Characterization and Distribution of Transferrin Receptors in the Rat Brain

The mechanism of calcium transport across the plasma membrane of chromaffin cells was studied using plasma membrane vesicles prepared from cells of adrenal medulla. Purification of the plasma membrane was about 30-fold, based on the alpha-bungarotoxin binding activity. The isolated membrane vesicles have both Na+/Ca2+ exchange and calcium pump activities. The Na+/Ca2+ exchange activity increased with the free calcium concentration and was not saturated at 1 mM, the highest concentration tried. The K1/2 of the calcium pump for calcium is 0.06 microM. Part of the Na+/Ca2+ exchange activity was inhibited by preincubation of the membrane vesicles with veratridine and the effect of veratridine was reversed by tetrodotoxin. The calcium taken up by the calcium pump was released by 0.005% saponin, but was not affected by oxalate. The calcium taken up by the calcium pump was released by exchanging with the external sodium, which suggests that the two calcium transport systems are located on the same population of membrane vesicles. The above evidence indicates that both calcium transport activities are located on the plasma membrane and not on contaminating organelle membranes. The significance of the two calcium transport systems in regulation of cytosolic calcium concentration of chromaffin cells is discussed.     

Cold resistance of the brain during hibernation. Temperature sensitivity of the partial reactions of the Na+, K+-ATPase.

The regulatory effect of calcium added in vitro on 25-hydroxycholecalciferol metabolism was studied in kidney mitochondria and in renal tubules from vitamin D-deficient chicks. The addition of calcium (0.05 – 0.2 mm) to mitochondrial suspensions prepared with calcium-chelating agents caused a marked and dose-related stimulation of 1-hydroxylation. A sharp decline in the activity was induced by higher concentrations of calcium (0.3 – 0.7 mm). A similar but less striking biphasic effect of calcium on 1-hydroxylation was observed in mitochondria prepared in the absence of calcium chelating agents. The effect of calcium was not a consequence of accelerated mitochondrial translocation of either exogenous NADP or Mg²⁺ but was related to mitochondrial calcium content. The addition of inhibitors of the calcium uptake, i.e., LaCl₃ or ruthenium red, or a calcium ionophore (A 23187) significantly inhibited the calcium-induced stimulation of the 1-hydroxylation reaction. Similar calcium effects were also observed in renal tubules isolated from intact, but not from parathyroidectomized, vitamin D-deficient chicks. These data strongly suggest that mitochondrial calcium plays an important role in the regulation of 1-hydroxylase activity in kidney.     

The effect of calcium load on the calcium permeability of sarcoplasmic reticulum

The effect of intravesicular and extravesicular calcium concentration on the passive efflux from sarcoplasmic reticulum (SR) vesicles isolated from cardiac and skeletal muscle was determined by measuring net efflux of calcium after stopping pump-mediated fluxes. The apparent permeability, calculated as the passive efflux divided by the total intravesicular calcium, depended on calcium load. This dependence of the apparent permeability on calcium load could be explained by the presence of intravesicular calcium-binding sites with a dissociation constant less than 10(-3) M. When the intravesicular bound calcium was taken into account, passive calcium efflux was found to be linearly related to the difference in calcium concentration across the SR membrane. Thus the permeability of the SR membrane is independent of intravesicular and extravesicular calcium concentration in the ranges investigated. The average first order rate constant for passive calcium efflux for six preparations was 0.8 +/- 0.2 min-1 for skeletal and 0.7 +/- 0.1 min-1 for cardiac SR. The amount of intravesicular bound calcium for the same preparations was 33 +/- 6 nmol mg-1 for skeletal and 13 +/- 2 nmol mg-1 for cardiac SR. The first order rate constants were unaffected by Mg concentration between 0.1 +/- 15.1 mM and by the presence of an ATP-regenerating system. The results suggest that some minimal calcium load may be required in order to observe a substantial passive calcium efflux, the passive calcium efflux is not carrier mediated, and passive calcium efflux is not a likely route of calcium release during excitation-contraction coupling.     

Increased intestinal calcium absorption from the ingestion of a phosphorylated guar gum hydrolysate independent of cecal fermentation in rats

The Apparent calcium absorption was increased in rats fed on P-GGH and GGH. However, this increase in calcium absorption from GGH feeding was cancelled by a cecectomy, whereas the corresponding increase from P-GGH feeding was not. The change in femoral calcium content was similar to that in calcium absorption. The calcium solubility in the ileum was increased in those rats fed on P-GGH. We conclude that cecal fermentation did not contribute to the increased calcium absorption by the rats fed on P-GGH.     

Calcium complexes in the xylem sap of apple shoots

Summary Concentrations of total calcium, ionic calcium, citric and malic acids have been measured in xylem sap extracted from apple shoots. Ionic calcium, as measured by an ion selective electrode, was about 50 per cent of the total calcium. The remainder of the soluble calcium was present as complexes with citric and malic acids. The implication of these findings on the mobility of calcium in the xylem is discussed.     

Endogenous prolactin modulated the calcium absorption in the jejunum of suckling rats

Prolactin has been reported to stimulate intestinal calcium absorption in young and mature, but not aging rats. The present study was performed on suckling rats to elucidate the actions of endogenous prolactin on calcium absorption in various intestinal segments. Before measuring the calcium fluxes, 9-day-old rats were administered for 7 days with 0.9% NaCl, s.c. (control), 3 mg/kg bromocriptine, i.p., twice daily to abolish secretion of endogenous prolactin, or bromocriptine plus exogenous 2.5 mg/kg prolactin, s.c. Thereafter, the 16-day-old rats were experimented upon by instilling the 45Ca-containing solution into the intestinal segments. The results showed that, under a physiological condition, the jejunum had the highest rate of calcium absorption compared with other segments (1.4 +/- 0.35 micromol.h-1.cm-1, p < 0.05). The duodenum and ileum also manifested calcium absorption, whereas the colon showed calcium secretion. Lack of endogenous prolactin decreased lumen-to-plasma and net calcium fluxes in jejunum from 2.07 +/- 0.31 to 1.19 +/- 0.12 and 1.40 +/- 0.35 to 0.88 +/- 0.18 micromol.h-1.cm-1 (p < 0.05), respectively, and exogenous prolactin restored the jejunal calcium absorption to the control value. Endogenous prolactin also had an effect on the duodenum but, in this case, exogenous prolactin did not reverse the effect of bromocriptine. However, neither ileal nor colonic calcium fluxes were influenced by prolactin. Because luminal sodium concentration has been demonstrated to affect calcium absorption in mature rats, the effect of varying luminal sodium concentrations on calcium fluxes in suckling rats was evaluated. The jejunum was used due to its highest rate of calcium absorption. After filling the jejunal segments with 124 (control), 80, 40 mmol/L Na+-containing or Na+-free solution, increases in calcium absorption were found to be inversely related to luminal sodium concentrations in both control and bromocriptine-treated rats. The plasma concentration of 45Ca under luminal sodium free condition was also higher than that of the control condition (2.26% +/- 0.07% vs. 2.01% +/- 0.09% administered dose, p < 0.05). However, 3H-mannitol, a marker of the widening of tight junction that was introduced into the lumen, had a stable level in the plasma during an increase in plasma 45Ca, suggesting that the widening of tight junction was not required for enhanced calcium absorption. In conclusion, calcium absorption in suckling rats was of the highest rate in the jejunum where endogenous prolactin modulated calcium absorption without increasing the paracellular transport of mannitol.     

Interaction of sodium with the sarcolemmal calcium system.

Sarcolemma isolated from guinea pig heart binds calcium in an ATP-dependent manner. Sodium ions decrease the total amount of calcium bound by the membranes. ATP-dependent calcium binding is more sensitive to sodium than the non-ATP-dependent calcium binding. The ATPase active during calcium binding is affected by sodium ions to the same extent as the ATP-dependent calcium binding process. The inhibition of the calcium binding process and of ATPase activity by sodium was more pronounced when the membranes were preincubated with sodium. The effect of sodium on calcium binding is dependent on both the time of contact between sodium and the membranes and the concentration of sodium. It is suggested that the effect of sodium on the calcium binding system in the sarcolemma may be a link between the inhibition of Na+K+-ATPase (EC 3.6.1.3) by cardiac glycosides and the subsequent increase in intracellular calcium.     

Functioning of the electromechanical connection in the course of the contracture contraction

The effects of calcium release blocker dantrolene was tested on electrically evoked twitches and on contractures induced by potassium depolarization, by acetylcholine or caffeine. It was shown that the first: developmental, stage of potassium or acetylcholine contracture is inhibited by dantrolene and is not influenced by calcium free medium, therefore we may interpret it as based on a "voltage-dependent Ca release" (VDCR) mechanism of activation, whereas depolarization directly opens the rhyanodin receptor calcium channels. On the contrary, the next stage: the long-lasting plateau of contracture, is directly dependent on external Ca2+ and inhibited by dantrolene, and therefore can be described as "calcium induced Ca-release" (CICR) activation mechanism. In this case stored calcium is also released by rhyanodine receptors, although by means of entering the extracellular Ca2+. Finally, the last stage of low amplitude is not influenced by dantrolene nor by calcium-free medium. Therefore the activation of contraction on this stage is not based on the Ca2+ release through the rhyanodin receptor calcium channels.     

Aspects of the mechanism of action of local anesthetics on the sarcoplasmic reticulum of skeletal muscle.

1. The effect was studied of local anesthetics (tetracaine, dibucaine, procaine and xylocaine) on the forward and the backward reactions of the calcium pump of skeletal muscle sarcoplasmic reticulum. 2. The inhibition of the rate of calcium uptake, the rate of calcium-dependent ATP splitting and the rate of calcium-dependent ATP-ADP phosphate exchange by sarcoplasmic reticulum in the presence of the above drugs is at least partially due to the inhibition of the phosphoprotein formation from ATP. 3. The rate of the ADP-induced calcium release from sarcoplasmic reticulum and the rate of ATP synthesis driven by the calcium efflux are inhibited on account of a reduction of the phosphoprotein formation by orthophosphate. 4. The phosphorylation of calcium transport ATPase by either ATP or orthophosphate is diminished by the local anesthetics owing to a reduction in the apparent calcium affinity of sarcoplasmic reticulum emmbranes on the outside and on the inside, respectively. 5. The drug-induced calcium efflux from calcium-preloaded sarcoplasmic reticulum vesicles, a reaction not requiring ADP, is probably not mediated by calcium transport ATPase.     

Localization of calcium in the pericarp cells of tomato fruits during the development of blossom-end rot

Summary. Blossom-end rot (BER) of tomato (Lycopersicon esculentum) fruits is considered to be a physiological disorder caused by calcium deficiency. We attempted to clarify the localization of calcium in the pericarp cells and the ultrastructural changes during the development of BER. Calcium precipitates were observed as electron-dense deposits by an antimonate precipitation method. Some calcium precipitates were localized in the cytosol, nucleus, plastids, and vacuoles at an early developmental stage of normal fruits. Calcium precipitates were increased markedly on the plasma membrane during the rapid-fruit-growth stage compared with their level at the early stage. Cell collapse occurred in the water-soaked region at the rapid-fruit-growth stage in BER fruits. There were no visible calcium precipitates on the traces of plasma membrane near the cell wall of the collapsed cells. The amount of calcium precipitates on plasma membranes near collapsed cells was smaller than that in the cells of normal fruits and normal parts of BER fruits, and the amount on cells near collapsed cells was small. The amount of calcium precipitates on the plasma membranes increased as the distance from collapsed cells increased. On the other hand, calcium precipitates were visible normally in the cytosol, organelles, and vacuoles and even traces of them in collapsed cells. The distribution pattern of the calcium precipitates on the plasma membrane was thus considerably different between normal and BER fruits. On the basis of these observations, we concluded that calcium deficiency in plasma membranes caused cell collapses in BER tomato fruits.Correspondence and reprints: National Institute of Vegetable and Tea Science, National Agricultural Research Organization, Ano, Mie, 514-2392, Japan.     

On the mechanisms of ATP-induced and succinate-induced redistribution of cations in isolated rat liver cells

1. The ability of external ATP to induce calcium uptake in isolated rat liver cells was further characterized. Stimulation of calcium uptake was specific for ATP, other nucleotides or ATP metabolites had no comparable effect. ATP was dephosphorylated while stimulating calcium uptake, but there was no stoichiometry between ATP hydrolysis and calcium uptake nor did dephosphorylation depend on calcium concentration. ATP acted from outside and was dephosphorylated by an ecto-ATPase of the cells. 2. In addition to its direct action, ATP enhanced succinate-dependent calcium uptake in a cooperative fashion. This is best explained by different sites of action. ATP increases cell membrane permeability while succinate stimulates uptake into mitochondria. 3. ATP was able to lower Na+ and K+ gradients and the pH gradient between cells and incubation medium. Increasing calcium concentration counteracted this effect though calcium uptake was then stimulated. 4. Succinate alone did not affect monovalent cation gradients but raised the pH gradient. It partially counteracted the ATP effects on these gradients. 5. Since catecholamine-like actions of ATP may be mediated by an increase in cytoplasmic calcium concentration, the action of extracellular ATP can be taken as a model to study the role of calcium as a transmitter of hormone actions. From interdependence between ATP-stimulated and succinate-stimulated calcium uptake, conclusions can be drawn on the resulting cytoplasmic calcium concentration and its effect on plasma membrane permeability.     

Channel formation in model membranes by the adenylate cyclase toxin of Bordetella pertussis: effect of calcium

Calmodulin-dependent adenylate cyclase toxin (ACT or CyaA) of Bordetella pertussis requires calcium ions for target cell binding, formation of hemolytic channels, and delivery of its enzyme component into cells. We examined the effect of calcium and calmodulin on toxin interaction with planar lipid bilayers. While calmodulin binding did not affect the properties of CyaA channels, addition of calcium ions and toxin to the same side of the membrane caused a steep increase of the channel-forming capacity of CyaA. The calcium effect was highly specific, since among other divalent cations only strontium caused some CyaA activity enhancement. The minimal stimulatory concentration of calcium ions ranged from 0.6 to 0.8 mM, depending on the ionic strength of the aqueous phase. Half-maximal channel activity of CyaA was observed at 2-4 mM, and saturation was reached at 10 mM calcium concentration, respectively. The unit size of single CyaA channels, assessed as single-channel conductance, was not affected by calcium ions, while the frequency of CyaA channel formation strongly depended on calcium concentration. The calcium effect was abrogated upon deletion of the RTX repeats of the toxin, suggesting that binding of calcium ions to the repeats modulates the propensity of CyaA to form membrane channels.     

Time-dependent restoration of the trigger pool of calcium after termination of angiotensin II action in adrenal glomerulosa cells

In adrenal glomerulosa cells, angiotensin II causes an immediate release of calcium from an intracellular trigger pool (Kojima, I., Kojima, K., and Rasmussen, H. (1985) Am. J. Physiol. 247, E36-E43). The present study was conducted to determine how the trigger pool of calcium is restored after cessation of the agonist action. Upon termination of angiotensin II action, calcium influx rate decreased immediately while total cell calcium increased rapidly. The increase in total cell calcium is not affected by 1 microM nitrendipine, which blocks angiotensin II-stimulated calcium influx without inhibiting basal influx of calcium. In contrast, total cell calcium did not increase in medium containing 1 microM calcium, in which basal calcium influx is negligible. A rapid increase in total cell calcium after an addition of the antagonist was not accompanied by changes in cytoplasmic free calcium concentration. A second stimulation of cells with either angiotensin II or carbachol did not cause calcium release when the interval of two stimulations was shorter than 20 min. The longer the interval, the greater the magnitude of calcium release in response to the second stimulator. The maximum response was obtained when the interval was 40 min or more. When exogenous arachidonic acid, which mobilized calcium by acting directly on the inositol trisphosphate-sensitive pool, was employed as a second stimulator, the magnitude of the decrease in total cell calcium was also dependent on the interval. These results suggest that, upon termination of angiotensin II action, calcium is rapidly accumulated first in an intracellular pool which is insensitive to either inositol 1,4,5-trisphosphate or arachidonic acid and that the trigger pool is restored gradually thereafter.     

Schistosoma mansoni: the role of calcium in the stimulation of cercarial proteinase release.

We investigated the role of calcium mobilization in the induction of proteinase release from cercarial preacetabular glands. Proteinase release was measured by the ability of cercariae to break down a 3H-labeled proline extracellular fibroblast matrix and calcium influx was measured using 45Ca2+. The role of calcium in the activation of cercarial proteinase was examined by investigating the effects of calcium addition and removal on linoleate-induced matrix degradation, the ability of various calcium modulators (Verapamil, fendiline, nifedipine, SK-525A, BAY K-8644, Ryanodine, and SK-7171A) to stimulate or inhibit linoleate-induced proteinase release, the ability of calcium modulators directly to induce cercarial proteinase release, and the ability of various stimulants of proteinase release to induce calcium influx or efflux from cercariae. The results of these studies indicate that proteinase release is dependent on external calcium concentration, voltage-operated channels are either nonexistent in cercariae or have a minimal role in overall calcium influx, and that activation of Ca2+ influx can be caused by both free fatty acids and calcium modulators by a hypothesized receptor-operated channel. Although calcium uptake is important in cercarial proteinase release, it is not the only factor involved. Calcium uptake alone does not guarantee that proteinase will be secreted. On the other hand, if Ca2+ influx does not occur, proteinase will not be secreted.     

Vanadate inhibits the calcium extrusion in rat pancreatic acinar cells

Our objective was to evaluate the role of vanadate on calcium extrusion in Fura-2-loaded rat pancreatic acinar cells by digital microscopic fluorimetry and spectrofluorimetry. In the absence of extracellular calcium, perfusion of pancreatic acinar cells with 1 nM CCK-8 and 1 mM vanadate did not significantly affect the typical transient calcium spike induced by CCK-8, but the plateau phase of calcium in response to CCK-8 remained elevated. In addition, vanadate was able to inhibit calcium efflux evoked by CCK-8 when we determined directly calcium transport across plasma membrane using Calcium Green-5N hexapotassium salt (cell impermeant form) in cell populations. The effect of vanadate on calcium extrusion was strongly blocked by the sulfhydryl-reducing agent dithiothreitol (DTT). The present results demonstrate that vanadate is able to irreversibly inhibit the calcium extrusion. This effect of vanadate can be blocked using DTT, indicating that its action is probably mediated by oxidation of sulfhydryl groups of Ca2+-ATPases.     

Effect of low or high dietary calcium on the morphology of the rat femur

The present study compared the effect of a calcium deficit or surfeit on femurs. Young female rats were fed with the normal (1.18%), low (0.05%), or high (2.00%) calcium diet for 3, 7, 15 or 30 days. Two groups received the low calcium diet for the first 15 days and then were followed by the normal (L-N) or high calcium diets (L-H) for the sequential 15 days. The morphology of the femur was studied together with serum calcium, parathyroid hormone (PTH), calcitonin and bone mineral density (BMD). We did not find any significant changes in the serum PTH level and bone morphology in the high calcium group. In the low calcium group, the serum PTH level increased, BMD of the whole body, the femoral weight and the femoral trabecular bone decreased as compared with the normal calcium group. There was a greater proportion of resorbing surface, less resting surface and larger vascular canal openings in the femoral endosteal surfaces in the low calcium group. In the L-N or L-H group, the femoral trabecular bone increased and the femoral resorbing surface decreased as compared with those of the low calcium group. These findings suggest that high calcium intakes do not affect the bone mass, and low calcium intakes have a deleterious effect on bone status, which may be related to vascular alternations of the bone. Reversing the low income calcium intake by a higher calcium diet can partially improve the bone alternations induced by low calcium intake.     

Calcium regulates the regeneration of cilia in Tetrahymena thermophila

A study of calcium metabolism in Tetrahymena during the regeneration of cilia evidenced that the process is inhibited by nifedipine and trifluoperazine. This suggests that calcium ions play an important regulatory role in this process. This was confirmed by studies on calcium uptake and efflux which showed that there was a net increase in calcium uptake prior to the reinitiation of motility. The increase coincided with a period of sensitivity to the calcium antagonist TMB-8 and with an increase in the intracellular level of cGMP. The process was also inhibited by neomycin and stimulated by phorbol esters, which suggests that hydrolysis of phosphatidylinositol phosphates may take place as part of the calcium regulatory network during the regeneration of cilia.     

Calcium Regulates the Regeneration of Cilia in Tetrahymena thermophila

A study of calcium metabolism in Tetrahymena during the regeneration of cilia evidenced that the process is inhibited by nifedipine and trifluoperazine. This suggests that calcium ions play an important regulatory role in this process. This was confirmed by studies on calcium uptake and efflux which showed that there was a net increase in calcium uptake prior to the reinitiation of motility. The increase coincided with a period of sensitivity to the calcium antagonist TMB-8 and with an increase in the intracellular level of cGMP. The process was also inhibited by neomycin and stimulated by phorbol esters, which suggests that hydrolysis of phosphatidylinositol phosphates may take place as part of the calcium regulatory network during the regeneration of cilia.     

Effect of the Calcium on Polygalacturonase (PG) Activity and Synthesis at Different Ripening Stages of Tomato Fruits

It has been reported that PG is a key enzyme related to the tomato fruit ripening and that the application of calcium can dramatically decrease the PG activity and delay the ripening of fruits. In this paper the effects of calcium treament at various ripening stages on the transformation of absorbed calcium, PG activity and PG synthesis in tomato fruits were studicd. According to the analysis of calcium by atomic absorption spectroscopy, it was shown that the soluble and total calcium contents in pericarp of fruits treated with calcium at mature-green stage were increased significantly, and that more soluble calcium was transformed into bound calcium. Both the absorption and transformation of calcium decreased in fruits treated with calcium at later stage of ripening. The inhibition of calcium on PG activity was most effective by treatment at mature-green stage, but less effective at later stage of ripening. One reason for the decrease of calcium inhibition was probably due to the decline of calcium absorption as fruit ripening. The polyacrylamide gel electrophoresis of PG showed that PG with a molecular weight of 46.7 kD was absent in mature-green fruits, and PG synthesis occurred only at the later stage of ripening. It seems that the earlier the treatment was done the more effective of the calcium inhibition of PG synthesis. Based on the above results, it was concluded that the PG plays a major role in ripening and senescence of tomato fruits, and both PG synthesis and its activity were inhibited by calcium. In order to delay the ripening and senescence of tomato fruits, the treatment with calcium should be done at mature-green stage.     

Skeletal sensitivity to dietary calcium deficiency is increased in the female compared with the male rat

We investigated potential sex differences in bone resorption and the conservation of whole body bone mass in 24-week-old Sprague-Dawley rats maintained on a 1.0% calcium diet and then fed diets containing 0.02, 0.5, 1.0, or 1.75% calcium for 31 days. Lowering dietary calcium from 1.00% to 0.02% doubled whole skeleton bone resorption (urinary 3H-tetracycline loss). Female rats were more sensitive to calcium stress, exhibiting the maximal resorptive response when fed the 0.5% calcium diet, whereas the 0.02% calcium diet was required to elicit this response in males. Despite the evidence of increased bone resorption, whole skeleton mass was unchanged in females and was significantly increased in males, indicating that switching to even the 0.02% calcium diet did not result in an overt loss of total body bone mass. Compared with controls, the skeleton mass of females (97+/-1.4%) maintained on the 0.02% calcium diet was significantly lower than males (107+/-2.4%), again suggesting a greater impact of calcium deficiency in females. The calculation of the average percentage growth of selected individual bones in male rats indicated a proportional increase in bone mass between the axial and appendicular skeleton of approximately +4% and +18% in animals maintained on 0.02 and 1.75% diets, respectively. By comparison, female rats consuming the 0.02% calcium diet showed an average 14% loss in axial bone and 7.5% gain in appendicular bone mass. The results indicate increased sensitivity to dietary calcium deficiency in female rats which involves a significant loss in axial bone mass not observed in male rats maintained under similar dietary conditions.