The ionic control of 1,25-dihydroxyvitamin D3 synthesis in isolated chick renal mitochondria. The role of calcium as influenced by inorganic phosphate and hydrogen-ion.
Isolated kidney mitochondria prepared from Vitamin D-deficient chicks catalyze the conversion of 25-hydroxyvitamin D3 to 1,25 dihydroxyvitamin D3. It wasfound that changes in the concentrations of Ca-2plus, HPO4-2minus, and Hplus altered synthesis in an interrelated fashion. Increasing the Ca-2plus concentration from 10-6 to 10-5 M caused a four- to fivefold increase in 1 alpha-hydroxylase activity when the medium pH was between 6.5 and 7.0. increasing the [Ca2+] to 10-4 M caused to furhter stimulation. At higher pH values, Ca-2plus had little effect upon 1 alpha-hydroxylase activity. In the absence of calcium [Ca2+] less than or equal to 10-7 M), a change in pH from 6.5 to 7.1 had no effect upon 1 alpha-hydroxylase activity in the presence of 10-5 M calcium, increasing the medium pH had a biphasic effect. An increase in pH from 6.5 to 6.9 caused a 1.5-fold increase in 1 alpha-hydroxylase activity, but a further increase of the pH to 7.1 caused a profound decrease in rate of hydroxylation to approximately 20% of the peak value. Neither 10-5 M LaC13 nor 10 mug/ml of oligomycin altered the effects of Ca2+ upon hydroxylate activity. However, the effect of calcium was blocked by 2.5 times 10-5 M ruthenium red, 0.83 mug/ml of antimycin A, and 500 muM dinitrophenol. The clcium ionophore, A23187, decreased but did not prevent the stimulatory effect of calcium. These data are consistent with the concept that the [Ca2+ in the mitochondrial matrix space is of importance in regulating the 1 alpha-hydroxylase. Phosphate exerted a biphasic effect on 1,25(OH)2D3 production with maximal stimulation (approximately twofold) at 1-3 mM. Calcium enhanced the stimulation by phosphate at all concentrations studied. The presence of potassium modified the interrelated effects of calcium and phosphate in two ways: 10-3 M calcium blocked the stimulation by phosphate; and in the presence of phosphate, 10-3 M calcium resulted in less 1,25(OH)2D3 production by production by isolated mitochondria are qualitatively similar to the effects of these ions on 1,25(OH)2D3 production yb isolated renal tubules.
D000818:Animals; D000968:Antimycin A / Q000494:pharmacology; D002118:Calcium / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D002645:Chickens; D004032:Diet; D004100:Dihydroxycholecalciferols / Q000096:biosynthesis*; D004140:Dinitrophenols / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D006887:Hydroxycholecalciferols / Q000096:biosynthesis*; D066298:In Vitro Techniques; D007668:Kidney / Q000201:enzymology* / Q000648:ultrastructure*; D008928:Mitochondria / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000378:metabolism; D009840:Oligomycins / Q000494:pharmacology; D010710:Phosphates / Q000494:pharmacology*; D012428:Ruthenium / Q000494:pharmacology; D014808:Vitamin D Deficiency / Q000378:metabolism
D004100:Dihydroxycholecalciferols; D004140:Dinitrophenols; D006887:Hydroxycholecalciferols; D009840:Oligomycins; D010710:Phosphates; D000968:Antimycin A; D012428:Ruthenium; D006899:Mixed Function Oxygenases; D002118:Calcium