Mechanisms through which ammonia regulates cortical collecting duct net proton secretion.
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 present studies examined whether this stimulation occurs through soluble N-ethylmaleimide-sensitive fusion attachment receptor (SNARE) 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 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester or by inhibiting microtubule polymerization with colchicine compared with parallel studies performed in the absence of these inhibitors. An inactive structural analog 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 subapical region of collecting duct intercalated cells. Similarly, antibodies to NSF protein, a protein involved in activation of SNARE proteins for subsequent vesicle fusion, localized to the apical and subapical region of collecting duct intercalated cells. These results indicate that ammonia stimulates CCD bicarbonate reabsorption through an 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.
Frank
Amy E
AE
Division of Nephrology, Hypertension, and Transplantation, University of Florida and Gainesville Veterans Affairs Medical Center, Gainesville, Florida 32610-0224, USA.
Wingo
Charles S
CS
Andrews
Peter M
PM
Ageloff
Shana
S
Knepper
Mark A
MA
Weiner
I David
ID
D000143:Acids / Q000378:metabolism; D000641:Ammonia / Q000378:metabolism*; D000818:Animals; D001639:Bicarbonates / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002614:Chelating Agents / Q000494:pharmacology; D003078:Colchicine / Q000494:pharmacology; D022162:Cytoplasmic Vesicles / Q000378:metabolism; D005260:Female; D066298:In Vitro Techniques; D007424:Intracellular Fluid / Q000378:metabolism; D007672:Kidney Cortex / Q000378:metabolism*; D007685:Kidney Tubules, Collecting / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism; D052066:N-Ethylmaleimide-Sensitive Proteins; D009928:Organ Specificity; D011522:Protons; D050683:R-SNARE Proteins; D011817:Rabbits; D050600:SNARE Proteins; D013744:Tetanus Toxin / Q000494:pharmacology; D033921:Vesicular Transport Proteins
D000143:Acids; D001639:Bicarbonates; D002352:Carrier Proteins; D002614:Chelating Agents; D008565:Membrane Proteins; D011522:Protons; D050683:R-SNARE Proteins; D050600:SNARE Proteins; D013744:Tetanus Toxin; D033921:Vesicular Transport Proteins; D000641:Ammonia; D052066:N-Ethylmaleimide-Sensitive Proteins; D003078:Colchicine; D002118:Calcium