Involvement of calcium/calmodulin-dependent protein kinase II to endotoxin-induced vascular hyporeactivity in rat superior mesenteric artery.
ABSTRACT Endotoxin causes impaired vascular contractility proposed to be mediated mainly by induction of inducible nitric oxide synthase (iNOS). Evidence suggests that calcium/calmodulin dependent protein kinase II (CaMKII) may lead to activation of cytosolic phospholipase A(2alpha) (cPLA(2alpha))/inducible cyclooxygenase (COX-2) pathway in response to endotoxin in vascular smooth muscle cells. This study was conducted to determine if CaMKII is involved in the endotoxin-induced vascular hyporeactivity by activating of iNOS and/or cPLA(2alpha)/COX-2 enzymes in rat isolated superior mesenteric artery with endothelium. Incubation with endotoxin (100 microg ml(-1)) for 4h caused vascular hyporeactivity to norepinephrine which was completely abolished by phenylene-1,3-bis[ethane-2-isothiourea] dihydrobromide (1,3-PBIT), a selective iNOS inhibitor, methyl arachidonyl fluorophosphonate (MAFP), a selective 85kDa cPLA(2alpha) inhibitor, DFU, a selective COX-2 inhibitor, and KN-93, a selective CaMKII inhibitor. Endotoxin-induced increase in tissue nitrite production was decreased by 1,3-PBIT and DFU, and further increased by MAFP. MAFP, DFU and KN-93 reversed the endotoxin-induced decrease in tissue 6-keto-PGF(1alpha). These data suggest that reversal of the endotoxin-induced vascular hyporeactivity by inhibition of CaMKII in rat superior mesenteric artery may be related to increased production of vasodilator arachidonic acid products by cPLA(2alpha)/COX-2 pathway rather than prostacyclin and nitric oxide.
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ABSTRACT: Cytosolic phospholipase A(2)α (cPLA(2)α) is the most widely studied member of the Group IV PLA(2) family. The enzyme is Ca(2+)-dependent with specificity for phospholipid substrates containing arachidonic acid. As the pinnacle of the arachidonic acid pathway, cPLA(2)α has a primary role in the biosynthesis of a diverse family of eicosanoid metabolites, with potent physiological, inflammatory and pathological consequences. cPLA(2)α activity is regulated by pro-inflammatory stimuli through pathways involving increased intracellular Ca(2+) levels, phosphorylation coupled to increased enzymatic activity and de novo gene transcription. This study addresses the signal transduction pathways for protein phosphorylation and gene induction following IL-1β stimulation in human fetal lung fibroblasts. Our results utilizing both inhibitors and kinase-deficient cells demonstrate that cPLA(2)α is phosphorylated within 10min of IL-1β treatment, an event requiring p38 MAPK as well as the upstream kinase, MKK3/MKK6. Inhibition of p38 MAPK also blocks the phosphorylation of a downstream, nuclear kinase, MSK-1. Our results further demonstrate that the activities of both cPLA(2)α and a downstream lipoxygenase (15-LOX2) are required for IL-1β-dependent induction of cPLA(2)α mRNA expression. Overall, these data support an MKK3/MKK6→p38 MAPK→MSK-1→cPLA(2)α→15-LOX2-dependent, positive feedback loop where a protein's enzymatic activity is required to regulate its own gene induction by a pro-inflammatory stimulus.Cellular Signalling 07/2011; 23(12):1944-51. · 4.47 Impact Factor
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ABSTRACT: The aim of the present study was to elucidate the direct effects of melatonin on bladder activity and to determine the mechanisms responsible for the detrusor activity of melatonin in the isolated rat bladder. We evaluated the effects of melatonin on the contractions induced by phenylephrine (PE), acetylcholine (ACh), bethanechol (BCh), KCl, and electrical field stimulation (EFS) in 20 detrusor smooth muscle samples from Sprague-Dawley rats. To determine the mechanisms underlying the inhibitory responses to melatonin, melatonin-pretreated muscle strips were exposed to a calcium channel antagonist (verapamil), three potassium channel blockers [tetraethyl ammonium (TEA), 4-aminopyridine (4-AP), and glibenclamide], a direct voltage-dependent calcium channel opener (Bay K 8644), and a specific calcium/calmodulin-dependent kinase II (CaMKII) inhibitor (KN-93). Melatonin pretreatment (10(-8)~10(-6) M) decreased the contractile responses induced by PE (10(-9)~10(-4) M) and Ach (10(-9)~10(-4) M) in a dose-dependent manner. Melatonin (10(-7) M) also blocked contraction induced by high KCl ([KCl](ECF); 35 mM, 70 mM, 105 mM, and 140 mM) and EFS. Melatonin (10(-7) M) potentiated the relaxation response of the strips by verapamil, but other potassium channel blockers did not change melatonin activity. Melatonin pretreatment significantly decreased contractile responses induced by Bay K 8644 (10(-11)~10(-7) M). KN-93 enhanced melatonin-induced relaxation. The present results suggest that melatonin can inhibit bladder smooth muscle contraction through a voltage-dependent, calcium-antagonistic mechanism and through the inhibition of the calmodulin/CaMKII system.Korean Journal of Physiology and Pharmacology 02/2012; 16(1):37-42. · 1.00 Impact Factor
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ABSTRACT: Up-regulation of cyclooxygenase (COX)-2 and prostaglandin E2 (PGE2) are implicated in lung inflammation. Adenosine 5'-triphosphate (ATP) has been shown to act via activation of P2 purinoceptors, leading to COX-2 expression in various inflammatory diseases. The mechanisms of ATP-induced COX-2 expression and PGE2 release remain unclear. We showed that pretreatment with the inhibitors of P2 receptors (PPADS and Suramin), Gq protein (GPA2A), phosphatidylcholine-phospholipase C (PC-PLC; D609), phosphoinositide-phospholipase C (PI-PLC; ET-18-OCH3), Ca(2+)/calmodulin-dependent protein kinase II (CaMKII; KN62), protein kinase C (PKC; Gö6976, Ro-318220, GF109203X, and rottlerin), MEK1/2 (PD98059), p38 MAPK (SB202190), and nuclear factor-kappaB (NF-κB; Bay11-7082) and the intracellular calcium chelator (BAPTA/AM) or transfection with siRNAs of these molecules and cPLA2 reduced ATPγS-induced COX-2 expression or PGE2 production in A549 cells. In addition, ATPγS-induced elevation of intracellular Ca(2+) concentration was attenuated by PPADS, Suramin, D609, or ET-18-OCH3. ATPγS-induced p38 MAPK, p42/p44 MAPK, and NF-κB p65 activation were inhibited by Gö6976, Ro-318220, GF109203X, or rottlerin. ATPγS also induced cPLA2 phosphorylation and activity, which were reduced via inhibition of P2 receptors, PKCs, p38 MAPK, and p42/p44 MAPK. ATPγS-induced cPLA2 expression was inhibited by SB202190, PD98059, or Bay11-7082. In the in vitro study, we established that ATPγS induced PGE2 generation via a cPLA2/COX-2-dependent pathway. In the in vivo study, we found that ATPγS induced COX-2 mRNA expression in the lungs and leukocyte (mainly eosinophils and neutrophils) count in bronchoalveolar lavage (BAL) fluid in mice via a P2 receptors-dependent signaling pathway. We concluded that ATPγS may induce lung inflammation via a cPLA2/COX-2/PGE2-dependent pathway.The international journal of biochemistry & cell biology 05/2013; · 4.89 Impact Factor