Abstract
Both cAMP- and cGMP-dependent protein kinases inhibit agonist-stimulated phospholipase C-beta (PLC-beta) 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-beta-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 microM) 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 () 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.
Highlights
Both cAMP- and cGMP-dependent protein kinases inhibit agonist-stimulated phospholipase C- (PLC-) activity and inositol 1,4,5-trisphosphate-dependent Ca2؉ release in vascular and visceral smooth muscle
The targets include the effector enzyme PLC-, the sarcoplasmic IP3 receptor/Ca2ϩ channel, plasmalemmal, and sarcoplasmic Ca2ϩ-ATPase pumps, and plasmalemmal Ca2ϩ and Kϩ channels, all of which are affected by both cAMP- and cGMP-dependent protein kinases, except for the sarcoplasmic Ca2ϩ-ATPase pump, which is selectively inhibited by cGMP-dependent protein kinase (31, 32, 38 – 42)
Ca2ϩ mobilization in smooth muscle from the intestinal longitudinal layer, differs markedly in that it is initiated by G protein-dependent activation of cytosolic PLA2 (cPLA2) and generation of arachidonic acid; the latter triggers Ca2ϩ influx and induces Ca2ϩ release from sarcoplasmic stores via ryanodine receptors/ Ca2ϩ channels [22, 23]
Summary
Both cAMP- and cGMP-dependent protein kinases inhibit agonist-stimulated phospholipase C- (PLC-) activity and inositol 1,4,5-trisphosphate-dependent Ca2؉ release in vascular and visceral smooth muscle. The initial steps in Ca2ϩ mobilization in vascular and intestinal circular smooth muscle (i.e. activation of PLC- and stimulation of Ca2ϩ release) are inhibited by cAMP- and cGMP-dependent protein kinases [27,28,29,30,31,32].
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