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G A Howard

Publications and source records attributed to G A Howard.

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

Development and reversibility of aluminum-induced bone lesion in the rat.

We studied the effect of aluminum injections on bones of rats after intervals of 3, 6, and 9 weeks. To study reversibility, we allowed one group to recover for 3 weeks. Both weanling and adult rats were examined to determine the influence of age. The calcium, phosphate, creatinine, and parathyroid hormone levels were similar in aluminum-treated rats and controls. Aluminum could be seen by histochemical stain after 6 weeks, but at that time the bone was otherwise normal. By 9 weeks the bone formation (as measured by tetracycline labeling) in aluminum-treated rats was severely decreased on trabecular and endosteal surfaces. The periosteal surfaces showed normal formation. After 3 weeks of recovery, the bone formation rate in the young aluminum-treated rats was similar to that in the controls, although the serum and bone aluminum values had not significantly decreased. A higher percentage of aluminum was seen in the cement lines. In the adult rats, the bones had more stainable aluminum, and increased osteoid was noted along trabecular and periosteal surfaces. The doses of aluminum used in these rats greatly exceeded those that cause toxicity in humans; thus these findings may not directly apply to clinical practice. We conclude that aluminum administration can lead to decreased rates of bone formation in the rat, despite normal calcium level and renal function, and without decreased parathyroid hormone levels. The peritoneal route of administration could also have contributed to bone lesions by causing peritonitis, malabsorption, or both. Adult rats showed signs of early osteomalacia.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗
PubMed1983

Calvarial cells synthesize 1 alpha,25-dihydroxyvitamin D3 from 25-hydroxyvitamin D3.

A metabolite of vitamin D has been isolated in pure form from incubation of 25-hydroxyvitamin D3 with embryonic chick calvarial cells that had been grown on Cytodex 1 microcarrier beads. The isolation involved dichloromethane extraction of the cells and incubation medium, followed by Sephadex LH-20 column chromatography and high-performance liquid chromatography of the extract. The metabolite was identified as 1 alpha,25-dihydroxyvitamin D3 by means of ultraviolet absorption spectroscopy, mass spectrometry, and sensitivity to oxidation by periodate. This metabolite was not produced by cell-free medium or by cells from embryonic chick liver, skin, or heart. In conclusion, (1) kidney cells are not unique in having 25-hydroxyvitamin D3:1 alpha-hydroxylase activity as previously believed and (2) vitamin D target tissues such as the skeleton may play a direct role in mediating the metabolism of 25-hydroxyvitamin D3 to 1 alpha,25-dihydroxyvitamin D3, a vitamin D metabolite active at those sites.

Animals↗
PubMed1983

Cells isolated from embryonic intestine synthesize 1,25-dihydroxyvitamin-D3 and 24,25-dihydroxyvitamin-D3 in culture.

Cells isolated from embryonic chick intestine in culture convert 25-hydroxyvitamin-D3 to a number of more polar metabolites. Two of these metabolites have been identified with chemical and chromatographic methods as 24,25-dihydroxyvitamin-D3 (24,25(OH)2D3) and 1,25-dihydroxyvitamin-D3 (1,25(OH)2D3). The enzymes conform to substrate saturation kinetics. The apparent Km for substrate for the synthesis of 1,25(OH)2D3 and 24,25(OH)2D3 is 70 nM and 29 nM, respectively.

24,25-Dihydroxyvitamin D 3↗
PubMed1982

Extracts of bone contain a potent regulator of bone formation.

We prepared aqueous extracts of whole femorae and tibiae of embryonic chicks. An amount of extract containing 25 microgram of protein resulted in a 500% increase in DNA synthesis in calvarial cell culture, and significant effects were detected with 5 microgram (55%). The time course for stimulation of DNA synthesis showed a peak occurring 16-20 h after addition of the extract. This matrix factor is nondialyzable, and fractionation on a column of Sephadex G-100 indicated a molecular weight of 60-80,000. At the maximum dose used, [3H]proline incorporation into total protein of calvarial cells was increased by 55%, and thus far, all fractions active in promoting DNA synthesis have been found to increase collagen synthesis in cultured chick tibiae. These data are consistent with an effect on osteoblasts as well as bone precursor cells. Extracts prepared from tibiae of 2-day-old chicks, from which the marrow had been removed, also stimulated DNA synthesis (280% increase), thus ruling out the possibility that the factor is a relatively nonspecific nitrogen from the hematopoietic cell line. We conclude that bone matrix contains a substance which could regulate bone formation in vitro by control of mitosis in osteogenic precursors and/or stimulation of osteoblast activity.

Animals↗
PubMed1982

An improved technique for rapid autoradiography of cells and tissue sections.

We have demonstrated a method for autoradiography of cell and tissue preparations which is both rapid and safe. The method utilizes only the primary scintillator, PPO, placed under the final emulsion to facilitate activation of the silver grains in the emulsion. Exposure of the autoradiographs is complete under the conditions described within 4 h at ambient temperature. The method is sensitive to exposure time and to the concentration of added radioisotope. The exclusion of volatile, toxic chemicals from the preparations allows the experiments to be performed without any health hazard to the investigator.

Animals↗
PubMed1981

Human bone cells in culture metabolize 25-hydroxyvitamin D3 to 1,25-dihydroxyvitamin D3 and 24,25-dihydroxyvitamin D3.

Bone cells respond to 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) for mineral mobilization and contain receptors for 1,25(OH)2D3. We report here the expression of 25-hydroxyvitamin D3 (25 (OH)D3) metabolizing enzymes in primary cultures of human bone cells, as well as n a human osteosarcoma cell line. Human bone cells were obtained by enzyme digestion of the extracellular matrix of bone from iliac crest biopsies from 3 male patients without primary bone disease. These cells were plated (5 X 10(4)/min) in medium with 10% fetal calf serum and proliferated to confluence in 10-14 days. At confluence, the medium was replaced with serum-free medium. The cells were preincubated in this serum-free medium for 24 h prior to incubating them 2-4 h with [3H]25(OH)D3 (10-20 nM). The vitamin D metabolites synthesized during this incubation were extracted from the medium and cells with dichloromethane, then separated by chromatography on Sephadex LH-20, followed by high performance liquid chromatography. The cells synthesized 1,25(OH)2D3 and 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) with the specific activities of the 1- and 24-hydroxylases similar in magnitude to those in kidney cells in vitro. The enzymes could be regulated by external perturbations, in that the activity of the 1-hydroxylase was inhibited by preincubation of the cells for 8 h with 1,25(OH)2D3 (10 nM), whereas the 24-hydroxylase was enhanced. Incubation of the cells in a low calcium medium (0.6 mM) depressed the 24-hydroxylase activity. We conclude: 1) normal human bone cells can produce 1,25(OH)2D3 and 24,25(OH)2D3 in vitro in amounts similar to kidney cells, suggesting a physiological significance and 2) this synthesis could account for the increase in osteoclast number in anephric patients with renal osteodystrophy.

24,25-Dihydroxyvitamin D 3↗
PubMed1981

Parathyroid hormone stimulates bone formation and resorption in organ culture: evidence for a coupling mechanism.

We have developed an in vitro system, using embryonic chicken tibiae grown in a serum-free medium, which exhibits simultaneous bone formation and resorption. Tibiae from 8-day embryos increased in mean (+/- SD) length (4.0 +/- 0.4 to 11.0 +/- 0.3 mm) and dry weight (0.30 +/- 0.04 to 0.84 +/- 0.04 mg) during 12 days in vitro. There was increased incorporation of [3H]proline into hydroxyproline (120 +/- 20 to 340 +/- 20 cpm/mg of bone per 24 hr) as a measure of collagen synthesis, as well as a 62 +/- 5% increase in total calcium and 45Ca taken up as an indication of active mineralization. A physiologic concentration (1 pM) of parathyroid hormone was found to stimulate bone resorption over control levels in this system. Parathyroid hormone stimulated the release of [3H]hydroxyproline from the bone shafts but not from the cartilage ends, indicating the specificity of the response. With 1 pM parathyroid hormone we observed an acute inhibition of bone formation, followed (after 12-16 hr) by a chronic stimulation of bone formation during the 12-day incubation. Both mineral uptake and matrix formation were enhanced at approximately the same rate during the 12-day incubation. The chronic enhancement of formation required parathyroid hormone only for the initial 8-10 hr of incubation. These results could be explained by the production or release of a factor from bone to stimulate formation in response to the acute increase in resorption--a "coupling factor." Indeed, dialyzed culture medium conditioned by actively resorbing bones stimulated bone formation over controls when added to organ cultures at a 1:20 dilution. The factor is larger than 12,000 daltons as determined by dialysis. The factor is specific for the bone shaft and did not affect the cartilage ends.

Animals↗
PubMed1980

In vitro synthesis of 1 alpha,25-dihydroxycholecalciferol and 24,25-dihydroxycholecalciferol by isolated calvarial cells.

The question of whether the skeleton metabolizes 25-hydroxycholecalciferol [25(OH)D3] to more-polar products was studied. Calvarial cells were dispersed from 16-day old chicken embryos by using collagenase and then grown in culture in serum-free medium. Confluent cell cultures were incubated with 7 nM 25(OH)[3H]D3 for 2 hr, and the vitamin D metabolites were then extracted. At least four polar metabolites were produced. Based on separation by Sephadex LH-20 chromatography followed by high-pressure liquid chromatography, two of these metabolites were identified as 1,25-dihydroxycholecalciferol [1,25(OH)2D3] and 24,25-dihydroxycholecalciferol [24,25(OH)2D3]. These metabolites were also produced by cultured kidney cells but not by liver, heart muscle, or skin cells isolated from the same embryos. The specific activities of the calvarial 1- and 24-hydroxylases were similar in magnitude to those in isolated kidney cells. The specific activity of the calvarial 25(OH)D3:1-hydroxylase was inhibited by an 8-hr preincubation with 1,25(OH)2D3, whereas the 24-hydroxylase was enhanced. It is concluded that (i) vitamin D metabolism by isolated cells is organ-specific, (ii) calvarial cells produce active metabolites of vitamin D in significant amounts, (iii) vitamin D metabolism by calvarial cells is regulated by 1,25(OH)2D3, and (iv) locally produced, active metabolites could act locally, thereby adding a new dimension to the regulation of mineral metabolism by vitamin D metabolites.

25-Hydroxyvitamin D3 1-alpha-Hydroxylase↗
PubMed1980

In vitro metabolism of 25-hydroxyvitamin D3 by isolated rat kidney cells.

Cells were dispersed from rat kidney after enzymatic digestion of the extracellular matrix. When the cells were suspended in a serum-free medium and incubated with (3)H-labeled 25-hydroxyvitamin D(3) (25-OH-D(3)) several polar metabolites, including 1,25-(OH)(2)[(3)H]D(3) and 24,25-(OH)(2)[(3)H]D(3) were produced. The specific activities of the 25-OH-D(3):1- and 24-hydroxylases in isolated rat kidney cells were 10-100 times greater than in avian kidney homogenates. The rates of production of 1,25-(OH)(2)D(3) and 24,25-(OH)(2)D(3) were linear over a wide range in cell densities (0.65-5.0 x 10(6) cells per ml) and substrate concentrations (3.5-70 nM). The rate of production of 24,25-(OH)(2)[(3)H]D(3) from 25-OH-[(3)H]D(3) by cells isolated from rats fed control diet was linear with time for up to 30 min, while the synthesis of 1,25-(OH)(2)[(3)H]D(3) was linear for over 90 min. The specific activity of the 25-OH-D(3):1-hydroxylase was increased in kidney cells from vitamin D-deficient rats (11.5 fmol/min per 10(6) cells) as well as calcium-deficient rats (8.1 fmol/min per 10(6) cells) when compared to cells from rats fed the control diet (2.0 fmol/min per 10(6) cells). Also, the specific activity of the 25-OH-D(3):24-hydroxylase was reduced in cells from the vitamin D-deficient rats (<0.2 fmol/min per 10(6) cells) and calcium-deficient rats (5.1 fmol/min per 10(6) cells) compared to the controls (15.2 fmol/min per 10(6) cells). On the basis of these results, as well as previous in vivo studies, we conclude that the metabolism of 25-OH-D(3) by freshly isolated rat kidney cells reflects the in vivo activities of the renal vitamin D-metabolizing enzymes and may prove useful as an assay.

25-Hydroxyvitamin D3 1-alpha-Hydroxylase↗
PubMed1979

Serum-free culture of Japanese quail kidney cells. Regulation of vitamin D metabolism.

Cells obtained from male quail kidneys by digestion with collagenase and hyaluronidase were plated and maintained in a chemically defined, serum-free medium. Culture dishes (35 mm) were inoculated with 1.5 . 10(6) cells which became confluent in 5 days. The cells maintained an epithelial-like morphology over the entire culture period. During a 2 h incubation the cells metabolized 25--30% of the 10 nM 25-hydroxyvitamin D-3 (25-OH-D-3) provided. Seven metabolites were chromatographically separated on Sephadex LH-20. Three have been identified as 1 alpha, 25-dihydroxyvitamin D-3 (1,25(OH)2D-3), 24,25-dihydroxyvitamin D-3 (24,25(OH)2D-3) and 1 alpha, 24,25-trihhydroxyvitamin D-3 (1,24,25(OH)3D-3). The activities of the 25-OH-D-3:1 alpha- and 24-hydroxylases increased eight times faster than the cell number in 5 days. Preincubation of the cells with 10 nM 25-OH-D-3 or 1,25(OH)2D-3 decreased 1,25(OH)2D-3 synthesis, and increased both 24,25(OH)2D-3 and metabolite IV synthesis. The decrease in 25-OH-D-3:1 alpha-hydroxylase activity required a 2 h preincubation with 25-OH-D-3, while stimulation of 25-OH-D-3:24-hydroxylase activity and metabolite IV production required a 6 h preincubation. Incubations of cells for 1 h with parathyroid hormone resulted in a 30-fold increase in cyclic AMP in the medium. A 6 h preincubation with parathyroid hormone decreased 24,25(OH)2D-3) synthesis 50% relative to control cells. These results demonstrate the amenability of this system for studying the regulation of 25-OH-D-3 metabolism, as well as its use for other in vitro studies on renal cell function in a chemically defined culture system.

Animals↗
PubMed1979

Bone mineralization and metabolism of 3H-25-hydroxyvitamin D3 in thyroparathyroidectomized rats treated with parathyroid extract.

Thyroparathyroidectomized rats fed a diet containing 1.2% calcium, 0.55% phosphorus maintain normal serum levels of these ions. Treatment of such rats with parathyroid extract (PTE; 20 U/100 g twice daily; 10 days) has no statistically significant effect on rates of bone formation, matrix apposition, or osteoid maturation. Significant decreases in osteoid width and mineralizing front width, as well as a 60% increase in the rate of initial mineralization were observed in the PTE-treated group. Conversion of 3H-25-hydroxyvitamin D3 to 3H-1,25-dihydroxyvitamin D3 was 4-fold higher in the PTE-treated group than in the untreated animals. Increased formation of 1,25-dihydroxyvitamin D3 in response to treatment with PTE may play a major role in correcting the mineralizing defect resulting from thyroparathyroidectomy.

Animals↗
PubMed1978

Specific binding of estrogen to nuclei from Japanese quail kidney cells.

The binding properties of estrogen to Japanese quail kidney cell nuclei were evaluated by 3H-17 beta-estradiol exchange. Kidney cell nuclei from estrogenized male, as well as laying female quail, had a single class of saturable (0.1-0.2 pmol/mg DNA), high affinity (Kd = 1-2 x 10-10M) nuclear binding sites for 17 beta-estradiol. High affinity estrogen binding sites were not found in kidney cell nuclei from untreated males. Various non-radioactive estrogens (17 beta-estradiol, estrone, estriol and diethylstilbestrol) competed effectively with 3H-17 beta-estradiol for the high affinity binding sites, whereas nonestrogens (testosterone and progesterone) did not complete. It is concluded that the 3H-estradiol binding characteristics of kidney cell nuclei provides strong evidence for a nuclear estrogen receptor in the avian kidney.

Animals↗