Studies of calcitonin cells and parathyroid gland of house shrew, Suncus murinus in response to experimental hypercalcemia

1. Hypercalcemia was induced in S. murinus by alternate day intramuscular injections of vitamin D (25 000 IU) and by providing them 1% CaCl2 solution (prepared in tap water) for drinking. 2. After such a treatment the serum calcium values recorded a rise as compared to those of the control specimens. 3. The histological picture of the thyroid of the treated specimens reveals increased number of calcitonin cells. This observation is supported by the occurrence of mitotic figures among them. 4. Perpetual calcium challenge results in degranulation of the secretory material (calcitonin) among these cells (at 26th and 30th day of treatment). 5. It also results in degenerative changes in certain number of C cells. 6. The blood capillaries around these cells get dilated and secretory granules of C cells tend to gather at the periphery of cytoplasm and towards vascular pole. 7. The parathyroid shows atrophic changes.     

Ultimobranchial body and parathyroid gland of the parrot Psittacula psittacula in response to experimental hypercalcemia

Psittacula psittacula when subjected to long term hypercalcemia by intramuscular injections of vitamin D2 (20,000 I.U.) on alternate days and by increasing dietary calcium, exhibit a rise in the serum calcium level after 10, 20 and 30 days of treatment as compared to their corresponding controls. The ultimobranchial cells show progressive hypertrophy up to 20th day of the treatment. From 20th day till the end of the experiment (30 days) these cells show feeble staining response. The parathyroid glands suffer from degenerative changes due to its inactivity under chronic hypercalcemia.     

Vitamin D Intoxication Treated with Porcine Calcitonin

Porcine calcitonin was used to treat three patients with hypercalcaemia due to vitamin D intoxication. In two patients a rapid and sustained fall to normal in serum calcium occurred within three days, in the third patient normocalcaemia was achieved in seven days. In view of its rapid and sustained effect calcitonin may be of value in the urgent treatment of hypercalcaemia due to vitamin D intoxication.     

Influence of parathormone on C cells, parathyroid glands, serum calcium and serum phosphate levels in the house shrew, Suncus murinus

A chronic hypercalcaemia was induced in Suncus murinus by the daily subcutaneous injections of parathormone for 15 days. Serum calcium increased 4 hours after the first injection (+ 0.88 mg/100 ml), it reached its highest value on day 10 (+ 2.18 mg/100 ml), and then decreased slightly at the end of the experiment (day 15). The serum phosphate level in treated animals showed a progressive fall throughout the experiment. Serial sections of thyroid-parathyroid complex were subjected to selective staining for C cells identification. The chronic hypercalcaemia increased the number of C cells. This was supported by the presence of mitotic figures. Degranulation of the secretory material was observed as the result of the chronic hypercalcaemia; degenerative changes were found in some C cells. The chronic hypercalcaemia also inhibited the activity of parathyroid "Chief-Cells" which ultimately degenerated.     

Ultimobranchial body of Notopterus notopterus in relation to calcium and sodium rich environments

The UBB of Notopterus notopterus is a paired structure situated between the oesophagus and sinus venosus. Both right and left lobes of the gland are enveloped by a common thick connective tissue which gets constricted between the lobes and separates them. Numerous follicles of varying sizes are encountered in each gland. In N. notopterus the effects of hypercalcaemia (caused by keeping the specimens in 0.5% of CaCl2 solution and by injecting 4000 I.U. of vitamin D2 on alternate days) on UBB has been observed. The effects of NaCl rich environment (created by keeping the fish in 0.5% NaCl solution) on this gland has also been studied. In the UBB of N. notopterus the activity of the gland is observed in terms of: 1. increase in the blood supply of the gland and the dilation of the blood vessel, 2. increase in the height of the follicular epithelium, 3. cytoplasmic hypertrophy resulting in the increase in secretory processes, 4. appearance of pseudostratified epithelium in place of single layered cuboidal follicular epithelium and 5. nuclear and cellular hypertrophy. According to these characteristics it is evident that the gland from group II shows gradual activity from the 2nd day onwards and is maximum on the 6th day. From 8th day to the close of the experiment gradual inactivity of the gland is discerned--follicles get atrophied and the cells appear in clumps. The gland from group III shows a good response to its environment and is more hypertrophied as compared to that of group II. The activity of the gland closely parallels serum sodium levels which increase up to the 8th day when UBB shows the maximum activity. The serum sodium level rises from a normal of 110 m eq/l to a peak of 180 m eq/l on 8th day. After 10 days onwards the gland shows gradual inactivity and degeneration. The serum sodium level is 130 m eq/l on 12th day. These observations support the view that the main role of UBB in N. notopterus lies in sodium metabolism and it is only partially responsible for calcium regulation.     

Structure and behaviour of ultimobranchial gland in response to vitamin D3--induced hypercalcemia in male Clarias batrachus

Hypercalcemia was induced in Clarias batrachus by treating them with vitamin D3 (5,000 I.U./100 g body wt.) and/or 0.5% solution of CaCl2. The animals were killed on 1st, 3rd, 5th, 9th, 13th and 17th days after the initiation of the experiment. Histological preparations of the ultimobranchial gland (UBG) were made. The gland exhibits nuclear hypertrophy, hyperplasia and loss of staining response corresponding to the rise in serum calcium levels. At later intervals, the UBG shows exhaustion and degeneration which is evident from vacuolization and nuclear shrinkage of the ultimobranchial cells after day 13 in groups B and C and day 9 in group D.     

Abolition by preventive action of calcitonin of vitamin D3-stimulated gluconeogenesis in rat kidney

The effects of vitamin D3, 1-alpha-OH D3 and calcitonin treatment on the rate of glucose synthesis in rat renal cortex slices were studied. The rate of glucose synthesis was significantly increased in animals treated with vitamin D3 and 1-alpha-OH D3 as compared to the control. The increase in the rat of glucose synthesis observed after vitamin D3 and 1-alpha-OH D3 treatment was abolished after administration of calcitonin in doses normalizing the serum calcium level. The obtained data point to a correlation between the serum calcium level observed in vivo and the ability to synthesize glucose measured in vitro in renal cortex slices.     

Metabolism of 25-hydroxyvitamin D3 to 24,25-dihydroxyvitamin D3 by blood derived macrophages from a patient with alveolar rhabdomyosarcoma during short-term culture and 1 alpha,25-dihydroxyvitamin D3 after long-term culture

We have examined the ability of blood-derived monocytes and macrophages isolated from a patient with alveolar rhabdomyosarcoma and hypercalcaemia, to form 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) or 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3) from 25-hydroxyvitamin D3 (25(OH)D3). Adherent monocyte-macrophage cells incubated with 25(OH)D3 over the initial 2 days in culture synthesized 1.9 pmol 24,25(OH)2D3/h/incubation (representing 0.63 pmol/h/10(6) cells), whereas macrophages synthesized 1.03 and 1.15 pmol 1 alpha,25(OH)2D3/h/incubation after 1 and 4 weeks in culture respectively. In a further experiment synthesis of 1 alpha,25(OH)2D3 by long-term cultured macrophages fell from 2.25 to 0.04 pmol/h/incubation following exposure to 10 nM 1 alpha,25(OH)2D3 for 7 days, whereas 24,25(OH)2D3 synthesis was induced (0.46 pmol/h/incubation). The vitamin D3 metabolites were identified by co-chromatography with authentic 24,25(OH)2D3 or 1 alpha,25(OH)2D3 in three different high-performance liquid chromatography systems. Serum 1 alpha,25(OH)2D3 in the patient was markedly suppressed at 5 pg/ml (normal 20-50 pg/ml) indicating that raised 1 alpha,25(OH)2D3 was not the cause of the hypercalcaemia, but rather, that raised calcium may have suppressed renal 1 alpha,25(OH)2D3 synthesis. Administration of APD (3-amino-1-hydroxypropylidine-1,1-bisphosphonate) corrected the hypercalcaemia in the patient suggesting that increased bone resorption was responsible for the raised calcium. The results of this study show for the first time that immature blood derived monocyte-macrophage cells can synthesize 24,25(OH)2D3 before they mature into macrophages able to synthesize 1 alpha,25(OH)2D3.     

Calcaemic responses in the yellow monitor, varanus flavescens to vitamin D3 administration

The effect of a daily intramuscular injection of vitamin D3 (2000 IU/100 g b.wt) on serum calcium level was investigated in Varanus flavescens. This treatment evoked hypercalcaemia on day 3 which progressed up to day 7. At day 14 a decline was noticed in the serum calcium level which was followed by a rise from day 21 to day 28.     

Pharmacokinetic study of 45Ca administered intravenously following intramuscular injection of vitamin D3 or 25-hydroxyvitamin D3 in early weaned lambs.

The objective of this study was to evaluate the pharmacokinetic parameters of 45Ca administered intravenously at a dose of 50 microCi in young lambs. The experiment was performed on 18 female lambs allocated to three treatments. Six animals received intravenous calcium only; six received intramuscularly vitamin D3 8 days prior to the administration of calcium; and six received intramuscularly 25-hydroxyvitamin D3 5 days prior to the administration of calcium. Blood samples were collected from 15 min up to 8 days after the administration of 45Ca. The different pharmacokinetic parameters (area under the curve, clearance, mean residence time and volume of distribution) were estimated using non-compartmental and compartmental approaches. Statistical analysis of the results showed no significant differences in the pharmacokinetic parameters of the three groups. It can be concluded that vitamin D does not modify either the elimination or the distribution of calcium in the lambs receiving adequate dietary vitamin D.     

Decrease in calcitonin and parathyroid hormone mRNA levels and hormone secretion under long-term hypervitaminosis D3 in rats

In calcium homeostasis, vitamin D3 is a potent serum calcium-raising agent which in vivo regulates both calcitonin (CT) and parathyroid hormone (PTH) gene expression. Serum calcium is the major secretagogue for CT, a hormone product whose biosynthesis is the main biological activity of thyroid C-cells. Taking advantage of this regulatory mechanism, long-term vitamin D3-induced hypercalcemia has been extensively used as a model to produce hyperactivation, hyperplasia and even proliferative lesions of C-cells, supposedly to reduce the sustained high calcium serum concentrations. We have recently demonstrated that CT serum levels did not rise after long-term hypervitaminosis D3. Moreover, C-cells did not have a proliferative response, rather a decrease in CT-producing C-cell number was observed. In order to confirm the inhibitory effect of vitamin D3 on C-cells, Wistar rats were administered vitamin D3 chronically (25,000 IU/d) with or without calcium chloride (CaCl2). Under these long-term vitamin D3-hypercalcemic conditions, calcium, active metabolites of vitamin D3, CT and PTH serum concentrations were determined by RIA; CT and PTH mRNA levels were analysed by Northern blot and in situ hybridization; and, finally, the ultrastructure of calciotrophic hormone-producing cells was analysed by electron microscopy. Our results show, that, in rats, long term administration of vitamin D3 results in a decrease in hormone biosynthetic activities of both PTH and CT-producing cells, albeit at different magnitudes. Based upon these results, we conclude that hypervitaminosis D3-based methods do not stimulate C-cell activity and can not be used to induce proliferative lesions of calcitonin-producing cells.     

Renal handling of calcium in hypoparathyroidism.

Treatment of hypoparathyroidism usually requires the use of pharmacological doses of parent vitamin D or near physiological amounts of the hydroxylated metabolites, calcitriol or alphacalcidol. Vitamin D intoxication and hypercalcaemia may be a problem but can be minimised by the use of small doses of vitamin D or its metabolites combined with large amounts of oral calcium. The response to treatment can be easily monitored by measuring serum and urinary calcium and creatinine concentrations. This allows the derivation of two simple indices reflecting calcium load presented to the kidney (calcium excretion in mmol/l glomerular filtrate) and renal tubular calcium reabsorption (TmCa/GFR). These can be used to predict the requirement of calcium supplements and also identify those patients at particular risk of hypercalcaemia.     

Compartmentalized mast cell degranulations in the ovarian hilum, fat pad, bursa and blood vessel regions of the cyclic hamster: relationships to ovarian histamine and blood flow.

Ovaries from hamsters on each day of the oestrous cycle at 09.00 h were observed for the number of mast cells, the pattern of mast cell degranulation, histamine concentration and blood flow. On day 4 (pro-oestrus), ovaries were also observed at 9.00, 15.00 and 21.00 h. Mast cell degranulation was evaluated by 3 criteria: (1) no degranulation = less than 5 granules dispersed from the cell; (2) moderate degranulation = 5 or more granules dispersed but less than 15, and (3) extensive degranulation = 15 or more granules released. Blood flow was determined using radio-active microspheres in anaesthetized animals. Mast cells were observed in fat pad (beyond 2 mm of the bursal mesothelium), bursa (within 2 mm of the bursal mesothelium), hilum and near ovarian blood vessels (these 4 regions are collectively called the ovarian complex). The distribution of ovarian mast cells was not uniform. Most mast cells were near ovarian blood vessels (42.2%) and in the fat pad (37.2%). A moderate number of cells were in the bursal wall (20%) and only a few cells were observed in the hilum (0.64%). Mast cell number remained unchanged on days 1-4 of the cycle in each ovarian compartment. However, summation of the number of mast cells in the entire ovarian complex revealed a significant decline in number at 15.00 h on pro-oestrus. Alterations in mast cell degranulation were primarily restricted to 2 periods of the cycle (pro-oestrus and di-oestrus). An increase in moderate but not extensive degranulation was observed in only the fat pad and bursa on day 2 when compared with day 1 values. In most ovarian compartments on pro-oestrus, degranulation was higher than on any other day of the cycle. At 15.00 h on pro-oestrus, extensive degranulation in bursa, fat pad and blood vessel regions (but not hilum) coincided with an increase in ovarian histamine and decline in number of mast cells; ovarian blood flow also increased at the time but remained unchanged the remainder of the cycle.(ABSTRACT TRUNCATED AT 250 WORDS)     

Model for skeletal resistance to vitamin D in renal failure.

Chronic renal disease in man and animals is associated with disturbances in calcium homeostasis which are resistant to vitamin D-therapy. Partially nephrectomized and intact rats were used to evaluate the effect of uremia on the response of bone to vitamin D. Serum calcium, serum phosphorus and blood urea nitrogen levels were higher in uremic rats than in intact rats, both given vitamin D. Metaphyseal bone in uremic rats was resistant to vitamin D-induced bone resorption; osteoblasts and osteocytes appeared less active ultrastructurally and osteoclass were infrequent. Calcitonin synthesis and release evaluated electron microscopically was greater in uremic rats. It is suggested that the altered response of bone to vitamin D in uremic rats was due in part to elevated serum phosphorus and increased calcitonin release. The present model does not refute experimental and clinical data that metabolism of vitamin D is altered in renal disease. It does, however, emphasize that in chronic renal failure other parameters (phosphorus levels, calcitonin release, uremia) are operating which may influence end organ response to pharmacologic doses of vitamin D. The partially nephrectomized rat may be a useful model for evaluating end-organ resistance to vitamin D in uremia.     

Ingestion of calcium solutions by common marmosets (Callithrix jacchus)

Callithrix jacchus, the common marmoset, is a small New World primate that feeds on insects, fruit, and gum, and has a high reproductive rate for a primate. Insects are generally a poor source of dietary calcium, while gums may contain significant levels of calcium. Their high reproductive rate also suggests that reproductive female C. jacchus might be susceptible to calcium deficiency. Thus, we hypothesized that common marmosets might find solutions containing calcium to be palatable. We used choice experiments to examine this phenomenon in 14 male and 19 female common marmosets. Animals were presented with identical water bottles containing either deionized water or a calcium lactate solution (either 2% or 4% calcium lactate by weight) and intake was measured. Serum 25-OH-vitamin D was monitored in a subset of the animals in the experiment to examine the role of vitamin D in this behavior. Our results demonstrated that C. jacchus can distinguish between plain water and a calcium lactate solution, and that in general they prefer calcium solutions. Although animals with marginal vitamin D status did have strong preferences for calcium solutions, they did not ingest significantly more of the calcium solutions than animals that were vitamin D-replete. Nulliparous females and males did not differ in the amounts of calcium solutions ingested. Lactating females ingested the greatest volume of the calcium solutions.     

1,25-Dihydroxycholecalciferol effects on plasma calcitonin levels and calcitonin mRNA in normal or partially vitamin D-depleted rats

The physiological effect of 1,25-(OH)2D3 on the regulation of calcitonin (CT) secretion was studied by measuring plasma CT levels and CT mRNAs extracted from thyroid glands of normal (D+) or partially vitamin D-depleted rats (D-). In both groups, acute 1,25-(OH)2D3 administration of 0.1 microgram/kg b.w. yielded an early drop in plasma calcium concentrations (around 0.6-1 mg/dl) with a maximum decrease 15 min after treatment. In spite of this hypocalcemia, a significant rise in plasma CT levels was observed within 5 min in D+ animals and within 30 min in D- animals after injection of the vitamin D metabolite. Nevertheless, the increased CT secretion was not associated with a marked and sustained rise in CT mRNA levels measured by dot-blot hybridization or CT mRNA activity evaluated by translation assay. By contrast to the observations made previously using supra-physiological doses of the vitamin D metabolites, no clear-cut effect on CT mRNA levels was found with lower doses. If we hypothesized that 1,25-(OH)2D3 plays a physiological role in CT secretion, our results suggest that this rapid control could be exerted at a post-translational level may be via an increase in the cytoplasmic ionized calcium concentration of C-cells.     

Organelle specific enzyme markers as indicators of methylmercury neurotoxicity and antidotal efficacy in mice

The efficacy of two monothiols, N-acetyl-DL-homocysteine thiolactone (NAHT) and glutathione (GSH) either alone or in combination with two vitamins, vitamin B complex and vitamin E were studied in 7 days methylmercury chloride (MMC; 1 mg kg) intoxicated male Swiss albino mice. Thirteen groups of animals, each containing 6 animals were used for the study. Three groups of animals were kept as control (treated either with vehicle, normal saline or olive oil). Rest of the ten groups were kept as treatment groups. All the animals were treated subcutaneously for 7 days with MMC and one group was sacrificed on the 8th day. The second group was kept without toxicant for another 7 days and were sacrificed on the 15th day. Two MMC pretoxicated groups were treated either with vitamin B complex (20 mg kg) or vitamin E (60 mg kg) and two other groups were treated with N-acetyl-DL-homocysteine thiolactone (40 mg kg) or glutathione (50 mg kg) for another 7 days. The rest of the four groups were treated with either N-acetyl-DL-homocysteine thiolactone or glutathione in combination with either vitamin B complex or vitamin E. All the animals were sacrificed on the 15th day, brain and spinal cord were dissected and estimated for acid phosphatase, alkaline phosphatase, succinic dehydrogenase and mannosidases. Some of the antidotes showed significant recovery of the enzymes in one tissue while some showed significant recovery in the other tissue depicting the need for treating methylmercury poisoned animals with multi-chelation therapy rather than as a monotherapy.     

Effect of vitamin E on follicular cell proliferation and expression of apoptosis-associated factors in rats with 6-N-propyl-2-thiouracil-induced goitrogenesis

We have investigated immunohistochemically the effect of dl-alpha-tocopherol (vitamin E) on thyroid gland with 6-n-propyl-2-thiouracil (PTU)-induced hypothyroidism in rats. The animals were divided into four groups. Rats in group I were designated as control, rats in group II were treated with injections of PTU (10 mg/kg) for 15 days, rats in group III were treated with injections of PTU+vitamin E (10 mg/100 g) for 15 days. Rats in group IV were treated with injections PTU for 15 days and kept for 15 next days after cessation of PTU treatment. At the end of experiment, the animals were killed by decapitation, blood samples were obtained, thyroid tissues were collected and processed for quantitative evaluation of immunohistochemical PCNA (marker of cell proliferation), Bax (pro-apoptotic marker) and Bcl-2 (anti-apoptotic marker) staining. There was an increase in the number of PCNA-immunopositive cells in follicular epithelial cells of group II rats compared with other groups (p<0.05). After vitamin E treatment, the number of PCNA-immunopositive cells decreased (p<0.05) while the number of Bax-immunopositive cells increased (p<0.05). The number of Bcl-2-positive follicular epithelial cells of group IV rats was higher than in those of other groups (p<0.05). The results of this study indicate that hypothyroidism induces cell proliferation in the thyroid gland and vitamin E may promote involution of the gland.     

Studies on the effect of vitamin D on calcium absorption and transport

1. Mucosal cells of the small intestine obtained from rats deprived of vitamin D or given excessive amounts of the vitamin accumulated significantly more calcium than did cells from control animals. 2. Mucosal cells from vitamin D-deficient rats released less calcium than did cells from normal or hypervitaminotic D animals. 3. Studies in vivo showed that the transfer of (45)Ca from the intestine to the blood was delayed in vitamin D deficiency, but was accelerated in hypervitaminosis D. 4. The findings support the thesis that vitamin D is involved in the release of calcium rather than in its uptake by mucosal cells. 5. Further evidence is presented suggesting that uptake of calcium by intestinal mucosal cells at 0 degrees is primarily passive, whereas at 38 degrees uptake and release are effected by an active process that depends on energy derived from glycolytic activity.     

The morphology and function of rat C-cells. 4. Determination of calcium, inorganic phosphorus and total nitrogen in the femora of untreated parathyroidectomized or thyreoparathyroidectomized and hormonally substituted female rats]

Female Wistar rats of a live weight of about 160 g and fed with a standard laboratory diet, were parathyroidectomized, or thyroparathyroidectomized and treated with thyroxine, parathyroid hormone, calcitonin. thyroxine and parathyroid hormone, or thyroxine and calcitonin. On the 15th day post operationem, and after twelve days of hormone treatment, the concentrations of calcium, inorganic phosphorus and total nitrogen were determined in the femur bone. Parathyroidectomy resulted in a decrease of phosphorus concentration in bone. After thyroparathyroidectomy (Tx), the concentrations of inorganic phosporus and nitrogen diminished during some days, whereas the calcium content decreased continuously. Thyroxine application normalized the concentration of inorganic phosphorus. The osteolytic and nitrogen-anabolic effect of parathyroid hormone took place only in simultaneous treatment with thyroxine. The injection of calcitonin had a nitrogen-anabolic effect on bone; the simultaneous treatment with thyroxine induced a loss of calcium out of bone, and a deposition of calcium phosphate in renal tissue. Calcitonin did not inhibit a significant decrease of calcium concentration in the femur bone; the hypophosphatemic effect was always present. The metabolism of bone tissue, influenced by hormonal actions, probably determined the localization of the deposition of inorganic phosphorus, deserting the serum under the influence of calcitonin.