GENERATION OF INOSITOL PHOSPHATES, DIACYLGLYCEROL AND CALCIUM FLUXES IN MYOBLASTS TREATED WITH 1,25-DIHYDROXYVITAMIN-D(3)

We have examined the effects of the seco-steroid hormone 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on membrane phosphoinositide metabolism, protein kinase C (PKC) activation and influx of extracellular Ca2+ in chick-embryo muscle-cell (myoblast) cultures. At physiological concentrations, the hormone induces a rapid (15 s) and transient release of inositol triphosphate (InsP3) and diacylglycerol (DAG). InsP3 release is maximal at 60 s (80% above controls) and then declines. The effects of 1,25(OH)2D3 on InsP production exhibited specificity, as 25-hydroxy-vitamin D3 and 24,25-dihydroxy-vitamin D. did not alter myoblast InsP3 levels. The stimulation of DAG is biphasic, with peaks at 60 s (+ 105%) and 5 min (+ 700%). The second phase of DAG release is not associated with changes in InsP3. 1,25(OH)2D3 induces a rapid (within 60 s) accumulation of InsP2, and its effect on InsP is delayed (120 s). The hormone rapidly activates myoblast PKC, with maximal translocation of activity from the cytosol to the cell membrane occurring at 60 s. Myoblast Ca-45 uptake significantly increases within 30 s of exposure to 1,25(OH)2D3. The response is time- (0.5-10 min) and dose- (1 pM-10 nM) dependent. The effects of the hormone are mimicked by the Ca2+-channel agonist Bay K 8644 and are effectively suppressed by nifedipine and extracellular EGTA. The results suggest that the rapid non-genomic actions of 1,25(OH)2D3 in myoblasts involve second-messenger systems associated with the generation of InsP3 and DAG and regulation of Ca2+ fluxes through voltage-operated channels.