Hepatic vasopressin receptor: Differential effects of divalent cations, guanine nucleotides, and n-ethylmaleimide on agonist and antagonist interactions with the v1 subtype receptor

Venkat Gopalakrishnan, J. Robert Mc Neill, Prakash V. Sulakhe, Christopher Triggle

Research output: Contribution to journalArticle

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Abstract

Previously, we reported that magnesium (Mg2+) enhanced the binding affinity of arginine vasopressin ([3H] AVP) to a single class of sites in rat liver microsomes. In the present study we have examined the effects of divalent cations and guanine nucleotides on the binding characteristics of both the nonselective agonist and the V1 receptor-selective antagonist, d(CH2)5Tyr(Me)-[3H]AVP, to microsomal and plasma membrane fractions of rat liver. At a subsaturating concentration (100 pM) of [3H]AVP, divalent cations increased specific binding in a concentration-dependent manner with the following rank order of potency: Co2+ > Mn2+ > Ni2+ > Mg2+ > Ca2+ = control. The maximal effect for Mg2+ was evident at 1 mM, a physiologically relevant concentration. In contrast, binding of the V1 receptor antagonist (at a subsaturating concentration of 10 pM) was inhibited by divalent cations, the rank order of potency being Mn2+ > Co2+ > Ca2+ > Mg2+ > Ni2+. The inhibitory effects of divalent cations were of lesser magnitude (up to 60%) compared to the stimulation of agonist binding (up to 700%). Mg2+ enhanced the affinity of [3H]AVP (Kd was decreased from -2 nM to 133 pM), while the affinity of the [3H]V1 antagonist was decreased (Kd was increased from 10 to 95 pM). Scatchard analysis of saturation data (Mg2+ present) revealed similar maximum binding values for the binding of radiolabeled agonist and antagonist, indicating that AVP receptors in rat liver are mostly of the V1 subtype. Competition experiments between V1/V2- specific AVP analogs with either the radiolabeled agonist or antagonist also indicated the presence of predominantly V1 receptor sites in rat liver microsomes. The properties of plasma membrane receptor sites were similar to those of the microsomal sites, except that the density of receptors was higher in the former. In both equilibrium and competitive inhibition experiments GTPase-resistant analogs of guanine nucleotides, GTPÎ3S and GDPÎ2S, decreased the affinity of the agonist for the receptor, but not that of the antagonist. Treatment of membranes with 0.2 mM N-ethylmaleimide (NEM) reduced the maximum binding of [3H]AVP and abolished the GTPÎ3S-evoked decrease in agonist-binding affinity. In contrast, antagonist binding was unaffected by NEM. NEM pretreatment failed to influence the divalent cation-dependent increase in agonist-binding affinity. The results provide direct evidence for the existence of a high and a low affinity state of the hepatic V1 receptor. These two affinity states appear to be differentially regulated by divalent cations and guanine nucleotides.

Original languageEnglish
Pages (from-to)922-931
Number of pages10
JournalEndocrinology
Volume123
Issue number2
DOIs
Publication statusPublished - 1 Aug 1988
Externally publishedYes

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Vasopressin Receptors
Ethylmaleimide
Guanine Nucleotides
Divalent Cations
Liver
Liver Microsomes
Cell Membrane
Arginine Vasopressin
GTP Phosphohydrolases
Magnesium
Membranes

ASJC Scopus subject areas

  • Endocrinology

Cite this

Hepatic vasopressin receptor : Differential effects of divalent cations, guanine nucleotides, and n-ethylmaleimide on agonist and antagonist interactions with the v1 subtype receptor. / Gopalakrishnan, Venkat; Mc Neill, J. Robert; Sulakhe, Prakash V.; Triggle, Christopher.

In: Endocrinology, Vol. 123, No. 2, 01.08.1988, p. 922-931.

Research output: Contribution to journalArticle

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abstract = "Previously, we reported that magnesium (Mg2+) enhanced the binding affinity of arginine vasopressin ([3H] AVP) to a single class of sites in rat liver microsomes. In the present study we have examined the effects of divalent cations and guanine nucleotides on the binding characteristics of both the nonselective agonist and the V1 receptor-selective antagonist, d(CH2)5Tyr(Me)-[3H]AVP, to microsomal and plasma membrane fractions of rat liver. At a subsaturating concentration (100 pM) of [3H]AVP, divalent cations increased specific binding in a concentration-dependent manner with the following rank order of potency: Co2+ > Mn2+ > Ni2+ > Mg2+ > Ca2+ = control. The maximal effect for Mg2+ was evident at 1 mM, a physiologically relevant concentration. In contrast, binding of the V1 receptor antagonist (at a subsaturating concentration of 10 pM) was inhibited by divalent cations, the rank order of potency being Mn2+ > Co2+ > Ca2+ > Mg2+ > Ni2+. The inhibitory effects of divalent cations were of lesser magnitude (up to 60{\%}) compared to the stimulation of agonist binding (up to 700{\%}). Mg2+ enhanced the affinity of [3H]AVP (Kd was decreased from -2 nM to 133 pM), while the affinity of the [3H]V1 antagonist was decreased (Kd was increased from 10 to 95 pM). Scatchard analysis of saturation data (Mg2+ present) revealed similar maximum binding values for the binding of radiolabeled agonist and antagonist, indicating that AVP receptors in rat liver are mostly of the V1 subtype. Competition experiments between V1/V2- specific AVP analogs with either the radiolabeled agonist or antagonist also indicated the presence of predominantly V1 receptor sites in rat liver microsomes. The properties of plasma membrane receptor sites were similar to those of the microsomal sites, except that the density of receptors was higher in the former. In both equilibrium and competitive inhibition experiments GTPase-resistant analogs of guanine nucleotides, GTP{\^I}3S and GDP{\^I}2S, decreased the affinity of the agonist for the receptor, but not that of the antagonist. Treatment of membranes with 0.2 mM N-ethylmaleimide (NEM) reduced the maximum binding of [3H]AVP and abolished the GTP{\^I}3S-evoked decrease in agonist-binding affinity. In contrast, antagonist binding was unaffected by NEM. NEM pretreatment failed to influence the divalent cation-dependent increase in agonist-binding affinity. The results provide direct evidence for the existence of a high and a low affinity state of the hepatic V1 receptor. These two affinity states appear to be differentially regulated by divalent cations and guanine nucleotides.",
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N2 - Previously, we reported that magnesium (Mg2+) enhanced the binding affinity of arginine vasopressin ([3H] AVP) to a single class of sites in rat liver microsomes. In the present study we have examined the effects of divalent cations and guanine nucleotides on the binding characteristics of both the nonselective agonist and the V1 receptor-selective antagonist, d(CH2)5Tyr(Me)-[3H]AVP, to microsomal and plasma membrane fractions of rat liver. At a subsaturating concentration (100 pM) of [3H]AVP, divalent cations increased specific binding in a concentration-dependent manner with the following rank order of potency: Co2+ > Mn2+ > Ni2+ > Mg2+ > Ca2+ = control. The maximal effect for Mg2+ was evident at 1 mM, a physiologically relevant concentration. In contrast, binding of the V1 receptor antagonist (at a subsaturating concentration of 10 pM) was inhibited by divalent cations, the rank order of potency being Mn2+ > Co2+ > Ca2+ > Mg2+ > Ni2+. The inhibitory effects of divalent cations were of lesser magnitude (up to 60%) compared to the stimulation of agonist binding (up to 700%). Mg2+ enhanced the affinity of [3H]AVP (Kd was decreased from -2 nM to 133 pM), while the affinity of the [3H]V1 antagonist was decreased (Kd was increased from 10 to 95 pM). Scatchard analysis of saturation data (Mg2+ present) revealed similar maximum binding values for the binding of radiolabeled agonist and antagonist, indicating that AVP receptors in rat liver are mostly of the V1 subtype. Competition experiments between V1/V2- specific AVP analogs with either the radiolabeled agonist or antagonist also indicated the presence of predominantly V1 receptor sites in rat liver microsomes. The properties of plasma membrane receptor sites were similar to those of the microsomal sites, except that the density of receptors was higher in the former. In both equilibrium and competitive inhibition experiments GTPase-resistant analogs of guanine nucleotides, GTPÎ3S and GDPÎ2S, decreased the affinity of the agonist for the receptor, but not that of the antagonist. Treatment of membranes with 0.2 mM N-ethylmaleimide (NEM) reduced the maximum binding of [3H]AVP and abolished the GTPÎ3S-evoked decrease in agonist-binding affinity. In contrast, antagonist binding was unaffected by NEM. NEM pretreatment failed to influence the divalent cation-dependent increase in agonist-binding affinity. The results provide direct evidence for the existence of a high and a low affinity state of the hepatic V1 receptor. These two affinity states appear to be differentially regulated by divalent cations and guanine nucleotides.

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