Vasopressin (V1) receptor characteristics in rat aortic smooth muscle cells

V. Gopalakrishnan, Y. Xu, P. V. Sulakhe, Christopher Triggle, J. R. McNeill

Research output: Contribution to journalArticle

31 Citations (Scopus)

Abstract

We report saturable, high-affinity, specific, reversible binding sites for both [3H]arginine vasopressin ([3H]AVP) and d(CH2)5Tyr(Me)-[3H]AVP, a V1-selective antagonist, in cultured smooth muscle cells obtained from rat aorta (RA) and rat mesenteric artery (RMA). Specific binding of [3H]AVP had the following characteristics in adherent monolayers of RA and RMA smooth muscle cells: dissociation constant (K(D)) = 1.42 and 1.23 nM and maximal binding capacity (B(max)) = 9,500 and 29,910 sites/cell, respectively. Lower K(D) and higher B(max) values were detected for 3H-labeled V1 antagonist binding to both types of cells. Complete dissociation of [3H]AVP binding to RA cells occurred in a biphasic manner with two rate constants of dissociation, suggesting high- and low-affinity states of the binding site for the agonist interaction. Partial dissociation of the antagonist-specific binding occurred, and it was monophasic, suggesting interaction of the 3H-labeled V1 antagonist radioligand to the high-affinity binding state. Inhibition constant (K(i)) values determined by competitive inhibition of [3H]AVP binding to RA cells by a series of AVP-related peptide analogues and antagonists were consistent with the saturation data. AVP in a concentration-dependent manner induced the accumulation of inositol phosphates [mean effective concentration (EC50) 1 nM] in the adherent RA cells. The free cytosolic Ca2+ level in the dispersed RA smooth muscle cells was increased by AVP (EC50 8.1 nM). Pretreatment with the V1 antagonist abolished these AVP-evoked responses. The data support the conclusion that the agonist binding occurs at a homogeneous population of V1-subtype receptors in the high-affinity (K(D)) = ~1 nM) state and that these receptors are functionally coupled to phospholipase C.

Original languageEnglish
JournalAmerican Journal of Physiology - Heart and Circulatory Physiology
Volume261
Issue number6 30-6
Publication statusPublished - 1991
Externally publishedYes

Fingerprint

Vasopressin Receptors
Smooth Muscle Myocytes
Aorta
Mesenteric Arteries
Binding Sites
Inositol Phosphates
Arginine Vasopressin
Type C Phospholipases
Peptides

Keywords

  • Angiotensin II
  • Endothelin-1
  • Fura-2 fluorescence
  • Inositol phosphates
  • Intracellular calcium ion concentration
  • Vascular smooth muscle cells
  • Vasopressin analogues and antagonists

ASJC Scopus subject areas

  • Physiology
  • Medicine(all)

Cite this

Vasopressin (V1) receptor characteristics in rat aortic smooth muscle cells. / Gopalakrishnan, V.; Xu, Y.; Sulakhe, P. V.; Triggle, Christopher; McNeill, J. R.

In: American Journal of Physiology - Heart and Circulatory Physiology, Vol. 261, No. 6 30-6, 1991.

Research output: Contribution to journalArticle

@article{1a33b1b94aa843febc026a63b977dbc7,
title = "Vasopressin (V1) receptor characteristics in rat aortic smooth muscle cells",
abstract = "We report saturable, high-affinity, specific, reversible binding sites for both [3H]arginine vasopressin ([3H]AVP) and d(CH2)5Tyr(Me)-[3H]AVP, a V1-selective antagonist, in cultured smooth muscle cells obtained from rat aorta (RA) and rat mesenteric artery (RMA). Specific binding of [3H]AVP had the following characteristics in adherent monolayers of RA and RMA smooth muscle cells: dissociation constant (K(D)) = 1.42 and 1.23 nM and maximal binding capacity (B(max)) = 9,500 and 29,910 sites/cell, respectively. Lower K(D) and higher B(max) values were detected for 3H-labeled V1 antagonist binding to both types of cells. Complete dissociation of [3H]AVP binding to RA cells occurred in a biphasic manner with two rate constants of dissociation, suggesting high- and low-affinity states of the binding site for the agonist interaction. Partial dissociation of the antagonist-specific binding occurred, and it was monophasic, suggesting interaction of the 3H-labeled V1 antagonist radioligand to the high-affinity binding state. Inhibition constant (K(i)) values determined by competitive inhibition of [3H]AVP binding to RA cells by a series of AVP-related peptide analogues and antagonists were consistent with the saturation data. AVP in a concentration-dependent manner induced the accumulation of inositol phosphates [mean effective concentration (EC50) 1 nM] in the adherent RA cells. The free cytosolic Ca2+ level in the dispersed RA smooth muscle cells was increased by AVP (EC50 8.1 nM). Pretreatment with the V1 antagonist abolished these AVP-evoked responses. The data support the conclusion that the agonist binding occurs at a homogeneous population of V1-subtype receptors in the high-affinity (K(D)) = ~1 nM) state and that these receptors are functionally coupled to phospholipase C.",
keywords = "Angiotensin II, Endothelin-1, Fura-2 fluorescence, Inositol phosphates, Intracellular calcium ion concentration, Vascular smooth muscle cells, Vasopressin analogues and antagonists",
author = "V. Gopalakrishnan and Y. Xu and Sulakhe, {P. V.} and Christopher Triggle and McNeill, {J. R.}",
year = "1991",
language = "English",
volume = "261",
journal = "American Journal of Physiology",
issn = "0363-6135",
publisher = "American Physiological Society",
number = "6 30-6",

}

TY - JOUR

T1 - Vasopressin (V1) receptor characteristics in rat aortic smooth muscle cells

AU - Gopalakrishnan, V.

AU - Xu, Y.

AU - Sulakhe, P. V.

AU - Triggle, Christopher

AU - McNeill, J. R.

PY - 1991

Y1 - 1991

N2 - We report saturable, high-affinity, specific, reversible binding sites for both [3H]arginine vasopressin ([3H]AVP) and d(CH2)5Tyr(Me)-[3H]AVP, a V1-selective antagonist, in cultured smooth muscle cells obtained from rat aorta (RA) and rat mesenteric artery (RMA). Specific binding of [3H]AVP had the following characteristics in adherent monolayers of RA and RMA smooth muscle cells: dissociation constant (K(D)) = 1.42 and 1.23 nM and maximal binding capacity (B(max)) = 9,500 and 29,910 sites/cell, respectively. Lower K(D) and higher B(max) values were detected for 3H-labeled V1 antagonist binding to both types of cells. Complete dissociation of [3H]AVP binding to RA cells occurred in a biphasic manner with two rate constants of dissociation, suggesting high- and low-affinity states of the binding site for the agonist interaction. Partial dissociation of the antagonist-specific binding occurred, and it was monophasic, suggesting interaction of the 3H-labeled V1 antagonist radioligand to the high-affinity binding state. Inhibition constant (K(i)) values determined by competitive inhibition of [3H]AVP binding to RA cells by a series of AVP-related peptide analogues and antagonists were consistent with the saturation data. AVP in a concentration-dependent manner induced the accumulation of inositol phosphates [mean effective concentration (EC50) 1 nM] in the adherent RA cells. The free cytosolic Ca2+ level in the dispersed RA smooth muscle cells was increased by AVP (EC50 8.1 nM). Pretreatment with the V1 antagonist abolished these AVP-evoked responses. The data support the conclusion that the agonist binding occurs at a homogeneous population of V1-subtype receptors in the high-affinity (K(D)) = ~1 nM) state and that these receptors are functionally coupled to phospholipase C.

AB - We report saturable, high-affinity, specific, reversible binding sites for both [3H]arginine vasopressin ([3H]AVP) and d(CH2)5Tyr(Me)-[3H]AVP, a V1-selective antagonist, in cultured smooth muscle cells obtained from rat aorta (RA) and rat mesenteric artery (RMA). Specific binding of [3H]AVP had the following characteristics in adherent monolayers of RA and RMA smooth muscle cells: dissociation constant (K(D)) = 1.42 and 1.23 nM and maximal binding capacity (B(max)) = 9,500 and 29,910 sites/cell, respectively. Lower K(D) and higher B(max) values were detected for 3H-labeled V1 antagonist binding to both types of cells. Complete dissociation of [3H]AVP binding to RA cells occurred in a biphasic manner with two rate constants of dissociation, suggesting high- and low-affinity states of the binding site for the agonist interaction. Partial dissociation of the antagonist-specific binding occurred, and it was monophasic, suggesting interaction of the 3H-labeled V1 antagonist radioligand to the high-affinity binding state. Inhibition constant (K(i)) values determined by competitive inhibition of [3H]AVP binding to RA cells by a series of AVP-related peptide analogues and antagonists were consistent with the saturation data. AVP in a concentration-dependent manner induced the accumulation of inositol phosphates [mean effective concentration (EC50) 1 nM] in the adherent RA cells. The free cytosolic Ca2+ level in the dispersed RA smooth muscle cells was increased by AVP (EC50 8.1 nM). Pretreatment with the V1 antagonist abolished these AVP-evoked responses. The data support the conclusion that the agonist binding occurs at a homogeneous population of V1-subtype receptors in the high-affinity (K(D)) = ~1 nM) state and that these receptors are functionally coupled to phospholipase C.

KW - Angiotensin II

KW - Endothelin-1

KW - Fura-2 fluorescence

KW - Inositol phosphates

KW - Intracellular calcium ion concentration

KW - Vascular smooth muscle cells

KW - Vasopressin analogues and antagonists

UR - http://www.scopus.com/inward/record.url?scp=0026294048&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0026294048&partnerID=8YFLogxK

M3 - Article

C2 - 1836312

AN - SCOPUS:0026294048

VL - 261

JO - American Journal of Physiology

JF - American Journal of Physiology

SN - 0363-6135

IS - 6 30-6

ER -