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

ANG II, ANG-(1-7), ALDO and AVP biphasic effects on Na+/H+ transport: the role of cellular calcium

“…Under these conditions, it inhibits NHE1 activity ( Figure 6). In more recent studies, it was demonstrated that in MDCK cells, the regulation of NHE1 and NHE3 activity by ANG II is mediated by the activation of the ANG II type I receptor/phospholipase C/calcium/ calmodulin pathway [118].…”

High Glucose Concentration Stimulates NHE-1 Activity in Distal Nephron Cells: the Role of the Mek/Erk1/2/p90RSKand p38MAPK Signaling Pathways

“…Cells at passage 55 were grown in 75-cm culture flasks containing 5 mM glucose- Dulbecco's Modified Eagle's Medium (DMEM) (Invitrogen, SP, Brazil, BR), as has been previously described [27]. For pH i experiments, cells were removed from the culture flasks using trypsin and 2.5x10 5 cells were seeded onto 25-mm-diameter glass coverslips inserted into 6-well plates containing complete DMEM medium.…”

“…As described in our previous studies [27,28,29], intracellular pH (pH i ) was monitored using the BCECF-AM fluorescent probe (Molecular Probes, Eugene, OR, USA). Cells grown to confluence on glass coverslips were fluorescently labeled using 5 µM BCECF-AM in control solution [28] for 3 minutes.…”

“…Control cells exposed to an extracellular glucose concentration of 5 mM or cells treated with a high glucose concentration of 25 mM and HOE-694 (NHE-1 inhibitor, 50 µM) [30], PD 98059 (Mek inhibitor, 1 µM) or SB 203580 (p38 MAPK inhibitor, 10 µM) (Merck-Millipore) were submitted to pH i recovery analysis. Cellular pH i recovery was examined after acid loading using the NH 4 Cl pulse techn0ique [27,31] by exposing the cells to 20 mM NH 4 Cl [(in mM) NaCl-125, KCl-5, MgCl 2 -1, NaH 2 PO 4 -0.8, Na 2 HPO 4 -0.83, CaCl 2 -1, HEPES-8, glucose-5 and NH 4 Cl-20; pH 7.4] for 2 minutes. As described in our previous studies [28,32].…”

“…Immunoblot analysis was performed on protein aliquots containing 50 µg/lane of proteins resolved in 10% SDS-PAGE as previously described [27,28]. The following primary antibodies were used in this study: rabbit anti-Glut2 (1:1000); mouse anti-Erk1/2 (1:1000); mouse anti-Erk1/2 with phosphorylated Thr and Tyr residues (1:1000) (Merck-Millipore, Darmstadt, Germany); rabbit anti-phospho-p90 ribosomal S6 kinase (p90 RSK -Thr 359 /Ser 363 ) (1:1000); rabbit anti-p38MAPK (1:1000); rabbit anti-p38MAPK with phospho-Thr 180 and -Tyr 182 (1:1000) (Cell Signaling Technology, Danvers, MA, USA); mouse anti-NHE-1 (1:1000); mouse anti-β actin (1:3000) (Abcam, Cambridge, UK); mouse anti-NHE-3-4F5 (1:750) (Santa Cruz Biotechnology, SC, CA, USA); and horseradish peroxidase-conjugated goat secondary antibodies against both rabbit and mouse (Jackson ImmunoResearch Laboratories, Baltimore, MD, USA ).…”