Cardiac L-type calcium channel α₁-subunit is increased by cyclic adenosine monophosphate: Messenger RNA and protein expression in intact bone

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 α₁-subunit of L-type calcium channels, (α(1C), α(1D), and α(1S)) are present in RNA extracted from ROS 1712.8 osteosarcoma cells, rat femur, and rat skull. Sequencing of most of the α(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 1α,25-dihydroxyvitamin D₃, increased the messenger RNA (mRNA) of the α(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; immunohistochemistry in ROS cells, intact bone, and cartilage, we found that the α(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.