PHARMACOLOGICAL AND BIOCHEMICAL-EVIDENCE FOR THE REGULATION OF OSTEOCALCIN SECRETION BY POTASSIUM CHANNELS IN HUMAN OSTEOBLAST-LIKE MG-63 CELLS

Previous reports have suggested the involvement of voltage-activated c alcium (Ca2+) channels in bone metabolism and in particular on the sec retion of osteocalcin by osteoblast-like cells,((1)) We now report tha t potassium (K+) channels can also modulate the secretion of osteocalc in by MG-SS cells, a human osteosarcoma cell line. When 1,25-dihydroxy vitamin D-3(1,25(OH)(2)D-3)-treated MG-63 cells were depolarized by st ep increases of the extracellular K+ concentration ([K+](out)) from 5- 30 mM, osteocalcin (OC) secretion increased from a control value of 21 8 +/- 13 to 369 +/- 18 ng/mg of protein/48 h (p < 0.005 by analysis of variance), In contrast, in the absence of 1,25(OH)(2)D-3, there is no osteocalcin secretion nor any effect of cell depolarization on this a ctivity, The depolarization-induced increase in 1,25(OH)(2)D-3-depende nt osteocalcin secretion was totally inhibited in the presence of 10 m u M Nitrendipine (a Ca2+ channel blocker p < 0.005) without affecting cellular all;aline phosphatase nor cell growth. Charybdotoxin, a selec tive blocker of Ca2+-dependent K+ channels (maxi-K) present in MG-63 c ells,((2)) stimulated 1,25(OH)(2)D-3-induced osteocalcin synthesis abo ut 2-fold (p < 0.005) after either 30, 60, or 120 minute; of treatment , However Charybdotoxin was without effect on basal release of osteoca lcin in the absence of 1,25(OH)(2)D-3 pretreatment, Using patch clamp technique, we occasionally observed the presence of a mall conductance K+ channel, compatible with an ATP-dependent KC channel (GK(ATP)) in nonstimulated cells, whereas multiple channel openings were observed w hen cells were treated with Diazoxide, a sulfonamide derivative which opens GK(ATP). Western blot analysis revealed the presence of the N-te rminal peptide of GK(ATP) in MG-63 cells, and its expression was regul ated with the proliferation rate of these cells, maximal detection by Western blots being observed during the logarithmic phase of the cycle , Glipizide and Glybenclamide, selective sulfonylureas which can black GK(ATP), dose-dependently enhanced 1,25(OH)(2)D-3-induced OG secretio n (p < 0.005), Reducing the extracellular calcium concentration with E GTA (mu M range) totally inhibited the effect of Glipizide and Glybenc lamide on osteocalcin secretion (p < 0.005), which remained at the sam e levels as controls, Diazoxide totally prevented the effect of these sulfonylureas, These results suggest that voltage-activated Ca2+ chann els triggered via cell depolarization can enhance 1,25(OH)(2)D-3-induc ed OC release by MG-63 cells. In addition, OC secretion is increased b y blocking two types of K+ channels: maxi-K channels, which normally h yperpolarize cells and close Ca2+ channels, and GK(ATP) channels, The role of these channels is closely linked to the extracellular Ca2+ con centration.