arachidonyltrifluoromethane and 1-6-bis(cyclohexyloximinocarbonyl)hexane

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

Since cytochrome P(450)-derived metabolites of arachidonic acid and K(+) have been implicated in endothelium-derived hyperpolarizing factor (EDHF)-dependent responses, the aim of this study was to determine whether such factors contribute to non-nitric oxide (NO), endothelium-dependent relaxation to bradykinin (BK) in bovine isolated coronary artery. In rings of artery contracted with U46619 and treated with indomethacin (3 microM) and N(G)-nitro-L-arginine (L-NOARG; 100 microM), relaxation to BK (0.01 nM-0.3 microM) was blocked by approximately 60% after inhibition of K(+) channels with either high extracellular K(+) (high [K(+)](o); 15 - 67 mM) or apamin (0.3 microM). Ouabain (1 microM), an inhibitor of Na(+)/K(+)-ATPase, decreased the sensitivity to BK without affecting the maximum response. In L-NOARG-treated rings, ouabain had no further effect on the relaxation to BK. An inhibitor of inward-rectifying K(+) channels, Ba(2+) (30 microM), had no effect on relaxations to BK in the absence or presence of either L-NOARG or ouabain. KCl (2.5 - 10 mM) elicited small relaxations ( approximately 20%) that were abolished by nifedipine (0.3 microM) and ouabain. Both the high [K(+)](o)/apamin-sensitive relaxation to BK, and the relaxation to the K(ATP) channel-opener, levcromakalim (0.6 microM), were unaffected by the cytochrome P(450) inhibitor, 7-ethoxyresorufin (10 microM), or by co-treatment with a phospholipase A(2) inhibitor, arachidonyl trifluoromethyl ketone (AACOCF(3); 3 microM) and a diacylglycerol (DAG)-lipase inhibitor, 1, 6-bis-(cyclohexyloximinocarbonylamino)-hexane (RHC 80267; 30 microM). The non-NO/high [K(+)](o)-insensitive, approximately 40% relaxation to BK was, however, abolished by these treatments. Therefore, neither cytochrome P(450)-derived metabolites of arachidonic acid nor K(+) appear to mediate the EDHF-like relaxation to BK (i.e the non-NO, high [K(+)](o)/apamin-sensitive component) in bovine coronary arteries. Cytochrome P(450)-derived metabolites may be released at higher BK concentrations to act in parallel with NO and the high [K(+)](o)/apamin-sensitive mechanism.

Topics: Animals; Apamin; Arachidonic Acid; Arachidonic Acids; Biological Factors; Bradykinin; Cattle; Coronary Vessels; Cromakalim; Cyclohexanones; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Endothelium, Vascular; Enzyme Inhibitors; In Vitro Techniques; Muscle Relaxation; Muscle, Smooth, Vascular; Nitric Oxide; Ouabain; Oxazines; Potassium; Potassium Chloride; Protease Inhibitors

Oxalate, a common constituent of kidney stones, is cytotoxic for renal epithelial cells. Although the exact mechanism of oxalate-induced cell death remains unclear, studies in various cell types, including renal epithelial cells, have implicated phospholipase A2 (PLA2) as a prominent mediator of cellular injury. Thus, these studies examined the role of PLA2 in the cytotoxic effects of oxalate.. The release of [3H]-arachidonic acid (AA) or [3H]-oleic acid (OA) from prelabeled Madin-Darby canine kidney (MDCK) cells was measured as an index for PLA2 activity. The cell viability was assessed by the exclusion of ethidium homodimer-1.. Oxalate exposure (175 to 550 microM free) increased the release of [3H]-AA in MDCK cells but had no effect on the release of [3H]-OA. Oxalate-induced [3H]-AA release was abolished by arachidonyl trifluoromethyl ketone (AACOCF3), a selective inhibitor of cytosolic PLA2 (cPLA2), but was not affected by selective inhibitors of secretory PLA2 and calcium-independent PLA2. The [3H]-AA release could be demonstrated within 15 minutes after exposure to oxalate, which is considerably earlier than the observed changes in cell viability. Furthermore, AACOCF3 significantly reduced oxalate toxicity in MDCK cells.. Oxalate increases AA release from MDCK cells by a process involving cPLA2. In addition, based on the evidence obtained using a selective inhibitor of this isoform, it would appear that the activity of this enzyme is responsible, at least in part, for the cytotoxic effects of oxalate. The finding that oxalate can trigger a known lipid-signaling pathway may provide new insight into the initial events in the pathogenesis of nephrolithiasis.

Topics: Anesthetics, Local; Animals; Arachidonic Acid; Arachidonic Acids; Biological Transport; Cell Line; Cyclohexanones; Dibucaine; Diglycerides; Dogs; Enzyme Inhibitors; Epithelial Cells; Free Radicals; Kidney Tubules, Distal; Oleic Acid; Oxalates; Phospholipases A; Phospholipases A2; Protease Inhibitors; Quinacrine; Tritium

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Arteries; Cyclohexanones; Enzyme Activation; Enzyme Inhibitors; Estrenes; In Vitro Techniques; Isoenzymes; Lipoprotein Lipase; Muscle, Smooth, Vascular; Naphthalenes; Norepinephrine; Phospholipases A; Pyrones; Pyrrolidinones; Rats; Terpenes; Type C Phospholipases

We have used an established cell line of rabbit cortical collecting duct (RCCD) epithelial cells representing a mixed population of principal and intercalated cell types to determine which phospholipase A2 (PLA2) enzyme therein is responsible for bradykinin (BK)-stimulated arachidonic acid (AA) release and how its activation is regulated. BK-stimulated AA release was reduced 92% by arachidonyl trifluoromethyl ketone, an inhibitor of cytosolic PLA2 (cPLA2). Examination of PLA2 activity in vitro demonstrated that BK stimulation resulted in a greater than twofold increase in PLA2 activity and that this activity was dithiothreitol insensitive and was inhibited by an antibody directed against cPLA2. To determine a possible role for protein kinase C (PKC) in the BK-mediated activation of cPLA2, we used the PKC-specific inhibitor Ro31-8220 and examined its effects on AA release, cPLA2 activity, and phosphorylation. Ro31-8220 reduced BK-stimulated AA release and cPLA2 activity by 51 and 58%, respectively. cPLA2 activity stimulated by phorbol ester [phorbol 12-myristate 13-acetate (PMA)] displayed a similar degree of activation and was associated with an increase in serine phosphorylation identical to that caused by BK. The phosphorylation-induced activation of this enzyme was confirmed by the phosphatase-mediated reversal of both BK- and PMA-stimulated cPLA2 activity. In addition, we have also found that PMA stimulation did not cause a synergistic potentiation of BK-stimulated AA release as did calcium ionophore. This occurred despite membrane PKC activity increasing 93% in response to PMA vs. 42% in response to BK. These data, taken together, indicate that cPLA2 is the enzyme responsible for BK-mediated AA release, and, moreover, they indicate that PKC is involved in the onset responses of cPLA2 to BK.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Bradykinin; Calcimycin; Cells, Cultured; Cyclohexanones; Cytosol; Enzyme Inhibitors; Kidney Cortex; Kidney Tubules, Collecting; Kinetics; Phospholipases A; Phospholipases A2; Phosphorylation; Protease Inhibitors; Protein Kinase C; Rabbits; Tetradecanoylphorbol Acetate