Evidence that stimulation of 1,25(OH)2D3 production in primary cultures of mouse kidney cells by cyclic AMP requires new protein synthesis.

When primary culture of C75BL6 mouse cortical kidney cells in serum-free medium were incubated with unlabeled 25(OH)D3, they produced a metabolite which co-migrated with authentic 1,25(OH)2D3 and which could be measured by competitive receptor assay. A metabolite co-migrating with authentic 10-oxo-19-nor-25-OH-D3 was also produced. However, when cultures were incubated with 25(OH)D3 for 1 hour or longer, 10-oxo-19-nor-25-OH-D accounted for less than 15% of the total 3H-1,25(OH)2D3 displacement activity. Production of 1,25(OH)2D3 increased with increasing content of the culture, with time of incubation, and with substrate concentration. The apparent Km was 1.4 +/- 0.6 microM and Vmax 2.6 +/- 0.4 pM/mg protein/hr. These cultures possessed a very high level of phosphodiesterase activity, as indicated by their high cyclic AMP (cAMP) response to IBMX. This high phosphodiesterase activity may have been responsible for the lack of stimulation of 1,25(OH)2D3 production by physiologic or near physiologic concentrations of parathyroid hormone (PTH) in the absence of IBMX. However, when IBMX 10(-6) M was present, bPTH 10(-9) M significantly increased production of both cAMP and 1,25(OH)2D3. There was a close correlation between 1,25(OH)2D3 production and cAMP content of the cultures (basal or stimulated). An incubation time of at least 4 hours was required for cAMP to increase 1,25(OH)2D3 production and was inhibited in the presence of cycloheximide and actinomycin D. This study further documents the regulation of renal 1,25(OH)2D3 synthesis by PTH in mammalian kidney and provides evidence for cAMP as a possibly important second messenger in this effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Reduced binding of [3H]1,25-dihydroxyvitamin D3 in the parathyroid glands of patients with renal failure.

This study examined the hypothesis that altered binding of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) to parathyroid receptors might be involved in the pathogenesis of secondary hyperparathyroidism associated with chronic renal failure. The binding of [3H]1,25-(OH)2D3 to hyperplastic parathyroid glands obtained from seven patients with chronic renal failure was measured. These values were compared with those for binding to hyperplastic parathyroid tissue obtained from six patients who had received renal transplants and for binding to parathyroid adenomas removed from five patients who had primary hyperparathyroidism. We found that Nmax (an estimate of the concentration of 1,25-(OH)2D3 receptors) was reduced (42 +/- 15 fmol per milligram of protein) in patients with chronic renal failure as compared with patients with transplanted kidneys (78 +/- 24 fmol per milligram of protein) and patients with primary hyperparathyroidism (114 +/- 30). Nmax correlated inversely with the severity of renal dysfunction, the serum level of phosphorus, and the logarithm of the serum level of immunoreactive parathyroid hormone. These observations suggest that 1,25-(OH)2D3 binding by parathyroid tissue is reduced in chronic renal failure. This may contribute to the pathogenesis of secondary hyperparathyroidism by reducing the inhibition by 1,25-(OH)2D of parathyroid hormone secretion. The low serum levels of 1,25-(OH)2D in chronic renal failure may accentuate this effect.

Immobilization hypercalcemia associated with Landry-Guillain-Barré syndrome. Successful therapy with combined calcitonin and etidronate.

Two patients with immobilization hypercalcemia associated with Landry-Guillain-Barré syndrome had marked hypercalciuria (890 and 1136 mg/d [22.2 and 28.3 mmol/d]) and radiologic evidence of generalized osteopenia. Parathyroid hormone levels were either low or normal by C-terminal radioimmunoassay. Subtotal parathyroidectomy was performed in the one patient, with no improvement in serum or urinary calcium levels. A bone biopsy specimen revealed decreased cellular activity in the first patient and increased bone resorption in the second patient. Treatment with intravenous saline, furosemide, oral phosphate supplementation, mithramycin, and calcitonin alone was ineffective in lowering serum or urinary calcium levels. However, when subcutaneous calcitonin combined with oral etidronate disodium was used, a reduction in the serum calcium level was observed within two days of therapy. Within one week of the start of this combined therapy, the calcium level returned to normal and urinary calcium excretion was substantially reduced.

Reduced absorption of 45calcium from isolated duodenal segments in vivo in juvenile but not adult X-linked hypophosphatemic mice.

In juvenile X-linked hypophosphatemic (Hyp) mice, whole body calcium balances are significantly lower than in genetically normal mice. This is associated with low duodenal vitamin D-dependent calcium-binding protein and a failure of skeletal mineralization. To seek more specific evidence of an intestinal defect in these mice, absorption of 45Ca was measured in isolated duodenal segments in vivo in mice from 2-13 weeks of age. The duodenum was isolated by sutures and 45Ca was injected into the lumen in 150 mM NaCl and 2 mM CaCl2 at pH = 7.2. Absorption was measured by the amount of isotope remaining in the lumen and by the plasma isotope level. Hemizygous Hyp male and heterozygous Hyp female mice absorbed significantly less 45Ca at 4 and 7 weeks of age than genetically normal mice while Hyp mice at 2, 10, and 13 weeks of age were not significantly affected. At 4 and 7 weeks of age, the Hyp mice also had significantly reduced plasma radioactivity midway through the collection period as well as at the end of the period. To explore a possible mechanism for this malabsorption, 1,25(OH)2-vitamin D receptors were measured in cytosol prepared from 4-week-old normal and Hyp duodenum. There were non-significant differences between the normal and Hyp mice in both binding affinity, Kd, and the number of receptors, nmax. In conclusion, juvenile Hyp mice at 4 and 7 weeks of ages malabsorbed calcium from their duodenum. Hyp mice younger than this period were not affected nor were adult Hyp mice.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of chronic prednisone administration on intestinal receptors for 1,25-dihydroxyvitamin D3 in the dog.

Glucocorticoid inhibits intestinal calcium absorption. To further explore the mechanism of this inhibition, we studied dogs during the administration of oral prednisone (1.2-1.5 mg/kg X day) for 20 to 28 weeks in comparison to untreated dogs. Prednisone administration had no effect on serum 25-hydroxyvitamin D concentrations, but was accompanied by a fall in serum 1,25-dihydroxyvitamin D [1,25-(OH)2D] concentrations from 87 +/- 20 pM (control) to 62 +/- 28 pM (prednisone-treated; P less than 0.01). Cytosol prepared from the duodenal, jejunal, and ileal mucosa of control dogs was found to contain a specific 3.2S [3H]1,25-(OH)2D3 binder analogous to the binder that has been observed in the intestine of other species and in other tissues. The apparent concentration of this binder decreased progressively from duodenum to ileum. Prednisone administration increased the apparent duodenal concentration of the binder from 170 +/- 91 (control) to 363 +/- 124 fmol/mg protein (prednisone-treated; P less than 0.025). The intestinal content of calcium-binding protein also declined progressively from the duodenum to the ileum, but was not affected by prednisone administration. These data suggest that events other than alterations in intestinal 1,25-(OH)2D3 receptors must mediate the inhibition of intestinal calcium absorption during chronic glucocorticoid administration.