ION CHANNELS CONTROLLING BONE REMODELING

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DESCRIPTION (Adapted from the Applicant's Abstract): The long-term goal of this research is to determine the molecular mechanisms underlying the signal transduction pathways in osteoblasts and to elucidate the physiological functions of Ca+ channels in bone. The Specific Aims are to: 1) Test the hypothesis that total [3H]-1,25D3 binding is predicted quantitatively by VDR message, the RO displaces plasma membrane (or cytosolic) binding sites, whereas EO displaces nuclear labeling. Using receptor binding techniques with [3H]-1,25D3, the pharmacology and distribution of RO and EO (an agent which acts at the nVDR to stimulate transcription but is not effective at activation of Ca+ channels below 10 nM) will be quantitated and correlated in cells lines which express different levels of VDR message; 2) Determine whether message of nuclear receptors of other steroid hormones predict the steroid hormone potentiation of Ca+ channels. ROS 17/2.8 cells show potentiation of Ca+ currents with addition of 0.1 nM testosterone, but doses of up to 100 nM 17B estradiol cause minimal effects. The principal investigator proposes to test the hypothesis that transfection of the normal estrogen receptor into "estrogen unresponsive" ROS 17/2.8 cells, will cause a rapid potentiation of Ca+ channels by low doses of 17B estradiol. The sensitivity of the non-genomic response will be quantitated to the level of expression of steroid receptor message; 3) Determine the Ca+ channel isoform in ROS 17/2.8 cells and mature rat bone which is associated with the response to steroid hormones. To answer which channel isoforms are present in mature rat bone or ROS cell cultures, oligonucleotides specific to the major L-type Ca+ channel isoforms, knockout technology will be used to specifically define which channel type is activated by steroid receptors. The steroid-activated Ca+ channel isoform will be cloned and expressed. Ca+ channels are involved in bone matrix production and bone resorption, and investigation of their pharmacology may yield new methods of drug treatment for bone disease.