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5HT2 receptors in the rat portal vein: Desensitization following cumulative serotonin addition
Life Sciences, Vol. 35, pp. 71-77 Printed in the U.S.A.
Pergamon Press
5HT2 RECEPTORSIN THE RAT PORTAL VEIN: DESENSITIZATIONFOLLOWING CUMULATIVE SEROTONINADDITION Harriet F. Lemberger, Norman Mason and Marlene L. Cohen Cardiovascular Pharmacology L i l l y Research Laboratories Eli L i l l y and Company Indianapolis, IN 46285 (Received in final form April 23, 1984)
SUMMARY Contractile responses to serotonin were examined in vitro in the longitudinal portal vein to determine whether sucB responses were mediated by the interaction of serotonin with 5HT1 receptors (those that preferentially bind [3H]serotonin) or 5HT2 receptors (those that preferentially bind [JH]spiperone). Using eight serotonin receptor antagonists (spiperone, metergoline, LY53857, ketanserin, trazodone, benzoctamine, 1-(1-naphthyl)piperazine, and 1-meta-methoxyphenylpiperazine), we found a significant correlation between the a f f i n i t y for serotonin receptors in the rat portal vein and the a b i l i t y to bind to 5HT2, but not 5HT1 receptors in rat frontal cortical membranes. Thus, the receptors mediating vascular contraction to serotonin in the rat portal vein were similar to those receptors defined in other vascular beds from the rat (aorta, jugular vein, and caudal artery). Furthermore, contraction resulting from the cumulative addition of serotonin in the rat portal vein was associated with desensitization (higher ED50 value) relative to contractions produced by the non-cumulative addition of serotonin. A f f i n i t i e s of serotonin receptor antagonists were also lower when determined by antagonism of cumulative serotonin concentrationresponse curves compared to a f f i n i t i e s obtained by antagonism of non-cumulative concentration-response curves. Thus, 5HT2 receptor a f f i n i t i e s of antagonists in the rat portal vein are best determined by the s h i f t of non-cumulative responses to serotonin. Vascular receptors mediating serotonin-induced contraction have been shown to be of the 5HT2 subtype in the caudal artery (1,2), aorta (3) and jugular vein (3,4) from the rat. However, the possibility that not all vascular serotonin receptors are of the 5HT2 type has been raised by Peroutka et al. (5) using the canine basilar artery. In the basilar artery, potent 51f~-~ceptor antagonists, such as ketanserin did not antagonize the contractile response to serotonin. Using a series of antagonists and agonists, Peroutka et al. (5) have proposed that the receptors mediating contraction to s e r o ~ n ~ in the basil ar artery may best be described as 5HTI. Furthermore, receptors mediating the contractile response to serotonin in several gastrointestinal preparations including the guinea pig ileum and the rat fundus (6,7) do not f i t the classical definition of 5HT2 receptors ( i . e . , inhibition did not correlate with [3H]spiperone binding data). The~ observations raise the possibility that some blood vessels and gastrointestinal smooth muscle may contract to serotonin via receptors other than 5HT2 . 0024-3205/84 $3.00 + .00 Copyright (c) 1984 Pergamon Press Ltd.
72
5HT2 Receptors
in Rat Portal Vein
Vol. 35, No. i, 1984
Since myogenic rhythmic a c t i v i t y occurs in the rat portal vein but not in other in v i t r o vascular preparations and the hepatic portal circulation is associated with the gastrointestinal tract which may not possess 5HT2 receptors, we asked whether serotonin receptors in the portal vein would resemble those receptors in other vascular beds from the rat (5HT2 receptors) or those in the gastrointestinal tract (not 5HT2). To examine this, we compared the a f f i n i t i e s of a series of serotonin receptor antagonists for vascular serotonin receptors with their a b i l i t y to bind to 5HTI or 5HT2 r~ceptors in the brain based on displacement of [3H]serotonin and [JH]spiperone, respectively (8). For these antagonists, a signiflcant correlation, occurred between the a f f l n i t y for serotonin receptors in the rat portal vein and their a b i l l t y to b~nd to 5HT2 receptors.
Methods Isolation of Portal veins: Male Sprague-Dawley rats (200-300 g) (Harlan Industries, Inc., Cumberland, IN) were s a c r l f i c e d by cervical d l s l o c a t i o n . Portal veins were dissected free of connective tissue, ligated with cotton thread, excised, and transferred to petri dishes containing Krebs' blcarbonate buffer. Longitudinal preparatlons were suspended in organ baths containing 10 ml of a modlfied Krebs' solution of the following composition (in milllmoles per l l t e r ) : NaCI, 118.2; KCl, 4.6; CAC12.2 H20, 1.6; KH2P04, 1.2; MgS04, 1.2; dextrose, 10.0, and NaHC03, 24.8. Tissue bath solutions were maintained at 37°C and e q u i l i b r a t e d with 95 percent 02 and 5 percent CO2. Preparations were stretched to 4 grams force and were allowed to e q u i l l b r a t e for 1-2 h before exposure to drugs. Isometric contractions were recorded as changes in grams of force on a Beckman Dynograph with Statham UC-3 transducers. Vascular contraction to serotonin. Cumulative contractile concentrationresponse curves for serotonin were obtalned by a stepwise increase in the concentration of serotonln after a steady response occurred to the preceding dose. For non-cumulative contractile concentration-response curves, increasing concentrations of serotonin were added to the bath after washout of the previous concentration with at least 15-20 minutes between successive additions. ED50 values were taken as the concentration of serotonin that produced half-maximal contraction. Determination of apparent dissociation constants for antagonists. After control responses to serotonin were obtained, vessels were incubated with appropriate concentrations of antagonists for 20-30 minutes. Responses to serotonin were then repeated in the presence of antagonist. In experiments in which serotonin was added non-cumulatively, antagonists were added in the wash buffer between serotonin additions.
Apparent antagonlst dissociation constants (KB) were determined for each concentration of antagonist according to the following equation (9):
[B]
KB = [dose r a t i o - I ] where [B] is the concentration of the antagonist and dose ratio is the ED50 of the agonist in the presence of the antagonist divided by the control ED50. These results were then expressed as the negative logarithm of the KB ( i . e . , -log KB). Calculations were performed with aid of a computer and d i g i t a l plotter as previously described (10). The data were also analyzed according to the procedure of Arunlakshana and Schlld (11). The dose ratio was determined at various concentrations of
Vol. 35, No. i, 1984
5HT 2 Receptors
in Rat Portal Vein
73
antagonist. According to Arunlakshana and Schild (11), i f blockade is competitive, under equilibrium conditions, a plot of the logarithm of (dose ratio - I) against the negative logarithm of the molar concentration of antagonist should yield a straight line whose slope is I and intercept along the abscissa is the pA2 which is equal to -log KB. Cortical bindin 9 to 5HTp and 5HT1 receptors. Brain tissue was obtained from 150 to 200 g male Wistar rats. The cerebral cortex was dissected, homogenized, and prepared according to the method described by Nelson et al. (12). Tissue was preincubated in buffer without monoamine oxidase i n h ~ i ~ r in order to eliminate endogenous serotonin. For receptor binding, an amount of membrane preparation equivalent to 250-350 mg of protein was used per sample in I ml of Tris buffer. The assay for serotonin binding was done following the method of Bennett and Snyder (13) and that for spi~erone binding according to Peroutka and Snyder (8). Non-specific binding of [JH]serotonin and [3H]spiperone was determined in the presence of 10-bM serotonin or lO-bM lysergic acid diethylamide (LSD), respectively. Specific binding was calculated as the difference between total binding (without added nonradioactive compound) and non-specific binding. The IC50 values were determined as the amount of substance causing 50 percent i n h i b l t i o n of ~he specific binding using ten d i f f e r e n t concentrations in the range of I0 -~ to IO-4M. The concentration of [3H]serotonin (sp. act. 17.6 Ci/mmole, Amersham ~orp., Arlington Heights, IL) in each sample was 2.3 to 2.6 nM and that of [JH]spiperone (sp. act. 20 Ci/mmole, Amersham) was 0.5 to 0.7 nM. Results Eight compounds (spiperone, metergoline, LY53857, ketanserin, trazodone, benzoctamine, 1-(l-naphthyl)piperazine, and 1-meta-methoxyphenylpiperazine) were evaluated for t h e i r a b i l i t y to antagonize serotonin-induced contractions in rat portal v~in and to displace the binding of [3H]spiperone (5HT2 receptors) or [JH]serotonin (5HTI receptors) in brain c o r t i c a l membranes. 5 JCORRELATIONCOEFFICIENT: 0.94 /
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5
FIG. 1 Correlation of the a f f i n i t i e s of several serotonin receptor antagonists for serotonin receptors in the rat portal vein to t h e i r a f f i n i t i e s f o r 5HT2 receptors (displacement of [JH]spiperone) in rat f r o n t a l c o r t i c a l membranes.
74
5HT2 Receptors in Rat Portal Vein
Vol. 35, No. i, 1984
CORRELATION COEFFICIENT : 0.1'12 1MMOPP
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-, 7
INF
BENZOCTAMIN|
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'=28 METERGOLINE
LY53857 • • KETANSERIN
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FIG. 2
Correlation of the a f f i n i t i e s of several serotonin receptor antagonists for serotonin receptors in the rat pQrtal vein to their a f f i n i t i e s for 5HT1 receptors (displacement of [JH]serotonin) in rat frontal cortical membranes. The receptor antagonists examined in the present study were potent inhibitors of the contractile response to serotonin in the rat portal vein. A significant correlation (correlation coefficient = 0.94) was found between the a f f i n i t i e s of these antagonists for serotonin receptors in the portal vein and their a f f i n i t i e s for 5HT2 receptors in brain cortical membranes (Fig. 1). In contrast, no correlation was found between receptors mediating contraction in the portal vein and their a f f i n i t y for 5HT1 receptors in brain cortical membranes (Fig. 2). Although a significant correlation was obtained with the a b i l i t y of antagonists to block serotonin contractions in the portal vein and their a b i l i t y to bind to 5HT2 receptors, the dissociation constants obtained for these antagonists in the rat portal vein were higher than in the rat jugular vein (4, 14, 15). Thus, although the relative rank order of the serotonin receptor antagonists was similar in the rat portal vein and jugular vein, the relative s e n s i t i v i t y to the antagonists was lower in the rat portal vein. Several explanations may be advanced for the lower pA2 values found in the rat portal vein relative to the jugular vein. Since alpha receptors occur in the rat portal vein but not in the rat jugular vein (16), we considered the possibility that serotonin might be interacting with alpha receptors in the portal vein complicating the interpretation of our data. However, prazosin (10-6M), a potent alpha I receptor antagonist, did not block the contractile response to serotonin in the rat portal vein. Prazosin was a potent antagonist of the contractile response to phenylephrine in this tissue (-log KB =8.59 * 0.31; n=5). Next, we considered the p o s s i b i l i t y that the cumulative addition of serotonin in the portal vein might be associated with a reduced s e n s i t i v i t y to serotonin. In fact, the ED50 determined after the cumulative administration of serotonin was higher (3-4 fold) than the ED50 determined after noncumulative addition of serotonin in the rat portal vein (Table I ) . Thus, tissues were more sensitive to the contractile responses of serotonin
Vol. 35, No. i, 1984
5HT 2 Receptors in Rat Portal Vein
following i t s non-cumulative addition. A small reduction in the s e n s i t i v i t y to serotonin could also be demonstrated following both the cumulative and non-cumulative addition of serotonin between the i n i t i a l and second response in each tissue (Table I ) . There was approximately 20-30 percent decrease in s e n s i t i v i t y of the second response r e l a t i v e to the i n i t i a l response to serotonin, based on a comparison of ED50 values. TABLE I
Sensitivity of Rat Portal Vein to Serotonin Following Cumulative and Non-Cumulative Addition to the Tissue Baths Serotonin I n i t i a l Response Second Response ED50(~M)a Cumulative (10)
5.4 * 1.4
7.0 * 1.7
Non-Cumulative (10)
1.6 ± 0.4 b
1.9 • 0.4 a
Ratio Cumulative/Non-Cumulative a b
3.4
3.7
Values are means • SE for the number of tissues indicated in parentheses. ED50was s i g n i f i c a n t l y lower r e l a t i v e to cumulative addition as determined by student's t - t e s t for unpaired data.
TABLE I / Comparison of the Negative Logarithm of the Dissociation Constants Determined for Ketanserin, Metergoline and LY53857 from the Antagonism of the Cumulative and Non-Cumulative Addition of Serotonin in the Rat Portal Vein Serotonin Addition Cumulative Non-Cumulative -Log KB * SEa Ketanserin
8.21 4. 0.08 (14)
9.73 * 0.06 (9)
Metergoline
8.75 • 0.15 (11)
9.28 * 0.16 (6)
LY53857
8.39 * 0.09 (12)
9.13 * 0.09 (11)
a
Valuesare means • SE for the number of tissues indicated in parentheses.
75
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5HTo Receptors
in Rat Portal Vein
Vol. 35, No.
i, 1984
Since tissue s e n s i t i v i t y to serotonin was greater when serotonin was administered non-cumulatlvely in the rat portal vein, we repeated the determination of the dlssociation constants for three 5HT2 receptor antagonists, ketanserln, metergoline, and LY53857. For a l l three antagonists, the dlSSociation constants determined following the non-cumulative addition of serotonin, were lower than the dissociation constants obtained following the cumulative addition of serotonin (Table I I ) . The d i s s o c i a t i o n constants obtalned followlng non-cumulative addition of serotonin approached those previously determined in the rat jugular vein (4,14). Discussion Although the c o n t r a c t i l e response to serotonin in several vascular beds from the rat (caudal a r t e r y , jugular vein, and aorta) is mediated by i n t e r a c tion with 5HT2 receptors, the canine b a s i l a r a r t e r y has been reported to contract to serotonin by a mechanism not involving 5HT2 receptors (5). Furthermore, serotonin-induced contraction in g a s t r o i n t e s t i n a l smooth muscle from the rat also does not involve i n t e r a c t i o n of serotonin with 5HT2 receptors (6,7). Nevertheless, the myogenlcally active rat portal vein from the g a s t r o i n t e s t i n a l area was s i m i l a r to other vascular beds from the r a t and d~fferent from g a s t r o i n t e s t i n a l smooth muscle since contractions to serotonin were mediated by i n t e r a c t i o n with 5HT2 receptors. This concluslon is based on the e x c e l l e n t c o r r e l a t i o n (Figure I) of the a f f i n i t l e s of several d i f f e r e n t serotonin antagonists for the serotonin receptor in the rat portal vein to the a f f i n i t y of these antagonists for 5HT2 receptors (based on the displacement of radiolabeled spiperone from f r o n t a l c o r t l c a l membranes). During the course of these experiments, i t was noted that the dlssoclation constants obtained for a l l the serotonin antagonlsts evaluated in the rat portal vein were higher ( i . e . , lower a f f i n i t y ) than those previously obtained for the same serotonin receptor antagonists in the rat jugular vein. Several potential explanatlons might account f o r the differences observed. I t is possible that there is a s i t e of loss of serotonin such as neuronal uptake that occurred in the portal vein which is innervated (17) but not in the jugular vein which lacks innervatlon (16). However, the portal vein lacks a s p e c i f i c neuronal uptake mechanism f o r serotonin since fluoxet~ne did not potentiate c o n t r a c t i l e responses to serotonln in t h l s tissue (17). Nevertheless, since the portal vein is innervated (17), the neuronal uptake of serotonin into adrenerglc nerves must be considered. However, nisoxetine, a s e l e c t i v e i n h i b i t o r of adrenergic uptake mechanisms, did not potentiate c o n t r a c t i l e responses to serotonin in concentrations that markedly potentiated the responses to norepinephrine in the rat portal vein (17). These data suggest that neuronal uptake mechanisms for serotonin probably play a minor role in the lower a f f i n i t y of antagonists that occurred following the cumulative addition of serotonin in t h i s tissue . We also considered the p o s s i b l i t y that serotonin may be i n t e r a c t i n g e i t h e r d i r e c t l y or i n d i r e c t l y with alpha receptors in the rat portal vein since the r a t jugular vein possess mnimal alpha receptors (16). However, the alpha I receptor antagonist, prazosin, did not i n h i b i t the c o n t r a c t i l e responses to serotonin suggestlng that serotonin was not i n t e r a c t i n g with alpha adrenergic receptors e i t h e r d i r e c t l y or by the i n d i r e c t release of norepinephrine from nerves in the rat portal vein. Thus, i t is u n l i k e l y that t h i s p o s s i b i l i t y provides an explanation for the lower a f f i n i t i e s demonstrated with the series of serotonin receptor antagonists in the r a t portal vein r e l a t i v e to the r a t jugular vein. L a s t l y , we considered the p o s s i b l i t y that receptor desensitization occurred both to the agonist, serotonin, and to the antagonists examined in
Vol. 35~ No. i, 1984
5HT 2 Receptors
in Rat Portal Vein
77
the present study when serotonin was added cumulatively. In f a c t , s e n s i t i v i t y to the cumulative addition of serotonin was 3-4 fold lower than the s e n s i t i v i t y of the receptors to the non-cumulative addition of serotonin in the rat portal vein. Such a s e n s i t i v i t y difference may be a r e f l e c t i o n of desensitization that occurred following cumulative administration. The fact that desensitization can occur is also consistent with the reduced s e n s i t i v i t y (higher ED50) of the second concentration-response to serotonin that occurred when serotonin was added cumulatively or non-cumulatively in the portal vein. Furthermore, desensitization to serotonin was associated with a reduction in receptor a f f i n i t y to all the serotonin receptor antagonists evaluated in the present study. Since desensitization was least when serotonin was added non-cumulatively, the a f f i n i t i e s of ketanserin, metergoline and LY53857 also were greater following non-cumulative addition of serotonin. Why desensitization to serotonin occurred in the rat portal vein but not in the rat jugular vein remains to be c l a r i f i e d . Future studies w i l l be directed toward understanding more thoroughly the mechanism for the desens i t i z a t i o n to serotonin and the reduction in a f f i n i t i e s of the various antagonists examined when serotonin was used cumulatively in the rat portal vein. Nevertheless, the present report c l e a r l y establishes 5HT2 receptors as responsible for mediating the c o n t r a c t i l e response to serotonin in the r a t portal vein. Thus, the rat portal vein may be added to the l i s t of other vascular tissues (aorta, jugular vein, and caudal artery) that possess 5HT2 receptors. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
J . E . LEYSEN, J. Physiol. Paris 77 351-362 (1981). J . E . LEYSEN, J. E. NIEMEGEERS, -~.. M. VAN NEUTEN, and P. M. LADURON, MoI. Pharmacol. 21 301-314 (1981). M . L . COHEN, R. ~TT.FULLER, and K. S. WILEY, J. Pharmacol. Exp. Ther. 218 421-425 (1981). M . L . COHEN, N. MASON, and K. S. WILEY, Biochem. Pharmacol. 32 567-570 (1983). S . J . PEROUTKA, M. NOGUCHI, D. J. TOLNER and G. S. ALLEN, Brain Res. 259 327-330 (1983). J . M . VAN NEUTEN, and P. M. VANHOUTTE, Angiology 32 476-484 (1981). J . M . VAN NEUTEN, J. E. LEYSEN, P. M. . .~. . . . .~. . . . d .. P. A. J. JANSSEN, Arc. Intern. Pharm. Ther. 256 331-334 (1982). S . J . PEROUTKA and S. H. SNYDER, Mol. Pharmacol. 16 687-699 (1979). R . F . FURCHGOTT, In Catecholamines, ed. by H. BlaTchko and C. Muscholl, pp. 283-335, Springer-Verlag, Berlin, (1972). B. R. ZABOROWSKY, W. C. MCMAHON, W. A. GRIFFIN, F. H. NORRIS and R. R. RUFFOLO, J. Pharmacol. Methods 4 165-178 (1980). O. ARUNLAK~HANAAND H. O. SCHILD, Br. J. Pharmacol. Chemother. 14 48-58 (1958). D. L. NELSON, A. HERBET, S. BOURGOIN, J. GLOWINSKI, AND M. HAMON, Mol. Pharmacol. 14 983-995 (1978). J. P. BENNETT, JR., AND S. H. SNYDER, Mol. Pharmacol. 12 373-389 (1976). M. L. COHEN, R. W. FULLER, and K. D. KI]IT~, Hypertensio-6-~, 5 676-681 (1983).. M. L. COHEN, R. W. FULLER, and K. D. KURZ, J. Pharmacol. Exp. Ther. 227 327-332 (1983). M. L. COHENAND K. S. WILEY, J. Pharmacol. Exp. Ther. 205 400-409 (1978). M. L. COHEN AND K. S. WILEY, Eur. J. Pharmacol. 44 219-229 (1977).
Pergamon Press
5HT2 RECEPTORSIN THE RAT PORTAL VEIN: DESENSITIZATIONFOLLOWING CUMULATIVE SEROTONINADDITION Harriet F. Lemberger, Norman Mason and Marlene L. Cohen Cardiovascular Pharmacology L i l l y Research Laboratories Eli L i l l y and Company Indianapolis, IN 46285 (Received in final form April 23, 1984)
SUMMARY Contractile responses to serotonin were examined in vitro in the longitudinal portal vein to determine whether sucB responses were mediated by the interaction of serotonin with 5HT1 receptors (those that preferentially bind [3H]serotonin) or 5HT2 receptors (those that preferentially bind [JH]spiperone). Using eight serotonin receptor antagonists (spiperone, metergoline, LY53857, ketanserin, trazodone, benzoctamine, 1-(1-naphthyl)piperazine, and 1-meta-methoxyphenylpiperazine), we found a significant correlation between the a f f i n i t y for serotonin receptors in the rat portal vein and the a b i l i t y to bind to 5HT2, but not 5HT1 receptors in rat frontal cortical membranes. Thus, the receptors mediating vascular contraction to serotonin in the rat portal vein were similar to those receptors defined in other vascular beds from the rat (aorta, jugular vein, and caudal artery). Furthermore, contraction resulting from the cumulative addition of serotonin in the rat portal vein was associated with desensitization (higher ED50 value) relative to contractions produced by the non-cumulative addition of serotonin. A f f i n i t i e s of serotonin receptor antagonists were also lower when determined by antagonism of cumulative serotonin concentrationresponse curves compared to a f f i n i t i e s obtained by antagonism of non-cumulative concentration-response curves. Thus, 5HT2 receptor a f f i n i t i e s of antagonists in the rat portal vein are best determined by the s h i f t of non-cumulative responses to serotonin. Vascular receptors mediating serotonin-induced contraction have been shown to be of the 5HT2 subtype in the caudal artery (1,2), aorta (3) and jugular vein (3,4) from the rat. However, the possibility that not all vascular serotonin receptors are of the 5HT2 type has been raised by Peroutka et al. (5) using the canine basilar artery. In the basilar artery, potent 51f~-~ceptor antagonists, such as ketanserin did not antagonize the contractile response to serotonin. Using a series of antagonists and agonists, Peroutka et al. (5) have proposed that the receptors mediating contraction to s e r o ~ n ~ in the basil ar artery may best be described as 5HTI. Furthermore, receptors mediating the contractile response to serotonin in several gastrointestinal preparations including the guinea pig ileum and the rat fundus (6,7) do not f i t the classical definition of 5HT2 receptors ( i . e . , inhibition did not correlate with [3H]spiperone binding data). The~ observations raise the possibility that some blood vessels and gastrointestinal smooth muscle may contract to serotonin via receptors other than 5HT2 . 0024-3205/84 $3.00 + .00 Copyright (c) 1984 Pergamon Press Ltd.
72
5HT2 Receptors
in Rat Portal Vein
Vol. 35, No. i, 1984
Since myogenic rhythmic a c t i v i t y occurs in the rat portal vein but not in other in v i t r o vascular preparations and the hepatic portal circulation is associated with the gastrointestinal tract which may not possess 5HT2 receptors, we asked whether serotonin receptors in the portal vein would resemble those receptors in other vascular beds from the rat (5HT2 receptors) or those in the gastrointestinal tract (not 5HT2). To examine this, we compared the a f f i n i t i e s of a series of serotonin receptor antagonists for vascular serotonin receptors with their a b i l i t y to bind to 5HTI or 5HT2 r~ceptors in the brain based on displacement of [3H]serotonin and [JH]spiperone, respectively (8). For these antagonists, a signiflcant correlation, occurred between the a f f l n i t y for serotonin receptors in the rat portal vein and their a b i l l t y to b~nd to 5HT2 receptors.
Methods Isolation of Portal veins: Male Sprague-Dawley rats (200-300 g) (Harlan Industries, Inc., Cumberland, IN) were s a c r l f i c e d by cervical d l s l o c a t i o n . Portal veins were dissected free of connective tissue, ligated with cotton thread, excised, and transferred to petri dishes containing Krebs' blcarbonate buffer. Longitudinal preparatlons were suspended in organ baths containing 10 ml of a modlfied Krebs' solution of the following composition (in milllmoles per l l t e r ) : NaCI, 118.2; KCl, 4.6; CAC12.2 H20, 1.6; KH2P04, 1.2; MgS04, 1.2; dextrose, 10.0, and NaHC03, 24.8. Tissue bath solutions were maintained at 37°C and e q u i l i b r a t e d with 95 percent 02 and 5 percent CO2. Preparations were stretched to 4 grams force and were allowed to e q u i l l b r a t e for 1-2 h before exposure to drugs. Isometric contractions were recorded as changes in grams of force on a Beckman Dynograph with Statham UC-3 transducers. Vascular contraction to serotonin. Cumulative contractile concentrationresponse curves for serotonin were obtalned by a stepwise increase in the concentration of serotonln after a steady response occurred to the preceding dose. For non-cumulative contractile concentration-response curves, increasing concentrations of serotonin were added to the bath after washout of the previous concentration with at least 15-20 minutes between successive additions. ED50 values were taken as the concentration of serotonin that produced half-maximal contraction. Determination of apparent dissociation constants for antagonists. After control responses to serotonin were obtained, vessels were incubated with appropriate concentrations of antagonists for 20-30 minutes. Responses to serotonin were then repeated in the presence of antagonist. In experiments in which serotonin was added non-cumulatively, antagonists were added in the wash buffer between serotonin additions.
Apparent antagonlst dissociation constants (KB) were determined for each concentration of antagonist according to the following equation (9):
[B]
KB = [dose r a t i o - I ] where [B] is the concentration of the antagonist and dose ratio is the ED50 of the agonist in the presence of the antagonist divided by the control ED50. These results were then expressed as the negative logarithm of the KB ( i . e . , -log KB). Calculations were performed with aid of a computer and d i g i t a l plotter as previously described (10). The data were also analyzed according to the procedure of Arunlakshana and Schlld (11). The dose ratio was determined at various concentrations of
Vol. 35, No. i, 1984
5HT 2 Receptors
in Rat Portal Vein
73
antagonist. According to Arunlakshana and Schild (11), i f blockade is competitive, under equilibrium conditions, a plot of the logarithm of (dose ratio - I) against the negative logarithm of the molar concentration of antagonist should yield a straight line whose slope is I and intercept along the abscissa is the pA2 which is equal to -log KB. Cortical bindin 9 to 5HTp and 5HT1 receptors. Brain tissue was obtained from 150 to 200 g male Wistar rats. The cerebral cortex was dissected, homogenized, and prepared according to the method described by Nelson et al. (12). Tissue was preincubated in buffer without monoamine oxidase i n h ~ i ~ r in order to eliminate endogenous serotonin. For receptor binding, an amount of membrane preparation equivalent to 250-350 mg of protein was used per sample in I ml of Tris buffer. The assay for serotonin binding was done following the method of Bennett and Snyder (13) and that for spi~erone binding according to Peroutka and Snyder (8). Non-specific binding of [JH]serotonin and [3H]spiperone was determined in the presence of 10-bM serotonin or lO-bM lysergic acid diethylamide (LSD), respectively. Specific binding was calculated as the difference between total binding (without added nonradioactive compound) and non-specific binding. The IC50 values were determined as the amount of substance causing 50 percent i n h i b l t i o n of ~he specific binding using ten d i f f e r e n t concentrations in the range of I0 -~ to IO-4M. The concentration of [3H]serotonin (sp. act. 17.6 Ci/mmole, Amersham ~orp., Arlington Heights, IL) in each sample was 2.3 to 2.6 nM and that of [JH]spiperone (sp. act. 20 Ci/mmole, Amersham) was 0.5 to 0.7 nM. Results Eight compounds (spiperone, metergoline, LY53857, ketanserin, trazodone, benzoctamine, 1-(l-naphthyl)piperazine, and 1-meta-methoxyphenylpiperazine) were evaluated for t h e i r a b i l i t y to antagonize serotonin-induced contractions in rat portal v~in and to displace the binding of [3H]spiperone (5HT2 receptors) or [JH]serotonin (5HTI receptors) in brain c o r t i c a l membranes. 5 JCORRELATIONCOEFFICIENT: 0.94 /
°I
7
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e ; / O O n"l ~a" 8 [
BENZOCTAMINE~IP
e;~AzoooN, O / OLY538S7
KETANSERIN /
~9
/ / • 5PIPERONE IO
9
,
,
8 7 6 "LOG ICso 5HT2 ( CORTICAL BINDING)
5
FIG. 1 Correlation of the a f f i n i t i e s of several serotonin receptor antagonists for serotonin receptors in the rat portal vein to t h e i r a f f i n i t i e s f o r 5HT2 receptors (displacement of [JH]spiperone) in rat f r o n t a l c o r t i c a l membranes.
74
5HT2 Receptors in Rat Portal Vein
Vol. 35, No. i, 1984
CORRELATION COEFFICIENT : 0.1'12 1MMOPP
A6
-, 7
INF
BENZOCTAMIN|
TRAZODONE
'=28 METERGOLINE
LY53857 • • KETANSERIN
~9 SPIPERONE
IO
8
*,6
i
- L O G ICso 5HTI (CORTICAL BINDING)
FIG. 2
Correlation of the a f f i n i t i e s of several serotonin receptor antagonists for serotonin receptors in the rat pQrtal vein to their a f f i n i t i e s for 5HT1 receptors (displacement of [JH]serotonin) in rat frontal cortical membranes. The receptor antagonists examined in the present study were potent inhibitors of the contractile response to serotonin in the rat portal vein. A significant correlation (correlation coefficient = 0.94) was found between the a f f i n i t i e s of these antagonists for serotonin receptors in the portal vein and their a f f i n i t i e s for 5HT2 receptors in brain cortical membranes (Fig. 1). In contrast, no correlation was found between receptors mediating contraction in the portal vein and their a f f i n i t y for 5HT1 receptors in brain cortical membranes (Fig. 2). Although a significant correlation was obtained with the a b i l i t y of antagonists to block serotonin contractions in the portal vein and their a b i l i t y to bind to 5HT2 receptors, the dissociation constants obtained for these antagonists in the rat portal vein were higher than in the rat jugular vein (4, 14, 15). Thus, although the relative rank order of the serotonin receptor antagonists was similar in the rat portal vein and jugular vein, the relative s e n s i t i v i t y to the antagonists was lower in the rat portal vein. Several explanations may be advanced for the lower pA2 values found in the rat portal vein relative to the jugular vein. Since alpha receptors occur in the rat portal vein but not in the rat jugular vein (16), we considered the possibility that serotonin might be interacting with alpha receptors in the portal vein complicating the interpretation of our data. However, prazosin (10-6M), a potent alpha I receptor antagonist, did not block the contractile response to serotonin in the rat portal vein. Prazosin was a potent antagonist of the contractile response to phenylephrine in this tissue (-log KB =8.59 * 0.31; n=5). Next, we considered the p o s s i b i l i t y that the cumulative addition of serotonin in the portal vein might be associated with a reduced s e n s i t i v i t y to serotonin. In fact, the ED50 determined after the cumulative administration of serotonin was higher (3-4 fold) than the ED50 determined after noncumulative addition of serotonin in the rat portal vein (Table I ) . Thus, tissues were more sensitive to the contractile responses of serotonin
Vol. 35, No. i, 1984
5HT 2 Receptors in Rat Portal Vein
following i t s non-cumulative addition. A small reduction in the s e n s i t i v i t y to serotonin could also be demonstrated following both the cumulative and non-cumulative addition of serotonin between the i n i t i a l and second response in each tissue (Table I ) . There was approximately 20-30 percent decrease in s e n s i t i v i t y of the second response r e l a t i v e to the i n i t i a l response to serotonin, based on a comparison of ED50 values. TABLE I
Sensitivity of Rat Portal Vein to Serotonin Following Cumulative and Non-Cumulative Addition to the Tissue Baths Serotonin I n i t i a l Response Second Response ED50(~M)a Cumulative (10)
5.4 * 1.4
7.0 * 1.7
Non-Cumulative (10)
1.6 ± 0.4 b
1.9 • 0.4 a
Ratio Cumulative/Non-Cumulative a b
3.4
3.7
Values are means • SE for the number of tissues indicated in parentheses. ED50was s i g n i f i c a n t l y lower r e l a t i v e to cumulative addition as determined by student's t - t e s t for unpaired data.
TABLE I / Comparison of the Negative Logarithm of the Dissociation Constants Determined for Ketanserin, Metergoline and LY53857 from the Antagonism of the Cumulative and Non-Cumulative Addition of Serotonin in the Rat Portal Vein Serotonin Addition Cumulative Non-Cumulative -Log KB * SEa Ketanserin
8.21 4. 0.08 (14)
9.73 * 0.06 (9)
Metergoline
8.75 • 0.15 (11)
9.28 * 0.16 (6)
LY53857
8.39 * 0.09 (12)
9.13 * 0.09 (11)
a
Valuesare means • SE for the number of tissues indicated in parentheses.
75
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5HTo Receptors
in Rat Portal Vein
Vol. 35, No.
i, 1984
Since tissue s e n s i t i v i t y to serotonin was greater when serotonin was administered non-cumulatlvely in the rat portal vein, we repeated the determination of the dlssociation constants for three 5HT2 receptor antagonists, ketanserln, metergoline, and LY53857. For a l l three antagonists, the dlSSociation constants determined following the non-cumulative addition of serotonin, were lower than the dissociation constants obtained following the cumulative addition of serotonin (Table I I ) . The d i s s o c i a t i o n constants obtalned followlng non-cumulative addition of serotonin approached those previously determined in the rat jugular vein (4,14). Discussion Although the c o n t r a c t i l e response to serotonin in several vascular beds from the rat (caudal a r t e r y , jugular vein, and aorta) is mediated by i n t e r a c tion with 5HT2 receptors, the canine b a s i l a r a r t e r y has been reported to contract to serotonin by a mechanism not involving 5HT2 receptors (5). Furthermore, serotonin-induced contraction in g a s t r o i n t e s t i n a l smooth muscle from the rat also does not involve i n t e r a c t i o n of serotonin with 5HT2 receptors (6,7). Nevertheless, the myogenlcally active rat portal vein from the g a s t r o i n t e s t i n a l area was s i m i l a r to other vascular beds from the r a t and d~fferent from g a s t r o i n t e s t i n a l smooth muscle since contractions to serotonin were mediated by i n t e r a c t i o n with 5HT2 receptors. This concluslon is based on the e x c e l l e n t c o r r e l a t i o n (Figure I) of the a f f i n i t l e s of several d i f f e r e n t serotonin antagonists for the serotonin receptor in the rat portal vein to the a f f i n i t y of these antagonists for 5HT2 receptors (based on the displacement of radiolabeled spiperone from f r o n t a l c o r t l c a l membranes). During the course of these experiments, i t was noted that the dlssoclation constants obtained for a l l the serotonin antagonlsts evaluated in the rat portal vein were higher ( i . e . , lower a f f i n i t y ) than those previously obtained for the same serotonin receptor antagonists in the rat jugular vein. Several potential explanatlons might account f o r the differences observed. I t is possible that there is a s i t e of loss of serotonin such as neuronal uptake that occurred in the portal vein which is innervated (17) but not in the jugular vein which lacks innervatlon (16). However, the portal vein lacks a s p e c i f i c neuronal uptake mechanism f o r serotonin since fluoxet~ne did not potentiate c o n t r a c t i l e responses to serotonln in t h l s tissue (17). Nevertheless, since the portal vein is innervated (17), the neuronal uptake of serotonin into adrenerglc nerves must be considered. However, nisoxetine, a s e l e c t i v e i n h i b i t o r of adrenergic uptake mechanisms, did not potentiate c o n t r a c t i l e responses to serotonin in concentrations that markedly potentiated the responses to norepinephrine in the rat portal vein (17). These data suggest that neuronal uptake mechanisms for serotonin probably play a minor role in the lower a f f i n i t y of antagonists that occurred following the cumulative addition of serotonin in t h i s tissue . We also considered the p o s s i b l i t y that serotonin may be i n t e r a c t i n g e i t h e r d i r e c t l y or i n d i r e c t l y with alpha receptors in the rat portal vein since the r a t jugular vein possess mnimal alpha receptors (16). However, the alpha I receptor antagonist, prazosin, did not i n h i b i t the c o n t r a c t i l e responses to serotonin suggestlng that serotonin was not i n t e r a c t i n g with alpha adrenergic receptors e i t h e r d i r e c t l y or by the i n d i r e c t release of norepinephrine from nerves in the rat portal vein. Thus, i t is u n l i k e l y that t h i s p o s s i b i l i t y provides an explanation for the lower a f f i n i t i e s demonstrated with the series of serotonin receptor antagonists in the r a t portal vein r e l a t i v e to the r a t jugular vein. L a s t l y , we considered the p o s s i b l i t y that receptor desensitization occurred both to the agonist, serotonin, and to the antagonists examined in
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5HT 2 Receptors
in Rat Portal Vein
77
the present study when serotonin was added cumulatively. In f a c t , s e n s i t i v i t y to the cumulative addition of serotonin was 3-4 fold lower than the s e n s i t i v i t y of the receptors to the non-cumulative addition of serotonin in the rat portal vein. Such a s e n s i t i v i t y difference may be a r e f l e c t i o n of desensitization that occurred following cumulative administration. The fact that desensitization can occur is also consistent with the reduced s e n s i t i v i t y (higher ED50) of the second concentration-response to serotonin that occurred when serotonin was added cumulatively or non-cumulatively in the portal vein. Furthermore, desensitization to serotonin was associated with a reduction in receptor a f f i n i t y to all the serotonin receptor antagonists evaluated in the present study. Since desensitization was least when serotonin was added non-cumulatively, the a f f i n i t i e s of ketanserin, metergoline and LY53857 also were greater following non-cumulative addition of serotonin. Why desensitization to serotonin occurred in the rat portal vein but not in the rat jugular vein remains to be c l a r i f i e d . Future studies w i l l be directed toward understanding more thoroughly the mechanism for the desens i t i z a t i o n to serotonin and the reduction in a f f i n i t i e s of the various antagonists examined when serotonin was used cumulatively in the rat portal vein. Nevertheless, the present report c l e a r l y establishes 5HT2 receptors as responsible for mediating the c o n t r a c t i l e response to serotonin in the r a t portal vein. Thus, the rat portal vein may be added to the l i s t of other vascular tissues (aorta, jugular vein, and caudal artery) that possess 5HT2 receptors. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
J . E . LEYSEN, J. Physiol. Paris 77 351-362 (1981). J . E . LEYSEN, J. E. NIEMEGEERS, -~.. M. VAN NEUTEN, and P. M. LADURON, MoI. Pharmacol. 21 301-314 (1981). M . L . COHEN, R. ~TT.FULLER, and K. S. WILEY, J. Pharmacol. Exp. Ther. 218 421-425 (1981). M . L . COHEN, N. MASON, and K. S. WILEY, Biochem. Pharmacol. 32 567-570 (1983). S . J . PEROUTKA, M. NOGUCHI, D. J. TOLNER and G. S. ALLEN, Brain Res. 259 327-330 (1983). J . M . VAN NEUTEN, and P. M. VANHOUTTE, Angiology 32 476-484 (1981). J . M . VAN NEUTEN, J. E. LEYSEN, P. M. . .~. . . . .~. . . . d .. P. A. J. JANSSEN, Arc. Intern. Pharm. Ther. 256 331-334 (1982). S . J . PEROUTKA and S. H. SNYDER, Mol. Pharmacol. 16 687-699 (1979). R . F . FURCHGOTT, In Catecholamines, ed. by H. BlaTchko and C. Muscholl, pp. 283-335, Springer-Verlag, Berlin, (1972). B. R. ZABOROWSKY, W. C. MCMAHON, W. A. GRIFFIN, F. H. NORRIS and R. R. RUFFOLO, J. Pharmacol. Methods 4 165-178 (1980). O. ARUNLAK~HANAAND H. O. SCHILD, Br. J. Pharmacol. Chemother. 14 48-58 (1958). D. L. NELSON, A. HERBET, S. BOURGOIN, J. GLOWINSKI, AND M. HAMON, Mol. Pharmacol. 14 983-995 (1978). J. P. BENNETT, JR., AND S. H. SNYDER, Mol. Pharmacol. 12 373-389 (1976). M. L. COHEN, R. W. FULLER, and K. D. KI]IT~, Hypertensio-6-~, 5 676-681 (1983).. M. L. COHEN, R. W. FULLER, and K. D. KURZ, J. Pharmacol. Exp. Ther. 227 327-332 (1983). M. L. COHENAND K. S. WILEY, J. Pharmacol. Exp. Ther. 205 400-409 (1978). M. L. COHEN AND K. S. WILEY, Eur. J. Pharmacol. 44 219-229 (1977).