VASOPRESSIN INCREASES CYTOSOLIC FREE [CA2+] IN THE NEONATAL RAT CARDIOMYOCYTE - EVIDENCE FOR V1-SUBTYPE RECEPTORS

Radioligand binding studies of the cardiac arginine vasopressin (AVP) receptor, together with studies on the AVP-evoked alterations in the [Ca2+]i levels, were undertaken using primary cultures of neonatal rat cardiomyocytes. Rapid, reversible, specific, high-affinity and low-capacity binding sites were detected for the agonist, [H-3]AVP, and the V1 selective antagonist, d(CH2)5 Tyr (Me)-[H-3]AVP (V1 antagonist), radioligands. The V2 selective antagonist radioligand, d(CH2)5 D-Ile Ile des-Gly NH2-[H-3]AVP, showed very little binding even at very high concentrations. [H-3]AVP and [H-3]V1 antagonist specific binding attained equilibrium in 10 minutes at 37-degrees-C. The K(d) and B(max) values (mean +/- SEM) were [H-3]AVP: K(d) 1.44 +/- 0.18 nM; B(max) 5,253 +/- 590 sites/cell; [H-3]V1 antagonist: K(d) 0.96 +/- 0.10 nM; B(max) 6,869 +/- 485 sites/cell. K(i) values for a series of AVP-related peptide analogues and antagonists determined by competitive inhibition of [H-3]AVP binding were consistent with the saturation data. The results suggest that these cells possess a homogeneous population of V1 subtype AVP receptors. AVP increased [Ca2+]i in a concentration-dependent manner as judged by fura-2 fluorescence. This was completely attenuated by inclusion of the V1 antagonist. The maximal increase in [Ca2+]i evoked by AVP from a resting level of 60 +/- 5 nM was less (250 +/- 35 nM) in comparison to the maximal response evoked by angiotensin II (2,337 +/- 640 nM). However, the EC50 value for AVP (0.8 +/- 0.2 nM)-evoked increase in [Ca2+]i was significantly lower than that for angiotensin II (2.3 +/- 0.9 nM). Thus, the identification of V1 receptors coupled to increases in myocyte [Ca2+]i appears to provide, at the cellular level, a direct role for AVP in the regulation of contractility of neonatal rat cardiomyocytes.