Protein kinase C α modulates the Ca2+ influx phase of the Ca2+ response to 1α,25-dihydroxy-vitamin-D3 in skeletal muscle cells

Treatment of chick skeletal muscle cells with 1 alpha ,25-dihydroxy-vitamin D-3 [1 alpha ,25(OH)(2)D-3] triggers a rapid and sustained increase in cytosolic Ca2+ ([Ca2+](i)), which depends on Ca2+ mobilization from inner stores and extracellular Ca2+ entry. Fluorimetric analysis of changes in [Ca2+](i) in Fura-2-loaded cells revealed that the hormone significantly stimulates the Ca2+ influx phase within the concentration range of 10(-12)-10(-6) M, with maximal effects (3.5-fold increase) at 10(-9) M 1 alpha ,25(OH)(2)D-3. The effects of the sterol on the Ca2+ entry pathway were abolished by the PKC inhibitors bisindolylmaleimide and calphostin. We have recently shown that, in these cells, 1 alpha ,25(OH)(2)D-3 activates and translocates PKC alpha to the membrane, suggesting that this isozyme accounts for PKC-dependent 1 alpha ,25(OH)(2)D-3 modulation of Ca2+ entry. The role of PKC alpha was specifically addressed here using antisense technology. When the expression of PKC alpha was selectively knocked out by intranuclear microinjection of an antisense oligonucleotide against PKC alpha mRNA, the Ca2+ influx component of the response to 1 alpha ,25(OH)(2)D-3 was markedly reduced (-60%). These results demonstrate that 1 alpha ,25(OH)(2)D-3-induced activation of PKC alpha enhances extracellular Ca2+ entry partially contributing to maintainance of the sustained phase of the Ca2+ response to the sterol.