Abstract
Angiotensin II (ANG II) stimulates renal tubular reabsorption of NaCl by targeting Na(+)/H(+) exchanger NHE3. We have shown previously that inositol 1,4,5-triphosphate receptor-binding protein released with inositol 1,4,5-triphosphate (IRBIT) plays a critical role in stimulation of NHE3 in response to elevated intracellular Ca(2+) concentration ([Ca(2+)](i)). In this study, we investigated the role of IRBIT in mediating NHE3 activation by ANG II. IRBIT is abundantly expressed in the proximal tubules where NHE3 is located. ANG II at physiological concentrations stimulates NHE3 transport activity in a model proximal tubule cell line. ANG II-induced activation of NHE3 was abrogated by knockdown of IRBIT, whereas overexpression of IRBIT enhanced the effect of ANG II on NHE3. ANG II transiently increased binding of IRBIT to NHE3 at 5 min but became dissociated by 45 min. In comparison, it took at least 15 min of ANG II treatment for an increase in NHE3 activity and NHE3 surface expression. The stimulation of NHE3 by ANG II was dependent on changes in [Ca(2+)](i) and Ca(2+)/calmodulin-dependent protein kinases II. Inhibition of CaMKII completely blocked the ANG II-induced binding of IRBIT to NHE3 and the increase in NHE3 surface abundance. Several serine residues of IRBIT are thought to be important for IRBIT binding. Mutations of Ser-68, Ser-71, and Ser-74 of IRBIT decreased binding of IRBIT to NHE3 and its effect on NHE3 activity. In conclusion, our current findings demonstrate that IRBIT is critically involved in mediating activation of NHE3 by ANG II via a Ca(2+)/calmodulin-dependent protein kinases II-dependent pathway.
Highlights
The renin-angiotensin system is critically involved in regulation of body blood pressure and fluid balance
The localization of IRBIT at the proximal tubules was confirmed by co-labeling of NHE3 (Fig. 1A, red), which is present in all sections of proximal tubules [1, 25]
In comparison with the potentiation of Angiotensin II (ANG II)-mediated NHE3 activity by overexpression of WT HA-IRBIT (34% for WT IRBIT versus 22% for pcDNA control), overexpression of either mutant IRBIT did not enhance NHE3 activity (Fig. 9B). These data demonstrate that the interaction of IRBIT with NHE3 is sensitive to the phosphorylation state of IRBIT, and the IRBITNHE3 interaction is important for the effect of ANG II on NHE3 activity
Summary
Biotinylation of NHE3 was performed as described previously [16, 19]. Briefly, OKP cells were. Calcium Imaging—OKP cells grown on coverslips were incubated with a perfusion buffer (in mM: 145 NaCl, 6 KCl, 1 MgSO4, 1 CaCl2, 10 glucose, 10 HEPES, pH 7.3) suppleblotted with an anti-NHE3 antibody, 3H3 [20]. The coverslips were mounted Briefly, a rat was anesthetized with pentobarbital sodium and on a perfusion chamber mounted on an inverted microscope and were superfused with NH4ϩ buffer (40 mM NH4Cl, 90 mM NaCl, 20 mM HEPES, 5 mM KCl, 1 mM TMA-PO4, 2 mM CaCl2, 1 mM MgSO4, and 18 mM glucose) and subsequently with was perfused via the ascending aorta with PBS, followed by paraformaldehyde lysine periodate fixative (2% paraformaldehyde, 75 mM lysine, and 10 mM sodium periodate, pH 7.4). Statistical significance was assessed by Student’s t test using the SPSS statistic program (SPSS, Inc). p Ͻ 0.05 was considered significant
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