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A 5-HT receptor in the central nervous system, positively coupled with adenylate cyclase, is antagonized by ICS 205 930
European Journal of Pharmacology, 146 (1988) 187-188
187
Elsevier EJP 0103R
Rapid communication
A 5-HT receptor in the central nervous system, positively coupled with adenylate cyclase, is antagonized by ICS 205 930 Aline Dumuis *, Rochdi Bouhelal, Mich~le Sebben and JoEl Bockaert Centre CNRS-INSERM de Pharmacologie-Endocrinologie. Rue de la Cardonille, 34094 Montpellier Cedex, France Received 28 December 1987, accepted 29 December 1987
Only two main 5-HT receptor classes have been clearly described in the central nervous system (CNS); the 5-HT~ and 5-HT2 receptors (Bradley et al., 1986). There are several 5-HT~ receptor subclasses and of these receptors, the 5-HTIA (De Vivo and Maayani, 1986; Bockaert et al., 1987; Shenker et al., 1987) and the 5-HT~B (Bouhelal et al., submitted) modulate cyclic AMP levels. A third class of excitatory 5-HT receptors has been described in the peripheral nervous system: the 5-HT3 receptors (Richardson et al., 1985; Bradley et al., 1986). Their pharmacological and functional characterization has long been hampered by the absence of selective agonists and antagonists. These drugs are now available and include 2methyl-5-hydroxytryptamine, a good specific agonist and MDL 72222 or ICS 205 930 which are good selective antagonists (Richardson et al., 1985; Bradley et al., 1986). 5-HT3 receptors have never been described in the CNS. We now provide evidence that a 5-HT receptor found in colliculi neurons in primary culture and that mediates cyclic AMP stimulation, does not have the same pharmacological profile as a 5-HTa or a 5-HT2 receptor. The pharmacological characterization of this receptor suggests that it could correspond to a 5-HT3 receptor type. All the experiments described here were performed with neuronal cell cultures generated from 14- or 15-day-old mouse embryo colliculi. Neuro-
* To whom all correspondence should be addressed.
nal cells were cultured as described previously (Bockaert et al., 1987) and were grown for six days. Intracellular cAMP production was determined using the [3H]adenine prelabelling technique (Bockaert et al., 1987). In colliculi neurons, 5-HT stimulated cAMP production in a concentration-dependent manner with an ECs0 of 109 + 17 nM (n = 5). The percent conversion of [3H]ATP to [3H]cAMP in the absence of 5-HT was 0.8_ 0.1% (n = 5) whereas it was 2_+0.3% (n = 5) in the presence of 1 /~M 5-HT. Several high affinity antagonists interacting with the various classes of 5-HT receptors (5-HT 1, 5HT2 and 5-HT3) were used to determine the nature of this 5-HT receptor that is positively coupled to adenylate cyclase. The neurons were stimulated with 1 ~tM 5-HT (2- to 2.5-fold increase of basal conversion [ 3H]ATP --* [3H]cAMP) and increasing concentration of four antagonists: spiroperidol (a 5-HTIA and 5-HT2 antagonist), methiothepin (a 5-HT~ and 5-HT2 antagonist), ketanserin (a 5-HT2 antagonist) and ICS 205 930 (a 5-HT3 antagonist). Classical 5-HT antagonists (ketanserin and methiothepin) failed to antagonize the 5-HTstimulated cAMP production (fig. I B). At high concentrations (100 /~M) spiroperidol (a potent selective 5-HTIA and 5HT2 antagonist) slightly inhibited (18%, P < 0.001) the stimulatory effect of 1 pM 5-HT. In contrast, ICS 205 930, a potent and specific 5-HT3 antagonist in functional studies, inhibited the 5-HT-stimulated activity in a
0014-2999/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
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Fig. 1. Stimulation by 5-HT of cAMP formation in mouse embryonic colliculi neurons. (A) Concentration-response curve for cAMP formation by 5-HT. Neurons in primary culture, generated from colliculi of 14- or 15-day-old mouse embryos were grown for 6 days in serum-free medium, in 12-well Costar dishes (106 cells/well) in a humidified atmosphere of 5% CO 2 and 95% 02 at 37°C. Before each experiment, the cells were washed and incubated with 2/~M of [3H]adenine (24/.tCi/mol, Amersham, U.K.) under the same culture conditions. The cultures were washed to remove free [3 H]adenine and the cells were then incubated with isobuthylmethylxanthine, 0.75 mM, forskolin, 0.1 /~M and increasing concentrations of 5-HT (10 nM to 100 /~M) in a 1 ml volume of culture medium. The reaction was stopped after 5 rain at 37 ° C by aspiration of the medium and addition of 1 ml ice-cold 5% trichloracetic acid. The cells were scraped with a rubber policeman; ATP, 5 mM and cAMP, 5 mM were then added to the mixture. Cellular protein was centrifuged at 5000× g. [~H]ATP and [3H]cAMP were separated by sequential chromatography on Dowex and alumina columns. Stimulation of cAMP formation is expressed as a percent stimulation above the basal activity level. The values are the means+ S.E.M. from five separate experiments, each performed with duplicate determinations. (B) Effect of 5-HT:, 5-HT2- and 5-HT3-related antagonists on 5-HT stimulation of cAMP formation in colliculi neurons. Neuronal cells were exposed in presence of forskolin, 0.1 btM and 5-HT, 1 I~M to increasing concentrations of methiothepin, ketanserin, spiroperidol and ICS 205 930. The results, expressed in percent stimulation over the basal activity level, are the means + S.E.M. from four separate experiments performed in duplicate. The basal and stimulated conversion of[ 3H]ATP to[ "~H]cAMP was 0.8 + 0.1 and 2 + 0.3% respectively. The differences between the values obtained in the presence of 10 -7 to 10 -4 M spiroperidol and the values for basal stimulation (1 /~M 5-HT) were compared using Student's t-test: * P < 0.02 compared to basal stimulation. * * P < 0.001.
concentration-dependent manner. The 5-HT effect was completely reversed only at high concentrations of ICS 205 930 (100 #M). An apparent K i value of 710 + 180 nM ( n - 4) was obtained for ICS 205 930 which was about 100-fold higher than the values obtained in functional studies with
guinea pig ileum (Richardson et al., 1985). ICS 205 930 competitively inhibited the 5-HT-induced cAMP production (data not shown). Our results indicate that this 5-HT receptor that is present in colliculi neurons is neither a 5-HT 1 nor a 5-HT2 receptor type. The effect of ICS 205 930 indicates that this receptor could belong to the 5-HT3 type according to the classification of Bradley et al. (1986). The difference between the pK, values for ICS 205 930 in our studies (6.15 +0.14) and the pA 2 values (7.7 to 13.5 depending on the systems tested) for its peripheral effects (Richardson et al., 1985) could be due to the existence of an embryonic form of 5-HT3 receptor in colliculi neurons. This idea is substantiated by our finding that ICS 205 930 potently antagonized (with a pK, of 7.6) the low affinity 5-HT receptor (RL) that mediates cAMP stimulation, and that was described by Shenker et al. (1987) in adult guinea pig hippocampal membranes (in preparation). In conclusion, the results presented here provide the first evidence for the existence of a 5-HT3 receptor type positively coupled to adenylate cyclase and located in the CNS. References Bockacrt, J., A. Dumuis, R. Bouhelal, M. Sebben and R.N. Cory, 1987, Piperazine derivatives including the putative anxiolytic drugs, buspirone and ipsapirone are agonists at 5-HTIA receptors negatively coupled with adenylate cyclase in hippocampal neurons, Naunyn-Schmiedeb. Arch. Pharmacol. 335, 588. Bradley, P.B., G. Engel, W. Feniuk, J.R. Fozard, P.P.A. Humphrey, D.N. Middlemiss, E.J. Mylecharane, B.P. Richardson and P.R. Saxena, 1986, Proposals for the classification and nomenclature of functional receptors for 5-hydroxytryptamine, Neuropharmacology 25, 563. De Vivo, M. and S. Maayani, 1986, Characterization of the 5-hydroxytryptanune~^ receptor mediated inhibition of forskolin-stimulated adenylate cyclase activity in guinea pig and rat hippocampal membranes, J. Pharmacol. Exp. Ther. 238, 248. Richardson, B.P., G. Engel, P. Donatsch and P.A. Stadler, 1985, Identification of serotonin M-receptor subtypes and their specific blockade by a new class of drugs, Nature 316, 126. Shenker, A., S. Maayani, H. Weinstein and J.P. Green. 1987. Pharmacological characterization of two 5-hydroxytryptamine receptors coupled to adenylate cyclase in guinea pig hippocampal membranes, Mol. Pharmacol. 31,357.
187
Elsevier EJP 0103R
Rapid communication
A 5-HT receptor in the central nervous system, positively coupled with adenylate cyclase, is antagonized by ICS 205 930 Aline Dumuis *, Rochdi Bouhelal, Mich~le Sebben and JoEl Bockaert Centre CNRS-INSERM de Pharmacologie-Endocrinologie. Rue de la Cardonille, 34094 Montpellier Cedex, France Received 28 December 1987, accepted 29 December 1987
Only two main 5-HT receptor classes have been clearly described in the central nervous system (CNS); the 5-HT~ and 5-HT2 receptors (Bradley et al., 1986). There are several 5-HT~ receptor subclasses and of these receptors, the 5-HTIA (De Vivo and Maayani, 1986; Bockaert et al., 1987; Shenker et al., 1987) and the 5-HT~B (Bouhelal et al., submitted) modulate cyclic AMP levels. A third class of excitatory 5-HT receptors has been described in the peripheral nervous system: the 5-HT3 receptors (Richardson et al., 1985; Bradley et al., 1986). Their pharmacological and functional characterization has long been hampered by the absence of selective agonists and antagonists. These drugs are now available and include 2methyl-5-hydroxytryptamine, a good specific agonist and MDL 72222 or ICS 205 930 which are good selective antagonists (Richardson et al., 1985; Bradley et al., 1986). 5-HT3 receptors have never been described in the CNS. We now provide evidence that a 5-HT receptor found in colliculi neurons in primary culture and that mediates cyclic AMP stimulation, does not have the same pharmacological profile as a 5-HTa or a 5-HT2 receptor. The pharmacological characterization of this receptor suggests that it could correspond to a 5-HT3 receptor type. All the experiments described here were performed with neuronal cell cultures generated from 14- or 15-day-old mouse embryo colliculi. Neuro-
* To whom all correspondence should be addressed.
nal cells were cultured as described previously (Bockaert et al., 1987) and were grown for six days. Intracellular cAMP production was determined using the [3H]adenine prelabelling technique (Bockaert et al., 1987). In colliculi neurons, 5-HT stimulated cAMP production in a concentration-dependent manner with an ECs0 of 109 + 17 nM (n = 5). The percent conversion of [3H]ATP to [3H]cAMP in the absence of 5-HT was 0.8_ 0.1% (n = 5) whereas it was 2_+0.3% (n = 5) in the presence of 1 /~M 5-HT. Several high affinity antagonists interacting with the various classes of 5-HT receptors (5-HT 1, 5HT2 and 5-HT3) were used to determine the nature of this 5-HT receptor that is positively coupled to adenylate cyclase. The neurons were stimulated with 1 ~tM 5-HT (2- to 2.5-fold increase of basal conversion [ 3H]ATP --* [3H]cAMP) and increasing concentration of four antagonists: spiroperidol (a 5-HTIA and 5-HT2 antagonist), methiothepin (a 5-HT~ and 5-HT2 antagonist), ketanserin (a 5-HT2 antagonist) and ICS 205 930 (a 5-HT3 antagonist). Classical 5-HT antagonists (ketanserin and methiothepin) failed to antagonize the 5-HTstimulated cAMP production (fig. I B). At high concentrations (100 /~M) spiroperidol (a potent selective 5-HTIA and 5HT2 antagonist) slightly inhibited (18%, P < 0.001) the stimulatory effect of 1 pM 5-HT. In contrast, ICS 205 930, a potent and specific 5-HT3 antagonist in functional studies, inhibited the 5-HT-stimulated activity in a
0014-2999/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
188 r", lh3 _
/
~:~'C_
/i
/ / E
/
_ _';_ " , ' _
-\
\
• Netll ;t*ge;:lf' • ~e'dnSer;n
\i -~
:/ - t0g [ 5 kiT ]
- tog [Antag0mst]
Fig. 1. Stimulation by 5-HT of cAMP formation in mouse embryonic colliculi neurons. (A) Concentration-response curve for cAMP formation by 5-HT. Neurons in primary culture, generated from colliculi of 14- or 15-day-old mouse embryos were grown for 6 days in serum-free medium, in 12-well Costar dishes (106 cells/well) in a humidified atmosphere of 5% CO 2 and 95% 02 at 37°C. Before each experiment, the cells were washed and incubated with 2/~M of [3H]adenine (24/.tCi/mol, Amersham, U.K.) under the same culture conditions. The cultures were washed to remove free [3 H]adenine and the cells were then incubated with isobuthylmethylxanthine, 0.75 mM, forskolin, 0.1 /~M and increasing concentrations of 5-HT (10 nM to 100 /~M) in a 1 ml volume of culture medium. The reaction was stopped after 5 rain at 37 ° C by aspiration of the medium and addition of 1 ml ice-cold 5% trichloracetic acid. The cells were scraped with a rubber policeman; ATP, 5 mM and cAMP, 5 mM were then added to the mixture. Cellular protein was centrifuged at 5000× g. [~H]ATP and [3H]cAMP were separated by sequential chromatography on Dowex and alumina columns. Stimulation of cAMP formation is expressed as a percent stimulation above the basal activity level. The values are the means+ S.E.M. from five separate experiments, each performed with duplicate determinations. (B) Effect of 5-HT:, 5-HT2- and 5-HT3-related antagonists on 5-HT stimulation of cAMP formation in colliculi neurons. Neuronal cells were exposed in presence of forskolin, 0.1 btM and 5-HT, 1 I~M to increasing concentrations of methiothepin, ketanserin, spiroperidol and ICS 205 930. The results, expressed in percent stimulation over the basal activity level, are the means + S.E.M. from four separate experiments performed in duplicate. The basal and stimulated conversion of[ 3H]ATP to[ "~H]cAMP was 0.8 + 0.1 and 2 + 0.3% respectively. The differences between the values obtained in the presence of 10 -7 to 10 -4 M spiroperidol and the values for basal stimulation (1 /~M 5-HT) were compared using Student's t-test: * P < 0.02 compared to basal stimulation. * * P < 0.001.
concentration-dependent manner. The 5-HT effect was completely reversed only at high concentrations of ICS 205 930 (100 #M). An apparent K i value of 710 + 180 nM ( n - 4) was obtained for ICS 205 930 which was about 100-fold higher than the values obtained in functional studies with
guinea pig ileum (Richardson et al., 1985). ICS 205 930 competitively inhibited the 5-HT-induced cAMP production (data not shown). Our results indicate that this 5-HT receptor that is present in colliculi neurons is neither a 5-HT 1 nor a 5-HT2 receptor type. The effect of ICS 205 930 indicates that this receptor could belong to the 5-HT3 type according to the classification of Bradley et al. (1986). The difference between the pK, values for ICS 205 930 in our studies (6.15 +0.14) and the pA 2 values (7.7 to 13.5 depending on the systems tested) for its peripheral effects (Richardson et al., 1985) could be due to the existence of an embryonic form of 5-HT3 receptor in colliculi neurons. This idea is substantiated by our finding that ICS 205 930 potently antagonized (with a pK, of 7.6) the low affinity 5-HT receptor (RL) that mediates cAMP stimulation, and that was described by Shenker et al. (1987) in adult guinea pig hippocampal membranes (in preparation). In conclusion, the results presented here provide the first evidence for the existence of a 5-HT3 receptor type positively coupled to adenylate cyclase and located in the CNS. References Bockacrt, J., A. Dumuis, R. Bouhelal, M. Sebben and R.N. Cory, 1987, Piperazine derivatives including the putative anxiolytic drugs, buspirone and ipsapirone are agonists at 5-HTIA receptors negatively coupled with adenylate cyclase in hippocampal neurons, Naunyn-Schmiedeb. Arch. Pharmacol. 335, 588. Bradley, P.B., G. Engel, W. Feniuk, J.R. Fozard, P.P.A. Humphrey, D.N. Middlemiss, E.J. Mylecharane, B.P. Richardson and P.R. Saxena, 1986, Proposals for the classification and nomenclature of functional receptors for 5-hydroxytryptamine, Neuropharmacology 25, 563. De Vivo, M. and S. Maayani, 1986, Characterization of the 5-hydroxytryptanune~^ receptor mediated inhibition of forskolin-stimulated adenylate cyclase activity in guinea pig and rat hippocampal membranes, J. Pharmacol. Exp. Ther. 238, 248. Richardson, B.P., G. Engel, P. Donatsch and P.A. Stadler, 1985, Identification of serotonin M-receptor subtypes and their specific blockade by a new class of drugs, Nature 316, 126. Shenker, A., S. Maayani, H. Weinstein and J.P. Green. 1987. Pharmacological characterization of two 5-hydroxytryptamine receptors coupled to adenylate cyclase in guinea pig hippocampal membranes, Mol. Pharmacol. 31,357.