calcimycin and Vitamin-D-Deficiency

Many biotransformation activities have absolute or modulated localization within the hepatic acinus. To investigate the intrahepatic acinar zonation of vitamin D3 (D3) metabolism, hepatic D3 extraction was investigated by antegrade or retrograde perfusion of normal livers and livers bearing selective periportal (PP) or perivenous (PV) destruction; D3 C-25 hydroxylation was studied after selective harvesting of PP or PV hepatocytes by digitonin-collagenase perfusion. Data indicate that hepatic D3 extraction is not regioselective and not perturbed by destruction of the proximal (PP) or distal (PV) part of the acinus, indicating that D3 extraction takes place in the most proximal hepatocytes being perfused. These observations suggest that, in vivo, D3 extraction will take place according to its concentration gradient within the hepatic acinus, thus resulting in a preferential PP extraction of the vitamin. D3 C-25 hydroxylation was higher in PP than in PV hepatocytes in the presence of 1.9 mM Ca2+, with 25-hydroxyvitamin D3 [25(OH)D3] formation of 34.6 +/- 3.9 and 24.4 +/- 1.1 fmol.h-1.(10(6) hepatocytes)-1, respectively (P less than 0.05). Modulators of extracellular [ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA)] or intracellular Ca2+ (parathyroid hormone, A23187), however, significantly influenced 25(OH)D3 formation with similar decreases in the PP (31%) and PV (26%) areas in the presence of EGTA but with increases in the presence of Ca2+ ionophore A23187 of 189 +/- 16% in PP and of 260 +/- 20% in PV hepatocytes, resulting in similar production in both regions of the acinus.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Biotransformation; Calcifediol; Calcimycin; Calcium; Cells, Cultured; Cholecalciferol; Digitonin; Egtazic Acid; Female; Hydroxylation; Liver; Male; Organ Specificity; Rats; Rats, Inbred Strains; Sucrose; Tritium; Vitamin D Deficiency

The role played by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and/or by calcium on the C-25 hydroxylation of vitamin D3 (D3) was studied in hepatocytes isolated from D-depleted rats which were divided into four treatment groups: Group 1 served as controls, Group 2 received calcium gluconate, Groups 3 and 4 were infused with 1,25(OH)2D3 at 7 and 65 pmol/24 h x 7 days respectively. The treatments normalized serum calcium in all but the controls which remained hypocalcaemic, while serum 1,25(OH)2D3 remained low in Groups 1 and 2 but increased to physiologic and supraphysiologic levels in Groups 3 and 4. The data show that basal D3-25 hydroxylase activities were not significantly affected by any of the treatments. Addition of CaCl2, EGTA, or Quin-2 in vitro revealed that relative to basal values, EGTA strongly inhibited the enzyme activity in all groups (P less than 0.0001), except in G 1; Quin-2 and CaCl2 had no significant effect on the activity of the enzyme in any of the groups. Addition of 1,25(OH)2D3 or A23187 in vitro in the presence of CaCl2 revealed that 1,25(OH)2D3 did not significantly affect enzyme activity, while A23187 was found to stimulate its activity in vitamin D-depleted animals, but most specifically in Group 2 (P less than 0.001); low serum calcium (Group 1) dampened (P less than 0.01), and 1,25(OH)2D3 treatment in vivo totally blunted (P less than 0.001) the response to A23187. The data suggest that 1,25(OH)2D3 supplementation in vivo has per se little or no effect on the basal D3-25 hydroxylase activity. The data show, however, that the magnitude of the response to various challenges in vitro is greatly influenced by the conditioning in vivo of the animals. They also show that A23187 can be a potent stimulator of the enzyme activity, which allowed us to demonstrate a significant reserve for the C-25 hydroxylation of D3 which is well expressed in hepatocytes obtained from D-depleted calcium-supplemented rats.

Topics: Animals; Calcifediol; Calcimycin; Calcitriol; Calcium Chloride; Cholecalciferol; Cholestanetriol 26-Monooxygenase; Egtazic Acid; Enzyme Induction; In Vitro Techniques; Liver; Male; Rats; Rats, Inbred Strains; Steroid Hydroxylases; Vitamin D Deficiency

Rat duodenal cells were isolated sequentially to give fractions enriched for villus and crypt cells. From each of these fractions, basolateral-enriched membrane vesicles were prepared and ATP-dependent calcium uptake was studied. Calcium uptake was sensitive to temperature, was inhibited by vanadate and by A23187, and was lower in vitamin D-deficient animals. In normal animals, calcium transport was approximately twofold greater in villus-tip than in crypt cell-fraction basolateral membranes though the affinity of the uptake for calcium was similar (Km = 0.3 microM). In vitamin D-deficient animals, the crypt-to-villus gradient was reduced, and in all fractions, calcium transport was similar to or lower than that in the crypts of normal animals. Six hours after vitamin D-deficient animals were repleted with 1,25-dihydroxycholecalciferol, a significant increase in calcium transport by everted gut sacs was present; however, basolateral calcium transport was significantly increased in only the mid-villus fractions, and no change was seen in the villus-tip fractions. Thus vitamin D appears necessary for the development of increased basolateral membrane calcium pump activity in duodenal villus cells, but not all cells in vitamin D-deficient rats are able to respond to 1,25-dihydroxycholecalciferol.

Topics: Adenosine Triphosphate; Animals; Biological Transport; Calcimycin; Calcitriol; Calcium; Cell Membrane; Cell Separation; Duodenum; Ion Channels; Kinetics; Male; Rats; Vanadates; Vanadium; Vitamin D Deficiency

The presence of an Na+/Ca2+ exchange system in basolateral plasma membranes from rat small intestinal epithelium has been demonstrated by studying Na+ gradient-dependent Ca2+ uptake and the inhibition of ATP-dependent Ca2+ accumulation by Na+. The presence of 75 mM Na+ in the uptake solution reduces ATP-dependent Ca2+ transport by 45%, despite the fact that Na+ does not affect Ca2+-ATPase activity. Preincubation of the membrane vesicles with ouabain or monensin reduces the Na+ inhibition of ATP-dependent Ca2+ uptake to 20%, apparently by preventing accumulation of Na+ in the vesicles realized by the Na+-pump.l It was concluded that high intravesicular Na+ competes with Ca2+ from intravesicular Ca2+ binding sites. In the presence of ouabain, the inhibition of ATP-dependent Ca2+ transport shows a sigmoidal dependence on the Na+ concentration, suggesting cooperative interaction between counter transport of at least two sodium ions for one calcium ion. The apparent affinity for Na+ is between 15 and 20 mM. Uptake of Ca2+ in the absence of ATP can be enhanced by an Na+ gradient (Na+ inside greater than Na+ outside). This Na+ gradient-dependent Ca2+ uptake is further stimulated by an inside positive membrane potential but abolished by monensin. The apparent affinity for Ca2+ of this system is below 1 microM. In contrast to the ATP-dependent Ca2+ transport, there is no significant difference in Na+ gradient-dependent Ca2+ uptake between basolateral vesicles from duodenum, midjejunum and terminal ileum. In duodenum the activity of ATP-driven Ca2+ uptake is 5-times greater than the greater than the Na+/Ca2+ exchange capacity but in the ileum both systems are of equal potency. Furthermore, the Na+/Ca2+ exchange mechanism is not subject to regulation by 1 alpha, 25-dihydroxy vitamin D-3, since repletion of vitamin D-deficient rats with this seco-steroid hormone does not influence the Na+/Ca2+ exchange system while it doubles the ATP-driven Ca2+ pump activity.

Topics: Adenosine Triphosphate; Animals; Calcimycin; Calcium; Cell Membrane; Intestine, Small; Kinetics; Male; Monensin; Ouabain; Rats; Rats, Inbred Strains; Sodium; Vitamin D Deficiency

The vitamin D metabolite 1,25-dihydroxyvitamin D [1,25-(OH)2D] given in vivo stimulates calcium accumulation by subsequently isolated duodenal brush border membrane vesicles (BBMV). Stimulation is rapid (within 2 h), reaching a maximum between 2-4 h. This effect occurs well before stimulation of in vivo calcium transport (2-4 h), cytosolic calcium-binding protein production (4-8 h, or alkaline phosphatase activity (8 h). No cytosolic calcium-binding protein was found in the BBMV at any time. The extent of calcium accumulation by BBMV exceeds by severalfold the predicted value based on the equilibrium distribution of glucose, indicating a substantial amount of binding. The ability of the calcium ionophore A23187 to increase the rate of accumulation suggests that this binding is intravesicular. The Eadie Hofstee analysis of the rate of calcium accumulation as a function of calcium concentration is nonlinear. At submillimolar calcium concentrations, the difference in the apparent Km for calcium accumulation by BBMV from vitamin D-deficient and 1,25-(OH)2D-treated chicks is nearly 2-fold (1.9 X 10(-4) vs. 1.1 X 10(-4)M, respectively), a difference that is not observed at higher calcium concentrations. Release of calcium from preloaded BBMV with the addition of EGTA is rapid but not complete (20-30% of the initial value after 60 min). The rapidity, but not the extent, of release is increased with A23187. BBMV from vitamin D-replete and vitamin D-deficient duodena do not differ in their rate or extent of calcium release, in contrast to their different rates of calcium accumulation. We conclude that the stimulation by 1,25-(OH)2D of calcium accumulation by BBMV is one of the earliest actions of 1,25-(OH)2D on the intestine, that this process does not involve alkaline phosphatase or cytosolic calcium-binding protein, and that influx, but not efflux, of calcium is regulated.

Topics: Alkaline Phosphatase; Animals; Calcimycin; Calcitriol; Calcium; Calcium-Binding Proteins; Chickens; Duodenum; Kinetics; Male; Microvilli; Vitamin D Deficiency