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 t1/2 and EC50 values were 68 s and 0.5 microM, 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 microM. Thapsigargin, a Ca(2+)-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 M3 muscarinic receptor antagonist, than by pirenzepine, an M1 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 mobilization (IC50 = 0.17 microM) in a concentration-dependent manner. Microsomal fractions isolated from SV-CISM-2 cells contained phospholipase C (PLC) which was stimulated by CCh (10 microM) and GTP gamma S (0.1 microM). Pretreatment of the cells with ISO or forskolin, 5 microM 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 M3 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.
Ding
K H
KH
Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta 30912-2100, USA.
Husain
S
S
Akhtar
R A
RA
Isales
C M
CM
Abdel-Latif
A A
AA
D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002217:Carbachol / Q000494:pharmacology*; D002415:Cats; D002458:Cell Fractionation; D002462:Cell Membrane / Q000201:enzymology; D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004963:Estrenes / Q000494:pharmacology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006868:Hydrolysis; D015544:Inositol 1,4,5-Trisphosphate / Q000096:biosynthesis; D007498:Iris / Q000166:cytology; D007545:Isoproterenol / Q000494:pharmacology; D007700:Kinetics; D018721:Muscarinic Agonists / Q000494:pharmacology*; D018727:Muscarinic Antagonists / Q000494:pharmacology; D009130:Muscle, Smooth / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism*; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D010766:Phosphorylation; D011760:Pyrrolidinones / Q000494:pharmacology; D012433:Ryanodine / Q000494:pharmacology; D019284:Thapsigargin / Q000494:pharmacology; D010738:Type C Phospholipases / Q000378:metabolism
D000318:Adrenergic beta-Agonists; D004791:Enzyme Inhibitors; D004963:Estrenes; D018721:Muscarinic Agonists; D018727:Muscarinic Antagonists; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates; D010726:Phosphodiesterase Inhibitors; D011760:Pyrrolidinones; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; D012433:Ryanodine; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D019284:Thapsigargin; D015544:Inositol 1,4,5-Trisphosphate; D002217:Carbachol; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D010738:Type C Phospholipases; D007545:Isoproterenol; D002118:Calcium