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Interactions of norepinephrine with subcellular fractions of rat brain I. Characteristics of norepinephrine uptake
298
ltRAi N RESI-ARCII
I N T E R A C T I O N S OF N O R E P I N E P H R I N E W I T H S U B C E L L U L A R F R A C T I O N S OF R A T B R A I N I. C H A R A C T E R I S T I C S OF N O R E P I N E P H R I N E U P T A K E
WILLIAM F. HERBLIN* AND RICHARD D. O'BRIEN Section of Neurobiology and Behavior, Cornell University, Ithaca, N. Y. (U.S.A.)
(Accepted November 25th, 1967)
INTRODUCTION It is generally accepted that synaptic transmission in the central nervous system is accomplished chemically. This implies that a chemical transmitter is released from the axon terminal, diffuses across the synaptic cleft, and combines with a specialized receptor site on the postsynaptic membrane. Strong evidence has been obtained for the existence of synapses mediated by acetylcholine 8 and norepinephrine~,4,5, t2, but the identity of the transmitter at the vast majority of synapses is unknown. Even less is known about the nature of the receptor and the combination of receptor and transmitter. Triggle 2z has suggested that receptors, like enzymes, are 'probably proteins or protein derivatives' and may be isolated, and their composition explored. Similarly, Ehrenpreis s states that 'receptor substance had to be a macromolecule of some sort: however, there is a school of thought that dissents even from this view', after which he documents some of the approaches to isolation which have been based upon the macromolecular view. The macromoleeular hypothesis is the starting point for our studies. Additional hypotheses (for each of which there are arguments both for and against) are that the receptor may be studied in broken cell preparations, in which it retains its ability to bind transmitter; that this binding has high affinity: and that the binding should be blocked by those drugs whose action in vivo is caused by receptor blockade. A corollary is that high-affinity, appropriately blockable binding should be present in subcellular fractions containing the synaptic apparatus and absent in fractions which lack the synaptic apparatus. One important problem arises from the fact that all known or hypothetical transmitters are bases, and therefore bind non-specifically to proteins, and other macromolecules with acidic groups. Such non-specific binding would be expected to have a low affinity, with values of dissociation constants in the order of 10-2 to 10 -4 M, as found, for instance, in the binding of organic cations to serum albumin * Present address: Central Research Department. E. 1. du Pont de Nemours and Co.. Wilmington. Del., U.S.A. Brain Research, 8 (1968) 298-309
NOREPINEPHRINE UPTAKE
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and other proteins t3. It has proved possible to distinguish such non-specific lowaffinity binding from high-affinity specific binding, by exploring the amount of binding over a large range of concentrations. Thus for serotonin, Marchbanks et al. 16,17 found that a high-affinity, LSD-sensitive binding was present in nerve-ending particles, but absent from liver mitochondria or serum albumin. An additional problem in any study of the binding of a transmitter to a receptor is the presence of presynaptic storage systems. In the case of norepinephrine, re-uptake by the presynaptic ending is the major mechanism in the inactivation of released transmitter at peripheral adrenergic synapses r~. This implies that there are sites on the presynaptic membrane which function as carriers, transporting norepinephrine from the external solution into the presynaptic ending. The binding of norepinephrine to these sites should have the same order of specificity and affinity as the binding to the receptor and can be distinguished only on the basis of inhibition data. It is therefore essential that the uptake of norepinephrine be studied under the same conditions which will later be used for the investigation of the binding to the receptor. Only in this way can a definite discrimination be made between the two processes. Binding studies are complicated if enzymic degradation of the transmitter occurs in the preparation under study; such degradation has to be evaluated in each new preparation. Fortunately, by performing the experiments at temperatures near 0°C, it is possible to slow down enzymic degradation; binding is relatively insensitive to low temperatures, for it results from a forward (k0 and a backward (k 1) reaction, the affinity being described inversely by the binding constant K.a ~ k - l / k 1 ; as both kl and k 1 will be reduced at lower temperatures, Ka is not greatly affected. One may suspect that he is examining binding to a receptor when the binding has a very low K~, is antagonized by appropriate drugs, and occurs primarily or exclusively in tissues or subfractions known to contain receptors. The present paper, then, is the first step toward the investigation of the binding of norepinephrine to its receptor. It should be emphasized that at this stage we are not claiming isolation of receptor or of receptor-transmitter complex, but asking questions whose answers are necessary to determine the validity of using the above approaches for norepinephrine. Specifically, we have sought the relation between the concentration and extent of norepinephrine uptake and/or binding in vitro, and how it is affected by a variety of drugs. At best, the answers may prove a starting point for more refined studies. At least, they should add to the available information about norepinephrine interactions with cell particulates under simplified conditions, and about the influence of drugs upon such interactions. The uptake of exogenous norepinephrine by adrenergic nerve granules has been studied extensivelygA0, as has the uptake by various tissues 6 and tissue homogenates ~1,2°. The interactions of norepinephrine with brain homogenates, however, have not received as much attention. Mirkin and Gillis 14 reported that brain homogenates would accumulate norepinephrine and reported a bimodal distribution of the exogenous amine in a sucrose density gradient. Potter and Axelrod 19 found a similar distribution. Mirkin et al. 15 reported certain characteristics of norepinephrine uptake by a crude subcellular fraction. Brain Research, 8 (1968) 298-309
300
w,
F. H E R B L I N ANI) R. 1). O ' B R | E N
We have therefore begun our investigations using a crude mitochondrial fraction obtained from rat brain by the methods of De Robertis et al. 7 and Whittaker et al.23, z4. This preparation should contain all of the synaptic apparatus and has been shown to accumulate norepinephrine. By successively fractionating this material into less complex preparations, and examining the interactions of norepinephrine with each fraction, it may be possible in the future to prepare a fraction containing onl~ the postsynaptic receptor. METHODS
Tissue preparation Female rats were sacrificed by decapitation and the brains were removed and placed in ice-cold 0.25 M sucrose. The pooled brains were homogenized in a glass Potter-Elvehjem homogenizer equipped with a Teflon-glass pestle and brought to a final concentration of 1 0 ~ , w/v. The homogenate was centrifuged at 1500 v g for 10 rain to remove the nuclear fraction and then at 20,000 × g for 30 min to isolate the crude mitochondrial fraction. This pellet was washed three times by rehomogenization in 0.25 M sucrose and centrifugation at 25.000 ~ g for 30 min. The washed pellet was suspended in sufficient 0.25 M sucrose to yield a 0.5 g/ml suspension based on weight of fresh brain. Further fractionation of the homogenate was accomplished on a sucrose density gradient made from successwe layers o f 1.3. 1.2. 1.0 and 0.7 M sucrose in a I " × 3" cellulose nitrate tube, which was then centrifuged at 25.000 rev./min at 4°C for 2 h in an SW 25.1 rotor on a Spinco model L-2 ultracentrifuge. This fractionation resulted in five fractions (A1-A5), which were isolated by means of a tube slicer. By analogy with the reports of Whittaker et al. 23,24 and De Robertis et aL 7, these fractions should contain myelin (A 1), debris and nerve terminals (A2), nerve terminals (A3 and A4), and mitochondria (A5).
Performance o f binding experiments After the addition of either 1.0 ml or 0.5 ml of the homogenate fraction, each tube contained 0,05 M Tris (hydroxymethyl) amino methane buffer at p H 714, 0.2 M NaCI, F7-14C~norepinephrine *, and distilled water to give a total volume of 3.0 mt. The tubes were shaken at 4°C for 16 h and centrifuged in a small table model Sorvall centrifuge. It was shown that this centrifugation yielded the same results as centrifuging the samples at 30,000 × g for 30 min. Aliquots (0.5 ml) were taken from each tube before and after centrifugation and placed in counting vials containing 10.0 ml of Bray's dioxane counting medium. The vials were counted for at least three separate
* The [7-14C]norepinephrine used in these studies was obtained from the New England Nuclear Corp. with a specific activity of 20.5 mC/mmole.
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NOREPINEPHRINE UPTAKE
3 rain intervals in a Packard Tri-Carb Series 314 E liquid scintillation counter. The amount of norepinephrine 'bound' was determined by difference.
Succinoxidase assay
The succinoxidase activity of various homogenates and fractions was determined manometrically on a G M E Warburg apparatus. The main compartment of the flasks contained 0.3 ml 0.05 M succinic acid sodium salt, 1.0 ml 0.15 M Tris buffer (pH 7.4) containing 0.6 M NaC1, and 1.2 ml HzO. The sidearms received 0.5 ml of either one of the fractions or of 0.32 M sucrose. A small piece of fluted Whatman No. 1 filter paper was inserted into the center well and moistened with 2 M NaOH. Standard techniques of control and correction of data were used to determine the oxygen utilization of the samples at 37°C over a 30 rain interval.
Cholinesterase assay
The cholinesterase activity of the fractions was determined by a titrimetric procedure using a Radiometer TTT1 pH stat. Aliquots (0.1-0.5 ml) of the fractions were added to a jacketed reaction vessel containing 8.0 ml 5.0 • 10-4 M acetylcholine chloride at p H 7.0. The amount of 0.005 M N a O H necessary to maintain the pH at 7.0 was recorded on a Radiometer Titrigraph for 10 min. The initial period (I rain) was often masked by a drastically increased or reduced rate caused by the addition of the fraction. This period was therefore not used in the calculation of the cholinesterase activity of the fraction.
to/,
I¢
2.e ---~
/S g
wE z~_ a_ ~ z
/
0.1
0,01
I0-'
,fl"
,
,
i
i
10-7 J0-6 I0 -5 10-4 FrNAL NOREPINEPHR[NE CONCENTRATION(M)
Fig. 1. The uptake of [14C]norepinephrine by crude mitochondrial fractions as a function of the final concentration of norepinephrine. All the data were taken from samples containing 0.5 g brain homogenate (fresh weight) in a total volume of 3.0 ml and incubated at 0-4°C. Vertical lines indicate ranges for the values where necessary. The data are expressed as m/*moles norepinephrine bound per ml of incubation mixture per g of tissue (fresh weight).
Brain Research, 8 (1968) 298-309
302
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RESULTS
The norepinephrine uptake curve The u p t a k e o f n o r e p i n e p h r i n e by a crude m i t o c h o n d r i a l fraction from rat b r a i n is shown in Fig. 1 as a l o g - l o g p l o t o f n o r e p i n e p h r i n e b o u n d vs. the final nore p i n e p h r i n e c o n c e n t r a t i o n . T h e Curve is not a s m o o t h curve, b u t exhibits several inflection points. These p o i n t s occur at final n o r e p i n e p h r i n e c o n c e n t r a t i o n s of 1.7 - 10 -7, 4 • 10 -7, 4 • 10 -~ a n d 2 - 10 -,5 M. The curve was n o t e x t e n d e d a b o v e
6.0 l[
_
k
"o E ~
4.C
3.0
~ 2.0 a:: I.OF
I
t~ 1.0 2.0 5.0 4.0 5.0 6.0 FINAL NOREPINEPHRINE CONCENTRA/ION(xiOSM)
Fig. 2. Linear plot of a portion of the data from Fig. 1 which has not been converted to uptake per g of tissue (fresh weight).
TABLE I THE EFFECTS OF D R U G S ON THE U P T A K E OF N O R E P I N E P H R I N E BY THE C R U D E M I T O C H O N D R I A L FRACTION
The values are listed as percentages of the control value. All values were rounded to the nearest percentage. Norepinephrine 4"10 -a M.
Drug,
Drug concentration (10 4 M)
Effect ( % of control)
D-Amphetamine Bretylium tosylate Bufotenin bioxalate Ergotamine Mephenesin Psilocybin Pyrogallol Serotonin Creatinine-H~SOa complex Hydrogen oxalate
2.0 2.0 2.0 4.0 4.0 1.0 1.0
65 73 47 17 74 165 35
4.0 4.0
150 122
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6 • l0 -5 M because it was felt that any phenomena of interest in this study would occur at lower concentrations. Log-log plots derive their usefulness from the great range of values which can be displayed on a single graph, but the compression of data to accomplish this can mask other relationships. Fig. 2 is a linear plot of one region of the uptake curve, which clearly exhibits an inflection at 2 • 10 .5 M norepinephrine. The vertical lines are ranges of values in multiple determinations. Inhibitor studies An extensive series of drugs has been examined for effects on the uptake of norepinephrine by the crude mitochondrial fraction. The most active of these are listed in Table ! along with their effects and concentrations. The variety of effects observed probably reflects the inhomogeneity of the fraction and indicates that a variety of phenomena are taking place. This is supported by the multicomponent appearance of the uptake curve. The three most potent inhibitors found in the course of the investigation were ergotamine, bufotenin, and pyrogallol. At concentrations of 2 • 10 4 M, these drugs gave over 40 % inhibition when 4.03 • 10-6 M norepinephrine was used. Harmine was of particular interest since this drug is known to inhibit monoamine oxidase. Enzymatic degradation could easily be mistaken for binding since both would remove norepinephrine from solution. Even at 6.7 • l0 -4 M, however, harmine resulted in only 5-10 % inhibition, indicating that oxidation of norepinephrine by monoamine oxidase is not a major factor under these experimental conditions. Certain of the drugs examined resulted in an apparent activation of the norepinephrine uptake. The most powerful of the series in this respect were serotonin and psilocybin. Two salts of serotonin were used, the creatinine-H2SO4 complex and the hydrogen oxalate. Both resulted in activation, though the creatinine-H2SO4 complex was slightly more active. Sodium oxalate alone resulted in a slight inhibition of the uptake. The activation by psilocybin was one of the most interesting results of the investigation, since in addition to activation of the norepinephrine uptake, a stable blue complex was formed. The inhibition by pyrogallol raised the suspicion that catechol-O-methyl transferase (COMT) was responsible for at least part of the observed uptake. Although COMT should be inactive at 0-4°C, the prolonged incubation could allow some degradation. Therefore homogenates were incubated with high levels of tritiated norepinephrine for 24 h at 0-4°C. The samples were centrifuged and the pellets extracted with 1.0 ml 95% ethanol. Both the supernatants and pellet extracts were examined by ascending chromatography in butanol acetic acid-water (12 : 3 : 5, v/v). A single radioactive peak was found with an R~, of 0.34-0.36, corresponding to norepinephrine. Four derivatives of lysergic acid diethylamide (LSD) were tested, D-LSD, L-LSD, bromo-LSD, and acetyl-LSD. These compounds all gave slight to moderate Brain Research,8 (1968)298-309
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H E R B L I N A N D R, I), O'BRII/N
inhibition ( 1 0 - 3 0 ~ ) at 6.7 . 10 :~ M, thus indicating no obvious correlation betwecn activity in this system and psychotropic potency. Three drugs that have been implicated in norepinephrine function bear special mention. Guanethidine, isoproterenol, and tyramine all showed peculiar concentration o/ effects. Guanethidine activated uptake (27 ~o) at 4 . 10-4 M but inhibited (261'o) at 6.7 • 10-4 M. Isoproterenol resulted in 32% inhibition at 2.0 • t0 -4 M and 41 '~o at 6.7 • 10-4 M, but only 8°/i, at 4.0 - IO 4 M. Tyramine had no effect at 4.0 - 10 -~ M, inhibited uptake (20~o) at 2.0 • 10 -4 M and activated uptake (22~o) at 6.7 - 10 4 M. These results are interpreted as further support for the multicomponent nature of the uptake phenomenon. Atropine, adenosine triphosphate, bulbocapnine, chlorpromazine, ergonovine, imipramine, a-methyl-m-tyrosine, pilocarpine, and strychnine were all inactive at 6 . 7 . 10-4 M. Acetytcholine, reserpine and phenoxybenzamine were inactive at 1.0- 10-4 M.
Subcellular distribution of norepinephrine uptake The appearance of the sucrose density gradient after centrifugation is shown in Fig. 3. Fractions A3 and A4 should contain nerve-ending particles with the myelin in fraction AI and the mitochondria in fraction A5. When these fractions were assayed for norepinephrine uptake activity, a bimodal distribution was observed. In Fig. 4. a histogram is presented which illustrates the relative activities of the fractions when assayed for norepinephrine uptake, succinoxidase, and cholinesterase. It can be seen that although the uptake activity is spread throughout the gradient, slightly increased activities are found in fraction AI and fractions A3. A4. and A5. Modifications in the composition of the gradient resulted in changes in the distribution which were consistent with the hypothesis that at least two distinct uptake components were present. In some cases a peak was observed in fraction A4 with fraction A5 being slightly lower. Further evidence for distinct components was obtained by determining the effects of inhibitors on the uptake activity of the fractions. Due to the small amounts FRACTIONS
SUCROSE 0.5~
I
0.7M
1. OM 1.2M
~
4
I.SM
Fig. 3. The appearance of the density gradient tube after centrifugation. Shaded areas indicate the location of the various fractions, Broken lines indicate the location of the boundari~ of the sucrose solutions and solid lines indicate where the tube was sliced for the isolation Of the fractions.
Brain Research, 8 (1968) 298-309
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I00
>- 8O
K I.o
< 60 0
40 I.Z tad t3 n~
uJ 20
Ix.
AI
Az
A5
A4
A5
Fig. 4. T h e distribution of cholinesterase (C), succinoxidase (S), a n d norepinephrine uptake activity (N) in fractions of the c r u d e m i t o c h o n d r i a . T h e values are plotted as the percentage of the total activity f o u n d in the gradient.
of each fraction produced by the gradient procedure, duplicates were impossible within any run. This led to considerable variation in the effects noted for particular drugs. Duplicate runs were made, however, and permitted the detection and deletion of inconsistent results. Only reproducible effects are reported and the figures given are averages of multiple runs. T A B L E 11 THE
EFFECTS
OF
DRUGS
ON
THE
UPTAKE
OF
NOREPINEPHRINE
BY SUBFRACTION
OF
THE
CRUDE
MITO-
CHONDRIA
The values are given as the percentage o f the control values using an initial n o r e p i n e p h r i n e concentration of 4.0 - 10 6 M. All values are r o u n d e d to the nearest percentage. Drug
Fraction A1
A2
A3
A4
A5
Bufotenin bioxalate 2.10 4M Ergotamine
78
102
101
139
172
1.10
36
36
36
60
42
Psilocybin 1 • 10 a M Pyrogallol
112
120
189
239
220
1 - 10 a M
43
54
87
104
78
1 - 10 4 M
53
65
64
138
150
Serotonin s o d i u m oxalate 2 • 10 4 M
75
52
69
78
67
aM
Serotonin h y d r o g e n oxalate
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ttERBI IN ANI) I~, t), ~ ' B R t l "-,
Table 11 summarizes the data obtained for the effects of drug,~ on the norepinephrine uptake activity of the individual gradient fractions. Serotonin gave consistent results on the gradient fractions, although the magnitude of the elfects varied. The hydrogen oxatate was used and this compound inhibited fractions AI-A3 and activated fractions A4 and A5. The results with bufotenin were not as definite. This compound seemed to have little effect on fraction A1, although slight inhibition was observed in certain runs. No consistent effect was observed on fraction A2. The effect on fraction A3 was extremely variable, but tended to approach zero ~hen averaged. Fractions A4 and A5 were activated in all instances by this drug. Ergotamine inhibited the uptake of all fractions. DISCUSSION
All facets of the results obtained for the uptake of norepinephrine by rat brain homogenates indicated the complexity of the phenomenon and inhomogeneity of the fractions. The curve obtained for the uptake of norepinephrine by the crude mitochondrial fraction exhibited four inflection points which indicated the parucipation of five separate components. The effects of the series of drugs demonstrated the mutticomponent nature of the system both by the lack of logical concentration dependence and the different effects on the separate gradient fractions. Once the crude mitochondrial fraction was recognized as containing multiple components, attempts at direct analysis were abandoned. It became obvious thal drugs which affected the various components of the curve non-uniformly would yield results which were extremely difficult to analyze. Therefore. further analysis was attempted by fractionating the crude mitochondria on a sucrose density gradient. When the more homogeneous fractions obtained from the gradient were investigated, a more meaningful determination of the uptake was possible. The data show that at least three distinct components are involved in the gross uptake curve. The component found in fraction A I could be due to either microsomes~ a population of small nerve-ending particles, or synaptic vesicles released from a small number of nerve-ending particles ruptured in preparation. Washing the crude mitochondrial fraction in 0.32 M sucrose before gradient fractionation reduces this component, a finding consistent with all of the proposed origins. This component is inhibited by serotonin, pyrogallol, and ergotamine, slightly inhibited by bufotenin. and activated by psilocybin. The component found in fraction A3 is most probably associated with isolated nerve-ending particles. Electron microscopic examinations of this fraction in other laboratories have indicated that nerve-ending particles are the primary constituent of this fraction, and similar studies of the fractions produced in our laboratory have confirmed this. This fraction would seem to correspond to the non-cholinergic nerveending particles fraction reported by De Robertis v. but in the absence of detailed assays for acetylcholine and choline acetylase, this correspondence is highly speculative. This component is also inhibited by serotonin, pyrogallol, and ergotamine. unaffected by bufotenin, and activated by psilocybin. Brain Research, 8 ( 1 9 6 8 ) 2 9 8 - 3 0 9
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The component found in fractions A4 and A5 could be either mitochondria or a slightly different population of nerve-ending particles. The distribution of norepinephrine uptake in these fractions does not follow the succinoxidase activity distribution, but it is possible that the presence of two additional uptake components in the gradient effectively mask a relationship otherwise present. This component is inhibited by ergotamine and strongly activated by serotonin, bufotenin, and psilocybin. One striking feature of the drug profile of the fractions is that although bufotenin strongly inhibits the uptake by the crude mitochondrial fraction, this drug does not greatly inhibit the uptake of norepinephrine by any of the isolated fractions. This finding indicates that in addition to the multicomponent nature of the gross uptake curve, there may be an interaction of certain components of the crude mitochondrial fraction with some factor which is eliminated when the homogenate is further fractionated. The activation phenomenon is difficult to explain on the basis of any of the present theories concerning norepinephrine uptake. Calcium ion has been implicated in the release of norepinephrine 3, but its involvement in the activation is doubtful in view of the small effect of sodium oxalate. The uptake, storage, and release of norepinephrine have been the object of extensive research as outlined earlier. The major portions of this research have been devoted to either the adrenal medulla or peripheral sympathetically innervated structures. Both adrenal medullary granules and adrenergic nerve granules have been isolated and studied and have been found to be similar but not identical 9. The results obtained with subcellular particles from brain indicate that these particles are not identical with adrenal medullary granules or adrenergic nerve granules. Chlorpromazine inhibits the uptake of norepinephrine by both adrenal medullary granules and adrenergic nerve granules at drug concentrations of 1-3 • 10-5 M (ref. 9). This compound failed to significantly inhibit the uptake of norepinephrine by brain particles even at a concentration of 6.7 • 10 -4 M. This is in general agreement with the conclusion of P:etscher et aL TM, that chlorpromazine had little effect on the storage of catecholamines in the brain. The uptake of norepinephrine by adrenergic nerve granules has been reported to require the addition of ATP and Mg 2+ when the amine concentration is below 5 . 10-'~ M. This uptake is inhibited by reserpine, chlorpromazine and phenoxybenzamine 9. Brain homogenates exhibited uptake of norepinephrine at concentrations as low as I • l0 7 M, and the uptake was not activated by the addition of ATP and Mg ~ . Moreover, the uptake at the low norepinephrine concentration was not inhibited by either reserpine or phenoxybenzamine. It becomes increasingly clear that the uptake of norepinephrine by brain homogenates is not identical with the norepinephrine uptake by other preparations. This could indicate a basic difference between peripheral and central norepinephrine stores or could reflect changes in the characteristics of the uptake caused by the presence of the presynaptic membrane. This membrane might be expected to exhibit its own characteristic drug profile. It will be necessary to systematically 'dissect' the crude mitochondria by successive subfractionation in order to isolate and identify Brain Research, 8 (1968) 298 309
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\V. F. HERBLIN ANI) R l), d,)'BRIIIN
the various uptake components and determine their individual drug profiles. Notable exceptions to the effectiveness of the indole nucleus are crgonovine and the LSD derivatives. LSD has been reported to block the activity of electrophoretically applied norepinephrine on certain central cells, and was lound to be a more effective blocker of norepinephrine than of serotonin zl. However, Marchbanks 1~ found that LSD inhibited serotonin binding by brain particles at low concentrations and LSD had little effect on the norepinephrine uptake investigated in this study. The interactions of serotonin and norepinephrine have been much discussed and little can be added here to the experimental observation that serotonin activates the uptake of norepinephrine. Conversely, norepinephrine inhibits serotonin uptake ~6. Brodie I has suggested that high levels of serotonin are tranquilizing whereas increased norepinephrine levels are excitatory. It becomes obvious that more information on the identity of uptake components and their drug profiles is required belbre a general explanation can be advanced. SUMMARY
The uptake of exogenous norepinephrine by subcellular fractions of rat brain has been investigated in a sucrose-NaCl-Tris buffer medium. Although the uptake of norepinephrine has been shown to be a temperature-dependent process, these studies were run at 0-4°C so that the results would be compatible with subsequent binding experiments. The uptake of this catecholamine which occurs at O-4°C is inhibited by pyrogallol, bufotenin, and ergotamine, and is activated by serotonin and psilocybin. Plots of norepinephrine uptake as a function of the equilibrium norepinephrine concentration have indicated that the uptake of norepinephrine is a multicomponent phenomenon, and this is supported by inhibition data. One component appears to be associated with the microsomal fraction and is inhibited by serotonin, pyrogallol, and ergotamine, and activated by psilocybin. A second component appears to be associated with nerve-ending particles and exhibits a very similar drug profile. A third component also appears to be associated with nerve,ending particles and is inhibited by ergotamine but activated by bufotenin, serotonin, and psilocybin. ACKNOWLEDGMENTS
This work was performed under the sponsorship of the Cognitive Systems Research Program and supported by Office of Naval Research Grant Nol NONR 401 (40) and National Science Foundation Grants No. GP-971 and GK,250. The authors would like to thank Dr. Frank Rosenblatt for guidanceand financial support and Miss Frances Fu-Ti Fan for technical assistance throughout the investigation. Bretylium tosylate was donated by Burroughs Wellcome and Co., chlorpromazine and phenoxybenzamine by the Smith, Kline and French Laboratories, imipramine by Geigy Pharmaceuticals, ismelin by the CIBA Pharmaceutical Co. mephenesin by E. R. Squibb and Sons, and the lysergic acid derivatives and psilocybin by Sandoz Pharmaceuticals. Brain Research, 8 (1968) 298-309
NOREPINEPHRINE UPTAKE
309
REFERENCES 1 BRODIE, B. B., Interaction of psychotropic drugs with physiological and biochemical mechanisms in the brain, Mod. Med. (Minneap.), 26 (1958) 69-80. 2 BURN, J. H., AND GIBBONS,W. R., The effect of phenoxybenzamine and of tolazoline on the response to sympathetic stimulation, Brit. J. Pkarmacol., 22 (1964) 527-539. 3 BURN, J. H., AND GIBBONS, W. R., The part played by calcium in determining the response to stimulation of sympathetic post-ganglionic fibers, Brit. J. Pharmacol., 22 (1964) 540 548. 4 CARLSSON, A., FALCK, B., AND HILLARP, N. A., Cellular localization of brain monoamines, Acta physiol, scand., 56, Suppl. 196 (1962) 1-27. 5 DAI-tLSTROM,A., FUXE, K., OLSON, L., AND UNGERSTEDT, U., Ascending systems of catecholamine neurons from the lower brain stem, Acta physiol, scand., 62 (1964) 485-486. 6 DEr~GLER, H. J., SPIEGEL, H. E., AND TITUS, E. O., Uptake of tritium labeled norepinephrine in brain and other tissues of cat in vitro, Science, 133 (1961) 1072-1073. 7 DE ROBERTIS, E., PELLEGR1NODE IRALD1,A., RODRIGUEZDE LORES ARNAIZ, G., AND SALGANICOI:F, L., Cholinergic and non-cholinergic nerve endings in rat brain. I. Isolation and subcellular distribution of acetylcholine and acetylcholinesterase, J. Neurochem., 9 (1962) 23 35. 8 EHREr~PREIS, S., Molecular aspects of cholinergic mechanisms. In A. BURGER (Ed.), Drugs Affecting the Peripheral Nervous System, Dekker, New York, 1967, pp. 1-78. 9 EULER, U. S. YON, AND LISHAJKO, F., Effect of drugs on the storage granules of adrenergic nerves, Proc. 2nd int. pharmacol. Meeting, Vol. 3, Macmillan, New York, 1965, pp. 245-259. 10 EULER, U. S. VON, STJARNE, L., AND LISHAJKO, F., Effects of reserpine, segontin, and phenoxybenzamine on the catecholamines and ATP of isolated nerve and adrenomedullary storage granules, LiJe Sci., 3 (1964) 35 40. I 1 G~LHs, C. N., Characteristics of norepinephrine retention by a subcellular fraction of rabbit heart, J. Pharmacol., 146 (1964) 54-60. 12 IVERSEN, L. L., The Uptake and Storage of Noradrenaline in Sympathetic Nerves, University Press, Cambridge, 1967. 13 Kx.oTz, I. M., GELEWlTZ, E. W., AND URQUHART, J. M., The binding of organic ions by proteins. Interactions with cations, J. Amer. chem. Soc., 74 (1952) 209. 14 MIRK1N, B. L., AND GILLIS, C. N., The subcellular localization of tritium after incubation of homogenates of rat brain with 3H-norepinephrine, Biochem. Pharmacol., 12 (1963) 1173-1179. 15 MIRKIN, B. L., GIARMAN, N. J., AND FREEDMAN, D. X., Factors influencing the uptake of noradrenaline by subcellular particles in homogenates of rat brain, Biochem. Pharmaeol., 13 (1964) 1027-1035. 16 M ARCHBANKS, R. M., Serotonin binding to nerve-ending particles and other preparations from rat brain, J. Neurochem., 13 (1966) 1481 1493. 17 MARCHI3ANKS, R. M., ROSENBLATT, F., AND O'BRIEN, R. D., Serotonin binding to nerve-ending particles of the rat brain and its inhibition by lysergic acid diethylamide, Science, 144 (1964) 1135 1136. 18 PL~TSCHER, A., GEv, K. F., AND KUNZ, E., Accumulation of exogenous monoamines in brain in vivo and its alteration by drugs. In H. E. HIMWICH AND W. A. HIMWlCH (Eds.), Biogenie Amines, Progress in Brain Research, Vol. 8, Elsevier, Amsterdam, 1964, pp. 45-52. 19 POTTER, L. T., AND AXELROO, J., Subcellular localization of catecholamines in tissues of the rat, J. Pharmacol., 142 (1964) 291-298. 20 POTTER, L. T., AND AXELROD, J., Properties of norepinephrine storage particles of the rat heart, J. Pharmacol., 142 (1964) 299-305. 21 SALMO1RAGHI,G. C., BLOOM, F. E., AND COSTA, E., Adrenergic mechanisms in rabbit olfactory bulb, Amer. J. Physiol., 207 (1964) 1417-1424. 22 TRIGGLE, D. J., Chemical Aspects of the Autonomic Nervous System, Academic Press, New York, 1965, pp. 307-309. 23 WHHTAKE~, V. P., MICHAELSON, I. A., AND KIRKLAND, R, J. A., The separation of synaptic vesicles from disrupted nerve-ending particles, Biochem. Pharmacol., 12 (1963) 300-302. 24 WHIVTAKER, V. P., MICHAELSON, I. A., AND KIRKLAND, R. J. A.. The separation of synaptic vesicles from nerve-ending particles (synaptosomes), Biockem. J., 90 (1964) 293-303.
Brain Researek, 8 (1968) 298-309
ltRAi N RESI-ARCII
I N T E R A C T I O N S OF N O R E P I N E P H R I N E W I T H S U B C E L L U L A R F R A C T I O N S OF R A T B R A I N I. C H A R A C T E R I S T I C S OF N O R E P I N E P H R I N E U P T A K E
WILLIAM F. HERBLIN* AND RICHARD D. O'BRIEN Section of Neurobiology and Behavior, Cornell University, Ithaca, N. Y. (U.S.A.)
(Accepted November 25th, 1967)
INTRODUCTION It is generally accepted that synaptic transmission in the central nervous system is accomplished chemically. This implies that a chemical transmitter is released from the axon terminal, diffuses across the synaptic cleft, and combines with a specialized receptor site on the postsynaptic membrane. Strong evidence has been obtained for the existence of synapses mediated by acetylcholine 8 and norepinephrine~,4,5, t2, but the identity of the transmitter at the vast majority of synapses is unknown. Even less is known about the nature of the receptor and the combination of receptor and transmitter. Triggle 2z has suggested that receptors, like enzymes, are 'probably proteins or protein derivatives' and may be isolated, and their composition explored. Similarly, Ehrenpreis s states that 'receptor substance had to be a macromolecule of some sort: however, there is a school of thought that dissents even from this view', after which he documents some of the approaches to isolation which have been based upon the macromolecular view. The macromoleeular hypothesis is the starting point for our studies. Additional hypotheses (for each of which there are arguments both for and against) are that the receptor may be studied in broken cell preparations, in which it retains its ability to bind transmitter; that this binding has high affinity: and that the binding should be blocked by those drugs whose action in vivo is caused by receptor blockade. A corollary is that high-affinity, appropriately blockable binding should be present in subcellular fractions containing the synaptic apparatus and absent in fractions which lack the synaptic apparatus. One important problem arises from the fact that all known or hypothetical transmitters are bases, and therefore bind non-specifically to proteins, and other macromolecules with acidic groups. Such non-specific binding would be expected to have a low affinity, with values of dissociation constants in the order of 10-2 to 10 -4 M, as found, for instance, in the binding of organic cations to serum albumin * Present address: Central Research Department. E. 1. du Pont de Nemours and Co.. Wilmington. Del., U.S.A. Brain Research, 8 (1968) 298-309
NOREPINEPHRINE UPTAKE
299
and other proteins t3. It has proved possible to distinguish such non-specific lowaffinity binding from high-affinity specific binding, by exploring the amount of binding over a large range of concentrations. Thus for serotonin, Marchbanks et al. 16,17 found that a high-affinity, LSD-sensitive binding was present in nerve-ending particles, but absent from liver mitochondria or serum albumin. An additional problem in any study of the binding of a transmitter to a receptor is the presence of presynaptic storage systems. In the case of norepinephrine, re-uptake by the presynaptic ending is the major mechanism in the inactivation of released transmitter at peripheral adrenergic synapses r~. This implies that there are sites on the presynaptic membrane which function as carriers, transporting norepinephrine from the external solution into the presynaptic ending. The binding of norepinephrine to these sites should have the same order of specificity and affinity as the binding to the receptor and can be distinguished only on the basis of inhibition data. It is therefore essential that the uptake of norepinephrine be studied under the same conditions which will later be used for the investigation of the binding to the receptor. Only in this way can a definite discrimination be made between the two processes. Binding studies are complicated if enzymic degradation of the transmitter occurs in the preparation under study; such degradation has to be evaluated in each new preparation. Fortunately, by performing the experiments at temperatures near 0°C, it is possible to slow down enzymic degradation; binding is relatively insensitive to low temperatures, for it results from a forward (k0 and a backward (k 1) reaction, the affinity being described inversely by the binding constant K.a ~ k - l / k 1 ; as both kl and k 1 will be reduced at lower temperatures, Ka is not greatly affected. One may suspect that he is examining binding to a receptor when the binding has a very low K~, is antagonized by appropriate drugs, and occurs primarily or exclusively in tissues or subfractions known to contain receptors. The present paper, then, is the first step toward the investigation of the binding of norepinephrine to its receptor. It should be emphasized that at this stage we are not claiming isolation of receptor or of receptor-transmitter complex, but asking questions whose answers are necessary to determine the validity of using the above approaches for norepinephrine. Specifically, we have sought the relation between the concentration and extent of norepinephrine uptake and/or binding in vitro, and how it is affected by a variety of drugs. At best, the answers may prove a starting point for more refined studies. At least, they should add to the available information about norepinephrine interactions with cell particulates under simplified conditions, and about the influence of drugs upon such interactions. The uptake of exogenous norepinephrine by adrenergic nerve granules has been studied extensivelygA0, as has the uptake by various tissues 6 and tissue homogenates ~1,2°. The interactions of norepinephrine with brain homogenates, however, have not received as much attention. Mirkin and Gillis 14 reported that brain homogenates would accumulate norepinephrine and reported a bimodal distribution of the exogenous amine in a sucrose density gradient. Potter and Axelrod 19 found a similar distribution. Mirkin et al. 15 reported certain characteristics of norepinephrine uptake by a crude subcellular fraction. Brain Research, 8 (1968) 298-309
300
w,
F. H E R B L I N ANI) R. 1). O ' B R | E N
We have therefore begun our investigations using a crude mitochondrial fraction obtained from rat brain by the methods of De Robertis et al. 7 and Whittaker et al.23, z4. This preparation should contain all of the synaptic apparatus and has been shown to accumulate norepinephrine. By successively fractionating this material into less complex preparations, and examining the interactions of norepinephrine with each fraction, it may be possible in the future to prepare a fraction containing onl~ the postsynaptic receptor. METHODS
Tissue preparation Female rats were sacrificed by decapitation and the brains were removed and placed in ice-cold 0.25 M sucrose. The pooled brains were homogenized in a glass Potter-Elvehjem homogenizer equipped with a Teflon-glass pestle and brought to a final concentration of 1 0 ~ , w/v. The homogenate was centrifuged at 1500 v g for 10 rain to remove the nuclear fraction and then at 20,000 × g for 30 min to isolate the crude mitochondrial fraction. This pellet was washed three times by rehomogenization in 0.25 M sucrose and centrifugation at 25.000 ~ g for 30 min. The washed pellet was suspended in sufficient 0.25 M sucrose to yield a 0.5 g/ml suspension based on weight of fresh brain. Further fractionation of the homogenate was accomplished on a sucrose density gradient made from successwe layers o f 1.3. 1.2. 1.0 and 0.7 M sucrose in a I " × 3" cellulose nitrate tube, which was then centrifuged at 25.000 rev./min at 4°C for 2 h in an SW 25.1 rotor on a Spinco model L-2 ultracentrifuge. This fractionation resulted in five fractions (A1-A5), which were isolated by means of a tube slicer. By analogy with the reports of Whittaker et al. 23,24 and De Robertis et aL 7, these fractions should contain myelin (A 1), debris and nerve terminals (A2), nerve terminals (A3 and A4), and mitochondria (A5).
Performance o f binding experiments After the addition of either 1.0 ml or 0.5 ml of the homogenate fraction, each tube contained 0,05 M Tris (hydroxymethyl) amino methane buffer at p H 714, 0.2 M NaCI, F7-14C~norepinephrine *, and distilled water to give a total volume of 3.0 mt. The tubes were shaken at 4°C for 16 h and centrifuged in a small table model Sorvall centrifuge. It was shown that this centrifugation yielded the same results as centrifuging the samples at 30,000 × g for 30 min. Aliquots (0.5 ml) were taken from each tube before and after centrifugation and placed in counting vials containing 10.0 ml of Bray's dioxane counting medium. The vials were counted for at least three separate
* The [7-14C]norepinephrine used in these studies was obtained from the New England Nuclear Corp. with a specific activity of 20.5 mC/mmole.
Brain Research, 8 (1968) 298-309
301
NOREPINEPHRINE UPTAKE
3 rain intervals in a Packard Tri-Carb Series 314 E liquid scintillation counter. The amount of norepinephrine 'bound' was determined by difference.
Succinoxidase assay
The succinoxidase activity of various homogenates and fractions was determined manometrically on a G M E Warburg apparatus. The main compartment of the flasks contained 0.3 ml 0.05 M succinic acid sodium salt, 1.0 ml 0.15 M Tris buffer (pH 7.4) containing 0.6 M NaC1, and 1.2 ml HzO. The sidearms received 0.5 ml of either one of the fractions or of 0.32 M sucrose. A small piece of fluted Whatman No. 1 filter paper was inserted into the center well and moistened with 2 M NaOH. Standard techniques of control and correction of data were used to determine the oxygen utilization of the samples at 37°C over a 30 rain interval.
Cholinesterase assay
The cholinesterase activity of the fractions was determined by a titrimetric procedure using a Radiometer TTT1 pH stat. Aliquots (0.1-0.5 ml) of the fractions were added to a jacketed reaction vessel containing 8.0 ml 5.0 • 10-4 M acetylcholine chloride at p H 7.0. The amount of 0.005 M N a O H necessary to maintain the pH at 7.0 was recorded on a Radiometer Titrigraph for 10 min. The initial period (I rain) was often masked by a drastically increased or reduced rate caused by the addition of the fraction. This period was therefore not used in the calculation of the cholinesterase activity of the fraction.
to/,
I¢
2.e ---~
/S g
wE z~_ a_ ~ z
/
0.1
0,01
I0-'
,fl"
,
,
i
i
10-7 J0-6 I0 -5 10-4 FrNAL NOREPINEPHR[NE CONCENTRATION(M)
Fig. 1. The uptake of [14C]norepinephrine by crude mitochondrial fractions as a function of the final concentration of norepinephrine. All the data were taken from samples containing 0.5 g brain homogenate (fresh weight) in a total volume of 3.0 ml and incubated at 0-4°C. Vertical lines indicate ranges for the values where necessary. The data are expressed as m/*moles norepinephrine bound per ml of incubation mixture per g of tissue (fresh weight).
Brain Research, 8 (1968) 298-309
302
\V
/.. H E R B I . I N
ANI)
~£ I), ()'t~IRIFb:
RESULTS
The norepinephrine uptake curve The u p t a k e o f n o r e p i n e p h r i n e by a crude m i t o c h o n d r i a l fraction from rat b r a i n is shown in Fig. 1 as a l o g - l o g p l o t o f n o r e p i n e p h r i n e b o u n d vs. the final nore p i n e p h r i n e c o n c e n t r a t i o n . T h e Curve is not a s m o o t h curve, b u t exhibits several inflection points. These p o i n t s occur at final n o r e p i n e p h r i n e c o n c e n t r a t i o n s of 1.7 - 10 -7, 4 • 10 -7, 4 • 10 -~ a n d 2 - 10 -,5 M. The curve was n o t e x t e n d e d a b o v e
6.0 l[
_
k
"o E ~
4.C
3.0
~ 2.0 a:: I.OF
I
t~ 1.0 2.0 5.0 4.0 5.0 6.0 FINAL NOREPINEPHRINE CONCENTRA/ION(xiOSM)
Fig. 2. Linear plot of a portion of the data from Fig. 1 which has not been converted to uptake per g of tissue (fresh weight).
TABLE I THE EFFECTS OF D R U G S ON THE U P T A K E OF N O R E P I N E P H R I N E BY THE C R U D E M I T O C H O N D R I A L FRACTION
The values are listed as percentages of the control value. All values were rounded to the nearest percentage. Norepinephrine 4"10 -a M.
Drug,
Drug concentration (10 4 M)
Effect ( % of control)
D-Amphetamine Bretylium tosylate Bufotenin bioxalate Ergotamine Mephenesin Psilocybin Pyrogallol Serotonin Creatinine-H~SOa complex Hydrogen oxalate
2.0 2.0 2.0 4.0 4.0 1.0 1.0
65 73 47 17 74 165 35
4.0 4.0
150 122
Brain Research, 8 (1968) 298-309
NOREPINEPHRINE UPTAKE
303
6 • l0 -5 M because it was felt that any phenomena of interest in this study would occur at lower concentrations. Log-log plots derive their usefulness from the great range of values which can be displayed on a single graph, but the compression of data to accomplish this can mask other relationships. Fig. 2 is a linear plot of one region of the uptake curve, which clearly exhibits an inflection at 2 • 10 .5 M norepinephrine. The vertical lines are ranges of values in multiple determinations. Inhibitor studies An extensive series of drugs has been examined for effects on the uptake of norepinephrine by the crude mitochondrial fraction. The most active of these are listed in Table ! along with their effects and concentrations. The variety of effects observed probably reflects the inhomogeneity of the fraction and indicates that a variety of phenomena are taking place. This is supported by the multicomponent appearance of the uptake curve. The three most potent inhibitors found in the course of the investigation were ergotamine, bufotenin, and pyrogallol. At concentrations of 2 • 10 4 M, these drugs gave over 40 % inhibition when 4.03 • 10-6 M norepinephrine was used. Harmine was of particular interest since this drug is known to inhibit monoamine oxidase. Enzymatic degradation could easily be mistaken for binding since both would remove norepinephrine from solution. Even at 6.7 • l0 -4 M, however, harmine resulted in only 5-10 % inhibition, indicating that oxidation of norepinephrine by monoamine oxidase is not a major factor under these experimental conditions. Certain of the drugs examined resulted in an apparent activation of the norepinephrine uptake. The most powerful of the series in this respect were serotonin and psilocybin. Two salts of serotonin were used, the creatinine-H2SO4 complex and the hydrogen oxalate. Both resulted in activation, though the creatinine-H2SO4 complex was slightly more active. Sodium oxalate alone resulted in a slight inhibition of the uptake. The activation by psilocybin was one of the most interesting results of the investigation, since in addition to activation of the norepinephrine uptake, a stable blue complex was formed. The inhibition by pyrogallol raised the suspicion that catechol-O-methyl transferase (COMT) was responsible for at least part of the observed uptake. Although COMT should be inactive at 0-4°C, the prolonged incubation could allow some degradation. Therefore homogenates were incubated with high levels of tritiated norepinephrine for 24 h at 0-4°C. The samples were centrifuged and the pellets extracted with 1.0 ml 95% ethanol. Both the supernatants and pellet extracts were examined by ascending chromatography in butanol acetic acid-water (12 : 3 : 5, v/v). A single radioactive peak was found with an R~, of 0.34-0.36, corresponding to norepinephrine. Four derivatives of lysergic acid diethylamide (LSD) were tested, D-LSD, L-LSD, bromo-LSD, and acetyl-LSD. These compounds all gave slight to moderate Brain Research,8 (1968)298-309
304
w.F.
H E R B L I N A N D R, I), O'BRII/N
inhibition ( 1 0 - 3 0 ~ ) at 6.7 . 10 :~ M, thus indicating no obvious correlation betwecn activity in this system and psychotropic potency. Three drugs that have been implicated in norepinephrine function bear special mention. Guanethidine, isoproterenol, and tyramine all showed peculiar concentration o/ effects. Guanethidine activated uptake (27 ~o) at 4 . 10-4 M but inhibited (261'o) at 6.7 • 10-4 M. Isoproterenol resulted in 32% inhibition at 2.0 • t0 -4 M and 41 '~o at 6.7 • 10-4 M, but only 8°/i, at 4.0 - IO 4 M. Tyramine had no effect at 4.0 - 10 -~ M, inhibited uptake (20~o) at 2.0 • 10 -4 M and activated uptake (22~o) at 6.7 - 10 4 M. These results are interpreted as further support for the multicomponent nature of the uptake phenomenon. Atropine, adenosine triphosphate, bulbocapnine, chlorpromazine, ergonovine, imipramine, a-methyl-m-tyrosine, pilocarpine, and strychnine were all inactive at 6 . 7 . 10-4 M. Acetytcholine, reserpine and phenoxybenzamine were inactive at 1.0- 10-4 M.
Subcellular distribution of norepinephrine uptake The appearance of the sucrose density gradient after centrifugation is shown in Fig. 3. Fractions A3 and A4 should contain nerve-ending particles with the myelin in fraction AI and the mitochondria in fraction A5. When these fractions were assayed for norepinephrine uptake activity, a bimodal distribution was observed. In Fig. 4. a histogram is presented which illustrates the relative activities of the fractions when assayed for norepinephrine uptake, succinoxidase, and cholinesterase. It can be seen that although the uptake activity is spread throughout the gradient, slightly increased activities are found in fraction AI and fractions A3. A4. and A5. Modifications in the composition of the gradient resulted in changes in the distribution which were consistent with the hypothesis that at least two distinct uptake components were present. In some cases a peak was observed in fraction A4 with fraction A5 being slightly lower. Further evidence for distinct components was obtained by determining the effects of inhibitors on the uptake activity of the fractions. Due to the small amounts FRACTIONS
SUCROSE 0.5~
I
0.7M
1. OM 1.2M
~
4
I.SM
Fig. 3. The appearance of the density gradient tube after centrifugation. Shaded areas indicate the location of the various fractions, Broken lines indicate the location of the boundari~ of the sucrose solutions and solid lines indicate where the tube was sliced for the isolation Of the fractions.
Brain Research, 8 (1968) 298-309
NOREPINEPHRINE UPTAKE
305
I00
>- 8O
K I.o
< 60 0
40 I.Z tad t3 n~
uJ 20
Ix.
AI
Az
A5
A4
A5
Fig. 4. T h e distribution of cholinesterase (C), succinoxidase (S), a n d norepinephrine uptake activity (N) in fractions of the c r u d e m i t o c h o n d r i a . T h e values are plotted as the percentage of the total activity f o u n d in the gradient.
of each fraction produced by the gradient procedure, duplicates were impossible within any run. This led to considerable variation in the effects noted for particular drugs. Duplicate runs were made, however, and permitted the detection and deletion of inconsistent results. Only reproducible effects are reported and the figures given are averages of multiple runs. T A B L E 11 THE
EFFECTS
OF
DRUGS
ON
THE
UPTAKE
OF
NOREPINEPHRINE
BY SUBFRACTION
OF
THE
CRUDE
MITO-
CHONDRIA
The values are given as the percentage o f the control values using an initial n o r e p i n e p h r i n e concentration of 4.0 - 10 6 M. All values are r o u n d e d to the nearest percentage. Drug
Fraction A1
A2
A3
A4
A5
Bufotenin bioxalate 2.10 4M Ergotamine
78
102
101
139
172
1.10
36
36
36
60
42
Psilocybin 1 • 10 a M Pyrogallol
112
120
189
239
220
1 - 10 a M
43
54
87
104
78
1 - 10 4 M
53
65
64
138
150
Serotonin s o d i u m oxalate 2 • 10 4 M
75
52
69
78
67
aM
Serotonin h y d r o g e n oxalate
Brain Research, 8 (1968) 298-309
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w.F.
ttERBI IN ANI) I~, t), ~ ' B R t l "-,
Table 11 summarizes the data obtained for the effects of drug,~ on the norepinephrine uptake activity of the individual gradient fractions. Serotonin gave consistent results on the gradient fractions, although the magnitude of the elfects varied. The hydrogen oxatate was used and this compound inhibited fractions AI-A3 and activated fractions A4 and A5. The results with bufotenin were not as definite. This compound seemed to have little effect on fraction A1, although slight inhibition was observed in certain runs. No consistent effect was observed on fraction A2. The effect on fraction A3 was extremely variable, but tended to approach zero ~hen averaged. Fractions A4 and A5 were activated in all instances by this drug. Ergotamine inhibited the uptake of all fractions. DISCUSSION
All facets of the results obtained for the uptake of norepinephrine by rat brain homogenates indicated the complexity of the phenomenon and inhomogeneity of the fractions. The curve obtained for the uptake of norepinephrine by the crude mitochondrial fraction exhibited four inflection points which indicated the parucipation of five separate components. The effects of the series of drugs demonstrated the mutticomponent nature of the system both by the lack of logical concentration dependence and the different effects on the separate gradient fractions. Once the crude mitochondrial fraction was recognized as containing multiple components, attempts at direct analysis were abandoned. It became obvious thal drugs which affected the various components of the curve non-uniformly would yield results which were extremely difficult to analyze. Therefore. further analysis was attempted by fractionating the crude mitochondria on a sucrose density gradient. When the more homogeneous fractions obtained from the gradient were investigated, a more meaningful determination of the uptake was possible. The data show that at least three distinct components are involved in the gross uptake curve. The component found in fraction A I could be due to either microsomes~ a population of small nerve-ending particles, or synaptic vesicles released from a small number of nerve-ending particles ruptured in preparation. Washing the crude mitochondrial fraction in 0.32 M sucrose before gradient fractionation reduces this component, a finding consistent with all of the proposed origins. This component is inhibited by serotonin, pyrogallol, and ergotamine, slightly inhibited by bufotenin. and activated by psilocybin. The component found in fraction A3 is most probably associated with isolated nerve-ending particles. Electron microscopic examinations of this fraction in other laboratories have indicated that nerve-ending particles are the primary constituent of this fraction, and similar studies of the fractions produced in our laboratory have confirmed this. This fraction would seem to correspond to the non-cholinergic nerveending particles fraction reported by De Robertis v. but in the absence of detailed assays for acetylcholine and choline acetylase, this correspondence is highly speculative. This component is also inhibited by serotonin, pyrogallol, and ergotamine. unaffected by bufotenin, and activated by psilocybin. Brain Research, 8 ( 1 9 6 8 ) 2 9 8 - 3 0 9
NOREPINEPHRINE UPTAKE
307
The component found in fractions A4 and A5 could be either mitochondria or a slightly different population of nerve-ending particles. The distribution of norepinephrine uptake in these fractions does not follow the succinoxidase activity distribution, but it is possible that the presence of two additional uptake components in the gradient effectively mask a relationship otherwise present. This component is inhibited by ergotamine and strongly activated by serotonin, bufotenin, and psilocybin. One striking feature of the drug profile of the fractions is that although bufotenin strongly inhibits the uptake by the crude mitochondrial fraction, this drug does not greatly inhibit the uptake of norepinephrine by any of the isolated fractions. This finding indicates that in addition to the multicomponent nature of the gross uptake curve, there may be an interaction of certain components of the crude mitochondrial fraction with some factor which is eliminated when the homogenate is further fractionated. The activation phenomenon is difficult to explain on the basis of any of the present theories concerning norepinephrine uptake. Calcium ion has been implicated in the release of norepinephrine 3, but its involvement in the activation is doubtful in view of the small effect of sodium oxalate. The uptake, storage, and release of norepinephrine have been the object of extensive research as outlined earlier. The major portions of this research have been devoted to either the adrenal medulla or peripheral sympathetically innervated structures. Both adrenal medullary granules and adrenergic nerve granules have been isolated and studied and have been found to be similar but not identical 9. The results obtained with subcellular particles from brain indicate that these particles are not identical with adrenal medullary granules or adrenergic nerve granules. Chlorpromazine inhibits the uptake of norepinephrine by both adrenal medullary granules and adrenergic nerve granules at drug concentrations of 1-3 • 10-5 M (ref. 9). This compound failed to significantly inhibit the uptake of norepinephrine by brain particles even at a concentration of 6.7 • 10 -4 M. This is in general agreement with the conclusion of P:etscher et aL TM, that chlorpromazine had little effect on the storage of catecholamines in the brain. The uptake of norepinephrine by adrenergic nerve granules has been reported to require the addition of ATP and Mg 2+ when the amine concentration is below 5 . 10-'~ M. This uptake is inhibited by reserpine, chlorpromazine and phenoxybenzamine 9. Brain homogenates exhibited uptake of norepinephrine at concentrations as low as I • l0 7 M, and the uptake was not activated by the addition of ATP and Mg ~ . Moreover, the uptake at the low norepinephrine concentration was not inhibited by either reserpine or phenoxybenzamine. It becomes increasingly clear that the uptake of norepinephrine by brain homogenates is not identical with the norepinephrine uptake by other preparations. This could indicate a basic difference between peripheral and central norepinephrine stores or could reflect changes in the characteristics of the uptake caused by the presence of the presynaptic membrane. This membrane might be expected to exhibit its own characteristic drug profile. It will be necessary to systematically 'dissect' the crude mitochondria by successive subfractionation in order to isolate and identify Brain Research, 8 (1968) 298 309
308
\V. F. HERBLIN ANI) R l), d,)'BRIIIN
the various uptake components and determine their individual drug profiles. Notable exceptions to the effectiveness of the indole nucleus are crgonovine and the LSD derivatives. LSD has been reported to block the activity of electrophoretically applied norepinephrine on certain central cells, and was lound to be a more effective blocker of norepinephrine than of serotonin zl. However, Marchbanks 1~ found that LSD inhibited serotonin binding by brain particles at low concentrations and LSD had little effect on the norepinephrine uptake investigated in this study. The interactions of serotonin and norepinephrine have been much discussed and little can be added here to the experimental observation that serotonin activates the uptake of norepinephrine. Conversely, norepinephrine inhibits serotonin uptake ~6. Brodie I has suggested that high levels of serotonin are tranquilizing whereas increased norepinephrine levels are excitatory. It becomes obvious that more information on the identity of uptake components and their drug profiles is required belbre a general explanation can be advanced. SUMMARY
The uptake of exogenous norepinephrine by subcellular fractions of rat brain has been investigated in a sucrose-NaCl-Tris buffer medium. Although the uptake of norepinephrine has been shown to be a temperature-dependent process, these studies were run at 0-4°C so that the results would be compatible with subsequent binding experiments. The uptake of this catecholamine which occurs at O-4°C is inhibited by pyrogallol, bufotenin, and ergotamine, and is activated by serotonin and psilocybin. Plots of norepinephrine uptake as a function of the equilibrium norepinephrine concentration have indicated that the uptake of norepinephrine is a multicomponent phenomenon, and this is supported by inhibition data. One component appears to be associated with the microsomal fraction and is inhibited by serotonin, pyrogallol, and ergotamine, and activated by psilocybin. A second component appears to be associated with nerve-ending particles and exhibits a very similar drug profile. A third component also appears to be associated with nerve,ending particles and is inhibited by ergotamine but activated by bufotenin, serotonin, and psilocybin. ACKNOWLEDGMENTS
This work was performed under the sponsorship of the Cognitive Systems Research Program and supported by Office of Naval Research Grant Nol NONR 401 (40) and National Science Foundation Grants No. GP-971 and GK,250. The authors would like to thank Dr. Frank Rosenblatt for guidanceand financial support and Miss Frances Fu-Ti Fan for technical assistance throughout the investigation. Bretylium tosylate was donated by Burroughs Wellcome and Co., chlorpromazine and phenoxybenzamine by the Smith, Kline and French Laboratories, imipramine by Geigy Pharmaceuticals, ismelin by the CIBA Pharmaceutical Co. mephenesin by E. R. Squibb and Sons, and the lysergic acid derivatives and psilocybin by Sandoz Pharmaceuticals. Brain Research, 8 (1968) 298-309
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Brain Researek, 8 (1968) 298-309