Regulation of Cytoplasmic pH in Osteoclasts

doi:10.1074/jbc.270.5.2203

Regulation of Cytoplasmic pH in Osteoclasts

CONTRIBUTION OF PROTON PUMPS AND A PROTON-SELECTIVE CONDUCTANCE (*)

From the (1)Division of Cell Biology, The Hospital for Sick Children, M5G 1X8 Toronto, the
(2)Department of Surgery, Toronto Hospital and University of Toronto, M5G 2C4 Toronto, and the
(3)Faculty of Dentistry, University of Toronto, M5G 1G6 Toronto, Canada

** International Scholar of the Howard Hughes Medical Institute. To whom correspondence should be addressed:
Div. of Cell Biology, Hospital for Sick Children, 555 University Ave., Toronto M5G 1X8, Canada.
Tel.: 416-813-5727; Fax: 416-813-5028.

Abstract

Osteoclasts resorb bone by secreting protons into an extracellular resorption zone through vacuolar-type proton pumps located in the ruffled border. The present study was undertaken to evaluate whether proton pumps also contribute to intracellular pH (pH) regulation. Fluorescence imaging and photometry, and electrophysiological methods were used to characterize the mechanisms of pH regulation in isolated rabbit osteoclasts. The fluorescence of single osteoclasts cultured on glass coverslips and loaded with a pH-sensitive indicator was measured in nominally HCO₃-free solutions. When suspended in Na-rich medium, the cells recovered from an acute acid load primarily by means of an amiloride-sensitive Na/H antiporter. However, rapid recovery was also observed in Na-free medium when K was used as the substitute. Bafilomycin-sensitive, vacuolar-type pumps were found to contribute marginally to pH regulation and no evidence was found for K/H exchange. In contrast, pH recovery in high K medium was largely attributed to a Zn-sensitive proton conductive pathway. The properties of this conductance were analyzed by patch-clamping osteoclasts in the whole-cell configuration. Depolarizing pulses induced a slowly developing outward current and a concomitant cytosolic alkalinization. Determination of the reversal potential during ion substitution experiments indicated that the current was due to H (equivalent) translocation across the membrane. The H current was greatly stimulated by reducing pH, consistent with a homeostatic role of the conductive pathway during intracellular acidosis. These results suggest that vacuolar-type proton pumps contribute minimally to the recovery of cytoplasmic pH from intracellular acid loads. Instead, the data indicate the presence of a pH- and membrane potential-sensitive H conductance in the plasma membrane of osteoclasts. This conductance may contribute to translocation of charges and acid equivalents during bone resorption and/or generation of reactive oxygen intermediates by osteoclasts.

Footnotes

↵^§ Scholar of the Medical Research Council of Canada.
↵^¶ Cross-appointed to the Dept. of Biochemistry of the University of Toronto.
↵* This work was supported in part by the Medical Research Council of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

V-ATPase

vacuolar-type adenosine triphosphatase

pH

cytoplasmic pH

pH

extracellular pH

BCECF

2′,7′-bis-carboxyethyl-5(6)-carboxyfluorescein

NMG

N-methyl-D-glucammonium

TRAP

tartrate-resistant acid phosphatase

HBSS

Hanks' balanced salt solution

ohm(s)

pF

picofarad(s)

MES

4-morpholineethanesulfonic acid

PIPES

1,4-piperazinediethanesulfonic acid.
↵²The free concentration of Zn, which can form stable complexes with certain anions, was not determined. For this reason, concentrations in the text are only nominal. The total concentration of Zn used in electrophysiological experiments (0.5 mM) was greater than that used in fluorescence determinations, to compensate for the ability of aspartate (the main anion in the patch-clamp experiments) to bind the divalent cation.
↵³We cannot rule out the possibility that plasmalemmal and endomembrane V-ATPases differ in their sensitivity to bafilomycin A₁.
↵⁴This value is an average of the whole cell pH. Because of the inordinately large size of these cells, it is most likely that the ΔpH near the membrane was considerably greater.
↵⁵Data were calculated from the bulk pH of the pipette and bath using the Nernst equation.
- Received July 21, 1994.
- Revision received October 20, 1994.