Increased level of calcitonin mRNA after 1,25-dihydroxyvitamin D3 injection in the rat

Vitamin D metabolites are able to change plasma calcitonin (CT) levels, but nothing is known about a possible effect at the CT gene level. Here we have investigated the acute effects of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) on the CT biosynthetic activity of thyroid glands from adult rats. Plasma CT levels were significantly increased (X2) 1 and 2 h after 1,25-(OH)2D3 injection in the face of unchanged plasma calcium values. The thyroidal CT content also was unchanged. A 2-fold increase in CT mRNA level measured by dot-blot hybridization occurred 1 and 2 h after 1,25-(OH)2D3 administration. Expression of CT gene products was examined in the rabbit reticulocyte lysate cell-free translation assay. After polyacrylamide gel electrophoresis, specific immunoprecipitates were autoradiographed and quantified by integration. A single precursor of Mr approximately equal to 15 000 could be specifically immunoprecipitated with CT antisera. A 3-4-fold rise in translatable CT mRNA activity was observed 1 and 2 h after 1,25-(OH)2D3 injection. Thus, parallel changes in CT mRNA level, CT mRNA activity and plasma CT levels were observed in adult female rats after administration of 1,25-(OH)2D3. These findings demonstrate for the first time that 1,25-(OH)2D3 enhanced CT gene expression in the face of unchanged plasma calcium levels.     

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.     

Expression of calcium-binding protein regucalcin mRNA in rat liver is stimulated by calcitonin: The hormonal effect is mediated through calcium

The involvement of a hypocalcemic hormone calcitonin (CT) in the expression of hepatic Ca²⁺-binding protein regucalcin mRNA was investigated. The change of regucalcin mRNA levels was analyzed by Northern blotting using liver regucalcin complementary DNA (0.9 kb). A single oral administration of calcium chloride (100 mg Ca/100 g body weight) to rats induced a remarkable increase in the serum calcium concentration and a corresponding elevation of the liver calcium content during 120 min after the administration. Thyroparathyroidectomy (TPTX) did not cause a significant increase in the liver calcium content after calcium administration. Hepatic regucalcin mRNA level was markedly elevated by calcium administration; the level was about 180% of controls at 60 min after the administration. This increase was completely abolished by TPTX. A single subcutaneous administration of CT (synthetic eel CT; 25–100 MRC mU/100 g) to TPTX rats received oral administration of calcium (100 mg/100 g) produced a remarkable increase in hepatic regucalcin mRNA levels; the level was about 280% of controls with the dose of 25 MRC mU CT/100 g. The present finding suggests that the expression of hepatic mRNA is stimulated by CT, and that the hormonal effect is mediated through Ca²⁺ in rat liver.     

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.     

Rapid changes in the concentration of phosphoenolpyruvate carboxykinase mRNA in rat liver and kidney. Effects of insulin and cyclic AMP

Starvation and diabetes both caused a marked increase in the concentration of hepatic phosphoenolpyruvate caroboxykinase mRNA while the administration of insulin to diabetic rats or refeeding glucose to starved animals caused a marked reduction in the levels of enzyme mRNA as measured by hybridization using a cDNA probe.l The Administration of dibutyryl cAMP to a starved-refed cat caused an 8-fold induction of phosphoenolpyruvate carboxykinase mRNA in 1 h. Triamcinolone plus acidosis induced the levels of enzyme mRNA in kidney 3-fold within 6 h, however, starvation for 24h had only marginal effects. In all of the above conditions, the levels of phosphoenolpyruvate carboxykinase mRNA measured by hybridization assay agreed well with the relative levels of translatable mRNA for the enzyme. The half-time of phosphoenolpyruvate carboxykinase mRNA, determined after the administration of either alpha-amanitin or cordycepin to starved animals, was approximately 40 min. However, cycloheximide either alone or together with cordycepin, not only prevented the decrease in phosphoenolpyruvate carboxykinase mRNA sequence abundance, but induced it 2-fold. Cycloheximide itself, when injected into 21-day fetal rats in utero caused an induction of enzyme mRNA equal to that noted when dibutyryl cAMP was administered. The mRNA for phosphoenolpyruvate carboxykinase is approximately 2.8 kb in length, but nuclei from the livers of diabetic rats contain a number of putative precursor RNA species for the enzyme, up to 6.5 kb in size, all containing a poly(A) tail. Two hours after refeedng glucose to a starved rat, these nuclear RNA species could no longer be detected by hybridization to our cDNA probe.     

Calcitonin mRNA activity in young obese (fa/fa) Zucker rats

Alterations in both calcitonin (CT) secretion and plasma calcium were recently described in adult obese Zucker rats. We have investigated the CT biosynthetic activity of thyroid glands in 30-day-old obese Zucker rats (fa/fa), and their controls (Lean). Plasma calcium level was significantly increased (+0.6 mg/dl) in obese animals, but plasma phosphate was unchanged. Plasma CT levels measured by radioimmunoassay (RIA) were significantly decreased in fatty (0.50 +/- 0.03 vs 0.68 +/- 0.03 ng/ml in Leans; P less than 0.001), but thyroidal hormone content was not different between Lean and fatty rats (68.7 +/- 5.1 in Leans vs 60.5 +/- 3.6 ng/gland in fatty rats). mRNA was extracted from 10 thyroids, and translated in a rabbit reticulocyte lysate (NEN) in the presence of [35S]methionine. After polyacrylamide gel electrophoresis, specific immunoprecipitates were autoradiographed and quantified by integration. A 50% decrease in translatable CT mRNA was observed in fatty rats. In basal conditions, the biosynthetic activity of C cells in obese rats correlates with the secretion rate of the hormone in the face of unchanged thyroidal CT contents.     

Expression of the calcitonin gene during migration of the Pacific salmon, Oncorhynchus gorbuscha

Plasma calcitonin levels increase during reproduction in female salmon. Whether these changes in circulating levels of the hormone are due to increased secretion and or increased biosynthesis is not established. We measured total and poly A+ RNAs and calcitonin content of ultimobranchial bodies of male and female pink salmon during the different stages of the reproductive cycle. Calcitonin mRNAs were analysed by hybridization of northern and dot blots with a specific probe for salmon calcitonin. Dot blot autoradiography indicated that female animals had higher levels of CT mRNA than males and these values were correlated with plasma calcitonin levels. In conclusion, sexual hormones still to be identified, are probably implicated in the expression of the calcitonin gene in females during the reproductive cycle.     

Elevated levels of calcitonin mRNA: a marker for the spontaneous development of medullary thyroid carcinoma in rats

The aging WAG/Rij rats (a Wistar derived strain) develop spontaneously medullary thyroid carcinoma with a high frequency (50%). We have studied calcitonin biosynthesis in Wistar and WAG/Rij rats strains in order to determine if early changes in this parameter occurred in the WAG/Rij strain. Thyroidal and plasma CT levels were measured in three months old WAG/Rij and Wistar rats before and after acute calcium challenge. Total RNA was extracted from thyroid glands and specific CT messenger RNA levels estimated by dot and Northern blot analysis with a 32P-labeled probe specific for CT mRNA. The capacity of mRNA to direct synthesis of CT precursor was also measured by translation in an in vitro system. Though mean basal circulating CT levels were equivalent in both strains, CT release after calcium stimulation was much increased in the WAG/Rij rat. CT content of the glands and CT mRNA levels were two fold higher in the WAG/Rij strain. Thus, in this strain, CT biosynthesis and secretion were increased long before the development of a C-cell carcinoma.     

Calcitonin secretion during postnatal development in the rat: beta-adrenergic agents and vitamin D3 metabolites

The possible contribution of catecholamines and vitamin D3 metabolites to the high plasma calcitonin (CT) levels in suckling baby rats is unknown. So, in vivo and in vitro (using a perifusion system) effects of beta-adrenergic agents and vitamin D3 metabolites on CT release were studied in the rat during the postnatal development. In 13-day-old rats, the increase in plasma CT levels induced by isoproterenol injection (0.1 micrograms/kg b.w.) was inhibited by a previous administration of propranolol. A significant decrease in plasma CT levels was observed after propranolol injection in baby rats (0.68 +/- 0.05 ng/ml vs. 0.93 +/- 0.01 ng/ml). A daily injection of 1,25-dihydroxycholecalciferol (1,25-(OH)2D3; 25 pmoles/rat/day during 4 days) induced a marked rise in plasma calcium (16.1 +/- 0.2 mg/dl), and a great decrease in thyroidal CT contents (approximately 70% of control values) in 13-day-old rats while no change was noted with 24,25-dihydroxycholecalciferol (24,25-(OH)2D3). A negative correlation between plasma calcium and thyroidal CT stores was found in suckling and in weaning rats treated with different doses of 1,25-(OH)2D3, suggesting an indirect effect of 1,25-(OH)2D3 on CT secretion. The mobilization of the thyroidal CT content was greater in weaning than in suckling rats in response to a given hypercalcemia. In vitro, 5 X 10(-5) M isoproterenol induced a rapid increase in CT secretion rate while 1,25-(OH)2D3 inhibited the rise in CT release induced by 3.0 mM calcium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of calcium-binding protein regucalcin mRNA in the kidney cortex of rats: The stimulation by calcium administration

The expression of calcium-binding protein regucalcin mRNA in the kidney cortex of rats was investigated. The change of regucalcin mRNA levels was analyzed by Northern blotting using liver regucalcin complementary DNA (0.9 kb of open-reading frame). Regucalcin mRNA was expressed in the kidney cortex, and this expression was clearly increased by a single intraperitoneal administration of calcium chloride solution (5–15 mg Ca/100 g body weight) in rats; this increase was remarkable at 60–120 min after the administration. Thyroparathyroidectomy (TPTX) caused a slight decrease of regucalcin mRNA levels in the kidney cortex. However, the administration of calcium (10 mg/100 g) in TPTX rats produced a clear increase of regucalcin mRNA levels in the kidney cortex. The subcutaneous administration of calcitonin (10–100 MRC mU/100 g) or parathyroid hormone [1–34] (1–10 U/100 g) in TPTX rats which received calcium (10 mg/100 g) administration did not cause an appreciable alteration of regucalcin mRNA levels in the kidney cortex, suggesting that the mRNA expression is not stimulated by calcium-regulating hormones. The administration of trifluoperazine (TFP; 5 mg/100 g), an inhibitor of Ca²⁺/calmodulin action, completely blocked the expression of regucalcin mRNA stimulated by calcium administration. Now, calcium content in the kidney cortex was significantly elevated by a single intraperitpneal administration of calcium (10 mg/100 g) in rats. The present study clearly demonstrates that the expression of regucalcin mRNA in the kidney cortex is stimulated by calcium administration in rats. This expression may be mediated through Ca²⁺/calmodulin action in the kidney cortex.     

Effect of calcitonin on fructose 1,6-diphosphatase activity in rat liver: role of cytosolic calcium concentration

The effect of calcitonin (CT) on fructose 1,6-diphosphatase activity in the hepatic cytosol was investigated after a single subcutaneous administration of the hormone to rats. Administration of CT (synthetic [Asu1,7] eel CT; 80 MRC mU/100 g body weight) produced significant increase in fructose 1,6-diphosphatase activity and calcium content in the hepatic cytosol of intact and thyroparathyroidectomized rats. Those alterations were also observed with the dose of CT at physiological level. The binding of calcium by 10 microM EGTA in the hepatic cytosol caused a clear reduction of the increase in fructose 1,6-diphosphatase activity produced by CT administration. The enzyme activity of CT-treated rats was markedly reduced by W-7 (100 microM), calmodulin inhibitor, while that of control rats was not significantly altered by the drug. Meanwhile, fructose 1,6-diphosphatase activity in the hepatic cytosol obtained from normal rats was significantly enhanced by addition of calcium ion (0.1-5.0 microM). This increase was also clearly inhibited by W-7. These results suggest that CT increases fructose 1,6-diphosphatase activity in the hepatic cytosol of rats, and that this hormonal regulation may depend on calmodulin, mediated through calcium increased in the cytosol.     

Calcitonin increases pyruvate carboxylase activity in hepatic mitochondria of rats

The effect of calcitonin (CT) on calcium content and enzyme activity in the hepatic mitochondria of intact rats was investigated. A single subcutaneous administration of CT (80 MRC mU/100 g BW) produced a significant increase in the content of calcium, the activity of pyruvate carboxylase, succinate dehydrogenase and ATPase 15 min after the hormone treatment. The significant increases in calcium content and pyruvate carboxylase activity were also observed 30 min after CT administration, while succinate dehydrogenase and ATPase activity began to decrease. A physiological dose of CT (20 MRC mU/100 g BW) caused a marked increase in calcium content and pyruvate carboxylase activity but not succinate dehydrogenase of ATPase-activity. The removal of calcium by 10 mM EGTA washing of the mitochondria produced a remarkable reduction in pyruvate carboxylase activity increased by CT administration. The addition of calcium ion of 2.5 x 10(-2) - 2.5 x 10(1) nmoles Ca2+ per mg mitochondrial protein produced a marked increase in pyruvate carboxylase activity. The present results suggest that calcium taken up by the hepatic mitochondria after CT administration activates pyruvate carboxylase.     

Plasma immunoreactive calcitonin levels in pregnant mares and newborn foals.

Plasma calcium and calcitonin levels were measured periodically during the two last months of pregnancy and at the time of parturition in 9 pregnant mares and their foals. In pregnant animals, there was an increase in plasma calcitonin levels in the days before parturition, which was not due to any change in plasma calcium. This result indicates that in the mare, as in the cow, in the days before parturition CT secretion escapes from its control by plasma calcium. In 0-day and 7-day-old foals plasma calcium levels were significantly higher than in their mothers, but plasma calcitonin levels were not significantly different from those observed in their dams at the time of parturition.     

Effect of calcitonin on Ca-ATPase activity of plasma membrane in liver of rats.

The effect of calcitonin (CT) on Ca-ATPase activity in the plasma membrane fraction of rat liver was investigated. CT (80 MRC mU/100 g BW) administered subcutaneously to rats, caused a significant decrease in serum calcium, while increasing liver calcium. The administration of CT produced a rapid decrease of Ca-ATPase activity in the plasma membrane fraction of liver, whereas CT did not cause a significant alteration of p-nitrophenyl phosphatase activity. The maximal response of CT was obtained with 80 MRC mU/100 g BW. Meanwhile, the administration of imidazole (30 mg/100 g BW) which has a hypocalcemic effect, like CT, produced a significant increase in liver calcium and a corresponding fall in Ca-ATPase activity of the plasma membrane fraction. The reduction of Ca-ATPase activity produced by imidazole was significantly potentiated by the simultaneous administration of CT, and the rise in liver calcium was enhanced slightly. The present results suggest that the action of CT on liver calcium involves the decrease of Ca-ATPase activity in the plasma membrane of rat liver.     

Comparison of calcitonin, epinephrine and glucagon effects on plasma membrane Ca-ATPase activity and calcium content in liver of rats

The effects of calcitonin (CT), epinephrine and glucagon on the plasma membrane Ca-ATPase activity and the calcium content in the liver were investigated 30 min after a single subcutaneous administration of hormones to rats. Ca-ATPase activity in the plasma membrane fraction was significantly decreased by CT (80 MRC mU/100 g BW), while it was not significantly lowered by insulin (100 mU/100 g BW), epinephrine (100 micrograms/100 g BW), glucagon (50 micrograms/100 g BW), or parathyroid hormone (25 U/100 g BW). The calcium content in the liver was markedly increased by CT, while it was not significantly elevated by epinephrine or glucagon. Meanwhile, the decrease of Ca-ATPase activity in the plasma membrane fraction produced by CT was significantly prevented by simultaneous administration of epinephrine or glucagon, and also the increase in liver calcium was noticeably interfered with. The present results suggests that the action of CT on liver calcium may differ from that of epinephrine or glucagon which causes an increase in cyclic AMP in the liver cells.     

Calcitonin increases fatty acid synthetase activity dependently of calcium-calmodulin in the hepatic cytosol of fed rats

The effect of calcitonin (CT) on fatty acid synthetase activity in the hepatic cytosol was investigated after a single subcutaneous administration of the hormone to fed rats. Administration of CT (synthetic [Asu1,7] eel CT; 80 MRC mU/100 g body weight) produced significant increases in fatty acid synthetase activity and calcium concentration in the hepatic cytosol of intact and thyroparathyroidectomized rats. The hormonal effect on the enzyme activity was not observed in rats fasted for 24 h. The increase in fatty acid synthetase activity by CT administration was completely inhibited by treatment with 10 microM EGTA. This enzyme activity was restored by addition of calcium ion (2.5-10 microM). The increased enzyme activity of CT-treated rats was markedly reduced by addition of W-7 (15 microM), a calmodulin inhibitor, in the enzyme assay system. Moreover, the cytosolic enzyme activity of normal rat liver was markedly raised by in vitro addition of both calcium ion (5 microM) and calmodulin (2.5 micrograms). These results suggest that CT increases fatty acid synthetase activity in the hepatic cytosol of fed rats, and that this hormonal regulation may depend on calmodulin, and be mediated through raised calcium in the cytosol.     

Reactivity of propylene oxides towards deoxycytidine and identification of reaction products

In order to elucidate the mechanism of the stimulative effect of molybdenum on mercury-mediated renal metallothionein induction, the levels of translatable metallothionein mRNA (MT mRNA) in the kidneys of rats treated with saline or Na2MoO4 or HgCl2 or Na2MoO4 and HgCl2 were measured by translation experiments in cell-free protein synthesizing systems. The time course of accumulation of mercury in renal nuclei of rats given HgCl2 with or without Na2MoO4-pretreatment was also investigated. Molybdenum, itself, did not elevate levels of MT mRNA compared to saline controls at all time points (0, 6 and 14 h after exposure to HgCl2) but rapidly elevated the levels of the mRNA more than Hg-dosed rats when HgCl2 was also administered. On the other hand, the time course study in renal nuclei showed that the mercury content of nuclei was consistently lower in Mo-Hg-dosed rats than in Hg-dosed rats at all time points (4, 8 and 24 h after exposure to HgCl2). These results suggest that the stimulative effect of molybdenum on mercury-mediated metallothionein induction is coupled with an increase of the mRNA coding for the low molecular weight protein and that such an increase in the levels of translatable MT mRNA is not due to the difference in uptake of mercury into renal nuclei.     

Effect of parathyroid hormone on the increase in serum glucose and insulin levels after a glucose load to thyroparathyroidectomized rats.

Thyroparathyroidectomy (TPTX) caused a significant increase in serum glucose and a corresponding fall in serum calcium in both fed and fasted rats. The increase in serum glucose, induced by TPTX, was markedly potentiated by a single intraperitoneal administration of calcium (2 mg/100 g BW) which caused a significant elevation of serum calcium in thyroparathyroidectomized rats. Parathyroid hormone (PTH; 20 U/100 g BW) administered subcutaneously to thyroparathyroidectomized rats, caused a significant decrease in serum glucose (0.1 g/100 g BW) to sham-operated rats significantly increased both serum glucose and insulin. The rise of serum glucose produced by a glucose load was markedly potentiated by TPTX, but the increase in serum insulin was not promoted significantly. The administration of PTH decreased both serum glucose and insulin levels increased by a glucose load to thyroparathyroidectomized rats, in a dose-dependent manner. The administration of calcitonin (80 MRC mU/100 g BW) significantly prevented the effect of PTH to decrease serum glucose after a glucose load to thyroparathyroidectomized rats, and calcitonin increased serum insulin. These results suggest that the effect of PTH on serum glucose does not involve insulin secretion.