Basolateral Na+/HCO3– cotransport activity is regulated by the dissociable Na+/H+ exchanger regulatory factor
| Autor: | Debra Steplock, F. T. Kear, Edward J. Weinman, Anna V.P. Santos, Jianfei Ma, A. A. Bernardo, R. Brooks Robey |
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| Rok vydání: | 1999 |
| Předmět: |
Sodium-Hydrogen Exchangers
Renal cortex Kidney Article Cell Line chemistry.chemical_compound medicine Colforsin Animals Protein kinase A Forskolin Sodium-Bicarbonate Symporters General Medicine Phosphoproteins Cyclic AMP-Dependent Protein Kinases Cell biology Sodium–hydrogen antiporter medicine.anatomical_structure Biochemistry chemistry Cell culture Rabbits Carrier Proteins Cotransporter |
| Zdroj: | Journal of Clinical Investigation. 104:195-201 |
| ISSN: | 0021-9738 |
| DOI: | 10.1172/jci5344 |
| Popis: | In the renal proximal tubule, the activities of the basolateral Na+/HCO3– 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 10–8M 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 μM 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. J. Clin. Invest. 104:195–201 (1999). |
| Databáze: | OpenAIRE |
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