concanamycin-a and ethylisopropylamiloride

Colony-stimulating factor-1 (CSF-1) promotes the survival of osteoclasts, short-lived cells that resorb bone. Although a rise in intracellular pH (pH(i)) has been linked to inhibition of apoptosis, the effect of CSF-1 on pH(i) in osteoclasts has not been reported. The present study shows that, in the absence of CO(2)/HCO(3)(-), CSF-1 causes little change in osteoclast pH(i). In contrast, exposing these cells to CSF-1 in the presence of CO(2)/HCO(3)(-) causes a rapid and sustained cellular alkalinization. The CSF-1-induced rise in pH(i) is not blocked by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, an inhibitor of HCO(3)(-) transporters but is abolished by removing extracellular sodium. This inhibition profile is similar to that of the electroneutral Na/HCO(3) cotransporter NBCn1. By RT-PCR, NBCn1 transcripts are present in both osteoclasts and osteoclast-like cells (OCLs), and by immunoblotting, the protein is present in OCLs. Moreover, CSF-1 promotes osteoclast survival in the presence of CO(2)/HCO(3)(-) buffer but not in its absence. Preventing the activation of NBCn1 markedly attenuates the ability of CSF-1 to 1) block activation of caspase-8 and 2) prolong osteoclast survival. Inhibiting caspase-3 or caspase-8 in OCLs prolongs osteoclast survival to the same extent as does CSF-1. This study provides the first evidence that osteoclasts express a CSF-1-regulated Na/HCO(3) cotransporter, which may play a role in cell survival.

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Alkalies; Amiloride; Animals; Bicarbonates; Carbon Dioxide; Caspase Inhibitors; Cell Separation; Cell Survival; Cells, Cultured; Enzyme Inhibitors; Hydrogen-Ion Concentration; Intracellular Fluid; Macrolides; Macrophage Colony-Stimulating Factor; Mice; Mice, Inbred Strains; Osteoclasts; Protons; Rats; Sodium; Sodium-Bicarbonate Symporters

Cells of the dopaminergically innervated salivary ducts in the cockroach Periplaneta americana have a vacuolar-type H(+)-ATPase (V-ATPase) of unknown function in their apical membrane. We have studied whether dopamine affects intracellular pH (pH(i)) in duct cells and whether and to what extent the apical V-ATPase contributes to pH(i) regulation. pH(i) measurements with double-barrelled pH-sensitive microelectrodes and the fluorescent dye BCECF have revealed: (1) the steady-state pH(i) is 7.3+/-0.1; (2) dopamine induces a dose-dependent acidification up to pH 6.9+/-0.1 at 1 micromol l(-1) dopamine, EC(50) at 30 nmol l(-1) dopamine; (3) V-ATPase inhibition with concanamycin A or Na(+)-free physiological saline (PS) does not affect the steady-state pH(i); (4) concanamycin A, Na(+) -free PS and Na(+)/H(+) exchange inhibition with 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) each reduce the rate of pH(i) recovery from a dopamine-induced acidification or an acidification induced by an NH(4)Cl pulse; (5) pH(i) recovery after NH(4)Cl-induced acidification is almost completely blocked by concanamycin A in Na(+)-free PS or by concanamycin A applied together with EIPA; (6) pH(i) recovery after dopamine-induced acidification is also completely blocked by concanamycin A in Na(+)-free PS but only partially blocked by concanamycin A applied together with EIPA. We therefore conclude that the apical V-ATPase and a basolateral Na(+)/H(+) exchange play a minor role in steady-state pH(i) regulation but contribute both to H(+) extrusion after an acute dopamine- or NH(4)Cl-induced acid load.

Topics: Acid-Base Equilibrium; Amiloride; Ammonium Chloride; Analysis of Variance; Animals; Cytophotometry; Dopamine; Dose-Response Relationship, Drug; Hydrogen-Ion Concentration; Intracellular Fluid; Macrolides; Microelectrodes; Periplaneta; Salivary Ducts; Sodium-Hydrogen Exchangers; Vacuolar Proton-Translocating ATPases