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

Kehong Ding, Shahid Husain, Rashid A. Akhtar, Carlos M Isales, Ata A. Abdel-Latif

Research output: Contribution to journalArticle

26 Citations (Scopus)

Abstract

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 μM, 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 μM. 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 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 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 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.

Original languageEnglish (US)
Pages (from-to)411-421
Number of pages11
JournalCellular Signalling
Volume9
Issue number6
DOIs
StatePublished - Sep 1 1997

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Phosphatidylinositol Phosphates
Carbachol
Iris
Cholinergic Agents
Smooth Muscle Myocytes
Cats
Hydrolysis
Type C Phospholipases
Cyclic AMP-Dependent Protein Kinases
Isoproterenol
Inhibitory Concentration 50
Muscarinic M3 Receptors
Inositol 1,4,5-Trisphosphate Receptors
Muscarinic Agonists
Muscarinic Antagonists
Inositol 1,4,5-Trisphosphate
Thapsigargin
Colforsin
GTP-Binding Proteins
Smooth Muscle

Keywords

  • Carbachol
  • Cyclic AMP
  • Intracellular calcium
  • Iris sphincter smooth muscle cells
  • Isoproterenol
  • Phosphoinositide metabolism

ASJC Scopus subject areas

  • Cell Biology

Cite this

Inhibition of muscarinic-stimulated polyphosphoinositide hydrolysis and Ca2+ mobilization in cat iris sphincter smooth muscle cells by cAMP-elevating agents. / Ding, Kehong; Husain, Shahid; Akhtar, Rashid A.; Isales, Carlos M; Abdel-Latif, Ata A.

In: Cellular Signalling, Vol. 9, No. 6, 01.09.1997, p. 411-421.

Research output: Contribution to journalArticle

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abstract = "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 μM, 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 μM. 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 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 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 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.",
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T1 - Inhibition of muscarinic-stimulated polyphosphoinositide hydrolysis and Ca2+ mobilization in cat iris sphincter smooth muscle cells by cAMP-elevating agents

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AU - Husain, Shahid

AU - Akhtar, Rashid A.

AU - Isales, Carlos M

AU - Abdel-Latif, Ata A.

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N2 - 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 μM, 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 μM. 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 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 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 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.

AB - 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 μM, 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 μM. 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 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 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 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.

KW - Carbachol

KW - Cyclic AMP

KW - Intracellular calcium

KW - Iris sphincter smooth muscle cells

KW - Isoproterenol

KW - Phosphoinositide metabolism

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