Murthy K S, Makhlouf G M
Departments of Physiology and Medicine, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia 23298-0711, USA.
J Biol Chem. 1998 Dec 18;273(51):34519-26. doi: 10.1074/jbc.273.51.34519.
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.
环磷酸腺苷（cAMP）依赖性蛋白激酶和环磷酸鸟苷（cGMP）依赖性蛋白激酶均能抑制激动剂刺激的磷脂酶C-β（PLC-β）活性以及血管和内脏平滑肌中肌醇1,4,5-三磷酸（IP3）依赖性Ca2+释放。然而，在小肠纵行肌中，Ca2+动员的起始步骤涉及胞质型磷脂酶A2（cPLA2）的激活以及花生四烯酸（AA）依赖性的Ca2+内流刺激。本研究还检测了cAMP依赖性蛋白激酶和cGMP依赖性蛋白激酶是否也能够调节这些过程。激活cAMP依赖性蛋白激酶的试剂（5,6-二氯-1-β-D-呋喃核糖基苯并咪唑3',5'-环单磷酸硫酯（Sp-异构体）和异丙肾上腺素）、cGMP依赖性蛋白激酶（8-（4-氯苯硫基）-鸟苷3',5'-环单磷酸和硝普钠）或两种激酶（血管活性肠肽和大于1μM的异丙肾上腺素）均可诱导cPLA2磷酸化并抑制激动剂刺激的cPLA2活性。cAMP依赖性蛋白激酶和cGMP依赖性蛋白激酶对cPLA2的磷酸化及活性抑制作用可被相应的选择性抑制剂阻断（cAMP依赖性蛋白激酶，N-[2-（对溴肉桂氨基）乙基]-5-异喹啉磺酰胺盐酸盐（H-89）和肉豆蔻酰化蛋白激酶抑制剂（）酰胺；cGMP依赖性蛋白激酶，（8R,9S,11S）-（-）-9-甲氧基甲酰基-8-甲基-2,3,9,10-四氢-8,11-环氧-1H,8H,11H-2,7b,11a-三氮杂二苯并（a,g）环辛（c,d,e）-三茚-1-酮（KT-5823））。相反，AA刺激的Ca2+内流仅被激活cGMP依赖性蛋白激酶的试剂所抑制；该抑制作用可被KT-5823选择性阻断。本研究首次提供了体内cAMP依赖性蛋白激酶和cGMP依赖性蛋白激酶对cPLA2进行抑制性磷酸化的证据。cPLA2活性的抑制以及AA诱导的Ca2+内流部分解释了cAMP依赖性蛋白激酶和/或cGMP依赖性蛋白激酶引起舒张的能力。它们的重要性在于其位于Ca2+信号级联反应的起始位置。
