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5-HT-stimulated [35S]guanosine-5′-O-(3-thio)triphosphate binding as an assay for functional activation of G proteins coupled with 5-HT1B receptors in rat striatal membranes

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Abstract

Receptor-mediated guanine nucleotide-binding regulatory protein (G protein) activation or functional coupling between receptors and G proteins has been investigated by means of agonist-induced [35S]guanosine-5′-O-(3-thio)triphosphate ([35S]GTPγS) binding, especially for the receptor subtypes negatively coupled to adenylyl cyclase through Gi type G proteins. In the present study, 5-HT-stimulated [35S]GTPγS binding to rat stritatal membranes was pharmacologically characterized in detail with the help of an extensive series of 5-HT receptor ligands. The optimum experimental conditions for the concentrations of GDP, MgCl2 and NaCl in the assay buffer were initially determined, and the standard assay was performed with 20 μM GDP, 5 mM MgCl2 and 100 mM NaCl. The specific [35S]GTPγS binding was stimulated by several compounds that had been shown to be agonists at 5-HT1B/1D receptors. The negative logarithmic values of the concentration eliciting half-maximal effect (pEC50) for these agonists were significantly correlated with their pK i’s reported in the previous study of 5-HT1B receptor binding in rat frontal cortical membranes. The increase in specific [35S]GTPγS binding in response to 1 μM 5-HT was potently inhibited by several 5-HT1B/1D receptor antagonists as well as β-adrenoceptor antagonists such as S(−)-cyanopindolol. On the other hand, 3-[4-(4-chlorophenyl)piperazin-1-yl]-1,1-diphenyl-2-propanol HCl (BRL15572), a selective antagonist against human 5-HT1D receptors, was inactive as an antagonist at least up to 1 μM. Additionally, the concentration-response curve for 2-[5-[3-(4-methylsulphonylamino)benzyl-1,2,4-oxadiazol-5-yl]-1H-indol-3-yl]ethanamine (L694247) was shifted rightward in parallel by the addition of S(−)-cyanopindolol at concentrations of 10 and 100 nM, indicative of the competitive inhibitory manner. The specific [35S]GTPγS binding was reduced by 1′-methyl-5-([2′-methyl-4′-(5-methyl-1,2,4-oxadiazol-3-yl)biphenyl-4-yl]carbonyl)-2,3,6,7-tetrahydrospirospiro(furo[2,3-f]indole-3,4′-piperidine) (SB224289) and methiothepin in a concentration-dependent manner. The inhibitory curve by either compound was shifted to the right by 10 and 100 nM S(−)-cyanopindolol, suggesting that these two drugs behaved as inverse agonists at 5-HT1B receptors in the present functional assay system. 5-HT-stimulated [35S]GTPγS binding to rat striatal membranes serves as a simple but useful method of investigating the functional interaction between the native 5-HT1B receptors and their coupled G proteins in this brain region.

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Abbreviations

Anpirtoline:

6-chloro-2[piperidinyl-4-thio]pyridine HCl

BMY7378:

8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4.5]decane-7,9-dione 2HCl

BRL15572:

3-[4-(4-chlorophenyl)piperazin-1-yl]-1,1-diphenyl-2-propanol HCl

BRL54443:

3-(1-methylpiperidine-4-yl)-1H-indol-5-ol maleate salt

CGS12066A:

7-trifluroromethyl-4(4-methyl-1-piperazinyl)-pyrrolo-[1,2-a] quinoxaline maleate salt

CHO:

Chinese hamster ovary

CP93129:

1,4-dihydro-3-(1,2,3,6-tetrahydro-4-pyridinyl)-5H-pyrrol[3,2-b]pyridin-5-one 2HCl

CP94253:

5-propoxy-3-(1,2,3,6-tetrahydro-4-pyridinyl)-1H-pyrrolo[3,2-b]pyridine HCl

5-CT:

5-carboxamidotryptamine maleate

(±)-DOI:

(±)-2,5-dimethoxy-4-iodoamphetamine HCl

EC50 :

the concentration eliciting half-maximal effect

G protein:

guanine nucleotide-binding regulatory protein

GR46611:

3-[3-(2-dimethylaminoethyl)-1H-indol-5-yl]-N-(4-methoxybenzyl)acrylamide

GR55562:

3-[3-(dimethylamino)propyl]-4-hydroxy-N-[4-(4-pyridinyl)phenyl]benzamide 2HCl

GR125743:

N-[4-methoxy-3-(4-methylpiperazin-1-yl)phenyl]-3-methyl-4-(4-pyridyl)-benzamide

GR127935:

N-[4-methoxy-3-(4-methyl-1-piperazinyl)phenyl]-2′-methyl-4′-(5-methyl-1,2,4-oxadiazol-3-yl)-1,1′-biphenyl-4-carboxamide HCl

GTPγS:

guanosine-5′-O-(3-thio)triphosphate

IC50 :

the concentration eliciting half-maximal inhibition

L694247:

2-[5-[3-(4-methylsulphonylamino)benzyl-1,2,4-oxadiazol-5-yl]-1H-indol-3-yl]ethanamine

LSD:

lysergic acid diethylamide

LY278584:

1-methyl-N-(8-methyl-8-azabicyclo[3.2.1]-oct-3-yl)-1H-indazole-3-carboxamide maleate

mCPP:

1-(3-chlorophenyl)piperazine HCl

5-MeO-N,N-DMT:

5-methoxy-N,N-dimethyltryptamine

5-MeOT:

5-methoxytryptamine HCl

N-methylquipazine:

2-[1-(4-methyl)piperazinyl]quinoline dimaleate salt

N,N-DP-5CT:

N,N-dipropyl-5-carboxamidotryptamine maleate

8-OH-DPAT:

8-hydroxy-2-(di-n-propylamino)tetralin hydrobromide

PAPP:

p-aminophenyl-m-trifluoromethylphenyl piperazine

pEC50 :

the negative logarithmic value of the EC50

RU24969:

5-methoxy-3-(1,2,5,6-tetrahydro-4-pyridinyl)-1H-indole hemisuccinate

SB224289:

1′-methyl-5-([2′-methyl-4′-(5-methyl-1,2,4-oxadiazol-3-yl)biphenyl-4-yl]carbonyl)-2,3,6,7-tetrahydrospirospiro(furo[2,3-f]indole-3,4(-piperidine)

TFMPP:

N-(3-trifluoromethylphenyl)piperazine HCl

WAY100635:

N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinylcyclohexanecarboxamide maleate salt

WB4101:

2-(2,6-dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane HCl

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Acknowledgements

The authors thank Mr. Masakazu Kinoshita for his technical assistance. This work was supported by a Grant-in-Aid for Scientific Research (C) (No. 13671030) (to Y.O.) from the Japan Society for the Promotion of Science.

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  1. Department of Psychiatry, Saitama Medical School, 38 Morohongo, Moroyama-machi, Iruma-gun, Staitama, 350-0495, Japan

    Yuji Odagaki & Ryoichi Toyoshima

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  1. Yuji Odagaki
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  2. Ryoichi Toyoshima
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Correspondence to Yuji Odagaki.

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Odagaki, Y., Toyoshima, R. 5-HT-stimulated [35S]guanosine-5′-O-(3-thio)triphosphate binding as an assay for functional activation of G proteins coupled with 5-HT1B receptors in rat striatal membranes. Naunyn Schmied Arch Pharmacol 372, 335–345 (2006). https://doi.org/10.1007/s00210-006-0041-x

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