MULTIPLE CALCIUM CHANNEL TRANSCRIPTS IN RAT OSTEOSARCOMA CELLS - SELECTIVE ACTIVATION OF ALPHA-1D ISOFORM BY PARATHYROID HORMONE

摘要

Osteoblasts express calcium channels that are thought to be involved in the transduction of extracellular signals regulating bone metabolism, The molecular identity of the pore-forming subunit alpha(1) of L-type calcium channel(s) was determined in rat osteosarcoma UMR-106 cells, which express an osteoblast phenotype, A homology-based reverse transcriptase-polymerase chain reaction cloning strategy was employed that used primers spanning the fourth domain. Three types of cDNAs were isolated, corresponding to the alpha(1S) (skeletal), alpha(1C) (cardiac); and alpha(1D) (neuroendocrine) isoforms. In the transmembrane segment IVS3 and the extracellular loop formed by the IVS3-S4 linker, a single pattern of mRNA splicing was found that occurs in all three types of calcium channel transcripts. Northern blot analysis revealed an 8.6-kb mRNA that hybridized to the alpha(1C) probe and 4.8- and 11.7-kb mRNAs that hybridized to the alpha(1S) and alpha(1D) probes, Antisense oligonucleotides directed to the calcium channel alpha(1D) transcript, but not those directed to alpha(1S) or alpha(1C) transcripts, inhibited the rise of intracellular calcium induced by parathyroid hormone, However, alpha(1D) antisense oligonucleotides had no effect on the accumulation of cAMP induced by parathyroid hormone, When L-type calcium channels were activated with Bay K 8644, antisense oligonucleotides to each of the three isoforms partially inhibited the rise of intracellular calcium, The present results provide evidence for the expression of three distinct calcium channel alpha(1)-subunit isoforms in an osteoblast-like cell line, We conclude that the alpha(1D) isoform is selectively activated by parathyroid hormone. [References: 44]