Relation of ATPases in rat renal brush-border membranes to ATP-driven H+ secretion

Abstract

In the presence of inhibitors for mitochondrial H+-ATPase, (Na+ + K+)- and Ca2+-ATPases, and alkaline phosphatase, sealed brush-border membrane vesicles hydrolyse externally added ATP demonstrating the existence of ATPases at the outside of the membrane ("ecto-ATPases"). These ATPases accept several nucleotides, are stimulated by Ca2+ and Mg2+, and are inhibited by N.N'-dicyclohexylcarbodiimide (DCCD), but not by N-ethylmaleimide (NEM). They occur in both brush-border and basolateral membranes. Opening of brush-border membrane vesicles with Triton X-100 exposes ATPases located at the inside (cytosolic side) of the membrane. These detergent-exposed ATPases prefer ATP, are activated by Mg2+ and Mn2+, but not by Ca2+, and are inhibited by DCCD as well as by NEM. They are present in brush-border, but not in basolateral membranes. As measured by an intravesicularly trapped pH indicator. ATP-loaded brush-border membrane vesicles extrude protons by a DCCD- and NEM-sensitive pump. ATP-driven H+ secretion is electrogenic and requires either exit of a permeant anion (Cl-) or entry of a cation, e.g., Na+ via electrogenic Na+/D-glucose and Na+/L-phenylalanine uptake. In the presence of Na+, ATP-driven H+ efflux is stimulated by blocking the Na+/H+ exchanger with amiloride. These data prove the coexistence of Na+-coupled substrate transporters, Na+/H+ exchanger, and an ATP-driven H+ pump in brush-border membrane vesicles. Similar location and inhibitor sensitivity reveal the identity of ATP-driven H+ pumps with (a part of) the DCCD- and NEM- sensitive ATPases at the cytosolic side of the brush-border membrane.

Vacuolar-type H+-ATPase regulates cytoplasmic pH in Toxoplasma gondii tachyzoites

“…These pumps occur in two forms : an electrogenic H + -ATPase and an electroneutral H + \K + -ATPase. The plasma membrane H + -ATPases present in lower eukaryotes, such as yeast and other fungi [8,9] is described as being of the Ptype, whereas that present in several normal [10][11][12][13][14] and tumoral [15] mammalian cells is of the vacuolar (V)-type. The H + -ATPase in fungal plasma membrane functions physiologically to hydrolyse ATP and to pump H + out of the cell ; the resulting electrochemical H + gradient provides energy for an array of secondary transport systems [16].…”

Expression and distribution of renal vacuolar proton-translocating adenosine triphosphatase in response to chronic acid and alkali loads in the rat.

“…Intercalated cells showing H' ATPase staining: A, apically stained; B, basolaterally stained; BA, basolateral and apical pole staining; D, diffusely stained; PPA, poorly polarized apically stained; PPB, poorly polarized basolaterally stained; WPA, wellpolarized apically stained; WPB, well-polarized basolaterally stained. ent in the apical membrane (2-4), contributes to up to 40% of the overall proximal H' secretion in rat (5)(6)(7). Physiologic studies have revealed increased or reduced Na+/H+ antiport activity and bicarbonate reabsorption in the proximal tubule in response, respectively, to metabolic acidosis (8)(9)(10)(11)(12)(13)(14) or metabolic alkalosis (12,13).…”

A Brush Border Membrane-Bound H⁺ -ATPase from the Dog Proximal Tubule

“…the effect of membrane solubiliza tion with 0.1% DOC was examined. The ATP-binding sites of endosomal H+-ATPascs are spontaneously exposed to the extravesicular environment [24], while those of BBM H+-ATPases are hidden within the intravesicular compartment [ 14], We observed a stim ulation of the NEM-sensitive H+-ATPase ac tivity of 6.6 times upon solubilization of ves icles with DOC. indicating that the contribu tion of H+-ATPases of endosomal origin to total ATPase activity is low compared to H+-ATPases of right-side-out BBMVs (598 nmol-min-'-mg protein-1; table 2).…”

“…Indeed, the Na+/H+ exchanger is thought to be inac tive at a pH above 7.2 [9], The maintenance of bicarbonate reabsorption during chronic metabolic alkalosis elevating the extracellular pH above 7.5. and the intracellular pH above 7.3-7.4 [10] may not be mediated by this exchanger, unless some compartmentation of cellular pH or an increase in the affinity of the exchanger for H+ [11] occurs within proximal cells. On the other hand, the proton pump identified in the renal cortex of different spe cies including the rat [12][13][14], pig [15] and beef [ 16] may be active at this pH. The quan titative importance of this second mechanism for bicarbonate reabsorption remains largely unclear.…”