Acid extrusion is induced by osteoclast attachment to bone. Inhibition by alendronate and calcitonin.

Acid extrusion is essential for osteoclast (OC) activity. We examined Na+ and HCO3(-)-independent H+ extrusion in rat- and mouse OCs by measuring intracellular pH (pHi) changes, with the pHi indicator BCECF (biscarboxyethyl-5-(6) carboxyfluorescein) after H+ loading with an ammonium pulse. 90% of OCs attached to glass do not possess HCO3- and Na(+)-independent H(+)-extrusion (rate of pHi recovery = 0.043 +/- 0.007 (SEM) pH U/min, n = 26). In contrast, in OCs attached to bone, the pHi recovery rate is 0.228 +/- 0.011 pHi U/min, n = 25. OCs on bone also possess a NH(4+)-permeable pathway not seen on glass. The bone-induced H+ extrusion was inhibited by salmon calcitonin (10(-8) M, for 2 h), and was not present after pretreating the bone slices with the aminobisphosphonate alendronate (ALN). At ALN levels of 0.22 nmol/mm2 bone, H+ extrusion was virtually absent 12 h after cell seeding (0.004 +/- 0.002 pH U/min) and approximately 50% inhibition was observed at 0.022 pmol ALN/mm2 bone. The Na(+)-independent H+ extrusion was not inhibited by bafilomycin A1 (up to 10(-7) M), although a bafilomycin A1 (10(-8) M)-sensitive H+ pump was present in membrane vesicles isolated from these osteoclasts. These findings indicate that Na(+)-independent acid extrusion is stimulated by osteoclast attachment to bone and is virtually absent when bone is preincubated with ALN, or when osteoclasts are treated with salmon calcitonin

Isolation of renal brush-border membrane vesicles by a low-speed centrifugation; effect of sex hormones on Na⁺-H⁺ exchange in rat and mouse kidney

Na+-H+ exchange in rat and mouse renal brush-border membrane vesicles was studied by fluorescence quenching of the ΔpH indicator, acridine orange. Brush-border membrane vesicles were isolated by a modified Mg/EGTA-precipitation method at low speed centrifugation (8000 × g). The enzymatic characteristics of these membrane vesicles were similar to those obtained by the original high-speed centrifugation method (Biber et al. (1981) Biochim. Biophys. Acta, 647, 169–176). The rates of Na+-H+ exchange in renal brush-border membrane vesicles from male and female rats were similar. Neither ovariectomy nor treatment of ovariectomized rats with estradiol or testosterone changed the activity of Na+-H+ exchanger. The rates of Na+-H+ exchange in the mouse were smaller than in the rat indicating the existence of species differences. Na+-H+ exchange in mouse renal brush-border membranes exhibit strong sex differences, the rates in the male being higher than in the female. Castration of male mice led to a decrease in Na+-H+ exchanges to values found in females. Treatment of castrated mice with estradiol had no effect. In contrast, treatment with testosterone increased the rat of the exchanger by more than 100%. The effect of testosterone was restricted to the Vmax of the Na+-H+ exchanger, whereas the apparent Km for Na+ remained unchanged. Na+-dependent d-glucose transport in mouse renal luminal membranes exhibited also sex differences due to the potent stimulatory effect of testosterone. Therefore, Na+-H+ exchange and Na+-dependent d-glucose transport in the mouse kidney are under control of androgen hormones. This effect could be in close connection with the wellknown renotropic action of androgens in the mouse

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

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 brushborder 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