Inhibition of muscarinic-stimulated polyphosphoinositide hydrolysis and Ca2+ mobilization in cat iris sphincter smooth muscle cells by cAMP-elevating agents

The effects of carbachol (CCh) on inositol 1,4,5-trisphosphate (IP3) production and intracellular calcium ([Ca2+](i)) mobilization, and their regulation by cAMP-elevating agents were investigated in SV-40 transformed cat iris sphincter smooth muscle (SV-CISM-2) cells. CCh produced time-and dose-dependent increases in IP3 production; the t(1/2) and EC50 values were 68 s and 0.5 mu M, respectively. The muscarinic agonist provoked a transient increase in [Ca2+](i) which reached maximum within 77 s, and increased [Ca2+](i) mobilization in a concentration-dependent manner with an EC50 of 1.4 mu M. Thapsigargin, a Ca2+-pump inhibitor, caused a rapid rise in [Ca2+](i) and subsequent addition of CCh was without effect. Both CCh-induced IP3 production and CCh: induced [Ca2+](i) mobilization were more potently antagonized by 4-DAMP, an M-3 muscarinic receptor antagonist, than by pirenzepine, an M-1 receptor antagonist, suggesting that both responses are mediated through the M3 receptor subtype. Treatment of the cells with U73122, a phospholipase C (PLC) inhibitor, resulted in a concentration-dependent-decrease in both CCh-stimulated IP3 production and [Ca2+]i mobilization. These data indicate close correlation between enhanced IP3 production and [Ca2+](i) mobilization in these smooth muscle cells and suggest that the CCh-stimulated increase in [Ca2+](i) could be mediated through increased IP3 production. Isoproterenol (ISO) inhibited CCh-induced IP3 production (IC50 = 80 nM) and [Ca2+](i) mobilisation (IC50 = 0.17 mu M) in a concentration-dependent manner. Microsomal fractions isolated from SV-CISM-2 cells contained phospholipase C (PLC) which was stimulated by CCh (10 mu M) and GTP gamma S (0.1 mu M) Pretreatment of the cells with ISO or forskolin, 5 mu M each, produced membrane fractions in which CCh-stimulated PLC activity was significantly attenuated. Furthermore, when microsomal fractions isolated from SV-CISM-2 cells were phosphorylated with Protein kinase A (PKA), the CCh- and GTP gamma S-stimulated IP3 production were significantly inhibited. It can be concluded from these studies that in SV-CISM-2 cells, activation of M-3 muscarinic receptors results in stimulation of PLC-mediated PIP2 hydrolysis, generating IP3 which mobilizes [Ca2+](i). Furthermore, elevation of cAMP may inhibit IP3 production and [Ca2+](i) mobilization through mechanisms involving PKA dependent phosphorylation of PLC, G-proteins, IP3 receptor and/or IP3 metabolizing enzymes. (C) 1991 Elsevier Science Inc.