Expression of NHE-3 in the apical membrane of rat renal proximal tubule and thick ascending limb

In the renal medullary thick ascending limb (MTAL), inhibiting the basolateral NHE1 Na؉ /H ؉ exchanger with amiloride or nerve growth factor (NGF) results secondarily in inhibition of the apical NHE3 Na ؉ /H ؉ exchanger, thereby decreasing transepithelial HCO 3 ؊ absorption. MTALs from rats were studied by in vitro microperfusion to identify the mechanism underlying cross-talk between the two exchangers. The basolateral addition of 10 M amiloride or 0.7 nM NGF decreased HCO 3 ؊ absorption by 27-32%. Jasplakinolide, which stabilizes F-actin, or latrunculin B, which disrupts F-actin, decreased basal HCO 3 ؊ absorption by 30% and prevented the inhibition by amiloride or NGF. Jasplakinolide had no effect on HCO 3 ؊ absorption in tubules bathed with amiloride or a Na ؉ -free bath to inhibit NHE1. Jasplakinolide and latrunculin B did not prevent inhibition of HCO 3 ؊ absorption by vasopressin or stimulation by hyposmolality, factors that regulate HCO 3 ؊ absorption through primary effects on apical Na ؉ /H ؉ exchange. Treatment of MTALs with amiloride or NGF for 15 min decreased polymerized actin with no change in total cell actin, as assessed both by fluorescence microscopy and by actin Triton X-100 solubility. Jasplakinolide prevented amiloride-induced actin remodeling. Vasopressin, which inhibits HCO 3 ؊ absorption by an amount similar to that observed with amiloride and NGF but does not act via NHE1, did not affect cellular F-actin content. These results indicate that basolateral NHE1 regulates apical NHE3 and HCO 3 ؊ absorption in the MTAL by controlling the organization of the actin cytoskeleton.

Section: Namentioning

confidence: 99%

Section: Namentioning

confidence: 99%

The Basolateral NHE1 Na+/H+ Exchanger Regulates Transepithelial HCO3− Absorption through Actin Cytoskeleton Remodeling in Renal Thick Ascending Limb

Watts¹,

George

Good³

2005

“…Polyclonal antiserum directed against human EBP50 was generated in rabbits as described previously (15). Rabbit antisera 1566 and 1568 specific for NHE3 were generated as described previously (16). Monoclonal antibodies against an R domain and a C terminus of CFTR were purchased from R & D Systems (Minneapolis, MN).…”

Section: Methodsmentioning

confidence: 99%

Regulatory Interaction between the Cystic Fibrosis Transmembrane Conductance Regulator and HCO 3− Salvage Mechanisms in Model Systems and the Mouse Pancreatic Duct

Ahn

Kim

Lee

et al. 2001

Self Cite

105

114

The pancreatic duct expresses cystic fibrosis transmembrane conductance regulator (CFTR) and HCO 3 ؊ secretory and salvage mechanisms in the luminal membrane. Although CFTR plays a prominent role in HCO 3 ؊ secretion, the role of CFTR in HCO 3 ؊ salvage is not known. In the present work, we used molecular, biochemical, and functional approaches to study the regulatory interaction between CFTR and the HCO 3 ؊ salvage mechanism Na ؉ /H ؉ exchanger isoform 3 (NHE3) in heterologous expression systems and in the native pancreatic duct. We found that CFTR regulates NHE3 activity by both acute and chronic mechanisms. In the pancreatic duct, CFTR increases expression of NHE3 in the luminal membrane. Thus, luminal expression of NHE3 was reduced by 53% in ducts of homozygote ⌬F508 mice. Accordingly, luminal Na ؉ -dependent and HOE694-sensitive recovery from an acid load was reduced by 60% in ducts of ⌬F508 mice. CFTR and NHE3 were coimmunoprecipitated from PS120 cells expressing both proteins and the pancreatic duct of wild type mice but not from PS120 cells lacking CFTR or the pancreas of ⌬F508 mice. The interaction between CFTR and NHE3 required the COOH-terminal PDZ binding motif of CFTR, and mutant CFTR proteins lacking the C terminus were not co-immunoprecipitated with NHE3. Furthermore, when expressed in PS120 cells, wild type CFTR, but not CFTR mutants lacking the C-terminal PDZ binding motif, augmented cAMP-dependent inhibition of NHE3 activity by 31%. These findings reveal that CFTR controls overall HCO 3 ؊ homeostasis by regulating both pancreatic ductal HCO 3 ؊ secretory and salvage mechanisms.Fluid secretion and the control of the ionic composition of biological fluids are essential for the function of many secretory epithelia, including the respiratory, digestive, and reproductive systems. HCO 3 Ϫ is an important constituent of secreted fluids by virtue of its function as the biological pH buffer and its effect on the solubility of proteins and ions in biological fluids. Accordingly, the mechanisms underlying HCO 3 Ϫ homeostasis at rest and during stimulation have attracted much attention in recent years. The importance of HCO 3 Ϫ homeostasis is evident from the marked reduction in Cl Ϫ -and HCO 3 Ϫ

“…All NHEs have a predicted N-terminal hydrophobic ion-translocating domain and a variable C-terminal hydrophilic domain that harbors regulatory sequences (15)(16)(17)(18). NHE3 is the isoform in the apical membrane based on antigenic (19,20) and functional data (21)(22)(23)(24). Non-NHE3-mediated Na ϩ /H ϩ exchange activity in the proximal tubule apical membrane (24) can theoretically be caused by NHE8 (14), but definitive data are still forthcoming.…”

mentioning

confidence: 99%

Acute Regulation of Na/H Exchanger NHE3 by Adenosine A1 Receptors Is Mediated by Calcineurin Homologous Protein

Sole

Cerull

Babich

et al. 2004

Self Cite