24R,25-(OH)(2) VITAMIN-D-3 INHIBITS 1-ALPHA,25-(OH)(2) VITAMIN-D-3 AND TESTOSTERONE POTENTIATION OF CALCIUM CHANNELS IN OSTEOSARCOMA CELLS

Calcium influx via L-type calcium channels in osteoblast cells causes a rapid (in seconds) elevation in intracellular calcium initiated by p lasma membrane receptors for 1 alpha,25-dihydroxyvitamin D-3 (1 alpha, 25-D-3). 24R,25-Dihydroxyvitamin D-3 (24,25-D-3) alone, in concentrati ons up to 200 nM, does not cause potentiation of calcium currents in o steoblasts, but it does inhibit the current potentiation by 1 alpha,25 -D-3. To determine how various steroids interact in their potentiation of calcium channels, the action of vitamin D-3 analogues and testoste rone with calcium channels in the rat osteoblast-like cell line ROS 17 /2.8 was investigated. Bath additions of both 1 alpha,25-D-3 and testo sterone at doses below K-1/2 (the dose causing 50% left shift in the c urrent-voltage relationship) are additive in their ability to potentia te calcium channels. When 1 alpha,25-D-3 and testosterone are added to gether at concentrations that would cause a maximal shift in the curre nt-voltage relationship by each agent alone (V-max), the effect of the se steroids is not additive. Taken together these data suggest one pop ulation of calcium channels is activated by 1 alpha,25-D-3 or testoste rone. The shift in the current-voltage relationship caused by 1 alpha, 25-D-3 is reduced by 1 beta,25-dihydroxvitamin D-3 (1 beta,25-D-3), an agent which is thought to act specifically on the plasma membrane rec eptor for 1 alpha,25-D-3, but the potentiation caused by testosterone is not blocked by 1 beta,25-D-3. However, 24,25-D-3 inhibits the left shift in the peak current-voltage relationship mediated by either 1 al pha,25-D-3 and testosterone. This result implies that 1) 1 beta,25-D-3 directly displaces 1 alpha,25-D-3 but not testosterone from its plasm a membrane receptor, and 2) the rapid (in seconds) stimulatory effects of 1 alpha,25-D-3 and testosterone on calcium channels are mediated b y separate plasma membrane receptors for testosterone and 1 alpha,25-D -3, which are blocked by another receptor for 24,25-D-3. The interacti on of these three receptors with L-type calcium channels is pertussis toxin-sensitive.