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Endothelin-1 activates p38 mitogen-activated protein kinase and cytosolic phospholipase A2 in cat iris sphincter smooth muscle cells.

ABSTRACT: We have shown previously that cytosolic phospholipase A(2) (cPLA(2)) is responsible for endothelin-1-induced release of arachidonic acid for prostaglandin synthesis in cat iris sphincter smooth muscle (CISM) cells [Husain and Abdel-Latif (1998) Biochim. Biophys. Acta 1392, 127-144]. Here we show that p38 mitogen-activated protein (MAP) kinase, but not p42/p44 MAP kinases, plays an important role in the phosphorylation and activation of cPLA(2) in endothelin-1-stimulated CISM cells. This conclusion is supported by the following findings. Both p38 MAP kinase and p42/p44 MAP kinases were present in the CISM cells and both were activated by endothelin-1. SB203580, a potent specific inhibitor of p38 MAP kinase, but not the p42/p44 MAP kinases specific inhibitor, PD98059, markedly suppressed endothelin-1-enhanced cPLA(2) phosphorylation, cPLA(2) activity and arachidonic acid release. The addition of endothelin-1 resulted in the phosphorylation and activation of cPLA(2). Endothelin-1 stimulated p38 MAP kinase activity in a time- and concentration-dependent manner, and these effects were mediated through the endothelin-A receptor subtype. The protein kinase C (PKC) inhibitor, RO 31-8220, had no inhibitory effect on endothelin-1-induced p38 MAP kinase activation, suggesting that endothelin-1 activation of p38 MAP kinase is independent of PKC. Pertussis toxin inhibited both endothelin-1 and mastoparan stimulation of p38 MAP kinase activity and arachidonic acid release. The inhibitory effects of pertussis toxin are not mediated through cAMP formation. Mastoparan-stimulated [(3)H]arachidonic acid release and cPLA(2) activation was inhibited by SB203580, but not by RO 31-8220. These data suggest that endothelin-1 binds to the endothelin-A receptor to activate the Gi-protein which, through a series of kinases, leads to the activation of p38 MAP kinase and subsequently to phosphorylation and activation of cPLA(2). Activation of cPLA(2) leads to the liberation of arachidonic acid from membrane phospholipids. The ability of the activated endothelin-A receptor, which is coupled to both Gq- and Gi-proteins, to recruit and activate this complex signal transduction pathway remains to be elucidated. Further studies on the mechanism of these relationships could provide important information about the functions of p38 MAP kinase in smooth muscle.

SUBMITTER: Husain S

PROVIDER: S-EPMC1220440 | biostudies-other | 1999 Aug

REPOSITORIES: biostudies-other

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Project description:Two forms (I and II) of phosphoinositide-specific phospholipase C (PLC) were purified from the cytosol of bovine iris sphincter by sequential chromatography on DEAE-Sepharose, EAH-Sepharose, heparin-Sepharose, Sephacryl S-200 gel filtration and Mono Q HR columns. The final step resulted in specific activities of PLC-I and PLC-II of 4.3 and 5.9 mumol of phosphatidylinositol (PI) cleaved/min per mg of protein, which represented up to 295-fold purification compared with that of the starting supernatant. The purified enzymes were further investigated for the presence of isoenzymes and characterized for molecular mass, substrate specificity, pH, Ca2+ requirements and kinetic parameters. Using monoclonal antibodies, PLC-I was identified as PLC-delta 1. The apparent molecular mass of PLC-I as determined by SDS/PAGE and gel filtration was 85 kDa. PLC-II contained an apparently invisible protein band that reacted with the antibody against PLC-gamma 1, and a major 109 kDa protein band that was not recognized by any of the PLC monoclonal antibodies. Further purification of PLC-II by size-exclusion h.p.l.c. resulted in elution of the enzyme activity as a single peak which corresponded to 109 kDa position. Again, this PLC activity was not recognized by any of the PLC monoclonal antibodies. However, the 109 kDa protein activity was recognized by a polyclonal antibody raised against a rat PLC-gamma 1 fragment (amino acids 1272-1287), thus suggesting that this protein is a proteolytic product of PLC-gamma 1. PLC-delta 1 and PLC-gamma 1 were identified in the supernatant fraction and PLC-beta 1 in the membrane fraction of the iris sphincter. Although immunologically different, the catalytic properties of PLC-I and PLC-II were quite similar. The Vmax and Km values for phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis were three to five times greater than those for PI hydrolysis. Both forms preferred PIP and PIP2 over PI and both were inactive against phosphatidylcholine. With PIP2 as substrate, the optimal pH values for PLC-I and PLC-II were 6.5 and 7.5 respectively. Unlike PIP2, PI hydrolysis by both forms was dependent on the presence of free Ca2+. The maximal hydrolysis of PI and PIP2 by both forms occurred at 200 and 5 microM Ca2+ respectively. Incubation of the purified enzymes with the catalytic subunit of protein kinase A (PKA) and [gamma-32P]ATP resulted in increased phosphorylation of PLC-I and PLC-II, but it had no inhibitory effect on their enzyme activities.(ABSTRACT TRUNCATED AT 400 WORDS)

Project description:1. The effects of endothelin-1 (ET-1) on guinea-pig lower oesophageal sphincter (LOS) circular smooth muscle were investigated by using intracellular microelectrodes and isometric tension recording techniques. 2. ET-1 produced biphasic mechanical responses; an initial transient relaxation followed by a sustained contraction. The initial relaxation was not inhibited by either tetrodotoxin (TTX, 1 microM) or L-N(G)-nitroarginine (L-NOARG, 100 microM). The sustained contraction was greatly attenuated by nifedipine (1 microM). 3. ET-1 (1 - 30 nM) induced a concentration-dependent hyperpolarisation that was unaffected by TTX or L-NOARG. The ET(A) receptor antagonist, BQ123 (0.3 microM) abolished the ET-1-induced hyperpolarisation, whereas the ET(B) receptor antagonist, BQ788 (0.3 microM) had no detectable effect. Sarafotoxin S6c (10 nM) did not change the membrane potential. 4. The ET-1-induced hyperpolarisation was abolished by apamin (0.1 microM). Interestingly, apamin abolished the ET-1-induced transient relaxation but potentiated the sustained contraction. 5. In Ca(2+)-free Krebs solution, the ET-1-induced hyperpolarisation was greatly attenuated and returned to the control value when the tissue was reperfused with Krebs solution containing Ca(2+). The ET-1-induced hyperpolarisation was insensitive to nifedipine but was attenuated by SK&F 96365 (1 - [beta-[3-(4 - methoxy - phenyl)propoxy] - 4 - methoxyphenethyl] - 1H-imidazole hydrochloride, 50 microM), an inhibitor of receptor-mediated Ca(2+) entry. The residual component of the ET-1-induced hyperpolarisation was sensitive to thapsigargin (1 microM). 6. These results demonstrate that, in guinea-pig LOS circular smooth muscle, ET-1 hyperpolarizes the membrane by activating apamin-sensitive K(+) channels, mainly as a result of receptor-mediated Ca(2+) entry and partly by Ca(2+) release from intracellular stores. The hyperpolarisation triggers the initial transient relaxation, which acts to oppose the sustained contraction.

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Endothelin-1 activates p38 mitogen-activated protein kinase and cytosolic phospholipase A2 in cat iris sphincter smooth muscle cells.

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