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The binding of 3H-acetylcholine to cholinergic receptors in bovine cerebral arteries
Life Sciences, Vol. 37, pp. 1887-1893 Printed in the U.S.A.
Pergamon Press
THE BINDING OF 3H-ACETYLCHOLINE TO CHOLINERGIC RECEPTORS IN BOVINE CEREBRAL ARTERIES
S. Shimohama, T. Tsukahara, T. Taniguchi and M. Fujiwara
Department of Pharmacology, Faculty of Medicine, Kyoto University, Kyoto 606, Japan (Received in final form September 4, 1985) Summary Cholinergic receptor sites in bovine cerebral arteries were analyzed using radioligand binding techniques with the cholinergic agonist, 3H-acetylcholine (ACh), as the ligand. Specific binding of 3H-ACh to membrane preparations of bovine cerebral arteries was saturable, of two binding sites, with dissociation constant (KD) values of 0.32 and 23.7 riM, and maximum binding capacity (Bmax) values of 67 and 252 fmol/mg protein, respectively. Specific binding of 3H-ACh was displaced effectively by muscarinic cholinergic agents and less effectively by nicotinic cholinergic agents. IC 50 values of cholinergic drugs for 3H-ACh binding were as follows: atropine, 38.5 nM; ACh, 59.8 nM; oxotremorine, 293 nM; scopolamine 474 nM; carbamylcholine, 990 nM. IC50 values of nicotinic cholinergic agents such as nicotine, cytisine and ~-bungarotoxin exceeded 50 pM. Choline acetyltransferase activity was 1.09 nmol/mg protein/hour in the cerebral arteries. These findings suggest that the cholinergic nerves innervate the bovine cerebral arteries and that there are at least two classes of ACh binding sites of different affinities on muscarinic receptors in these arteries. Cholinergic innervation to cerebral blood vessels is considered to play an important role in regulation of cerebral blood flow (1-5). Although muscarinic cholinergic receptors in the cerebral blood vessels were analyzed with a radioligand binding assay using a specific muscarinic antagonist, 3H-quinuclidinyl benzilate (QNB), as ligand (6-8), data regarding the 3H-agonist binding to cholinergic receptors in the cerebral blood vessels have apparently not been reported. 3H-Acetylcholine (ACh) is a specific cholinergic agonist and has been used to identify nicotinic and muscarinic cholinergic receptor sites in the mouse brain (9), Torpedo electric tissue (I0,II) and rat brain (12,13). We have now characterized the cholinergic receptor sites in bovine cerebral arteries using 3H-ACh as a ligand.
Methods Membrane preparations Pia-arachnoid (pial) arteries were carefully removed from the bovine brain within 30 min of obtaining the brains from the local slaughterhouse and were kept in a freezer (-80°C). Membranes from the arteries were prepared as 0024-3205/85 $3.00 + .00 Copyright (c) 1985 Pergamon Press Ltd.
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follows: the arteries were minced with scissors and then homogenized in i0 volumes of Ice-cold 50 raM sodium phosphate buffer (pH 7.4) with a glass homogenizer. The artery homogenates were filtrated through two layers of gauze, then were homogenized at a setting of i0 on a Polytron (Brlnkmann Instrument) with 20 second bursts. The homogenate was centrifuged at 1,000 x g for I0 mln, and an aliquot of the supernatant was preserved for analysis of choline acetyltransferase (CHAT) activity. The residual supernatant was carefully removed and centrifuged at i00,000 x g for 60 mln. The resulting pellet was resuspended in 50 mM sodium phosphate buffer (pH 7.4). Protein concentrations were determined by the method of Lowry et al. (14). 3H-ACh binding assay The 3H-ACh binding assay was performed according to the procedure of Schwartz et al. (13). In brief, aliquots of the bovine cerebral artery membranes were incubated at 0°C for 40 mln in 250 ~i of 50 rmM sodium phosphate buffer (pH 7.4), containing 3H-ACh, in the absence or presence of a high concentration of carbamylchollne (I00 ~M). To prevent hydrolysis of the 3H_ACh by chollnesterase, i00 ~M diisopropylfluorophosphate (DFP) was added to the assay mixture. The assay was terminated by the addition of 3 ml of ice-cold buffer and then filtrated under reduced pressure through Whatman GF/C glass fiber filters, which had been soaked in a 0.I % polyethyleneimine solution to eliminate nonspecific binding to the filter. After three washings with the buffer, the filters were dried in an oven and transferred to counting vials, after which 8 ml of scintillation fluid was added. Radioactivity was counted in a Packard Tri-Carb scintillation spectrometer (Model 3255). Each assay was performed in duplicate. Nonspecific binding was defined as 3 H-ACh binding in the presence of I00 ~M carbamylcholine, whereas total binding was defined as that in the absence of carbamylcholine. Specific binding was defined as the difference between total and nonspecific bindings. H, qNB binding Binding of 3H-QNB was assayed by the method of Yamamura and Snyder (15). In brief, atropine (I0 ~M) was used to define nonspeciflc binding. The tubes, containing 3H-QNB, the tissue membranes and 50 raM sodium phosphate buffer (pH 7.4) to a final volume of 250 ~i, were incubated at 37°C for I hr. The assay was terminated by the addition of 3 ml of ice-cold buffer and then filtrated under reduced pressure through Whatman GF/C glass fiber filters. Filters were then counted for radioactivity in 8 ml of scintillation fluid. ChAT assay ChAT activity in the 1,000 x g supernatant fractions of the bovine cerebral arteries was measured according to the method of Fonnum (16), as modified by Florence and Bevan (17). ChAT activity was expressed as nmol/mg protein/hour. Chemicals and Reagents 3H-acetylchollne (ACh) (86 Ci/mmol) was obtained from Amersham Inter national plc, Bu c klnghamshire, England, and 3 H-l-quinuclldlnyl benzilate (QNB) (36 Ci/mmol) an d i~ 4 C-acetyl-coenzyme A (53 mCi/~mol) were obtained from New England Nuclear, Boston, Mass., and cold QNB was a gift from Dr. S. Spector, Roche Institute of Molecular Biology, Nutley, N.J.. All other chemicals were of reagent grade and were obtained commercially.
Vol. 37, No. 20, 1985
3H-Ach Binding to Bovine Cerebral Artery
1889
20
15
1C c.-,= o ~D
I
0
100
200
I
300
3H-ACh Bound (fn~l/n~ protein) FIG. 1 A typical Scatchard plot of 3H-ACh saturation binding. Analysis of the data was made by the computer best-fit two-site model, with the two individual binding components represented by the lower two lines, including a high affinity site (KDI = 0.26 nM, Bmax = 53 fmol/mg protein) and a low affinity site ( ~ 2 = 18.8 nM, Bmax = 252 fmol/mg protein).
Results 3H-ACh bindin~ to bovine cerebral artery membranes Specific 3H-ACh binding increased linearly with increasing concentrations of the membranes over the range of 25 ~g to I00 pg of protein per assay (data not shown). Specific binding of increasing concentrations of 3H-ACh (0.26 to 32.5 nM) was saturable. Theoretical binding curves were fitted to the twosite model by a non-linear least squared regression analysis using a computer program described by Olsen et al. (18). The best fit gives values of the affinity constant for the high affinity site of 0.26 nM, and for the low affinity site of 18.8 nM with maximum bindings of 53 fmol/mg protein and 252 fmol/mg protein, respectively (Fig.l). The K D and Bmax in three experiments are summarized in Table I. Time course of association and dissociation of the 3H-ACh binding was studied in bovine cerebral artery membranes. Association of 3H-ACh was time-dependent and the binding reached equilibrium within 30 min. The binding was rapidly reversed by addition of a high concentration (I00 ~M) of carbamylcholine. The rate constant of association (K+I) was 0.0185 x 109 M - ~ i n -I. The rate constant of dissociation (K_I) was 0.34 min -I (Fig. 2). The dissociation constant calculated from the equation K D = K_I/K+I was 18.3 nM, such being in close agreement with the K D of low affinity site determined by Scatchard analyses. Time course of association and
1890
3H-Ach Binding to Bovine Cerebral Artery
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~
Vol. 37, No. 20, 1985
~
so
-r
0
10
20
30
Time
40
50
60
(min)
FIG. 2 Time course of association (0) and dissociation ~ ) of specific 3H-ACh binding. At 40 min i00 ~M unlabelled earbamylchollne was added. Concentration of 3H_ACh was 13 nM. One hundred percent specific 3H-ACh binding was 160 fmol/mg protein. Figures are the mean of three experiments.
TABLE I
3H-ACh and 3H-QNB BINDING IN BOVINE CEREBRAL ARTERY MEMBRANES
Ligand
a KDI
Bb maxl
3H-ACh
0.32 + 0.14
67 + I0 a KD
3H-QNB
0.55 + 0.04
Ka D2
Bb max2
23.7 + 6.6
252 + 20
Bb max
514 + 20
a: nM b: fmol/mg protein Results are the mean + S.E.M. of three experiments.
Vol. 37, No. 20, 1985
3H-Ach Binding to Bovine Cerebral Artery
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TABLE II
INHIBITION BY VARIOUS DRUGS OF SPECIFIC 3H-ACh BINDING TO BOVINE CEREBRAL ARTERY MEMBRANES a
Drug
Atropine Acetylcholine Oxotremorlne Scopolamine Carbamylcholine (-)-Nicotine Cytislne e-bungatotoxin
IC~0(M)
3.85 5.98 2.93 4.74 9.90
+ ~ ~ ~ ~ ~ > >
1.52 2.97 0.79 1.88 4.81 5 5 5
x x x x x x x x
I0-8 I0-~ I0-~ i0-~ I0-~ I0-~ I0-~ I0 -D
Hill coefficient
N
0.91 0.90 0.87 0.94 0.87
6 6 4 3 6 3 3 3
+ ~ + ~ ~ --
0.17 0.07 0.07 0.33 0.I0
a: 3H-ACh binding was performed as described under "Methods". Concentration of 3H-ACh was 13 nM. b: IC50 is the concentration of drug that reduces the specific 3H-ACh binding by 50 %. The results are the mean + S.E.M. of the number of experiments indicated.
dissociation of the 3H-ACh binding was also studied using low 3H-ACh concentration (1.2 nM) (data not shown). The half-tlme for association was 3 min, and the binding reached equilibrium within 30 mln. The half-tlme for dissociation was 3 min. K + l W a S 0.5 x 10 9 M - ~ I n - l . K _ l W a S 0.17 mln The dissociation constant calculated from the equation K D = K_I/K+I was 0.34 ruM, such being in good agreement with the K D of high affinity site determined by Scatchard analyses. The specificity of 3H-ACh binding was studied using chollnerglc agonists and antagonists. Values of IC50 of muscarinic agonists, acetylcholine, oxotremorine and carbamylcholine and the specific muscarinlc antagonists, atropine and scopolamine were less than 1.26 pM (Table II). In contrast, the IC50 values of nicotinic agents such as ~-bungarotoxln, cytisine and nicotine exceeded 50 ~M. The data on the inhibition were analyzed using Hill plots (Table II). 3H-QNB bindin~ to bovine cerebral artery membranes Specific binding of increasing concentrations of 3H-QNB (0.15 to 5 ruM) was saturable (data not shown). Scatchard analysis indicated a single class of binding site. The K D and Bmax in four experiments are summarized in Table I. ChAT activity in bovine cerebral artery membranes ChAT activity was detected in all bovine cerebral artery membranes studied. The activity was linear with time at lease up to 20 mln (data not shown). The ChAT activity in bovine cerebral arteries was 1.09 + 0.14 nmol/mg protein/hour (N=3).
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Discussion Cholinergic receptor sites in bovine cerebral arteries were characterized using 3H_ACh as the ligand. Specific binding of 3H-ACh to the cerebral arteries was saturable, of high affinity and reversible. Scatchard plot analysis of the data indicated that 3H-ACh binding sites in the cerebral arteries were of two classes. Cholinergic drugs competed for this binding in the order of atropine > ACh > oxotremorine > scopolamine > carbamylcholine. The potent nicotinic drugs, such as nicotine, cytisine and a-bungarotoxin, failed to inhibit 3H-ACh binding even at I0 pM. These results suggest that 3H-ACh binding sites exhibit properties of muscarinic cholinergic receptors in the bovine cerebral arteries. The characteristics of the muscarinic receptors in bovine cerebral arteries have been studied using 3H-QNB, an antagonist, as ligand (6-8). These workers reported that 3H-QNB binding site appeared to be of a single class, on the basis of Scatchard plot analyses. In the present study, Scatchard analysis of specific 3H-QNB binding to the cerebral arteries also indicated one class of binding site. However, 3H_ACh binding sites in the cerebral arteries in this study, which appeared to be muscarinic cholinergic receptor sites, indicated two classes of binding sites. Indeed, Estrada and Krase (6) suggested the presence of more than one class of binding sites, as deduced from °H-QNB competition binding studies. Using 3 H-muscarinic agonists such as 3H-ACh and 3H-oxotremorine, Birdsall et al. obtained curved Scatchard plots, in case of the rat cerebral cortex (12). They suggested the presence of two major populations of agonist binding sites which do not interconvert during the binding experiments and have the same affinity constants for antagonist, in the rat cerebral cortex. Since the Scatchard analysis showed two binding sites, the Hill coefficients would be assumed to be less than 1.0. However, the calculated Hill coefficients for muscarinic agonist and antagonist competition with 13 nM 3H-ACh were close to 1.0 in bovine cerebral arteries in the present study. Birdsall et al. (12) showed that the Hill coefficients for agonist displacement of 3H-agonist binding were among 0.8 and 1.0 in the rat cerebral cortex. Indeed, the Hill coefficients of oxotremorine and ACh were 1.00 and 1.06, respectively, in their experiments (12). As the kineticall~-derived dissociation constant deduced from the experiments using 13 nM OH-ACh was in good agreement with the K D of low affinity binding sites determined by the Scatchard analysis, the displacement studies using 13 nM 3H-ACh seemed to reflect mainly low affinity binding sites in the present study. So in the displacement studies the presence of high affinity binding sites did not have large influence on the result. The Hill coefficients near 1.0 may be explained by the use of high concentration of 3H-ACh. We showed in the present work that there were multiple agonist binding sites in bovine cerebral arteries. Further work is required to understand the functional role of these binding sites. Acknowledgments This work was supported by a Grant-in-Aid for Cooperative Research (No. 60304046) from the Ministry of Education, Science and Culture, Japan, and by a grant from the Japan Tobacco Inc. We thank M.Ohara for reading the manuscript.
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References I. 2. 3. 4. 5. 6. 7. 8. 9. I0. II. 12. 13. 14. 15. 16. 17. 18.
L. EDVINSSON, B. FALCK and C. OWMAN, J. Pharmaeol. Exp. Ther. 200 117-126 (1977). L. EDVINSSON, C. OWMAN and N. SJOBERG, Brain Res. 115 377-393 (1976). N. TODA, J. Pharmacol. Exp. Ther. 209 352-358 (1979~. T.J.F. Lee, Eur. J. Pharmacol. 68 393-394 (1980). H. USUI, K. KURAHASHI, K. ASHIDA and M. FUJIWARA, IRCS Med. Sci. II 418419 (1983). C. ESTRADA and D.N. KRASE, J. Pharmacol. Exp. Ther. 221 85-90 (1982). C. ESTRADA, E. HAMEL and D.N. KRASE, Brain Res. 266 261-270 (1983). P. GRAMMAS, C.A. DIGLIO, B.H. MARKS, F. GIACOMELLI and J. WIENER, J. Neurochem. 40 645-651 (1983). L.S. SCHLEITER and M.E. ELDEFRAWI, Neuropharmacology 1 3 5 3 - 6 3 (1974). N.D. BOYD and J.B. COHEN, Biochemistry 195344-5353 (1980). N.D. BOYD and J.B. COHEN, Biochemistry 1 9 5 3 5 3 - 5 3 5 8 (1980). N.J.M. BIRDSALL, A.S.V. BURGEN and E.C. HULME, Mol. Pharmacol. 14 723-736 (1978). R.D. SCHWARTZ, R. McGEE and K.J. KELLAR, Mol. Pharmacol. 22 56-62 (1982). O.H. LOWRY, M.J. ROSEBROUGH, A.L. FARR and R.J. RANDALL, J. Biol. Chem. 193 265-275 (1951). H.I. YAMAMURA and S.H. SNYDER, Proc. Natl. Acad. Sci. USA 71 1725-1729 (1974). F. FONNUM, Biochem. J. 115 465-472 (1969). V.M. FLORENCE and J.A. BEVAN, Cir. Res. 45 212-218 (1979). R.W. OLSEN, M.O. VERGMAN, P.C.V. NESS, S.C. LUMMITS, A.E. WATKINS, C. NALIAS and D.V. GREENLEE, Mol. Pharmacol. 19 217-227 (1981).
Pergamon Press
THE BINDING OF 3H-ACETYLCHOLINE TO CHOLINERGIC RECEPTORS IN BOVINE CEREBRAL ARTERIES
S. Shimohama, T. Tsukahara, T. Taniguchi and M. Fujiwara
Department of Pharmacology, Faculty of Medicine, Kyoto University, Kyoto 606, Japan (Received in final form September 4, 1985) Summary Cholinergic receptor sites in bovine cerebral arteries were analyzed using radioligand binding techniques with the cholinergic agonist, 3H-acetylcholine (ACh), as the ligand. Specific binding of 3H-ACh to membrane preparations of bovine cerebral arteries was saturable, of two binding sites, with dissociation constant (KD) values of 0.32 and 23.7 riM, and maximum binding capacity (Bmax) values of 67 and 252 fmol/mg protein, respectively. Specific binding of 3H-ACh was displaced effectively by muscarinic cholinergic agents and less effectively by nicotinic cholinergic agents. IC 50 values of cholinergic drugs for 3H-ACh binding were as follows: atropine, 38.5 nM; ACh, 59.8 nM; oxotremorine, 293 nM; scopolamine 474 nM; carbamylcholine, 990 nM. IC50 values of nicotinic cholinergic agents such as nicotine, cytisine and ~-bungarotoxin exceeded 50 pM. Choline acetyltransferase activity was 1.09 nmol/mg protein/hour in the cerebral arteries. These findings suggest that the cholinergic nerves innervate the bovine cerebral arteries and that there are at least two classes of ACh binding sites of different affinities on muscarinic receptors in these arteries. Cholinergic innervation to cerebral blood vessels is considered to play an important role in regulation of cerebral blood flow (1-5). Although muscarinic cholinergic receptors in the cerebral blood vessels were analyzed with a radioligand binding assay using a specific muscarinic antagonist, 3H-quinuclidinyl benzilate (QNB), as ligand (6-8), data regarding the 3H-agonist binding to cholinergic receptors in the cerebral blood vessels have apparently not been reported. 3H-Acetylcholine (ACh) is a specific cholinergic agonist and has been used to identify nicotinic and muscarinic cholinergic receptor sites in the mouse brain (9), Torpedo electric tissue (I0,II) and rat brain (12,13). We have now characterized the cholinergic receptor sites in bovine cerebral arteries using 3H-ACh as a ligand.
Methods Membrane preparations Pia-arachnoid (pial) arteries were carefully removed from the bovine brain within 30 min of obtaining the brains from the local slaughterhouse and were kept in a freezer (-80°C). Membranes from the arteries were prepared as 0024-3205/85 $3.00 + .00 Copyright (c) 1985 Pergamon Press Ltd.
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follows: the arteries were minced with scissors and then homogenized in i0 volumes of Ice-cold 50 raM sodium phosphate buffer (pH 7.4) with a glass homogenizer. The artery homogenates were filtrated through two layers of gauze, then were homogenized at a setting of i0 on a Polytron (Brlnkmann Instrument) with 20 second bursts. The homogenate was centrifuged at 1,000 x g for I0 mln, and an aliquot of the supernatant was preserved for analysis of choline acetyltransferase (CHAT) activity. The residual supernatant was carefully removed and centrifuged at i00,000 x g for 60 mln. The resulting pellet was resuspended in 50 mM sodium phosphate buffer (pH 7.4). Protein concentrations were determined by the method of Lowry et al. (14). 3H-ACh binding assay The 3H-ACh binding assay was performed according to the procedure of Schwartz et al. (13). In brief, aliquots of the bovine cerebral artery membranes were incubated at 0°C for 40 mln in 250 ~i of 50 rmM sodium phosphate buffer (pH 7.4), containing 3H-ACh, in the absence or presence of a high concentration of carbamylchollne (I00 ~M). To prevent hydrolysis of the 3H_ACh by chollnesterase, i00 ~M diisopropylfluorophosphate (DFP) was added to the assay mixture. The assay was terminated by the addition of 3 ml of ice-cold buffer and then filtrated under reduced pressure through Whatman GF/C glass fiber filters, which had been soaked in a 0.I % polyethyleneimine solution to eliminate nonspecific binding to the filter. After three washings with the buffer, the filters were dried in an oven and transferred to counting vials, after which 8 ml of scintillation fluid was added. Radioactivity was counted in a Packard Tri-Carb scintillation spectrometer (Model 3255). Each assay was performed in duplicate. Nonspecific binding was defined as 3 H-ACh binding in the presence of I00 ~M carbamylcholine, whereas total binding was defined as that in the absence of carbamylcholine. Specific binding was defined as the difference between total and nonspecific bindings. H, qNB binding Binding of 3H-QNB was assayed by the method of Yamamura and Snyder (15). In brief, atropine (I0 ~M) was used to define nonspeciflc binding. The tubes, containing 3H-QNB, the tissue membranes and 50 raM sodium phosphate buffer (pH 7.4) to a final volume of 250 ~i, were incubated at 37°C for I hr. The assay was terminated by the addition of 3 ml of ice-cold buffer and then filtrated under reduced pressure through Whatman GF/C glass fiber filters. Filters were then counted for radioactivity in 8 ml of scintillation fluid. ChAT assay ChAT activity in the 1,000 x g supernatant fractions of the bovine cerebral arteries was measured according to the method of Fonnum (16), as modified by Florence and Bevan (17). ChAT activity was expressed as nmol/mg protein/hour. Chemicals and Reagents 3H-acetylchollne (ACh) (86 Ci/mmol) was obtained from Amersham Inter national plc, Bu c klnghamshire, England, and 3 H-l-quinuclldlnyl benzilate (QNB) (36 Ci/mmol) an d i~ 4 C-acetyl-coenzyme A (53 mCi/~mol) were obtained from New England Nuclear, Boston, Mass., and cold QNB was a gift from Dr. S. Spector, Roche Institute of Molecular Biology, Nutley, N.J.. All other chemicals were of reagent grade and were obtained commercially.
Vol. 37, No. 20, 1985
3H-Ach Binding to Bovine Cerebral Artery
1889
20
15
1C c.-,= o ~D
I
0
100
200
I
300
3H-ACh Bound (fn~l/n~ protein) FIG. 1 A typical Scatchard plot of 3H-ACh saturation binding. Analysis of the data was made by the computer best-fit two-site model, with the two individual binding components represented by the lower two lines, including a high affinity site (KDI = 0.26 nM, Bmax = 53 fmol/mg protein) and a low affinity site ( ~ 2 = 18.8 nM, Bmax = 252 fmol/mg protein).
Results 3H-ACh bindin~ to bovine cerebral artery membranes Specific 3H-ACh binding increased linearly with increasing concentrations of the membranes over the range of 25 ~g to I00 pg of protein per assay (data not shown). Specific binding of increasing concentrations of 3H-ACh (0.26 to 32.5 nM) was saturable. Theoretical binding curves were fitted to the twosite model by a non-linear least squared regression analysis using a computer program described by Olsen et al. (18). The best fit gives values of the affinity constant for the high affinity site of 0.26 nM, and for the low affinity site of 18.8 nM with maximum bindings of 53 fmol/mg protein and 252 fmol/mg protein, respectively (Fig.l). The K D and Bmax in three experiments are summarized in Table I. Time course of association and dissociation of the 3H-ACh binding was studied in bovine cerebral artery membranes. Association of 3H-ACh was time-dependent and the binding reached equilibrium within 30 min. The binding was rapidly reversed by addition of a high concentration (I00 ~M) of carbamylcholine. The rate constant of association (K+I) was 0.0185 x 109 M - ~ i n -I. The rate constant of dissociation (K_I) was 0.34 min -I (Fig. 2). The dissociation constant calculated from the equation K D = K_I/K+I was 18.3 nM, such being in close agreement with the K D of low affinity site determined by Scatchard analyses. Time course of association and
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3H-Ach Binding to Bovine Cerebral Artery
100
~
Vol. 37, No. 20, 1985
~
so
-r
0
10
20
30
Time
40
50
60
(min)
FIG. 2 Time course of association (0) and dissociation ~ ) of specific 3H-ACh binding. At 40 min i00 ~M unlabelled earbamylchollne was added. Concentration of 3H_ACh was 13 nM. One hundred percent specific 3H-ACh binding was 160 fmol/mg protein. Figures are the mean of three experiments.
TABLE I
3H-ACh and 3H-QNB BINDING IN BOVINE CEREBRAL ARTERY MEMBRANES
Ligand
a KDI
Bb maxl
3H-ACh
0.32 + 0.14
67 + I0 a KD
3H-QNB
0.55 + 0.04
Ka D2
Bb max2
23.7 + 6.6
252 + 20
Bb max
514 + 20
a: nM b: fmol/mg protein Results are the mean + S.E.M. of three experiments.
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TABLE II
INHIBITION BY VARIOUS DRUGS OF SPECIFIC 3H-ACh BINDING TO BOVINE CEREBRAL ARTERY MEMBRANES a
Drug
Atropine Acetylcholine Oxotremorlne Scopolamine Carbamylcholine (-)-Nicotine Cytislne e-bungatotoxin
IC~0(M)
3.85 5.98 2.93 4.74 9.90
+ ~ ~ ~ ~ ~ > >
1.52 2.97 0.79 1.88 4.81 5 5 5
x x x x x x x x
I0-8 I0-~ I0-~ i0-~ I0-~ I0-~ I0-~ I0 -D
Hill coefficient
N
0.91 0.90 0.87 0.94 0.87
6 6 4 3 6 3 3 3
+ ~ + ~ ~ --
0.17 0.07 0.07 0.33 0.I0
a: 3H-ACh binding was performed as described under "Methods". Concentration of 3H-ACh was 13 nM. b: IC50 is the concentration of drug that reduces the specific 3H-ACh binding by 50 %. The results are the mean + S.E.M. of the number of experiments indicated.
dissociation of the 3H-ACh binding was also studied using low 3H-ACh concentration (1.2 nM) (data not shown). The half-tlme for association was 3 min, and the binding reached equilibrium within 30 mln. The half-tlme for dissociation was 3 min. K + l W a S 0.5 x 10 9 M - ~ I n - l . K _ l W a S 0.17 mln The dissociation constant calculated from the equation K D = K_I/K+I was 0.34 ruM, such being in good agreement with the K D of high affinity site determined by Scatchard analyses. The specificity of 3H-ACh binding was studied using chollnerglc agonists and antagonists. Values of IC50 of muscarinic agonists, acetylcholine, oxotremorine and carbamylcholine and the specific muscarinlc antagonists, atropine and scopolamine were less than 1.26 pM (Table II). In contrast, the IC50 values of nicotinic agents such as ~-bungarotoxln, cytisine and nicotine exceeded 50 ~M. The data on the inhibition were analyzed using Hill plots (Table II). 3H-QNB bindin~ to bovine cerebral artery membranes Specific binding of increasing concentrations of 3H-QNB (0.15 to 5 ruM) was saturable (data not shown). Scatchard analysis indicated a single class of binding site. The K D and Bmax in four experiments are summarized in Table I. ChAT activity in bovine cerebral artery membranes ChAT activity was detected in all bovine cerebral artery membranes studied. The activity was linear with time at lease up to 20 mln (data not shown). The ChAT activity in bovine cerebral arteries was 1.09 + 0.14 nmol/mg protein/hour (N=3).
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3H-Ach Binding to Bovine Cerebral Artery
Vol. 37, No. 20, 1985
Discussion Cholinergic receptor sites in bovine cerebral arteries were characterized using 3H_ACh as the ligand. Specific binding of 3H-ACh to the cerebral arteries was saturable, of high affinity and reversible. Scatchard plot analysis of the data indicated that 3H-ACh binding sites in the cerebral arteries were of two classes. Cholinergic drugs competed for this binding in the order of atropine > ACh > oxotremorine > scopolamine > carbamylcholine. The potent nicotinic drugs, such as nicotine, cytisine and a-bungarotoxin, failed to inhibit 3H-ACh binding even at I0 pM. These results suggest that 3H-ACh binding sites exhibit properties of muscarinic cholinergic receptors in the bovine cerebral arteries. The characteristics of the muscarinic receptors in bovine cerebral arteries have been studied using 3H-QNB, an antagonist, as ligand (6-8). These workers reported that 3H-QNB binding site appeared to be of a single class, on the basis of Scatchard plot analyses. In the present study, Scatchard analysis of specific 3H-QNB binding to the cerebral arteries also indicated one class of binding site. However, 3H_ACh binding sites in the cerebral arteries in this study, which appeared to be muscarinic cholinergic receptor sites, indicated two classes of binding sites. Indeed, Estrada and Krase (6) suggested the presence of more than one class of binding sites, as deduced from °H-QNB competition binding studies. Using 3 H-muscarinic agonists such as 3H-ACh and 3H-oxotremorine, Birdsall et al. obtained curved Scatchard plots, in case of the rat cerebral cortex (12). They suggested the presence of two major populations of agonist binding sites which do not interconvert during the binding experiments and have the same affinity constants for antagonist, in the rat cerebral cortex. Since the Scatchard analysis showed two binding sites, the Hill coefficients would be assumed to be less than 1.0. However, the calculated Hill coefficients for muscarinic agonist and antagonist competition with 13 nM 3H-ACh were close to 1.0 in bovine cerebral arteries in the present study. Birdsall et al. (12) showed that the Hill coefficients for agonist displacement of 3H-agonist binding were among 0.8 and 1.0 in the rat cerebral cortex. Indeed, the Hill coefficients of oxotremorine and ACh were 1.00 and 1.06, respectively, in their experiments (12). As the kineticall~-derived dissociation constant deduced from the experiments using 13 nM OH-ACh was in good agreement with the K D of low affinity binding sites determined by the Scatchard analysis, the displacement studies using 13 nM 3H-ACh seemed to reflect mainly low affinity binding sites in the present study. So in the displacement studies the presence of high affinity binding sites did not have large influence on the result. The Hill coefficients near 1.0 may be explained by the use of high concentration of 3H-ACh. We showed in the present work that there were multiple agonist binding sites in bovine cerebral arteries. Further work is required to understand the functional role of these binding sites. Acknowledgments This work was supported by a Grant-in-Aid for Cooperative Research (No. 60304046) from the Ministry of Education, Science and Culture, Japan, and by a grant from the Japan Tobacco Inc. We thank M.Ohara for reading the manuscript.
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3H-Ach Binding to Bovine Cerebral Artery
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