A role for intracellular calcium and calmodulin in the release of triiodothyronine from human thyroid-cell monolayer cultures

Human thyroid cells in monolayer responded to acute stimulation by TSH with an increase in the secretion of T₃. This process appeared to be dependent on a rise in the cytosolic calcium concentration since the antagonist of intraceliular calcium mobilization, TMB-8, was found to inhibit the release of T₃ in response to TSH. The importance of intracellular calcium was further shown using the agent veratridine which increases the free calcium level within cells; veratridine potentiated the stimulation of T₃ secretion by TSH and itself stimulated the release of T₃ to a level higher than that seen in the presence of TSH alone. The calcium ionophore A23197 produced a biphasic effect on T₃ secretion from human thyroid monolayers; at low concentrations, A23187 caused a decrease in both unstimulated and TSH-stimulated T₃ secretion but above a concentration of 1 M, T₃ secretion was increased. The calmodulin antagonist W7 was found to inhibit T₃ release in response to TSH, indicating a role for calmodulin in mediating the effects of intracellular calcium on T₃ secretion.     

Calmodulin-binding proteins of bovine thyroid plasma membranes

Calmodulin binding proteins in bovine thyroid plasma membranes were investigated using the ¹²⁵I-labeled calmodulin gel overlay technique. The purified thyroid plasma membranes contained two calmodulin binding proteins with molecular weights of approx. 220 000 and 150 000 respectively. The binding of ¹²⁵I-labeled calmodulin to the calmodulin binding proteins was inhibited by excess unlabeled calmodulin, 100 μM trifluoperazine or 1 mM EGTA, indicating that the binding was calmodulin-specific and calcium-dependent. The calmodulin binding proteins appear to be components of the cytoskeleton since they remained in the pellet after treatment of the thyroid plasma membranes with 1% Triton X-100. Similar calmodulin binding proteins were present in rat liver plasma membranes, but not in human red blood cell plasma membranes. These two calmodulin binding proteins may interact with other components of the cytoskeleton and regulate endocytosis, exocytosis and hormone secretion in thyroid cells.     

Stimulation of calcitonin secretion in the pig by calcitonin gene-related peptide

Pig thyroid glands were surgically isolated in situ and perfused with autologous blood to which was added known concentrations of calcitonin gene-related peptide (αCGRP). When thyroids were perfused with measured concentrations of CGRP within the range of 0.6–600 nM, the secretion rate of calcitonin (CT) was stimulated while the release of T₃, T₄, and somatostatin remained unchanged. Specific binding of ¹²⁵I-CGRP to pig thyroid plasma membranes was demonstrated, and binding was inhibited by unlabelled CGRP but not by CT or by other peptides unrelated structurally to CGRP. The findings indicate that the pig thyroid gland contains plasma membrane binding sites for CGRP and that CGRP is capable of stimulating the secretion of CT.     

Studies on the involvement of calcium and calmodulin in the action of growth-hormone-releasing factor

A possible role for Ca 2⁺ and calmodulin in the action of growth-hormone-releasing factor (GHRF) was investigated . Low extracellular Ca²⁺ (<100 M), methoxyverapamil, flunarizine, cinnarizine, and Co²⁺ decreased both basal and GHRF-stimulated growth-hormone secretion, but did not totally inhibit GHRF-stimulation secretion. A calmodulin antagonist, W7, abolished GHRF-stimulated GH secretion, with no effect on basal secretion. It is suggested that GHRF may act primarily by elevating cellular cyclic AMP, which may then modulate calcium mobilization or flux; the increased intracellular Ca²⁺ concentrations may then activate calmodulin.     

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.     

Calmodulin modulates prolactin secretion in vitro: studies with calmodulin containing liposomes

The control of prolactin secretion by Ca calmodulin and cyclic AMP was studied. Ca++ ionophore A23187 stimulated both cyclic AMP accumulation and prolactin release by primary culture of anterior pituitary cells in vitro. The increase of cyclic AMP formation by A23187 preceded that of prolactin release. To test the calmodulin involvement in these processes we used either selective calmodulin antagonist, the naphthalene sulphonamide derivative W7, or calmodulin containing liposomes. W7 dose dependently inhibited both basal or A23187 stimulated cyclic AMP accumulation and prolactin secretion. Insertion of Ca calmodulin within the cells stimulated prolactin secretion without modifying cyclic AMP accumulation. W7 inhibited the Ca calmodulin containing liposomes stimulation of prolactin release. These results suggest that calmodulin participates to the process of prolactin release.     

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.     

Effect of Extracellular Calmodulin on the Cytosolic Ca2+ Concentration in Lily Pollen Grains

Using Lilium davidii Duchartre pollen as material, the calcium ion-fluorescence indicator fluo-3AM was loaded successfully into the pollen grains by low temperature loading method. Laser confocal scanning microscopy was used to study the effect of extracellular calmodulin on intracellular calcium. It is found that the purified exogenous calmodulin could elevate the intracellular calcium ion concentration, and the effect was correlated with the concentration of exogenous calmodulin to a certain extent. Cell membrane nonpermeable inhibitor of calmodulin, W 7-agarose, and the anti-serum of calmodulin could decrease the cytosolic calcium level. The results show that the endogenous extracellular calmodulin may play an important role in maintaining and increasing the cytosolic calcium level in pollen grain cell.     

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.     

Plasma concentrations of immunoreactive calcitonin in rats during lactation

A mild hypocalcemia (0.5 mg/dl) is observed in rats after 14 days of lactation, but plasma and thyroidal calcitonin (CT) levels are both increased on day 7 of lactation. Plasma CT levels are higher (x2) in lactating females than those found in virgin females from day 7 to the end of lactation (21 days). In vitro, the CT secretion rate after calcium stimulation(3 mM) is not different between lactating and virgin females. The rapid removal of pups after parturition in control females induces a rebound in plasma calcium (+1.4 mg/dl) and plasma phosphate (+1.6 mg/dl) associated with elevated plasma CT values. Our results suggest, that the transient mild hypocalcemia of lactation is preceded by increased plasma CT levels; and that it is not the cause of elevated plasma parathyroid hormone levels already reported by us.     

Phase-shifts of the Bulla ocular circadian pacemaker in the presence of calmodulin antagonists

Previous work has shown that light-induced phase shifts of the Bulla ocular circadian pacemaker require extracellular calcium, suggesting the possibility that the action of calcium as a second messenger via calmodulin is an element in the phase shifting mechanism. The calmodulin antagonists calmidazolium, trifluoperazine (TFP) and W7 were applied with phase shifting light pulses. Light phase shifts were not blocked by calmidazolium or TFP, suggesting that calmodulin does not mediate light-induced phase shifts. Period changes were observed with treatments of both TFP and W7, but not with calmidazolium and are probably not calmodulin-mediated.     

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.     

Effect of plasma calcium concentration on the metabolic clearance rate of calcitonin in the dog

Plasma immunoreactive calcitonin (iCT) levels generally are not elevated in chronic, nonmalignant, hypercalcemic states. Normocalcitoninemia might occur despite increased CT secretion if hypercalcemia itself accelerated the clearance of CT from the circulation. Therefore, we have measured the metabolic clearance rate (MCR) of human CT (hCT), infused to constant plasma levels in thyroparathyroidectomized dogs, under conditions of acute and chronic hypo- and hypercalcemia. Acute increases of plasma calcium were without significant effect on the MCR of hCT, but chronic hypercalcemia, induced by dihydrotachysterol treatment, reduced the MCR of hCT by 30% (P less than 0.05) and glomerular filtration rate (GFR) by 40% (P less than 0.02). There was a significant, positive correlation (r = 0.60, P less than 0.02) between GFR and the MCR of CT. The data suggest that failure to detect elevated iCT levels in chronic hypercalcemia is not the result of an increased MCR of CT. In fact, hypercalcemia should exaggerate the effect of increased CT secretion by decreasing the renal clearance of the hormone.     

Calcitonin action on ATP citrate lyase activity in the hepatic cytosol of fed rats and its calcium-calmodulin dependency

The effect of calcitonin (CT) on ATP citrate lyase activity in the hepatic cytosol was investigated after a single subcutaneous administration of the hormone to fed rats. Administration of CT (synthetic [Asu107] eel CT; 80 MRC mU/100 g body weight) produced significant increases in ATP citrate lyase 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 increased cytosolic ATP citrate lyase activity resulting from CT administration was prevented by treatment with 10 microM EGTA. This enzyme activity was restored by addition of calcium ion (2.5-10 microM). The rise in enzyme activity of CT-treated rats was markedly reduced by the presence of W-7 (10 and 100 microM), a calmodulin inhibitor, in the enzyme assay system, while that of control rats was not significantly altered by the drug. These results suggest that CT increases ATP citrate lyase 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.     

Differential regulation of smooth muscle contraction in rabbit internal anal sphincter by substance P and bombesin.

Substance P and bombesin induce contraction of isolated IAS smooth muscle cells by different intracellular mechanisms. The cells contracted in a dose dependent manner to both peptides. The kinetics of contraction were different. Substance P induced contraction peaked at 30 seconds and declined in a time dependent manner while bombesin induced contraction peaked at 30 seconds and was maintained for up to 8 minutes. The absence of extracellular calcium in the medium (0 calcium and 2 mM EGTA) had no affect on substance P induced contraction while it blocked bombesin induced contraction. Substance P induced contraction was blocked by the calmodulin antagonist W7 (10(-9)M) and was not affected by the PKC antagonist H7 (10(-6)M). Bombesin induced contraction was blocked by the PKC antagonist H7 and was not affected by the calmodulin antagonist W7. Our data indicate that substance P induces a transient contraction utilizing intracellular calcium and a calmodulin dependent pathway, while bombesin induces a sustained contraction utilizing calcium from extracellular sources and a calmodulin independent pathway.     

Comparison of the properties of active Ca2+ transport by the islet-cell endoplasmic reticulum and plasma membrane

The properties of active or ATP-dependent calcium transport by islet-cell endoplasmic reticulum and plasma membrane-enriched subcellular fractions were directly compared. These studies indicate that the active calcium transport systems of the two membranes are fundamentally distinct. In contrast to calcium uptake by the endoplasmic reticulum-enriched fraction, calcium uptake by islet-cell plasma membrane-enriched vesicles exhibited a different pH optimum, was not sustained by oxalate, and showed an approximate 30-fold greater affinity for ionized calcium. A similar difference in affinity for calcium was exhibited by the Ca²⁺-stimulated ATPase activities which are associated with these islet-cell subcellular fractions. Consistent with the effects of calmodulin on calcium transport, calmodulin stimulated Ca²⁺-ATPase in the plasma membranes, but did not increase calcium-stimulated ATPase activity in the endoplasmic reticulum membranes. The physiological significance of the differences observed in calcium transport by the endoplasmic reticulum and plasma membrane fractions relative to the regulation of insulin secretion by the islets of Langerhans is discussed.     

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.     

Calcium and calmodulin regulate atrial natriuretic factor stimulation of cyclic GMP in a human renal cell line.

We examined calcium and calmodulin regulation of atrial natriuretic factor stimulation of particulate-membrane guanylate cyclase (ANF-s-GC) in SK-NEP-1 cells. W7 and trifluoropiperazine, but not W5, inhibited whole cellular ANF-stimulated cyclic GMP accumulation (ANF-s-cGMP). EGTA and LaCl3 decreased ANF-s-GC and calmodulin reversed this inhibition. A23187-induced inhibition of ANF-s-cGMP was only partly reversible by IBMX. H7 or staurosporine counteracted the inhibitory effect of A23187. Calcium inhibited basal and ANF-s-GC. These data suggest that at low concentrations of calcium, ANF-s-GC was calcium-calmodulin dependent but high concentrations of calcium inhibited ANF-s-GC through phosphodiesterase, through inhibition of GC, and probably through protein kinase C.