ACTIVATION OF MAXI-K CHANNELS BY PARATHYROID-HORMONE AND PROSTAGLANDIN E(2) IN HUMAN OSTEOBLAST BONE-CELLS

Patch damp experiments were performed on two human osteosarcoma cell l ines (MG-63 and SaOS-2 cells) that show an osteoblasticlike phenotype to identify and characterize the specific K channels present in these cells. In case of MG-63 cells, in the cell-attached patch configuratio n (CAP) no channel activity was observed in 2 mM Ca Ringer (control co ndition) at resting potential. In contrast, a maxi-K channel was obser ved in previously silent CAP upon addition of 50 nM parathyroid hormon e (PTH), 5 nM prostaglandin E(2) (PGE(2)) or 0.1 mM dibutyryl cAMP + 1 mu M forskolin to the bath solution. However, maxi-K channels were pr esent in excised patches from both stimulated and nonstimulated cells in 50% of total patches tested. A similar K channel was also observed in SaOS-2 cells. Characterization of this maxi-K channel showed that i n symmetrical solutions (140 mM K) the channel has a conductance of 24 6 +/- 4.5 pS (n = 7 patches) and, when Na was added to the bath soluti on, the permeability ratio (P-K/P-Na) was 10 and 11 for MG-63 and SaOS -2 cells respectively. In excised patches from MG-63 cells, the channe l open probability (P-0) is both voltage- (channel opening with depola rization) and Ca-dependent; the presence of Ca shifts the P-0 vs. volt age curve toward negative membrane potential. Direct modulation of thi s maxi-K channel via protein kinase A (PKA) is very unlikely since in excised patches the activity of this channel is not sensitive to the a ddition of 1 mM ATP + 20 U/ml catalytic subunit of PKA. We next evalua ted the possibility that PGE(2) or PTH stimulated the channel through a rise in intracellular calcium. First, calcium uptake (Ca-45(++)) by MG-63 cells was stimulated in the presence of PTH and PGE(2), an effec t inhibited by Nitrendipine (10 mu M). Second, whereas PGE(2) stimulat ed the calcium-activated maxi-g channel in 2 mM Ca Ringer in 60% of pa tches studied, in Ca-free Ringer bath solution, PGE(2) did not open an y channels (n = 10 patches) nor did cAMP + forskolin(n = 3 patches), a lthough K channels were present under the patch upon excision. In addi tion, in the presence of 2 mM Ca Ringer and 10 mu M Nitrendipine in CA P configuration, PGE(2) (n = 5 patches) and cAMP + forskolin (n = 2 pa tches) failed to open K channels present under the patch. As channel a ctivation by phosphorylation with the catalytic subunit of PKA was not observed, and Nitrendipine addition to the bath or the absence of cal cium prevented the opening of this channel, it is concluded that activ ation of this channel by PTH, PGE(2) or dibutyryl cAMP + forskolin is due to an increase in intracellular calcium concentration via Ca influ x.