Five nanometer size highly positive silver nanoparticles are bactericidal targeting cell wall and adherent fimbriae expression.

To tackle growing antibiotic resistance (AR) and hospital-acquired infections (HAIs), novel antimicrobials are warranted that are effective against HAIs and safer for human use. We hypothesize that small 5 nm size positively charged nanoparticles could specifically target bacterial cell wall and adherent fimbriae expression, serving as the next generation antibacterial agent. Herein we show highly positively charged, 5 nm amino-functionalized silver nanoparticles (NH2-AgNPs) were bactericidal; highly negatively charged, 45 nm citrate-functionalized AgNPs (Citrate-AgNPs) were nontoxic; and Ag+ ions were bacteriostatic forming honeycomb-like potentially resistant phenotype, at 10 µg Ag/mL in E. coli. Further, adherent fimbriae were expressed with Citrate-AgNPs (0.5-10 µg/mL), whereas NH2-AgNPs (0.5-10 µg/mL) or Ag+ ions (only at 10 µg/mL) inhibited fimbriae expression. Our results also showed no lipid peroxidation in human lung epithelial and dermal fibroblast cells upon NH2-AgNPs treatments, suggesting NH2-AgNPs as a biocompatible antibacterial candidate. Potent bactericidal effects demonstrated by biocompatible NH2-AgNPs and the lack of toxicity of Citrate-AgNPs lend credence to the hypothesis that small size, positively charged AgNPs may serve as a next-generation antibacterial agent, potentially addressing the rising HAIs and patient health and safety.

Functional modulation of the ATP-sensitive potassium channel by extracellular signal-regulated kinase-mediated phosphorylation.

ATP-sensitive potassium (K(ATP)) channels play an important role in controlling insulin secretion and vascular tone as well as protecting neurons under metabolic stress. We have previously demonstrated that stimulation of the K(ATP) channel by nitric oxide (NO) requires activation of Ras- and extracellular signal-regulated kinase (ERK) of the mitogen-activated protein kinase (MAPK) family. However, the mechanistic link between ERK and the K(atp) channel remained unknown. To investigate how ERK modulates the function of K(ATP) channels, we performed single-channel recordings in combination with site-directed mutagenesis. The Kir6.2/SUR1 channel, a neuronal K(ATP) channel isoform, was expressed in human embryonic kidney (HEK) 293 cells by transient transfection. Direct application of the activated ERK2 to the cytoplasmic surface of excised, inside-out patches markedly enhanced the single-channel activity of Kir6.2/SUR1 channels. The normalized open probability (NPo) and opening frequency were significantly increased, whereas the mean closed duration was reduced. The single-channel conductance level was not affected. The ERK2-induced stimulation of Kir6.2/SUR1 channels was prevented by heat-inactivation of the enzyme. Furthermore, alanine substitutions of T341 and S385 to disrupt the potential ERK phosphorylation sites present in the Kir6.2 subunit significantly abrogated the stimulatory effects of ERK2, while aspartate substitutions of T341 and S385 to mimic the (negative) charge effect of phosphorylation rendered a small yet significant reduction in the ATP sensitivity of the channel. Taken together, here we report for the first time that ERK2/MAPK activates neuronal-type K(ATP) channels, and this stimulation requires ERK phosphorylation of the Kir6.2 subunit at T341 and S385 residues. The ERK2-induced K(ATP) channel stimulation can be accounted for by changes in channel gating that destabilize the closed states and by reduction in the ATP sensitivity. As Kir6.2 is the pore-forming subunit of K(ATP) channels, ERK2-mediated phosphorylation may represent a common mechanism for K(ATP) channel regulation in different tissues.

1,25(OH)(2)D(3) blocks TNF-induced monocytic tissue factor expression by inhibition of transcription factors AP-1 and NF-kappaB.

An essential coagulation factor, tissue factor (TF), is rapidly expressed by human monocytes when exposed to a variety of agonists, such as lipopolysaccharide or tumor necrosis factor (TNF). We previously found that 1alpha,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) and its potent synthetic analogs downregulate TF and upregulate thrombomodulin expression on monocytic cells, counteracting the effects of TNF at the level of transcription. The human TF gene has characteristic binding sequences for activator protein-1 (AP-1) (c-Jun/c-Fos), nuclear factor-kappaB (NF-kappaB), Sp-1, and early growth response factor-1 (Egr-1). In this study, we investigated the regulatory mechanisms by which 1,25(OH)(2)D(3) inhibits TNF-induced TF expression in human monocytic cells. 1,25(OH)(2)D(3) reduced basal and TNF-induced TF activities. Gel-shift assay and luciferase assay with the respective reporter vectors showed that 1,25(OH)(2)D(3) reduced basal and TNF-induced activities of the nuclear proteins AP-1 and NF-kappaB, but not Egr-1. 1,25(OH)(2)D(3) inhibited TNF-induced phosphorylation of c-Jun without affecting phosphorylation of the other pathways. On the other hand, 1,25(OH)(2)D(3) directly inhibited nuclear binding and activities of NF-kappaB in the nucleus without affecting phosphorylation of the NF-kappaB activation pathway. These results indicate that 1,25(OH)(2)D(3) suppresses basal and TNF-induced TF expression in monocytic cells by inhibition of AP-1 and NF-kappaB activation pathways, but not of Egr-1. Our results may help to elucidate the regulatory mechanisms of 1,25(OH)(2)D(3) in TF induction, and may have physiological significance in the clinical challenge to use potential 1,25(OH)(2)D(3) analogs in antithrombotic therapy as well as immunomodulation and antineoplastic therapy of leukemia.

Calpain-mediated AQP2 proteolysis in inner medullary collecting duct.

Vitamin D-elicited hypercalcemia/hypercalciuria is associated with polyuria in humans and in animal models. In rats, dihydrotachysterol (DHT) induces AQP2 water channel downregulation despite unaltered AQP2 mRNA expression and thus we investigated the mechanism of AQP2 degradation. Incubation of AQP2-containing inner medullary collecting duct (IMCD) endosomes with Ca(2+) or calpain elicited AQP2 proteolysis, an effect abolished by leupeptin. This endogenous, Ca(2+)-sensitive protease activity exhibited a different proteolytic digest pattern from trypsin, which also degraded AQP2 in vitro. IMCDs contain abundant micro-calpain protein and functional calpain proteolytic activity as demonstrated by immunohistochemistry, immunoblotting, and gel zymography. Furthermore, by small particle flow cytometry we demonstrated that micro-calpain colocalizes with apical IMCD endosomes. DHT does not appear to elicit general proteolysis, however, in addition to AQP2 degradation, DHT treatment also diminished micro-calpain and calpastatin expression although whether these changes contributed to the AQP2 instability remains unclear. Together, these data show for the first time that AQP2 is a substrate for calpain-mediated proteolysis and that furthermore, micro-calpain, like AQP2, is both highly expressed in renal inner medulla and localized to apical IMCD endosomes.

Potent suppression of the parathyroid glands by hydroxylated metabolites of dihydrotachysterol(2).

BACKGROUND: Dihydrotachysterol(2), a licensed pharmaceutical, is hydroxylated to 25-hydroxydihydrotachysterol(2) (25(OH)DHT(2)) and 1 alpha,25-dihydroxydihydrotachysterol(2) (1 alpha,25(OH)(2)DHT(2)) in man. We have compared the biological activity of these metabolites with calcitriol and the 'non-calcaemic' analogue, 22-oxacalcitriol (OCT) in bovine parathyroid cell cultures and in rats. METHODS: The effect of each sterol on parathyroid hormone (PTH) secreted by primary bovine parathyroid cells was measured. High-performance liquid chromotography and gas chromotography-mass spectrometry were used to investigate in vitro 25(OH)DHT(2) metabolism. Rats were given a single intraperitoneal injection or five daily injections of each sterol, and changes in ionized calcium and PTH were measured. RESULTS: In vitro, all sterols suppressed PTH significantly. Calcitriol and OCT were of similar potency, but 1 alpha, 25(OH)(2)DHT(2) and 25(OH)DHT(2) required higher concentrations to suppress PTH equally. We were unable to detect metabolism of 25(OH)DHT(2) to 1 alpha,25(OH)(2)DHT(2) in vitro. In rats, a single dose of 0.5 microg/rat of calcitriol increased ionized calcium at 30 and 40 h (statistically significant at 48 h). 50 microg of OCT and 1 alpha,25(OH)(2)DHT(2) did not cause significant hypercalcaemia at 48 h, although 1 alpha,25(OH)(2)DHT(2) caused hypercalcaemia at 30 h. In contrast, 50 microg of 25(OH)DHT(2) caused hypercalcaemia at 48 h but not at 30 h. Five daily doses of 0.001 microg/rat of calcitriol caused a significant rise in calcium and a 50% fall in PTH. OCT and 1 alpha,25(OH)(2)DHT(2) at 0.025 and 0.5 microg/rat respectively caused similar suppression of PTH but without hypercalcaemia. CONCLUSION: 1 alpha,25(OH)(2)DHT(2) and 25(OH)DHT(2) are potent suppressors of PTH in vitro and in vivo. 25(OH)DHT(2) may be active by virtue of its pseudo-1 alpha-hydroxyl group. Hypercalcaemia caused by a single dose of 1 alpha,25(OH)(2)DHT(2) appeared to be more transient than calcitriol. Five daily doses of 1 alpha, 25(OH)(2)DHT(2) and OCT could achieve 50% suppression of PTH without significant increments in ionized calcium. In contrast, suppression of PTH by calcitriol was associated with significant increments in ionized calcium. These data suggest that like OCT, 1 alpha, 25(OH)(2)DHT(2) can dissociate calcaemic actions from parathyroid-suppressing actions in a manner that may be therapeutically useful.

Effects of new analogues of vitamin D on bone cells: implications for treatment of uremic bone disease.

BACKGROUND: The use of calcitriol in the treatment of uremic hyperparathyroidism and renal osteodystrophy is limited in many patients by hypercalcemic side-effects. New less calcemic analogues of calcitriol are being developed, and some are under clinical evaluation. To investigate whether these compounds possess important differences in their action on bone cells, we have studied their effects [with and without parathyroid hormone (PTH)] on the release and synthesis of the resorptive osteotropic cytokine, interleukin-6 (IL-6). METHODS: MG 63 and SaOS-2 human osteoblastic cell lines were cultured for 6 or 24 hours in media containing calcitriol, the sterols of interest, or 1-34 synthetic PTH. IL-6 release was assayed by commercially available enzyme-linked immunosorbent assay. IL-6 mRNA levels were assessed by reverse transcriptase-polymerase chain reaction. RESULTS: We found that calcitriol and paricalcitol behaved in a similar fashion, resulting in increased IL-6 release only at higher concentrations (10(-7) to 10(-9) M). In contrast, 22-oxacalcitriol and 1,25-dihydroxydihydrotachysterol2 stimulated release to a similar extent but at concentrations three to four orders of magnitude lower (10(-11) to 10(-13) M), despite being less potent as suppressers of parathyroid function than calcitriol. Studies of IL-6 mRNA showed a similar pattern of concentration and cell line-dependent transcription. CONCLUSIONS: Compounds stimulating IL-6 release at concentrations achievable during the treatment of uremic hyperparathyroidism might favor continuing linked bone formation and resorption and thereby avoid adynamic bone disease while still allowing profound suppression of PTH.

Hypercalcemia stimulates expression of intrarenal phospholipase A2 and prostaglandin H synthase-2 in rats. Role of angiotensin II AT1 receptors.

In chronic hypercalcemia, inhibition of thick ascending limb sodium chloride reabsorption is mediated by elevated intrarenal PGE2. The mechanisms and source of elevated PGE2 in hypercalcemia are not known. We determined the effect of hypercalcemia on intrarenal expression of cytosolic phospholipase A2 (cPLA2), prostaglandin H synthase-1 (PGHS-1), and prostaglandin H synthase-2 (PGHS-2), enzymes important in prostaglandin production. In rats fed dihydrotachysterol to induce hypercalcemia, Western blot analysis revealed significant upregulation of both cPLA2 and PGHS-2 in the kidney cortex and the inner and outer medulla. Immunofluorescence localized intrarenal cPLA2 and PGHS-2 to interstitial cells of the inner and outer medulla, and to macula densa and cortical thick ascending limbs in both control and hypercalcemic rats. Hypercalcemia had no effect on intrarenal expression of PGHS-1. To determine if AT1 angiotensin II receptor activation was involved in the stimulation of cPLA2 and PGHS-2 in hypercalcemia, we treated rats with the AT1 receptor antagonist, losartan. Losartan abolished the polydipsia associated with hypercalcemia, prevented the increase in cPLA2 protein in all regions of the kidney, and diminished PGHS-2 expression in the inner medulla. In addition, losartan completely prevented the increase in urinary PGE2 excretion in hypercalcemic rats. Intrarenal levels of angiotensin II were unchanged in hypercalcemia. These data indicate that hypercalcemia stimulates intrarenal cPLA2 and PGHS-2 protein expression. Our results further support a role for angiotensin II, acting on AT1 receptors, in mediating this stimulation.

Calcification of polyurethanes implanted subdermally in rats is enhanced by calciphylaxis.

Calcification complicates the use of the polymer polyurethane in cardiovascular implants. To date only costly experimental circulatory animal models have been useful for investigating this disease process. In this paper we report that polyurethane calcification in rat subdermal implants is enhanced by overdosing with a vitamin-D analog. The calcification-prone state, known as calciphylaxis, was induced in 4-week old rats by oral administration of a vitamin-D analog, dihydrotachysterol. We studied two commercially available polyurethanes (Biomer and Mitrathane) and two proprietary polyurethanes (PEU-2000 and PEU-100). PEU-100 is unique because it is derivatized with ethanehydroxy-bisphosphonate (EHBP) for calcification resistance. Polyurethane calcium and phosphate levels and morphological changes due to calciphylaxis were compared with those of control rat subdermal explants in 60-day studies. Increased polyurethane mineralization was observed due to calciphylaxis with 60-day rat subdermal explants of Biomer, Mitrathane, and PEU-2000 (calcium levels, respectively, 4.13 +/- 0.56, 18.61 +/- 2.73, and 3.37 +/- 0.22 microgram/mg, mean +/- standard error) as compared to control explants (calcium levels, respectively, 1.22 +/- 0.1, 12.57 +/- 0.86, and 0.20 +/- 0.86 microgram/mg). The study also demonstrated that with 60-day implants calciphylaxis had no side effects on somatic growth and serum calcium levels. Explant surface morphology of these polyurethane explants examined by scanning electron microscopy, back scattering electron imaging coupled with energy dispersive X-ray spectroscopy, and light microscopy demonstrated the presence of predominantly surface-oriented calcification. PEU-100, derivatized with 100 n.moles/ mg of EHBP, resisted calcification with explant calcium levels 0.51 +/- 0.01 (calciphylaxis) and 0.38 +/- 0.01 (control) microgram/mg. It is concluded that calciphylaxis enhances superficial polyurethane calcification in rat subdermal implants and that an EHBP-modified polyurethane resists calcification despite calciphylaxis. Rat subdermal implants using calciphylaxis may be generally useful for evaluating the calcification potential of various biomedical polymers.

In vivo dihydrotachysterol2 metabolism in normal man: 1 alpha- and 1 beta-hydroxylation of 25-hydroxydihydrotachysterol2 and effects on plasma parathyroid hormone and 1 alpha,25-dihydroxyvitamin D3 concentrations.

It has recently been shown that in the rat, dihydrotachysterol (DHT) is extensively metabolized in the side-chain in vivo along pathways similar to those of vitamin D. In addition 25-hydroxy-DHT2 [25OHDHT2] is hydroxylated at C1, producing both 1 alpha- and 1 beta- hydroxy compounds. An in vivo study in 1988 demonstrated that in normal adult subjects receiving oral DHT2, plasma 1 alpha,25-dihydroxyvitamin D [1,25-(OH)2D] concentrations fell, but with unchanged plasma PTH levels. Down-regulation of 1,25-(OH)2D3 production by 25-(OH)DHT2 or some other unknown metabolite was also suggested as an explanation for these observations. To investigate whether either of the newly characterized 1 alpha,25- or 1 beta,25-(OH)2DHT2 was formed in vivo in normal man, DHT2 (approximately 1 mg/day, orally) was administered to healthy volunteers (three males and one female). Plasma was analyzed by high performance liquid chromatography and gas chromatography-mass spectrometry, demonstrating the formation of both 1 alpha,25- and 1 beta,25-(OH)2DHT2 in vivo in normal human subjects. Plasma levels of 1,25-(OH)2D3, PTH, ionized and total calcium, inorganic phosphate, and alkaline phosphatase were monitored. The plasma concentrations of DHT2, 25OHDHT2, and 1 alpha,25- and 1 beta,25-(OH)2DHT2 were measured by gas chromatography-mass spectrometry. In all volunteers, plasma ionized calcium increased slightly during DHT2 administration; 1,25-(OH)2D3 and PTH concentrations fell. Plasma levels of DHT2 and its metabolites rose over the same period. The average fall in the level of plasma 1,25-(OH)2D (60-70 pmol/L) was mirrored by a rise in the concentration of 1 alpha,25-(OH)2DHT2 (550 pmol/L). This ratio is appropriate, because it has previously been shown that in a reconstituted COS cell, 1 alpha,25-(OH)2DHT3 has roughly one tenth the potency of 1,25-(OH)2D3. At maximum concentration, the ratios of DHT2/25OHDHT2/1 beta,25-(OH)2DHT2/1 alpha,25-(OH)2DHT2 were approximately 10:1:2:0.1. The concentration of 1 beta,25-(OH)2DHT2 was greater than that of 25OHDHT2, and the ratio of 1 alpha,25- to 1 beta,25-(OH)2DHT2 (1:20) was substantially lower than that in rat plasma (3:10). The data presented here suggest that the active DHT2 metabolite in man is 1 alpha,25-(OH)2DHT2 and that the fall in plasma 1,25-(OH)2D seen during DHT therapy may be partly the result of suppressed PTH secretion.

A prospective, double-blind study of growth failure in children with chronic renal insufficiency and the effectiveness of treatment with calcitriol versus dihydrotachysterol. The Growth Failure in Children with Renal Diseases Investigators.

Because controlled trials in adults have shown accelerated deterioration of renal function in a small number of patients receiving calcitriol for renal osteodystrophy, we initiated a prospective, randomized, double-blind study of the use of calcitriol versus dihydrotachysterol in children with chronic renal insufficiency. We studied children aged 1 1/2 through 10 years, with a calculated glomerular filtration rate between 20 and 75 ml/min per 1.73 m2, and with elevated serum parathyroid hormone concentrations. Ninety-four patients completed a mean of 8.0 months of control observations and were randomly assigned to a treatment period; 82 completed the treatment period of at least 6 months while receiving a calcitriol dosage (mean +/- SD) of 17.1 +/- 5.9 ng/kg per day or a dihydrotachysterol dosage of 13.8 +/- 3.3 micrograms/kg per day. With treatment the height z scores for both calcitriol- and dihydrotachysterol-treated groups showed no differences between the two groups. In relation to cumulative dose, there was a significant decrease in glomerular filtration rate for both calcitriol and dihydrotachysterol; for calcitriol the rate of decline was significantly steeper (p = 0.0026). The treatment groups did not differ significantly with respect to the incidence of hypercalcemia (serum calcium concentration > 2.7 mmol/L (> 11 mg/dl)). We conclude that careful follow-up of renal function is mandatory during the use of either calcitriol or dihydrotachysterol because both agents were associated with significant declines in renal function. There was no significant difference between calcitriol and dihydrotachysterol in promoting linear growth or causing hypercalcemia in children with chronic renal insufficiency. Dihydrotachysterol, the less costly agent, can be used with equal efficacy.

[Plasma renin activity in tachistin-stimulated hypercalcemia and under the effect of chlorazine]. / Plazmena reninova aktivnost pri stimulirana chrez takhistin khiperkaltsiemiia i pri prilozhenie na khlorazin.

The aim of the present study was to elucidate the correlation between the Renin secretion and increased Plasma Calcium concentration and the role of Calmodulin in this process. Plasma Renin activity was determined radioimmunologically in 31 white rats, that were grouped as follows: group I - 7 controls loaded for 6 days perorally with 0.5 ml/200 g b.w. glycerin and injected i.m. for 6 days with 0.1 ml/200 g b.w. with 0.9% NaCl; group II - 8 rats, treated for 6 days with Tachistin 0.0025 mg/200 g b.w., dissolved in glycerin 0.5 ml/200 g b.w.; group III - 5 rats, treated with Tachistin 0.005 mg/200 g b.w. in the same manner; group IV - 5 rats injected i.m. with Chlorazin 0.5 mg/200 g b.w. for 6 days; group V - 6 rats, loaded with double dose Tachistin and with Chlorazin 0.5 mg/200 g b.w. for 6 days. Blood samples were taken intracardially on the seventh day from the beginning of the experiment and were analyzed with kits of Sorin-Biomedica-Italy. Our results suggest that the hypercalcemia induced by Tachistin caused a dose-dependent increase of PRA and Ca-Calmodulin complex is the dominant second messenger of Renin secretion.

Endogenous prostaglandin E2 mediates inhibition of rat thick ascending limb Cl reabsorption in chronic hypercalcemia.

The hypothesis that endogenous PGE2 mediates defective thick ascending limb (TAL) Cl reabsorption (percent delivered load: FRCl%) in rats with vitamin D-induced chronic hypercalcemia (HC) was tested by measuring FRCl% in loop segments microperfused in vivo in HC and control rats treated acutely with indomethacin (Indo) or its vehicle, and obtaining the corresponding outer medullary [PGE2]. Microperfusion conditions were developed in which FRCl% was exclusively furosemide sensitive. To determine the cellular mechanism, tubules were perfused acutely with forskolin (FSK), cAMP, or the protein kinase C inhibitor staurosporine (SSP). Outer medullary [PGE2] in HC rats was 9 to 10 times greater than control and could be normalized by Indo. FRCl% was 20% lower in HC rats infused with vehicle, and Indo, FSK, and cAMP returned FRCl% to normal despite sustained HC. Indo or FSK had no effect on FRCl% in control rats and Indo did not prevent inhibition of FRCl% by luminal PGE2 (1 microM). Luminal SSP (10(-7), 10(-8) M) in HC did not return FRCl% to control values. We conclude that impaired TAL FRCl% in HC occurs at a pre-cAMP site and is due to endogenous PGE2 and not to HC.

[Effect of dihydrotachysterol on the periodontal tissues in rats].

The purpose of the present study was to investigate the histological changes of the periodontal tissues in rats produced by oral administration of dihydrotachysterol (DHT). Forty-five rats, 6 weeks old, were divided equally into 9 groups (Groups A-I). Groups A, D and G, control groups, received an administration of corn oil (0.5 ml) once a day for 1, 2 and 4 weeks, respectively. Groups B, E and H received an administration of DHT (25 micrograms) once a day for 1, 2 and 4 weeks, respectively. Groups C, F and I had an administration of DHT (50 micrograms) once a day for 1, 2 and 4 weeks, respectively. The animals of all groups were killed 24 hours after the last administration. The periodontal tissues of the maxillary first molar of all animals were examined histologically and histometrically. Histological and histometrical findings: 1. In Groups A, D and G, control groups, the periodontal ligament, alveolar bone and cementum appeared normal during the entire experimental period. 2. In Group B, the peridontal tissues were almost similar to those of the control group. 3. In Groups C and E, osteoid tissue was observed in the alveolar bone surface. The thickness of the periodontal ligament decreased and the bone marrow spaces were slightly enlarged. The volmetric density of the alveolar bone decreased compared with that of the control groups. 4. In Groups F, H and I, the thickness of the periodontal ligament decreased significantly. In the periodontal ligament, destruction of collagen fibers was observed, and fibroblasts, collagen fibers and oxytalan fibers decreased in number. Dental ankylosis was also found. An abnormal darkly-stained layes (using the PAS staining method) was observed in the cementum surface. The alveolar bone marrow spaces were enlarged and replaced by fibrous tissue. In the histometrical findings, the thickness of the periodontal ligament and the volumetric density of the alveolar bone had decreased, and the volume of the cementum had increased significantly. The result of the present study suggested that oral administration of dihydrotachysterol to the rats induced the changes of the periodontal tissues similar to those seen in aging humans.

Effect of dihydrotachysterol on bone induction in ovariectomized rats.

We have demonstrated via marrow stromal cell cultures and the osteoinductive response to demineralized bone grafts (DBM) that the cortical bone deficit in the ovariectomized (OVX) rat (6 weeks postop) is primarily due to impaired osteoprogenitor cell proliferation, and that dihydrotachysterol (DHT) treatment can be protective. In cultured marrow stromal cells from OVX rats, short-term DHT-Rx exaggerated the already subnormal pattern of marrow stromal cell proliferation. However, in DBM grafts, DHT treatment benefited the time-course of mesenchymal cell DNA synthesis as measured by tritiated thymidine incorporation and osteogenic cell maturation as measured by alkaline phosphatase concentration, and established a suggestive trend toward normalization of bone formation/mineralization (24 h 45Ca incorporation). The data from this animal model infer that DHT could moderate the bone loss normally seen in ovariectomized rats via an activation of the osteoprogenitor cell population.

Serum dihydrotachysterol levels and biological action in normal man.

Dihydrotachysterol (DHT2) has been a safe and effective treatment for hypocalcemic disorders for many years, but few assays for quantitation of DHT2 have been developed. Thus little is known about its pharmacokinetics. The 2-fold purpose of this study was 1) to develop a practical method for quantitating DHT2 after oral dosing in normal subjects, and 2) to assess changes in serum DHT2 levels and calcium and phosphorus metabolism after DHT2 administration for 8 days. Peak serum DHT2 levels in six normal subjects, assayed by high performance liquid chromatography were achieved 4 h after administration of 0.4-0.8 mg DHT2; at 24 h, levels had declined by 70% whether DHT2 had been given for 1 or 8 days. These data indicate that a standard approach is needed to interpret the results of serum DHT2 measurements in treated patients. Interfering substances were detected in lipemic serum. The major biological effects of DHT2 administration were hypercalciuria in two subjects and a fall in serum 1,25-dihydroxyvitamin D[1,25-(OH)2D] levels, including free levels when measured, in all subjects. Possible explanations for this fall in serum 1,25-(OH)2D levels include decreased 1,25-(OH)2D production because of competition for the 1 alpha-hydroxylase enzyme by a metabolite(s) of DHT or increased metabolic clearance of 1,25-(OH)2D.

Aluminum metabolism in rats: effects of vitamin D, dihydrotachysterol, 1,25-dihydroxyvitamin D and phosphate binders.

In order to study the effects of vitamin D on aluminium balance when different forms of vitamin D and phosphate binders are used simultaneously for therapeutic purposes, 30 Sprague-Dawley weanling rats, weighing 44-66 g, were randomly assigned to 5 groups: (A) control, (B) aluminum hydroxide, (C) dihydrotachysterol at 16 micrograms/kg/day, (D) 1,25-dihydroxyvitamin D at 16 ng/kg/day and (E) vitamin D at 2,000 IU/kg/day. Aluminum hydroxide (60 mg/kg/day) in the feed was provided to all except the control group. The vitamin D or metabolites were fed by stomach tube daily for a period of 10 days. At the end of the study, the mean (+/- SEM) serum aluminum concentration, as determined by flameless atomic absorption spectrophotometry, was 5.0 +/- 2.4 micrograms/l; there were no significant differences in these results between groups. During the last three days of the study, 24-hour urine and stool collections were made with the usual precautions against trace mineral contamination. The means (+/- SEM) of aluminum balances for groups A, B, C, D and E were -388 +/- 261, 1,121 +/- 331; 2,316 +/- 304; 2,387 +/- 245, and 1,968 +/- 337 micrograms/day, respectively. We conclude that at therapeutic doses of aluminum hydroxide and vitamin D or its metabolites, hyperaluminemia was not observed. However, the positive aluminum balances imply retention, and the use of vitamin D, especially its potent metabolites dihydrotachysterol and 1,25-dihydroxyvitamin D, intensified this risk.

Action and metabolism of dihydrotachysterol2.

Dihydrotachysterol2 (DHT2) is a synthetic analogue of vitamin D2. DHT2 is used extensively in the treatment of renal osteodystrophy and hypoparathyroidism. It is equally efficacious as 1 alpha,25-dihydroxyvitamin D3 and 1 alpha-hydroxyvitamin D3. Moreover, it offers interesting therapeutical advantages and it is surprising that until recently little was known of its metabolism and sites of action. This paper deals with studies on the pharmacology of DHT2 in rats. Following the synthesis of [3H]DHT2 and oral administration, evidence was obtained that DHT2 is metabolized extensively; three of the major metabolites could be identified as 25-hydroxy-DHT2, 1 alpha,25- and 1 beta,25-dihydroxy-DHT2.

Prevention of hypercalcemia-induced renal concentrating defect and tissue calcium accumulation.

The mechanism of the concentrating defect of hypercalcemia is explored by examining the effect of concomitant phosphate restriction. Rats were pair fed a normal phosphorus diet, without (group 1) or with dihydrotachysterol (group 2), or a low-phosphorus diet (group 3). Hypercalcemia was comparable in groups 2 (12.1 +/- 0.6 mg/dl) and 3 (11.8 +/- 0.4 mg/dl), but serum phosphate was lower in group 3 than group 2 (3.8 +/- 0.7 vs. 7.1 +/- 1.1 mg/dl, P less than 0.005). Group 2 rats had impaired maximum urinary concentration after 24 h of fluid deprivation (2,441 +/- 450 mosmol/kg H2O, P less than 0.001) compared with group 1 (3,263 +/- 466 mosmol/kg H2O) or group 3 (3,332 +/- 515 mosmol/kg H2O) animals. Polydipsia and polyuria were found in group 2 rats only. Tubular calcium reabsorption was higher in group 2 (83.1 +/- 33.5 mg/24 h, P less than 0.001) than group 1 (47.0 +/- 26.1 mg/24 h) or group 3 (52.8 +/- 19.3 mg/24 h) animals, and medullary calcium concentration was higher in group 2 (7.57 +/- 3.08 nmol/mg dry wt, P less than 0.05) as compared to group 1 (5.04 +/- 1.37 nmol/mg dry wt) or group 3 (5.32 +/- 0.98 nmol/mg dry wt) rats. Total medullary solute concentration was significantly higher in group 3 than group 2 animals. Thus phosphate restriction prevents the defect of urinary concentrating ability of chronic hypercalcemia, probably by decreasing tubular uptake and tissue accumulation of calcium.

The possible role of reticulo endothelial system in hepatic regeneration.

Calciphylactic blockade of Reticulo endothelial system (REB) was used to study the role of reticulo-endothelial system (RES) in hepatic regeneration. REB was induced by i.v. egg yolk administration in DHT sensitized animals, which were subsequently partially hepatectomized. Mitotic index and percentage regeneration were studied in these animals 48 hr after partial hepatectomy. REB produced a significant depression of the mitotic index and percentage regeneration in regenerating livers. This suggests that functional integrity of the RES in essential hepatic parenchymal proliferation after partial hepatectomy.