The regulation of NHE₁ and NHE₃ activity by angiotensin II is mediated by the activation of the angiotensin II type I receptor/phospholipase C/calcium/calmodulin pathway in distal nephron cells.
Angiotensin II (Ang II), acting via the AT1 receptor, induces an increase in intracellular calcium [Ca(2+)]i that then interacts with calmodulin (CaM). The Ca(2+)/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 Ang II-mediated regulation of NHE1 and NHE3 in Madin-Darby canine kidney (MDCK) cells. The NHE1- and NHE3-dependent pHi 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 (1pM or 1 nM) for 30 min induced, via the AT1 but not the AT2 receptor, an equal increase in NHE1 and NHE3 activity that was reduced by the specific inhibitors HOE 694 and S3226, respectively. Ang II (1 nM) did not change the total expression of NHE1, NHE3 or calmodulin, but it induced CaMKII, cRaf-1, Erk1/2 and p90(RSK) phosphorylation. The stimulatory effects of Ang II (1 nM) on NHE1 or NHE3 activity or protein abundance was reduced by ophiobolin-A (CaM inhibitor), KN93 (CaMKII inhibitor) or PD98059 (Mek inhibitor). These results indicate that after 30 min, Ang II treatment may activate G protein-dependent pathways, including the AT1/PLC/Ca(2+)/CaM pathway, which induces CaMKII phosphorylation to stimulate NHE3 and induces cRaf-1/Mek/Erk1/2/p90(RSK) activity to stimulate NHE1.
Department of Physiology and Biophysics, Instituto de Ciências Biomédicas. University of São Paulo, São Paulo 05508-900, Brazil.
Juliana Martins da
JM
Costa-Pessoa
Claudia Ferreira Dos Santos Ruiz
CF
Figueiredo
Karina
K
Thieme
Maria
M
Oliveira-Souza
D000804:Angiotensin II / Q000494:pharmacology*; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism*; D004285:Dogs; D005786:Gene Expression Regulation / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D042541:Intracellular Space / Q000737:chemistry / Q000187:drug effects; D061985:Madin Darby Canine Kidney Cells; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009399:Nephrons / Q000166:cytology*; D044140:Receptor, Angiotensin, Type 1 / Q000378:metabolism*; D044139:Receptor, Angiotensin, Type 2 / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism*; D010738:Type C Phospholipases / Q000378:metabolism*
D002147:Calmodulin; D044140:Receptor, Angiotensin, Type 1; D044139:Receptor, Angiotensin, Type 2; D017923:Sodium-Hydrogen Exchangers; D000804:Angiotensin II; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D020928:Mitogen-Activated Protein Kinases; D010738:Type C Phospholipases; D002118:Calcium
Angiotensin II; CaMKII; Erk1/2; NHE(1); NHE(3)