Effects Of Calcium Deprivation Research Articles

Role of polyamines in regulating silymarin production in Silybum marianum (L.) Gaertn (Asteraceae) cell cultures under conditions of calcium deficiency

As part of our efforts to identify the possible role of polyamines (PAs) in silymarin (Sm) production, the effects of calcium deprivation on cell growth and on endogenous PAs levels and Sm production by milk thistle (Silybum marianum (L.) Gaertn) grown in cell cultures were examined. Young cultured cells of the H2 line of S. marianum were transferred to a medium without calcium and with ethylene glycol-bis-(β-aminoethyl) ether-N,N,N′,N′-tetraacetic acid present to chelate any free calcium in order to analyze the effects of this medium on the levels of PAs and Sm produced by the cells. During the 17 days of exposure to this calcium-free medium most of the cell populations were in the G0/G1 phase (from day 7 to day 14 of culture) while PA levels underwent a progressive decline up to day 17, after which they were no longer detectable. We observed that putrescine (Put) accumulation was always lower than that observed under normal conditions. The lack of calcium in the MS medium advances the onset of the stationary phase, whose beginning is marked by an increase in the Put/spermidine (Spd) index, raising the production of Sm; the suspensions were productive for a longer time and hence produced more of the substance. Our results indicate that under stress conditions the production of Sm in young-cell suspensions of S. marianum is not associated with high levels of PAs in the medium – contrary to what one would expect – allowing us to conclude that growth inhibition appears to be the factor responsible for the maximum Sm accumulation while PAs are not directly involved in the Sm synthesis pathway by milk thistle grown in culture.

Calcium restriction inhibits cancer cell proliferation in vitro

Prostate and breast cancer cells metastasize to the bone. Strontium‐89 (Sr‐89) is a radioisotope that is used for treating bone metastases. The effect of strontium on calcium uptake was studied in human fetal osteoblast (hFOB) as well as androgen independent PC‐3 human prostate and MDA‐MB231 human breast cancer cells using digitized fluorescence microscopy in cells preloaded with Fura‐2AM. The effect of calcium deprivation on cell growth was studied using the MTT assay. When compared to cultures grown in complete medium, calcium and serum deprivation for 24 hours decreased cell growth by 80, 58 and 52 percent in hFOB, MDA‐MB231 and PC‐3 cells respectively. Addition of strontium chloride (up to 2 mM) did not cause further growth inhibition in calcium and serum starved medium. Calcium deprivation caused a G1 block and decreased the proportion of cells in the S phase. PC‐3 cells cultured for 24 hours in complete calcium containing medium supplemented with fetal bovine serum had a cell cycle distribution of 56% G1, 18.4% G2 and 25.6% S phase cells. These parameters shifted to 74.5% G1, 12.8% G2 and 12.6 % S phase cells in calcium and serum starved medium. Although strontium did not affect cell growth, it inhibited uptake of calcium by PC‐3 and MDA‐MB231 cells. Uptake of calcium from the medium by cells was inhibited by the anti diarrhea drug loperamide, which is a calcium channel blocker. Interestingly, loperamide inhibited hFOB and MDA‐MB231 cell growth even in complete medium. Preferential inhibition of calcium uptake by cancer cells may be an option in cancer therapy.(Supported in part by USMRMC grants DAMD17‐03‐1‐0759, DAMD17‐03‐1‐0123, 2 G12 RR003048 from RCMI, NCRR, NIH and R 25HL003679 from HLBI, NIH)

Effect of calcium restriction on cardenolide accumulation in two cell lines of Digitalis thapsi grown under different light regimes

The effect of calcium-deprivation on growth and the production of cardenolides in two undifferentiated cell lines of Digitalis thapsi maintained under three different light regimes (16 h photoperiod, darkness, or continuous light) was investigated. Growth was stimulated by continuous light in both cell lines cultured in complete medium. The light regime did not affect cardenolide accumulation in the cells of the hypocotyl-derived line; by contrast, continuous light or darkness increased the production in the leaf-derived line. The elimination of calcium favoured cardenolide production independently of the origin of the suspensions and the light regime, this beneficial effect being predominantly manifested under continuous light.

Drosophila nerve cord culture: a tool for studying neural development

The culture of explanted neural tissues has been a useful tool for the study of cellular and molecular neurobiology in vertebrates. We have developed a technique for the culture of explanted ventral nerve cords from Drosophila embryos. We have examined the morphology and dynamic behaviour of the growth cones that extend from these nerve cords, and the effect of calcium deprivation on the bundling of axons that are regenerated from the explanted tissue. This technique offers a unique opportunity to combine in vitro techniques for neuronal cell culture with the powerful techniques of genetic analysis that are available with Drosophila.

Different dietary calcium intake and relative supersaturation of calcium oxalate in the urine of patients forming renal stones.

1. Dietary calcium restriction, an efficient practice in reducing urinary calcium excretion, has been reported to induce either an increase or no change in oxalate excretion, questioning its use in hypercalciuric stone-forming patients. In addition, calcium restriction has been previously demonstrated to induce other urinary changes which might influence the relative supersaturation of calcium oxalate. So the overall effect of calcium deprivation on the relative supersaturation of calcium oxalate is unpredictable. 2. The aim of the study was to evaluate the effect of dietary calcium restriction on the relative supersaturation of calcium oxalate in the urine of stone-forming patients utilizing a computer methodology which takes into account the main soluble complex species of oxalate. 3. We studied 34 stone-forming patients on both a free-choice diet, whose Ca and oxalate content (24 and 1.2 mmol respectively) was assessed by dietary inquiry, and after 30 days on a prescribed low-calcium and normal oxalate diet (11 and 1.1 mmol respectively). Under both conditions, the excretion of the main urinary parameters related to dietary composition, electrolytes, oxalate and daily citrate urinary excretion, were measured. The relative supersaturation of calcium oxalate was calculated by means of an iterative computer method which takes into account the main soluble complex species on which the solubility of calcium oxalate is dependent. In addition, intact parathyroid hormone and 1,25-dihydroxyvitamin D blood levels were also evaluated. In 13 of the patients intestinal calcium absorption was evaluated during both a free- and a low-calcium diet, utilizing kinetics methodology. 4. The low-calcium diet induced, together with an expected reduction of calcium excretion, a marked increase in oxalate urinary output. This finding was independent of the presence or otherwise of hypercalciuria and of the serum levels of parathyroid hormone and vitamin D. Intestinal calcium absorption was also stimulated by calcium deprivation and its levels were well correlated with oxalate excretion. Minor changes in magnesium and citrate excretion were also observed. The overall effect on the relative supersaturation of calcium oxalate consisted in a substantial increase in this parameter during the low-calcium diet. 5. In conclusion, our data reinforce the concept that dietary calcium restriction has potentially deleterious effects on lithogenesis, by increasing the relative supersaturation of calcium oxalate.

Glucose modulation of sodium in pancreatic islets. Effects of calcium deprivation

Glucose modulation of sodium in pancreatic islets. Effects of calcium deprivation

Basal and gonadotrophin-releasing hormone-induced biosynthesis and release of luteinizing hormone: Effect of calcium deprivation

The present study examines the basal and gonadotrophin-releasing hormone (GnRH)-stimulated biosynthesis and release of luteinizing hormone (LH) by pituitary cells in primary culture, and the effect of extracellular calcium deprivation on these events. Pituitaries from ovariectomized adult rats were enzymatically dispersed and cultured for 96 h. The cells were then incubated for 5 h (Expts. 1 and 3) or for different time intervals between 0 and 5 h (Expt. 2), in medium containing [ 14C]leucine ([ 14C]leu) and [ 3H]glucosamine ([ 3H]gln), with or without GnRH. Total immunoreactive LH (iLH) was measured in the medium and the cell extract by radioimmunoassay. LH translation (as estimated by [ 14C]leu incorporation into LH; [ 14C]LH) and LH glycosylation (as estimated by [ 3H]gln incorporation into LH; [ 3H]LH) were measured by immunoprecipitation with specific LHβ antiserum in both medium and cell extract. Treating the cells with GnRH caused both time- and dose-dependent increases of iLH in the medium as well as in total (cells plus medium) content, with an approximate ED 50 of 0.7 nM. GnRH also stimulated LH biosynthesis by increasing both LH polypeptide chain synthesis and LH glycosylation. The effect of GnRH on LH glycosylation was detected earlier than that on translation, the [ 3H]LH rates of production and release being higher than those of [ 14C]LH. These findings suggest that GnRH-induced translation and glycosylation of LH are independently regulated. Removal of extracellular calcium resulted in the loss of cellular responsiveness to GnRH, preventing not only the stimulatory effects of GnRH on total and released iLH but also the GnRH-induced incorporation of both [ 14C]leu and [ 3H]gln into newly synthesized LH. These observations suggest that GnRH-stimulated LH glycosylation and LH translation involve calcium-dependent mechanisms. Neither the uptake of radiolabeled precursors nor their incorporation into total protein were affected by GnRH or Ca 2+-deficient (no added calcium) medium. The results also suggest that the release of newly synthesized LH is regulated differently from previously synthesized stored hormone.

Effects of calcium deprivation on the metabolism of acellular bone in tilapia, Oreochromis niloticus

1. 1. Effects of calcium deprivation from ambient water or from both diet and ambient water on the metabolism of fish acellular bone were examined by histomorphometry of the pharyngeal bone in tilapia ( Oreochromis niloticus). 2. 2. Calcium-deficient ambient water activated bone resorptive cells and suppressed osteoblasts, irrespective of dietary calcium supplementation. 3. 3. Hypercalcemia and decreased contents of body calcium and phosphate were observed only under the combined condition of ambient water and dietary calcium deficiency. 4. 4. Results indicated that the activity of acellular bone cells changed to withdraw calcium from bone into the extracellular fluid. This occurred when fish faced conditions of low calcium stress, such as ambient water calcium deficiency. Dietary calcium alone seemed to be insufficient for normal metabolism of bone in tilapia.

Effects of calcium deprivation on n-6 fatty acid metabolism in growing rats.

Two separate experiments examining the effects of calcium deficiency on plasma and liver fatty acids in rats were conducted. In Experiment I, weanling male Sprague-Dawley rats were fed a calcium-deficient diet with or without the supplementation of 5 or 20 g/kg calcium for 22 days. There were no significant differences in plasma and liver fatty acid distribution between the two calcium-supplemented groups. However, calcium deficiency significantly elevated the levels of 18:3n-6 in plasma and liver cholesteryl esters and liver phospholipids, while it reduced the levels of 20:3n-6 in plasma cholesteryl esters. In Experiment II, weanling rats were fed a calcium-deficient diet supplemented with 5 g/kg calcium for 22 days. After overnight fast, animals were given by intragastric feeding a dose of 4 g/kg body wt gamma-linolenic acid concentrate (containing 92% 18:3n-6 ethyl ester), and were killed 22 hr later. The levels of 18:3n-6 were significantly higher, whereas the levels of 20:3n-6 were either not changed or lower than those in calcium-supplemented group. In both experiments, the ratios of (20:3n-6 + 20:4n-6)/18:3n-6 in plasma and liver lipids were significantly reduced in calcium-deficient rats. These results suggest that calcium may play an important and specific role in the process of elongation of 18:3n-6 to 20:3n-6.

Effect of verapamil on cytoplasmic distribution of granules and microfilaments in amphibian urinary bladder

The amphibian urinary bladder has been used as a ‘model’ system for studies of the mechanism of action of antidiuretic hormone (ADH) in stimulating transepithelial water flow. The increase in water permeability is accompanied by morphological changes that include the stimulation of apical microvilli, mobilization of microtubules and microfilaments and vesicular membrane fusion events . It has been shown that alterations in the cytosolic calcium concentrations can inhibit ADH transmembrane water flow and induce alterations in the epithelial cell cytomorphology, including the cytoskeletal system . Recently, the subapical granules of the granular cell in the amphibian urinary bladder have been shown to contain high concentrations of calcium, and it was suggested that these cytoplasmic constituents may act as calcium storage sites for intracellular calcium homeostasis. The present study utilizes the calcium antagonist, verapamil, to examine the effect of calcium deprivation on the cytomorphological features of epithelial cells from amphibian urinary bladder, with particular emphasis on subapical granule and microfilament distribution.

Effect of calcium deprivation on rat dams on fetuses and newborn offspring.

Calcium-deficient rats were put to breeding, and fetuses and offsprings were collected at 18 days in utero, at birth, and at 5-10 days post partum. Furthermore, blood and milk were collected from the dams, and blood from the offsprings. The rat pups had normal weights at 18 days in utero and at birth, whereas at 5-10 days post partum the growth was significantly reduced. The relative amount of total solids in the body was also decreased at 5 and 10 days post partum (increased water content). Despite the fact that serum Ca in the dams was severely reduced, the calcium content in the serum of the offspring was only moderately lowered, and there was no correlation between serum Ca in dams and offspring. The content of total solids in the rat milk was increased, whereas the calcium content remained unchanged. The body content of Ca and P in the rat pups was decreased only at 5 and 10 days post partum. The degree of reduction was nearly the same for all these elements, amounting to 15-19% at 5 days post partum and 38-40% at 10 days post partum. If, however, the content of the elements was calculated as percentage of total body solids, a significant increase was found for all of them at 5 and 10 days post partum. Significant differences in Ca:P ratios were not found at any age.

Growth hormone secretion in chicks during dietary calcium deficiency

The role of calcium in the secretion of growth hormone (GH) has been examined in vivo in immature domestic fowl. Chicks reared on a low calcium (0.16% calcium in the feed) diet showed a reduced growth rate, compared with those on a normal (0.86%) calcium diet and had lower basal levels of plasma GH 1, 5, 10 and 15 d after calcium deprivation, but not after 20, 25, 30 or 35 d of calcium deficiency. No consistent changes in the concentration of immunoreactive somatomedin C were observed during calcium restriction. In both the control and low calcium fed birds the plasma concentrations of GH were elevated by the intravenous administration of human pancreatic GH releasing factor (hpGRF) and by thyrotropin releasing hormone (TRH). The GH responses to these provocative stimuli were not reduced in magnitude by calcium deficiency. It is suggested, therefore, that the effect of calcium deprivation on the secretion of GH is mediated via the reduced release of stimulatory hypophysiotrophic factors from the hypothalamus. Pharmacological alterations in calcium status also suggest that calcium deprivation inhibits GH secretion. Plasma concentrations of GH were acutely depressed in young chicks following the administration of a calcium chelator (ethylene diaminotetraacetic acid) a calcium channel blocker (verapamil) and after calmodulin inhibition (by chlordiazapine and trifluorpenazine administration). These data therefore demonstrate the importance of calcium in the stimulation of GH secretion in the domestic fowl.

The Pathogenesis of Idiopathic Hypercalciuria: Evidence for Parathyroid Hyperfunction

We studied the effect of calcium deprivation and loading in 17 healthy subjects and 76 patients with renal calculi. Five had primary hyperparathyroidism with an elevated plasma ionised calcium and detectable plasma parathyroid hormone. Forty-nine had idiopathic hypercalciuria, defined by a urine calcium greater than 7 mmol/day on a free diet. Twenty-two were normocalciuric. Fasting plasma calcium, corrected for albumin, was higher in the patients with idiopathic hypercalciuria (2.40 +/- 0.10 mmol/l) than in controls (2.28 +/- 0.05 mmol/l, p less than 0.005). Plasma calcium was intermediate in the normocalciuric stone formers (2.35 +/- 0.08 mmol/l) and elevated in the patients with primary hyperparathyroidism (2.62 +/- 0.07 mmol/l). Nephrogenous cyclic adenosine monophosphate (cAMP) and parathyroid hormone levels were highest in the primary hyperparathyroid group and did not differ significantly within the other groups. Nephrogenous cAMP correlated positively with plasma calcium in the patients with primary hyperparathyroidism and negatively in controls; there was no correlation in the idiopathic hypercalciuria group. Following an oral calcium load, plasma calcium rose and nephrogenous cAMP fell similarly in all groups. Fasting urinary calcium and its increase after load were greatest in the idiopathic hypercalciuria and primary hyperparathyroid groups, with intermediate results in the normocalciuric patients. Neither the initial metabolic patterns nor the response to thiazide fitted with the previously described patterns of absorptive and renal hypercalciuria. Increased parathyroid gland activity is the most probable cause of idiopathic hypercalciuria.

Calcium deprivation enhances glucagon release in the presence of 2-ketoisocaproate.

The effect of calcium deprivation upon glucagon and insulin release was studied in the rat pancreas perfused in the presence of 2-ketoisocaproate (10 mM). Control perfusions were conducted in the presence of glucose (11.1 mM). In the presence of 2-ketoisocaproate, the decrease in the extracellular concentration of calcium provoked a dramatic, but reversible, enhancement of glucagon release. Such a secretory response was similar in pattern to that seen upon calcium omission in the presence of glucose. Bio-Gel P-30 chromatographic studies showed that only true glucagon (mol wt, 3500) was liberated in the pancreatic effluent during the period of calcium deprivation. On the other hand, the secretion of insulin induced by 2-ketoisocaproate was, like that elicited by glucose, markedly inhibited upon the decrease in extracellular calcium concentration (80% and 65% inhibition, respectively). The results are interpreted in support of the hypothesis that calcium may play an inhibitory role in the control of glucagon release. It is suggested that such a role is somehow linked to the metabolism of exogenous nutrients in the A2 cells.

Renin release from isolated rat glomeruli: seasonal variations and effects of D600 on the response to calcium deprivation.

1. The effects of calcium deprivation and D600 on the rate of renin release and seasonal variations in the response were studied on juxtaglomerular cells from a preparation of isolated rat glomeruli superfused in vitro. 2. Reduction of superfusate calcium concentration caused an increase in renin release, which was significantly higher during the summer (May-August) than during the rest of the year. 3. Addition of D600 (2 X 10(-4) M) to a calcium-free medium in the low responsive period caused a markedly increased renin release. In the high responsive period renin release increased more rapidly and to a higher level initially than observed in the control lines without D600. 4. It is suggested that the effect of calcium on renin release predominantly is mediated by changes in calcium bound to the plasma membrane of the juxtaglomerular cell. The sensitivity of this cell to changes in the extracellular calcium concentration seems to be regulated and varies with season, possibly due to regulation of the amount of calcium bound to the membrane.

Calcium ATPase and intestinal calcium transport in uremic rats.

Calcium ATPase, an enzyme involved in intestinal calcium transport, was measured in homogenates of duodenal mucosal scrapings of normal and uremic rats. The effects of calcium deprivation and treatment with 1 alpha,25-dihydroxycholecalciferol [1,25-(OH)2D3] were investigated as well. Uremia decreased the enzyme activity and impaired the rise after calcium deprivation as observed in intact rats. The 1,25-(OH)2D3 treatment increased the enzyme activity in uremic animals and resulted in an identical response to calcium deprivation as observed in intact rats; parathyroidectomy abolished this effect. A striking correlation between everted duodenal gut sac calcium transport and calcium ATPase activity could be demonstrated for all groups of rats studied. It is concluded that the calcium ATPase activity is linked to the production of 1,25-(OH)2D3 as well as to an additional factor, probably parathyroid hormone. The close relationship between enzyme activity and in vitro calcium transport, even during constant physiological supplementation with 1,25-(OH)2D3, suggests an autonomous role of the calcium ATPase activity for mediation of calcium transport in the duodenum in addition to the well-known mechanisms related to vitamin D and its metabolites.

The effects of calcium deprivation upon mechanical and electrophysiological parameters in skeletal muscle fibres of the frog.

1. The effects of Ca2+ deprivation upon mechanical and electrophysiological parameters of single muscle fibres from the m. semitendinosus and the m. iliofibularis of the frog were investigated. 2. When the external free Ca concentration was reduced in steps of one order of magnitude from 10(-3) to 10(-9) M, using up to 10 mM-EGTA and in the presence of 3 mM-Mg2+, the maximum force of K contractures declined by 5-15%, the plateau of maximum force shortened, and in most cases the phase of spontaneous relaxation lengthened. 3. In Ringer solution containing 10(-9) M-Ca2+ and 1 mM-Mg2+ 85% of maximum tetanic force could be maintained for at least 15 sec (5 Hz; 3 degrees C). 4. The reduction of external Ca2+ from 10(-3) to 10(-9) M and its replacement by Mg2+ induced a 20-30 mV shift towards more negative potentials of the 'steady state' inactivation curve (which relates maximum force upon full depolarization to the logarithm of the K concentration or the corresponding membrane potential during the conditioning period). 5. The same alteration in concentrations of divalent cations caused little or no change in the shape and potential dependence of the activation curve (which relates maximum force to the logarithm of the external K concentration of the corresponding membrane potential). 6. The threshold potential for the onset of delayed rectification (point voltage clamp) and that for the initiation of an action potential did not change when external Ca2+ was reduced to 10(-9) M and replaced by Mg2+. 7. When the concentration of EGTA2- was increased to 80 mM (in the presence of 5 mM-Mg2+) twitch height dropped to very small values within a few minutes. However, tetanic force (50 Hz) reaching 20-85% of the original value could still be induced after 1 hr in high EGTA2-. 8. The experiments show that external Ca2+ acts upon excitation-contraction coupling mainly by impeding 'inactivation'. A hypothesis is proposed in which the plateau of maximum force during a contracture is the consequence of a regenerative Cai2+-dependent shift of the inactivation curve towards more positive potentials.

Effects of calcium deprivation and orchidectomy on bone composition in the rat.

The effects of calcium deprivation and of orchidectomy, separately and combined, on body growth and bone composition have been examined in mature male rats. Calcium deprivation had no significant effect on the rate of body growth but femoral bone weight, bone ash weight and total calcium and phosphorus contents of the femora were reduced while the water content increased. However, there were no significant changes in the ratio of bone weight to body weight, in the amounts of calcium or phosphorus per unit weight of bone, or in the ratio of calcium to phosphorus in the bone. Orchidectomy reduced the rate of body growth, the mean weight of the femora and the ratio of bone weight to body weight. There was a 60% decrease in bone water and a 30--35% reduction in the calcium and phosphorus content of the femora but the amounts of ash, calcium and phosphorus per unit weight of bone fell by only 6--10%. The combination of orchidectomy and calcium deprivation had no greater effect on body weight, bone weight or bone composition than had orchidectomy alone. It is concluded that calcium deprivation and orchidectomy both cause osteoporosis in mature rats but the effects of orchidectomy are more severe than those of calcium deprivation and there appears to be some demineralization of the bone remaining after orchidectomy.

The Effects of Calcium Deprivation and Inhibition of Intracellular Metabolism on the Formation of Intracellular Slow Reacting Substance of Anaphylaxis in passively Sensitized Human Lung Challenged with Antigen

The Effects of Calcium Deprivation and Inhibition of Intracellular Metabolism on the Formation of Intracellular Slow Reacting Substance of Anaphylaxis in passively Sensitized Human Lung Challenged with Antigen

Inhibition of the acetylcholine-induced secretion of bovine growth hormone by somatostatin

The effects of calcium deprivation, somatostatin and verapamil on the stimulation of growth-hormone release and the alteration of pituitary metabolism in response to acetylcholine were investigated. Calcium deprivation decreased the rises in growth-hormone secretion and in cyclic GMP content in response to 25 μM acetylcholine but did not prevent the increased incorporation of 32P into phosphatidyl inositol. Somatostatin (1 μg/ml) prevented the rise in growth-hormone secretion and inhibited the efflux of 45Ca in response to acetylcholine (25 μM) but did not modify the increase in cyclic GMP content or phosphatidyl inositol labelling. Verapamil (50 μM) did not affect any of the responses to acetylcholine (25 μM). The data suggest that acetylcholine stimulates calcium mobilization from tissue stores and that somatostatin can prevent this mobilization. The relevance of this to the inhibition of secretion by somatostatin is discussed.