The regulation of NHE1 and NHE3 activity by angiotensin ll is mediated by the activation of the angiotensin ll type I receptor/phospholipase C/calcium/calmodulin pathway in distal nephron cells

Angiotensin II (Ang II), acting via the AT(1) receptor, induces an increase in intracellular calcium [Ca2+ I; that then interacts with calmodulin (CaM). The Ca2+/Cam complex directly or indirectly activates sodium hydrogen exchanger 1 (NHE1) and phosphorylates calmodulin kinase II (CaMKII), which then regulates sodium hydrogen exchanger 3 (NHE3) activity. In this study, we investigated the cellular signaling pathways responsible for Aug II mediated regulation of N1-IE1 and NHE3 in Madin-Darby canine kidney (MDCK) cells. The NHE1- and NHE3 dependent pH, recovery rates were evaluated by fluorescence microscopy using the fluorescent probe BCECF/AM, messenger RNA was evaluated with the reverse transcription polymerase chain reaction (RT-PCR), and protein expression was evaluated by immunoblot. We demonstrated that treatment with Ang II (1 pM or 1 nM) for 30 min induced, via the AT3 but not the AT(2) receptor, an equal increase in NHE1 and NHE3 activity that was reduced by the specific inhibitors HOE 694 and S3226, respectively. Ang (1 nM) did not change the total expression of NHE1, NHE3 or calmoclulin, but it induced CaMKii, cRaf-1, Erk1/2 and p9(ORsK) phosphorylation. The stimulatory effects of Ang 11 (1 nM) on NHE1 or NHE3 activity or protein abundance was reduced by ophiobolin-A (CaM inhibitor), KN93 (CaMKII inhibitor) or P098059 (Mek inhibitor). These results indicate that after 30 min, Ang II treatment may activate G protein dependent pathways, including the ATI/PLC/Ca2+/CaM pathway, which induces CaMKII phosphorylation to stimulate NHE3 and induces cRaf-1/Mek/Erk1/2/p90(RSK) activity to stimulate NHE1. (C) 2013 Elsevier RN. All rights reserved.