Abstract
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 HCO3- 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 microM amiloride or 0.7 nM NGF decreased HCO3- absorption by 27-32%. Jasplakinolide, which stabilizes F-actin, or latrunculin B, which disrupts F-actin, decreased basal HCO3- absorption by 30% and prevented the inhibition by amiloride or NGF. Jasplakinolide had no effect on HCO3- absorption in tubules bathed with amiloride or a Na(+)-free bath to inhibit NHE1. Jasplakinolide and latrunculin B did not prevent inhibition of HCO3- absorption by vasopressin or stimulation by hyposmolality, factors that regulate HCO3- 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 HCO3- 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 HCO3- absorption in the MTAL by controlling the organization of the actin cytoskeleton.
Highlights
Membrane of epithelial cells, where it mediates housekeeping functions such as regulation of intracellular pH and cell volume [1,2,3, 6]
The results demonstrate that NHE1 regulates the organization of the actin cytoskeleton in the medullary thick ascending limb (MTAL) and that actin remodeling is involved in mediating NHE1-induced regulation of apical NHE3 and HCO3Ϫ absorption
The regulation of HCO3Ϫ absorption by AVP and hyposmolality is preserved in the presence of the F-actin modifier. These results demonstrate that the effect of jasplakinolide to block HCO3Ϫ transport regulation is specific for factors that act via the basolateral NHE1 Naϩ/Hϩ exchanger
Summary
Vasopressin, which inhibits HCO3؊ 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 HCO3؊ absorption in the MTAL by controlling the organization of the actin cytoskeleton. When considered in the context of epithelial cells such as the MTAL that express NHE1 in the basolateral membrane and NHE3 in the apical membrane, the above findings raise the possibility that the cytoskeleton could mediate crosstalk between the two exchangers, whereby NHE1 could induce changes in the actin network that in turn modulate NHE3. The purpose of the present experiments was to investigate the role of the actin cytoskeleton in transepithelial HCO3Ϫ absorption in the MTAL and to determine whether the cytoskeleton mediates the regulatory interaction between the basolateral NHE1 and apical NHE3 Naϩ/Hϩ exchangers. The results demonstrate that NHE1 regulates the organization of the actin cytoskeleton in the MTAL and that actin remodeling is involved in mediating NHE1-induced regulation of apical NHE3 and HCO3Ϫ absorption
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