Cardiac L-Type Calcium Channel α1-Subunit Is Increased by Cyclic Adenosine Monophosphate: Messenger RNA and Protein Expression in Intact Bone

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Abstract
L-type calcium channels have been identified previously in both osteoblast-like osteosarcoma cell lines and primary cultures of osteoblasts using numerous techniques such as patch clamp recording, drug inhibited 45Ca2+ uptake, and Fura-2 measurements, but intact bone has not been investigated. Using reverse-transcription polymerase chain reaction (RT-PCR) we found that the three major isoforms of the alpha 1-subunit of L-type calcium channels, (alpha 1C, alpha 1D, and alpha 1S) are present in RNA extracted from ROS 17/2.8 osteosarcoma cells, rat femur, and rat skull. Sequencing of most of the alpha 1C-subunit from rat femur and ROS cells revealed that the splice variants in osteosarcoma cells and intact bone differ, but there are no unique sequence variations compared with those found in other tissues. Northern blot analysis of ROS cell RNA indicated that cyclic adenosine monophosphate (cAMP), but not 1 alpha, 25-dihydroxyvitamin D3, increased the messenger RNA (mRNA) of the alpha 1C-subunit. Western blot of ROS cell lysates revealed a band of more then 220 kDa, the amount of which increased in cells treated with cAMP. Using confocal microscopy combined with immunohistochemistry in ROS cells, intact bone, and cartilage, we found that the alpha 1C-subunit of this channel is expressed in osteoblasts and chondrocytes suggesting this channel may be a pathway for signal transduction in intact tissue, because it is in osteosarcoma cell lines and primary osteoblasts grown in tissue culture.