Role of Na-H exchangers and vacuolar H+ pumps in intracellular pH regulation in neonatal rat osteoclasts.

Abstract

Osteoclasts resorb bone by pumping of H + into a compartment between the cell and the bone surface. Intracellular pH (pHi) homeostasis requires that this acid extrusion, mediated by a vacuolar-type H + ATPase, be complemented by other acid-base transporters. We investigated acid-extrusion mechanisms of single, freshly isolated, neonatal rat osteoclasts. Cells adherent to glass coverslips were studied in the nominal absence of CO2/HCO~, using the pH-sensitive dye BCECF and a digital imaging system. Initial pHi averaged 7.31 and was uniform throughout individual cells. Intrinsic buffering power (l~l) decreased curvilinearly from ~ 25 mM at pHi = 6.4 to ~ 6.0 mM at pHi = 7.4. In all polygonally shaped osteoclasts, and ~ 60% of round osteoclasts (~ 20% of total), pHi recovery from acid loads was mediated exclusively by Na-H exchange. In these pattern-1 cells, pHi recovery was 95% complete within 200 s, and was blocked by removing Na +, or by applying 1 mM amiloride, 50 IxM ethylisopropylamiloride (EIPA), or 50 ~M hexamethyleneamiloride (HMA). The apparent K~/2 for HMA ([Na+]o = 150 mM) was 49 nM, and the apparent Kl/2 for Na § was 45 mM. Na-H exchange, corrected for amiloride-insensitive fluxes, was half maximal at pHi 6.73, with an apparent Hill coefficient for intracellular H § of 2.9. Maximal Na-H exchange averaged 741 IxM/s. In the remaining ~40% of round osteoclasts (pattern-2 cells), pHi recovery from acid loads was brisk even in the absence of Na § or presence of amiloride. This Na+-independent pHi recovery was blocked by 7-chloro-4-nitrobenz-2-oxa-l,3-diazol (NBD-C1), a vacuolar-type H § pump inhibitor. Corrected for NBD-C1 insensitive fluxes, H § pump fluxes decreased approximately linearly from 96 at pHi 6.8 to 11 txM/s at pHi 7.45. In ~45% of pattern-2 cells, Na + readdition elicited a further pHi recovery, suggesting that H § pumps and Na-H exchangers can exist

pH regulation in single CA1 neurons acutely isolated from the hippocampi of immature and mature rats.

“…This is about twice the S.D. of other mammalian cells studied by our group (Boyarsky et al 1988; Ransom, Schlue, Davis & Boron, 1993;Ravesloot, Eisen, Baron & Boron, 1995). Although, for mature rats, the mean initial pHi (7 08) Figure 1B shows that mature neurons were more likely to have initial pHi values corresponding to those at the far ends of the Gaussian distribution for immature neurons (Fig.…”

“…In three experiments on neurons with a relatively high initial pH, (mean, 7-29; range, 7-17-7-48), we also have found that applying 10 or 50/uM EIPA during a pH1 recovery from an NH4,-induced acid load either had no effect or paradoxically increased the rate of pH1 recovery (data not shown). (Kaplan & Boron, 1994), and single rat osteoclasts (Ravesloot et al 1995). In both osteoclasts and forebrain astrocytes, the paradoxical effect of EIPA occurs in the absence of Na+.…”

“…In the astrocytes, it is unaffected by the organic anion transport inhibitor probenecid. In rat osteoclasts, another derivative of amiloride, hexamethyleneamiloride (HMA), has an alkalinizing effect similar to that of EIPA, and yet does not interfere with the measurement of BCECF fluorescence (Ravesloot et al 1995 . This rapid, segment f-g pH1 recovery is presumably due to the combination of Na+-H+ and Nae-dependent Cl--HCO3-exchange, as opposed by such acid loading mechanisms as Cl--HCO3-exchange.…”

Effect of isolated removal of either basolateral HCO3-or basolateral CO2on HCO3-reabsorption by rabbit S2 proximal tubule

“…We calculated intracellular pH (pHi) from the fluorescence excitation ratio of BCECF using the approach outlined previously for osteoclasts (21). We loaded perfused tubules with the dye by exposing them from the bath at room temperature for ϳ5 min to solution 7 containing 10 M of BCECF-AM (B-1170, Molecular Probes, Eugene, OR).…”

L’ostéoclaste et les mécanismes moléculaires de la résorption osseuse

“…Cela entraîne une inhibition inside-out de l'affinité des intégrines et un détachement du point focal d'adhérence, assurant ainsi la mobilité cellulaire [35,41]. [50,51]. Les protons et le bicarbonate sont produits par la forte concentration d'anhydrase carbonique (CAII) présente dans le cytoplasme de l'ostéoclaste.…”