PARATHYROID-HORMONE, PROSTAGLANDIN E-2, AND 1,25-DIHYDROXYVITAMIN D-3DECREASE THE LEVEL OF NA-CA2+ EXCHANGE PROTEIN IN OSTEOBLASTIC CELLS()

We previously described Na+-Ca2+ exchange in osteoblastic rat osteosar coma cells (UMR-106) and demonstrated that Na+-dependent Ca2+ transpor t was inhibited by 24-hour treatment of cells with parathyroid hormone (PTH), prostaglandin E-2 (PGE(2)), or 1,25(OH)(2)D-3. To determine wh ether this inhibition of Na+-Ca2+ exchange is at the level of exchange r protein synthesis we have examined exchanger protein levels using im munoblot analysis. UMR-106 cells were treated for 24 hours with or wit hout PTH, PGE(2), or 1,25(OH)(2)D-3. Plasma membrane fractions (7500 g ) were obtained and proteins were separated by SDS-PAGE transferred to nylon membranes, and immunoblotted with a polyclonal antibody to the canine cardiac Na+-Ca2+ exchanger. In rat cardiac membranes, we detect ed 125 and 75 kD bands, similar to findings for the canine exchanger. In the osteoblastic UMR cell membranes, a specific band was detected a t 90 kD that decreased 65% after treatment of cells with PTH. Inhibiti on by PTH was dose dependent, was maximal with 10(-7) M PTH, and requi red 16-24 hour treatment time. Similar inhibition was observed after a 24 hour treatment with 10(-6) M PGE(2) or 10(-8) M 1,25(OH)(2)D-3. Th ese results demonstrate the presence of a specific protein in UMR cell s that cross-reacts with antibody directed against the cardiac Na+-Ca2 + exchanger. Thus, the previously reported inhibition of Na+-Ca2+ exch ange activity by calcemic agents in osteoblasts appears to be due to r egulation of exchanger protein levels in these osteoblastic cells.