Osteoclast ATP receptor activation leads to a transient decrease in intracellular pH.
Application of extracellular adenosine triphosphate (ATP) induces a pulsed decrease in osteoclast intracellular pH (pHi), as measured with seminaphthofluorescein (SNAFL)-calcein on a laser scanning confocal microscope. Adenosine diphosphate also produces a pHi decrease, but adenosine monophosphate, uridine triphosphate, 2-methylthio-ATP, and beta, gamma-methylene-ATP have little effect on pHi. The ATP-induced pHi decrease is largely inhibited by suramin, a P2 purinergic receptor blocker. Clamping intracellular free [Ca2+] ([Ca2+]i) with BAPTA/AM does not affect the ATP-induced pHi change, showing that this pHi decrease is not caused by the increased intracellular [Ca2+]i that is produced by activation of osteoclast purinergic receptors. We show that an increase in [Ca2+]i by itself will produce a pHi increase. The ATP effect is not blocked by inhibition of Na+/H+ exchange by either Na(+)-free bathing medium or amiloride. Two inhibitors of the osteoclast cell membrane proton pump, N-ethylmaleimide and vanadate, produce partial inhibition of the ATP-induced pHi decrease. Two other proton pump inhibitors, bafilomycin and N,N'-dicyclohexylcarbodiimide, have no influence on the ATP effect. None of the proton pump inhibitors but vanadate has a direct effect on pHi. Vanadate produces a transient pHi increase upon application to the bathing medium, possibly as a result of its known effect of stimulating the Na+/H+ exchanger. Inhibition of Cl-/HCO3- exchange by decreasing extracellular Cl- gives a pronounced long-term pHi increase, supporting the hypothesis that this exchange has an important role in osteoclast pHi homeostasis. In Cl(-)-free extracellular medium, there is a greatly reduced effect of extracellular ATP on pHi.(ABSTRACT TRUNCATED AT 250 WORDS)
Yu
H
H
Medical Research Council Group in Periodontal Physiology, University of Toronto, Ontario, Canada.
Ferrier
J
J
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D018613:Microscopy, Confocal; D010010:Osteoclasts / Q000187:drug effects* / Q000378:metabolism; D054328:Proton Pump Inhibitors; D058910:Purinergic P2 Receptor Agonists; D011817:Rabbits; D018048:Receptors, Purinergic P2 / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism; D013997:Time Factors
D005452:Fluoresceins; D005456:Fluorescent Dyes; D054328:Proton Pump Inhibitors; D058910:Purinergic P2 Receptor Agonists; D018048:Receptors, Purinergic P2; D017923:Sodium-Hydrogen Exchangers; C094271:seminaphthofluorescein-calcein; D000244:Adenosine Diphosphate; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; D000255:Adenosine Triphosphate; D002118:Calcium