engleski

Phospholipase D: A new transduction pathway for angiotensin II in cortical tubules from rat kidney

We have shown previosuly that 1) Angiotensin II (ANGII) activates luminal Na/H activity in a suspension of cortical tubule fragments through a protein kinase C (PKC)-signalling pathway;2) PKC alfa, delta, eta and zeta are expressed in cortical tubules and detected in the luminal membranes. In the present study the PKC acitvated by low ANGII concentrations was identified first. Then, the involvement of phospholipase D (PLD) as an alternative pathway to stimulate PKCs has been investigated. Using the Mg precipitation method, luminal membrane vesicles (LMV) fractions were isolated from suspensions of cortical tubules free of glomeruli, treated or not by ANGII and used for immunoblot analysis of PKCs. PLD activity was assessed by the production of phosphatidic acid (PtdOH) or phosphatidyethanol (PtdEtn) by (3H)alkyl-lyso-phosphatidylcholine labelled tubules. The abundance of the atypical PKC zeta, insensitive to Ca and to diglycerides, was increased by 49+10% (p<0.01) by ANGII 10(-10) M, 4 min pretreatement while that of other PKCs was not. In addition to phospatidylserine, PKC zeta co-activators content of PtdOH was enhanced by 22.57+7.53% (p<0.01) by ANG II 10(-10) M, 2.5 min. All subsequent experiments were performed in the presence of 1% ethanol in the incubation medium to shift PLD activity towards the transphosphatidylation pathway which gives PtdEtn, the unequivocal mark of PLD activity. ANG II elicited a time- and dose-dependent increase in PtdEtn accumulation (by 19.72+7.46 % (p<0.02) and 40.60+8.86% (p<0.001) for 10 (-10) M and 10 (-7) M respectively after 2.5 min and 63.49+19.46% (p<0.01) for 10(-7) M after 5 min). The effect of ANGII 10(-7) M was not modified by pretreating the tubles by cytochalasin D 10(-6) M, 10 min which indicated that ANG II-induced PLD activation was not under the contorol of cytoskeleton elements. Conclusions: 1) only the abundance of the atypical PKC zeta is enhanced in LMV by 10(-10) M ANG II, 2) a PLD acting on phosphatidylcholine and activated by ANGII could participate to PKC zeta regulation by providing one of its coactivtors, namely PtdOH.

phospholipase D; angiotensin II; protein kinase C; phosphatidic acid; rat kidney; cortical tubules

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