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Purinergic inhibition of diazepam binding to rat brain (in vitro )
Life Sciences, Vol. 24, pp. 851-8S8 Printed in the U.S.A.
Pergamon Press
PURINERGIC INHIBITION OF DIAZEPAM BINDING TO RAT BRAIN (IN VITRO) + P.J. Marangos , S.M. Paul, A.M. Parma, F.K. Goodwin P. Syapin and P. Skolnick National Institute of Mental Health and National Institute on Alcohol Abuse and Alcoholism 9000 Rockville Pike Bethesda, Maryland 20014 (Received in final form January 2S, 1979) SUMMARY Our recent report that the endogenous purines inosine and hypoxanthine competitively inhibit [3H] diazepam binding to rat brain synaptosomal membranes (1,2) has now been confirmed (3). We now report that a wide spectrum of purines are able to inhibit specific [3H] diazepam binding while pyrimidines are inactive. Preliminary structure activity relationships indicate that the 2'-deoxypurines are more potent in diazepam binding inhibition as are the 1-methyl compounds, whereas the 7-methyl purines are inactive. Data are also presented which show that the xanthine stimulants caffeine, theophylline, and theobromine as well as the central nervous system convulsant pentylenetetrazol all competitively inhibit [3H] diazepam binding. The recent finding that stereospecific, saturable benzodiazepine receptor sites exist in mammalian central nervous tissue has provided new insights into the possible mechanism of action of this class of tranquilizers (4-9). The existence of specific receptors in brain for benzodiazepines has raised the intriguing question of whether endogeneus compounds exist that interact with these sites in a physiologically relevant manner. We have recently purified two factors from bovine brain acetone extracts which competitively inhibit the specific binding of [3HI diazepam to rat brain synaptosomal membranes (i). These two fractions have been identified as the purines inosine and hypoxanthine (2,3). The present report examines a broad s~ectrum of purine and pyrmidine compounds for their abilities to inhibit [aH] diazepam binding to rat brain synaptosomal membranes. In addition, data are presented establishing that the methylxanthines caffeine, theophylline, and theobromine as well as the convulsant pentylenetetrazol also compete with diazepam for benzodiazepine receptors in vitro. METHODS Assay for specific 3H diazepam binding inhibition: Crude synaptosomal membranes were prepared from whole rat brains (Sprague-Dawley 150g) by h o m o g e n i z a tion in lO volumes of ice-cold 0.32 M sucrose using a teflon glass apparatus.
+To whom requests for reprints should be addressed.
0024-3205/79/090851-07502.00/0
852
Purinergic Inhibition of Diazepam Binding
Vol. 24, No. 9, 1979
The crude syT~aDtosomal P2 fraction was obtained as previously described (i), and carefully suspended in 50 tissue volumes of 50 mM tris-chloride buffer pH 7.4. The membrane suspension was stored at -20 ° until ready for use. No decrease in specific [3H] diazepam binding was observed during storage under these conditions for at least 2 months. The receptor binding assay employed was similar to the previously described procedure (6). The assay contained 0.5 ml of P2 suspension, 1.26 nM [3HI diazepam (New England Nuclear 80 Ci/mmole), and the compound to be tested. The total assay volume was i ml and the final tris-chloride (pH 7.4) buffer concentration was 50 mM. The assay was initiated by the addition of [3H] diazepam followed by a 15-minute incubation in ice. The assay was terminated by filtration of GF-B (Whatman) filters with 4 x 5 ml buffer washes. The filters were dried and counted in i0 ml of Aquasol (New England Nuclear) by liquid scintillation counting. Non-specific binding was determined by incorporation of 3 ~M unlabeled diazepam in the assay and routinely represented 5% of the total binding. The compounds tested for their ability to inhibit [3H] diazepam binding were prepared as 5 mM stock solutions in 50 mM tris-chloride pH 7.4 and added to the binding assay at 5 different concentrations. The pH of each stock solution was determined with no variations from pH 7.4 observed. The limited solubility of several of the compounds tested necessitated heating them to 60 ° . All heated solutions were cooled in ice after they dissolved and before addition into the binding assay. The IC50 and IC20 values for compounds inhibiting [3H] diazepam binding were determined by probit analysis. Inhibition kinetics were determined by Lineweaver Burke reciprocal analysis at one or two concentrations of the inhibitor. RESULTS Inhibition of 3H diazepam binding by purines: The ability of a variety of purine and pyrimidine compounds to inhibit specific [3H] diazepam binding was tested by their incorporation in the binding assay at concentrations between 125-2000 ~M. An example of the log-probit plots obtained is illustrated in Fig. i. Analysis of a series of purines and pyrimidines by this method produced the results shown in Table I. Pyrimidines such as cytosine, thymine, and uracil are not active inhibitors of binding, whereas the purines are active. Inosine, guanosine, and hypoxanthin~ display approximately equal potencies as inhibitors of [~H] diazepambinding with IC50 values of approximately i mM. Adenine is the most aeitve of the purine bases with an IC50 of 630 ~M. Removal of the 2'-hydroxyl group from the ribose moiety of inosine, guanosine, and adenosine (the 2'-deoxypurines) markedly increases inhibitory activity. The 2',3'-isopropylidene derivatives of inosine, guanosine and adenosine also exhibit greater inhibitory potency. Methyl substitution in the I position increases the potency in the case of both guanosine and adenosine while it decreases the potency of inosine. Methyl substitution in the 7 position decreases the inhibitory potency of both inosine and guanosine. Phosphorylated purines such as IMP, IDP, and ITP are inactive. Competitive inhibition of [3HI diazepam binding by methylxanthines and pentylenetetrazol: As shown in Table II the xanthine stimulants caffeine, theophylline, and theobromine all inhibit [3H] diazepam binding. The rank order potency observed is: caffeine > theophylline > theobromine. The convulsant pentylenetetrazol was also found to be active in displacing [3H] diazepam binding displaying an IC50 of 3.2 mM and an IC20 of 620 ~m. The nature of the [3H] diazepam binding inhibition exhibited by caffeine,
Vol. 24, No. 9, 1979
Purinergic
Inhibition of, Diazepam Binding
853
EmL "r" 95 Z I
"eo
125
I
250'500
I
I
I
I
1000
2000
5000
[h,m Figure 1 Log-probit analysis of [3H] diazepam binding inhibition exhibited by pentylenetetrazol and various purines. Compounds were incorporated into the [3H] diazen~m receptor binding assay at concentrations ranging from 125 ~m to 2 mM. The compounds are A pentylenetetrazol, ~ hypoxanthine, 0 inosine, 0 2'-deoxyinosine, A caffeine.
theophylline and pentylenetetrazol was determined by Lineweaver Burke analysis in the presence of fixed concentrations of each compound. The results shown in Fig. 2 indicate that in all three cases the Km for diazepam binding is increased while the maximal binding remains unchanged for each of the tested compounds, indicating that all three compounds are inhibiting specific diazepam binding in a competitive manner. DISCUSSION The demonstration of specific receptor sites in brain for benzodiazepines has stimulated interest in possible endogenous substances that may serve as ligands for these receptors (1,2,10). The isolation and characterization of several such compounds that are able to competitively inhibit [3H] diazepam binding to rat brain synaptosomes has recently been reported (2). These endogenous compounds have been identified as the purines, inosine and hypoxanthine. The present report examines the reactivity of a wide range of purine and pyrimidine compounds in displacing specific diazepam binding. The results clearly establish that a variety of purine compounds and their derivatives are active, whereas the pyrimidine are not. These studies also establish some preliminary structure-activity relationships for the purines concerning their
854
Purinergic
Inhibition of Diazepam Binding
Vol. 24, No. 9, 1979
TABLE I
COMPOUND Inosine 2'-Deoxyinosine 2',3'-Isopropylidene 1-Methyl Inosine 7-Methyl Inosine Guanosine 2'-Deoxyguanosine 2',3'-Isopropylidene 1-Methyl Guanosine 7-Methyl Guanosine Adenosine 2'-Deoxyadenosine 2',3'-Isopropylidene 1-Methyl Adenosine Hypoxanthine 1-Methyl Hypoxanthine Adenine Allopurinol Cytosine Uracil Thymine 2'-Deoxyribose
IC20 (~M)
Inosine
Guanosine
Adenosine
IC50 (~M)
Ki (~M)
165 65 82 1,520 --
1,150 395 360 >2,000 >2,000
836 287 262 3,673 --
240 75 83 160 --
1,000 280 325 610 >2,000
727 204 236 440 --
1,050 400 340 240
>2,000 2,000 1,750 1,100
4,291 -1,273 800
220 250 182 215 -----
1,350 1,300 630 >2,000 >2,000 >2,000 >2,000 >2,000
982 945 458 538 -----
Inhibition of [3H] diazepam binding by various purine compounds. Each of the compounds listed was tested at five different concentrations (125 ~M 2 mM) for inhibition of specific [3HI diazepam binding. The ICs0 and IC20 value~ were estimated by probit analysis. The Ki was calculated using the formula Ki = IC50 where [L] = the concentration of diazepam used (1.26 nM) and 1 + [L] K D = 3.4.
KD
TABLE II
COMPOUND Caffeine Theophylline Theobromine Pentylenetetrazol
IC20 (uM)
IC50 (~M)
62 95 425 620
390 620 2,500 3,200
Ki (DM) 284 451 1,818 2,327
Inhibition of [3HI diazepam binding by xanthine stimulants and pentylenetetrazol. Each of the compounds were tested for inhibition of [3H] diazepam binding at 5 concentrations ranging from 125 ~M to 4 mM. IC50 , IC20, and Ki ~alues were calculated as described in Table I.
Vol. 24, No. 9, 1979
Puringeric Inhibition of Diazepam Binding
855
CAFFEINE
16
:o: .-:?-
14
';'o x
12
/
/
PENTYLENETETRAZOL
4
2mM
6
0
8 ~
6
Kmt.M) o 1mM
2raM
1 (nM x 10') =H Diazepa~ x.
/
/ ImM
8.0
11.1
6
emykmetlmaz~
-li 4
2
THEOPHYLLINE
~ / ~ -2 -1
o
14
12 x c •~
1
a. E
m
"TheophyUine Km(nM)//2,~OHM 0 3.70 Jl( Theophylline / 2~,M 7.14 / ~
~
/
~
~ 1
'H Dizepam
6
(nM x 10')
. 0 Theophylhne
o
1 [nM x 10') =H Diazepam
Figure 2 Lineweaver inhibition porated at assay at 4
Burke analysis of caffeine, theophyl!ine, and pentylenetetrazol of [3HI diazepam binding. Each of the compounds tested was incorthe indicated concentration in the [3H] diazepam receptor binding to 6 different concentrations of [3H] diazepam.
6
856
Purinergic Inhibition of Diazepam Binding
Vol. 24, No. 9, 1979
potencies as inhibitors of [OH] diazepam binding. Purine bases such as hypoxanthine and adenine are as potent as nucleosides containing the ribose moiety. Compared to the hydroxylated purines tested, the 2'-deoxy derivatives which are also endogenous compounds display a marked increase in potency. Specifically, 2'-deoxyguanosine is 4 times more potent than guanosine while 2'-deoxyadenosine and 2'-deoxyinosine are 3 times more potent than adenosine and inosine respectively. Methylation of the purine ring also has marked effects on binding inhibition with 1-methyl substitutions causing an increase in potency and the 7-methyl compounds being completely inactive. The exception to this trend is 1-methyl inosine which is less active than inosine. The structural similarity between the purines and the xanthine stimulants suggested that these latter compounds might interact with the diazepam receptor. The rank order potencies of the methylxanthines as inhibitors of diazepam binding is in good agreement with their potencies in brain as cortical stimulants and conwllsants (ii) but not as adenosine antagonists (theophylline > caffeine > theobromine) or phosphodiesterase inhibitors (caffeine = theophyl]ine > theobromine). The IC50 for caffeine inhibition of diazepam binding is well below the concentration expected in brain of a lethal convulsant dose in humans (I0 grams caffeine p.o. distributed in whole body water would yield a brain concentration of 740 ~M). The finding that the convulsant pentylenetetrazol also inhibits diazepam binding coupled with the observation that diazepam specifically inhibits pentylenetetrazol-induced seizures suggests that the convulsant action of this drug involves an interaction with the benzodiazepine receptor. Consistent with this hypothesis is the observation that the IC20 for pentylenetetrazol inhibition of [3H] diazepam binding is in the range of in vivo concentrations known to elicit seizure activity (12). It has recently been shown that only 20-30% of benzodiazepine receptors must be occupied by diazepam to prevent pentylenetetrazol-induced seizures (unpublished observations). Also, inosine and 2'-deoxyinosine increase the latency of pentylenetetrazol-induced seizures, whereas 7-methyl inosine is without effect (13). It, therefore, appears that purine compounds are not only active in the in vitro receptor binding assay but that they share some of the pharmacological effects of diazepam. These results suggest that both xanthine stimulants (which have convulsant effects at high doses) and pentylenetetrazol exert pharmacologic effects opposite to those of diazepam and the purines and that these effects are mediated by the benzodiazepine receptor. These preliminary results further suggest an agonist-antagonist relationship exists between these two groups of compounds. Although the relatively high IC50 values for the purines raise questions about physiological relevance, the combined concentrations of inosine and hypoxanthine in brain are relatively high (approximately 50 lJM (14)) and local concentrations may be considerably higher. The existence of high affinity ligands may therefore be unnecessary. In this context it is interesting to note that both inosine and hypoxanthine rise markedly following electrical or chemical depolarization of brain (15,16). Although inosine and hypoxanthine have been isolated and identified as endogenous inhibitors of diazepam binding in brain (1,2), the multiplicity of active purines makes it premature to assign any particular compound(s) as the physiologically active benzodiazepine receptor ligand(s). However, the data indicate that a purine or purine-like compound may prove to be an endogenous ligand(s) or modulator of the benzodiazepine receptor in brain. Furthermore, the wide variations in potency brought about by slight modifications in the purine nucleus indicates that chemical substitution or modification of the purine nucleus may yield compounds of potential therapeutic benefit.
Vol. 24, No. 9, 1979
Purinergic Inhibition of Diazepam Binding
857
REFERENCES i. 2. 3. 4. 5. 6. 7. 8. 9. i0.
P.J. MARANGOS, S.M° PAUL, P. GREENLAW, F.K. GOODWIN and P. SKOLNICK, Life Sci. 2 0 : 1 8 9 3 (1978). P. SKOLNICK, P.J. MARANGOS, F.K. GOODWIN, M. EDWARDS and S.M. PAUL, Life Sci. 2 3 : 1 4 7 3 (1978). T. ASANO and S. SPECTOR, Proc. Nat. Acado Sci., in press. R.F. SQUIRES and C. BRAESTRUP, Nature 2 6 6 : 7 3 2 (1977). C. BRAESTRUP and R.F. SQUIRES, Proc. Nat° Acad. Sci. 7 4 : 3 8 0 5 (1977). H. MOHLER and T. OKADA, Life Sci. 2 0 : 2 1 0 1 (1977). H.B. BOSMANN, K.R. CASE, and P. DIS--TEFANO, FEBS Letters 8 2 : 3 6 8 (1977). C. BRAESTRUP, R. ALBRECHTSEN, and R.F. SQUIRES, Nature 2 6 9 : 7 0 2 (1977). M.J. WILLIAMSON, S.M. PAUL, and P. SKOLNICK, Nature 2 7 5 : 5 5 1 (1978). M. KAROBATH, G. SPERK, and G. SCHONBECK, Europ. J. Pharmacol. 4 9 : 3 2 3
(1978). ii. 12. 13. 14. 15. 16.
L.S. GOODMAN, and A. GILMAN, in The Pharmacolosical Basis of Therapeutics, Macmillan, London, p. 358 (1978), 4th Edition. F. MARCUCCI, M.L. AIROLDI, E. MUSSINI and S. GARATTINI, Europ. J. Pharmacol. 1 6 : 2 1 9 (1971). P. SKOLNICK, P.J. SYAPIN, B.A. PAUGH, V. MONCADA, P.J. MARANGOS and S.M. PAUL, Proc. Nat. Acad. Sci., in press. P. KLEIHUES, K. KOBAYASHI, and K.A. HOSSMAN, J. Neurochem. 2 3 : 4 1 7 (1974). I. PULL and H. MclLWAIN, Biochem. J. 1 2 6 : 9 7 5 (1972). M.C. SUN, H. MclLWAIN, and I. PULL, J. Neurobiol. 7 : 1 0 9 (1977).
Pergamon Press
PURINERGIC INHIBITION OF DIAZEPAM BINDING TO RAT BRAIN (IN VITRO) + P.J. Marangos , S.M. Paul, A.M. Parma, F.K. Goodwin P. Syapin and P. Skolnick National Institute of Mental Health and National Institute on Alcohol Abuse and Alcoholism 9000 Rockville Pike Bethesda, Maryland 20014 (Received in final form January 2S, 1979) SUMMARY Our recent report that the endogenous purines inosine and hypoxanthine competitively inhibit [3H] diazepam binding to rat brain synaptosomal membranes (1,2) has now been confirmed (3). We now report that a wide spectrum of purines are able to inhibit specific [3H] diazepam binding while pyrimidines are inactive. Preliminary structure activity relationships indicate that the 2'-deoxypurines are more potent in diazepam binding inhibition as are the 1-methyl compounds, whereas the 7-methyl purines are inactive. Data are also presented which show that the xanthine stimulants caffeine, theophylline, and theobromine as well as the central nervous system convulsant pentylenetetrazol all competitively inhibit [3H] diazepam binding. The recent finding that stereospecific, saturable benzodiazepine receptor sites exist in mammalian central nervous tissue has provided new insights into the possible mechanism of action of this class of tranquilizers (4-9). The existence of specific receptors in brain for benzodiazepines has raised the intriguing question of whether endogeneus compounds exist that interact with these sites in a physiologically relevant manner. We have recently purified two factors from bovine brain acetone extracts which competitively inhibit the specific binding of [3HI diazepam to rat brain synaptosomal membranes (i). These two fractions have been identified as the purines inosine and hypoxanthine (2,3). The present report examines a broad s~ectrum of purine and pyrmidine compounds for their abilities to inhibit [aH] diazepam binding to rat brain synaptosomal membranes. In addition, data are presented establishing that the methylxanthines caffeine, theophylline, and theobromine as well as the convulsant pentylenetetrazol also compete with diazepam for benzodiazepine receptors in vitro. METHODS Assay for specific 3H diazepam binding inhibition: Crude synaptosomal membranes were prepared from whole rat brains (Sprague-Dawley 150g) by h o m o g e n i z a tion in lO volumes of ice-cold 0.32 M sucrose using a teflon glass apparatus.
+To whom requests for reprints should be addressed.
0024-3205/79/090851-07502.00/0
852
Purinergic Inhibition of Diazepam Binding
Vol. 24, No. 9, 1979
The crude syT~aDtosomal P2 fraction was obtained as previously described (i), and carefully suspended in 50 tissue volumes of 50 mM tris-chloride buffer pH 7.4. The membrane suspension was stored at -20 ° until ready for use. No decrease in specific [3H] diazepam binding was observed during storage under these conditions for at least 2 months. The receptor binding assay employed was similar to the previously described procedure (6). The assay contained 0.5 ml of P2 suspension, 1.26 nM [3HI diazepam (New England Nuclear 80 Ci/mmole), and the compound to be tested. The total assay volume was i ml and the final tris-chloride (pH 7.4) buffer concentration was 50 mM. The assay was initiated by the addition of [3H] diazepam followed by a 15-minute incubation in ice. The assay was terminated by filtration of GF-B (Whatman) filters with 4 x 5 ml buffer washes. The filters were dried and counted in i0 ml of Aquasol (New England Nuclear) by liquid scintillation counting. Non-specific binding was determined by incorporation of 3 ~M unlabeled diazepam in the assay and routinely represented 5% of the total binding. The compounds tested for their ability to inhibit [3H] diazepam binding were prepared as 5 mM stock solutions in 50 mM tris-chloride pH 7.4 and added to the binding assay at 5 different concentrations. The pH of each stock solution was determined with no variations from pH 7.4 observed. The limited solubility of several of the compounds tested necessitated heating them to 60 ° . All heated solutions were cooled in ice after they dissolved and before addition into the binding assay. The IC50 and IC20 values for compounds inhibiting [3H] diazepam binding were determined by probit analysis. Inhibition kinetics were determined by Lineweaver Burke reciprocal analysis at one or two concentrations of the inhibitor. RESULTS Inhibition of 3H diazepam binding by purines: The ability of a variety of purine and pyrimidine compounds to inhibit specific [3H] diazepam binding was tested by their incorporation in the binding assay at concentrations between 125-2000 ~M. An example of the log-probit plots obtained is illustrated in Fig. i. Analysis of a series of purines and pyrimidines by this method produced the results shown in Table I. Pyrimidines such as cytosine, thymine, and uracil are not active inhibitors of binding, whereas the purines are active. Inosine, guanosine, and hypoxanthin~ display approximately equal potencies as inhibitors of [~H] diazepambinding with IC50 values of approximately i mM. Adenine is the most aeitve of the purine bases with an IC50 of 630 ~M. Removal of the 2'-hydroxyl group from the ribose moiety of inosine, guanosine, and adenosine (the 2'-deoxypurines) markedly increases inhibitory activity. The 2',3'-isopropylidene derivatives of inosine, guanosine and adenosine also exhibit greater inhibitory potency. Methyl substitution in the I position increases the potency in the case of both guanosine and adenosine while it decreases the potency of inosine. Methyl substitution in the 7 position decreases the inhibitory potency of both inosine and guanosine. Phosphorylated purines such as IMP, IDP, and ITP are inactive. Competitive inhibition of [3HI diazepam binding by methylxanthines and pentylenetetrazol: As shown in Table II the xanthine stimulants caffeine, theophylline, and theobromine all inhibit [3H] diazepam binding. The rank order potency observed is: caffeine > theophylline > theobromine. The convulsant pentylenetetrazol was also found to be active in displacing [3H] diazepam binding displaying an IC50 of 3.2 mM and an IC20 of 620 ~m. The nature of the [3H] diazepam binding inhibition exhibited by caffeine,
Vol. 24, No. 9, 1979
Purinergic
Inhibition of, Diazepam Binding
853
EmL "r" 95 Z I
"eo
125
I
250'500
I
I
I
I
1000
2000
5000
[h,m Figure 1 Log-probit analysis of [3H] diazepam binding inhibition exhibited by pentylenetetrazol and various purines. Compounds were incorporated into the [3H] diazen~m receptor binding assay at concentrations ranging from 125 ~m to 2 mM. The compounds are A pentylenetetrazol, ~ hypoxanthine, 0 inosine, 0 2'-deoxyinosine, A caffeine.
theophylline and pentylenetetrazol was determined by Lineweaver Burke analysis in the presence of fixed concentrations of each compound. The results shown in Fig. 2 indicate that in all three cases the Km for diazepam binding is increased while the maximal binding remains unchanged for each of the tested compounds, indicating that all three compounds are inhibiting specific diazepam binding in a competitive manner. DISCUSSION The demonstration of specific receptor sites in brain for benzodiazepines has stimulated interest in possible endogenous substances that may serve as ligands for these receptors (1,2,10). The isolation and characterization of several such compounds that are able to competitively inhibit [3H] diazepam binding to rat brain synaptosomes has recently been reported (2). These endogenous compounds have been identified as the purines, inosine and hypoxanthine. The present report examines the reactivity of a wide range of purine and pyrimidine compounds in displacing specific diazepam binding. The results clearly establish that a variety of purine compounds and their derivatives are active, whereas the pyrimidine are not. These studies also establish some preliminary structure-activity relationships for the purines concerning their
854
Purinergic
Inhibition of Diazepam Binding
Vol. 24, No. 9, 1979
TABLE I
COMPOUND Inosine 2'-Deoxyinosine 2',3'-Isopropylidene 1-Methyl Inosine 7-Methyl Inosine Guanosine 2'-Deoxyguanosine 2',3'-Isopropylidene 1-Methyl Guanosine 7-Methyl Guanosine Adenosine 2'-Deoxyadenosine 2',3'-Isopropylidene 1-Methyl Adenosine Hypoxanthine 1-Methyl Hypoxanthine Adenine Allopurinol Cytosine Uracil Thymine 2'-Deoxyribose
IC20 (~M)
Inosine
Guanosine
Adenosine
IC50 (~M)
Ki (~M)
165 65 82 1,520 --
1,150 395 360 >2,000 >2,000
836 287 262 3,673 --
240 75 83 160 --
1,000 280 325 610 >2,000
727 204 236 440 --
1,050 400 340 240
>2,000 2,000 1,750 1,100
4,291 -1,273 800
220 250 182 215 -----
1,350 1,300 630 >2,000 >2,000 >2,000 >2,000 >2,000
982 945 458 538 -----
Inhibition of [3H] diazepam binding by various purine compounds. Each of the compounds listed was tested at five different concentrations (125 ~M 2 mM) for inhibition of specific [3HI diazepam binding. The ICs0 and IC20 value~ were estimated by probit analysis. The Ki was calculated using the formula Ki = IC50 where [L] = the concentration of diazepam used (1.26 nM) and 1 + [L] K D = 3.4.
KD
TABLE II
COMPOUND Caffeine Theophylline Theobromine Pentylenetetrazol
IC20 (uM)
IC50 (~M)
62 95 425 620
390 620 2,500 3,200
Ki (DM) 284 451 1,818 2,327
Inhibition of [3HI diazepam binding by xanthine stimulants and pentylenetetrazol. Each of the compounds were tested for inhibition of [3H] diazepam binding at 5 concentrations ranging from 125 ~M to 4 mM. IC50 , IC20, and Ki ~alues were calculated as described in Table I.
Vol. 24, No. 9, 1979
Puringeric Inhibition of Diazepam Binding
855
CAFFEINE
16
:o: .-:?-
14
';'o x
12
/
/
PENTYLENETETRAZOL
4
2mM
6
0
8 ~
6
Kmt.M) o 1mM
2raM
1 (nM x 10') =H Diazepa~ x.
/
/ ImM
8.0
11.1
6
emykmetlmaz~
-li 4
2
THEOPHYLLINE
~ / ~ -2 -1
o
14
12 x c •~
1
a. E
m
"TheophyUine Km(nM)//2,~OHM 0 3.70 Jl( Theophylline / 2~,M 7.14 / ~
~
/
~
~ 1
'H Dizepam
6
(nM x 10')
. 0 Theophylhne
o
1 [nM x 10') =H Diazepam
Figure 2 Lineweaver inhibition porated at assay at 4
Burke analysis of caffeine, theophyl!ine, and pentylenetetrazol of [3HI diazepam binding. Each of the compounds tested was incorthe indicated concentration in the [3H] diazepam receptor binding to 6 different concentrations of [3H] diazepam.
6
856
Purinergic Inhibition of Diazepam Binding
Vol. 24, No. 9, 1979
potencies as inhibitors of [OH] diazepam binding. Purine bases such as hypoxanthine and adenine are as potent as nucleosides containing the ribose moiety. Compared to the hydroxylated purines tested, the 2'-deoxy derivatives which are also endogenous compounds display a marked increase in potency. Specifically, 2'-deoxyguanosine is 4 times more potent than guanosine while 2'-deoxyadenosine and 2'-deoxyinosine are 3 times more potent than adenosine and inosine respectively. Methylation of the purine ring also has marked effects on binding inhibition with 1-methyl substitutions causing an increase in potency and the 7-methyl compounds being completely inactive. The exception to this trend is 1-methyl inosine which is less active than inosine. The structural similarity between the purines and the xanthine stimulants suggested that these latter compounds might interact with the diazepam receptor. The rank order potencies of the methylxanthines as inhibitors of diazepam binding is in good agreement with their potencies in brain as cortical stimulants and conwllsants (ii) but not as adenosine antagonists (theophylline > caffeine > theobromine) or phosphodiesterase inhibitors (caffeine = theophyl]ine > theobromine). The IC50 for caffeine inhibition of diazepam binding is well below the concentration expected in brain of a lethal convulsant dose in humans (I0 grams caffeine p.o. distributed in whole body water would yield a brain concentration of 740 ~M). The finding that the convulsant pentylenetetrazol also inhibits diazepam binding coupled with the observation that diazepam specifically inhibits pentylenetetrazol-induced seizures suggests that the convulsant action of this drug involves an interaction with the benzodiazepine receptor. Consistent with this hypothesis is the observation that the IC20 for pentylenetetrazol inhibition of [3H] diazepam binding is in the range of in vivo concentrations known to elicit seizure activity (12). It has recently been shown that only 20-30% of benzodiazepine receptors must be occupied by diazepam to prevent pentylenetetrazol-induced seizures (unpublished observations). Also, inosine and 2'-deoxyinosine increase the latency of pentylenetetrazol-induced seizures, whereas 7-methyl inosine is without effect (13). It, therefore, appears that purine compounds are not only active in the in vitro receptor binding assay but that they share some of the pharmacological effects of diazepam. These results suggest that both xanthine stimulants (which have convulsant effects at high doses) and pentylenetetrazol exert pharmacologic effects opposite to those of diazepam and the purines and that these effects are mediated by the benzodiazepine receptor. These preliminary results further suggest an agonist-antagonist relationship exists between these two groups of compounds. Although the relatively high IC50 values for the purines raise questions about physiological relevance, the combined concentrations of inosine and hypoxanthine in brain are relatively high (approximately 50 lJM (14)) and local concentrations may be considerably higher. The existence of high affinity ligands may therefore be unnecessary. In this context it is interesting to note that both inosine and hypoxanthine rise markedly following electrical or chemical depolarization of brain (15,16). Although inosine and hypoxanthine have been isolated and identified as endogenous inhibitors of diazepam binding in brain (1,2), the multiplicity of active purines makes it premature to assign any particular compound(s) as the physiologically active benzodiazepine receptor ligand(s). However, the data indicate that a purine or purine-like compound may prove to be an endogenous ligand(s) or modulator of the benzodiazepine receptor in brain. Furthermore, the wide variations in potency brought about by slight modifications in the purine nucleus indicates that chemical substitution or modification of the purine nucleus may yield compounds of potential therapeutic benefit.
Vol. 24, No. 9, 1979
Purinergic Inhibition of Diazepam Binding
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