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Differential Regulation of Phospholipase A2(PLA2)-dependent Ca2+ Signaling in Smooth Muscle by cAMP- and cGMP-dependent Protein Kinases

1998; Elsevier BV; Volume: 273; Issue: 51 Linguagem: Inglês

10.1074/jbc.273.51.34519

ISSN

1083-351X

Autores

Karnam S. Murthy, Gabriel M. Makhlouf,

Tópico(s)

Calcium signaling and nucleotide metabolism

Resumo

Both cAMP- and cGMP-dependent protein kinases inhibit agonist-stimulated phospholipase C-β (PLC-β) activity and inositol 1,4,5-trisphosphate-dependent Ca2+release in vascular and visceral smooth muscle. In smooth muscle of the intestinal longitudinal layer, however, the initial steps in Ca2+ mobilization involve activation of cytosolic PLA2 (cPLA2) and arachidonic acid (AA)-dependent stimulation of Ca2+ influx. The present study examined whether cAMP- and cGMP-dependent protein kinases are capable of regulating these processes also. Agents that activated cAMP-dependent protein kinase (5,6-dichloro-1-β-d-ribofuranosylbenzimidazole 3′,5′-cyclic monophosphothioate (Sp-isomer) and isoproterenol), cGMP-dependent protein kinase (8-(4-chlorophenylthio)-guanosine 3′,5′-cyclic monophosphate and Na nitroprusside), or both kinases (vasoactive intestinal peptide and isoproterenol >1 μm) induced phosphorylation of cPLA2 and inhibition of agonist-stimulated cPLA2 activity. Phosphorylation and inhibition of cPLA2 activity by cAMP- and cGMP-dependent protein kinases were blocked by the corresponding selective inhibitors (cAMP-dependent protein kinase,N-[2(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide hydrochloride (H-89) and myristoylated protein kinase inhibitor (14Lin L.L. Lin A.Y. Knopf J.L. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6147-6151Crossref PubMed Scopus (518) Google Scholar, 15Lin L.L. Wartman M. Lin A.Y. Knopf J.L. Seth A. Davis R.J. Cell. 1993; 72: 269-278Abstract Full Text PDF PubMed Scopus (1643) Google Scholar, 16Nemonoff R.A. Winitz S. Qian N.-X. Van Putten V. Johnson G.L. Heasley L.E. J. Biol. Chem. 1993; 268: 1960-1964PubMed Google Scholar, 17Qui Z.-H. de Carvalho M.S. Leslie C.C. J. Biol. Chem. 1993; 268: 24506-24513Abstract Full Text PDF PubMed Google Scholar, 18de Carvalho M.G.S. McCormack A.L. Olson E. Ghomashchi F. Gelb M.H. Yates III, J.R. Leslie C.C. J. Biol. Chem. 1996; 271: 6987-6997Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 19Qui Z.-H. Leslie C.C. J. Biol. Chem. 1994; 269: 19480-19487Abstract Full Text PDF PubMed Google Scholar, 20Leslie C.C. J. Biol. Chem. 1997; 272: 16709-16712Abstract Full Text Full Text PDF PubMed Scopus (738) Google Scholar, 21Borsch-Haubold A.G. Bartoli F. Asselin J. Dudler T. Kramer R.M. Apitz-Castro R. Watson S.P. Gelb M.H. J. Biol. Chem. 1998; 273: 4449-4458Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 22Murthy K.S. Kuemmerle J.F. Makhlouf G.M. Am. J. Physiol. 1995; 269: G93-G102Crossref PubMed Google Scholar) amide; cGMP-dependent protein kinase, (8R,9S,11S)-(−)-9-methoxy-carbamyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,11H,-2,7b,11a-trizadizobenzo(a,g)cycloocta(c,d,e)-trinden-1-one (KT-5823)). In contrast, AA-stimulated Ca2+ influx was inhibited by agents that activated cGMP-dependent protein kinase only; the inhibition was selectively blocked by KT-5823. The study provides the first evidence of inhibitory phosphorylation of cPLA2 in vivo by cAMP- and cGMP-dependent protein kinases. Inhibition of cPLA2 activity and AA-induced Ca2+ influx partly account for the ability of cAMP-dependent protein kinase and/or cGMP-dependent protein kinase to cause relaxation. Their importance resides in their location at the inception of the Ca2+ signaling cascade. Both cAMP- and cGMP-dependent protein kinases inhibit agonist-stimulated phospholipase C-β (PLC-β) activity and inositol 1,4,5-trisphosphate-dependent Ca2+release in vascular and visceral smooth muscle. In smooth muscle of the intestinal longitudinal layer, however, the initial steps in Ca2+ mobilization involve activation of cytosolic PLA2 (cPLA2) and arachidonic acid (AA)-dependent stimulation of Ca2+ influx. The present study examined whether cAMP- and cGMP-dependent protein kinases are capable of regulating these processes also. Agents that activated cAMP-dependent protein kinase (5,6-dichloro-1-β-d-ribofuranosylbenzimidazole 3′,5′-cyclic monophosphothioate (Sp-isomer) and isoproterenol), cGMP-dependent protein kinase (8-(4-chlorophenylthio)-guanosine 3′,5′-cyclic monophosphate and Na nitroprusside), or both kinases (vasoactive intestinal peptide and isoproterenol >1 μm) induced phosphorylation of cPLA2 and inhibition of agonist-stimulated cPLA2 activity. Phosphorylation and inhibition of cPLA2 activity by cAMP- and cGMP-dependent protein kinases were blocked by the corresponding selective inhibitors (cAMP-dependent protein kinase,N-[2(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide hydrochloride (H-89) and myristoylated protein kinase inhibitor (14Lin L.L. Lin A.Y. Knopf J.L. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6147-6151Crossref PubMed Scopus (518) Google Scholar, 15Lin L.L. Wartman M. Lin A.Y. Knopf J.L. Seth A. Davis R.J. Cell. 1993; 72: 269-278Abstract Full Text PDF PubMed Scopus (1643) Google Scholar, 16Nemonoff R.A. Winitz S. Qian N.-X. Van Putten V. Johnson G.L. Heasley L.E. J. Biol. Chem. 1993; 268: 1960-1964PubMed Google Scholar, 17Qui Z.-H. de Carvalho M.S. Leslie C.C. J. Biol. Chem. 1993; 268: 24506-24513Abstract Full Text PDF PubMed Google Scholar, 18de Carvalho M.G.S. McCormack A.L. Olson E. Ghomashchi F. Gelb M.H. Yates III, J.R. Leslie C.C. J. Biol. Chem. 1996; 271: 6987-6997Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 19Qui Z.-H. Leslie C.C. J. Biol. Chem. 1994; 269: 19480-19487Abstract Full Text PDF PubMed Google Scholar, 20Leslie C.C. J. Biol. Chem. 1997; 272: 16709-16712Abstract Full Text Full Text PDF PubMed Scopus (738) Google Scholar, 21Borsch-Haubold A.G. Bartoli F. Asselin J. Dudler T. Kramer R.M. Apitz-Castro R. Watson S.P. Gelb M.H. J. Biol. Chem. 1998; 273: 4449-4458Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 22Murthy K.S. Kuemmerle J.F. Makhlouf G.M. Am. J. Physiol. 1995; 269: G93-G102Crossref PubMed Google Scholar) amide; cGMP-dependent protein kinase, (8R,9S,11S)-(−)-9-methoxy-carbamyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,11H,-2,7b,11a-trizadizobenzo(a,g)cycloocta(c,d,e)-trinden-1-one (KT-5823)). In contrast, AA-stimulated Ca2+ influx was inhibited by agents that activated cGMP-dependent protein kinase only; the inhibition was selectively blocked by KT-5823. The study provides the first evidence of inhibitory phosphorylation of cPLA2 in vivo by cAMP- and cGMP-dependent protein kinases. Inhibition of cPLA2 activity and AA-induced Ca2+ influx partly account for the ability of cAMP-dependent protein kinase and/or cGMP-dependent protein kinase to cause relaxation. Their importance resides in their location at the inception of the Ca2+ signaling cascade. phospholipase A 8-(4-chlorophenylthio)-guanosine 3′,5′-cyclic monophosphate 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole 3′,5′-cyclic monophosphothioate, Sp-isomer N-[2(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide hydrochloride (8R,9S,11S)-(−)-9-methoxy-carbamyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,11H,-2,7b,11a-trizadizobenzo(a,g)cycloocta(c,d,e)-trinden-1one protein kinase inhibitor (14Lin L.L. Lin A.Y. Knopf J.L. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6147-6151Crossref PubMed Scopus (518) Google Scholar, 15Lin L.L. Wartman M. Lin A.Y. Knopf J.L. Seth A. Davis R.J. Cell. 1993; 72: 269-278Abstract Full Text PDF PubMed Scopus (1643) Google Scholar, 16Nemonoff R.A. Winitz S. Qian N.-X. Van Putten V. Johnson G.L. Heasley L.E. J. Biol. Chem. 1993; 268: 1960-1964PubMed Google Scholar, 17Qui Z.-H. de Carvalho M.S. Leslie C.C. J. Biol. Chem. 1993; 268: 24506-24513Abstract Full Text PDF PubMed Google Scholar, 18de Carvalho M.G.S. McCormack A.L. Olson E. Ghomashchi F. Gelb M.H. Yates III, J.R. Leslie C.C. J. Biol. Chem. 1996; 271: 6987-6997Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 19Qui Z.-H. Leslie C.C. J. Biol. Chem. 1994; 269: 19480-19487Abstract Full Text PDF PubMed Google Scholar, 20Leslie C.C. J. Biol. Chem. 1997; 272: 16709-16712Abstract Full Text Full Text PDF PubMed Scopus (738) Google Scholar, 21Borsch-Haubold A.G. Bartoli F. Asselin J. Dudler T. Kramer R.M. Apitz-Castro R. Watson S.P. Gelb M.H. J. Biol. Chem. 1998; 273: 4449-4458Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 22Murthy K.S. Kuemmerle J.F. Makhlouf G.M. Am. J. Physiol. 1995; 269: G93-G102Crossref PubMed Google Scholar) amide cytosolic PLA arachidonic acid inositol 1,4,5-trisphosphate sodium nitroprusside cholecystokinin octapeptide vasoactive intestinal peptide. Phospholipases A2(PLA2s),1, which catalyze the hydrolysis of the Sn2 fatty acyl bond of phospholipids to yield free fatty acids and lysophospholipids, have been divided into two main categories comprising up to nine groups (1Dennis E.A. Boyer P.D. 3rd Ed. The Enzymes. 16. Academic Press, New York1983: 307-353Google Scholar, 2Balsinde J. Dennis E.A. J. Biol. Chem. 1997; 272: 16069-16072Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar). One category (Groups I, II, III, V, VII, and IX) includes the small molecular weight, secretory PLA2s, which are Ca2+-dependent except for Group VII PLA2 (3Davidson F.F. Dennis E.A. J. Mol. Evol. 1990; 31: 228-238Crossref PubMed Scopus (292) Google Scholar, 4Dennis E.A. Trends Biochem. Sci. 1997; 22: 1-2Abstract Full Text PDF PubMed Scopus (754) Google Scholar, 5McIntosh J.M. Ghomashchi F. Gelb M.H. Dooley D.J. Stoehr S.R. Giordani A.B. Naisbitt S.R. Olivera B.M. J. Biol. Chem. 1995; 270: 3518-3526Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 6Tjoelker L.W. Eberhardt C. Unger J. Trong H.L. Zimmerman G.A. McIntyre T.M. Stafforini D.M. Presott S.M. Gray P.W. J. Biol. Chem. 1995; 270: 25481-25487Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). The other category includes large molecular weight, Ca2+-dependent (Group IV) and Ca2+-independent (Groups VI and VIII) cytosolic PLA2s (7Clark J.D. Milona N. Knopf J.L. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 7708-7712Crossref PubMed Scopus (420) Google Scholar, 8Wolf M.J. Gross R.W. J. Biol. Chem. 1996; 271: 20989-20992Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 9Hazen S.L. Stuppy R.J. Gross R.W. J. Biol. Chem. 1990; 265: 10622-10630Abstract Full Text PDF PubMed Google Scholar). The Ca2+-independent, Group VII secretory PLA2 and the Ca2+-dependent, Group VIII cytosolic PLA2s are specific for platelet-activating factor and may be viewed as platelet-activating factor acetyl hydrolases (2Balsinde J. Dennis E.A. J. Biol. Chem. 1997; 272: 16069-16072Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar, 10Hattori M. Adachi H. Tsujimoto M. Arai H. Inoue K. J. Biol. Chem. 1994; 269: 23150-23155Abstract Full Text PDF PubMed Google Scholar). The Ca2+-independent, Group VI cytosolic PLA2 is involved in the continuous recycling of phospholipids and incorporation of arachidonic acid (11Balsinde J. Bianco I.D. Ackermann E.J. Conde-Frieboes K. Dennis E.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 92: 8527-8531Crossref Scopus (255) Google Scholar, 12Chilton F.H. Fonteh A.N. Surette M.E. Triggiani M. Winkler J.D. Biochim. Biophys. Acta. 1996; 1299: 1-15Crossref PubMed Scopus (208) Google Scholar). Group IV cytosolic PLA2 (cPLA2) possesses several distinctive features, including a dependence on submicromolar concentrations of Ca2+ essential for translocation of the enzyme via a Ca2+ and phospholipid binding domain, a preference for hydrolysis of arachidonate-containing phospholipids, and a susceptibility to regulatory phosphorylation by mitogen-activated protein kinase and protein kinase C (13Kramer R.M. Roberts E.F. Manetta J. Putman J.E. J. Biol. Chem. 1991; 266: 5268-5272Abstract Full Text PDF PubMed Google Scholar, 14Lin L.L. Lin A.Y. Knopf J.L. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6147-6151Crossref PubMed Scopus (518) Google Scholar, 15Lin L.L. Wartman M. Lin A.Y. Knopf J.L. Seth A. Davis R.J. Cell. 1993; 72: 269-278Abstract Full Text PDF PubMed Scopus (1643) Google Scholar, 16Nemonoff R.A. Winitz S. Qian N.-X. Van Putten V. Johnson G.L. Heasley L.E. J. Biol. Chem. 1993; 268: 1960-1964PubMed Google Scholar, 17Qui Z.-H. de Carvalho M.S. Leslie C.C. J. Biol. Chem. 1993; 268: 24506-24513Abstract Full Text PDF PubMed Google Scholar, 18de Carvalho M.G.S. McCormack A.L. Olson E. Ghomashchi F. Gelb M.H. Yates III, J.R. Leslie C.C. J. Biol. Chem. 1996; 271: 6987-6997Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). There is evidence that phosphorylation by protein kinase C may be mediated indirectly by mitogen-activated protein kinase (19Qui Z.-H. Leslie C.C. J. Biol. Chem. 1994; 269: 19480-19487Abstract Full Text PDF PubMed Google Scholar). Phosphorylation by mitogen-activated protein kinase on serine residues (chiefly Ser505) or tyrosine residues is associated with increase in cPLA2 activity (14Lin L.L. Lin A.Y. Knopf J.L. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6147-6151Crossref PubMed Scopus (518) Google Scholar, 17Qui Z.-H. de Carvalho M.S. Leslie C.C. J. Biol. Chem. 1993; 268: 24506-24513Abstract Full Text PDF PubMed Google Scholar, 18de Carvalho M.G.S. McCormack A.L. Olson E. Ghomashchi F. Gelb M.H. Yates III, J.R. Leslie C.C. J. Biol. Chem. 1996; 271: 6987-6997Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 20Leslie C.C. J. Biol. Chem. 1997; 272: 16709-16712Abstract Full Text Full Text PDF PubMed Scopus (738) Google Scholar, 21Borsch-Haubold A.G. Bartoli F. Asselin J. Dudler T. Kramer R.M. Apitz-Castro R. Watson S.P. Gelb M.H. J. Biol. Chem. 1998; 273: 4449-4458Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). Phosphorylation by cAMP- or cGMP-dependent protein kinase in vivohas not been characterized. Agonist-induced, G protein-dependent activation of cPLA2 has been demonstrated in dispersed intestinal smooth muscle cells and is the initial trigger for Ca2+mobilization in muscle cells of the longitudinal layer (22Murthy K.S. Kuemmerle J.F. Makhlouf G.M. Am. J. Physiol. 1995; 269: G93-G102Crossref PubMed Google Scholar). An initial transient increase in cPLA2 activity occurs only in smooth muscle cells from the intestinal longitudinal layer and coincides with the initial Ca2+ transient. This is followed by a sustained increase in cPLA2 activity, which is partly dependent on activation of protein kinase C. The initial increase in cPLA2 activity in longitudinal muscle leads to arachidonic acid (AA)-induced stimulation of Ca2+ influx, which triggers Ca2+ release from sarcoplasmic stores via ryanodine receptor/Ca2+ channels and stimulates the activity of ADP-ribosylcyclase. The resultant increase in cADP ribose enhances Ca2+-induced Ca2+ release (22Murthy K.S. Kuemmerle J.F. Makhlouf G.M. Am. J. Physiol. 1995; 269: G93-G102Crossref PubMed Google Scholar, 23Kuemmerle J.F. Murthy K.S. Makhlouf G.M. Am. J. Physiol. 1995; 266: C1421-C1431Crossref Google Scholar, 24Kuemmerle J.F. Makhlouf G.M. J. Biol. Chem. 1995; 270: 25488-25494Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Unlike longitudinal smooth muscle, agonist-stimulated Ca2+mobilization in smooth muscle of the circular layer is mediated by phospholipase C-β1 and/or -β3, resulting in inositol 1,4,5-trisphosphate (IP3)-dependent Ca2+ release (25Bitar K.N. Bradford P.G. Putney Jr., J.W. Makhlouf G.M. J. Biol. Chem. 1986; 261: 16591-16596Abstract Full Text PDF PubMed Google Scholar, 26Murthy K.S. Grider J.R. Makhlouf G.M. Am. J. Physiol. 1991; 261: G937-G944PubMed Google Scholar). Only small amounts of IP3 are formed in longitudinal smooth muscle, which does not possess high affinity IP3 receptor/Ca2+channels (23Kuemmerle J.F. Murthy K.S. Makhlouf G.M. Am. J. Physiol. 1995; 266: C1421-C1431Crossref Google Scholar, 26Murthy K.S. Grider J.R. Makhlouf G.M. Am. J. Physiol. 1991; 261: G937-G944PubMed Google Scholar). The initial steps in Ca2+ mobilization in vascular and intestinal circular smooth muscle (i.e. activation of PLC-β and stimulation of Ca2+ release) are inhibited by cAMP- and cGMP-dependent protein kinases (27Cornwell T.L. Lincoln T.M. J. Biol. Chem. 1989; 264: 1146-1155Abstract Full Text PDF PubMed Google Scholar, 28Szewczak S.M. Behar J. Billet G. Hillemeier C. Rhim R.Y. Biancani P. Am. J. Physiol. 1990; 259: G239-G244PubMed Google Scholar, 29Tachado S.D. Akthar R.A. Abdel-Latif A.A. Invest. Ophthalmol. & Visual Sci. 1989; 30: 2232-2239PubMed Google Scholar, 30Komalavilas P. Lincoln T.M. J. Biol. Chem. 1996; 271: 21933-21938Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, 31Murthy K.S. Severi C. Grider J.R. Makhlouf G.M. Am. J. Physiol. 1993; 262: G967-G974Google Scholar, 32Murthy K.S. Makhlouf G.M. Am. J. Physiol. 1995; 268: C171-C180Crossref PubMed Google Scholar). It is not known, however, whether the initial steps in Ca2+mobilization in intestinal longitudinal smooth muscle (i.e.activation of cPLA2 and stimulation of Ca2+influx) are influenced by cAMP- and cGMP-dependent protein kinases. In this study, we have examined the initial steps in Ca2+ signaling in longitudinal intestinal smooth muscle to determine the ability of cAMP- and cGMP-dependent protein kinases (a) to phosphorylate cPLA2 and influence its activity and (b) to modulate AA-induced stimulation of Ca2+ influx. The results provide the first evidence of inhibitory phosphorylation of cPLA2 by both cAMP- and cGMP-dependent protein kinases and demonstrate their ability to regulate the initial step in Ca2+ signaling. The next step, i.e. AA-stimulated Ca2+ influx, is selectively inhibited by cGMP-dependent protein kinase. Smooth muscle cells were isolated from the longitudinal muscle layer of rabbit intestine by sequential enzymatic digestion, filtration, and centrifugation as described previously (22Murthy K.S. Kuemmerle J.F. Makhlouf G.M. Am. J. Physiol. 1995; 269: G93-G102Crossref PubMed Google Scholar, 23Kuemmerle J.F. Murthy K.S. Makhlouf G.M. Am. J. Physiol. 1995; 266: C1421-C1431Crossref Google Scholar, 24Kuemmerle J.F. Makhlouf G.M. J. Biol. Chem. 1995; 270: 25488-25494Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Muscle strips were incubated for 30 min at 31 °C in 15 ml of HEPES medium containing 0.1% collagenase (type II) and 0.1% soybean trypsin inhibitor with no added Ca2+. The composition of the medium was 120 mm NaCl, 4 mm KCl, 2.6 mmKH2PO4, 0.6 mm MgCl2, 25 mm HEPES, 14 mm glucose, and 2.1% Eagle's essential amino acid mixture. The partly digested tissue was washed with 100 ml of enzyme-free medium and reincubated for 30 min to allow spontaneous dispersion of muscle cells. The cells were harvested by filtration through 500-μm Nitex mesh, centrifuged twice for 10 min at 350 × g, and resuspended in HEPES medium containing 2 mm Ca2+. Contraction of dispersed muscle cells was measured by scanning micrometry as described previously (22Murthy K.S. Kuemmerle J.F. Makhlouf G.M. Am. J. Physiol. 1995; 269: G93-G102Crossref PubMed Google Scholar, 23Kuemmerle J.F. Murthy K.S. Makhlouf G.M. Am. J. Physiol. 1995; 266: C1421-C1431Crossref Google Scholar, 24Kuemmerle J.F. Makhlouf G.M. J. Biol. Chem. 1995; 270: 25488-25494Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). An aliquot (0.5 ml) of cells (104 cells/ml) was added to 0.2 ml of medium containing 1 μm AA in the presence of the cyclooxygenase inhibitor indomethacin (10 μm) and the lipoxygenase inhibitor nordihydroguaiaretic acid (10 μm). The reaction was terminated after 30 s with acrolein. Inhibition of contraction (i.e. relaxation) was measured in muscle cells maximally contracted for 30 s with AA (1 μm). Relaxation was expressed as the increase in the length of AA-contracted muscle cells (mean resting muscle cell length, 118 ± 5 μm; mean length of AA-contracted muscle cells, 83 ± 2 μm). Ca2+ influx in dispersed muscle cells was measured from the initial uptake of45Ca2+ in the presence of anti-mycin and thapsigargin to prevent uptake into mitochondrial and sarcoplasmic Ca2+ stores. Dispersed muscle cells were suspended in 10 ml of HEPES medium containing 2 mm Ca2+ and45Ca2+ (10 μCi/ml), with thapsigargin (2 μm) and anti-mycin (10 μm). The muscle cells were treated for 60 s with a relaxant agent (Na nitroprusside (SNP) and isoproterenol) or protein kinase activator (cBIMPS and 8-pcCPT-cGMP), followed by addition of 1 μmAA for 2 min. Samples (0.5 ml) were withdrawn at intervals for measurement of 45Ca2+ cell content. The muscle cells were centrifuged and washed twice with HEPES medium, and the45Ca2+ cell content was measured and expressed as cpm/106 cells. Phosphorylation of cPLA2 was measured from the amount of [32P]ATP incorporated into the enzyme after immunoprecipitation with specific cPLA2 antibody. Dispersed smooth muscle cells (10 ml, 4 × 106 cells/ml) were prelabeled with 0.5 mCi/ml [32P]orthophosphate for 3 h. Samples (0.5 ml) were incubated with various relaxant agents for 60 s in the presence or absence of cAMP-dependent protein kinase inhibitors (H-89 or myristoylated PKI) or cGMP-dependent protein kinase inhibitor (KT-5823), and the reaction was terminated with an equal volume of lysis buffer (final concentrations, 1% Triton X-100, 0.5% SDS, 0.75% deoxycholate, 10 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 100 μg/ml aprotinin, 10 mmNa4P2O7, 50 mm NaF, 0.2 mm Na3VO4) and placed on ice for 30 min. The cell lysates were separated from the insoluble material by centrifugation at 13,000 × g for 15 min at 4 °C, precleared with 40 μl of protein A-Sepharose CL-4B, and incubated with polyclonal rabbit cPLA2 antibody for 2 h at 4 °C and with 40 μl of protein A-Sepharose CL-4B for another 1 h. The immunoprecipitates were collected, washed five times with 1 ml of wash buffer (0.5% Triton X-100, 150 mm NaCl, 10 mm Tris-HCl, pH 7.4), extracted with Laemmli sample buffer, boiled for 15 min, and separated on 10% SDS-polyacrylamide gel electrophoresis. After transfer to polyvinylidene difluoride membranes,32P-labeled cPLA2 was visualized by autoradiography, and the amount of radioactivity in the band was measured. cPLA2 activity in dispersed muscle cells was measured by an adaptation of the method of Damron et al.(33Damron D.S. Van Wagoner D.R. Moravec C.S. Bond M. J. Biol. Chem. 1993; 268: 27335-27344Abstract Full Text PDF PubMed Google Scholar) as described previously (22Murthy K.S. Kuemmerle J.F. Makhlouf G.M. Am. J. Physiol. 1995; 269: G93-G102Crossref PubMed Google Scholar). Twenty ml of cell suspension (106 cells/ml) were incubated with [3H]AA (1 μCi/ml) at 31 °C for 3 h. The cells were diluted with 50 ml of HEPES medium, centrifuged at 350 × g for 15 min, and then resuspended in 10 ml of fresh medium containing 10 μm indomethacin and 10 μmnordihydroguaiaretic acid to inhibit AA metabolism via the lipoxygenase and cyclooxygenase pathways. Duplicate samples (106cells/0.5 ml) were incubated at 31 °C with 1 nmcholecystokinin octapeptide (CCK-8) for 30 s, and the reaction was terminated with 1.8 ml of chloroform/methanol/HCl (100:200:2, v/v/v). The phases were separated with 0.6 ml of chloroform and 0.6 ml of 2 mm HCl. The organic phase was dried under nitrogen, resuspended in 50 μl of chloroform/methanol (9:1), and spotted on silica gel plates for thin layer chromatography using hexane/ethylether/acetic acid (70:30:3.5). The radioactivity in spots corresponding to AA was counted, and the results were expressed as cpm/106 cells above basal levels. [3H]arachidonic acid (210 Ci/mmol), carrier-free [32P]Pi, and45Ca2+ were obtained from NEN Life Science Products; indomethacin, nordihydroguaiaretic acid, and PKI were from Biomol; polyclonal antibodies to cPLA2 were from Santa Cruz; HEPES was from Research Biochemicals; thapsigargin and H-89 were from Calbiochem; CCK-8 and vasoactive intestinal peptide (VIP) were from Bachem; KT-5823 was from Kamiya Biomedical (Thousand Oaks, CA); 8-pcCPT-cGMP and cBIMPS were from Alexis Corp. (San Diego, CA); and all other reagents were from Sigma. Selective activators of cGMP-dependent protein kinase (8-pcCPT-cGMP) and cAMP-dependent protein kinase (cBIMPS) and three relaxant agents were used to determine the ability of cAMP- and cGMP-dependent protein kinases to phosphorylate cPLA2 in smooth muscle: SNP, which stimulates cGMP and selectively activates cGMP-dependent protein kinase at concentrations <1 μm; isoproterenol, which stimulates cAMP and preferentially activates cAMP-dependent protein kinase at low concentrations (<1 μm) but can cross-activate cGMP-dependent protein kinase at higher concentrations; and VIP, which stimulates both cAMP and cGMP in gastric smooth muscle and activates cAMP- and cGMP-dependent protein kinases at all concentrations (32Murthy K.S. Makhlouf G.M. Am. J. Physiol. 1995; 268: C171-C180Crossref PubMed Google Scholar, 34Lincoln T.M. Cornwell T.L. Taylor A.E. Am. J. Physiol. 1990; 258: C399-C407Crossref PubMed Google Scholar, 35Francis S.H. Noblett B.D. Todd B.W. Wells J.N. Corbin J.D. Mol. Pharmacol. 1988; 34: 506-517PubMed Google Scholar, 36Jiang H. Colbran J.L. Francis S.H. Corbin J.D. J. Biol. Chem. 1992; 267: 1015-1019Abstract Full Text PDF PubMed Google Scholar). The cAMP-dependent protein kinase inhibitors H-89 and myristoylated PKI and the cGMP-dependent protein kinase inhibitor KT-5823 were used to evaluate the involvement of each kinase in cPLA2 phosphorylation. Previous studies (32Murthy K.S. Makhlouf G.M. Am. J. Physiol. 1995; 268: C171-C180Crossref PubMed Google Scholar) of cAMP- and cGMP-dependent protein kinase activity in dispersed muscle cells had shown that at concentrations of ≤1 μm, H-89 and KT-5823 were selective inhibitors of cAMP- and cGMP-dependent protein kinases, respectively. Both 8-pcCPT-cGMP and cBIMPS increased cPLA2phosphorylation in dispersed muscle cells by 406 ± 58% (p < 0.01) and 343 ± 40% (p < 0.01), respectively. Phosphorylation induced by 8-pcCPT-cGMP was abolished by KT-5823 (98 ± 2% inhibition) but was not affected by H-89 or myristoylated PKI, whereas phosphorylation induced by cBIMPS was abolished by H-89 (97 ± 2% inhibition) and myristoylated PKI (99 ± 3% inhibition) but was not affected by KT-5823 (Fig.1). SNP, isoproterenol (1 μm), and VIP also increased cPLA2phosphorylation by 515 ± 67% (p < 0.01), 262 ± 23% (p < 0.01), and 459 ± 52% (p < 0.01), respectively (Fig.2). The phosphorylation induced by SNP was abolished by KT-5823 (98 ± 2% inhibition) but was not affected by H-89 or myristoylated PKI, whereas phosphorylation induced by 1 μm isoproterenol was abolished by H-89 (99 ± 4% inhibition) and myristoylated PKI (98 ± 3% inhibition) but was not significantly affected by KT-5823. Phosphorylation induced by 1 μm VIP was weakly inhibited by myristoylated PKI and H-89 (17 ± 3% inhibition; p < 0.02) but was not affected by KT-5823; however, a combination of H-89 and KT-5823 virtually abolished cPLA2 phosphorylation (87 ± 7% inhibition). Because VIP activates both cAMP- and cGMP-dependent protein kinases, minimal inhibition by each kinase inhibitor separately and virtually complete inhibition by a combination of both inhibitors suggest interplay of the two kinases when activated concurrently.Figure 2Phosphorylation of cPLA2 by relaxant agents in smooth muscle. Intestinal smooth muscle cells labeled with 32P were incubated with cAMP-dependent protein kinase inhibitors H-89 (1 μm) and myristoylated PKI (1 μm) or the cGMP-dependent protein kinase inhibitor KT-5823 (1 μm) for 10 min and treated with isoproterenol (ISOP; 1 μm), SNP (0.1 μm), or VIP (1 μm) for 1 min. Immunoprecipitates using polyclonal cPLA2 antibody were separated on SDS-polyacrylamide gel electrophoresis, 32P-labeled cPLA2 was identified by autoradiography (upper panel), and the measured radioactivity was expressed as cpm/mg protein (lower panel). Values are means ± S.E. of three experiments. Inhibition of phosphorylation: ∗∗, p < 0.01; ∗,p < 0.05.View Large Image Figure ViewerDownload (PPT) Previous studies have shown that activation of cPLA2 by contractile agonists (e.g. CCK-8) is biphasic, with an initial peak of activity during the first minute followed by a sustained phase of activity that is partly mediated by protein kinase C (22Murthy K.S. Kuemmerle J.F. Makhlouf G.M. Am. J. Physiol. 1995; 269: G93-G102Crossref PubMed Google Scholar). Phosphorylation of cPLA2 was not present during the initial phase (1 min) of cPLA2 activity but increased significantly during the sustained phase (10 min after treatment with CCK-8). Preincubation of the cells with the protein kinase C inhibitor calphostin C (1 μm) abolished the increase in cPLA2phosphorylation during the sustained phase and, as shown previously (22Murthy K.S. Kuemmerle J.F. Makhlouf G.M. Am. J. Physiol. 1995; 269: G93-G102Crossref PubMed Google Scholar), inhibited cPLA2 activity by 40% (Fig.3). The initial peak of cPLA2 activity induced by CCK-8 (431 ± 41 cpm arachidonic acid/106 cells in the presence of cyclooxygenase and lipoxygenase inhibitors) was inhibited by 1 μm isoproterenol (36 ± 8% inhibition;p < 0.01) and by the selective cAMP-dependent protein kinase activator cBIMPS (54 ± 2%; p < 0.001). The inhibition of cPLA2activity by either agent was completely reversed by H-89 and PKI but was not affected by KT-5823 (Fig. 4). The initial peak of CCK-stimulated cPLA2 activity was also inhibited by SNP (75 ± 5%, p < 0.01) and the selective cGMP-dependent protein kinase activator 8-pcCPT-cGMP (51 ± 2%, p < 0.001); the inhibition by either agent was completely reversed by KT-5823 but was not affected by H-89 or PKI (Fig. 5). A higher concentration of isoproterenol (100 μm), which activates cAMP-dependent protein kinase and cross-activates cGMP-dependent protein kinase, inhibited CCK-stimulated cPLA2 activity by 83 ± 4% (p < 0.001); the inhibition was partly reversed by H-89 and PKI to 55 ± 3 and 56 ± 6%, respectively, and by KT-5823 to 66 ± 5% and was completely reversed by a combination of KT-5823 with either H-89 or PKI (Fig.6). Similarly, VIP (1 μm), which activates both cAMP- and cGMP-dependent protein kinases, inhibited CCK-stimulated cPLA2 activity by 88 ± 4% (p < 0.001); the inhibition was partly reversed by H-89 and PKI to 58 ± 6 and 55 ± 7%, respectively, and to a lesser extent by KT-5823 (73 ± 6%) and was completely reversed by a combination of both inhibitors (Fig. 6).Figure 5Inhibition of agonist-stimulated cPLA2 activity by selective activation of cGMP-dependent protein kinase. Muscle cells labeled with [3H]AA were incubated for 60 s with SNP (0.1 μm) or 8-pcCPT-cGMP (10 μm) in the presence and absence of H-89, myristoylated PKI, or KT-5823, followed by addition of 1 nm CCK-8 for 30 s. Formation of [3H]AA was expressed as cpm/106 cells above basal levels. Results are means ± S.E. of four experiments. ∗∗, inhibition of cPLA2 activity, p < 0.01.View Large Image Figure ViewerDownload (PPT)Figure 6Inhibition of agonist-stimulated cPLA2 activity by combined activation of cGMP-dependent protein kinase and cAMP-dependent protein kinase. Muscle cells labeled with [3H]AA were incubated for 60 s with 100 μm isoproterenol (ISOP) or 1 μmVIP in the presence and absence of cAMP-dependent protein kinase inhibitors (H-89 and myristoylated PKI), cGMP-dependent protein kinase inhibitor (KT-5823), or both cAMP- and cGMP-dependent protein kinase inhibitors, followed by addition of 1 nm CCK-8 for 30 s. Formation of [3H]AA was expressed as cpm/106 cells above basal levels. Results are means ± S.E. of four experiments. ∗∗, inhibition of cPLA2 activity, p < 0.01.View Large Image Figure ViewerDownload (PPT) We have previously shown that endogenous formation of AA stimulates Ca2+influx in intestinal longitudinal muscle cells; the effect could be reproduced by addition of nanomolar concentrations of AA (22Murthy K.S. Kuemmerle J.F. Makhlouf G.M. Am. J. Physiol. 1995; 269: G93-G102Crossref PubMed Google Scholar). In the present study, the rate of Ca2+ influx was measured from the initial uptake of 45Ca2+ (2869 ± 510 cpm/106 cells) induced by exogenous AA in the presence of cyclooxygenase and lipoxygenase inhibitors. AA-induced Ca2+influx was not affected by selective activation of cAMP-dependent protein kinase with cBIMPS (3115 ± 321 cpm/106 cells) or 1 μm isoproterenol (2816 ± 636 cpm/106 cells). A higher concentration of isoproterenol (100 μm) inhibited AA-induced Ca2+ influx by 84 ± 4% (p < 0.001); the inhibition was completely reversed by KT-5823 but was not affected by H-89 (Fig. 7), implying that inhibition was dependent on cross-activation of cGMP-dependent protein kinase by higher concentrations of isoproterenol. Consistent with selective inhibition of Ca2+ influx by cGMP-dependent protein kinase, SNP and 8-pcCPT-cGMP inhibited AA-induced Ca2+ influx by 83 ± 4% (p < 0.001) and 62 ± 8% (p < 0.01), respectively; the inhibition by both SNP and 8-pcCPT-cGMP was completely reversed by KT-5823 but was not affected by H-89 (Fig.8). AA-stimulated Ca2+ influx in intestinal longitudinal smooth muscle cells leads to Ca2+-induced Ca2+ release from sarcoplasmic stores in intestinal longitudinal smooth muscle cells; the resultant increase in [Ca2+]i triggers an initial muscle contraction (22Murthy K.S. Kuemmerle J.F. Makhlouf G.M. Am. J. Physiol. 1995; 269: G93-G102Crossref PubMed Google Scholar, 23Kuemmerle J.F. Murthy K.S. Makhlouf G.M. Am. J. Physiol. 1995; 266: C1421-C1431Crossref Google Scholar, 24Kuemmerle J.F. Makhlouf G.M. J. Biol. Chem. 1995; 270: 25488-25494Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). AA-induced muscle contraction (36.2 ± 2.9 μm decrease in muscle cell length) was inhibited by activators of cGMP-dependent protein kinase (SNP and 8-pcCPT-cGMP) and cAMP-dependent protein kinase (1 μmisoproterenol and cBIMPS) (Figs. 9 and10). The inhibition of contraction (i.e. relaxation) induced by SNP and 8-pcCPT-cGMP was selectively blocked by KT-5823 (Fig. 9), whereas the inhibition of contraction induced by 1 μm isoproterenol and cBIMPS was selectively blocked by H-89 (Fig. 10). Thus, although cAMP-dependent protein kinase had no effect on AA-induced Ca2+ influx, it inhibited contraction by acting at one or more loci distal to AA-induced Ca2+ influx.Figure 10Blockade of relaxation in dispersed smooth muscle cells by cAMP-dependent protein kinase inhibitors. Contraction of dispersed intestinal smooth muscle cells treated with AA for 30 s was measured by scanning micrometry. Inhibition of AA-induced contraction (i.e.relaxation) by isoproterenol (1 μm) or cBIMPS (10 μm) measured in the presence and absence of H-89 and/or KT-5823 was expressed as the mean change in muscle cell length (μm). Results are means ± S.E. of three experiments. ∗∗, blockade of relaxation, p < 0.01.View Large Image Figure ViewerDownload (PPT) Agonist-induced activation of PLC-β and generation of IP3 are the initial steps in Ca2+ mobilization in most cell types. In vascular and visceral smooth muscle, including smooth muscle of the intestinal circular layer (26Murthy K.S. Grider J.R. Makhlouf G.M. Am. J. Physiol. 1991; 261: G937-G944PubMed Google Scholar, 27Cornwell T.L. Lincoln T.M. J. Biol. Chem. 1989; 264: 1146-1155Abstract Full Text PDF PubMed Google Scholar, 28Szewczak S.M. Behar J. Billet G. Hillemeier C. Rhim R.Y. Biancani P. Am. J. Physiol. 1990; 259: G239-G244PubMed Google Scholar, 29Tachado S.D. Akthar R.A. Abdel-Latif A.A. Invest. Ophthalmol. & Visual Sci. 1989; 30: 2232-2239PubMed Google Scholar, 30Komalavilas P. Lincoln T.M. J. Biol. Chem. 1996; 271: 21933-21938Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, 37Somlyo A.P. Somlyo A.V. Nature. 1994; 372: 231-236Crossref PubMed Scopus (1715) Google Scholar), relaxant agents inhibit Ca2+ mobilization by activating cAMP-dependent protein kinase and/or cGMP-dependent protein kinase. The kinases act on various molecular targets to inhibit Ca2+ release from sarcoplasmic stores and Ca2+ influx into the cell, and to stimulate Ca2+ efflux from the cell. The targets include the effector enzyme PLC-β, the sarcoplasmic IP3receptor/Ca2+ channel, plasmalemmal, and sarcoplasmic Ca2+-ATPase pumps, and plasmalemmal Ca2+ and K+ channels, all of which are affected by both cAMP- and cGMP-dependent protein kinases, except for the sarcoplasmic Ca2+-ATPase pump, which is selectively inhibited by cGMP-dependent protein kinase (31Murthy K.S. Severi C. Grider J.R. Makhlouf G.M. Am. J. Physiol. 1993; 262: G967-G974Google Scholar, 32Murthy K.S. Makhlouf G.M. Am. J. Physiol. 1995; 268: C171-C180Crossref PubMed Google Scholar, 38Rashatwar S.S. Cornwell T.L. Lincoln T.M. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 5685-5689Crossref PubMed Scopus (127) Google Scholar, 39Twort C.H.C. Van Breemen C. Circ. Res. 1988; 62: 961-964Crossref PubMed Scopus (154) Google Scholar, 40Yoshida Y. Sun H.-T. Cai J.-Q. Imai S. J. Biol. Chem. 1991; 266: 19819-19825Abstract Full Text PDF PubMed Google Scholar, 41Chen X.-L. Rembold C.M. Am. J. 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Ca2+ mobilization in smooth muscle from the intestinal longitudinal layer, however, differs markedly in that it is initiated by G protein-dependent activation of cPLA2 and generation of arachidonic acid; the latter triggers Ca2+influx and induces Ca2+ release from sarcoplasmic stores via ryanodine receptors/Ca2+ channels (22Murthy K.S. Kuemmerle J.F. Makhlouf G.M. Am. J. Physiol. 1995; 269: G93-G102Crossref PubMed Google Scholar, 23Kuemmerle J.F. Murthy K.S. Makhlouf G.M. Am. J. Physiol. 1995; 266: C1421-C1431Crossref Google Scholar). Minimal amounts of IP3 are produced in this smooth muscle, which is virtually devoid of IP3 receptors/Ca2+ channels (23Kuemmerle J.F. Murthy K.S. Makhlouf G.M. Am. J. Physiol. 1995; 266: C1421-C1431Crossref Google Scholar, 26Murthy K.S. Grider J.R. Makhlouf G.M. Am. J. Physiol. 1991; 261: G937-G944PubMed Google Scholar). In this study we show that by analogy with other types of smooth muscle, the initial steps in Ca2+ mobilization in longitudinal smooth muscle, i.e. activation of cPLA2 and arachidonic acid-induced Ca2+ influx, are inhibited by cAMP- or cGMP-dependent protein kinase. It is possible that more distal targets involved in Ca2+mobilization in intestinal longitudinal muscle, such as ryanodine receptors/Ca2+ channels, Ca2+-ATPase pumps, and plasmalemmal Ca2+ and K+ channels, are also susceptible to regulatory phosphorylation by cAMP- and cGMP-dependent protein kinases, but these were not examined in the present study. Agonist-induced phosphorylation of cPLA2 in various cell types is accompanied by increase in cPLA2 activity, which appears to be mediated by protein kinase C-dependent and -independent activation of mitogen-activated protein kinase (19Qui Z.-H. Leslie C.C. J. Biol. Chem. 1994; 269: 19480-19487Abstract Full Text PDF PubMed Google Scholar, 20Leslie C.C. J. Biol. 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In the present study we show a delayed, protein kinase C-dependent, stimulatory phosphorylation of cPLA2 induced by the contractile agonist CCK and demonstrate for the first time an inhibitory phosphorylation induced by relaxant agonists (SNP, isoproterenol, and VIP) and activators of cAMP-dependent protein kinase (cBIMPS) and cGMP-dependent protein kinase (8-pcCPT-cGMP). The different patterns of phosphorylation by contractile and relaxant agonists suggest that the residues phosphorylated by cAMP- and cGMP-dependent protein kinases are distinct from the residues (chiefly Ser505) phosphorylated by protein kinase C-dependent mechanisms (14Lin L.L. Lin A.Y. Knopf J.L. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6147-6151Crossref PubMed Scopus (518) Google Scholar, 17Qui Z.-H. de Carvalho M.S. Leslie C.C. J. Biol. Chem. 1993; 268: 24506-24513Abstract Full Text PDF PubMed Google Scholar, 18de Carvalho M.G.S. McCormack A.L. Olson E. Ghomashchi F. Gelb M.H. Yates III, J.R. Leslie C.C. J. Biol. Chem. 1996; 271: 6987-6997Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 19Qui Z.-H. Leslie C.C. J. Biol. Chem. 1994; 269: 19480-19487Abstract Full Text PDF PubMed Google Scholar, 20Leslie C.C. J. Biol. Chem. 1997; 272: 16709-16712Abstract Full Text Full Text PDF PubMed Scopus (738) Google Scholar). Phosphorylation of cPLA2 induced by relaxant agents that preferentially activated cAMP-dependent protein kinase (i.e. low concentrations of isoproterenol) was blocked by the selective cAMP-dependent protein kinase inhibitors H-89 and myristoylated PKI, whereas phosphorylation induced by relaxant agents that preferentially activated cGMP-dependent protein kinase (i.e. SNP) was blocked by the selective cGMP-dependent protein kinase inhibitor KT-5823. Phosphorylation induced by agents that activated both cAMP- and cGMP-dependent protein kinases (i.e. VIP) was partially blocked by cAMP- and cGMP-dependent protein kinase inhibitors and abolished by a combination of both inhibitors (32Murthy K.S. Makhlouf G.M. Am. J. Physiol. 1995; 268: C171-C180Crossref PubMed Google Scholar). The pattern of inhibition of cPLA2 activity by cAMP- and cGMP-dependent protein kinases paralleled the pattern of phosphorylation of cPLA2, suggesting that inhibition of activity was probably mediated by phosphorylation. The initial peak (1 min) of cPLA2 activity elicited by the contractile agonist CCK-8 was inhibited by both cAMP- and cGMP-dependent protein kinases. The inhibition induced by cBIMPS and low concentrations of isoproterenol was selectively blocked by the cAMP-dependent protein kinase inhibitors H-89 and myristoylated PKI, whereas the inhibition induced by 8-pcCPT-cGMP and SNP was selectively blocked by the selective cGMP-dependent protein kinase inhibitor KT-5823. Inhibition induced by VIP and high concentrations of isoproterenol, which activate both cAMP- and cGMP-dependent protein kinases, was partially blocked by cAMP- and cGMP-dependent protein kinase inhibitors and completely blocked by a combination of both inhibitors (32Murthy K.S. Makhlouf G.M. Am. J. Physiol. 1995; 268: C171-C180Crossref PubMed Google Scholar). The next step in Ca2+ mobilization in intestinal longitudinal smooth muscle, i.e. arachidonic acid-induced Ca2+ influx (22Murthy K.S. Kuemmerle J.F. Makhlouf G.M. Am. J. Physiol. 1995; 269: G93-G102Crossref PubMed Google Scholar, 23Kuemmerle J.F. Murthy K.S. Makhlouf G.M. Am. J. Physiol. 1995; 266: C1421-C1431Crossref Google Scholar, 24Kuemmerle J.F. Makhlouf G.M. J. Biol. Chem. 1995; 270: 25488-25494Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar), was inhibited by agents that activated cGMP-dependent protein kinase only (i.e. SNP, 8-pcCPT-cGMP, and high concentrations of isoproterenol). The molecular target of cGMP-dependent protein kinase in this instance could be either Cl−channels or voltage-sensitive Ca2+ channels. Previous studies (46Kuemmerle J.F. Murthy K.S. Makhlouf G.M. Cell Biochem. Biophys. 1998; 28: 31-44Crossref PubMed Scopus (26) Google Scholar) have shown that arachidonic acid activates primarily Cl− channels, resulting in membrane depolarization and opening of voltage-sensitive Ca2+ channels. Consistent with this notion, depolarization and Ca2+ influx induced by contractile agonists was abolished by cPLA2 inhibitors and stilbene Cl− channel blockers; nifedipine, on the other hand, abolished Ca2+ influx but only partially inhibited depolarization, implying that Ca2+ influx was only a minor contributor to membrane depolarization. Depolarization and Ca2+ influx induced by nanomolar concentrations of exogenous arachidonic acid was also abolished by stilbene Cl− channel blockers (46Kuemmerle J.F. Murthy K.S. Makhlouf G.M. Cell Biochem. Biophys. 1998; 28: 31-44Crossref PubMed Scopus (26) Google Scholar). The present study addressed only the initial steps in Ca2+mobilization and showed that inhibition of the effector enzyme cPLA2, which mirrors inhibition of PLC-β in other smooth muscle cell types, was mediated by cAMP- and cGMP-dependent protein kinases, whereas inhibition of AA-induced Ca2+influx was exclusively mediated by cGMP-dependent protein kinase. Blockade of either step by cAMP-dependent protein kinase and/or cGMP-dependent protein kinase should lead to a decrease in [Ca2+]i and inhibition of muscle contraction. A decrease in [Ca2+]i, however, could also result from inhibition of Ca2+-induced Ca2+ release, stimulation of Ca2+ uptake into intracellular stores, and Ca2+ extrusion from the cell. Furthermore, inhibition of muscle contraction could also reflect phosphorylation by cAMP- and cGMP-dependent protein kinases of targets distal to those involved in Ca2+ mobilization, such as myosin light chain kinase and/or phosphatase (37Somlyo A.P. Somlyo A.V. Nature. 1994; 372: 231-236Crossref PubMed Scopus (1715) Google Scholar, 47Wu X. Somlyo A.V. Somlyo A.P. Biochem. Biophys. Res. Commun. 1996; 220: 658-663Crossref PubMed Scopus (137) Google Scholar). The importance of the inhibitory processes examined in the present study resides in their location at the start of the signaling cascade.

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