Mechanism of endothelin activation of phospholipase A2 in rat renal medullary interstitial cells

Abstract

Previous studies from this laboratory have demonstrated that endothelin-1 (ET) stimulates phosphatidylinositol (PI) hydrolysis, activates dihydropyridine-insensitive Ca2+ channels, and promotes prostaglandin E2 (PGE2) accumulation in cultured rat renal medullary interstitial cells (RMIC). The mechanism whereby ET augments PGE2 production was explored in the current study. ET-evoked PGE2 accumulation proceeded independent of large increments in cytosolic free Ca2+ concentration ([Ca2+]i), derived from either extracellular or intracellular sources. Chelation of intracellular Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid eliminated ET-evoked PGE2 production, indicating that eicosanoid production was nonetheless a Ca(2+)-requiring process. Nanomolar concentrations of phorbol 12-myristate 13-acetate (PMA) alone did not stimulate PGE2 production, nor did PMA alter ET-stimulated PGE2 accumulation. Furthermore, downregulation of protein kinase C (PKC) by prolonged exposure of cells to PMA did not mitigate ET-mediated PGE2 production, demonstrating that PKC stimulation was not required for PGE2 production. ET stimulated PGE2 accumulation despite PI-specific phospholipase C (PI-PLC) inhibition by nanomolar concentrations of PMA, indicating that eicosanoid production was not a downstream event of PI hydrolysis. ET stimulated arachidonic acid metabolite release in parallel with a loss of label from membrane phospholipids. Phosphatidylethanolamine was the preferred substrate for ET-mediated activation of phospholipase A2 (PLA2). Immunocytochemical studies including immunostaining, immunoblotting, and immunoprecipitation confirmed the presence of cytosolic PLA2 (cPLA2) in RMIC. In summary, ET stimulation of PGE2 production in RMIC is mediated via agonist activation of cPLA2 independent of activation of PI-PLC, suggesting direct coupling to the ET receptor. Constitutive levels of [Ca2+]i rather than abrupt increments in [Ca2+]i are sufficient for activation of this receptor-effector system, with no obligatory requirement for PKC.

Cytochrome P450 and Cyclooxygenase Metabolites Contribute to the Endothelin-1 Afferent Arteriolar Vasoconstrictor and Calcium Responses

“…3,7,9 Actions of ET-1, via ET A and ET B receptors, include phospholipase A 2 stimulation and the resultant increase in arachidonic acid-derived mediators. 1,7,15,21 Previous studies have investigated the involvement of CYP450 hydroxylase and COX metabolites in the renal vasoconstrictor response to ET-1. 7,9,10,16, 22 Oyekan et al, using in vitro and in vivo techniques, have shown that the CYP450 hydroxylase pathway contributes to the ET-1-evoked decrease in renal blood flow, but the role of the COX pathway appears to be complex.…”

“…Second messengers responsible for the ET-1-mediated vascular response include phospholipase C activation and subsequent calcium mobilization from intracellular stores, as well as phospholipase A 2 stimulation, resulting in production of arachidonic acid metabolites. 1,14,21,25 In freshly isolated smooth muscle cells from large-caliber renal vessels, Gordienko et al 25 demonstrated that ET-1-induced mobilization of intracellular calcium was accompanied by membrane depolarization. ET-1 also stimulated Ca 2ϩ -activated K ϩ channels in renal vascular smooth muscle cells.…”

“…16 ET-1 also has the capacity to increase production of vasodilatory prostaglandins (PGs), PGI 2 and PGE 2 , as well as vasoconstrictor COX metabolites, PGF 2␣a , PGH 2 , and TX. 1,10,14,21 Interestingly, the CYP450 hydroxylase metabolite 20-HETE can be transformed by COX to a vasoconstrictor PGH 2 analogue, 20-OH PGH 2 . 31,32 Consistent with the hypothesis that 20-HETE conversion to a TX/PGH 2 (TP) receptor agonist contributes to its action, the aortic vasoconstriction in response to 20-HETE can be blocked by COX inhibition or TP receptor antagonism.…”

Epstein-Barr virus infection of renal proximal tubule cells: possible role in chronic interstitial nephritis

“…Immunocytochemical staining for CD21 was carried out on acetone-fixed, 5-µm isopentane-liquid nitrogen frozen sections according to a procedure adapted from methods described previously by this laboratory (21). Optimal dilutions of the first antibody, determined by checkerboard titration, were incubated with the tissue at room temperature for 60-90 minutes followed by 3 rinses in buffered saline at 3°-5°C.…”

Altered Endothelin-1 Signaling in Production of Thromboxane A2 in Kupffer Cells from Bile Duct Ligated Rats

“…ET-1 induced the phosphorylation of cPLA 2 at ser505 in KCs from BDL and sham animals; however, the effect was significantly greater in KCs from BDL animals. In other cell types, ET-1 has been demonstrated to activate cPLA 2 and the release of various eicosanoids including TXA 2 , via binding to Gq/G11 and Gi coupled ET (A) and ET (B) receptors, subsequently inducing the activation of ERK1/2 or p38 MAPK through PLC or PKC mediated signaling pathways (38,39,41,42,44,45,47,(55)(56)(57)(58)(59)(60)(61). Therefore, the purposes of the present study were to delineate the signaling pathways induced via stimulation of Gq/G11 and Gi coupled ETBRs and the relative roles of intermediates in the activation of cPLA 2 and production of TXA 2 in KCs in response to ET-1, and determine whether the hepatic stress of early stage fibrosis alters the signaling pathways through modifications in the expressions or activities of signaling intermediates.…”