Micromolar aluminum levels reduce 3H-thymidine incorporation by cell line UMR 106-01.

Aluminum-induced osteomalacia is a frequent complication observed in patients on maintenance hemodialysis. However, it is not known whether there are direct effects of aluminum on osteoblasts, or alternatively, whether the observed changes are due to changes in PTH or other factors. We sought to determine the effect of micromolar levels of aluminum on osteoblasts using a well-defined cell line derived from a 32P induced osteosarcoma of rat, UMR 106-01, which is alkaline-phosphatase positive, responds to PTH, and synthesizes type I collagen. Aluminum exposure was controlled using tissue culture media with [Al ] less than 1 microgram/liter (40 nM), produced by precipitation of aluminum salts at pH 8.5. The effect of defined [Al ], from 20 to 800 micrograms/liter (0.7 to 30 microM), was then determined by adding back aluminum while measuring DNA and protein synthesis. We found that aluminum depressed DNA synthesis, as determined by 3H-thymidine incorporation, by 60%, with half maximal effect at 20 micrograms/liter (740 nM) in cells at a density of 20,000/cm2. Alternatively, protein synthesis, as determined by 3H-leucine incorporation, did not decline, and in some cases increased. However, qualitative analysis of matrix proteins produced with and without 800 micrograms/liter (30 mM) [Al ] showed no differences. Direct measurements of cell number and protein synthesis confirmed these findings. Al does not alter the PTH-induced cAMP response of these cells. Thus, aluminum has a direct effect on cell division, and probably on protein synthesis, in this osteoblast-like cell line. These effects occur at levels of aluminum below those commonly contaminating tissue culture media, and thus are seen reproducibly only in media of defined [Al ].(ABSTRACT TRUNCATED AT 250 WORDS)

Enhancement of parathyroid hormone-responsive renal cortical adenylate cyclase activity by a cytosol protein activator from rat reticulocytes.

The effects of the cytosol activator protein obtained from rat reticulocytes (RCAP) were investigated in a heterologous membrane system--partially purified cell membranes from dog renal cortex. RCAP enhanced the response of dog renal cortical adenylate cyclase to bovine parathyroid hormone (1-34) [bPTH (1-34)] from two- to three-fold. RCAP also enhanced the response to 5 microM arginine vasopressin, 10 microM glucagon, and 10 microM isoproterenol. Analysis of double-reciprocal plots of substrate concentration and enzyme activity indicated that bPTH (1-34) alone and together with RCAP increased the Vmax of the adenylate cyclase enzyme and did not alter the apparent Km of the enzyme for MgATP. Membranes from dog renal cortex contain 42K and 39K proteins that are ADP-ribosylated by cholera toxin and pertussis toxin, respectively, and appear to be the stimulatory (Ns) and inhibitory (Ni) guanine nucleotide binding proteins described in many other hormone-responsive membrane preparations. Similar to its effects in rat reticulocytes, RCAP inhibited ADP-ribosylation of Ns and enhanced ADP-ribosylation of Ni. The muscarinic agonist, carbachol, inhibited PTH-responsive adenylate cyclase activity in dog renal cortical membranes and this inhibition was reversed by RCAP. These results indicate that RCAP enhances stimulation of adenylate cyclase by a variety of hormones in a heterologous membrane preparation and supports the hypothesis that RCAP's site of action is common to all adenylate cyclase systems. RCAP may facilitate coupling between Ns and the catalytic unit of adenylate cyclase by a pertussis toxin-like effect to inactivate Ni. The dual effects of RCAP upon ADP-ribosylation of Ni and Ns alpha subunits suggest that a binding site for RCAP may exist at a site of homology between Ns alpha and Ni alpha.

Extrarenal metabolism of 25-hydroxycholecalciferol in the rat: regulation by 1,25-dihydroxycholecalciferol.

To determine the role of the kidney in regulation of 25-hydroxycholecalciferol (25OHD3, metabolism, the effects of 1,25-dihydroxycholecalciferol [1,25(OH)2D3] on 3H-25OHD3 were compared in intact and nephrectomized vitamin D-deficient rats. Sixteen hours after the intravenous administration of 3H-25OHD3, extracts of serum and pooled small intestinal mucosa were fractionated by Sephadex LH-20 column chromatography followed by high performance liquid chromatography. In intact rats, 1,25(OH)2D3 (50 ng/day i.p. for 7 days) increased mean serum 3H-24,25-dihydroxycholecalciferol [3H-24,25(OH)2D3] from 2 +/- 2-210 +/- 80 fmol/ml (mean +/- 1 SD), increased mean serum 3H-25,26-dihydroxycholecalciferol [3H-25,26(OH)2D3] from 2 +/- 2-12 +/- 6 fmol/ml and lowered mean serum 3H-1,25(OH)2D3 from 210 +/- 40-4 +/- 4 fmol/ml. Similarly, in nephrectomized animals, 1,25(OH)2D3 increased mean serum 3H-24,25-(OH)2D3 from 6 +/- 11-115 +/- 30 fmol/ml and increased mean serum 3H-25,26(OH)2D3 from 3 +/- 3-26 +/- 10 fmol/ml. Nephrectomy increased serum 3H-25(OH)D3 in untreated (from 1450 +/- 225-2675 +/- 225 fmol/ml serum) and 1,25(OH)2D3 treated rats (from 1600 +/- 175-3075 +/- 100 fmol/ml). 3H-1,25(OH)2D3 averaged 74 +/- 16% of total radioactivity in intestinal mucosa of untreated intact rats and was not detected in either the serum or intestinal mucosa of nephrectomized animals. The results suggest that in intact animals, extrarenal synthesis can account for substantial 24,25(OH)2D3 production and for most 25,26(OH)2D3 production.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for extrarenal production of 1 alpha ,25-dihydroxyvitamin D in man.

Recent studies provide evidence for extrarenal production of 1 alpha ,25-dihydroxyvitamin D [1 alpha ,25(OH)2D]. To investigate this possibility, serum vitamin D, 25-hydroxyvitamin D (25-OHD), 24,25-dihydroxyvitamin D [24,25(OH)2D], and 1 alpha ,25(OH)2D were measured in eight adult anephric subjects. All were undergoing hemodialysis and three of them were receiving vitamin D, 50,000 or 100,000 U/d. Serum vitamin D was elevated in two of the patients given vitamin D and was abnormally low in the others. Mean serum 25-OHD was increased in patients given vitamin D (94.0 +/- 7.6 ng/ml) and was normal in the others (16.4 +/- 0.9 ng/ml, P less than 0.001). Mean serum 24,25(OH)2D was normal in patients given vitamin D (1.38 +/- 0.27 ng/ml) and was low in the others (0.25 +/- 0.08 ng/ml, P less than 0.001). Serum 24,25(OH)2D correlated significantly with serum 25-OHD (r = 0.848, P less than 0.01). Mean serum 1 alpha ,25(OH)2D determined by receptor assay was 5.8 +/- 1.9 pg/ml in patients who were not given vitamin D and was 14.1 +/- 0.6 in those who were given vitamin D (P less than 0.001). Serum 1 alpha ,25(OH)2D correlated significantly with serum 25-OHD (r = 0.911, P less than 0.01). Mean serum 1 alpha ,25(OH)2D, measured by bioassay, was 8.3 +/- 1.9 pg/ml in patients who were given vitamin D and was 15.9 +/- 2.4 pg/ml in those who were given vitamin D (P less than 0.05). There was a significant correlation between the values for serum 1 alpha ,25(OH)2D obtained with the two methods (r = 0.728, P less than 0.01). The results (a) provide evidence in man for extrarenal production of both 24,25(OH)2D and, by two independent assays, of 1 alpha , 25(OH)2D, and (b) indicate that serum values of the two dihydroxy metabolites of vitamin D in anephric subjects vary with the serum concentration of the precursor 25-OHD.

Intestinal phosphate transport and alkaline phosphatase activity in the chick.

The initial rates of phosphate accumulation by isolated chick intestinal epithelial cells have been examined. At high concentrations of phosphate (1.5 mM), phosphate uptake is relatively independent of sodium and demonstrates a pH optimum of 8.0. At pH 8.0, 56% of the uptake is dependent on the presence of Ca in the uptake medium compared to 28% at pH 6.8. Membranes prepared from these same intestinal epithelial cells contain a Ca-dependent phosphatase that can be distinguished from the more abundant Mg-dependent alkaline phosphatase. The Ca-dependent phosphatase has a pH optimum between 8.5 and 9.0 and, compared to the Mg-dependent activity, is more readily inactivated at 58 degrees C and is relatively resistant to L-phenylalanine inhibition but more sensitive to ethane-1-hydroxy-1,1-diphosphonate (EHDP). Both activities are distributed in a constant proportion between the brush border and basal lateral membranes and at various segments along the intestine. Vitamin D in vivo and 25-hydroxyvitamin D [25(OH)D] in vitro stimulated both activities. In vitro, utilizing the isolated intestinal cells, the stimulation of phosphate uptake paralleled the increase in Ca-dependent alkaline phosphatase activity. The role of alkaline phosphatase in intestinal phosphate transport is discussed.

Characterization of phosphate uptake in isolated chick intestinal cells.

A preparation of isolated intestinal cells has been developed which permits the incubation of these cells in culture for 90 min. with preservation of viability and responsiveness to vitamin D analogues added in vitro. Characterization of phosphate transport suggested the presence of two transport processes which differed in their affinity for phosphate, calcium stimulation, and pH dependence. Both processes were facilitated by sodium transport. This cell preparation promises to be a valuable experimental model for the elucidation of the action of vitamin D on phosphate transport.