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Articles in PresS, published online ahead of print January 8, 2002
Am J Physiol Renal Physiol, 10.1152/ajprenal.00266.2001
Submitted on August 27, 2001
Accepted on January 5, 2002
1 Nephrology, University of Florida, Gainesville, FL, USA
2 Nephrology, University of Florida, Gainesville, FL, USA; Nephrology, Gainesville VAMC, Gainesville, FL, USA
3 Cell Biology, Georgetown University, Washington, DC, USA
4 NHLBI, NIH, Bethesda, MD, USA
* To whom correspondence should be addressed. E-mail: WeineID{at}ufl.edu.
Ammonia stimulates cortical collecting duct (CCD) net bicarbonate reabsorption by activating an apical H+-K+-ATPase through mechanisms that are independent of ammonia's known effects on intracellular pH and active sodium transport. The current studies examined whether this stimulation occurs through SNARE (soluble NSF protein attachment receptor) protein-mediated vesicle fusion. Rabbit CCD segments were studied using in vitro microperfusion and transepithelial bicarbonate transport was measured using microcalorimetry. Ammonia's stimulation of bicarbonate reabsorption was blocked by either chelating intracellular calcium, with BAPTA-AM, or by inhibiting microtubule polymerization, with colchicine, as compared with parallel studies performed in the absence of these inhibitors. An inactive structural analogue of colchicine, lumicolchicine, did not alter ammonia's stimulation of bicarbonate reabsorption. Tetanus toxin, a zinc endopeptidase specific for vesicle-associated SNARE (v-SNARE) proteins prevented ammonia from stimulating net bicarbonate reabsorption. Consistent with the functional evidence for v-SNARE involvement, antibodies directed against a conserved region of isoforms 1-3 of the tetanus toxin-sensitive, vesicle-associated membrane protein (VAMP) members of v-SNARE proteins labeled the apical and sub-apical region of collecting duct intercalated cells. Similarly, antibodies to NEM-sensitive fusion protein (NSF), a protein involved in activation of SNARE proteins for subsequent vesicle fusion, localized to the apical and sub-apical region of collecting duct intercalated cells. These results indicate that ammonia stimulates CCD bicarbonate reabsorption through intracellular calcium-dependent, microtubule-dependent, and v-SNARE-dependent mechanism that appears to involve insertion of cytoplasmic vesicles into the apical plasma membrane of CCD intercalated cells.
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