Atrial Natriuretic Peptide Provokes a Dramatic Increase in Cyclic GMP Formation and Markedly Inhibits Muscarinic-Stimulated Ca 2+ Mobilisation in SV-40 Transformed Cat Iris Sphincter Smooth Muscle (SV-CISM-2) Cells

Abstract

We investigated the effects of cGMP-elevating agents, including atrial natriuretic peptide (ANP), C-type natriuretic peptide (CNP) and sodium nitroprusside (SNP), on cGMP accumulation and on carbachol (CCh)-stimulated intracellular calcium ([Ca 2+] i) mobilisation in SV-40 transformed cat iris sphincter smooth muscle (SV-CISM-2) cells and in primary cultured cat iris sphincter smooth muscle (CISM) cells. The stimulatory effects of the natriuretic peptides on cGMP production correlated well with their inhibitory effects on CCh-induced [Ca 2+] i mobilisation, and these effects were significantly more pronounced in the SV-CISM-2 cells than in the CISM cells. Thus, ANP (1 μM) increased cGMP production in the SV-CISM-2 cells and CISM cells by 487- and 1.7-fold, respectively, and inhibited CCh-induced [Ca 2+] i mobilisation by 95 and 3%, respectively. In the SV-CISM-2 cells, ANP and CNP dose dependently inhibited CCh-induced [Ca 2+] i mobilisation with ic 50 values of 156 and 412 nM, respectively, and dose dependently stimulated cGMP formation with ec 50 values of 24 and 88 nM, respectively, suggesting that the inhibitory actions of the peptides are mediated through cGMP. Both ANP and CNP stimulated cGMP accumulation in a time-dependent manner. The potency of the cGMP-elevating agents were in the following order: ANP>>CNP>>SNP; these agents had no effect on cAMP accumulation. The inhibitory effects of the natriuretic peptides were mimicked by 8-Br-cGMP, a selective activator of cGMP-dependent protein kinase. LY83583, a soluble guanylyl cyclase inhibitor, significantly inhibited SNP-induced cGMP formation but had no effect on those of ANP and CNP. The basal activities of the guanylyl cyclase and the dissociation constant ( K d) and total receptor density ( B max) values of the natriuretic peptide receptor for [ 125I]ANP binding were not significantly different between the two cell types. The cGMP system, as with the cAMP system, has a major inhibitory influence on the muscarinic responses in the iris sphincter smooth muscle cells, and SV-CISM-2 cells can serve as an excellent model for investigating the cross talk between cGMP and the Ca 2+ signalling system.