Antagonistic effects of 24R,25-dihydroxyvitamin D3 and 25-hydroxyvitamin D3 on L-type Ca2+ channels and Na+/Ca 2+ exchange in enterocytes from Atlantic cod (Gadus morhua)

There is mounting evidence that vitamin D and its metabolites play important roles in regulating plasma calcium concentrations in teleost fish as in other vertebrates. The aims of the present study were to elucidate the possible cellular target mechanisms for the rapid actions of 24R,25(OH)(2)D-3, 25(OH)D-3 and 1,25(OH)(2)D-3 in Atlantic cod enterocytes at physiological doses, and to establish the concentration and thus the physiological range of circulating 24R,25(OH)(2)D-3, 25(OH)D-3 and 1,25(OH)(2)D-3, in the Atlantic cod. The plasma concentrations of 25(OH)D-3, 1,25(OH)(2)D-3 and 24R,25(OH)(2)D-3 were 15.3 +/- 2.7 nM, 125.1 +/- 12.3 pM and 10.1 +/- 23.5 nM respectively. Exposure of enterocytes to 10 mM calcium (Ca2+) evoked an increase in intracellular Ca2+ concentrations ([Ca2+](i)). This increase was suppressed by 24R,25(OH)(2)D-3 dose-dependently, with an EC50 of 4.9 nM and a maximal inhibition of 60%. 24R,25(OH)(2)D-3 (20 nM) abolished an increase in [Ca2+](i) (similar to252%) in the control enterocytes exposed to 10 muM S(-)-BAYK-8644, suggesting that the hormone acts by inhibiting Ca2+ entry through L-type voltage-gated Ca2+ channels. Administration of 20 nM 24R,25(OH)(2)D-3 to enterocytes in the absence of extracellular Ca2+ increased [Ca2+](i) by similar to20%, indicating a release of Ca2+ from intracellular stores. Administration of 25(OH)D, (20 nM) resulted in a biphasic change in the enterocyte [Ca2+](i): within 1-5 s, it decreased to 87 +/- 12 nM below its mean basal [Ca2+](i) (334 +/- 13 nM), followed by a rapid recovery of [Ca2+](i) to a new level, 10% lower than the initial [Ca2+](i). The rapid decrease, the recovery rate and the final [Ca2+](i) were all affected dose-dependently by 25(OH)D-3 with EC50 values of 8.5, 17.0 and 18.9 nM respectively. Furthermore, the effects of 25(OH)D-3 were sensitive to sodium (Na+), bepridil (110 muM) and nifedipine (5 pM), suggesting that 25(OH)D-3 regulates the activity of both basolateral membrane-associated Na+/Ca2+ exchangers and brush border membrane-associated L-type Ca2+ channels. Administration of 25(OH)D-3 (10 nM) to enterocytes in the absence of extracellular Ca2+ increased [Ca2+](i) by similar to18%, indicating a release of Ca2+ from intracellular stores. 1,25(OH)(2)D-3 also affected enterocyte [Ca2+](i) in a biphasic manner: the rapid decrease, the recovery rate, and the mean final [Ca2+](i) were all affected dose-dependently, with EC50 values of 8.3, 24.5 and 7.7 nM respectively. The high EC50 values for 1,25(OH),D, compared with circulating concentrations of 1,25(OH)(2)D-3 (130 pM) suggest that this effect is pharmacological, rather than of physiological relevance in enterocyte Ca2+ homeostasis of the Atlantic cod. It is concluded that 24R,25(OH)(2)D-3 has a physiological role in decreasing intestinal Ca2+ uptake via inactivation of L-type Ca2+ channels, whereas the physiological role of 25(OH)D-3 is to increase enterocyte Ca2+ transport via activation of Na+/Ca2+ exchangers, concurrent with activation of L-type Ca2+ channels.