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Publications and source records attributed to Y Nishii.
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Catabolism of 22-oxacalcitriol (OCT) in parathyroid cells was compared to that of the parent hormone, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. Catabolism of both compounds was greatly accelerated by pretreatment of the cells with 1,25-(OH)2D3 or OCT. The rate of degradation of OCT was slightly greater than that of 1,25-(OH)2D3. Excess unlabeled OCT or 1,25-(OH)2D3 inhibited metabolism of both tritiated substrates. Ketoconazole, a cytochrome P450 inhibitor, blocked catabolism of both compounds. The major OCT metabolite appeared to be 1,20-dihydroxy-22,23,24,25,26,27-hexanor-vitamin D3 which was not active in suppressing PTH secretion. We conclude that OCT appears to be metabolized by the same vitamin D-inducible side chain oxidation pathway that catabolizes other vitamin D compounds and that its higher than expected suppression of PTH secretion is not due to slower cellular metabolism.
22-oxacalcitriol can decrease the serum level of 1,25-dihydroxycholecalciferol by increasing its metabolic clearance rate and decreasing its production rate. To determine whether 22-oxacalcitriol suppressed the renal 25-hydroxycholecalciferol-1 alpha- hydroxylase we treated rats with 200 ng of 22-oxacalcitriol daily for 1 week. Enzyme activity was measured in vitro by measurement of production of 1,25-dihydroxycholecalciferol by renal slices incubated with 25-hydroxycholecalciferol. 22-oxacalcitriol significantly decreased the 25-hydroxycholecalciferol-1 alpha-hydroxylase activity from 797 +/- 208 pg of 1,25-dihydroxycholecalciferol/g of kidney/h in the control group to 257 +/- 150 pg of 1,25-dihydroxycholecalciferol/g of kidney/h (p less than 0.05). This is the first demonstration of suppression of the renal 25-hydroxycholecalciferol-1 alpha-hydroxylase by the vitamin D3 analog, 22-oxacalcitriol.
Rat hepatic t protein that is negatively regulated by thyroid hormone in nuclear globulin extract was characterized by the antibodies. The following evidence indicated that t protein is a peroxisomal enoyl-CoA hydratase-3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme (bifunctional enzyme). 1. Both proteins had an identical molecular size, and were immunologically indistinguishable from each other. 2. The t protein was abundant in mitochondrial fraction which contained abundant peroxisomes. 3. The amount of the t protein was increased by a peroxisomal proliferator. 4. The activity of the peroxisomal bifunctional enzyme corresponded to the t protein in CM-Sephadex column chromatography. The amount of peroxisomal bifunctional enzyme was increased by thyroidectomy and decreased by 3,5,3'- triiodo-L-thyronine treatment in the whole homogenate of rat liver. These results indicate that the levels of peroxisomal bifunctional enzyme were regulated by thyroid hormone in vivo.
We previously showed that OCT, an analog of 1,25-(OH)2D3 with little calcemic activity, can decrease PTH mRNA levels in normal rats and inhibit PTH secretion in cultured bovine parathyroid cells with the same potency as 1,25-(OH)2D3 and that in normal rats fed a normal calcium diet, administration of OCT (500 ng) for 5 days did not increase plasma Ca. Thus, to determine if PTH suppression by OCT contributes to its lack of calcemic activity and to further characterize the effects of OCT on Ca metabolism, we performed several studies in parathyroidectomized (PTX) rats. PTX rats, maintained on a normal diet (0.9% Ca), received daily injections of vehicle, 1,25-(OH)2D3 (200 ng/day), or OCT (200 ng/day) for 6 days. Plasma Ca was measured daily. Plasma Ca in control rats stayed between 6.60 and 7.40 mg/dl, whereas Ca increased to 12.9 +/- 0.42 mg/dl in 1,25-(OH)2D3-treated rats and to 9.53 +/- 0.35 mg/dl in OCT-treated rats after 6 days. With a Ca-deficient diet, control rats maintained a plasma Ca between 4.25 and 4.60 mg/dl, but Ca increased to 13.7 +/- 0.24 mg/dl with 1,25-(OH)2D3 and to 7.29 +/- 0.17 mg/dl with OCT. Since the elevation in Ca by OCT was similar with both diets, OCT appears to act primarily on bone. PTX rats were infused with PTH (1.84 micrograms/kg/day) via an Alzet pump to achieve normal plasma Ca and then treated daily with either vehicle or OCT (200 ng/day). After 6 days, OCT increased serum Ca to 10.7 +/- 0.21 mg/dl over a control value of 8.58 +/- 0.29 mg/dl.(ABSTRACT TRUNCATED AT 250 WORDS)
A new, highly sensitive radioreceptor assay, which does not require high-performance liquid chromatography, has been developed for the determination of 1,25-dihydroxyvitamin D3 (1,25-(OH2)D3) in serum. The assay involves rapid extraction of serum, Sep Pak silica purification, and addition of 1,25-dihydroxyvitamin D3 receptor, radiolabeled 1,25-dihydroxyvitamin D3, bovine serum albumin, and monoclonal antibody to specifically precipitate the receptor. This method is sensitive to 0.3-0.6 pg/tube, with B50 occurring at 5.8 pg/tube. This sensitivity combined with overall recovery of 1,25-dihydroxyvitamin D3 (81.5 +/- 5.2%, n = 50, mean +/- SD) allows the measurement of serum 1,25-(OH)2D3 in duplicates with only 0.5 ml of serum. Intra- and interassay coefficient of variation were 9.5 and 14.6%, respectively. Dilution analysis, analytical recovery of added 1,25-dihydroxyvitamin D3, and comparison with a standard method using HPLC have been used to validate the assay. Serum 1,25-dihydroxyvitamin D3 level was for normal adults, 36.6 +/- 10.5 pg/ml (n = 14); in primary hyperparathyroidism, 98.9 +/- 19.9 pg/ml (n = 16); in chronic renal failure, 17.8 +/- 5.1 pg/ml (n = 12). This method allows large numbers of samples to be processed at once. Further, the method is rapid and provides an accurate assay using small amounts of serum.
We investigated the effect of 22-oxa-1,25-dihydroxyvitamin D3, a synthetic analogue of vitamin D3, on the production of prostacyclin by vascular tissues using rat aortic rings and A7r5 cells derived from fetal rat aortic smooth muscle. Prostacyclin synthesis by aortic rings of rats treated with 22-oxa-1,25-dihydroxyvitamin D3 was much higher than that of non-treated controls, but did not cause any significant hypercalcemia. Treatment with 22-oxa-1,25-dihydroxyvitamin D3 significantly increased the production of prostacyclin by A7r5 cells for 48 hours in a dose-dependent manner. In time-course studies, cells incubated with 22-oxa-1,25-dihydroxyvitamin D3 or 1,25-dihydroxyvitamin D3 produced prostacyclin progressively over a period of 48 hours. The shortest period of incubation that produced a significant amount of prostacyclin compared with control cultures was 24 hours. We observed that treatment with 22-oxa-1,25-dihydroxyvitamin D3 induced cyclooxygenase mRNA in A7r5 cells. Our data suggest that 22-oxa-1,25-dihydroxyvitamin D3 may possibly be a protective substance against the development of atherosclerosis by modulating prostaglandin metabolism.
Thyroid hormone, T3, enters cells through an energy-dependent, saturable process and/or passive diffusion. Although the nucleus is the primary site of T3 action, exact mechanisms by which T3 is transported to nucleus are uncertain. In this report, initial cellular and nuclear uptake of T3 was determined using a rat liver cell line (clone 9), in which energy-dependent cellular T3 uptake was demonstrable. One to 5 mM sodium butyrate enhanced cellular T3 uptake with a concomitant increase in nuclear T3 uptake after 30 h. Increased cellular T3 uptake was associated with the increase in the maximum velocity of saturable cellular T3 uptake systems without affecting the Michaelis-Menten constant. Sodium butyrate, however, elicited a reduction in nuclear thyroid hormone receptor levels. On the other hand, heat shock (42 C for 60 min) reduced nuclear T3 uptake without affecting the Michaelis-Menten constant and maximum velocity of saturable cellular T3 uptake. Although nuclear receptor levels were reduced transiently after the heat shock, decreased nuclear T3 transport was not caused by the changes in the receptor levels. NADPH-dependent cytosolic T3 binding protein was undetectable in clone 9 cells before and after butyrate treatment or heat shock. In conclusion, sodium butyrate enhanced nuclear T3 uptake through the increase in cellular T3 uptake that is prerequisite to nuclear T3 transport. However, nuclear T3 uptake mechanisms independent of the cellular uptake system exist and are sensitive to heat shock. Nuclear receptors and cytosolic T3 binding proteins do not seem to be involved in the alteration of nuclear T3 uptake after sodium butyrate or heat shock. These findings suggest intracellular regulatory mechanisms of thyroid hormone transport.
1,25-Dihydroxyvitamin D3 (1,25D) regulates its own levels in circulation by affecting its rates of synthesis and degradation, 22-Oxacalcitriol (OCT), a vitamin D analog with low calcemic activity, decreases circulating PTH levels, one of the regulators of renal 1 alpha-hydroxylase, and stimulates vitamin D degradation in vitro. The purpose of this study was to examine the effects of OCT administration on serum levels of 1,25D. In normal rats, OCT administration (4-200 ng, ip, daily for 5 days) caused a dose-dependent reduction in serum calcitriol levels. At a dose of 200 ng, OCT reduced serum 1,25D from 34.5 +/- 2.7 to 10.9 +/- 0.7 pg/ml (P less than or equal to 0.01) without significant changes in ionized Ca or phosphorus levels. The contribution of the suppression of PTH by OCT to the reduction of serum 1,25D was examined by administering OCT to parathyroidectomized (PTX) rats. Two hundred nanograms of OCT, ip, daily for 5 days significantly reduced serum calcitriol from 29.7 +/- 7.6 to 9.1 +/- 0.5 pg/ml (P less than or equal to 0.01) in rats fed a normal calcium diet. Because OCT increased total calcium (TCa) in this group from 7.4 +/- 0.1 to 9.5 +/- 0.3 mg/dl, similar doses of OCT were given to PTX rats fed a calcium-deficient diet. OCT decreased 1,25D from 58.9 +/- 8.9 to 10.3 +/- 0.4 pg/ml and increased TCa from 4.8 +/- 0.2 to 7.4 +/- 0.1 mg/dl. Comparison of serum 1,25D for identical TCa levels in PTX rats (normal calcium diet controls vs. calcium-deficient diet, OCT-treated) clearly indicates that OCT per se reduced serum 1,25D. Further support for a direct effect of OCT was provided by studies in PTX rats fed a low phosphorus diet. OCT decreased serum 1,25D from 125.8 +/- 15.6 to 10.9 +/- 0.6 pg/ml without significant changes in TCa. To further characterize the mechanisms involved in this effect, similar studies were performed in six normal dogs. Intravenous administration of 0.75 micrograms OCT every other day for 1 week decreased serum calcitriol from 25.4 +/- 3.2 to 12.2 +/- 1.3 pg/ml (P less than or equal to 0.002). Ionized Ca and phosphorus remained unchanged. Despite the short half-life of OCT in the circulation, 1,25D levels returned to basal concentrations 96 h after the last dose of OCT.(ABSTRACT TRUNCATED AT 400 WORDS)
In our previous study, we reported that the administration of T4 to patients with Graves' disease who were under treatment with methimazole (MMI) decreased the level of antibodies to thyroid-stimulating hormone (TSH) receptors and the rate of recurrence of hyperthyroidism. In this study, the effect of T4 administration on the rate of postpartum recurrence of hyperthyroidism was examined. Seventy-eight patients with Graves' disease had been treated with MMI for 1-3 yr before pregnancy, and MMI was discontinued 5-6 months after the onset of pregnancy because the levels of antibodies to TSH receptors decreased during early pregnancy. The patients were then divided into two groups. Group A (n = 40) was given T4 (100 micrograms/day) and group B (n = 38) was not given any drugs from 5 months after the onset of pregnancy until 1 yr after delivery. The levels of the antibodies to TSH receptors and serum concentrations of thyroxine-binding globulin (TBG) and T4 were not different between the two groups before and during pregnancy, although a transient increase in serum T4 and TBG concentrations were observed during the pregnancy in both groups. After delivery, levels of antibodies to TSH receptors increased in both groups. The rate of increase, however, was more rapid in group B than in group A. The levels were significantly higher in group B than A at 3, 6, 9, and 12 months after delivery. Serum T4 and TBG concentrations decreased after delivery in both groups. Serum concentrations of T4 increased after delivery in group B but not in group A. The concentration of T4 was significantly higher in group B than in group A at 9 and 12 months after delivery. Postpartum recurrence of hyperthyroidism was 5.0% in group A and 31.6% in group B, respectively, during the first year after delivery. These results suggest that administration of T4 during pregnancy and after delivery is effective in decreasing the level of antibodies to TSH receptors and to prevent the postpartum recurrence of hyperthyroidism.
Human beta thyroid hormone receptor (c-erb A beta protein) produced by an Escherichia coli expression system was purified by sequential column chromatography followed by electroelution from an electrophoresis gel and an antibody was prepared. The antibody recognized a 56 kDa protein band in a partially purified rat hepatic nuclear thyroid hormone receptor fraction on Western blotting. Although multiple bands appeared on Western blotting of crude rat hepatic receptor preparations, a 56 kDa band was the most prominent and preadsorption of the antibody by purified c-erb A protein resulted in almost complete disappearance of the 56 kDa band, indicating that the 56 kDa band was formed by a specific antigen-antibody interaction. Furthermore, the 56 kDa protein appeared to co-elute with 3, 5, 3'-triiodo-L-thyronine binding activity in hydroxylapatite, Sephacryl S-200, and DNA-cellulose column chromatography of rat hepatic nuclear receptor, and sequential column purification resulted in selective enrichment of the 56 kDa band. These results suggest that the 56 kDa protein may be the major component of the rat hepatic thyroid hormone receptor.
Patients with autoimmune insulin antibody are characterized by hypoglycemic attacks and antibodies to insulin in serum without prior insulin administration. In the present report, a patient with hypoglycemia due to autoimmune insulin antibody associated with primary empty sella syndrome and polymyositis appeared to have high urinary immunoreactive insulin (IRI) in the face of normal urinary C peptide. Consequently, the urinary IRI/C peptide ratio was apparently high. The amelioration of hypoglycemic attacks and polymyositis by prednisolone treatment was accompanied by the disappearance of the antibodies and complete normalization of the urinary IRI and IRI/C peptide ratio. No comparable rise in the urinary IRI and IRI/C peptide ratio was observed in the patients with other disorders studied. Glucose clamp and glucose tolerance study showed decreased sensitivity to exogenous or newly secreted insulin, prolonged half disappearance time of serum insulin, and normal disappearance of blood glucose. These results were consistent with the idea that autoantibodies buffered the effect of exogenous or newly secreted insulin and maintained a relatively constant level of serum free insulin which was not high enough when a large amount of glucose was loaded, but was too high after prolonged fasting, which eventually caused hypoglycemic attacks.
During the process of enhancing monocytic differentiation of the human leukemia line HL-60, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) also "primes" the cell for respiratory burst by increasing the uptake of Ca2+ across the plasma membrane (Hruska, K.A., Bar-Shavit, Z., Malone, J.D., and Teitelbaum, S.L. (1988) J. Biol. Chem. 263, 16039-16044). The present study asked if the maturational effect of vitamin D is dependent upon this "priming" phenomenon. To this end, we exposed HL-60 to either 1,25(OH)2D3 or its synthetic analogue (1 alpha, 3 beta, 5Z, 7E)-9-10-Secocholesta-5,7,10(19)-triene-1, 3, 25-triol (22-oxa). We found that 22-oxa induced HL-60 maturation as effectively as does the natural steroid. As expected, 48 h of 1,25(OH)2D3 exposure more than doubles (p less than 0.005) HL-60 basal cytosolic Ca2+ and increases inositol triphosphate-sensitive Ca2+ stores approximately 4-fold (p less than 0.01). 22-oxa in contrast alters neither Ca(2+)- nor inositol triphosphate-mobilizable deposits. Moreover, 1,25(OH)2D3 treatment prompts a transient Ca2+ "spike" in response to formyl-methionyl-leucyl-phenylalanine (fMLP) and a marked increase in superoxide (O-2) generation when exposed to the chemotactic peptide (p less than 0.01) or phorbol ester (p less than 0.02). Treatment with 22-oxa does not enable HL-60 to respond to fMLP with a Ca2+ spike or prime the cell for respiratory burst unless it is co-incubated with the Ca2+ ionophore, ionomycin. Similarly, phorbol ester impacts more profoundly on O-2 generation by 1,25(OH)2D3 than 22-oxa preincubated cells (p less than 0.02), unless the latter is added with ionomycin. Our findings indicate that the maturational effects of vitamin D sterols are independent of their capacity to prime cells for respiratory burst and that the Ca2+ ionophoretic effects of 1,25(OH)2D3 play a major role in such priming.
The localization of synapsin I in the rat adrenal medulla was studied using the light- and electronmicroscopic immunohistochemistry. By light microscopy, many dot-like reaction products for synapsin I were recognized to be distributed throughout the medullary tissue. The immunoelectron microscopy clearly revealed that gold particles for synapsin I accumulated in abundance in the nerve terminals forming synapses with the chromaffin cell, while the particles were not localized in the chromaffin cells at all. In the nerve terminal, the gold particles were localized exclusively in the region occupied by synaptic vesicles except for the region just beneath the presynaptic plasma membrane. The synaptic vesicles were frequently linked with the adjacent ones by filamentous structures implicated in synapsin I. It is concluded morphologically that synapsin I is a highly-specific protein for the genuine neuron, and is not detected even in the chromaffin cell which originates from the neural crest.
The fluorogenic dienophile 1,2,4-triazoline-3,5-dione with a highly fluorescent quinoxalinone group at the 4-position (DMEQ-TAD) was synthesized and exploited as a reagent to assay vitamin D metabolites. 25-Hydroxyvitamin D3, 1 alpha,25-dihydroxyvitamin D3, and 24(R),25-dihydroxyvitamin D3 reacted quantitatively with DMEQ-TAD when the two substrates were mixed in dichloromethane at room temperature to yield the corresponding 6,19-cycloadduct. The reaction was very fast so that 1 alpha,25-dihydroxyvitamin D3 at a concentration as low as 10(-8) M could be quantitatively labeled with the fluorescent reagent within 30 min at room temperature. With this reagent, down to 10 fmol of vitamin D metabolites could be quantified linearly. The detection limit of the labeled vitamin D using high-performance liquid chromatography was usually about 1 fmol. Thus, it was shown in a model system that the fluorometric method using the new reagent (DMEQ-TAD) can be applied to the assay of the three major vitamin D metabolites in 1 ml of plasma. This is the first practical fluorometric method for assaying the active vitamin D metabolite.
A novel vitamin D3 analogue, [2 beta-(3-hydroxypropoxy)-calcitriol: ED-71] showed a similar Ca-regulating activity as calcitriol in the in vivo and in vitro Ca mobilization test and ex vivo intestinal Ca absorption assay using vitamin D-deficient rats. The differentiation-inducing activity of ED-71 in mouse myeloid leukemia cell line (WEHI-3 cell) was slightly less than that of calcitriol. ED-71 distributes predominantly in plasma as an intact form and its half-life plasma was twice as long as that of calcitriol. Further study revealed that the higher binding potency of ED-71 to plasma-specific vitamin D-binding protein (DBP) compared with that of calcitriol accounts for its stability in the blood circulation. The pharmacological effect of ED-71 for the animal models with osteoporosis seemed to be better than that calcitriol. These results suggest that ED-71 should become a valuable therapeutic long-acting drug for patients with osteoporosis.
OCT, a non-calcemic analogue of 1,25(OH)2D3 has been found to have a more potent activity than that of 1,25(OH)2D3 regarding cell differentiation and immunopotentiation activity, and to prolong the average life span of MRL/l mice. Recently, we found that OCT effectively suppressed the secretion and synthesis of PTH without inducing hypercalcemia. In primary cultures of bovine parathyroid cells, OCT was capable of suppressing PTH release in a dose-dependent manner. OCT was also active in vivo, and, like 1,25(OH)2D3, decreased the pre-pro(PTH) mRNA levels. In a group of rats with CRF, daily administration of OCT, 8 ng i.p. for 2 weeks returned PTH levels to normal without changes in serum calcium. Preliminary results in dogs with CRF indicated that after the administration of OCT 5 micrograms i.v., N-terminal PTH decreased by 76% without changes in Ca. In conclusion, OCT may provide a unique contribution to the treatment of secondary hyperparathyroidism.
The binding potencies of OCT to chick intestinal calcitriol receptor and vitamin DBP were approximately 1/8 and 1/600 of the respective values of calcitriol. OCT is circulating mainly as an intact form bound to chylomicrons and/or lipoproteins. Intravenously injected [3H]-OCT to normal rats was quickly decreased from blood and rapid excretion of OCT as a glucuronate into bile was observed. However, significant amounts of radioactivities were recovered in the intact form in liver and intestine even after 24 h. The separation of calcemic and immune and/or differentiation activities may be derived from the rapid turnover and the nonspecific transporting system.