Basolateral Na(+)/HCO(3)(-) cotransport activity is regulated by the dissociable Na(+)/H(+) exchanger regulatory factor.
In the renal proximal tubule, the activities of the basolateral Na(+)/HCO(3)(-) cotransporter (NBC) and the apical Na(+)/H(+) exchanger (NHE3) uniformly vary in parallel, suggesting that they are coordinately regulated. PKA-mediated inhibition of NHE3 is mediated by a PDZ motif-containing protein, the Na(+)/H(+) exchanger regulatory factor (NHE-RF). Given the common inhibition of these transporters after protein kinase A (PKA) activation, we sought to determine whether NHE-RF also plays a role in PKA-regulated NBC activity. Renal cortex immunoblot analysis using anti-peptide antibodies directed against rabbit NHE-RF demonstrated the presence of this regulatory factor in both brush-border membranes (BBMs) and basolateral membranes (BLMs). Using a reconstitution assay, we found that limited trypsin digestion of detergent solubilized rabbit renal BLM preparations resulted in NBC activity that was unaffected by PKA activation. Co-reconstitution of these trypsinized preparations with a recombinant protein corresponding to wild-type rabbit NHE-RF restored the inhibitory effect of PKA on NBC activity in a concentration-dependent manner. NBC activity was inhibited 60% by 10(-8)M NHE-RF; this effect was not observed in the absence of PKA. Reconstitution with heat-denatured NHE-RF also failed to attenuate NBC activity. To establish further a physiologic role for NHE-RF in NBC regulation, the renal epithelial cell line B-SC-1, which lacks detectable endogenous NHE-RF expression, was engineered to express stably an NHE-RF transgene. NHE-RF-expressing B-SC-1 cells (B-SC-RF) exhibited markedly lower basal levels of NBC activity than did wild-type controls. Inhibition of NBC activity in B-SC-RF cells was enhanced after 10 microM of forskolin treatment, consistent with a postulated role for NHE-RF in mediating the inhibition of NBC activity by PKA. These findings not only suggest NHE-RF involvement in PKA-regulated NBC activity, but also provide a unique molecular mechanism whereby basolateral NBC and apical NHE3 activities may be coordinately regulated in renal proximal tubule cells.
Bernardo
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Section of Nephrology, Department of Medicine, College of Medicine, University of Illinois-Chicago, Chicago, Illinois 60612-7315, USA. aabernar@uic.edu
Kear
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Santos
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Ma
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Steplock
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Robey
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Weinman
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D000818:Animals; D002352:Carrier Proteins / Q000032:analysis*; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology; D007668:Kidney / Q000378:metabolism*; D010750:Phosphoproteins / Q000502:physiology*; D011817:Rabbits; D027982:Sodium-Bicarbonate Symporters; D017923:Sodium-Hydrogen Exchangers / Q000032:analysis*
D002352:Carrier Proteins; D010750:Phosphoproteins; D027982:Sodium-Bicarbonate Symporters; D017923:Sodium-Hydrogen Exchangers; C093376:sodium-hydrogen exchanger regulatory factor; D005576:Colforsin; D017868:Cyclic AMP-Dependent Protein Kinases