Characterization of a specific, high affinity [3H]arginine8 vasopressin-binding site on liver microsomes from different strains of rat and the role of magnesium

Venkat Gopalakrishnan, Chris R. Triggle, Prakash V. Sulakhe, J. Robert McNeill

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Abstract

A single class of high affinity, low capacity, specific binding sites for [3H]arginine8 vasopfessin (AVP) has been characterized in a plasma membrane-enriched microsomal fraction of the rat liver. Specific binding was saturable, linear with protein concentration, reversible, and 40–65% of the total binding. Binding at 25 C achieved a plateau after 30 min of incubation, whereas at 4 C, equilibrium was reached more slowly, and the level of binding was reduced. The presence of magnesium (Mg2+) in the assay medium enhanced the affinity of specific binding, while calcium and higher levels of sodium and potassium decreased binding. Scatchard analysis of binding in the presence of Mg2+ (5 mM) revealed an apparent mean ± SE equilibrium dissociation constant (Kd) of 0.29 ± 0.08 nM, with a maximal site density (Bmax) of 150.4 ± 25.0 fmol/mg; in contrast, the Kd was 1.93 ± 0.33 nM and the Bmax was 113.4 ± 40.0 fmol/mg in the absence of Mg2+. No significant differences in Kd and Bmax were observed among membrane fractions derived from spontaneously hypertensive rats, Wistar-Kyoto rats, Long-Evans rats, and Brattleboro rats. Plasma levels of AVP were similar in spontaneously hypertensive, Wistar-Kyoto, and Long-Evans rats, but AVP was not detectable in the plasma from DI rats. Competitive inhibition of specific [3H]AVP binding by unlabeled AVP and related peptides showed the following Ki values: AVP, 0.19 nM; LVP, 1.7 nM; oxytocin, 41.4 nM; desamino AVP, 0.38 nM; [1-(β-mercapto-β,β-cyclopentamethylene propionic acid) 4-Val,8-D-Arg]VP2-(O-methyl)tyrosine]AVP, 1.8 nM; desglycinamide AVP, 2.2 μM. The neuropeptide metabolite of AVP [[pGlu4, Cyt6] AVP-(4–9)], angiotensin II, and other unrelated peptides did not displace [3H] AVP, demonstrating the specificity of AVP and its related biologically active peptides for this binding site. Moreover, the rank order of potency for displacement of [3H]AVP binding by these various peptides parallels their reported glycogenolytic activity in liver and/or their agonistic or antagonistic potency in vascular smooth muscle. Finally, the Mg2+-induced increase in the affinity of [3H]AVP for this liver binding site is similar to the reported effect of Mg2+ on the contractile responses of vascular smooth muscle to AVP (i.e. increased affinity). The results are consistent with the interpretation that the high affinity receptor site characterized in rat liver microsomes is of the V1 type.

Original languageEnglish
Pages (from-to)990-997
Number of pages8
JournalEndocrinology
Volume118
Issue number3
DOIs
Publication statusPublished - Mar 1986

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ASJC Scopus subject areas

  • Endocrinology

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