endothelin-1 and arachidonyltrifluoromethane

The present study investigates whether endothelin-1 (ET-1), like noradrenaline (NA), stimulates the release of arachidonic acid (AA) via cytosolic phospholipase A2 (cPLA2) in rat tail artery. In tail artery segments labelled with [3H]AA, ET-1-induced AA release in a concentration-dependent manner with an EC50 of 1.3 nM. The effect of ET-1 was inhibited by bosentan and was insensitive to BQ788, suggesting the involvement of ETA receptor. The stimulation of AA release induced by ET-1 was prevented by arachydonyl trifluoromethyl ketone (AACOCF3), a selective inhibitor of cPLA2 and not by RHC80267, a diacylglycerol lipase inhibitor. Furthermore, PD98059, inhibitor of mitogen-activated protein kinase kinase (MEK) cascade and calphostin C, a protein kinase C (PKC) inhibitor, prevented the stimulation of AA release induced by ET-1 and NA. Immunoblotting of the cytosolic fraction of rat tail arteries stimulated with ET-1 or NA showed an increase in extracellular signal-regulated kinases (ERKs) phosphorylation and this effect was abolished by calphostin C treatment. These findings show that in rat tail artery ET-1 and NA induce a sequential activation of protein kinase C and extracellular signal-regulated kinases that results in stimulation of AA release via cPLA2 activation. This may represent a general pathway by which G-proteins coupled receptors stimulate AA release and its metabolites in vascular smooth muscle.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Arteries; Carrier Proteins; Cyclohexanones; Drug Interactions; Endothelin Receptor Antagonists; Endothelin-1; Enzyme Activation; Enzyme Inhibitors; Intracellular Signaling Peptides and Proteins; Male; Mitogen-Activated Protein Kinases; Muscle, Smooth, Vascular; Norepinephrine; Phospholipases A; Phospholipases A2; Phosphorylation; Rats; Rats, Wistar; Tail; Tritium

We have investigated the role and mechanism of protein kinase C (PKC) isoforms in endothelin-1 (ET-1)-induced arachidonic acid (AA) release in cat iris sphincter smooth muscle (CISM) cells. ET-1 increased AA release in a concentration (EC50=8 nM) and time-dependent (t1/2=1.2 min) manner. Cytosolic phospholipase A2 (cPLA2), but not phospholipase C (PLC), is involved in the liberation of AA in the stimulated cells. This conclusion is supported by the findings that ET-1-induced AA release is inhibited by AACOCF3, quinacrine and manoalide, PLA2 inhibitors, but not by U-73122, a PLC inhibitor, or by RHC-80267, a diacylglycerol lipase inhibitor. A role for PKC in ET-1-induced AA release is supported by the findings that the phorbol ester, PDBu, increased AA release by 96%, that prolonged treatment of the cells with PDBu resulted in the selective down regulation of PKCalpha and the complete inhibition of ET-1-induced AA release, and that pretreatment of the cells with staurosporine or RO 31-8220, PKC inhibitors, blocked the ET-1-induced AA release. Gö-6976, a compound that inhibits PKCalpha and beta specifically, blocked ET-1-induced AA release in a concentration-dependent manner with an IC50 value of 8 nM. Thymeatoxin (0.1 microM), a specific activator of PKCalpha, beta, and gamma induced a 150% increase in AA release. Treatment of the cells with ET-1 caused significant translocation of PKCalpha, but not PKCbeta, from cytosol to the particulate fraction. These results suggest that PKCalpha plays a critical role in ET-1-induced AA release in these cells. Immunochemical analysis revealed the presence of cPLA2, p42mapk and p44mapk in the CISM cells. The data presented are consistent with a role for PKCalpha, but not for p42/p44 mitogen-activated protein kinase (MAPK), in cPLA2 activation and AA release in ET-1-stimulated CISM cells since: (i) the PKC inhibitor, RO 31-8220, inhibited ET-1-induced AA release, cPLA2 phosphorylation and cPLA2 activity, but had no inhibitory effect on p42/p44 MAPK activation, (ii) genistein, a tyrosine kinase inhibitor, inhibited ET-1-stimulated MAPK activity but had no inhibitory effect on AA release in the ET-1-stimulated cells. We conclude that in CISM cells, ET-1 activates PKCalpha, which activates cPLA2, which liberates AA for prostaglandin synthesis.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Biological Transport; Calcium-Calmodulin-Dependent Protein Kinases; Carbazoles; Cats; Cells, Cultured; Dose-Response Relationship, Drug; Down-Regulation; Endothelin-1; Genistein; Indoles; Iris; Isoenzymes; Lipoprotein Lipase; Muscle, Smooth; Phorbol 12,13-Dibutyrate; Phorbol Esters; Phospholipases A; Phospholipases A2; Prostaglandin-Endoperoxide Synthases; Protein Kinase C; Protein Kinase C-alpha