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Evidence for muscarinic cholinergic receptors in dog portal vein: Binding of [3H] quinuclidinyl benzilate
Life Sciences, Vol. 32, pp. 1757-1763 Printed in the U.S.A.
Pergamon Press
EVIDENCE FOR MUSCARINIC CHOLINERGIC RECEPTORS IN DOG PORTAL VEIN : BINDING OF [3H]QUINUCLIDINYL BENZILATE. Takashi Taniguchi, Tetsuya Tsukahara and Motohatsu Fujiwara Department of Pharmacology, Faculty of Medicine, Kyoto University, Kyoto 606, Japan (Received in final form January i0, 1983) Summary Muscarinic cholinergic receptor sites in dog portal veins were analyzed directly using [3H]quinuclidinyl benzilate (QNB) as a ligand. Specific [3H]QNB binding to crude membrane preparations from the isolated veins was saturable, reversible and of high affinity (KD = 15.5 + 2.8 pM) with a Bmax of ii0 ~ 14.7 fmol/mg protein. Scatch~rd and Hill plot analyses of the data indicated one class of binding sites. From kinetic analysis of the data, association and dissociation rate constants of 1.91 x 109 M -I min -I and 0.016 min -I, respectively, were calculated. The dissociation constant calculated from the equation K D = K_I/K+I was 8.3 pM, such being in good agreement with the Scatchard estimate of K D (15.5 pM). Specific binding of [3H]QNB was displaced by muscarinic agents. Nicotinic cholinergic agents, ~-bungarotoxin, nicotine and hexamethonium, were ineffective in displacing [3H]QNB binding at i0 ~M. Our findings provide direct evidence for the existence of muscarinic cholinergic receptors in dog portal veins.
It is generally considered that the portal vein receives sympathetic adrenergic innervation (1-3). Although little information is available regarding the parasympathetic cholinergic nervous system in the portal vein, the isolated rabbit portal vein is known to contract in response to acetylcholine (3,4) and the so-induced response is blocked by hyoscine ( 3 ) . Thus, the possibility that the portal vein may have muscarinic cholinergic receptors had to be considered. The muscarinic cholinergic receptors of the dog portal vein have now been analyzed directly with radioligand binding assay using a specific muscarinic antagonist, [3H]quinuclidinyl benzilate (QNB), as ligand. [3H]QNB is a potent muscarinic cholinergic agent which has been used to identify muscarinic cholinergic receptor binding sites in the rat brain (5), guinea-pig ileum (6), rabbit heart (7) and bovine cerebral blood vessels (8).
Methods Membrane preparation. Dogs of either sex weighing 4.5 to 15 kg were anesthetized with sodium pentobarbital (35 mg/kg, i.p.), and exsanguinated from the common carotid arteries. The portal vein was carefully removed and kept at -80 ° C for up to one week. Membranes from the vessels were prepared as 0024-3205/83/151757-07503.00/0 Copyright (c) 1983 Pergamon Press Ltd.
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follows: thawed portal veins were minced with scissors and then homogenized in i0 volumes of ice-cold 50 mM sodium phosphate buffer (pH 7.4) with a glass homogenizer. The vein homogenates were filtrated through two layers of gauze then were homogenized at setting i0 on a Polytron (Brinkmann Instruments) with 20 second bursts. The homogenate was centrifuged at 1,000 x g for I0 min, the supernatant was carefully removed and then centrifuged at i00,000 x g for 60 min. The resulting pellet was resuspended at a protein concentration of 1 to 1.6 mg/ml in 50 ~nM sodium phosphate buffer (pH 7.4). Protein concentrations were determined by the method of Lowry et al. (9). Binding assay. The [3H]QNB binding assay was performed by incubating aliquots of the portal vein homogenates (0.05 to 0.08 mg protein) at 37 ° C for 60 min in 250 ~i of the 50 mM sodium p h o s p h a t e buffer, containing 34 pM [3H]QNB in the absence or presence of a high c o n c e n t r a t i o n of atropine (i0 ~M). The assay was terminated by the addition of 3 ml of ice-cold buffer and rapid filtration through W h a t m a n GF/B glass fiber filters under suction. After washing twice with 3 ml of the buffer, the filters were dried in an oven and transferred to counting vials, then 8 ml of scintillation fluid was added. R a d i o a c t i v i t y was counted in a Packard Tri-Carb s c i n t i l l a t i o n spectrometer (Model 3255). To assay the reversal of [3H]QNB binding by i0 ~M atropine, the homogenate was incubated with [3H]QNB for 60 min at 37 ° C. Atropine was then added to a final concentration of i0 ~M, and specifically bound counts were d e t e r m i n e d at the incubated time. [3H]QNB bound in the presence of I0 ~M atropine was termed "nonspecific" and was subtracted from that obtained in the absence of i0 ~M atropine (total binding) to obtain the binding termed "specific". Scatchard analysis, Hill plot and d e t e r m i n a t i o n of the rate constants were p e r f o r m e d according to Bennett (i0). Chemicals and Reagents. [3H] £-quinuclidinyl b e n z i l a t e (QNB) (36 Ci/mmol) was obtained from New England Nuclear, Boston, Mass. and cold QNB was a gift from Dr. S.Spector, Roche Institute of M o l e c u l a r Biology, Nutley, N.J. All other chemicals were of reagent grade and were obtained commercially.
Results Protein d e p e n d e n c y of specific [3H]QNB binding to dog portal vein homogenates. Specific [3H]QNB binding increased linearly with increasing homogenate concentrations over the range of 5 to 90 ~g of protein (Fig. i). Binding assays were, therefore, conducted with less than 90 Dg of protein. Saturability of [3H]QNB binding. Specific binding of increasing concentrations of [3H]QNB (3 to 80 pM) was saturable (Fig. 2). Scatchard analysis indicated a single class of binding sites with an apparent equilibrium dissociation constant (KD) of 16.7 pM, and m a x i m u m binding capacity (Bmax) was 133 fmol/mg p r o t e i n (Fig. 3). The K D and Bmax in 5 experiments are summarized (K D = 15.5 + 2.8 pM; Bmax = ii0 + 14.7 fmol/mg protein). Hill coefficient for the inhibition of [3H]QNB b i n d i n g by the muscarinic cholinergic antagonist, atropine, was 1.06 ~ 0.03 (N=5), such being indicative homogeneity and noncoo p e r a t i v i t y of the b i n d i n g sites. Kinetics of [3H]QNB binding. The binding of [3H]QNB was time d e p e n d e n t and e q u i l i b r i u m was achieved after 40 min (Fig. 4). The rate constant of association (K+I) was 1.91 x 109 M -I min -I. Dissociation of [3H]QNB binding followed first order kinetics, with t 1/2 = 40 min and a rate constant of d i s s o c i a t i o n (K_ l) of 0.016 min -I (Fig. 5). The d i s s o c i a t i o n constant calculated from the equation K D = K_I/K+I was 8.3 pM, such being in good agreement with the Scatchard estimate of K D.
Vol. 32, No.
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[3H]QNB Binding to Dog Portal Vein
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6
~4 m >, 3
|
i
0
!
|
100 Protein
200
( pglassay
)
FIG. i Protein dependency of specific [3H]QNB binding to dog portal vein homogenates. Specific [3H]QNB binding was determined as described under "Methods". Total concentration of [3H]QNB was 34 pM. All points were determined in duplicate.
150
.S
E
100
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0
,
i
5O
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H] QNB ( pM ) FIG. 2 A typical experiment of specific and nonspecific [3H]QNB binding to portal vein homogenates. Specific [3H]QNB binding (Q) was experimentally determined as the difference between total binding and nonspecific binding (O) in parallel assays in the absence and presence of i0 ~M atropine.
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[3H]QNB Binding to Dog Portal Vein
Vol. 32, No. 15, 1983
10
u"o
5
c
o
rn
50 100 150 [3H] QNB Bound ( fmol/mg protein )
FIG. 3 A Scatchard plot derived of figure 2. The slope regression analysis (r = maximum binding capacity
from the specific [3H]QNB binding data of the plot was determined by linear 0.990); dissociation constant K D = 16.7 pM; Bmax = 133 fmol/mg protein.
Specificity of [3H]QNB binding. The specificity of [3H]QNB binding was studied using cholinergic agonists and antagonists (Fig. 6). Acetylcholine (ACh) exhibits an IC50 of 4.9 ~ 1.7 DM (N=3) in displacing [3H]QNB binding in the presence of physostigmine (i DM). Physostigmine (i NM) had no effect on the binding. Other muscarinic agonists such as oxotremorine, pilocarpine and arecoline, gave IC50 values of 1.5 + 0.6 ~M, 3.7 + 0.7 NM and 8.3 + 2.8 ~M, respectively (N=3), while the specific muscarinic--antagonists, atropine and QNB, had IC50 values of 3.4 ~ 0.7 nM and 1.7 ~ 0.4 pM, respectively (N=5). In contrast, the nicotinic agents such as ~-bungarotoxin, nicotine and hexamethonium did not displace [3H]QNB binding at I0 ~M. The inhibition data were also analyzed with Hill plot. The antagonists tested had Hill coefficients close to 1.0 (QNB, 0.94 ~ 0.04; atropine, 0.85 + 0.05), whereas the agonists tested had Hill coefficients less than 1.0 (ACh, 0.53 + 0.05; oxotremorine, 0.70 + 0.05; pilocarpine, 0.41 + 0.05; arecoline, 0.42--+ 0.07). Seven different-compounds (norepinephrine, propranolol, phenylephrine, phentolamine, serotonin, histamine and dopamine) at i0 DM each had no significant effect of [3H]QNB binding.
Vol. 32, No. 15, 1983
[3H]QNB Binding to Dog Portal Vein
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A --¢ ¢= 70
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.
.
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.
.
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FIG. 4. Time course for association of specific [3H]QNB binding to portal vein homogenates ( O ) . The inset shows a kinetic plot for association of specific binding. Beq = f moles specifically bound [3H]QNB at equilibrium and Bt = f moles specifically bound [3H]QNB at time t. Kobs is the slope of the line and is 0.078 min -I. K+I can be calculated from the equation K+I = (Kobs - K_I)/[3H]QNB, where K 1 is the rate constant of dissociation (Fig. 5). [3H]QNB = 34 pM?
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FIG. 5 Time course for dissociation of specific [3H]QNB binding to portal vein homogenates ( O ) . At the arrow, atropine (i0 uM) was added and dissociation of the [3H]QNB-receptor complex ( O ) was monitored for an additional 60 min. The inset shows a kinetic plot for the dissociation of binding. K_I is the slope of this line and is 0.016 min -I.
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[3H]QNB Binding
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6
The inhibition of specific [3H]QNB binding to portal vein homogenares by cholinergic agonists and antagonists. Each point represents the mean of 3 to 5 experiments.
Discussion Using a radioligand binding assay, we d e m o n s t r a t e d the presence of muscarinic cholinergic receptor sites in the dog portal vein. The binding characteristics of these receptors are similar to those of the muscarinic receptors in the rat brain (5), guinea-pig ileum (6) and rabbit heart (7). Thus, specific [3H]QNB binding was saturable, of high affinity and reversible. Scatchard and Hill plot analyses of the data indicated that [3H]QNB binding sites in the portal vein appear to be of a single p o p u l a t i o n and do not exhibit cooperativity. Potent nicotinic cholinergic drugs such as ~-bungarotoxin, nicotine and h e x a m e t h o n i u m failed to inhibit [3H]QNB binding, even at a dose of i0 ~M. In agreement with previous o b s e r v a t i o n s (5-8), the muscarinic cholinergic antagonists, atropine and QNB, were more potent in inhibiting [3H]QNB binding than were muscarinic agents such as ACh, oxotremorine, p i l o c a r p i n e and arecoline. The Hill coefficients calculated from the inhibition curves of agonists to muscarinic receptors in smooth muscles (ii), heart (7,12), cerebral blood vessels (8) and portal vein (the present experiment) were less than 1.0, while those of antagonists were close to 1.0. These results suggest either the presence of more than one binding site or negatively cooperative site-site interactions between identical sites. Histochemical studies revealed the presence of a c e t y l c h o l i n e s t e r a s e activities in the guinea-pig hepatic vein (13) and in the vicinity of rat liver sinusoids (14), and also d e m o n s t r a t e d that a d m i n i s t r a t i o n of ACh produces significant vasodilator effects in the rat liver sinusoids (15). Thus, cholinergic innervation does have an influence on blood flow in the liver. In our present studies, the substantial number of m u s c a r i n i c cholinergic receptors d e m o n s t r a t e d in the portal vein suggests a functional role of these sites in
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r e g u l a t i o n of portal circulation. Our results offer s u p p o r t i v e evidence for the view that autonomic i n n e r v a t i o n can influence b l o o d flow in liver veins. The existence of m u s c a r i n i c cholinergic receptors in the dog portal v e i n were e v i d e n c e d herein.
Acknowledgements This w o r k was supported by a G r a n t - i n - A i d for C o - o p e r a t i v e Research (No. 00537006) from the M i n i s t r y of Education, Science and Culture, Japan, and by a Grant from the Nippon Tobacco and Salt Public Corporation. We thank M. Ohara for p r e p a r i n g the manuscript.
References i.
2. 3. 4. 5. 6. 7. 8. 9. i0.
ii. 12. 13. 14. 15.
S.E. MAYER, in The P h a r m a c o l o g i c a l Basis of Therapeutics, ed. by A.F. GILMAN, L.S. G O O D M A N and A. GILMAN, Sixth edition: pp. 56-90, M a c m i l l a n P u b l i s h i n g Co. Inc., New York (1980). M.E. HOLMAN, C.B. KASBY, M.B. SUTHERS and J.A.F. WILSON, J. Physiol. (London) 196: 111-132 (1968). J. HUGHES and J.R. VANE, Br. J. Pharmacol. 30: 46-66 (1967). B.P. BROWN, S. ANURAS and D.D. HEISTAD, Am. J. Physiol. 242: G498-G503 (1982). H.I. Y A M A M U R A and S.H. SNYDER, Proc. Natl. Acad. Sci. USA 71: 1725-1729 (1974). H.I. Y A M A M U R A and S.H. SNYDER, Mol. Pharmacol. i0: 861-867 (1974). J.Z. FIELDS, W.R. ROESKE, E. M O R K I N and H.I. YAMAMURA, J. Biol. Chem. 253: 3251-3258 (1978). C. ESTRADA and D. KRAUSE, J. Pharmacol. Exp. Ther. 221: 85-90 (1982). O.H. LOWRY, N.J. ROSEBROUGH, A.L. FARR and R.J. RANDALL, J. Biol. Chem. 193: 265-275 (1951). J.P. BENNETT, Jr., in N e u r o t r a n s m i t t e r Receptor Binding, ed. by H.I. YAMAMURA, S.J. ENNA and M.J. KUHAR, pp. 57-90, Raven Press, New York (1978). J.M. YOUNG, FEBS Lett. 46: 354-356 (1974). J.B. GALPER, W. KLEIN and W.A. CATTERALL, J. Biol. Chem. 252: 8692-8699 (1977). S.D. SUTHERAND, J. Anat. Lond. 98: 321-326 (1964). I.Y.S. LIANG, A. KOO and H.C. LIU, Conf. Eur. Soc. Microcirc., Cagliari, Italy, 10th, 1978. (Abstract). A. KOO and I.Y.S. LIANG, Am. J. Physiol. 236: E728-E732 (1979).
Pergamon Press
EVIDENCE FOR MUSCARINIC CHOLINERGIC RECEPTORS IN DOG PORTAL VEIN : BINDING OF [3H]QUINUCLIDINYL BENZILATE. Takashi Taniguchi, Tetsuya Tsukahara and Motohatsu Fujiwara Department of Pharmacology, Faculty of Medicine, Kyoto University, Kyoto 606, Japan (Received in final form January i0, 1983) Summary Muscarinic cholinergic receptor sites in dog portal veins were analyzed directly using [3H]quinuclidinyl benzilate (QNB) as a ligand. Specific [3H]QNB binding to crude membrane preparations from the isolated veins was saturable, reversible and of high affinity (KD = 15.5 + 2.8 pM) with a Bmax of ii0 ~ 14.7 fmol/mg protein. Scatch~rd and Hill plot analyses of the data indicated one class of binding sites. From kinetic analysis of the data, association and dissociation rate constants of 1.91 x 109 M -I min -I and 0.016 min -I, respectively, were calculated. The dissociation constant calculated from the equation K D = K_I/K+I was 8.3 pM, such being in good agreement with the Scatchard estimate of K D (15.5 pM). Specific binding of [3H]QNB was displaced by muscarinic agents. Nicotinic cholinergic agents, ~-bungarotoxin, nicotine and hexamethonium, were ineffective in displacing [3H]QNB binding at i0 ~M. Our findings provide direct evidence for the existence of muscarinic cholinergic receptors in dog portal veins.
It is generally considered that the portal vein receives sympathetic adrenergic innervation (1-3). Although little information is available regarding the parasympathetic cholinergic nervous system in the portal vein, the isolated rabbit portal vein is known to contract in response to acetylcholine (3,4) and the so-induced response is blocked by hyoscine ( 3 ) . Thus, the possibility that the portal vein may have muscarinic cholinergic receptors had to be considered. The muscarinic cholinergic receptors of the dog portal vein have now been analyzed directly with radioligand binding assay using a specific muscarinic antagonist, [3H]quinuclidinyl benzilate (QNB), as ligand. [3H]QNB is a potent muscarinic cholinergic agent which has been used to identify muscarinic cholinergic receptor binding sites in the rat brain (5), guinea-pig ileum (6), rabbit heart (7) and bovine cerebral blood vessels (8).
Methods Membrane preparation. Dogs of either sex weighing 4.5 to 15 kg were anesthetized with sodium pentobarbital (35 mg/kg, i.p.), and exsanguinated from the common carotid arteries. The portal vein was carefully removed and kept at -80 ° C for up to one week. Membranes from the vessels were prepared as 0024-3205/83/151757-07503.00/0 Copyright (c) 1983 Pergamon Press Ltd.
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follows: thawed portal veins were minced with scissors and then homogenized in i0 volumes of ice-cold 50 mM sodium phosphate buffer (pH 7.4) with a glass homogenizer. The vein homogenates were filtrated through two layers of gauze then were homogenized at setting i0 on a Polytron (Brinkmann Instruments) with 20 second bursts. The homogenate was centrifuged at 1,000 x g for I0 min, the supernatant was carefully removed and then centrifuged at i00,000 x g for 60 min. The resulting pellet was resuspended at a protein concentration of 1 to 1.6 mg/ml in 50 ~nM sodium phosphate buffer (pH 7.4). Protein concentrations were determined by the method of Lowry et al. (9). Binding assay. The [3H]QNB binding assay was performed by incubating aliquots of the portal vein homogenates (0.05 to 0.08 mg protein) at 37 ° C for 60 min in 250 ~i of the 50 mM sodium p h o s p h a t e buffer, containing 34 pM [3H]QNB in the absence or presence of a high c o n c e n t r a t i o n of atropine (i0 ~M). The assay was terminated by the addition of 3 ml of ice-cold buffer and rapid filtration through W h a t m a n GF/B glass fiber filters under suction. After washing twice with 3 ml of the buffer, the filters were dried in an oven and transferred to counting vials, then 8 ml of scintillation fluid was added. R a d i o a c t i v i t y was counted in a Packard Tri-Carb s c i n t i l l a t i o n spectrometer (Model 3255). To assay the reversal of [3H]QNB binding by i0 ~M atropine, the homogenate was incubated with [3H]QNB for 60 min at 37 ° C. Atropine was then added to a final concentration of i0 ~M, and specifically bound counts were d e t e r m i n e d at the incubated time. [3H]QNB bound in the presence of I0 ~M atropine was termed "nonspecific" and was subtracted from that obtained in the absence of i0 ~M atropine (total binding) to obtain the binding termed "specific". Scatchard analysis, Hill plot and d e t e r m i n a t i o n of the rate constants were p e r f o r m e d according to Bennett (i0). Chemicals and Reagents. [3H] £-quinuclidinyl b e n z i l a t e (QNB) (36 Ci/mmol) was obtained from New England Nuclear, Boston, Mass. and cold QNB was a gift from Dr. S.Spector, Roche Institute of M o l e c u l a r Biology, Nutley, N.J. All other chemicals were of reagent grade and were obtained commercially.
Results Protein d e p e n d e n c y of specific [3H]QNB binding to dog portal vein homogenates. Specific [3H]QNB binding increased linearly with increasing homogenate concentrations over the range of 5 to 90 ~g of protein (Fig. i). Binding assays were, therefore, conducted with less than 90 Dg of protein. Saturability of [3H]QNB binding. Specific binding of increasing concentrations of [3H]QNB (3 to 80 pM) was saturable (Fig. 2). Scatchard analysis indicated a single class of binding sites with an apparent equilibrium dissociation constant (KD) of 16.7 pM, and m a x i m u m binding capacity (Bmax) was 133 fmol/mg p r o t e i n (Fig. 3). The K D and Bmax in 5 experiments are summarized (K D = 15.5 + 2.8 pM; Bmax = ii0 + 14.7 fmol/mg protein). Hill coefficient for the inhibition of [3H]QNB b i n d i n g by the muscarinic cholinergic antagonist, atropine, was 1.06 ~ 0.03 (N=5), such being indicative homogeneity and noncoo p e r a t i v i t y of the b i n d i n g sites. Kinetics of [3H]QNB binding. The binding of [3H]QNB was time d e p e n d e n t and e q u i l i b r i u m was achieved after 40 min (Fig. 4). The rate constant of association (K+I) was 1.91 x 109 M -I min -I. Dissociation of [3H]QNB binding followed first order kinetics, with t 1/2 = 40 min and a rate constant of d i s s o c i a t i o n (K_ l) of 0.016 min -I (Fig. 5). The d i s s o c i a t i o n constant calculated from the equation K D = K_I/K+I was 8.3 pM, such being in good agreement with the Scatchard estimate of K D.
Vol. 32, No.
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[3H]QNB Binding to Dog Portal Vein
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6
~4 m >, 3
|
i
0
!
|
100 Protein
200
( pglassay
)
FIG. i Protein dependency of specific [3H]QNB binding to dog portal vein homogenates. Specific [3H]QNB binding was determined as described under "Methods". Total concentration of [3H]QNB was 34 pM. All points were determined in duplicate.
150
.S
E
100
E v "o to m
50
Z O T D
|
0
,
i
5O
|
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I
100
H] QNB ( pM ) FIG. 2 A typical experiment of specific and nonspecific [3H]QNB binding to portal vein homogenates. Specific [3H]QNB binding (Q) was experimentally determined as the difference between total binding and nonspecific binding (O) in parallel assays in the absence and presence of i0 ~M atropine.
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[3H]QNB Binding to Dog Portal Vein
Vol. 32, No. 15, 1983
10
u"o
5
c
o
rn
50 100 150 [3H] QNB Bound ( fmol/mg protein )
FIG. 3 A Scatchard plot derived of figure 2. The slope regression analysis (r = maximum binding capacity
from the specific [3H]QNB binding data of the plot was determined by linear 0.990); dissociation constant K D = 16.7 pM; Bmax = 133 fmol/mg protein.
Specificity of [3H]QNB binding. The specificity of [3H]QNB binding was studied using cholinergic agonists and antagonists (Fig. 6). Acetylcholine (ACh) exhibits an IC50 of 4.9 ~ 1.7 DM (N=3) in displacing [3H]QNB binding in the presence of physostigmine (i DM). Physostigmine (i NM) had no effect on the binding. Other muscarinic agonists such as oxotremorine, pilocarpine and arecoline, gave IC50 values of 1.5 + 0.6 ~M, 3.7 + 0.7 NM and 8.3 + 2.8 ~M, respectively (N=3), while the specific muscarinic--antagonists, atropine and QNB, had IC50 values of 3.4 ~ 0.7 nM and 1.7 ~ 0.4 pM, respectively (N=5). In contrast, the nicotinic agents such as ~-bungarotoxin, nicotine and hexamethonium did not displace [3H]QNB binding at I0 ~M. The inhibition data were also analyzed with Hill plot. The antagonists tested had Hill coefficients close to 1.0 (QNB, 0.94 ~ 0.04; atropine, 0.85 + 0.05), whereas the agonists tested had Hill coefficients less than 1.0 (ACh, 0.53 + 0.05; oxotremorine, 0.70 + 0.05; pilocarpine, 0.41 + 0.05; arecoline, 0.42--+ 0.07). Seven different-compounds (norepinephrine, propranolol, phenylephrine, phentolamine, serotonin, histamine and dopamine) at i0 DM each had no significant effect of [3H]QNB binding.
Vol. 32, No. 15, 1983
[3H]QNB Binding to Dog Portal Vein
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A --¢ ¢= 70
~
N
60
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50
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FIG. 4. Time course for association of specific [3H]QNB binding to portal vein homogenates ( O ) . The inset shows a kinetic plot for association of specific binding. Beq = f moles specifically bound [3H]QNB at equilibrium and Bt = f moles specifically bound [3H]QNB at time t. Kobs is the slope of the line and is 0.078 min -I. K+I can be calculated from the equation K+I = (Kobs - K_I)/[3H]QNB, where K 1 is the rate constant of dissociation (Fig. 5). [3H]QNB = 34 pM?
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120
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FIG. 5 Time course for dissociation of specific [3H]QNB binding to portal vein homogenates ( O ) . At the arrow, atropine (i0 uM) was added and dissociation of the [3H]QNB-receptor complex ( O ) was monitored for an additional 60 min. The inset shows a kinetic plot for the dissociation of binding. K_I is the slope of this line and is 0.016 min -I.
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[3H]QNB Binding
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1983
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6
The inhibition of specific [3H]QNB binding to portal vein homogenares by cholinergic agonists and antagonists. Each point represents the mean of 3 to 5 experiments.
Discussion Using a radioligand binding assay, we d e m o n s t r a t e d the presence of muscarinic cholinergic receptor sites in the dog portal vein. The binding characteristics of these receptors are similar to those of the muscarinic receptors in the rat brain (5), guinea-pig ileum (6) and rabbit heart (7). Thus, specific [3H]QNB binding was saturable, of high affinity and reversible. Scatchard and Hill plot analyses of the data indicated that [3H]QNB binding sites in the portal vein appear to be of a single p o p u l a t i o n and do not exhibit cooperativity. Potent nicotinic cholinergic drugs such as ~-bungarotoxin, nicotine and h e x a m e t h o n i u m failed to inhibit [3H]QNB binding, even at a dose of i0 ~M. In agreement with previous o b s e r v a t i o n s (5-8), the muscarinic cholinergic antagonists, atropine and QNB, were more potent in inhibiting [3H]QNB binding than were muscarinic agents such as ACh, oxotremorine, p i l o c a r p i n e and arecoline. The Hill coefficients calculated from the inhibition curves of agonists to muscarinic receptors in smooth muscles (ii), heart (7,12), cerebral blood vessels (8) and portal vein (the present experiment) were less than 1.0, while those of antagonists were close to 1.0. These results suggest either the presence of more than one binding site or negatively cooperative site-site interactions between identical sites. Histochemical studies revealed the presence of a c e t y l c h o l i n e s t e r a s e activities in the guinea-pig hepatic vein (13) and in the vicinity of rat liver sinusoids (14), and also d e m o n s t r a t e d that a d m i n i s t r a t i o n of ACh produces significant vasodilator effects in the rat liver sinusoids (15). Thus, cholinergic innervation does have an influence on blood flow in the liver. In our present studies, the substantial number of m u s c a r i n i c cholinergic receptors d e m o n s t r a t e d in the portal vein suggests a functional role of these sites in
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r e g u l a t i o n of portal circulation. Our results offer s u p p o r t i v e evidence for the view that autonomic i n n e r v a t i o n can influence b l o o d flow in liver veins. The existence of m u s c a r i n i c cholinergic receptors in the dog portal v e i n were e v i d e n c e d herein.
Acknowledgements This w o r k was supported by a G r a n t - i n - A i d for C o - o p e r a t i v e Research (No. 00537006) from the M i n i s t r y of Education, Science and Culture, Japan, and by a Grant from the Nippon Tobacco and Salt Public Corporation. We thank M. Ohara for p r e p a r i n g the manuscript.
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2. 3. 4. 5. 6. 7. 8. 9. i0.
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