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5-HT1D receptor-mediated inhibition of forskolin-stimulated adenylate cyclase activity in calf substantia nigra
European Journal of Pharmacology, 147 (1988) 145-147
145
Elsevier EJP 0107R
Rapid communication
5-HT o receptor-mediated inhibition of Iorskolin-stimulated adenylate cyclase activity in calf substantia nigra Daniel Hoyer * and P~llppe Schoeffter Prechmcal Research, 386/525, Sandoz Ltd, CH-4002 Basel, Swztzerland
Received 18 January 1988, accepted 19 January 1988
[3H]5-HT (5-hydroxytryptamlne, serotonln) labels at least four different 5-HT 1 recogmtlon sites, designated 5-HTIA, 5-HT1B, 5-HTlc and 5HT1D, m brain membranes of various species (Hoyer et al., 1985; Heurlng and Peroutka, 1987). In terms of second messengers, activation of 5HTIA receptors may result in either sUmulaUon or mtubxUon of adenylate cyclase actlvaty in rat and guinea pig tuppocampus (De Vlvo and Maayanx, 1986). 5-HTIB receptors are negatwely coupled to adenylate cyclase m rat substantla mgra (Bouhelal et al, submitted) and 5-HT~c receptor actwatlon leads to sumulatlon of phosphohpase C m rat chorold plexus 5-HTIA and 5-HTlc sites have been ldenUfied m various species including man. In contrast, 5HT~B recogmUon sites appear to be specific to rat and mouse whereas 5-HTID sites are found in bovine, pig and human brain membranes, which seem to be devoid of 5-HTIB sites (Hoyer et al., 1985; 1986, Heurlng and Peroutka, 1987; Hoyer et al, in press) At present, no funcUonal correlate of the 5-HT~D binding site has been identified We now report that activation of 5-HT~D receptors results in inhibition of f o r s k o h n - s u m u l a t e d adenylate cyclase activity in homogenates of calf substantm mgra. Calf brains were obtained from a local slaughterhouse and were kept on ~ce until the Ume when
* To whom all correspondence should be addressed
the substantla mgra was dissected and transferred to 8 volumes of ice-cold Tns-sucrose buffer (comp o s m o n in mM: Trls-HC1 20, sucrose 300, E G T A 1, N a 2 E D T A 5 and dlthlothreltol 5, p H 7 4) The tissues were then minced and homogemzed by hand with a glass/glass Potter apparatus The pellet was d~scarded after a first centrlfugauon at about 50 × g for 5 mln and the supernatant was centrifuged again at 40000 x g for 10 nun. The pellet from this second centrlfugatlon was resuspended m 4 volumes of Tns-sucrose buffer and 1 ml ahquots were stored at - 7 0 ° C until used Adenylate cyclase actxwty was determined by measuring the formation of [32p]cychc A M P from a-[32p]ATP as described by DeVlvo and M a a y a m (1986) with some minor modifications. The xncubatlon m e d m m contained Trls-HC1 80 m M (pH 7 4), M g A T P 0 1 mM, MgC12 2 mM, G T P 10/~M, cychc A M P 1 mM, NaC1 100 mM, 3-1sobutyl-1methylxanthlne 2 mM, ascorblc acid 0 25 mM, phosphocreatme 5 mM, creatme phosphoklnase 0 2 m g / m l a-[32p]ATP (30 C l / m m o l ) about 1 /~C1/assay and the substances as indicated The reaction was started by the a d d m o n of membrane protems (about 30/~g) to the incubation medium, after a 2-mln equlhbrauon period at 30 o C. The assays were run m tnphcate for 5 nun at 30 ° C in a final volume of 200 /~1 Concentration-effect curves were analysed with a non-linear regression computer program (Hoyer et al., 1985). Emax,ECs0 and K B values were derived from this analysis The results are gtven as means + S.E M of 3-9 independent expenments
0014-2999/88/$03 50 © 1988 Elsevier Science Pubhshers B V (BlomedacalDlwslon)
146
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i
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10
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8
7
i
i
6
5
4
[AGONIST] (-log mol/1)
Fig 1 The mlubltion of forskohn-stlmulated adenylate cyclase actlvaty was measured in homogenates of calf substanua mgra with 5-HT (O) and 5-CT (A) or 5-HT in the presence of 10/~M methaothepm (D) The curves were fitted by non-linear regression The data are expressed as % maximal mlubmon of adenylate cyclase activity and are representative of 3-9 independent experiments
Forskohn (0.1-100 ~tM) stimulated adenylate cyclase act~vaty concentration dependently in homogenates of calf substantm mgra. At 10 /~M, forskohn increased the basal adenylate cyclase actlwty 6-7 fold. The effect developed with time (up to 10 nun) and was dependent on the protein concentration (up to 45 /~g per assay) (data not shown). The effects of agomsts and antagonists were studied in the presence of 10 /~M forskohn 5-HT caused a concentration-dependent inhibition of forskohn-stlmulated adenylate cyclase activity (fig. 1): Emax = 23 3 _ 1 3%, pECs0 = 7.56 _ 0.24. The effect of 5-HT was dependent on the presence of GTP (10/~M) since 5-HT was wrtually lneffectwe in the absence of GTP (data not shown). 5-Carboxarnldotrytamme (5-CT) mlrmcked the effects of 5-HT" Emax = 25.8 _ 0.8% inhibition, pECs0 7.71 + 0.14 (fig. 1) whereas the potent and selective 5-HTIA receptor agonlst 8-OH-DPAT had no effect at 100 nmol/1. MethlothepIn (10/~M) antagomsed the 5-HT effect on adenylate cyclase surmountably, and a p K a value of 7.00 + 0.10 was calculated (fig 1). In contrast, the potent 5-HTIA/ 5-HT1B receptor hgand, cyanopmdolol, chd not antagomse the effects of 5-HT at 1/xM (data not shown) The 5 - H T I A / 5 - H T2 receptor antagomst splperone and the 5 - H T l c / 5 - H T 2 receptor antagonist rmanserln were also devoid of sigmfl-
cant effects on 5-HT-lnduced inhibition of adenylate cyclase activity at 0.1/LM (data not shown). The lack of antagomsm by cyanoplndolol of 5-HT-lnduced inhibition of forskohn-stlmulated adenylate cyclase activity rules out a 5-HT1B receptor-mediated effect, and the lack of activity of 8-OH-DPAT, splperone and mlanserln suggests that 5-HT1A, 5-HTac or 5-HT 2 receptors are not responsible for the effects described The receptor can nevertheless be classified as being of the 5-HT 1 type, since 5-HT and 5-CT act as potent agonlsts and since the effects of 5-HT are antagomsed by methlothepm The pharmacological characteristics of the effects described here are similar to the pharmacological profile of the 5-HTID sites described by H e u n n g and Peroutka (1987) m bovine brain membranes, which suggests that a 5-HTID receptor mediates the inhibition of adenylate cyclase acUvlty m calf substantla nlgra. This suggestion receives further support from the resuits of radiohgand binding studies in which 5HT1D but not 5-HTIA, 5-HT1B or 5-HTlc recognition sites could be documented in calf substantla mgra (Waeber et al, submitted). Thus, there is a close parallel between the 5HT1D receptors of calf substantia mgra and the 5-HTIB sites of rat substantla mgra since in each case the site is of the predolnlnant subtype (Pazos and Palaclos, 1985) and is hnked negatively to adenylate cyclase.
Acknowledgment We thank J R Fozard for critical reading of the manuscript
References De Vwo, M and S Maayam, 1986, Characterization of 5-hydroxytryptamlnelA-receptor-medlated mlubxtlon of forskolm-stlrnulated adenylate cyclase activity m guinea-pig and rat bappocampalmembranes, J Pharmacol Exp Ther 238, 248 Heunng, R E and S J Peroutka, 1987, Charactertzatlon of a novel 3H-5-hydroxytryptamme binding site subtype m bovine brain membranes, J Neuroscl 7, 894 Hoyer, D, G Engel and H O Kalkman, 1985, Molecular pharmacologyof 5-HT1 and 5-HT2 recogmUonsites m rat and pig brain membranes radiohgandbinding studies vath
147 [3H]5-HT, [3H]8-OH-DPAT, (-)[125I]lodocyanopmdolol, [3H]mesulergme and [3H]ketansenn, European J Pharmacol 118, 13 Hoyer, D , A Pazos, A Probst and J M Palaclos, 1986, Serotonm receptors m the human brmn I Characterization and autora&ographlc locahzatlon of 5-HT1A recogmtlon
sites Apparent absence of 5-HT1B recogmtlon sites, Brain Res 376, 85 Pazos, A and J M Palaclos, 1985, Quantitative autora&ographic mapping of serotonln receptors in the rat brmn, I Serotomn-1 receptors, Brain Res 346, 205
145
Elsevier EJP 0107R
Rapid communication
5-HT o receptor-mediated inhibition of Iorskolin-stimulated adenylate cyclase activity in calf substantia nigra Daniel Hoyer * and P~llppe Schoeffter Prechmcal Research, 386/525, Sandoz Ltd, CH-4002 Basel, Swztzerland
Received 18 January 1988, accepted 19 January 1988
[3H]5-HT (5-hydroxytryptamlne, serotonln) labels at least four different 5-HT 1 recogmtlon sites, designated 5-HTIA, 5-HT1B, 5-HTlc and 5HT1D, m brain membranes of various species (Hoyer et al., 1985; Heurlng and Peroutka, 1987). In terms of second messengers, activation of 5HTIA receptors may result in either sUmulaUon or mtubxUon of adenylate cyclase actlvaty in rat and guinea pig tuppocampus (De Vlvo and Maayanx, 1986). 5-HTIB receptors are negatwely coupled to adenylate cyclase m rat substantla mgra (Bouhelal et al, submitted) and 5-HT~c receptor actwatlon leads to sumulatlon of phosphohpase C m rat chorold plexus 5-HTIA and 5-HTlc sites have been ldenUfied m various species including man. In contrast, 5HT~B recogmUon sites appear to be specific to rat and mouse whereas 5-HTID sites are found in bovine, pig and human brain membranes, which seem to be devoid of 5-HTIB sites (Hoyer et al., 1985; 1986, Heurlng and Peroutka, 1987; Hoyer et al, in press) At present, no funcUonal correlate of the 5-HT~D binding site has been identified We now report that activation of 5-HT~D receptors results in inhibition of f o r s k o h n - s u m u l a t e d adenylate cyclase activity in homogenates of calf substantm mgra. Calf brains were obtained from a local slaughterhouse and were kept on ~ce until the Ume when
* To whom all correspondence should be addressed
the substantla mgra was dissected and transferred to 8 volumes of ice-cold Tns-sucrose buffer (comp o s m o n in mM: Trls-HC1 20, sucrose 300, E G T A 1, N a 2 E D T A 5 and dlthlothreltol 5, p H 7 4) The tissues were then minced and homogemzed by hand with a glass/glass Potter apparatus The pellet was d~scarded after a first centrlfugauon at about 50 × g for 5 mln and the supernatant was centrifuged again at 40000 x g for 10 nun. The pellet from this second centrlfugatlon was resuspended m 4 volumes of Tns-sucrose buffer and 1 ml ahquots were stored at - 7 0 ° C until used Adenylate cyclase actxwty was determined by measuring the formation of [32p]cychc A M P from a-[32p]ATP as described by DeVlvo and M a a y a m (1986) with some minor modifications. The xncubatlon m e d m m contained Trls-HC1 80 m M (pH 7 4), M g A T P 0 1 mM, MgC12 2 mM, G T P 10/~M, cychc A M P 1 mM, NaC1 100 mM, 3-1sobutyl-1methylxanthlne 2 mM, ascorblc acid 0 25 mM, phosphocreatme 5 mM, creatme phosphoklnase 0 2 m g / m l a-[32p]ATP (30 C l / m m o l ) about 1 /~C1/assay and the substances as indicated The reaction was started by the a d d m o n of membrane protems (about 30/~g) to the incubation medium, after a 2-mln equlhbrauon period at 30 o C. The assays were run m tnphcate for 5 nun at 30 ° C in a final volume of 200 /~1 Concentration-effect curves were analysed with a non-linear regression computer program (Hoyer et al., 1985). Emax,ECs0 and K B values were derived from this analysis The results are gtven as means + S.E M of 3-9 independent expenments
0014-2999/88/$03 50 © 1988 Elsevier Science Pubhshers B V (BlomedacalDlwslon)
146
--
°
==
i
11
10
9
8
7
i
i
6
5
4
[AGONIST] (-log mol/1)
Fig 1 The mlubltion of forskohn-stlmulated adenylate cyclase actlvaty was measured in homogenates of calf substanua mgra with 5-HT (O) and 5-CT (A) or 5-HT in the presence of 10/~M methaothepm (D) The curves were fitted by non-linear regression The data are expressed as % maximal mlubmon of adenylate cyclase activity and are representative of 3-9 independent experiments
Forskohn (0.1-100 ~tM) stimulated adenylate cyclase act~vaty concentration dependently in homogenates of calf substantm mgra. At 10 /~M, forskohn increased the basal adenylate cyclase actlwty 6-7 fold. The effect developed with time (up to 10 nun) and was dependent on the protein concentration (up to 45 /~g per assay) (data not shown). The effects of agomsts and antagonists were studied in the presence of 10 /~M forskohn 5-HT caused a concentration-dependent inhibition of forskohn-stlmulated adenylate cyclase activity (fig. 1): Emax = 23 3 _ 1 3%, pECs0 = 7.56 _ 0.24. The effect of 5-HT was dependent on the presence of GTP (10/~M) since 5-HT was wrtually lneffectwe in the absence of GTP (data not shown). 5-Carboxarnldotrytamme (5-CT) mlrmcked the effects of 5-HT" Emax = 25.8 _ 0.8% inhibition, pECs0 7.71 + 0.14 (fig. 1) whereas the potent and selective 5-HTIA receptor agonlst 8-OH-DPAT had no effect at 100 nmol/1. MethlothepIn (10/~M) antagomsed the 5-HT effect on adenylate cyclase surmountably, and a p K a value of 7.00 + 0.10 was calculated (fig 1). In contrast, the potent 5-HTIA/ 5-HT1B receptor hgand, cyanopmdolol, chd not antagomse the effects of 5-HT at 1/xM (data not shown) The 5 - H T I A / 5 - H T2 receptor antagomst splperone and the 5 - H T l c / 5 - H T 2 receptor antagonist rmanserln were also devoid of sigmfl-
cant effects on 5-HT-lnduced inhibition of adenylate cyclase activity at 0.1/LM (data not shown). The lack of antagomsm by cyanoplndolol of 5-HT-lnduced inhibition of forskohn-stlmulated adenylate cyclase activity rules out a 5-HT1B receptor-mediated effect, and the lack of activity of 8-OH-DPAT, splperone and mlanserln suggests that 5-HT1A, 5-HTac or 5-HT 2 receptors are not responsible for the effects described The receptor can nevertheless be classified as being of the 5-HT 1 type, since 5-HT and 5-CT act as potent agonlsts and since the effects of 5-HT are antagomsed by methlothepm The pharmacological characteristics of the effects described here are similar to the pharmacological profile of the 5-HTID sites described by H e u n n g and Peroutka (1987) m bovine brain membranes, which suggests that a 5-HTID receptor mediates the inhibition of adenylate cyclase acUvlty m calf substantla nlgra. This suggestion receives further support from the resuits of radiohgand binding studies in which 5HT1D but not 5-HTIA, 5-HT1B or 5-HTlc recognition sites could be documented in calf substantla mgra (Waeber et al, submitted). Thus, there is a close parallel between the 5HT1D receptors of calf substantia mgra and the 5-HTIB sites of rat substantla mgra since in each case the site is of the predolnlnant subtype (Pazos and Palaclos, 1985) and is hnked negatively to adenylate cyclase.
Acknowledgment We thank J R Fozard for critical reading of the manuscript
References De Vwo, M and S Maayam, 1986, Characterization of 5-hydroxytryptamlnelA-receptor-medlated mlubxtlon of forskolm-stlrnulated adenylate cyclase activity m guinea-pig and rat bappocampalmembranes, J Pharmacol Exp Ther 238, 248 Heunng, R E and S J Peroutka, 1987, Charactertzatlon of a novel 3H-5-hydroxytryptamme binding site subtype m bovine brain membranes, J Neuroscl 7, 894 Hoyer, D, G Engel and H O Kalkman, 1985, Molecular pharmacologyof 5-HT1 and 5-HT2 recogmUonsites m rat and pig brain membranes radiohgandbinding studies vath
147 [3H]5-HT, [3H]8-OH-DPAT, (-)[125I]lodocyanopmdolol, [3H]mesulergme and [3H]ketansenn, European J Pharmacol 118, 13 Hoyer, D , A Pazos, A Probst and J M Palaclos, 1986, Serotonm receptors m the human brmn I Characterization and autora&ographlc locahzatlon of 5-HT1A recogmtlon
sites Apparent absence of 5-HT1B recogmtlon sites, Brain Res 376, 85 Pazos, A and J M Palaclos, 1985, Quantitative autora&ographic mapping of serotonln receptors in the rat brmn, I Serotomn-1 receptors, Brain Res 346, 205