Mechanisms through which ammonia regulates cortical collecting duct net proton secretion

Abstract

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.

The ammonia transporter RhCG modulates urinary acidification by interacting with the vacuolar proton-ATPases in renal intercalated cells

“…RhCG may modulate H + -ATPase activity through at least two different mechanisms. NH 3 stimulates H + /K + -ATPase possibly involving increased insertion of the H + /K + -ATPase via a SNARE-dependent process 52 . RhCG participates in basolateral NH 3 uptake 11 and loss of RhCG activity may reduce intracellular NH 3 levels and NH 3 dependent regulation of luminal H + -extrusion.…”

Roles of renal ammonia metabolism other than in acid–base homeostasis

“…Further studies have shown that ammonia activates H-K-ATPase through mechanisms involving intracellular calcium, microtubules, SNARE proteins and activation of MAPK ([69] and unpublished observations). Although ammonia altered intracellular pH, equivalent intracellular pH changes induced by an alternative, non-specific mechanism did not alter H-K-ATPase activity [63].…”

“…Because ammonia is reabsorbed in the thick ascending limb by transport of NH 4 + at the K + -binding site of NKCC2 [70], hypokalemia increases ammonia reabsorption, leading to increased interstitial ammonia concentrations, particularly in the cortex and outer medulla [71], the major sites of collecting duct K + transport. This ammonia can then act in the collecting duct to regulate net K + transport, inhibiting unidirectional potassium secretion and stimulating unidirectional potassium reabsorption [60, 63, 66, 69, 72]. Thus, ammonia meets all criteria necessary to be identified as a renal paracrine signaling molecule.…”

Apical ammonia transport by the mouse inner medullary collecting duct cell (mIMCD-3)

“…Electroneutral NH 4 ϩ /H ϩ exchange enables NH 4 ϩ secretion against this NH 4 ϩ gradient by coupling NH 4 ϩ secretion to H ϩ movement from the highly acidic luminal fluid to the relatively alkaline 4 ϩ secretion by collecting duct cells and supports the conclusion that this transport activity mediates an important role in transepithelial ammonia secretion. Extracellular ammonia stimulates collecting duct H ϩ secretion (19,20,31,58), and this involves, at least in the cortical collecting duct, stimulation of apical H ϩ -K ϩ -ATPase (20). Moreover, this occurs through effects of ammonia that are independent from ammonia's effects as a transported molecule (18 -20).…”