NMR spectra of norepinephrine (NE), Met-enkephalin (Met-enk), and combinations at pH 7.0. (A) 0.25 ml Met-enk + 0.04 ml NE (see text for concentrations); (B) 0.25 ml Met-enk + 0.16 ml NE. Progressive shifts occur in Tyr’ alpha Hs and Tyr’ beta Hs. Splitting of the Ne non-aromatic Hs is apparent. At the higher concentration of NE, some broadening of the Gly and Phe peaks becomes evident.
Met’ c H,S \
Met5 CH,s
Met -EnkephaMn
+.02
2:0----------I
2.0
Norepi
+.04 2.0
Norepi
+ .08 2.0
Norepi
+ .I6 Norepi
Saturated
77-----v
FIG. 2. 1H NMR spectra of the Met CHZ Hs and the Met CH, Hs of Met-enk at pH 7.0 kn the presence of increasing aliquots of NE. While only very small changes are apparent in the CH, Hs, significant changes in peak width occur in the CH, Hs, finally resulting in a diminution in peak height accompanied by splitting.
RC)O’I‘-HFRNS’I‘EIN
FIG. 3. Scatchard plot of the shifts in Met-enk beta Tyr Hs (Fig. I) with increasing concentrations of NE. The diminution in peak width of the Met CH, Hs (Fig. 2) yielded the same plot. The resulting association constant is 5.4X IO31mole.
,
, 4.0
1
3.0
*
,
PPM FIG. 4. IH NMR spectra of NE, Leu-enk, and combinations at pH 7.0. (A) 0.25 ml Leu-enk + 0.01 ml NE; (B) 0.25 mol Leu-enk + 0.04 ml NE; (C) 0.25 ml Leu-enk + 0.16 ml NE. As in the Met-et& f NE spectra (Fig. I), progressive shifts are apparent in the Tyr’ alpha Hs and Tyr’ beta Hs, accompanied by peak broadening as the concentration of NE is increased. Some broadening of the Leu-enk Gly Hs and Phe beta Hs, as well as the Ne non-aromatic Hs, is also apparent.
CATECHOLAMINE
515
BINDING
Leu- Enkephalin .:.\JLJLil Leu 5CH,s
I
2.0
h
+ 66 Norepl
+ .04 Norepl
+ .02 Norepi
I.“‘,‘...,...,
PPM
2.0
2.0
2.0
FIG. 5. 1H NMR spectra of Leu-enk Leu CH, Hs showing progressive changes in peak width with increasing concentrations of NE. As with the Met-enk Met CH, Hs, the peak eventually shortens ahd splits (Fig. 2). The similarity in the changes observed in the Leu CH, Hs and the Met CH3 Hs suggest that these two residues play the same role in binding NE.
Significant spectral shifts accompanied the addition of NE to morphine (Fig. 11). Splitting of the NE peaks occurred, and shifts occurred in virtually all non-aromatic morphine peaks. These shifts may indicate conformational change or direct interaction with NE residues. These shifts were found to be concentration dependent, and a Scatchard plot of the shift-concentration relationship was made yielding a binding constant of 4x10* per mole (Fig. 12). Three different peaks of morphine all yielded the same binding constant, indicating that the shifts are all orchestrated by the same mechanism. No such shifts occurred in any peak of the naloxone spectrum at any concentration of NE, nor were similar spectral changes observed in the other drugs tested with the exception of apomorphine. The results of pH titration generally confirm the NMR results to the extent that both indicate the presence or absence of binding. An equimolar combination of morphine with NE results in a buffer, as is apparent from Fig. 14, and the resulting titration values differ significantly (up to 0.9 PI-I) from the values predicted from the NE and morphine solutions alone. On the other hand, the predicted and experimentally determined values for an equimolar combination of naloxone with NE are virtually identical, the largest variance being approximately 0.05 pH. These results were reproduced several times with different solutions, and although the absolute values differ somewhat from combination to combination (probably due to differences in the absolute concentration of the independent solutions and in their relative concentrations upon addition), nonetheless the fact of either differing significantly from the predicted value or not is con-
stant. The same effect is apparent with a combination of apomorphine with NE (Fig. 15). The titration results for peptides with NE are not as clear as drug-catecholamine combinations, presumably because peptides tend to be amphoteric. Thus, in Fig. 13 it is apparent that some binding probably occurs between Gly-GlyPhe-Leu (des-Tyr-Leu-enkephalin) and NE below pH 7.0, although the NMR analysis of this combination showed no significant shifts at pH 7.0. One possible explanation is that the interaction is purely ionic, and so invisible to the NMR. Despite these results, it is clear that the experimentally obtained values and the predicted values differ to a much greater degree at pH 7.0 and above for Met-enkephalin + NE than for Gly-Gly-Phe-Leu + NE. Much the same results were obtained for NE + Tyr-Gly-Gly (Met-enkephalin l-3) (not shown). Tyrosine showed essentially no affinity for Met-enkephalin according to this method of analysis (i.e., the expected and predicted values were consistently within 0.1 pH), and NE showed no affinity for Phe-Gly-Gly-Phe according to the same criteria (not shown). A morphiceptin-NE combination differed from the predicted value by approximately 0.5 pH over the range of pH from 6.8 to 7.8 (not shown). One unexpected surprise was that both Tyr-Gly-Gly and Phe-Met displayed some affinity for NE or DA according to the pH titration technique (not shown). Since only very slight changes were observed in the NMR spectra, the pH titration data probably indicate ionic bonding, which would be invisible to the NMR. And since there is, at least at present, no way to calculate a binding constant from the pH data,
+ Dopamine
Gl y Gly Phe leu
NH
NH
TyrGly GlyPheLw
+ Dopamine
FIG. 6. IH NMR spectra of Leu-enk and des-Tyr-Leu-enk and their combinations t 1:I by molarity) with DA at pH 7.0. While a small shift in one of the Gly NHs is apparent in the Leu-Enk + DA combination and in some of the inverted aromatic peaks (at 6.5 ppm), no changes are apparent in the des-Tyr-Leu-enk + DA combinations. The Tyr residue would therefore appear to be essential for catecholamine binding.
(Leu-Enk=SxlO-?W) FIG. 7. Scatchard plot of the shifts in the Tyr’ beta Hs peak of Leu-enk + NE spectra (Fig. 4). The calculated association constant is 5.8X 1OYmole.
CATECHOLAMINE
517
BINDING
TYR-GLY-GLY
~.___
._..- _c_ “___
_. v
-- .--..
_._._l_
-_- -.--_ _
,. ,.__ ” .__
NOREPINEPHRINE .‘-+-_. .~“-..;r~%4~
%.
-
-..-
I_.. _ _. ___~ ..._. -I..
__,
1:l ---c
__-.
_._-_._-_.-- _____..._-
Phe b”S PHE -MET_ v----
FIG. 8. 1H NMR spectra of Tyr-Gly-Gly, Phe-Met, and their equimolar combinations with NE at pH 7.0. Very small upfield shifts are apparent in the Tyr alpha and beta Hs in combination with NE. These shifts are about l/10 those observed in the Met-enk and Leu-enk spectra at the same molar ratios (Figs. 1 and 4). No observable changes appear in the Phe-Met + NE combination. Thus, the Phe-Met (or Phe-Leu) residues are necessary to binding (Figs. 2 and 9, as is the Tyr-Gly-Gly sequence (see also Fig. 6).
it is unclear how important this observation is vis-a-vis the NMR results. Color reaction studies also indicate that opiatecatecholamine interaction takes place. Plate 1 shows the results of one of the color reaction experiments demonstrating that at pH 7 in phosphate buffer, a slight molar excess of morphine sulfate retards the oxidatio~~lyme~zation of DA, NE and EPI into their colored products. Plate 2 shows the results of combining morphiceptin and NE (2: 1 molarity) at pH 7 in phosphate buffer. The same results were achieved with the enkephalins, but a ten fold excess of enkephalin:NE was needed to observe the effect clearly. This result indicates that morphine and mo~~ceptin binding to NE is probably of the same order, and about ten times stronger than enkephalin-NE binding (which is also in accord with the binding constants derived from the NMR study [Figs. 3, 7,
121). It must be stressed that the color reaction experiments demonstrate that the catecholamine opioid interaction is reversible: if the solutions are let stand several weeks, the tubes or welis containing catecholam~e-opioid combinations eventually achieve the same color as the catecholamine tubes and wells. In other words, the interaction serves to retard, but not to prevent, the color reaction. Tyr-Gly-Gly also retarded the color change significantly, whereas Phe-Met appeared to catalyze the color reaction, perhaps by p~ici~ting in it. These observations will need further analysis before definitive interpretations are possible. Given that the origins and mechanism of melanins is unknown, the possibility that the enkephalins or enkephalin fragments might play a role in their formation is well worth investigation. DISCUSSiON
The results
obtained
in this study must be interpreted
from a number of interrelated points of view. First, the data permit models to be constructed of the probable form of binding that occurs between the catecholamines and the opiates and opioids. Second, this chemical binding needs to be examined in light of the anatomical distribution of catecholamines, enkephalins, and opiate receptors in vivo, and with regard to the phenomenon of co-tr~smission (that is, the co-storage and co-release of monoamines with peptide neurotransmitters). And finally, the binding must be discussed with regard to possible mechanisms of opiate and opioid action in vivo (e.g., the mechanisms of addiction and withdrawal), and vis-a-vis the problem of drug design. Chemical Models of the Binding To begin with, the NMR data, in conjunction with the pH titration and calorimetric data, are sufficient to provide quite strict criteria for building models of the mechanism by which NE and related catecholamines may bind to the enkephalins, mo~hi~eptin, and morphine. To interpret the NMR data, however, something must first be known about the conformation of the opiates and opiate peptides in solution. There is little difficulty with the preferred conformation of morphine, since it is a relatively rigid molecule. This is not the case with the enkephalins. Both NMR studies [41,70] and minimum energy conformation studies [38,46,48] agree that Met-enkeph~in has a large number of fairly stable states. The NMR data indicate that as many as 27 “rotomers,” or combinations of cis-trans pairs are not only possible between the Tyr, Phe, and Met side chains, but are actually present in substantial proportions in solution [41]. NMR studies of enkephalins is non-aqueous solutions (mimicking insertion of the molecule into a lipid membrane, for example) demonstrate yet other preferred coronations [4], and the crystallographic structure differs yet again f371. Energy calculations
NOREPI AROMATIC ti 5
COMBINATION
(1 1)
,
NOREPI HYDROXYLS
I
I 75
I
r
MORPHlCEPTlN I I PPM
I 70
AROMATIC 1
Hs 1
I
FIG. 9. 1H NMR spectra of morphiceptin (Tyr-Pro-Phe-Pro-NH,), NE, and their combination (1: 1by molarity) at pH 7.0. The NE hydroxyl peak completely disappears, and significant broadening of the NE aromatic Hs occurs (see particularly peak at 6.76 ppm). Small changes in the peak shapes of the morphiceptin aromatic Hs (Tyr and Phe residues) occur at 7.2 ppm, 7.14 ppm, 6.96 ppm and 6.84 ppm. These peak changes suggest the possibility of pi-pi overlap bonding as a significant feature of morphiceptin-NE binding.
tend to present a more limited number of low-energy conformers, but all of these studies have been made by ignoring peptide-solvent interactions [38, 46, 481. Even so, most studies indicate that at least half-a-dozen different low energy conformers may reasonably exist. In short, the enkephalins are highly flexible molecules in solution. One very important limitation exists on the flexibility of the enkephalins. Both NMR and energy calculation studies agree that a beta bend occurs in the Gly3-Phe4 region, most probably stabilized by a hydrogen bond between the Gly2 hydroxyl and the Met5 amide. Based upon these considerations, two possible models for NE binding to Metenkephalin are presented here. Figure 16 shows one low energy conformer for Metenkephalin. A caveat to remember in interpreting this picture is that the NMR data indicate that although this is one of the most preferred conformations of the molecule, the Tyr, Phe, and Met side chains are constantly rotating into all of the other possible cis-trans conformers. In other words, Fig. 16 must not be interpreted as being a static model of the molecule, but rather a dynamic one. Figure 18 shows a similar conformer, to which the same caveat applies. CPK model building resulted in two models that adhered to most of the criteria set by the experiments reported here and which could also be constructed with in accordance with the published data concerning the conformation of the enkephalins in solution. The first model presents a binding
mechanism that requires a minimum of conformational changes, but has the problem that it does not explain all of the NMR data presented here. The second model presents a binding mechanism that requires considerably greater conformational changes, but accounts much more completely for the NMR data reported here. In effect, the second model carries the implication that NE binding by the enkephalins induces a large conformational change. Model 1 (Fig. 17) shows one possible mechanism of NE binding to Met-enkephalin. Essentially all that is necessary for binding to occur is for the Tyr’ residue to rotate into a new position, which will then be stabilized by pi-pi overlap bonds to NE. NE will also form bonds to the N-terminal amine and to the Tyr hydroxyl. The Met5 CH, will interact with the pi system of NE. The main feature of this model is its simplicity. It accounts for the NMR peak shifts observed in the Tyr alpha hydrogens and the Met CHB peaks. The same general model would explain the analogous peak shifts observed in the Leu-enkephalin-NE spectra. The model does not, however, suggest why concentration dependent changes were observed in the Tyr and Phe beta hydrogen peaks, the shifts in the Gly2-Gly3 region, or the splitting of the nonaromatic peaks of NE. Thus, while this model is plausible, it is not fully consistent with the existing data. The second model (Fig. 19) is of the “molecular sandwich” type [63,64], which was found in earlier work on the mechanism of serotonin binding to peptides 1661. It accounts
CATECHOLAMINE
519
BINDING
MORPHICEPTIN + NOREPI (1 :l)
MORPHICEPTIN PRO YHs
I
z.tlI
I
,
I
2.88
,
1.90
,
,
I.&l
I
r
1.70
PPII
FIG. 10. 1H NMR spectra of mo~hiceptiR and mo~hiceptin + NE combination (1:l by molarity) at pH 7.0. (The NE spectrum is not shown because there are no peaks in this region.) This figure shows the only peak shift observed in the 1.0 to 4.5 ppm region of the spectra, indicating that virtually no hydrophobic interactions are involved in the morphiceptin + NE binding, save for the single interaction with one of the pair of Pro gamma Hs.
for the NMR data completely, but requires a quite large change from the lowest energy conformations to do so. The major features of this second model are that NE intercalates between the Tyr’ and Phe* side chains resulting in extensive pi-pi overlap bonds, an NE hydroxyl-Gly3 carbonyl hydrogen bond, a bond between the NE amine and the Tyr’ hydroxyl, and van der Waals (hydrophobic) interactions between the Met5 CH3 and the non-aromatic hydrogens of NE. The formation of the hydroxyl-carbonyl bond requires twisting of the peptide backbone in the Gly2-Gly3 region, which would account for the amide shift in the Gly region of the spectrum (Fig. 6). The proposed model also places the Tyr’ beta hydrogens facing NE so that van der Waals (hydrophobic) interactions are probable. The only observed peak shifts not accounted for directly in this model are the Phe4 beta hydrogens. These shifts are not extensive (compared to the beta hydrogens of Tyr’ for example), and may simply reflect some strain between the beta carbon and the Phe ring carbon atoms due to the formation of the pi-pi over-
lap bonds with NE. Once again, this model works equally well for Leu-enkephalin. Both model 1 and model 2, by predicting that the NE amine and at least one NE hydroxyl are involved in the binding to the enkephalins, explain the ability of the enkephalins to retard oxidation of NE, and the differences between the predicted and experimentally determined pH titration values. Both models also explain why NE, EPI, and DA bind reasonably well to the enkephalins, whereas serotonin, histamine, and acetylcholine do not. Model 2 is the more stereospecific of the two, however, and for the reasons given above, seems preferable. The differences between the two models are testable, although the peptides do not exist commercially to do so at present. If model 1 is correct (or possible), then NE should bind to some sequence Tyr-Gly-Gly-X-Met(or Leu), where -X- is some non-aromatic side chain other than Pro (which would not allow the formation of the required beta bend). The N-terminal amine, however, would be indispensable for
FIG. 11. 1H NMR spectra of morphine sulfate, Ne, and various combinations (by molar&y) at pH 7.0. Progressive shifts occur in virtually all morphine peaks. All of the non-aromatic NE peaks split. Some broadening of the aromatic peaks also occurred (not shown).
5-
O-l 0
0.1
0.2
0.3
0.4
0.5
0.6
0.1
0.9
0.9
1.0
S (MORPHINE) FIG. 12. Scatchard plot of morphine + NE combinations (some of which are shown in Fig. 1l), using the 2.8 ppm morphine peak shifts as a measure of binding. The 3.98 ppm morphine peak (Fig. 11) yielded the same results. The calculated association constant is 4X 104/mole.
CATECHOLAMINE
521
BINDING
1413@
12-
g
11-
d x
10-
l MET-ENKEPHALIN
+
NGREPI
fPREDlCTEDf
0 MET~NKEPHAL~
+
NGREPl
(EX~R~ENTAL)
0
l GLY-GLY-PHE-LEU
+
NOFtEPl
(PREDICTED)
0 GLY-GLY-PHE-LEU
+
NOREPI
(EXPERIMENTAL)
g
&
a
0
l l
7 6-
s *
5
ii
4-
+
3-
0
slo -
m m
0
l
mu m
0
9
l
cla
0
l
1-
on
0
n
0
l
CID
0
0
2-
OR
0
0
F
a
m
0
-
-8 SlD
0 0
l
0 0
08
0
0
!iii
om
0
l
om
0
0
l 2”
008
8
157
I
7.0
8.0
FIG. 13. pH titration study of Met-enk and des-Tyr-Leu-enk + NE free acid. Predicted values are averages of the titration results obtained for each compound separatety. Experimental values are actual measurements of I:1 combinations of the solutions used for the individual measurements. (See text for detailed description of the technique.) The Met-enk + NE experimental and predicted values clearly differ to a much greater degree than do the des-Tyr-Leu-enk + NE values, especially close to pH 7.0. These results suggest that NE is bound to Met-enk between pH 6.0 and 7.0, whereas very little NE binds to des-Tyr-Leu-enk above pH 6.5. These results accord with the NMR results (Figs. l-7).
binding. If model 2 is correct, then -X- would have to be Phe, Tyr, or possibly even Trp, so that a “molecular sandwich” can be formed, and the N-terminal amine could be substituted without affecting binding. The testing of these two models is important, since it is necessary to determine whether or not NE binding capacity is an absolute correlate of opiate activity or only an approximate one. One indication that opiate activity is a correlate of catecholamine binding is that morphiceptin (Tyr-Pro-Phe-ProNH&, another opiate peptide, also binds DA and NE. The same sort of “molecular sandwich” modei just proposed for NE binding to enkephalins is also the most probable model for NE binding to morphiceptin. Unfo~unately, much less is known about the preferred conformations of morphiceptin in solution so that less can be said about the observed shifts. The model presented in Fig. 20 is therefore tentative. The major feature of the NE-morphiceptin spectra were that no shifts occurred in any Pro peak with the exception of a single gamma hydrogen; there were no changes in the amide peaks; and most of the aromatic peaks, including those of NE, became significantly less sharp. These features are all accounted for by the model in terms of pi-pi overlap bonds (which would delocalize the aromatic hydrogen peaks), a NE hydroxyl-morphiceptin carbonyl hydrogen bond, an NE amine-morphiceptin Tyr hydroxyl bond, and an NE hydroxyl-mo~hiceptin terminal amine bond. In terms of the number of bonds formed, the proposed mode1 is, in fact, a
more stable one than either of the proposed NE-enkephalin models. This is consistent with the observation that even a slight excess of morphiceptin suffices to retard NE and DA color reactions, whereas a ten-fold excess of enkephalins is necessary. The NMR, pH titration data, and coiorimetric data afso provide criteria for building possible models of morphine-NE binding. Once again, CPK models were built. Both the pH titration data and the calorimetric data indicate that the mechanism of binding between morphine and NE is such that it involves a significant number of hydrophilic groups. This observation is p~icularly stren~hened by the ability of the rno~~ne-NE combination to act like a buffer (that is to share electrons). This consideration severely limits possible models. A second, key observation is that morphine (Fig. 21) binds NE, but that the opiate antagonist, and chemical cousin of morphine, naloxone (Fig. 22), does not. The most logical explanation of these observations is that the hydroxyl that is present on naloxone, but not on morphine, is sufficient to interfere with the NE binding site, by encroaching upon the binding site, or that the alteration of a hydroxyl to a carbonyl alters the ring conformation sufficiently to make binding to NE less likely. In fact, model building suggests that both effects contribute to the inability of naioxone to bind NE, as can be seen by comparison with the model of NE-morphine binding
ROOT-BERNS’l‘i:IN
/
l
Morphine+Norepinephrine(expected)
o Morphine+Norepinephrine(exptl.)
1
a Naloxone+Norepinephrine(expected)
q
12
Naloxone+Norepinephrine(exptl.) .
ml0
.
11’ I
n
.
0
0
cm
0
e
0
l l
q
.cl.
0
0
0.
I
6.8
7.0
7.2
7.4
0
.7.6
0
w 7.8
I 8.0
FIG. 14. pH titration study of morphine sulfate and its antagonist, naloxone hydrochloride + NE free acid. Expected values are the averages of the titration values obtained for each compound separately. Experimental values are actual measurements of 1: 1 combinations of the solutions used for the individual measurements. (See text for detailed description of the technique.) The naloxone + NE expected and experimental values are virtually identical, suggesting that no binding occurs. This result accords with the NMR study of naloxone + NE (Table 1). The morphine + NE values, however, differ almost 1.0 pH at pH 7.0, and the combination behaves like a buffer. Since buffers share electrons, these results strongly suggest that morphine binds to NE in an ionic complex. The NMR results also indicate that binding occurs between morphine and NE (Fig. 11).
shown in Fig. 23. The major features of this model are extensive pi-pi overlap bonds, a hydroxyl-hydroxyl hydrogen bond, and an amine-hydroxyl bond. Pi-pi overlap bonding is severiely impaired by the addition of the hydroxyl to naloxone, and the amine-hydroxyl bond is eliminated by the alteration of the morphine hydroxyl to a carbonyl, which not only alters the affinity, but causes a conformational shift in the ring that puts the carbonyl out of bonding range of the NE amine. (This latter effect is not obvious from the drawings, but becomes evident with the CPK models.) The most striking feature of the proposed models of enkephalin, morphiceptin, and morphine binding to catecholamines is the possibility that such binding ability is a common feature of all opiates and opioids, which differentiates them from their structurally similar, but inactive analogues, and from their antagonists. Some support for this hypothesis comes from the fact that of the enkephalins and enkephalin fragments, and enkephalin-like peptides studied in these experiments, binding or lack of binding correlates exactly with observed opiate activity [ 161. Nonetheless, the hypothesis will require a great deal more research before it can be considered valid. At least it provides a testable hypothesis that could be of great value in the design of novel opiates and peptides possessing opiate activity.
Co-Trunsmission and the Physiological oj’opiate Activity
Correlates
Now, what may be the physiological meaning of the binding of catecholamines to enkephalins and opiates? To properly consider this question requires some basic knowledge of the anatomical distribution of the catecholamine releasing neurons, catecholamine receptors, enkephalin releasing neurons, and opiate receptors, as well as the physiological effects of opiate treatment. Unfortunately the literature on these subjects is so vast and so full of contradictions that no attempt will be made to summarize it all here. Several salient features of the results relating to these topics will suffice to indicate how the present chemical study may complement previous approaches to these questions. To begin with, a considerable degree of overlap exists between the anatomical distribution of catecholamine neurons and receptors, and enkephalin releasing neurons and opiate receptors (Table 3). Within the brain, DA and NE are present in unusually high concentrations in the hypothalamus, caudate nuclei, and putamen, striatum, septum, and nucleus accumbens [19,36, 39,51,60,80]. These same brain regions also contain unusually high levels of enkephahns and/or opioid receptors [8, 12, 25,28, 32,34,58,60,77]. For
CATECHOLAMINE
523
BINDING
. APOMORPHINE+NOREPI
(EXPERIMENTAL)
o APOMORPHINE+NOREPI
(PREDICTED)
0
0
16
0
0
15
0
0
14
0
0
13
0
0
12
0
0 0
0
0
0
a
0 0
0 0
0 0
0
7.6
7.8
PH
FIG. 15. pH titration study of apomorphine + NE free acid. The predicted values are the average of the titrations of each compound separately. The experimental values are actual measurements of 1: 1 combinations of the solutions used for the individual measurements. (See text for detailed description of the method.) Clearly, the predicted and experimental values differ considerably, indicating that apomorphine binds to NE. This result accords with the results of the NMR study (Table 1).
example, opiate receptor densities are highest in regions such as the amygdala, which is densely ennervated by NE and DA releasing neurons [19]. Other organs, such as the
adrenal medulla, display a similar correlation of catecholamines and opioid receptors [57,81]. Furthermore, the three tissues most often used to test for opiate activity, the iris, vas deferens, and ileum, are all extremely high in either NE or DA compared to other body tissues [39]. There is very little correlation between opiate peptide concentrations and/or opiate receptors densities and serotonin or acetylcholine concentrations in particular brain regions or other organs [19, 36, 801. Not only are catecholamines and opiates or opiate receptors correlated anatomically, they are also, in some cases at least, co-stored and co-transmitted. Among the tissues or regions in which co-storage and co-release of NE and enkephalins have been reported are the sympathetic ganglia, adrenal medulla, and SIF cells [ll, 33, 45, 57, 811. DA is known to be co-stored with enkephalins in the carotid body [33,57]. Physiological evidence also links catecholamine and opiate activity. Extensive evidence exists to indicate that
DA neurons regulate the level of Met-enkephalin and conversely that Met-enkephalin levels regulate catecholamines in various regions of the brain including the striatum, the caudate nucleus, the nucleus accumbens, and tuberoinfundibular neurons [6, 23, 26, 35, 52, 56, 59, 791. The enkephalins appear to mediate stress-induced increases in dopamine levels associated with pain in a naloxonereversible manner [50]; Met-enkephalin inhibits dopamine induced LHRH release [71]; and Met-enkephalin antagonizes Jopamine control of MSH activity [72]. Similarly, large doses of morphine appear to increase synthesis, release, and turnover of catecholamines 11, 14, 15, 24, 29, 43, 62, 751; catecholamine neurons mediate the effects of morphine [3,5, 18, 27, 441; and the catecholamine levels in the brain appear to rise as a function of withdrawal of opiates [2,7,40, 621. It is believed that the effects of morphine on catecholamine release and turnover occur pre-synaptically [9,75]. These opiate-catecholamine interactions have correlates observable in temperature changes and dilation of the pupils and other physiological functions. In short, the chemical complementarity of the opioids and catecholamines is reflected in an anatomical and physiolog-
R001‘-BERNSTEIN
H2O MOR EPI NE DA PLATE I. One of several experiments utilizing the fact that the catecholamines DA, EPI. and NE oxidize and polymerize into colored products such as melanins. Addition of morphine (MOR) (2:1 by molarity) greatly retarded the color reaction for about a week, whereas water (H,O) and tyrosine (TYR) had no effect. Experiments were carried out at pH 7.0 in phosphate buffer. Photograph was taken one week after the experiment was set up. After a month, the morphine-treated catecholamines had also turned dark brown, indicating that morphine retards but does not prevent the color reactions from occurring. In short. the binding is reversible. (See text for discussion.)
ical complementarity of function. Can this be by chance, or has the evolution of the opioid and catecholamine systems been regulated by their chemical cooperativity? There are a number of possible responses to these questions, none of which are mutually exclusive. First, it is possible that the co-storage and co-release has a simple chemical function: to stabilize the catecholamines against oxidation and against melanin formation, and to stabilize the enkephalins against enzymatic degradation. The ability of enkephalins to retard oxidative color reactions involving the catecholamines has been demonstrated by the color reactions reported here; and the binding of a catecholamine by an enkephalin would most likely retard enzymatic cleavage by making the cleavage sites unavailable. The latter effect has yet to be tested, but should not be difficult to demonstrate. These chemical protection functions may be manifested in storage vesicles or intrasynaptically. A second possible explanation for co-storage and corelease may be to modulate the activity intrasynaptically or at the receptor. The effect of the binding would be to allow only a relatively slow release of either the catecholamine or enkephalin to the receptor, as compared with the amount of either that would reach the receptor if released alone. Similarly, release of a chemically complementary pair of neurotransmitters from separate neurons into a common synapse would also result in modulation by binding. A third possible explanation of the co-transmission may be an evolutionary one: that the complex release, re-uptake, and receptor systems that we observe today are highly elaborated forms of extremely simple chemical transmitting
PLATE 2. Experiment showing the ability of morphiceptin to retard NE oxidation-polymerization into colored products. Experiment carried out at pH 7.0 in phosphate buffer. Photograph taken at one week. By one month, the morphiceptin-NE combination had also turned dark brown, to match the NE tube. Thus. morphiceptin retards but does not prevent the color reactions from occurring. In short, the binding is reversible. Notably. about ten times as much enkephalin was necessary to produce the equivalent retardation of the color reaction, indicating that the binding of morphiceptin to NE is on the order of ten times that of the enkephalins for NE. and so on the order of morphine binding to NE (i.e., 4~ IO’lmole).
met
MET FIG.
(1)
16. A model of one of the many preferred
Met-enk (Tyr-Gly-Gly-Phe-Met). [411.
- ENK
_
conformations of After Jones, Gibbons and Garsky
CATECHOLAMINE
525
BINDING
tyr
MET-ENK
(2)
FIG. 18. A second model of one of the preferred conformations of Met-enk, based upon calculations by Isogai, Nemethy and Scheraga
[381. MET-
ENK
+ NOREPI (model
1)
FIG. 17. One possible model for a Met-enk + NE complex, involvamine bonding; ing NE-hydroxyl + Met-enk-N-terminal hydrophobic interaction between the Met-enk Met-CH, and the NE ring; pi-pi overlap bonds between the Met-enk Tyr’ and the NE ring; and a Met-enk Tyr’ hydroxyl + NE amine bond. In a Leu-enk + NE complex, the only difference would be that the Met CH,-NE ring interaction would be replaced by a Leu CH,-Ne ring interaction. This model explains many of the shifts observed in Figs. 1, 2,4 and 5, but does not account for the non-aromatic peak splitting in the NE spectra, the shifts and broadening of the- Tyr beta Hs, or the broadening of Gly and Phe peaks of Met- and Leu-enkephalins at higher concentrations of NE.
utilized by the very first living organisms. If so, the observed binding of co-transmitters may have no particular function today, but may simply be an artifact of the evolutionary history of the particular system. Nonetheless, the observation of binding between the two co-transmitters would still be a valuable piece of information for piecing together the chemical criteria that may have shaped the evolutionary process. One way to test this sub-hypothesis would be to determine the phylogeny of co-transmission throughout the vertebrates and into the invertebrates. It is possible that the lower invertebrates may utilize a simplified chemical version of the complex receptors systems found in vertebrates, and therefore may mimic more closely the direct chemical modulation suggested here. Fourth, it is possible that the binding of catecholamines and opiates is neither chance, modulatory, nor an artifact of history, but essential to the function of the opiates as blockers of neurotransmission. What if the chemical binding of the opiates to catecholamines were to effectively hinder either the release of the catecholamine, their reuptake, or their ability to reach their appropriate receptor? Then the opiates would cause functional blockade of neurotransmission. Anaesthesia is, in fact, one of the effects of high doses of morphine, which explains its use in treating chronic pain. This final possibility necessitates consideration of the mechanism of opiate activity and, in consequence, questions of drug design. systems
MET-
ENK + NOREPI (model
2)
FIG. 19. A second model of a possible Met-enk + NE complex. Extensive conformational shifts are required from the preferred conformations illustrated in Figs. 16 and 18, but it must be remembered that the enkephalins are extremely flexible in solution, so that all possible rotamers of the Tyr, Phe, and Met side chains are present at any given time [41]. This second model of the complex is preferable to the first (Fig. 17), although both may exist at the same time, for the following reasons: greater pi-pi overlaps are produced by this model; the splitting of the NE non-aromatic Hs is explained by van der Waals interactions with the Met CH, (or Leu CH,); the Tyr beta Hs are now facing into the interior of the complex, explaining the shifts in their peaks; and the conformational shifts are appropriate to explaining shifts in the Tyr and Phe alpha Hs. In short, all of the observed spectral changes can be accounted for by this “molecular sandwich” model [63, 64, 661.
Possible Mechanisms and Withdrawal
of Opiate Activity Vis-a-Vis Addiction
The usual explanation of opiate activity is that opiates and opioid peptides bind to a number of distinct classes of opiate receptors [12, 16, 47, 541. While this may be true, it in fact merely pushes back a step the question of how the opiates
RW’I
-HERNSl‘l
NALOXONE MORPHICEPTIN
+ NOREPI
FIG. 20. A “molecular sandwich” model [63, 64, 661 (Fig. 19) of morphiceptin (Tyr-Pro-Phe-Pro-NH,) binding of NE. Since only a single hydrophobic interaction (involving a Pro gamma H peak) is indicated by the NMR data (Figs. 9 and lo), the model accounts for this by proposing that the conformation induced by NE binding requires that a Phe aromatic H interacts with the Pro” gamma Hs. Since peak broadening is also observed in the aromatic region, this interaction is acceptable. Otherwise, the model postulates that NE binds to morphiceptin by extensive pi-pi overlap bonds (which would account for the other observed shifts in the aromatic region of both molecules); an NE terminal amine bond to the Tyr’ hydroxyl; a morphiceptin terminal amine bond to the NE hydroxyl (which may be shared with a morphiceptin hydroxyl as well, to stabilize the conformation: indicated by the open circles); and a morphiceptin hydroxyl hydrogen bond to one of the NE ring hydroxyls. The total number of bonds formed in the morphiceptin-NE complex is greater than in the enkephalin-NE complexes (Figs. 17 and 19), suggesting that the bonding should be stronger.
FIG. 22. A structural model of naloxone, a morphine antagonist. Note the moditications compared with morphine (Fig. 21).
“,C
MORPHINE-
NOREPI.
COMPLEX
FIG. 23. A structural model of NE binding to morphine based upon CPK model building. The interaction involves extensive pi-pi overlap bonds, as well as a pair of hydrogen bonds from the NE to the morphine hydroxyls. Note that the small structural modifications made to morphine to produce naloxone are such as to interfere seriously with NE binding to the latter compound. The additional hydroxyl prevents pi-pi overlap bonding, and the NE amine bond to the morphine hydroxyl is no longer possible since the hydroxyl has been replaced by a carbonyl, which also alters the conformation of that portion of the ring in such a way as to shift the oxygen radical out of the range of the NE amine.
H,C
MORPHINE FIG. 21. A structural model of morphine.
and opioid peptides function. How, for example, does the opiate receptor cause anaesthesia? Why does it bind naloxone, but fail to respond to naloxone binding as if it were an opiate? Furthermore, the receptor hypothesis certainly does not address how such a complex system could have evolved. From what simpler polypeptides did receptors originate?
CATECHOLAMINE
527
BINDING TABLE 3 COMFARISON
OF RELATIVE
CATECHOLAMINE, ENKEPHALIN, AND OPIATE RECEPTOR DEW%TIES FOR SELECXED BRAIN AREAS AND ORGANS
Monkeyl
Human*
Rat* Brain Area
NE
DA
OP. Rec.
NE
DA
5HT
ENK
OP. Rec.
Cerebral cortex Caudate nuclei Hypothalamus Amygdala Cerebellum Globus pallidus
0.18 0.27 1.79 0.07 -
0.01 6.39 0.14 0.03 -
1.1 il.5 3.8 6.5 0.8 -
0.09 1.25 0.21 0.01 0.15
3.50 0.29 0.26 0.23
0.04 0.33 0.80 0.60 0.00 0.50
0.46 5.28 4.20 1.68 0.45 4.94
6.0 19.4 24.3 49.0 <2.0 7.7
Organ or Tissue* Whole brain Whole brain Vas deferens Iris Skeletal muscle Skeletal muscle Heart Heart
Species
NE
Dog Rat Guinea Pig Rabbit Rabbit Cat Rat Rabbit
0.16 0.49 10.0 5.20 0.10 0.10 0.96 2.00
Catechola~nes in microg~s/~m of tissue; enkephalin in units per epigram of tissue; and * opiate receptor densities in terms of amount oflabelied morphine bound per mg of protein (monkey brain), or per 100 square microns of tissue slice (rat). Sources: *[39], 1[58], $[36], 8[77].
In fact, it appears that Vichy nothing is known about the opiate receptors [76] except their reiative affinities for different opiates (e.g., [16]). Without questioning the existence of such receptors and their functions, it may nonetheless be useful to consider some other chemical features of the opiates that may influence opiate activity. As I mentioned above, it appears possible that a common feature of opiates and opioid peptides is their ability to bind to catecholamines. If true, this phenomenon raises the possibility that opioids may produce some of their effects, such as interference with nerve transmission, by directly binding up of the neurotransmitter. What evidence exists to support this speculation? First, as detailed above, the effects of opioids are highly correlated with brain regions and other organs that utilize catecholamines as their primary neurotransmitters. Thus, direct interference with catecholamine neurotransmission is an anatomical possibility. In addition, at least two enkephalin analogues (D-AlaZ-Met-enkephali and N-CH,-Metenkephalin) both, “show considerable resistance to inactivation by peptidases and after intravent~cu~r or intracerebral admin~st~~on exhibit antinociceptive activity n>hich therefore does not seem to be closely and exclusively related to their aflnity to receptors represented by either naloxone or leucine-enkephalin binding” [47]. If activity is not a direct function of receptor affinity, then what controls it? Furthermore, the activity of any given opiate or opioid peptide differs with the brain region or the type of tissue (e.g., mouse vas deferens or guinea pig ileum) upon which it
is tested 112, 16, 471. Why should opiate or opioid peptide activity vary from one brain region or organ to another? Certainly a diversity of receptors is a possibility (though not one in accord with Occam’s razor), but might not there be other possibilities as well? These observations raise the possibility that opiate activity is mediated, at least in Part, not on& by receptor types but by the co~en~tions of resident catecholamines. Different opiates and opioids will display different afRmities for the resident catecholamines, just as morphine differs from the enkephahns in its affinity for NE and DA. There is even the possibility that so-called receptor types are actually comparative measures of the mix of catecholamines being used as neurot~smitters in the particular brain region or organ being tested: some morphine derivatives may have significantiy greater affinity for NE than for EPI or DA, for example. The presence or absence of serotonin or acetylcholine may affect affinities. Regardless of the validity of the speculations just presented, it is certainly true that the presence of very high concen~ations of catechoiamines in the tissues used to measure opioid binding (e.g., vas deferens and ileum) must affect the observed receptor binding constants. This is a necessary consequence of the demonstration that opiates and opioid peptides bind to the catecholamines. This binding to the catecholamines will, perforce, create a competition between the catecholamines and the opiate receptors for the opiate molecules. It is therefore highly unlikely that any reported a.@mity of an opiate for its receptor is accurate, and
ROOT-BERNSTEIN further studies will have to be undertaken to determine to what extent competitive binding from the catecholamines affects the reported values. A second characteristic of the opiates and opioids must also be considered. Both morphine and the enkephalins are highly hydrophobic (the water solubility of Met-enkephalin is <0.7%~ [70]) [4]. In combination with the catecholamines, the solubility is probably even lower, because most of the hydrophilic residues of both compounds are involved in the binding that forms the combination (N.B.: this effect remains to be demonstrated). In short, opioids, and opioids combined with catecholamines, are very likely to be lipophilic. In fact, studies of the interactions of enkephalins with lipid-like media have been performed, demonstrating that very signilicant interactions take place, correlated with extensive alterations in enkephalin conformation [4]. Indeed, it is important to note that of the several hundred substituted forms of enkephalins that have been synthesized, the most active are those multiply substituted with hydrophobic residues. The most potent enkephalin yet synthesized is (Me-Tyr)1-(D-Ala)2-Gly”-(Me-Phe)~-(Met-(0)-ol)Z, which is 1000 times as potent as morphine and 68,000 times as potent as Met-enk in the tail flick test. Eliminating the methyl group from the Tyr’ reduces the potency in half. Eliminating the D-Ala’ residue further decreases potency. Also, hydrophilic substitutions, such as pCl-Phel, decrease potency to below the level of unsubstituted enkephalins [16]. In short, as long as the residues necessary for catecholamine binding are respected (e.g., the Tyr’ hydroxyl, the Gly”. the Phe” aromatic ring, and a hydrophobic fifth side chain), activity appears to be maintained; and the more hydrophobic substitutions that can be made, the more potent the resulting molecule. The lipophilicity of opioids is an important characteristic for the following reason. Franks and Lieb [22] have noted that the anaesthetic properties of compounds increase with lipophilicity, and have suggested that one general mechanism for anaesthetic activity may be incorporation into the lipid membrane of neurons in such a way as to interfere with neurotransmission. The lipophilicity of the opioids would allow at least some of their activity to be explained by the Franks-Lieb mechanism, particularly if the lipophilicity is increased by combination with a catecholamine. The combination would be highly stable, for the reasons discussed above, and incorporation into neuron membranes would further stabilize the combination. Incorporation into the membrane of the releasing neuron could interfere with reuptake and release; incorporation into the receptor neuron would interfere with the function of the receptor. Now, what would be the biochemical consequences of the binding up of the catecholamines by opiates and opioids, particularly if release and reuptake were impaired? If we assume that the function of metabolism is to maintain homeostasis, then the result would be that the system would act as if it were producing too little catecholamines. Production of catecholamines would increase to a level sufficient to counter-balance the amount of opiate or opioid added to the system. Under normal physiological conditions, a balance would be reached between catecholamine and enkephalin (and endorphin?) metabolism. Exogenous additions of opiates or opioids would result in abnormally increased catecholamine production. Subsequent withdrawal of the opiate or opioid would then leave the production of catecholamines too high, resulting in the symptoms associated with narcotic withdrawal. In fact, these metabolic interactions between
catecholamines and opioid peptides, and these metabolic changes produced by exogenous opiates, are exactly what has been reported in the literature [I, 2, 7. 9. 14. 15. 17. 18. 24. 26, 27, 29, 40, 44. 54, 55. 751. Indeed. the metabolic scenario just sketched exactly fits the theoretical descriptions made previously by several investigators to explain narcotic addiction and withdrawal. For example. in 1943 Himmelsbach suggested that physical dependence to opiates might be a “condition of autonomic hyperreaction” characterized as follows: I. The prime function of autonomic (hypothalamic) centers is to maintain homeostasis and to make proper adjustments in the face of stress. 2. Morphine affects homeostasis through its action on these centers. 3. Autonomic reaction to this effect takes place. (Biological reactions generally are greater than necessary to overcome an effect.) 4. With repetition the ability to offset the opiate effect improves. (Physiological tolerance.) 5. An extension of this process of improved reaction results eventually (with larger and more frequent dosage) in disproportionate autonomic strength in checks and balances. 6. Thus a condition is created wherein a chemical is needed to maintain homeostasis; such reactive power having been developed that to preserve equilibrium there must be present an effect to counteract. 7. Since the body is unable to supply a counter-effect promptly it must be furnished from without, else equilibrium will be lost temporarily. Such loss of equilibrium results in an abstinence syndrome (withdrawal) [31]. Very similar models have since been suggested by Kauffman, Koski and Peat [42], and by Nichols [53]. Given appropriate adjustments, these models of addiction and withdrawal could be explained by the opiatecatecholamine binding mechanism proposed here, with one important additional feature: if the opiate-catecholamine complex actually incorporates into lipid membranes and is stabilized there, then an ever-increasing amount of catecholamine metabolism disruption would ensue as more and more opiate became incorporated into the membrane. The result would be an ever increasing amount of catecholamine production. In turn, ever-increasing amounts of opiate would be needed to offset withdrawal symptoms caused by ever higher production of catecholamines. One extremely important point must be stressed at this point. While the synthesis and turn-over of catecholamines would increase significantly with long-term, high-dose morphine treatment, it is not at all clear that the measurable amounts of catecholamines in brain (or elsewhere) would increase. According to the binding mechanism proposed here, the more opiate that is present in the system, the more catecholamine will be bound. Thus, the active, measurable levels of catecholamines will remain fairly constant at normal levels unless the opiate is withdrawn, in which case there will be no counter-balance to the increased catecholamine synthesis. In other words, tolerance to opiates should be accompanied by what appears to be normal levels of rrvailahlr catecholaminas. Tolerance is a return to homeostasis. Another apparent exception to the model just proposed must also be examined. Nichols [53] notes that patients treated for surgery or cancer rarely become addicted to opiates. He suggests a psychological difference between
CATECHOLAMINE
medical patients and abusers. There may also be a chemical difference: since catecholamine neurons are associated with many pain pathways, it is likely that patients in pain have unusually active catecholamine neurons. In consequence, opiate treatment may simply counter-balance an increased catecholamine metabolism induced by the pain. Instead of exogenous opiates creating a disequilibrium in the body’s homeostasis, in this case they return to normal the disequilibrium caused by the pain. In consequence, as the pain decreases with healing, catecholamine metabolism returns to normal and opiate treatment can be tapered off. In short, the patient in pain is in exactly the opposite metabolichomeostasis condition as is the abuser. In summary, opiate-catecholamine complexation provides a simple mechanism for explaining economically the existing literature concerning the anaesthetic, addictive, and metabolic effects of opiates. Drug Design in Light Complexation
529
BINDING
ofOpiate-Catecholamine
The opiate-catecholamine binding phenomenon may also have implications for the design of new opiate drugs. To date, most researchers who have compared the opiates and opioid peptides have attempted to find structural similarities between the two [46, 70, 741, with regard to receptors the characteristics of which are still totally hypothetical [76]. While it is possible to obtain some overlap between certain morphine derivatives (but not morphine itself) and some conformations of enkephalins, none of these investigators have been able to explain why naloxone should act as an antagonist rather than as an agonist. It is also very difftcult to make morphiceptin conform to the proposed structural models of the “ideal opiate,” and no one has apparently reported doing so. I would therefore like to propose a different set of criteria for opiate activity: (1) ability to bind catecholamine neurotransmitters; (2) ability to disrupt neurotransmission by incorporating into lipid membranes in combination with catecholamine neurotransmitters; (3) naloxone antagonism. These criteria move the search for opiate drugs away from specitlc structural similarities to a set of similar chemical properties. For example, it is possible that a peptide sequence such as Leu-Tyr-Thr-Phe-Leu-NH,, although quite different from the enkephalin sequences, might be highly active as an opioid; or that increasing the lipophilicity of morphine derivatives by adding methyl groups while adding another residue capable of forming a hydrogen bond to the amine ring hydroxyls might increase activity. Another possible advantage of these criteria would be to make analogous criteria possible for other sets of anaesthetits and antinociceptive drugs. For example, it is quite likely that there is a set of lipophilic compounds that selectively bind to and interfere with serotonin and its metabolism, or to acetylcholine interfering with its metabolism. Thus, the opiate-catecholamine complexation model may provide a paradigm for the elucidation of the activities of other drugs. Regarding this last point, it is worth mentioning again that at least two other cases of complexation between a biologically active chemical and an antagonistic or modulatory drug have been reported 165,731. In both cases, the reported binding is virtually identical to the morphine-catecholamine model suggested here: pi-pi bonding associated with ionic or hydrogen bonds between specific side chains or residues. Thus, it may be possible to specifically design drugs having
the appropriate stereochemical properties to bind to and inactivate any given aromatic neurotransmitter or related compound. The “molecular sandwiching” model of intercalation of aromatic amines into pockets of aromatic side chains on peptides may also provide a generalizable model for synthesizing peptides possessing specific modulatory activities. Co-Transmission Hypothesis
and Complementarity:
A General
The issue of “molecular sandwiching” raises some other interesting possibilities. Note that cholecystokinin, which contains the DA binding sequence Trp-Met-Asp-Phe-NH, (gastrin tetrapeptide: Table 2), is known to be modulated by DA and, in turn, to modulate DA activity in vivo [20, 21,49, 821. Also, substance P, which was found to bind DA and NE (Table 2), has been shown to produce analgesia [32], and is a reasonable substitute for morphine in tolerant rats [78]. Might these peptides represent further examples of peptides that bind catecholamines into “molecular sandwiches”? Although further research will be necessary to confirm the DA-cholecystokinin interaction, and the specificity of substance P for DA and NE (or SHT?), I would like to make a general prediction based upon the present study and previous research concerning serotonin-binding peptides. Many other instances of co-transmission have been reported, including co-storage or co-release of substance P-serotonin, cholecystokinin-DA, somatostatin-NE, TRH-serotonin, and neurotensin-NE [33,57]. I would like to suggest that a general property of all co-transmitters is that they are chemically complementary in such a manner that they can modulate one another’s activity by direct chemical interaction. Thus, physiological complementarity of function will be mirrored in stereochemical complementarity of molecular forms. No doubt this prediction will turn out to be overly simplistic, but it represents a powerful tool for inventing hypotheses amenable to straightforward experimental analysis. It is better to search for simplicity and discover that you are wrong than to remain inactive in the face of a vast, disordered array of confused observations. If the present research leads to greater understanding by providing clues to the order of Nature, it shall be successful, whether the outcome is to validate or refute the initial concept. FINAL CONCLUSIONS
To summarize, my general conclusions are as follows. The experiments reported here demonstrate clearly that morphine, the enkephalins, and morphiceptin bind to the catecholamines DA, EPI, and NE. This observation suggests the possibility that all opiates and opioids have catecholamine-binding capacity, and that one of the mechanisms of their activity is to modulate catecholamine neurotransmission and biosynthesis by direct binding. This binding mechanism, along with the observed lipophilicity of opiates and opioid peptides, provides a model for explaining the biochemical changes observed during addiction and withdrawal, and the differences observed between the use of * opiates for treating pain and as abused substances. The binding may provide a paradigm of the elucidation of the mechanisms (or partial mechanisms) of action of other drugs affecting neurotransmitters. The binding may also provide a simple chemical paradigm for understanding and explaining other instances of co-transmission such as those involving
570
ROOT-BERNSTEIN
DA-cholecystokinin, TRH-serotonin and substance P-serotonin. The basic concept underlying the interpretation of all of the above features of co-transmission is that physi-
ological complementarity of function stems from stereochemical complementarity of the molecular forms.
ACKNOWLEDGEMENTS 1 thank
Dr. Jonas Salk of the Salk Institute for Biological Studies in La Jolla, CA and Dr. Arthur Yuwiler of the Brentwood Veterans Administration Hospital, Los Angeles for the use of their laboratory facilities; the Southern California Regional NMR Facility at the California Institute of Technology, Pasadena, CA which operates under NSF Grant No. CHE 79-16324; the Institute for Disease Research (IDR) for chemicals and equipment; and Dr. Fred C. Westall of the IDR for his suggestions. David Felten. M.D. of the University of Rochester School of Medicine made the original suggestion that I look into the question of co-transmission, thus beginning the train of thought and research elaborated here. This research was made possible by a MacArthur Prize Fellowship to the author from the John D. and Catherine T. MacArthur Foundation, Chicago, IL.
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L. C.. S. P. Mennin, R. Selinfreund and P. Rasmussen. Interaction of beta-endorphin, naloxone and dopamine: effects on melanocyte-stimulating hormone secretion of amphibian pituitaries in vitro. Rc,yu/ Pep! 3: 371-381. 1983. Sayer. J. M.. H. Yagi, A. W. Wood. A. H. Conney and D. M. Jerina. Extremely facile reaction between the ultimate carcinogen benzolalpyrene-7,8diol-Y.lO-epoxide and ellagic acid. .I Am C~iicm Sot, 104: 5X2-5564. 1982. Schiller. P. W.. C. F. Yam and M. Lis. Evidence for topographical analogy between methionine-enkephalin and morphine Jerivativea. Rioc~lrc~mi.\rr~ 16: 1831-1838. 1977. Smialowski. M. and A. Bal. The effect of morphine on 5hydroxytryptamine and catecholamine neurons in the rat brain. Histofluorescence studies. .I h’~rrrcll Trtr/t.\nr 59: 25-41. 19x4. Smythies. J. R. On the molecular structure of the opiate receptot I’.\> c~h~~r~c~~rr~~rr~t~~~~ri~~~~l~~~~ 2: 71-72. 1977. Snyder, S. H., R. Simantov and G. W. Pasternak. The brain’s own morphine, “enkephalin”: a peptide neurotransmitter? ~l’C,//~lI\(~i .S~rn/, I: 82-98, 1976.
7X. Stewart. J. M.. C. J. Getto. K. Neldner, b.. 13, Reeve. W. A. Krivoy and E. Zimmerman. Substance P and analgesia. \~~rrrr<’ 262: 784-785. 1976. 79. Thal. L. J.. N. S. Sharpless, I. D. Hirschhortt. 5. ti. Horowttz and M. H. Makman. Striatal met-enkephalin concentration inHioc /I(,,,~ f’hrrr-L:reases following nigrostriatal denervation ,tcric~cd 32: 3297-3301. 1983. 80. Ungerstedt. U. Striatal mapping of monoamine pathways in rat brain. Ac,rtr Ph~.sio/ .Sc,trnd [Supp/] 367: I-122, 1971. XI. Viveros. 0. H. and S. P. Wilson. The adrenal medulla: a model for studying synthesis, co-storage and co-secretion of peptides and catecholamines. In: ~o-7~cr~1.smi.t.sion. edited hy A. C. Cuello. London: Macmillan, 1982, pp. 127-150. 82. Voigt. M. M. and R. Y. Wang. In vivo release of dopamine in the nucleus accumbens of the rat: modulation by cholecystokinin. Hrtritr Kc.\ 2Y6: 189-193. 1984.
Bulkfin,
Vol. 18, pp. 5-532.
0361-9230/87 $3.00 + .@I
B Pergamon Journals Ltd., 1987.Printed in the U.S.A.
Catecholamines Bind to Enkephalins, Morphiceptin, and Morphine ROBERT
SCOTT
ROOT-BERNSTEIN’
Neurobiochemistry, T-85, Veterans Administration Hospital, Brentwood 11301 Wilshire Boulevard, Los Angeles, CA 90073 Received
17 September
1986
ROOT-BERNSTEIN, R. S. Catecholamines bind tu enkephalins, morphiceptin, and morphine. BRAIN RES BULL lS(4) 509-532, 1987.-Nuclear magnetic resonance spectroscopy, pH titration, and color reactions demonstrate that the catecholamines dopamine, epinephrine, and norepinephrine bind to the enkephalins. Binding constants are c. 6~ 103,per mole. Catecholamines also bound to the mu opiate receptor agonist morphiceptin (Tyr-Pro-Phe-Pro-NH,). Very little binding was found to enkephalin and morphiceptin fragments and analogues, indicating that the entire molecules are necessary. Serotonin binding peptides do not bind the catecholamines. Morphine and apomorphine, however, do bind these catecholamines (with a binding constant for morphine of c. 4x lo* per mole). The opiate antagonist naloxone and a number of other drugs do not bind catecholamines. Morphine, morphiceptin, and the enkephalins also retard the formation of colored reaction products by catecholamines in vitro. These results may help to explain observations that the enkephalins are co-stored and co-transmitted with dopamine and norepinephrine, and may provide a basis for the elucidation of other known cases of peptide-monoamine co-transmission. Possible implications for understanding opiate effects on catecholamines during addiction and withdrawal are discussed, and suggestions concerning drug design are made. Co-transmission
Opiate addiction
NMR
Drug design
binding sites
specifically to the serotonin and SHIAA precursor, 5-hydroxytryptophan [65]. The enkephalins, which were used as controls in previous experiments, did not appreciably bind serotonin or related indoles [66]. Preliminary experiments using nuclear magnetic resonance spectroscopy, however, yielded evidence that both Met-enkephalin and Leu-enkephalin bind dopamine (DA), epinephrine (EPI), and norepinephrine (NE) [@I. Further experiments demonstrated that these catecholamines also bound to morphiceptin (a mu opiate receptor agonist, Tyr-Pro-Phe-Pro-NH* [ 13,30]), and morphine [69,71]. Since the enkephalins are often co-stored and co-released with DA and NE, and since both peptide and alkaloid opiates are known to affect catecholamine metabolism (see the Discussion section), I have investigated these chemical interactions in detail with the goal of possibly elucidating a simple, underlying chemical basis for the observed correlations of anatomical localization and physiological interaction. The purpose of this paper is to report the results of some 120 combinations of peptides and drugs with catecholamines demonstrating the specificity of the binding of NE and DA to the enkephalins, morphiceptin and morphine.
WHY are some peptides co-stored or co-released with particular monoamines? Why do such co-transmitters behave as
modulators for each other? Must neurotransmitter activity be expressed solely through complex cellular receptor systems? If so, how did such complex systems evolve? How do exogenous drugs affect endogenous mechanisms of neurotransmitter regulation? Questions such as these have led me to consider the possibility that a simple chemical basis might exist for the anatomical distribution and interactive functions of many neurotransmitters and hormones, as well as for the mechanisms by which some drugs affect neurotransmitter systems. My initial thoughts on this subject were formed by the elucidation of serotonin (S-hydroxytryptamine [SHT]) binding sites on the tryptophan peptide of myelin basic protein, luteinizing hormone-releasing hormone (LHRH), and a sequence common to alpha melanocyte stimulating hormone (MSH) and adrenocorticotropic hormone (ACTH) [63, 64, 661. The activities of each of these peptides are known to be modulated by serotonin. Similar binding sites were located by sequence analysis of serum albumins, which are known to transport tryptophan and serotonin, and the red pigment stimulating hormone of crustaceans, which is also modulated by serotonin [64,66]. Furthermore, it is thought that several drugs may produce their effects by direct binding to a metabolite involved in the pathway disrupted by the drug 1731. An example is the anorexigenic agent fenfluramine, which lowers blood serotonin and S-hydroxyindoleacetic acid (SHIAA) levels in man and animals, and which binds ‘Requests for reprints should be addressed
Catecholamine
METHOD
Seventeen peptides (Table 1) were used to investigate the specificity of binding of catecholamines to peptides. These peptides were chosen for their structural similarities to enkephalins, to fragments of enkephalins, or to peptides previously shown to bind serotonin [66]. All peptides were
to Robert Scott Root-Bernstein,
509
3514 Veteran Avenue, Los Angeles, CA 90034.
ROoT-BERNSTf:IN
obtained from Vega Biochemicals (Tucson, AZ) or BAChem (Torrance, CA), or both. Nine drugs having varied physiological effects were tested for binding to neurotransmitters, Drugs were obtained from the following manufacturers: morphine sulphate and apomorphine hydrochloride (Merck, Sharpe and Dohme, Rahway, NJ); naloxone hydrochloride (Endo, Garden City, NY); desiprimine (USV Pharmaceuticals, Tackahoe, NY); trifluoperazine dihydrochioride (Smith. Kline and French, Philadelphia, PA); pargyline (Regis. Morton Grove, IL); amphetamine D,L sulphate and 3-methoxy-4-hydroxyphenethanol (MOPET) (Sigma. St. Louis, MO). All other chemicals were from either Sigma or Calbiochem (San Diego, CA), or both. All peptides were dissolved in distilled water, 5.0 mg per ml, and then adjusted to pH 7.0 using minute amounts ofO.01 N NaOH and 0.01 N HCI. All other chemicals were dissolved in distilled water, 2.0 mg per ml, and similarly adjusted to pH 7.0. All peptide and other chemical soiutions were immediately frozen at -40 degrees C and kept in the dark until immediately prior to use (usually within a few hours). Nuclear magnetic resonance (NMR) spectroscopy was zarried out using a Brucker WM~~ very high field multinuclear spectrometer at the Southern California Regional NMR Facility, California Institute of Technology. To facilitate ease in preparation of the samples, and so as to have the ability to examine the amide region of the spectra, IH spectra were obtained utilizing the Redfield solvent suppression proce;lure 1611. Usually, spectra for both the region O-4.5 ppm and :he region 5.5-10 ppm were obtained in separate runs using newly mixed samples each time. This procedure assured that most results were verified at least once. All positive results have been obtained at least four times using freshly prepared samples, often from different suppliers as an added control. No internal reference was used in any sample so as to obviate the possibility of unexpected interaction with peptides, drugs, or catecholamines. (This is also the reason that a standard buffer was not employed.) In all cases, the middle af the water peak, which was never completely damped by the Redfield procedure, was used as a reference set at 4.65 ppm. The resulting ppm assignments are not, therefore, exact, but the results were consistent within the study, and previous utilization of this method yielded results in good accord with published spectra [66]. One drop 10.5 ml} of D?O ;99.98% pure, Aldrich, St. Louis, MO) was added to each 0.35 ml of sample to permit spectrometer lock. Standard Five-mm NMR tubes (Gold Label, Aldrich) were employed. At the concentrations utilized here, 500 MHz spectra typically required 256 to 400 scans at 32K data points to achieve the signal-to-noise ratio displayed in the accompanying figures. All spectra obtained were of this quality. or were rerun until they were. Spectra of various concentrations of the enkephalins were obtained, and within the limits of the dilutions employed in these experiments, no peak shifts due to dilution were observed. Further controls were obtained for this particular variable as the various catecholamines and related compounds were added to the peptides. Thus, there is no doubt that the peak shifts that result from the addition of DA, EPI, and NE are not due to dilution effects or to non-specific interactions with sulphate, chloride or other ions. Since these catechols had no effect upon the spectra of most of the ather peptides tested, it is very unlikely that the peak shifts observed in the enkephalins are due to unexpected contaminants such as paramagnetic ions.
Initially, 0.15 ml DA, NE, and the other catechoiamines and neurotransmitters were added to 0.15 ml of peptidc (making in most cases about a one-to-one combination by molarity). If no shifts in any peaks were observed. then the interaction was reported to be negative. If peak shil’[\ did appear, then the interaction was measured a4 ii fraction of the amount of peak shift that was caused by NE. which caused the greatest peak-shift-per-amount-of-chemicaladded (Table 2). The binding constants of the various chemi.. cals to the peptides and morphine was determined by adding 0.01. 0.02, 0.04, 0.08, 0.16. etc. ml aliquots to 0.30 ml of peptide or morphine until saturation was achieved. The percent of the shift compared to saturation at each concentrntion was then plotted in a Scatchard plot (Figs. 3, 7. 17). Peak assignments for the enkephalins were made with regard to 270 MHz NMR spectra previously assigned by Jones, Gibbons and Garsky [4lj, 300 MHz spectra of Rocques c,t crl. 1701, NMR studies of Gly-Gly-X-Ala peptides by Bundi and Wuthrich [IO]. and the spectra of enkephalin fragments such as Gly-Gly-Phe-Leu, Tyr-Gly-Gly. and Phe-Met, which were obtained during the present study. Peak assignments for morphiceptin were made with regard to the enkephalin spectra and the spectrum of Gly-Pro-Arg-Pro 1671. pH titration studies were also performed for a number of the catecholamine-peptide and catecholamine-opiate combinations. Ten mgiml solutions of peptides, opiates, and other drugs were made in distilled water; and approximately equimolar solutions of catecholamines or other neurotransmitters. Aliquots (0.20 m1) of each compound were titrated using 0.04 ml aliquots of 0.0025 N NaOH additively. The pH was measured after each addition of NaOH. Predicted values for the combinations of peptides or drugs with catecholamines were then calculated by averaging the pH values of the individual compounds. Combinations of the original solutions of the compounds were then made by adding 0.20 ml of each. The combinations were titrated using 0.04 ml aliquots of 0.0050 V NaOH. The experimental values were then compared with the predicted values. Theoretically, if the two compounds do not interact in any way, then the experimental values should match the predicted values, since the titration will proceed in the same manner whether the two compounds are in solution together or not. However. if the compounds interact so that hydrophilic residues or side chains are involved, then the bonding of these residues will change their susceptibility to titration and the experimental value will differ significantly from the predicted value. The one difficulty with this technique is that the titration values cannot differentiate between interactions between the peptide-monoamine or drug pairs and the ion pairs if the peptides and monoamines were salted. Thus. all experiments were run with NE or DA free acid. A variety of experiments were also made utilizing the fact that DA, EPI, and NE will oxidize into colored products and polymerize into very dark melanins. Molar excesses of morphine, morphiceptin, Met-enkephalin, Met-enkephalin fragments, and naioxone were added to 1.0 mgiml solutions of DA, EPI, and NE and allowed to stand open to the air for several days at room temperature. (Both test tubes and wells were employed with no difference in results.) A set of controls was also prepared of the individual solutions. The combinations were then compared in color with the individual solutions as a general, qualitative measure of the ability of the various opiates and peptides to prevent and/or catalyze oxidation and melanin formation.
CATECHOLAMINE
BINDING
TABLE 1 SUMMARYOFNMRRESULTSOFBINDINGOF DOPAMINE (DA), NOREPINEPHRINE (NE), AND SEROTONIN TO A NUMBER OF PEnIDES AND DRUGS Norepinephrine Binding + + -
Dopamine Binding
Serotonin Binding
+ + _ _
_ _
_ _ _ _ 2 +
_ _ _ -
_ -
Sequence
Peptide or Drug Met-enkephalin Leu-enkephalin Enkephalin (l-3) Met-enkephalin (2-5) Leu-enkephalin (2-5) Met-enkephalin (4-5) Morphiceptin Substance P Arg
Tyr Tyr Tyr
Pro
Lys
-
+
_ _
_
+
_
-
+
_
+
+
5
+ _ _ _ _
+ _ _ _
_ _
+
_ & -
Proctolin Gas&in tetrapeptide MSH-ACTH tetrapeptide LHRH Trp peptide Morphine Apomorphine Naloxone Desiprimine Trifluoperazine Pargyline Amphetamine Fenfluramine MOPET
Phe Phe Tyr Tyr Pro
Gly Gly Gly Gly
Gly Gly Gly Gly
Phe Phe
Met Leu
Phe
Met
Gly
Gly
Phe Phe
Leu Met
Gly Gly Tyr Pro Gln
Phe Gly Tyr Phe Gln
Gly Phe
Pro
Pro
Gly Pro Thr Phe
Arg
Gly Tyr Trp
Pro Leu Met
Arg Pro Asp
His
Phe
Arg
Trp
His
Trp Phe
Ser Ser
Tyr Trp
Pro Phe
Gly Gly
Phe
Gly
Leu Met NH,
Leu
Arg
Ala
Glh
Pro Gly Gly Gln
NH,
Arg
The results of serotonin binding are from Root-Bernstein and Westall [65,66], in which many other controls may be found. Plus signs indicate large shifts in peaks occur in an equimolar combination. Minus signs indicate that no or only very small shifts were observed. Plus-minus signs indicate that significant shifts occurred, but were not as large as those produced by one of the other neurotransmitters
tested. The major conclusion to be drawn from this table is that catecholamine binding is highly correlated with opiate or opioid peptides, whereas serotonin binding is not. With the exception of morphine, catecholamine and serotonin binding peptides and compounds appear to be distinct.
RESULTS
The results of the NMR study are summarized in Tables 1 and 2. Significant shifts appeared in the spectra of only a limited number of peptides and drugs upon addition of NE and DA (Table 1). These were Met- and Leu-enkephalins (Figs. 1, 2, 4, 5, 6), morphiceptin (Figs. 9 and lo), possibly substance P (although there was some difficulty in obtaining consistent results), gastrin tetrapeptide (which is a sequence common to cholecystokinin as well), morphine, and apomorphine. No significant shifts occurred in the spectra of the enkephalin fragments tested (e.g., Figs. 6 and 8), several enkephalin-like peptides, proctolin, a sequence common to melanocyte stimulating hormone and adrenocorticotropic hormone, luteinizing hormone-releasing hormone, or the tryptophan peptide of myelin basic protein, naloxone, des-
ipramine, tritluoperazine, pargyline, amphetamine, or MOPET. Other controls for the interactions of these hormones and drugs and their analogues with monoamines may be found in previous publications [65,66]. Further research demonstrated that binding constants for NE binding to Met- and Leu-enkephalin were virtually identical: 5.3 and 5.8~ 103/mole, respectively (Figs. 3 and 7). The binding constant for NE binding to morphine was determined to be 4x104/mole (Fig. 12). Using these values, the peak shifts caused by NE at a given concentration of peptide or drug were arbitrarily assigned a unitary value, and the peak shift caused by other catecholamines, indoleamines, and drugs was determined under the same conditions. These peak shifts were assigned a value between zero and one according to the percent of peak shift caused by NE at the same concentration (Table 2). Thus, EPI was found to bind
ROO’I -HERNSTElN
TABLE 2 SUMMARY OF NMR RESULTS OF BINDING OF A LARGE NUMBER OF CATECHOLAMINES, CATECHOLAMINE PRECURSORS AND METABOLITES, OTHER NEUROTRANSMIlTERS, AND VARIOUS DRUGS TO MET-ENKEPHALIN, LEU-ENKEPHALIN. AND MORPHINE
MetEnk*
Phenylalanine Tyrosine L-DOPA D-DOPA Dopamine Epinephrine Norepinephrine 3-OH-4-OCH,,-phenethylamine 3,4-diOH-phenethylamine 3-OH-tyramine 3,4-diOH-mandellic acid Octopamine Homovanillic acid 3-OH-4-OCH,-phenylacetic acid S-OH-indoleacetic acid Serotonin Histamine Acetylcholine Amphetamine Fenfluramine
0 0 0.3 I.0 0.6 0.8 I.0 0.6 0.2 0.4 0 0.2 0.2 0 0.2 0 0 0.4 0.2 0.5
LetIEnkt
Morphine*
0 0 0.4
0 0 0
0.8 0.9
0.8 0.9
I .o
I .o
0 0.1 0.2 0 0 0 0 0.2
0 0.2 0 0 0.7 0 0 0.5
*Binding constant, K=5.3x lW/mole= 1.0. tBinding constant, K=5.8x lOVmole= I .O. *Binding constant, K=4x lOYmole= 1.0. Binding constants were determined by Scatchard plots from shifts produced by varied concentrations of NE with these opioids (see Figs. 3. 7. 12). NE binding was arbitrarily assigned a value of one. and the binding of the other compounds was measured as a percent of the shift in a given peak (see Figs. 3, 7. I2 for identification) caused by NE at the same concentration and pH. Related controls for binding (or lack thereof) of many of the same compounds to serotonin binding peptides (LHRH, MSH tetrapeptide, and the tryptophan peptide of myelin basic protein) may be found in RootBernstein
and Westall
1661.
to the peptides and morphine whereas other neurotransmitters
very
nearly
as well as NE,
such as histamine and acetylcholine had very little affinity. One interesting difference between the affinities of the enkephalins and morphine is the latter’s afftnity for serotonin (SHT) and amphetamine. Examination of the NMR spectra yields specific information about the nature of the observed binding beginning with the Met-enkephalin spectra (Figs. 1 and 2). Peak shifts occur in several regions of the spectrum with addition of NE. The Tyr alpha hydrogen peaks shift, possibly indicating a change in the position of the Tyr residue relative to the rest of the molecule. The Tyr beta hydrogen peaks also shift and split, indicating at the least a significant change of conformation, and possibly direct interaction with NE. Small changes occur in the Gly peaks, although it was not possible to determine whether these changes occurred in the Gly* peaks, the Gly3 peaks, or both. No shifts or other changes occurred in any of the Phe peaks at any concentration of NE except for splitting of the beta hydrogens, suggesting that a conformational change involving the angle of the Phe residue
relative to its alpha carbon may occur. There I> no evtdence interaction between the NE hydrogens and any 01 the Phe hydrogens. Large changes did OCCUI. however, ic the Met CH,, peak, which alters its width considerably, eventually diminishing in height, and splitting. These changes probably indicate direct interaction with NE. None of the other Met peaks displayed any significant changes, suggesting that the interaction with NE is limited to the CH:, portion of the side chain. No significant changes were observed in the amide or aromatic portion of the spectra tnot shown). The spectral changes in the Tyr beta H\ and Met CH,,s were used to determine the binding constant. Both \ets of peak shifts yielded the same number: 5.3 y IO:’per mole ( Fig. of direct
3).
The only peak changes observed in the NE spectra m the presence of either Met- or Leu-enkephalin were a splitting of the beta hydrogen peaks (Figs. I and 4). There were no noticeable changes in the aromatic region of the NE spectrum. It is unlikely therefore that van der Waals forces play a significant role in the binding. The results of the NMR analysis of NE binding to Leuenkephalin are broadly similar to those just described for Met-enkephalin (Figs. 4 and 5). Shifts and splitting of Tyt alpha and beta hydrogen peaks, splitting of Phe beta hydrogen peaks, splitting of the Leu CH,, peak. Also shown (Fig. 6) is the spectrum of DA binding to Leu-enkephalin, which clearly shows a shift in one of the Gly amide peaks. The most reasonable interpretation of this shift is a conformational change involving the twisting of the peptide backbone near a Gly amide. No such conformational change was apparent when DA was added to Gly-Gly-Phe-Leu (Des-Tyr-Leuenkephalin) at the same concentrations (Fig. 6). The Tyr beta Hs were used to calculate the binding constant to NE, and yielded the number: 5.8~ IO: per mole (Fig. 7). Also shown are a pair of the dozens of control spectra obtained (Fig. 8). An equimolar combination of NE with Phe-Met produced no spectral changes whatsoever in either molecule. Very slight (c. one tenth of the corresponding changes in Met-enkephalin) shifts occurred when an equimolar combination of NE was made with Tyr-Gly-Gly (enkephalin l-3). Such spectra clearly indicate that NE binding to enkephalins requires the complete pentapeptide. In comparison with the enkephalins, the spectral changes induced in morphiceptin by NE were more limited (Figs. 9 and 10). The NE hydroxyl peak completely disappeared, indicating probable hydrogen or ionic bonding. Peak broadening occurred in most of the aromatic peaks of both morphiceptin and NE. And only one hydrophobic interaction was apparent: a shift in one of the two Pro gamma CH peaks of morphiceptin. No shifts or other changes were observed in the morphiceptin amide peaks, nor in any of the alpha, beta, gamma, or delta hydrogens, with the single exception already noted. Thus, it is unlikely that van der Waals forces are important to the binding. No significant changes were observed in the spectra of a number of peptides similar in sequence to morphiceptin, including Gly-Pro-Arg-Pro, ProPro-Gly, and Tyr-Tyr-Tyr. The type of spectral changes observed m the morphiceptin-NE interaction were not appropriate for calculating a binding constant, as the changes were not easily measurable. Evidence to be presented below suggests, however, that the binding constant for morphiceptin for NE may be of the same order of magnitude as that of morphine for NE, which is discussed next.
CATECHOLAMINE
513
BINDING
aH
aH
aHs
.
.
.
.
410
.
.
.
.
. 3Io
PPM
.
.
.
FIG. 1. 1H NMR spectra of norepinephrine (NE), Met-enkephalin (Met-enk), and combinations at pH 7.0. (A) 0.25 ml Met-enk + 0.04 ml NE (see text for concentrations); (B) 0.25 ml Met-enk + 0.16 ml NE. Progressive shifts occur in Tyr’ alpha Hs and Tyr’ beta Hs. Splitting of the Ne non-aromatic Hs is apparent. At the higher concentration of NE, some broadening of the Gly and Phe peaks becomes evident.
Met’ c H,S \
Met5 CH,s
Met -EnkephaMn
+.02
2:0----------I
2.0
Norepi
+.04 2.0
Norepi
+ .08 2.0
Norepi
+ .I6 Norepi
Saturated
77-----v
FIG. 2. 1H NMR spectra of the Met CHZ Hs and the Met CH, Hs of Met-enk at pH 7.0 kn the presence of increasing aliquots of NE. While only very small changes are apparent in the CH, Hs, significant changes in peak width occur in the CH, Hs, finally resulting in a diminution in peak height accompanied by splitting.
RC)O’I‘-HFRNS’I‘EIN
FIG. 3. Scatchard plot of the shifts in Met-enk beta Tyr Hs (Fig. I) with increasing concentrations of NE. The diminution in peak width of the Met CH, Hs (Fig. 2) yielded the same plot. The resulting association constant is 5.4X IO31mole.
,
, 4.0
1
3.0
*
,
PPM FIG. 4. IH NMR spectra of NE, Leu-enk, and combinations at pH 7.0. (A) 0.25 ml Leu-enk + 0.01 ml NE; (B) 0.25 mol Leu-enk + 0.04 ml NE; (C) 0.25 ml Leu-enk + 0.16 ml NE. As in the Met-et& f NE spectra (Fig. I), progressive shifts are apparent in the Tyr’ alpha Hs and Tyr’ beta Hs, accompanied by peak broadening as the concentration of NE is increased. Some broadening of the Leu-enk Gly Hs and Phe beta Hs, as well as the Ne non-aromatic Hs, is also apparent.
CATECHOLAMINE
515
BINDING
Leu- Enkephalin .:.\JLJLil Leu 5CH,s
I
2.0
h
+ 66 Norepl
+ .04 Norepl
+ .02 Norepi
I.“‘,‘...,...,
PPM
2.0
2.0
2.0
FIG. 5. 1H NMR spectra of Leu-enk Leu CH, Hs showing progressive changes in peak width with increasing concentrations of NE. As with the Met-enk Met CH, Hs, the peak eventually shortens ahd splits (Fig. 2). The similarity in the changes observed in the Leu CH, Hs and the Met CH3 Hs suggest that these two residues play the same role in binding NE.
Significant spectral shifts accompanied the addition of NE to morphine (Fig. 11). Splitting of the NE peaks occurred, and shifts occurred in virtually all non-aromatic morphine peaks. These shifts may indicate conformational change or direct interaction with NE residues. These shifts were found to be concentration dependent, and a Scatchard plot of the shift-concentration relationship was made yielding a binding constant of 4x10* per mole (Fig. 12). Three different peaks of morphine all yielded the same binding constant, indicating that the shifts are all orchestrated by the same mechanism. No such shifts occurred in any peak of the naloxone spectrum at any concentration of NE, nor were similar spectral changes observed in the other drugs tested with the exception of apomorphine. The results of pH titration generally confirm the NMR results to the extent that both indicate the presence or absence of binding. An equimolar combination of morphine with NE results in a buffer, as is apparent from Fig. 14, and the resulting titration values differ significantly (up to 0.9 PI-I) from the values predicted from the NE and morphine solutions alone. On the other hand, the predicted and experimentally determined values for an equimolar combination of naloxone with NE are virtually identical, the largest variance being approximately 0.05 pH. These results were reproduced several times with different solutions, and although the absolute values differ somewhat from combination to combination (probably due to differences in the absolute concentration of the independent solutions and in their relative concentrations upon addition), nonetheless the fact of either differing significantly from the predicted value or not is con-
stant. The same effect is apparent with a combination of apomorphine with NE (Fig. 15). The titration results for peptides with NE are not as clear as drug-catecholamine combinations, presumably because peptides tend to be amphoteric. Thus, in Fig. 13 it is apparent that some binding probably occurs between Gly-GlyPhe-Leu (des-Tyr-Leu-enkephalin) and NE below pH 7.0, although the NMR analysis of this combination showed no significant shifts at pH 7.0. One possible explanation is that the interaction is purely ionic, and so invisible to the NMR. Despite these results, it is clear that the experimentally obtained values and the predicted values differ to a much greater degree at pH 7.0 and above for Met-enkephalin + NE than for Gly-Gly-Phe-Leu + NE. Much the same results were obtained for NE + Tyr-Gly-Gly (Met-enkephalin l-3) (not shown). Tyrosine showed essentially no affinity for Met-enkephalin according to this method of analysis (i.e., the expected and predicted values were consistently within 0.1 pH), and NE showed no affinity for Phe-Gly-Gly-Phe according to the same criteria (not shown). A morphiceptin-NE combination differed from the predicted value by approximately 0.5 pH over the range of pH from 6.8 to 7.8 (not shown). One unexpected surprise was that both Tyr-Gly-Gly and Phe-Met displayed some affinity for NE or DA according to the pH titration technique (not shown). Since only very slight changes were observed in the NMR spectra, the pH titration data probably indicate ionic bonding, which would be invisible to the NMR. And since there is, at least at present, no way to calculate a binding constant from the pH data,
+ Dopamine
Gl y Gly Phe leu
NH
NH
TyrGly GlyPheLw
+ Dopamine
FIG. 6. IH NMR spectra of Leu-enk and des-Tyr-Leu-enk and their combinations t 1:I by molarity) with DA at pH 7.0. While a small shift in one of the Gly NHs is apparent in the Leu-Enk + DA combination and in some of the inverted aromatic peaks (at 6.5 ppm), no changes are apparent in the des-Tyr-Leu-enk + DA combinations. The Tyr residue would therefore appear to be essential for catecholamine binding.
(Leu-Enk=SxlO-?W) FIG. 7. Scatchard plot of the shifts in the Tyr’ beta Hs peak of Leu-enk + NE spectra (Fig. 4). The calculated association constant is 5.8X 1OYmole.
CATECHOLAMINE
517
BINDING
TYR-GLY-GLY
~.___
._..- _c_ “___
_. v
-- .--..
_._._l_
-_- -.--_ _
,. ,.__ ” .__
NOREPINEPHRINE .‘-+-_. .~“-..;r~%4~
%.
-
-..-
I_.. _ _. ___~ ..._. -I..
__,
1:l ---c
__-.
_._-_._-_.-- _____..._-
Phe b”S PHE -MET_ v----
FIG. 8. 1H NMR spectra of Tyr-Gly-Gly, Phe-Met, and their equimolar combinations with NE at pH 7.0. Very small upfield shifts are apparent in the Tyr alpha and beta Hs in combination with NE. These shifts are about l/10 those observed in the Met-enk and Leu-enk spectra at the same molar ratios (Figs. 1 and 4). No observable changes appear in the Phe-Met + NE combination. Thus, the Phe-Met (or Phe-Leu) residues are necessary to binding (Figs. 2 and 9, as is the Tyr-Gly-Gly sequence (see also Fig. 6).
it is unclear how important this observation is vis-a-vis the NMR results. Color reaction studies also indicate that opiatecatecholamine interaction takes place. Plate 1 shows the results of one of the color reaction experiments demonstrating that at pH 7 in phosphate buffer, a slight molar excess of morphine sulfate retards the oxidatio~~lyme~zation of DA, NE and EPI into their colored products. Plate 2 shows the results of combining morphiceptin and NE (2: 1 molarity) at pH 7 in phosphate buffer. The same results were achieved with the enkephalins, but a ten fold excess of enkephalin:NE was needed to observe the effect clearly. This result indicates that morphine and mo~~ceptin binding to NE is probably of the same order, and about ten times stronger than enkephalin-NE binding (which is also in accord with the binding constants derived from the NMR study [Figs. 3, 7,
121). It must be stressed that the color reaction experiments demonstrate that the catecholamine opioid interaction is reversible: if the solutions are let stand several weeks, the tubes or welis containing catecholam~e-opioid combinations eventually achieve the same color as the catecholamine tubes and wells. In other words, the interaction serves to retard, but not to prevent, the color reaction. Tyr-Gly-Gly also retarded the color change significantly, whereas Phe-Met appeared to catalyze the color reaction, perhaps by p~ici~ting in it. These observations will need further analysis before definitive interpretations are possible. Given that the origins and mechanism of melanins is unknown, the possibility that the enkephalins or enkephalin fragments might play a role in their formation is well worth investigation. DISCUSSiON
The results
obtained
in this study must be interpreted
from a number of interrelated points of view. First, the data permit models to be constructed of the probable form of binding that occurs between the catecholamines and the opiates and opioids. Second, this chemical binding needs to be examined in light of the anatomical distribution of catecholamines, enkephalins, and opiate receptors in vivo, and with regard to the phenomenon of co-tr~smission (that is, the co-storage and co-release of monoamines with peptide neurotransmitters). And finally, the binding must be discussed with regard to possible mechanisms of opiate and opioid action in vivo (e.g., the mechanisms of addiction and withdrawal), and vis-a-vis the problem of drug design. Chemical Models of the Binding To begin with, the NMR data, in conjunction with the pH titration and calorimetric data, are sufficient to provide quite strict criteria for building models of the mechanism by which NE and related catecholamines may bind to the enkephalins, mo~hi~eptin, and morphine. To interpret the NMR data, however, something must first be known about the conformation of the opiates and opiate peptides in solution. There is little difficulty with the preferred conformation of morphine, since it is a relatively rigid molecule. This is not the case with the enkephalins. Both NMR studies [41,70] and minimum energy conformation studies [38,46,48] agree that Met-enkeph~in has a large number of fairly stable states. The NMR data indicate that as many as 27 “rotomers,” or combinations of cis-trans pairs are not only possible between the Tyr, Phe, and Met side chains, but are actually present in substantial proportions in solution [41]. NMR studies of enkephalins is non-aqueous solutions (mimicking insertion of the molecule into a lipid membrane, for example) demonstrate yet other preferred coronations [4], and the crystallographic structure differs yet again f371. Energy calculations
NOREPI AROMATIC ti 5
COMBINATION
(1 1)
,
NOREPI HYDROXYLS
I
I 75
I
r
MORPHlCEPTlN I I PPM
I 70
AROMATIC 1
Hs 1
I
FIG. 9. 1H NMR spectra of morphiceptin (Tyr-Pro-Phe-Pro-NH,), NE, and their combination (1: 1by molarity) at pH 7.0. The NE hydroxyl peak completely disappears, and significant broadening of the NE aromatic Hs occurs (see particularly peak at 6.76 ppm). Small changes in the peak shapes of the morphiceptin aromatic Hs (Tyr and Phe residues) occur at 7.2 ppm, 7.14 ppm, 6.96 ppm and 6.84 ppm. These peak changes suggest the possibility of pi-pi overlap bonding as a significant feature of morphiceptin-NE binding.
tend to present a more limited number of low-energy conformers, but all of these studies have been made by ignoring peptide-solvent interactions [38, 46, 481. Even so, most studies indicate that at least half-a-dozen different low energy conformers may reasonably exist. In short, the enkephalins are highly flexible molecules in solution. One very important limitation exists on the flexibility of the enkephalins. Both NMR and energy calculation studies agree that a beta bend occurs in the Gly3-Phe4 region, most probably stabilized by a hydrogen bond between the Gly2 hydroxyl and the Met5 amide. Based upon these considerations, two possible models for NE binding to Metenkephalin are presented here. Figure 16 shows one low energy conformer for Metenkephalin. A caveat to remember in interpreting this picture is that the NMR data indicate that although this is one of the most preferred conformations of the molecule, the Tyr, Phe, and Met side chains are constantly rotating into all of the other possible cis-trans conformers. In other words, Fig. 16 must not be interpreted as being a static model of the molecule, but rather a dynamic one. Figure 18 shows a similar conformer, to which the same caveat applies. CPK model building resulted in two models that adhered to most of the criteria set by the experiments reported here and which could also be constructed with in accordance with the published data concerning the conformation of the enkephalins in solution. The first model presents a binding
mechanism that requires a minimum of conformational changes, but has the problem that it does not explain all of the NMR data presented here. The second model presents a binding mechanism that requires considerably greater conformational changes, but accounts much more completely for the NMR data reported here. In effect, the second model carries the implication that NE binding by the enkephalins induces a large conformational change. Model 1 (Fig. 17) shows one possible mechanism of NE binding to Met-enkephalin. Essentially all that is necessary for binding to occur is for the Tyr’ residue to rotate into a new position, which will then be stabilized by pi-pi overlap bonds to NE. NE will also form bonds to the N-terminal amine and to the Tyr hydroxyl. The Met5 CH, will interact with the pi system of NE. The main feature of this model is its simplicity. It accounts for the NMR peak shifts observed in the Tyr alpha hydrogens and the Met CHB peaks. The same general model would explain the analogous peak shifts observed in the Leu-enkephalin-NE spectra. The model does not, however, suggest why concentration dependent changes were observed in the Tyr and Phe beta hydrogen peaks, the shifts in the Gly2-Gly3 region, or the splitting of the nonaromatic peaks of NE. Thus, while this model is plausible, it is not fully consistent with the existing data. The second model (Fig. 19) is of the “molecular sandwich” type [63,64], which was found in earlier work on the mechanism of serotonin binding to peptides 1661. It accounts
CATECHOLAMINE
519
BINDING
MORPHICEPTIN + NOREPI (1 :l)
MORPHICEPTIN PRO YHs
I
z.tlI
I
,
I
2.88
,
1.90
,
,
I.&l
I
r
1.70
PPII
FIG. 10. 1H NMR spectra of mo~hiceptiR and mo~hiceptin + NE combination (1:l by molarity) at pH 7.0. (The NE spectrum is not shown because there are no peaks in this region.) This figure shows the only peak shift observed in the 1.0 to 4.5 ppm region of the spectra, indicating that virtually no hydrophobic interactions are involved in the morphiceptin + NE binding, save for the single interaction with one of the pair of Pro gamma Hs.
for the NMR data completely, but requires a quite large change from the lowest energy conformations to do so. The major features of this second model are that NE intercalates between the Tyr’ and Phe* side chains resulting in extensive pi-pi overlap bonds, an NE hydroxyl-Gly3 carbonyl hydrogen bond, a bond between the NE amine and the Tyr’ hydroxyl, and van der Waals (hydrophobic) interactions between the Met5 CH3 and the non-aromatic hydrogens of NE. The formation of the hydroxyl-carbonyl bond requires twisting of the peptide backbone in the Gly2-Gly3 region, which would account for the amide shift in the Gly region of the spectrum (Fig. 6). The proposed model also places the Tyr’ beta hydrogens facing NE so that van der Waals (hydrophobic) interactions are probable. The only observed peak shifts not accounted for directly in this model are the Phe4 beta hydrogens. These shifts are not extensive (compared to the beta hydrogens of Tyr’ for example), and may simply reflect some strain between the beta carbon and the Phe ring carbon atoms due to the formation of the pi-pi over-
lap bonds with NE. Once again, this model works equally well for Leu-enkephalin. Both model 1 and model 2, by predicting that the NE amine and at least one NE hydroxyl are involved in the binding to the enkephalins, explain the ability of the enkephalins to retard oxidation of NE, and the differences between the predicted and experimentally determined pH titration values. Both models also explain why NE, EPI, and DA bind reasonably well to the enkephalins, whereas serotonin, histamine, and acetylcholine do not. Model 2 is the more stereospecific of the two, however, and for the reasons given above, seems preferable. The differences between the two models are testable, although the peptides do not exist commercially to do so at present. If model 1 is correct (or possible), then NE should bind to some sequence Tyr-Gly-Gly-X-Met(or Leu), where -X- is some non-aromatic side chain other than Pro (which would not allow the formation of the required beta bend). The N-terminal amine, however, would be indispensable for
FIG. 11. 1H NMR spectra of morphine sulfate, Ne, and various combinations (by molar&y) at pH 7.0. Progressive shifts occur in virtually all morphine peaks. All of the non-aromatic NE peaks split. Some broadening of the aromatic peaks also occurred (not shown).
5-
O-l 0
0.1
0.2
0.3
0.4
0.5
0.6
0.1
0.9
0.9
1.0
S (MORPHINE) FIG. 12. Scatchard plot of morphine + NE combinations (some of which are shown in Fig. 1l), using the 2.8 ppm morphine peak shifts as a measure of binding. The 3.98 ppm morphine peak (Fig. 11) yielded the same results. The calculated association constant is 4X 104/mole.
CATECHOLAMINE
521
BINDING
1413@
12-
g
11-
d x
10-
l MET-ENKEPHALIN
+
NGREPI
fPREDlCTEDf
0 MET~NKEPHAL~
+
NGREPl
(EX~R~ENTAL)
0
l GLY-GLY-PHE-LEU
+
NOFtEPl
(PREDICTED)
0 GLY-GLY-PHE-LEU
+
NOREPI
(EXPERIMENTAL)
g
&
a
0
l l
7 6-
s *
5
ii
4-
+
3-
0
slo -
m m
0
l
mu m
0
9
l
cla
0
l
1-
on
0
n
0
l
CID
0
0
2-
OR
0
0
F
a
m
0
-
-8 SlD
0 0
l
0 0
08
0
0
!iii
om
0
l
om
0
0
l 2”
008
8
157
I
7.0
8.0
FIG. 13. pH titration study of Met-enk and des-Tyr-Leu-enk + NE free acid. Predicted values are averages of the titration results obtained for each compound separatety. Experimental values are actual measurements of I:1 combinations of the solutions used for the individual measurements. (See text for detailed description of the technique.) The Met-enk + NE experimental and predicted values clearly differ to a much greater degree than do the des-Tyr-Leu-enk + NE values, especially close to pH 7.0. These results suggest that NE is bound to Met-enk between pH 6.0 and 7.0, whereas very little NE binds to des-Tyr-Leu-enk above pH 6.5. These results accord with the NMR results (Figs. l-7).
binding. If model 2 is correct, then -X- would have to be Phe, Tyr, or possibly even Trp, so that a “molecular sandwich” can be formed, and the N-terminal amine could be substituted without affecting binding. The testing of these two models is important, since it is necessary to determine whether or not NE binding capacity is an absolute correlate of opiate activity or only an approximate one. One indication that opiate activity is a correlate of catecholamine binding is that morphiceptin (Tyr-Pro-Phe-ProNH&, another opiate peptide, also binds DA and NE. The same sort of “molecular sandwich” modei just proposed for NE binding to enkephalins is also the most probable model for NE binding to morphiceptin. Unfo~unately, much less is known about the preferred conformations of morphiceptin in solution so that less can be said about the observed shifts. The model presented in Fig. 20 is therefore tentative. The major feature of the NE-morphiceptin spectra were that no shifts occurred in any Pro peak with the exception of a single gamma hydrogen; there were no changes in the amide peaks; and most of the aromatic peaks, including those of NE, became significantly less sharp. These features are all accounted for by the model in terms of pi-pi overlap bonds (which would delocalize the aromatic hydrogen peaks), a NE hydroxyl-morphiceptin carbonyl hydrogen bond, an NE amine-morphiceptin Tyr hydroxyl bond, and an NE hydroxyl-mo~hiceptin terminal amine bond. In terms of the number of bonds formed, the proposed mode1 is, in fact, a
more stable one than either of the proposed NE-enkephalin models. This is consistent with the observation that even a slight excess of morphiceptin suffices to retard NE and DA color reactions, whereas a ten-fold excess of enkephalins is necessary. The NMR, pH titration data, and coiorimetric data afso provide criteria for building possible models of morphine-NE binding. Once again, CPK models were built. Both the pH titration data and the calorimetric data indicate that the mechanism of binding between morphine and NE is such that it involves a significant number of hydrophilic groups. This observation is p~icularly stren~hened by the ability of the rno~~ne-NE combination to act like a buffer (that is to share electrons). This consideration severely limits possible models. A second, key observation is that morphine (Fig. 21) binds NE, but that the opiate antagonist, and chemical cousin of morphine, naloxone (Fig. 22), does not. The most logical explanation of these observations is that the hydroxyl that is present on naloxone, but not on morphine, is sufficient to interfere with the NE binding site, by encroaching upon the binding site, or that the alteration of a hydroxyl to a carbonyl alters the ring conformation sufficiently to make binding to NE less likely. In fact, model building suggests that both effects contribute to the inability of naioxone to bind NE, as can be seen by comparison with the model of NE-morphine binding
ROOT-BERNS’l‘i:IN
/
l
Morphine+Norepinephrine(expected)
o Morphine+Norepinephrine(exptl.)
1
a Naloxone+Norepinephrine(expected)
q
12
Naloxone+Norepinephrine(exptl.) .
ml0
.
11’ I
n
.
0
0
cm
0
e
0
l l
q
.cl.
0
0
0.
I
6.8
7.0
7.2
7.4
0
.7.6
0
w 7.8
I 8.0
FIG. 14. pH titration study of morphine sulfate and its antagonist, naloxone hydrochloride + NE free acid. Expected values are the averages of the titration values obtained for each compound separately. Experimental values are actual measurements of 1: 1 combinations of the solutions used for the individual measurements. (See text for detailed description of the technique.) The naloxone + NE expected and experimental values are virtually identical, suggesting that no binding occurs. This result accords with the NMR study of naloxone + NE (Table 1). The morphine + NE values, however, differ almost 1.0 pH at pH 7.0, and the combination behaves like a buffer. Since buffers share electrons, these results strongly suggest that morphine binds to NE in an ionic complex. The NMR results also indicate that binding occurs between morphine and NE (Fig. 11).
shown in Fig. 23. The major features of this model are extensive pi-pi overlap bonds, a hydroxyl-hydroxyl hydrogen bond, and an amine-hydroxyl bond. Pi-pi overlap bonding is severiely impaired by the addition of the hydroxyl to naloxone, and the amine-hydroxyl bond is eliminated by the alteration of the morphine hydroxyl to a carbonyl, which not only alters the affinity, but causes a conformational shift in the ring that puts the carbonyl out of bonding range of the NE amine. (This latter effect is not obvious from the drawings, but becomes evident with the CPK models.) The most striking feature of the proposed models of enkephalin, morphiceptin, and morphine binding to catecholamines is the possibility that such binding ability is a common feature of all opiates and opioids, which differentiates them from their structurally similar, but inactive analogues, and from their antagonists. Some support for this hypothesis comes from the fact that of the enkephalins and enkephalin fragments, and enkephalin-like peptides studied in these experiments, binding or lack of binding correlates exactly with observed opiate activity [ 161. Nonetheless, the hypothesis will require a great deal more research before it can be considered valid. At least it provides a testable hypothesis that could be of great value in the design of novel opiates and peptides possessing opiate activity.
Co-Trunsmission and the Physiological oj’opiate Activity
Correlates
Now, what may be the physiological meaning of the binding of catecholamines to enkephalins and opiates? To properly consider this question requires some basic knowledge of the anatomical distribution of the catecholamine releasing neurons, catecholamine receptors, enkephalin releasing neurons, and opiate receptors, as well as the physiological effects of opiate treatment. Unfortunately the literature on these subjects is so vast and so full of contradictions that no attempt will be made to summarize it all here. Several salient features of the results relating to these topics will suffice to indicate how the present chemical study may complement previous approaches to these questions. To begin with, a considerable degree of overlap exists between the anatomical distribution of catecholamine neurons and receptors, and enkephalin releasing neurons and opiate receptors (Table 3). Within the brain, DA and NE are present in unusually high concentrations in the hypothalamus, caudate nuclei, and putamen, striatum, septum, and nucleus accumbens [19,36, 39,51,60,80]. These same brain regions also contain unusually high levels of enkephahns and/or opioid receptors [8, 12, 25,28, 32,34,58,60,77]. For
CATECHOLAMINE
523
BINDING
. APOMORPHINE+NOREPI
(EXPERIMENTAL)
o APOMORPHINE+NOREPI
(PREDICTED)
0
0
16
0
0
15
0
0
14
0
0
13
0
0
12
0
0 0
0
0
0
a
0 0
0 0
0 0
0
7.6
7.8
PH
FIG. 15. pH titration study of apomorphine + NE free acid. The predicted values are the average of the titrations of each compound separately. The experimental values are actual measurements of 1: 1 combinations of the solutions used for the individual measurements. (See text for detailed description of the method.) Clearly, the predicted and experimental values differ considerably, indicating that apomorphine binds to NE. This result accords with the results of the NMR study (Table 1).
example, opiate receptor densities are highest in regions such as the amygdala, which is densely ennervated by NE and DA releasing neurons [19]. Other organs, such as the
adrenal medulla, display a similar correlation of catecholamines and opioid receptors [57,81]. Furthermore, the three tissues most often used to test for opiate activity, the iris, vas deferens, and ileum, are all extremely high in either NE or DA compared to other body tissues [39]. There is very little correlation between opiate peptide concentrations and/or opiate receptors densities and serotonin or acetylcholine concentrations in particular brain regions or other organs [19, 36, 801. Not only are catecholamines and opiates or opiate receptors correlated anatomically, they are also, in some cases at least, co-stored and co-transmitted. Among the tissues or regions in which co-storage and co-release of NE and enkephalins have been reported are the sympathetic ganglia, adrenal medulla, and SIF cells [ll, 33, 45, 57, 811. DA is known to be co-stored with enkephalins in the carotid body [33,57]. Physiological evidence also links catecholamine and opiate activity. Extensive evidence exists to indicate that
DA neurons regulate the level of Met-enkephalin and conversely that Met-enkephalin levels regulate catecholamines in various regions of the brain including the striatum, the caudate nucleus, the nucleus accumbens, and tuberoinfundibular neurons [6, 23, 26, 35, 52, 56, 59, 791. The enkephalins appear to mediate stress-induced increases in dopamine levels associated with pain in a naloxonereversible manner [50]; Met-enkephalin inhibits dopamine induced LHRH release [71]; and Met-enkephalin antagonizes Jopamine control of MSH activity [72]. Similarly, large doses of morphine appear to increase synthesis, release, and turnover of catecholamines 11, 14, 15, 24, 29, 43, 62, 751; catecholamine neurons mediate the effects of morphine [3,5, 18, 27, 441; and the catecholamine levels in the brain appear to rise as a function of withdrawal of opiates [2,7,40, 621. It is believed that the effects of morphine on catecholamine release and turnover occur pre-synaptically [9,75]. These opiate-catecholamine interactions have correlates observable in temperature changes and dilation of the pupils and other physiological functions. In short, the chemical complementarity of the opioids and catecholamines is reflected in an anatomical and physiolog-
R001‘-BERNSTEIN
H2O MOR EPI NE DA PLATE I. One of several experiments utilizing the fact that the catecholamines DA, EPI. and NE oxidize and polymerize into colored products such as melanins. Addition of morphine (MOR) (2:1 by molarity) greatly retarded the color reaction for about a week, whereas water (H,O) and tyrosine (TYR) had no effect. Experiments were carried out at pH 7.0 in phosphate buffer. Photograph was taken one week after the experiment was set up. After a month, the morphine-treated catecholamines had also turned dark brown, indicating that morphine retards but does not prevent the color reactions from occurring. In short. the binding is reversible. (See text for discussion.)
ical complementarity of function. Can this be by chance, or has the evolution of the opioid and catecholamine systems been regulated by their chemical cooperativity? There are a number of possible responses to these questions, none of which are mutually exclusive. First, it is possible that the co-storage and co-release has a simple chemical function: to stabilize the catecholamines against oxidation and against melanin formation, and to stabilize the enkephalins against enzymatic degradation. The ability of enkephalins to retard oxidative color reactions involving the catecholamines has been demonstrated by the color reactions reported here; and the binding of a catecholamine by an enkephalin would most likely retard enzymatic cleavage by making the cleavage sites unavailable. The latter effect has yet to be tested, but should not be difficult to demonstrate. These chemical protection functions may be manifested in storage vesicles or intrasynaptically. A second possible explanation for co-storage and corelease may be to modulate the activity intrasynaptically or at the receptor. The effect of the binding would be to allow only a relatively slow release of either the catecholamine or enkephalin to the receptor, as compared with the amount of either that would reach the receptor if released alone. Similarly, release of a chemically complementary pair of neurotransmitters from separate neurons into a common synapse would also result in modulation by binding. A third possible explanation of the co-transmission may be an evolutionary one: that the complex release, re-uptake, and receptor systems that we observe today are highly elaborated forms of extremely simple chemical transmitting
PLATE 2. Experiment showing the ability of morphiceptin to retard NE oxidation-polymerization into colored products. Experiment carried out at pH 7.0 in phosphate buffer. Photograph taken at one week. By one month, the morphiceptin-NE combination had also turned dark brown, to match the NE tube. Thus. morphiceptin retards but does not prevent the color reactions from occurring. In short, the binding is reversible. Notably. about ten times as much enkephalin was necessary to produce the equivalent retardation of the color reaction, indicating that the binding of morphiceptin to NE is on the order of ten times that of the enkephalins for NE. and so on the order of morphine binding to NE (i.e., 4~ IO’lmole).
met
MET FIG.
(1)
16. A model of one of the many preferred
Met-enk (Tyr-Gly-Gly-Phe-Met). [411.
- ENK
_
conformations of After Jones, Gibbons and Garsky
CATECHOLAMINE
525
BINDING
tyr
MET-ENK
(2)
FIG. 18. A second model of one of the preferred conformations of Met-enk, based upon calculations by Isogai, Nemethy and Scheraga
[381. MET-
ENK
+ NOREPI (model
1)
FIG. 17. One possible model for a Met-enk + NE complex, involvamine bonding; ing NE-hydroxyl + Met-enk-N-terminal hydrophobic interaction between the Met-enk Met-CH, and the NE ring; pi-pi overlap bonds between the Met-enk Tyr’ and the NE ring; and a Met-enk Tyr’ hydroxyl + NE amine bond. In a Leu-enk + NE complex, the only difference would be that the Met CH,-NE ring interaction would be replaced by a Leu CH,-Ne ring interaction. This model explains many of the shifts observed in Figs. 1, 2,4 and 5, but does not account for the non-aromatic peak splitting in the NE spectra, the shifts and broadening of the- Tyr beta Hs, or the broadening of Gly and Phe peaks of Met- and Leu-enkephalins at higher concentrations of NE.
utilized by the very first living organisms. If so, the observed binding of co-transmitters may have no particular function today, but may simply be an artifact of the evolutionary history of the particular system. Nonetheless, the observation of binding between the two co-transmitters would still be a valuable piece of information for piecing together the chemical criteria that may have shaped the evolutionary process. One way to test this sub-hypothesis would be to determine the phylogeny of co-transmission throughout the vertebrates and into the invertebrates. It is possible that the lower invertebrates may utilize a simplified chemical version of the complex receptors systems found in vertebrates, and therefore may mimic more closely the direct chemical modulation suggested here. Fourth, it is possible that the binding of catecholamines and opiates is neither chance, modulatory, nor an artifact of history, but essential to the function of the opiates as blockers of neurotransmission. What if the chemical binding of the opiates to catecholamines were to effectively hinder either the release of the catecholamine, their reuptake, or their ability to reach their appropriate receptor? Then the opiates would cause functional blockade of neurotransmission. Anaesthesia is, in fact, one of the effects of high doses of morphine, which explains its use in treating chronic pain. This final possibility necessitates consideration of the mechanism of opiate activity and, in consequence, questions of drug design. systems
MET-
ENK + NOREPI (model
2)
FIG. 19. A second model of a possible Met-enk + NE complex. Extensive conformational shifts are required from the preferred conformations illustrated in Figs. 16 and 18, but it must be remembered that the enkephalins are extremely flexible in solution, so that all possible rotamers of the Tyr, Phe, and Met side chains are present at any given time [41]. This second model of the complex is preferable to the first (Fig. 17), although both may exist at the same time, for the following reasons: greater pi-pi overlaps are produced by this model; the splitting of the NE non-aromatic Hs is explained by van der Waals interactions with the Met CH, (or Leu CH,); the Tyr beta Hs are now facing into the interior of the complex, explaining the shifts in their peaks; and the conformational shifts are appropriate to explaining shifts in the Tyr and Phe alpha Hs. In short, all of the observed spectral changes can be accounted for by this “molecular sandwich” model [63, 64, 661.
Possible Mechanisms and Withdrawal
of Opiate Activity Vis-a-Vis Addiction
The usual explanation of opiate activity is that opiates and opioid peptides bind to a number of distinct classes of opiate receptors [12, 16, 47, 541. While this may be true, it in fact merely pushes back a step the question of how the opiates
RW’I
-HERNSl‘l
NALOXONE MORPHICEPTIN
+ NOREPI
FIG. 20. A “molecular sandwich” model [63, 64, 661 (Fig. 19) of morphiceptin (Tyr-Pro-Phe-Pro-NH,) binding of NE. Since only a single hydrophobic interaction (involving a Pro gamma H peak) is indicated by the NMR data (Figs. 9 and lo), the model accounts for this by proposing that the conformation induced by NE binding requires that a Phe aromatic H interacts with the Pro” gamma Hs. Since peak broadening is also observed in the aromatic region, this interaction is acceptable. Otherwise, the model postulates that NE binds to morphiceptin by extensive pi-pi overlap bonds (which would account for the other observed shifts in the aromatic region of both molecules); an NE terminal amine bond to the Tyr’ hydroxyl; a morphiceptin terminal amine bond to the NE hydroxyl (which may be shared with a morphiceptin hydroxyl as well, to stabilize the conformation: indicated by the open circles); and a morphiceptin hydroxyl hydrogen bond to one of the NE ring hydroxyls. The total number of bonds formed in the morphiceptin-NE complex is greater than in the enkephalin-NE complexes (Figs. 17 and 19), suggesting that the bonding should be stronger.
FIG. 22. A structural model of naloxone, a morphine antagonist. Note the moditications compared with morphine (Fig. 21).
“,C
MORPHINE-
NOREPI.
COMPLEX
FIG. 23. A structural model of NE binding to morphine based upon CPK model building. The interaction involves extensive pi-pi overlap bonds, as well as a pair of hydrogen bonds from the NE to the morphine hydroxyls. Note that the small structural modifications made to morphine to produce naloxone are such as to interfere seriously with NE binding to the latter compound. The additional hydroxyl prevents pi-pi overlap bonding, and the NE amine bond to the morphine hydroxyl is no longer possible since the hydroxyl has been replaced by a carbonyl, which also alters the conformation of that portion of the ring in such a way as to shift the oxygen radical out of the range of the NE amine.
H,C
MORPHINE FIG. 21. A structural model of morphine.
and opioid peptides function. How, for example, does the opiate receptor cause anaesthesia? Why does it bind naloxone, but fail to respond to naloxone binding as if it were an opiate? Furthermore, the receptor hypothesis certainly does not address how such a complex system could have evolved. From what simpler polypeptides did receptors originate?
CATECHOLAMINE
527
BINDING TABLE 3 COMFARISON
OF RELATIVE
CATECHOLAMINE, ENKEPHALIN, AND OPIATE RECEPTOR DEW%TIES FOR SELECXED BRAIN AREAS AND ORGANS
Monkeyl
Human*
Rat* Brain Area
NE
DA
OP. Rec.
NE
DA
5HT
ENK
OP. Rec.
Cerebral cortex Caudate nuclei Hypothalamus Amygdala Cerebellum Globus pallidus
0.18 0.27 1.79 0.07 -
0.01 6.39 0.14 0.03 -
1.1 il.5 3.8 6.5 0.8 -
0.09 1.25 0.21 0.01 0.15
3.50 0.29 0.26 0.23
0.04 0.33 0.80 0.60 0.00 0.50
0.46 5.28 4.20 1.68 0.45 4.94
6.0 19.4 24.3 49.0 <2.0 7.7
Organ or Tissue* Whole brain Whole brain Vas deferens Iris Skeletal muscle Skeletal muscle Heart Heart
Species
NE
Dog Rat Guinea Pig Rabbit Rabbit Cat Rat Rabbit
0.16 0.49 10.0 5.20 0.10 0.10 0.96 2.00
Catechola~nes in microg~s/~m of tissue; enkephalin in units per epigram of tissue; and * opiate receptor densities in terms of amount oflabelied morphine bound per mg of protein (monkey brain), or per 100 square microns of tissue slice (rat). Sources: *[39], 1[58], $[36], 8[77].
In fact, it appears that Vichy nothing is known about the opiate receptors [76] except their reiative affinities for different opiates (e.g., [16]). Without questioning the existence of such receptors and their functions, it may nonetheless be useful to consider some other chemical features of the opiates that may influence opiate activity. As I mentioned above, it appears possible that a common feature of opiates and opioid peptides is their ability to bind to catecholamines. If true, this phenomenon raises the possibility that opioids may produce some of their effects, such as interference with nerve transmission, by directly binding up of the neurotransmitter. What evidence exists to support this speculation? First, as detailed above, the effects of opioids are highly correlated with brain regions and other organs that utilize catecholamines as their primary neurotransmitters. Thus, direct interference with catecholamine neurotransmission is an anatomical possibility. In addition, at least two enkephalin analogues (D-AlaZ-Met-enkephali and N-CH,-Metenkephalin) both, “show considerable resistance to inactivation by peptidases and after intravent~cu~r or intracerebral admin~st~~on exhibit antinociceptive activity n>hich therefore does not seem to be closely and exclusively related to their aflnity to receptors represented by either naloxone or leucine-enkephalin binding” [47]. If activity is not a direct function of receptor affinity, then what controls it? Furthermore, the activity of any given opiate or opioid peptide differs with the brain region or the type of tissue (e.g., mouse vas deferens or guinea pig ileum) upon which it
is tested 112, 16, 471. Why should opiate or opioid peptide activity vary from one brain region or organ to another? Certainly a diversity of receptors is a possibility (though not one in accord with Occam’s razor), but might not there be other possibilities as well? These observations raise the possibility that opiate activity is mediated, at least in Part, not on& by receptor types but by the co~en~tions of resident catecholamines. Different opiates and opioids will display different afRmities for the resident catecholamines, just as morphine differs from the enkephahns in its affinity for NE and DA. There is even the possibility that so-called receptor types are actually comparative measures of the mix of catecholamines being used as neurot~smitters in the particular brain region or organ being tested: some morphine derivatives may have significantiy greater affinity for NE than for EPI or DA, for example. The presence or absence of serotonin or acetylcholine may affect affinities. Regardless of the validity of the speculations just presented, it is certainly true that the presence of very high concen~ations of catechoiamines in the tissues used to measure opioid binding (e.g., vas deferens and ileum) must affect the observed receptor binding constants. This is a necessary consequence of the demonstration that opiates and opioid peptides bind to the catecholamines. This binding to the catecholamines will, perforce, create a competition between the catecholamines and the opiate receptors for the opiate molecules. It is therefore highly unlikely that any reported a.@mity of an opiate for its receptor is accurate, and
ROOT-BERNSTEIN further studies will have to be undertaken to determine to what extent competitive binding from the catecholamines affects the reported values. A second characteristic of the opiates and opioids must also be considered. Both morphine and the enkephalins are highly hydrophobic (the water solubility of Met-enkephalin is <0.7%~ [70]) [4]. In combination with the catecholamines, the solubility is probably even lower, because most of the hydrophilic residues of both compounds are involved in the binding that forms the combination (N.B.: this effect remains to be demonstrated). In short, opioids, and opioids combined with catecholamines, are very likely to be lipophilic. In fact, studies of the interactions of enkephalins with lipid-like media have been performed, demonstrating that very signilicant interactions take place, correlated with extensive alterations in enkephalin conformation [4]. Indeed, it is important to note that of the several hundred substituted forms of enkephalins that have been synthesized, the most active are those multiply substituted with hydrophobic residues. The most potent enkephalin yet synthesized is (Me-Tyr)1-(D-Ala)2-Gly”-(Me-Phe)~-(Met-(0)-ol)Z, which is 1000 times as potent as morphine and 68,000 times as potent as Met-enk in the tail flick test. Eliminating the methyl group from the Tyr’ reduces the potency in half. Eliminating the D-Ala’ residue further decreases potency. Also, hydrophilic substitutions, such as pCl-Phel, decrease potency to below the level of unsubstituted enkephalins [16]. In short, as long as the residues necessary for catecholamine binding are respected (e.g., the Tyr’ hydroxyl, the Gly”. the Phe” aromatic ring, and a hydrophobic fifth side chain), activity appears to be maintained; and the more hydrophobic substitutions that can be made, the more potent the resulting molecule. The lipophilicity of opioids is an important characteristic for the following reason. Franks and Lieb [22] have noted that the anaesthetic properties of compounds increase with lipophilicity, and have suggested that one general mechanism for anaesthetic activity may be incorporation into the lipid membrane of neurons in such a way as to interfere with neurotransmission. The lipophilicity of the opioids would allow at least some of their activity to be explained by the Franks-Lieb mechanism, particularly if the lipophilicity is increased by combination with a catecholamine. The combination would be highly stable, for the reasons discussed above, and incorporation into neuron membranes would further stabilize the combination. Incorporation into the membrane of the releasing neuron could interfere with reuptake and release; incorporation into the receptor neuron would interfere with the function of the receptor. Now, what would be the biochemical consequences of the binding up of the catecholamines by opiates and opioids, particularly if release and reuptake were impaired? If we assume that the function of metabolism is to maintain homeostasis, then the result would be that the system would act as if it were producing too little catecholamines. Production of catecholamines would increase to a level sufficient to counter-balance the amount of opiate or opioid added to the system. Under normal physiological conditions, a balance would be reached between catecholamine and enkephalin (and endorphin?) metabolism. Exogenous additions of opiates or opioids would result in abnormally increased catecholamine production. Subsequent withdrawal of the opiate or opioid would then leave the production of catecholamines too high, resulting in the symptoms associated with narcotic withdrawal. In fact, these metabolic interactions between
catecholamines and opioid peptides, and these metabolic changes produced by exogenous opiates, are exactly what has been reported in the literature [I, 2, 7. 9. 14. 15. 17. 18. 24. 26, 27, 29, 40, 44. 54, 55. 751. Indeed. the metabolic scenario just sketched exactly fits the theoretical descriptions made previously by several investigators to explain narcotic addiction and withdrawal. For example. in 1943 Himmelsbach suggested that physical dependence to opiates might be a “condition of autonomic hyperreaction” characterized as follows: I. The prime function of autonomic (hypothalamic) centers is to maintain homeostasis and to make proper adjustments in the face of stress. 2. Morphine affects homeostasis through its action on these centers. 3. Autonomic reaction to this effect takes place. (Biological reactions generally are greater than necessary to overcome an effect.) 4. With repetition the ability to offset the opiate effect improves. (Physiological tolerance.) 5. An extension of this process of improved reaction results eventually (with larger and more frequent dosage) in disproportionate autonomic strength in checks and balances. 6. Thus a condition is created wherein a chemical is needed to maintain homeostasis; such reactive power having been developed that to preserve equilibrium there must be present an effect to counteract. 7. Since the body is unable to supply a counter-effect promptly it must be furnished from without, else equilibrium will be lost temporarily. Such loss of equilibrium results in an abstinence syndrome (withdrawal) [31]. Very similar models have since been suggested by Kauffman, Koski and Peat [42], and by Nichols [53]. Given appropriate adjustments, these models of addiction and withdrawal could be explained by the opiatecatecholamine binding mechanism proposed here, with one important additional feature: if the opiate-catecholamine complex actually incorporates into lipid membranes and is stabilized there, then an ever-increasing amount of catecholamine metabolism disruption would ensue as more and more opiate became incorporated into the membrane. The result would be an ever increasing amount of catecholamine production. In turn, ever-increasing amounts of opiate would be needed to offset withdrawal symptoms caused by ever higher production of catecholamines. One extremely important point must be stressed at this point. While the synthesis and turn-over of catecholamines would increase significantly with long-term, high-dose morphine treatment, it is not at all clear that the measurable amounts of catecholamines in brain (or elsewhere) would increase. According to the binding mechanism proposed here, the more opiate that is present in the system, the more catecholamine will be bound. Thus, the active, measurable levels of catecholamines will remain fairly constant at normal levels unless the opiate is withdrawn, in which case there will be no counter-balance to the increased catecholamine synthesis. In other words, tolerance to opiates should be accompanied by what appears to be normal levels of rrvailahlr catecholaminas. Tolerance is a return to homeostasis. Another apparent exception to the model just proposed must also be examined. Nichols [53] notes that patients treated for surgery or cancer rarely become addicted to opiates. He suggests a psychological difference between
CATECHOLAMINE
medical patients and abusers. There may also be a chemical difference: since catecholamine neurons are associated with many pain pathways, it is likely that patients in pain have unusually active catecholamine neurons. In consequence, opiate treatment may simply counter-balance an increased catecholamine metabolism induced by the pain. Instead of exogenous opiates creating a disequilibrium in the body’s homeostasis, in this case they return to normal the disequilibrium caused by the pain. In consequence, as the pain decreases with healing, catecholamine metabolism returns to normal and opiate treatment can be tapered off. In short, the patient in pain is in exactly the opposite metabolichomeostasis condition as is the abuser. In summary, opiate-catecholamine complexation provides a simple mechanism for explaining economically the existing literature concerning the anaesthetic, addictive, and metabolic effects of opiates. Drug Design in Light Complexation
529
BINDING
ofOpiate-Catecholamine
The opiate-catecholamine binding phenomenon may also have implications for the design of new opiate drugs. To date, most researchers who have compared the opiates and opioid peptides have attempted to find structural similarities between the two [46, 70, 741, with regard to receptors the characteristics of which are still totally hypothetical [76]. While it is possible to obtain some overlap between certain morphine derivatives (but not morphine itself) and some conformations of enkephalins, none of these investigators have been able to explain why naloxone should act as an antagonist rather than as an agonist. It is also very difftcult to make morphiceptin conform to the proposed structural models of the “ideal opiate,” and no one has apparently reported doing so. I would therefore like to propose a different set of criteria for opiate activity: (1) ability to bind catecholamine neurotransmitters; (2) ability to disrupt neurotransmission by incorporating into lipid membranes in combination with catecholamine neurotransmitters; (3) naloxone antagonism. These criteria move the search for opiate drugs away from specitlc structural similarities to a set of similar chemical properties. For example, it is possible that a peptide sequence such as Leu-Tyr-Thr-Phe-Leu-NH,, although quite different from the enkephalin sequences, might be highly active as an opioid; or that increasing the lipophilicity of morphine derivatives by adding methyl groups while adding another residue capable of forming a hydrogen bond to the amine ring hydroxyls might increase activity. Another possible advantage of these criteria would be to make analogous criteria possible for other sets of anaesthetits and antinociceptive drugs. For example, it is quite likely that there is a set of lipophilic compounds that selectively bind to and interfere with serotonin and its metabolism, or to acetylcholine interfering with its metabolism. Thus, the opiate-catecholamine complexation model may provide a paradigm for the elucidation of the activities of other drugs. Regarding this last point, it is worth mentioning again that at least two other cases of complexation between a biologically active chemical and an antagonistic or modulatory drug have been reported 165,731. In both cases, the reported binding is virtually identical to the morphine-catecholamine model suggested here: pi-pi bonding associated with ionic or hydrogen bonds between specific side chains or residues. Thus, it may be possible to specifically design drugs having
the appropriate stereochemical properties to bind to and inactivate any given aromatic neurotransmitter or related compound. The “molecular sandwiching” model of intercalation of aromatic amines into pockets of aromatic side chains on peptides may also provide a generalizable model for synthesizing peptides possessing specific modulatory activities. Co-Transmission Hypothesis
and Complementarity:
A General
The issue of “molecular sandwiching” raises some other interesting possibilities. Note that cholecystokinin, which contains the DA binding sequence Trp-Met-Asp-Phe-NH, (gastrin tetrapeptide: Table 2), is known to be modulated by DA and, in turn, to modulate DA activity in vivo [20, 21,49, 821. Also, substance P, which was found to bind DA and NE (Table 2), has been shown to produce analgesia [32], and is a reasonable substitute for morphine in tolerant rats [78]. Might these peptides represent further examples of peptides that bind catecholamines into “molecular sandwiches”? Although further research will be necessary to confirm the DA-cholecystokinin interaction, and the specificity of substance P for DA and NE (or SHT?), I would like to make a general prediction based upon the present study and previous research concerning serotonin-binding peptides. Many other instances of co-transmission have been reported, including co-storage or co-release of substance P-serotonin, cholecystokinin-DA, somatostatin-NE, TRH-serotonin, and neurotensin-NE [33,57]. I would like to suggest that a general property of all co-transmitters is that they are chemically complementary in such a manner that they can modulate one another’s activity by direct chemical interaction. Thus, physiological complementarity of function will be mirrored in stereochemical complementarity of molecular forms. No doubt this prediction will turn out to be overly simplistic, but it represents a powerful tool for inventing hypotheses amenable to straightforward experimental analysis. It is better to search for simplicity and discover that you are wrong than to remain inactive in the face of a vast, disordered array of confused observations. If the present research leads to greater understanding by providing clues to the order of Nature, it shall be successful, whether the outcome is to validate or refute the initial concept. FINAL CONCLUSIONS
To summarize, my general conclusions are as follows. The experiments reported here demonstrate clearly that morphine, the enkephalins, and morphiceptin bind to the catecholamines DA, EPI, and NE. This observation suggests the possibility that all opiates and opioids have catecholamine-binding capacity, and that one of the mechanisms of their activity is to modulate catecholamine neurotransmission and biosynthesis by direct binding. This binding mechanism, along with the observed lipophilicity of opiates and opioid peptides, provides a model for explaining the biochemical changes observed during addiction and withdrawal, and the differences observed between the use of * opiates for treating pain and as abused substances. The binding may provide a paradigm of the elucidation of the mechanisms (or partial mechanisms) of action of other drugs affecting neurotransmitters. The binding may also provide a simple chemical paradigm for understanding and explaining other instances of co-transmission such as those involving
570
ROOT-BERNSTEIN
DA-cholecystokinin, TRH-serotonin and substance P-serotonin. The basic concept underlying the interpretation of all of the above features of co-transmission is that physi-
ological complementarity of function stems from stereochemical complementarity of the molecular forms.
ACKNOWLEDGEMENTS 1 thank
Dr. Jonas Salk of the Salk Institute for Biological Studies in La Jolla, CA and Dr. Arthur Yuwiler of the Brentwood Veterans Administration Hospital, Los Angeles for the use of their laboratory facilities; the Southern California Regional NMR Facility at the California Institute of Technology, Pasadena, CA which operates under NSF Grant No. CHE 79-16324; the Institute for Disease Research (IDR) for chemicals and equipment; and Dr. Fred C. Westall of the IDR for his suggestions. David Felten. M.D. of the University of Rochester School of Medicine made the original suggestion that I look into the question of co-transmission, thus beginning the train of thought and research elaborated here. This research was made possible by a MacArthur Prize Fellowship to the author from the John D. and Catherine T. MacArthur Foundation, Chicago, IL.
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