Journal
AMERICAN JOURNAL OF PHYSIOLOGY-RENAL PHYSIOLOGY
Volume 282, Issue 6, Pages F1120-F1128Publisher
AMER PHYSIOLOGICAL SOC
DOI: 10.1152/ajprenal.00266.2001
Keywords
intracellular calcium; 1,2-bis(2-aminophenoxy)ethane-N,N,N '; N '-tetraacetic acid acetoxymethyl ester; microtubule; soluble N-ethylmaleimide-sensitive fusion attachment receptor protein; hydrogen-potassium-adenosine triphosphatase
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Funding
- Intramural NIH HHS [Z01 HL001285-21, Z99 HL999999] Funding Source: Medline
- NIDDK NIH HHS [R01 DK049750, DK-45788, DK-49750, R01 DK045788] Funding Source: Medline
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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.
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