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R W Gray

Publications and source records attributed to R W Gray.

At least 55 records · Page 3Linked to original sources
PubMed1987

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)

1-Methyl-3-isobutylxanthine↗
PubMed1987

Evidence that somatomedins mediate the effect of hypophosphatemia to increase serum 1,25-dihydroxyvitamin D3 levels in rats.

The present studies were undertaken in an effort to determine whether somatomedins (SMs) play a role in the elevation of serum 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] levels during dietary phosphate deprivation. Serum 1,25-(OH)2D3,SM-C, and phosphate levels were measured in rats fed diets containing adequate or very low levels of dietary phosphorus under circumstances known to affect SM levels, including hypophysectomy with and without GH replacement, normal protein vs. low protein diets, and streptozotocin-induced diabetes with and without insulin replacement. In all circumstances, serum 1,25-(OH)2D3 concentrations were directly related to serum SM-C levels. However, the slope for the relationship was increased 2- to 10-fold in animals fed the low phosphorus diets. As observed previously, serum 1,25-(OH)2D3 levels were inversely related to serum phosphate levels, but the slope for this relationship was deceased in the presence of low SM levels and absent in animals with very low SM levels. These results suggest that SM are required for elevation of serum 1,25-(OH)2D3 levels in response to phosphate deprivation.

Animals↗
PubMed1986

25-Hydroxyvitamin D3 metabolism in a human leukemia cell line.

1,25-dihydroxyvitamin D3 (1,25(OH)2D3) is a potent inducer of monocytic differentiation of the human promyelocytic leukemia cell line, HL-60. We have noted that 25-hydroxyvitamin D3 (25(OH)D3) in high doses is also capable of promoting monocytic differentiation of this cell line. To test the possibility that the latter activity is due to conversion of 25OHD3 to 1,25(OH)2D3 by HL-60, we exposed HL-60 cells to 25OHD3 and analyzed the products by HPLC and radioreceptor assay. When chromatographed in the traditional solvent system (isopropanol-hexane), a new peak appears which migrates with authentic 1,25(OH)2D3. However, in a solvent system containing dichloromethane, 90% of the peak migrates with another metabolite, 19-Nor-10-Keto-25OHD3 (19-Nor-25OHD3). Production of this metabolite is enhanced by living cells and is synthesized by both virgin HL-60 and those which have undergone differentiation. We next determined if authentic 19-Nor-25OHD3 also promotes differentiation of this cell. As assessed by appearance of the monocyte-specific surface antigen (63D3) and macrophage-specific esterase activity, we find that this metabolite does, in fact, induce monocytic differentiation of HL-60 with a potency of approximately 1/200 that of 1,25(OH)2D3 and similar to that of 25OHD3. In agreement with the effect upon cell maturation, 19-Nor-25OHD3 displaces 3H-1,25(OH)2D3 from its HL-60 receptor with an efficiency comparable to 25OHD3. Hence, HL-60 cells convert 25OHD3 to 19-Nor-25OHD3, and 19-Nor-25OHD3 induces monocytic differentiation of HL-60 with comparable efficiency to its precursor, 25OHD3.

Calcifediol↗
PubMed1986

The importance of renal net acid excretion as a determinant of fasting urinary calcium excretion.

To evaluate the effects of changing rates of fixed acid production on fasting urine Ca/creatinine, we studied five healthy men fed constant diets during control conditions (serum HCO3 27.3 +/- 2.6 SD mEq/liter and blood H+ 40.4 +/- 1.5 microEq/liter) and then during the administration of NH4Cl 3.0 mEq/kg/day (serum HCO3 22.5 +/- 4.9 mEq/liter; P less than 0.025, and H+ 46.8 +/- 2.3 mEq/liter; P less than 0.005). In addition to the expected increase in daily urinary Ca excretion from 5.2 +/- 2.0 to 12.5 +/- 3.0 mmole/day; P less than 0.001 as daily urinary net acid excretion was increased from 48 +/- 32 to 257 +/- 33 mEq/day; P less than 0.001 we observed that fasting urinary net acid/creatinine excretion also increased from 2.9 +/- 1.2 to 11.1 +/- 1.2 mEq/mmole creatinine; P less than 0.001 and fasting urine Ca/creatinine increased from 0.158 +/- 0.111 to 0.456 +/- 0.109 mmole/mmole creatinine; P less than 0.005. The additional Ca appearing in the urine during acidosis ultimately reflected augmented net bone resorption since daily urinary hydroxyproline excretion was increased from 0.232 +/- 0.062 to 0.377 +/- 0.108 mmole/day; P less than 0.01. Since variations in diet composition can cause fixed acid production and thus renal net acid excretion to vary from about zero to 200 mEq/day, such a range could cause fasting Ca/creatinine to vary from 0.09 to 0.37 mmole/mmole (0.03 to 0.13 mg/mg) and should be taken into account in the evaluation of fasting Ca/creatinine.

Acid-Base Equilibrium↗
PubMed1985

Picric acid methods greatly overestimate serum creatinine in mice: more accurate results with high-performance liquid chromatography.

The creatinine levels of blood and urine from humans, rats, and mice were measured by high-performance liquid chromatography. These were compared to the alkaline picrate analysis of creatinine performed by standard colorimetric, kinetic, and AutoAnalyzer techniques. For human serum and urine the values obtained using the HPLC technique gave good agreement with four out of five alkaline picrate techniques. For black or white mice, the serum creatinine concentration was 8.7 +/- 0.4 microM by HPLC but 44.9 +/- 1.9 microM by the lowest alkaline picrate method. Mouse urine creatinine concentrations were 3.24 +/- 0.19 mM by HPLC and 4.59 +/- 0.39 mM by the nearest alkaline picrate method. Rat serum creatinine concentrations analyzed by HPLC were about half the values obtained by AutoAnalyzer. Mouse and rat samples seemed to have substances which gave nonspecific color and thus interfered with the analysis of creatinine by the alkaline picrate methods. While the alkaline picrate analysis of creatinine was adequate for human samples, it was necessary to use HPLC to accurately measure rodent creatinine. The fractional excretion of creatinine was determined by measuring creatinine in mouse urine and plasma by both the kinetic and HPLC methods and comparing these values to urine and plasma inulin. Using the kinetic method, creatinine was cleared at 43 +/- 3% of the rate of inulin. Using the HPLC method, creatinine was cleared at 170 +/- 11% of the rate of inulin.

Animals↗
PubMed1985

Hydrochlorothiazide inhibits bone resorption in men despite experimentally elevated serum 1,25-dihydroxyvitamin D concentrations.

We evaluated the effects of hydrochlorothiazide administration in relation to Ca balance, the PTH and vitamin D endocrine systems, acid-base balance, and bone. We studied six healthy men fed constant diets providing only 5.1 +/- 0.7 SD mmoles Ca/day. Three of the men were also given calcitriol, 0.5 microgram 6-hrly throughout their studies. All subjects were observed during 18 control days and then during 18 days of hydrochlorothiazide (HTZ) administration, 25 mg 12-hrly. Observations during control days 11 through 16 were compared to those during days 7 through 18 of HTZ administration, inclusively. Directional changes during HTZ did not differ among subjects not given or given calcitriol. For all six subjects, control net intestinal Ca absorption, serum 1,25-(OH)2-D concentrations, serum iPTH concentrations, and daily urine cAMP excretion averaged 0.5 +/- 2.2 mmoles/day, 162 +/- 51 pM, 4.3 +/- 2.2 microliter Eq/ml and 4.2 +/- 0.9 mumoles/day, respectively; none changed during HTZ. As expected, HTZ administration was accompanied by a fall in urinary Ca excretion, averaging -1.4 +/- 0.8 mmoles/day; P less than 0.01. HTZ administration was also accompanied by less negative Ca balances, averaging +1.6 +/- 1.0 mmoles/day; P less than 0.025, and by a fall in daily urinary hydroxyproline excretion averaging -0.13 +/- 0.09 mmoles/day; P less than 0.025. We interpret these data to indicate that HTZ administration is accompanied by an inhibition of bone resorption. HTZ administration also raised serum HCO3 concentrations by +2.7 +/- 0.5 mEq/liter; P less than 0.001 and blood pH by + 0.05 +/- 0.02 units; P less than 0.005.(ABSTRACT TRUNCATED AT 250 WORDS)

Acid-Base Equilibrium↗
PubMed1985

Activation of renal 1,25-dihydroxyvitamin D3 synthesis by phosphate deprivation: evidence for a role for growth hormone.

In vitro 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] production in kidney slices from normal intact rats averaged 16 +/- 4 pmol/g . h and was increased about 8-fold by phosphate deprivation and 5-fold by calcium deprivation to levels averaging 128 +/- 12 and 84 +/- 19 pmol/g x h, respectively. Hypophysectomy in phosphate-deprived rats completely abolished any increase in 1 alpha-hydroxylase activity, while calcium deprivation in hypophysectomized (hypox) rats resulted in a 4-fold increase in 1 alpha-hydroxylase activity. Replacement of hypox rats fed a low phosphorus diet with pituitary extracts resulted in a 4-fold stimulation of 1 alpha-hydroxylase activity in response to the hypophosphatemic stimulus. However, replacement of hypox rats fed a normal phosphorus diet with pituitary extract stimulated 1 alpha-hydroxylase activity only 2-fold. Replacement of hypox rats fed a low phosphorus diet with GH resulted in a 3.5-fold elevation in plasma 1,25-(OH)2D3 levels, while no such elevation in plasma 1,25-(OH)2D3 levels was observed in similarly treated animals replaced with PRL, ACTH, TSH, or T3. Replacement of hypox rats eating a normal diet with GH resulted in no significant change in plasma 1,25-(OH)2D3 levels. These results suggest that GH is required for maintenance of elevated plasma 1,25-(OH)2D3 levels during dietary phosphate deprivation and that this effect is mediated by increased renal 1,25-(OH)2D3 synthesis.

Animals↗
PubMed1985

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.

Animals↗
PubMed1984

Effects of weight loss on serum 1,25-(OH)2-vitamin D concentrations in adults: a preliminary report.

During a review of 42 metabolic studies in healthy women and men we observed that serum 1,25-(OH)2-D concentrations were directly correlated to the observed daily changes in body weight (r = 0.68; P less than 0.001) and to caloric intake/kg/day (r = 0.39; P = 0.01). These relationships could not be accounted for by related and physiologically expected changes in serum Ca or iPTH concentrations. However, serum 1,25-(OH)2-D concentrations were observed to be inversely correlated to serum PO4 levels (r = -0.44; P = 0.004). In addition, serum PO4 levels were inversely correlated to the daily changes in body weight (r = -0.40; P = 0.009). Since dietary sodium intake averaged 142 mmol/day, it is unlikely that the observed changes in weight were the result of changes in salt and water balance. Thus it seems reasonable to speculate that serum 1,25-(OH)2-D concentrations may vary directly with energy balance, as reflected by changes in body weight. This effect may be mediated by alterations in PO4 metabolism. The accurate assessment of serum 1,25-(OH)2-D levels thus appears to require several measurements over time periods during which body weight is stable.

Adult↗
PubMed1984

Metabolites of vitamin D in normal and X-linked hypophosphatemic mice.

Hyp mice are a model for human X-linked hypophosphatemia (vitamin D-resistant rickets.) To determine whether an abnormality of vitamin D metabolism exists in this disease, the profiles of the metabolites of vitamin D were determined in normal and Hyp mouse plasma. Hyp and normal mice were fed a vitamin D-deficient diet and received 1,2 3H-vitamin D3 at 16 Ci/mmol by stomach tube at 5 ng/g body weight (0.21 microCi/g b.w.) on alternate days for 14 days. The dose of vitamin D given maintained near normal plasma 25-OH-vitamin D. Thus the mice were in a vitamin D-replete state with all metabolite pools labeled with 3H. Plasma was collected from 4 normal and 4 Hyp mice. The plasma was extracted, and the extracts were chromatographed separately for each mouse on an LH-20 column. Each major peak of radioactivity was rechromatographed using high performance liquid chromatography on a Zorbax-Sil column using solvent systems known to resolve several vitamin D metabolites. Twenty-one radio-active peaks were identified. The disintegrations per minute of 3H in each peak were quantified and converted to plasma concentration using the known specific activity of the administered vitamin D. The 25-OH-vitamin D accounted for 55% of the circulating radioactivity, and 24,25-(OH)2-vitamin D accounted for 22%. The plasma levels of 24,25-(OH)2-vitamin D were similar to levels previously reported by us using protein binding assays. No peaks of radioactivity were missing in the plasma extracts of the Hyp mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

Dietary calcium and serum 1,25-(OH)2-vitamin D concentrations as determinants of calcium balance in healthy men.

We previously reported that experimental elevations of serum 1,25-(OH)2-vitamin D [1,25-(OH)2-D] concentrations produced by the chronic oral administration of calcitriol, 0.75 micrograms every 6 hr, to healthy human males eating diets providing only 4 mmoles Ca/day stimulate net bone resorption as evidenced by more negative Ca balances and higher rates of urinary hydroxyproline excretion. To determine whether increased dietary Ca intake modifies this response we have compared serum 1,25-(OH)2-D and iPTH concentrations, Ca and PO4 balances, and urinary hydroxyproline excretion in three healthy human males adapted to diets providing 22.3 +/- 1.3 mmoles Ca/day and three healthy human males adapted to diets providing 9.3 +/- 0.7 mmoles Ca/day before and during the continuous oral administration of calcitriol 0.5 micrograms every 6 hr. For all six subjects, serum 1,25-(OH)2-D levels averaged 89 +/- 25 pM during control and 143 +/- 26 pM during calcitriol. Net intestinal Ca absorption and urinary Ca excretion rose during calcitriol but Ca balances did not change, averaging +2.2 +/- 2.2 mmoles/day during control and +4.3 +/- 2.2 mmoles/day during calcitriol for the subjects fed 22 mmoles Ca/day and -1.6 +/- 1.5 mmoles Ca/day during control and -1.7 +/- 2.0 mmoles Ca/day during calcitriol for the subjects fed 9 mmoles Ca/day. Urinary hydroxyproline excretion also did not change. Thus, when serum 1,25-(OH)2-D levels are elevated, the availability of dietary Ca appears to prevent more negative Ca balances and increased urinary hydroxyproline excretion suggesting that net bone resorption is not stimulated.

Adult↗
PubMed1984

Phosphate deprivation increases serum 1,25-(OH)2-vitamin D concentrations in healthy men.

To re-evaluate whether phosphate-deprivation alters serum 1,25-(OH)2-D concentrations in men, we measured serum 1,25-(OH)2-D levels in seven healthy men while they ate constant diets providing 52.2 +/- 4.3 sd mmoles PO4/day for 12 days and then while they ate a diet providing 29.4 +/- 6.0 mmoles PO4/day and also were given Al(OH)3 for 18 days. Serum PO4 levels fell only transiently. Net intestinal PO4 absorption became indistinguishable from zero but, because of renal PO4 conservation, PO4 balances did not change. Nevertheless, serum 1,25-(OH)2-D concentrations increased from 80 +/- 21 pM during control to 104 +/- 26 pM; during PO4 deprivation; P less than 0.01. Net intestinal calcium (Ca) absorption tended to rise during PO4 deprivation, the individual increments being correlated to the increments in serum 1,25-(OH)2-D concentrations (r = 0.78; P less than 0.05). Urinary Ca excretion rose; P less than 0.001, the increments exceeding the increments in net intestinal Ca absorption so that Ca balances became more negative; P less than 0.001. Urinary hydroxyproline excretion did not change. Thus, as in women and animals, PO4 deprivation appears to cause enhanced net bone resorption apparently by reducing bone formation.

Adult↗
PubMed1983

Dietary phosphate deprivation increases 1,25-dihyroxyvitamin D3 synthesis in rat kidney in vitro.

A sensitive radioreceptor assay has been used to measure in vitro 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) synthesis in vitamin D-replete rats. Incubation of kidney cortical slices with 25-hydroxyvitamin D3 produced a product which co-migrated on high performance liquid chromatography with authentic 1,25(OH)2D3 in two different solvent systems and displaced 1,25(OH)2D3 from its intestinal receptor. In addition, mass spectral analysis of the product produced a mass fragmentation consistent with that of authentic 1,25(OH)2D3. Endogenous renal cortical 1,25(OH)2D3 content in phosphate-deprived rats averaged 1.1 +/- 0.3 pmol/g (n = 11), which was significantly greater than the renal cortical 1,25(OH)2D3 content of age-matched rats eating a normal diet which averaged 0.44 +/- 0.21 pmol/g (n = 8, p less than 0.001). After incubation, net 1,25(OH)2D3 synthesis in renal slices from phosphate-deprived rats averaged 51 pmol/g/h, about 13-fold greater than the mean of 3.8 pmol/g/h observed in renal slices from rats eating the normal diet. These results indicate that the elevated plasma 1,25(OH)2D3 levels observed in rats during dietary phosphate deprivation are due to increased renal synthesis of the hormone.

Animals↗
PubMed1983

Regulation of plasma 1,25-(OH)2-D3 by phosphate: evidence against a role for total or acid-soluble renal phosphate content.

In order to evaluate a possible role for tissue phosphate or phosphorylated compounds in mediating the increase in plasma 1,25-(OH)2-D3 levels during dietary phosphate deprivation, measurements of total and acid-soluble renal cortical phosphate content have been made in both intact and hypophysectomized (hypox) rats eating a normal diet and also after four days of dietary phosphate deprivation. Similar measurements were also made in phosphate-deprived hypophysectomized rats replaced with growth hormone (GH). Total and acid-soluble renal cortical phosphate content averaged 81 +/- 8 mumol/g and 4.1 +/- 0.6 mumol/g, respectively, in intact rats eating the normal diet and were not significantly altered after phosphate deprivation despite a fall in plasma phosphate of about 40% and a fourfold increase in plasma 1,25-(OH)2-D3 levels. Total and acid-soluble renal cortical phosphate content levels were higher in hypox rats, averaging 92 +/- 8 mumol/g and 4.9 +/- 0.7 mumol/g, respectively, but also did not change after phosphate deprivation. Replacement of phosphate-deprived hypox rats with GH resulted in a further fall in plasma phosphate and a significant increase in plasma 1,25-(OH)2-D3 levels, but there was no change in either total or acid-soluble renal cortical phosphate content. The distribution of organophosphorus compounds in the acid-soluble phosphate fraction in these experiments was also evaluated using 31P NMR spectrometry. Although there appeared to be an increase in the total concentration of organophosphorus compounds after phosphate deprivation, this effect was not altered by hypophysectomy or by replacement of phosphate-deprived hypox rats with GH.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1983

Bone resorption stimulated by elevated serum 1,25-(OH)2-vitamin D concentrations in healthy men.

We evaluated whether calcitriol administration to healthy men stimulates bone resorption. We compared serum 1,25-(OH)2-D concentrations, Ca and PO4 balances, and urinary hydroxyproline excretion rates in four healthy men adapted to a low Ca diet providing only 4.0 +/- 0.2 SD mmoles Ca/day to those in four healthy men eating a comparable diet (4.2 +/- 0.9 mmoles Ca/day) during the chronic oral administration of calcitriol, 0.75 micrograms every 6 hr. Serum 1,25-(OH)2-D levels averaged 94 +/- 16 pM during the control studies and 209 +/- 35 pM during calcitriol administration. Net intestinal Ca absorption averaged 0.5 +/- 0.3 mmoles/day during control and 1.8 +/- 0.5 mmoles/day during calcitriol (P less than 0.005), but urinary Ca excretion averaged 8.7 +/- 2.0 mmoles/day during calcitriol as compared to 2.9 +/- 1.4 mmoles/day during control (P less than 0.005). Thus, mean Ca balance, which averaged -2.4 +/- 1.2 mmoles/day during control, was more negative during calcitriol at -6.3 +/- 2.4 mmoles/day (P less than 0.05). Average daily PO4 balances averaged +7.7 +/- 1.5 mmoles/day during control but only tended to be negative during calcitriol at -1.1 +/- 5.4 mmoles/day, (NS). Urinary hydroxyproline excretion averaged 0.26 +/- 0.03 mmoles/day during control and 0.49 +/- 0.06 during calcitriol (P less than 0.001). We conclude that elevated serum 1,25-(OH)2-D concentrations in healthy men eating low Ca diets stimulate bone resorption.

Bone Resorption↗