Inhibition of Muscarinic-Stimulated Polyphosphoinositide Hydrolysis and Ca 2+ Mobilization in Cat Iris Sphincter Smooth Muscle Cells By cAMP-Elevating Agents

Abstract

The effects of carbachol (CCh) on inositol 1,4,5-trisphosphate (IP 3) production and intracellular calcium ([Ca 2+] 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 IP 3 production; the t 1/2 and EC 50 values were 68 s and 0.5 μM, respectively. The muscarinic agonist provoked a transient increase in [Ca 2+] i which reached maximum within 77 s, and increased [Ca 2+] i mobilization in a concentration-dependent manner with an EC 50 of 1.4 μM. Thapsigargin, a Ca 2+-pump inhibitor, caused a rapid rise in [Ca 2+] i and subsequent addition of CCh was without effect. Both CCh-induced IP 3 production and CCh- induced [Ca 2+] 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 IP 3 production and [Ca 2+] i mobilization. These data indicate close correlation between enhanced IP 3 production and [Ca 2+] i mobilization in these smooth muscle cells and suggest that the CCh-stimulated increase in [Ca 2+] i could be mediated through increased IP 3 production. Isoproterenol (ISO) inhibited CCh-induced IP 3 production (IC 50 = 80 nM) and [Ca 2+] i mobilization (IC 50 = 0.17 μM) in a concentration-dependent manner. Microsomal fractions isolated from SV-CISM-2 cells contained phospholipase C (PLC) which was stimulated by CCh (10 μM) and GTPγS (0.1 μM). Pretreatment of the cells with ISO or forskolin, 5 μM 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 GTPγS-stimulated IP 3 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 PIP 2 hydrolysis, generating IP 3 which mobilizes [Ca 2+] i. Furthermore, elevation of cAMP may inhibit IP 3 production and [Ca 2+] i mobilization through mechanisms involving PKA-dependent phosphorylation of PLC, G-proteins, IP 3 receptor and/or IP 3 metabolizing enzymes.