CARBONIC-ANHYDRASE-II PLAYS A MAJOR ROLE IN OSTEOCLAST DIFFERENTIATION AND BONE-RESORPTION BY EFFECTING THE STEADY-STATE INTRACELLULAR PH AND CA2+

Carbonic anhydrase II (CA II) expression is characteristic for the ear ly stage of osteoclast differentiation. To study how CA II, which is c rucial in proton generation in mature osteoclasts, influences the oste oclast differentiation process we performed rat bone marrow cultures. In this model, acetazolamide, a specific CA inhibitor, decreased the 1 ,25(OH)(2)D-3-induced formation of multinucleated tartrate-resistant a cid phosphatase (TRAP)-positive cells, in a dose-dependent manner. We then performed intracellular pH (pH(i)) and Ca2+ (Ca-i(2+)) measuremen ts for cultured osteoclasts and noticed that addition of acetazolamide caused a rapid, transient increase of both parameters. The increase i n pH(i) was dependent neither on the culture substrate nor on the extr acellular pH (pH(e)) but the increase could be diminished by DIDS or b y bicarbonate removal. Membrane-impermeable CA inhibitors (benzolamide and pd5000) did not have this effect. Addition of CA II antisense oli gonucleotides into the cultures reduced the pH(i) increase significant ly. CA II inhibition was also found to neutralize the intracellular ve sicles at extracellular pH (pH(e)) of 7.4, but at less extent at pH(e) 7.0. In mouse calvaria cultures, bone resorption was inhibited dose d ependently by acetazolamide at pH(e) 7.4 while inhibition was smaller at pH(e) 7.0. We conclude that CA II is essential not only in bone res orption but also in osteoclast differentiation. In both processes, how ever, the crucial role of CA II is at least partially due to the effec t on the osteoclast pH(i) regulation, (C) 1998 Academic Press.