Effects of amiloride analogues on AVP binding and activation of V1-receptor-expressing cells

We tested the interactions between amiloride analogues and V1 vascular arginine vasopressin (AVP) receptors of human platelets, rat glomerular mesangial cells, and A7r5 smooth muscle cells by using radioligand binding techniques, intracellular calcium monitoring, platelet aggregation, and cell contraction techniques. Amiloride analogues were competitive inhibitors of both the agonist [3H]AVP and the tritiated V1 antagonist [1-(beta-mercapto-beta,beta-cyclopentamethylenepropionic acid), 2-O-methyl)tyrosine]AVP ([3H]d(CH2)5Tyr(Me)AVP) binding to V1 AVP receptors in the three different cell types used. The order of potency was ethylisopropyl amiloride (EIPA) greater than Benzamil greater than amiloride. AVP mobilization of intracellular calcium was blocked by the V1 antagonist d(CH2)5Tyr(Me)AVP and was reduced by EIPA in a dose-dependent manner. Moreover, EIPA also inhibited prostaglandin F2 alpha mobilization of intracellular calcium. Alkalinization of the intracellular pH with ammonium chloride reversed the inhibitory effect of EIPA but not that of the V1 antagonist on AVP-induced calcium mobilization. Both amiloride and EIPA blocked AVP-induced aggregation of human platelets and contraction of mesangial cells and glomeruli preparations independently of receptor site antagonism. In conclusion, amiloride analogues interfere with activation of V1 vascular receptors by AVP at different levels including binding to the receptor site, mobilization of intracellular calcium, cell contraction or aggregation, and presumably alteration of intracellular ion transports.