Various Concentrations Of Calcium Research Articles

A calorimetric study of the influence of calcium on the stability of bovine α-lactalbumin

Bovine α-lactalbumin has been studied by differential scanning calorimetry with various concentrations of calcium to elucidate the effect of this ligand on its thermal properties. In the presence of excess calcium, α-lactalbumin unfolds upon heating with a single heat-absorption peak and a significant increase of heat capacity. Analysis of the observed heat effect shows that this temperature-induced process closely approximates a two-state transition. The transition temperature increases in proportion with the logarithm of the calcium concentration, which results in an increase in the transition enthalpy as expected from the observed heat capacity increment of denaturation. As the total concentration of free calcium in solution is decreased below that of the proteins, there are two temperature-induced heat absorption peaks whose relative area depends on the calcium concentration, such that further decrease of calcium concentration results in a increase of the low-temperature peak and a decrease of the high-temperature one. The high-temperature peak occurs at the same temperature as the unfolding of the holo-protein, while the low-temperature peak is within the temperature range associated with the unfolding of the apo-protein. Statistical thermodynamic modeling of this process shows that the bimodal character of the thermal denaturation of bovine α-lactalbumin at non-saturated calcium concentrations is due to a high affinity of Ca 2+ for α-lactalbumin and a low rate of calcium exchange between the holo- and apo-forms of this protein. Using calorimetric data, the calcium-binding constant for α-lactalbumin has been determined to be 2.9×10 8 M −1.

Dynamic study of cell mechanical and structural responses to rapid changes of calcium level

Cell shape control is complex since it may involve multiple cytoskeletal components and metabolic pathways. Here we present a kinetic study of the mechanical and structural responses of cells from the monocytic THP-1 line to a rapid increase of cytosolic calcium level. Cells were exposed to ionomycin in a medium of varying calcium concentration and they were probed at regular intervals for (1) cortical rigidity as determined with micropipette aspiration, and (2) content and distribution of polymerized actin, myosin or ABP-280, as determined with flow cytometry and/or confocal microscopy. An increase of free intracellular calcium level induced: (1) a biphasic deformability change with marked stiffening within a second, and significant softening a minute later; (2) a biphasic change of actin polymerization with initial decrease (within less than a second) and rapid recovery (within a few seconds); (3) a topographical redistribution of microfilaments with an oscillatory behavior of the cortical fraction, while no substantial redistribution of myosin or ABP-280 was detected. It is suggested that a regulation of cell rigidity might be achieved without any structural change by suitable modulation of the lifetime of bridges formed between microfilaments by actin binding proteins.

Dynamic study of cell mechanical and structural responses to rapid changes of calcium level

Cell shape control is complex since it may involve multiple cytoskeletal components and metabolic pathways. Here we present a kinetic study of the mechanical and structural responses of cells from the monocytic THP-1 line to a rapid increase of cytosolic calcium level. Cells were exposed to ionomycin in a medium of varying calcium concentration and they were probed at regular intervals for (1) cortical rigidity as determined with micropipette aspiration, and (2) content and distribution of polymerized actin, myosin or ABP-280, as determined with flow cytometry and/or confocal microscopy. An increase of free intracellular calcium level induced: (1) a biphasic deformability change with marked stiffening within a second, and significant softening a minute later; (2) a biphasic change of actin polymerization with initial decrease (within less than a second) and rapid recovery (within a few seconds); (3) a topographical redistribution of microfilaments with an oscillatory behavior of the cortical fraction, while no substantial redistribution of myosin or ABP-280 was detected. It is suggested that a regulation of cell rigidity might be achieved without any structural change by suitable modulation of the lifetime of bridges formed between microfilaments by actin binding proteins. Cell Motil. Cytoskeleton 45:93–105, 2000. © 2000 Wiley-Liss, Inc.

Mitochondrial Oxygen Consumption Is Synergistically Inhibited by Metallothionein and Calcium

Objective.To determine the combined effect of metallothionein and calcium on the oxygen consumption of mitochondria.Background.We previously showed that mitochondrial oxygen consumption was inhibited by the intracellular acute-phase reactant, metallothionein, which is rapidly induced by nearly all stressed cells. Other investigators have demonstrated that calcium also inhibited oxygen consumption. However, the calcium concentrations used in their experiments were supraphysiologic. Our hypothesis was that metallothionein would enhance the effect of calcium.Methods.We conducted these experiments, of a paired design, on the effects of combinations of metallothionein and calcium using mitochondria isolated from rat liver. An oxygen electrode implanted into a 600-μl chamber with a stir bar was used to measure oxygen consumption. Various concentrations of calcium, metallothionein, and other reagents were added while oxygen consumption was being continuously recorded. Metals were removed from metallothionein by gel filtration to produce apometallothionein.Results.Physiological levels of metallothionein synergistically enhanced the inhibitory effect of calcium so that its action occurred at physiological concentrations. Metallothionein devoid of its metals, zinc and cadmium, had no effect on oxygen consumption.Conclusion.In isolated mitochondria metallothionein inhibits ADP-initiated oxygen consumption. This effect is synergistic with the inhibitory action of calcium. These observations suggest a possible enhancement by metallothionein of the effect of fluxes in intracellular calcium in stressed cells.

Pharmacologic Levels of Heparin Do Not Destabilize Neonatal Parenteral Nutrition

Calcium and heparin are known to destabilize the lipid emulsion of total parenteral nutrition (TPN). However, these observations were made over long periods of time, using 5 to 10 times the amount of heparin used in a neonatal intensive care unit. We investigated the effects of lower heparin concentrations with lipid-to-nutrient ratios normally administered to premature infants. Lipid emulsion stability was assessed over 30 minutes by measuring viscosity at 10 mmol/L calcium after the addition of 0, 0.5, 1, and 5 IU heparin/mL. This was done at a range of lipid-to-nutrient ratios in clinical use. The effect of varying calcium concentration and different multivitamin preparations on emulsion stability also was observed. Heparin caused an immediate increase in viscosity of pure Intralipid 20% (Intralipid; Kabi Pharmacia AB, Stockholm, Sweden), which eventually separated into two phases. Although changes in viscosity were observed at 1:1 lipid-to-nutrient ratios, no effect was seen at a 1:9 ratio. With the 1:1 ratio, the multivitamin preparations, MVI Pediatric (Rhone-Poulec Rorer, Montreal, Canada) and Vitlipid (Kabi Pharmacia AB), reduced the increase in viscosity. Heparin and calcium destabilize Intralipid. This is unlikely to cause problems for most infants receiving TPN, provided low heparin concentrations are used. In all cases, especially where the lipid ratio is high, the risk of the lipid phase separating out can be further minimized by (1) the addition of multivitamin preparations containing detergent or an emulsifying agent and (2) by having the shortest possible delivery tube between the point of mixing the lipid and amino acid solutions of TPN and entry into the infant.

Intraoperative crystallization on the intraocular lens surface

PURPOSE: To report a physician survey, laboratory studies, and clinical observations of intraoperative crystallization on the surface of the intraocular lens (IOL). METHOD: We sent a survey to all ophthalmologists in the states of Wyoming, Idaho, Montana, Utah, and Colorado asking whether crystallization on the IOL surface had occurred in any of their patients and what viscoelastics, IOLs, and other solutions were used. All returned surveys were tabulated and analyzed by standard statistical means. A sample of crystallization from an IOL on a glass slide submitted by a physician was analyzed to ascertain the relative elemental composition. During in vitro laboratory studies, BSS Plus (Alcon Surgical, Fort Worth, Texas) and BSS (Alcon Surgical) were measured and analyzed for precipitation. Healon GV (Pharmacia/Upjohn, Kalamazoo, Michigan) and calcium chloride were combined in various solutions and examined for precipitate formation. Silicone IOLs and plate silicone were placed in different BSS and BSS Plus solutions with different viscoelastics and varying calcium concentrations. In seven patients, prominent crystallization on an IOL surface was examined, photographed, and followed for up to 3 years. RESULTS: Two hundred six surveyed ophthalmologists returned 181 surveys (88%) and reported 29,609 cataract surgeries, with IOL implantation with 22 eyes (0.07%) (22 patients) in which intraoperative crystallization was observed on the IOL surface during 1993. The survey indicated there was a correlation with BSS Plus (chi-square = 4.9, P = .0268) and silicone IOLs (chi-square = 6.8, P = .0093). The sample showed the cation to be calcium. CONCLUSION: Crystallization on the IOL surface during cataract surgery is a rare occurrence that may be associated with calcium as the cation related to an osmotic gradient around the IOL with increased calcium concentration. If encountered surgically, the lens should be exchanged in the operating theater after irrigating the anterior chamber with BSS and completely filling the capsular bag with a low molecular weight viscoelastic.

Growth and characterization of ferroelectric Ba1-x Ca x TiO3 single crystals

Barium Calcium Titanate (Ba1-x Ca x TiO3) single crystals with various concentrations of calcium have been grown by high temperature solution growth technique using KF as solvent. The growth conditions were optimized for 12, 16 and 20 mole % of calcium. Concentric spiral growth and hopper structure were observed on the grown crystals. The variation in dielectric constant, refractive index and absorption coefficient for different Ca concentration has been reported.

Regulation of Intracellular Calcium Concentrations by Calcium and Magnesium in Cardioplegic Solutions Protects Rat Neonatal Myocytes from Simulated Ischemia

The effects of calcium and magnesium ions in cardioplegic solutions on cardioprotection and intracellular calcium ion handling during ischemia and reoxygenation were investigated in cultured neonatal rat myocardial cells. Myocytes were subjected to simulated ischemia for 60 min at 37°C in hyperkalemic cardioplegic solutions containing various concentrations of calcium and magnesium ions, followed by 30 min of reoxygenation. For each Ca2+concentration (0.1, 0.6, 1.2, or 2.4 mm), the Mg2+concentration was either 0, 1.2, 8, or 16 mm. The increase in intracellular Ca2+concentration during ischemia and reoxygenation was suppressed by the addition of magnesium ion, independent of cardioplegic Ca2+concentration. The recovery of spontaneous contraction rate and enzyme leakage (creatine phosphokinase and lactate dehydrogenase) during both ischemia and reoxygenation correlated with the degree of inhibition of intracellular Ca2+accumulation. However, in the 0.1 mmCa2+groups in which the Mg2+concentration was greater than 8 mm, the intracellular Ca2+concentration increased during reoxygenation in a dose-dependent fashion of Mg2+, and was associated with increased enzyme leakage. The findings suggest that in immature cardiac myocytes, the concentrations of Ca2+and Mg2+present in cardioplegic solutions control the intracellular Ca2+concentration during ischemia and reoxygenation, which, in turn, influences the cardioprotective effect of the cardioplegic solution.

Regulation of the 11 beta-hydroxysteroid dehydrogenase in the rat adrenal. Decrease enzymatic activity induced by ACTH.

Patients with ectopic ACTH syndrome often develop hypertension and hypokalemic alkalosis with an abnormal increase in the ratio of plasma cortisol to cortisone, indicating that 11 beta-hydroxysteroid dehydrogenase (11 beta HSD) activity is inhibited. Inhibition of 11 beta HSD allows access of cortisol or corticosterone to the mineralocorticoid receptor where it act as a mineralocorticoid. Two isozymes, 11 beta HSD-1 and 11 beta HSD-2, have been cloned and characterized. The rat adrenal expresses the mRNAs for 11 beta HSD-2 and, in lesser amounts, 11 beta HSD-1. We investigated the effect of ACTH on the 11 11 beta HSD-2 activity in the rat adrenal. Rat adrenal cells zone fasciculata (ZF) were dispersed and incubated separately with increasing concentrations of ACTH for 90 min, and secretion of corticosterone (B) and 11-dehydrocorticosterone (A) in the media was measured by enzyme-linked immunoabsorbent assays (ELISA). The conversion of [3H]B to [3H]A in the presence of 0.5 mM NAD+ was evaluated in microsomes prepared from dispersed cells preincubated for 30 min with cyanoketone and metyrapone followed by incubation for 30 min with the same inhibitors, with and without 10 nM ACTH. The dispersed cells of the ZF produced significant amounts of A which increased with ACTH. The basal B/A ratio was 0.97 +/- 0.05. ACTH caused a concentration-dependent increase in the ratio of B/A with a maximum ratio of 9.58 +/- 0.20. ACTH also inhibited the conversion of [3H]B to [3H]A in microsomes in which endogenous B production was inhibited by cyanoketone and metyrapone. ACTH did not change the K(m) for B conversion, but the Vmax was reduced significantly (1.73 +/- 0.43 pmol/min. mg protein), indicating that ACTH suppressed the 11 beta HSD-2 in a noncompetitive fashion. Dibutyryl cyclic AMP (dcAMP) also produced a concentration-dependent increase in the B/A ratio, but various concentrations of calcium did not affect the enzyme activity. In summary, adrenal cells treated with ACTH results in a significant increase in the ratio of B/A in the ZF owing a noncompetitive inhibition of the 11 beta HSD-2 via the ACTH receptor.

Aspects of calcium transport by the ovine placenta: Studies based on the interplacentomal region of the chorion

An in vitro technique for the measurement of calcium uptake into the maternal-facing fetal chorionic membrane (apical trophoblast) was used to study the relationship between calcium uptake and stage of pregnancy in the sheep. The effects on calcium uptake of varying calcium concentration and temperature of the incubation medium, of adding calcium channel blockers or heavy metals (lanthanum and nickel) or calcium ionophore/agonist were also studied. The data indicate a saturable calcium uptake process, plateauing after 15 min incubation. This uptake remained constant throughout the last third of gestation until a significant fall in uptake was noted during the final week prior to parturition. This uptake was not due to extracellular cellular diffusion since there was no significant uptake of tritiated inulin over the same period in each case. Calcium uptake in this system was also shown to be a temperature dependent process which was abolished at temperatures of 0-4 degrees C. A decrease in calcium concentration to 0.12 mM in the incubation medium also caused a corresponding decrease in calcium uptake to 21 per cent of control (1.2 mM). The addition of the heavy metals lanthanum and nickel also significantly reduced calcium uptake as did the calcium channel blockers verapamil, metoprolol and diltiazem. The calcium channel ionophore A23187 increased calcium uptake into the material facing chorion. Although the interplacentomal chorion may not be representative of the whole of the placental unit, it clearly contains a specific calcium uptake process under local physiological control. The blocking of calcium uptake by the specific I-type calcium channel blocker verapamil may indicate the presence of I-type channels of unusually low sensitivity since the concentration needed to block them was much higher than would be required for excitable I-type channels in isolated cells.

Effect of low pH on nitrogen fixation of common bean grown at various calcium and nitrate levels

The influence of low pH (4.0–4.5) of the medium on nodulation, nodule biomass, nodule nitrogenase activity, and ultrastructure at various concentrations of calcium (Ca) (0.127 mM and 0.508 mM) and nitrate (NO3) (0.390 mM and 1.560 mM) at different stages of development (2nd, 3rd, 6th, and 7th compound leaf) of the common bean plants was studied. The experiment was conducted using Hellriegel's nutrient solution under greenhouse conditions. Bean root nodule nitrogen (N2) fixation was influenced favorably at a decrease of Ca (0.127 mM) and NO3 (0.390 mM) concentrations in the growth solution. In contrast, the biomass of the roots, stems, leaves, and pods decreased at low Ca and NO3 levels while the most sensitive were the leaves and pods of the plants. The number of nodules, their fresh and dry weight, nitrogenase activity, and ultrastructure was affected to a greater extent at a low pH of the medium when Ca and NO3 concentrations were low (0.127 mM and 0.390 mM, respectively). Under these conditions, the roots, stems, leaves, and pods were slightly affected. Nodule N2 fixation was more sensitive to low pH at the 2nd and 3rd leaf stage of plant development. The root and pod biomass had the same tendency of change as the nodules. On the contrary, at low pH and decreased Ca and NO3 levels (0.127 mM and 0.390 mM, respectively), the stem and leaf biomass increased significantly at the later stages of bean plant development (6th and 7th leaf). Our conclusion was that the stress effects of low pH of the medium had different affects at various Ca and NO3 concentrations in the nutrient solution and stages of common bean plant development.

Effect of calcium marination on biochemical and textural properties of peri-rigor chicken breast meat

The objective of this study was to evaluate effects of marinades containing varying calcium concentrations on the biochemical and texture characteristics of peri-rigor chicken breast fillets. Breast muscles from 200 broiler chickens were excised immediately post-mortem and marinated in 0, 50, 100, 150, or 200 mM CaCl2. The treatments had no effect on meat pH either before or after cooking, but as calcium concentration increased, the normal post-mortem conversion of adenosine triphosphate (ATP) to inosine monophosphate (IMP) increased, according to the IMP:ATP ratios (R-values). Calcium treatment at all levels tested improved meat tenderness, but both marinade absorption and cooking losses increased as the calcium concentration in the marinades increased. It was concluded that although treating peri-rigor breast muscle with calcium might be useful in reducing or eliminating the conditioning period to assure tender chicken, methods must be developed for restoring the moisture binding properties that are damaged by the calcium.

Dye coupling in the muscles controlling squid chromatophore expansion

Dye coupling between the cone-shaped radial muscle fibres, which control the expansion and closing of a squid chromatophore organ, was investigated in the squid Loligo vulgaris. Particular attention was paid to the role of the myomuscular junctions located between the muscle fibres. Lucifer Yellow was injected ionophoretically into single muscle fibres under normal artificial sea water (ASW) and under various concentrations of calcium in ASW. Under ASW, 44% of muscle fibres examined were dye-coupled, 82% were coupled under calcium-free sea water and 67% were coupled under sea water containing high concentrations of calcium. Dye transfer was blocked by octanol. Muscle fibres were never seen to link adjacent chromatophore organs. Results are discussed in terms of the role of the myomuscular junctions in the regulation of chromatophore expansion in the living animal.

Voltammetric analysis of the competition between calcium and heavy metals for complexation by humic material

The competition between calcium and the heavy metals zinc(II) and cadmium(II) in their interaction with humic substances has been studied by differential pulse anodic stripping voltammetry at constant ionic strength. Two experimental methodologies were employed: (1) addition of humic acid to a solution of heavy metal; and (2) addition of heavy metals to a humic acid solution, both using various concentrations of calcium. The former method leads to complexation curves from which an overall mean stability for the heavy metal complex can be obtained. In the latter procedure, mean stability values are obtained at each titration point from the fractions of free and bound heavy metal ions. In the presence of competing ions like calcium, the association of heavy metals with the humic material can be well understood by incorporation of an additional equilibrium reaction for calcium with the humate polyanion. Hence, we are able to obtain information successfully on heavy metal speciation in more complex systems like natural waters, which contain competing cations such as calcium.

Regulation of intracellular cyclic GMP concentration by light and calcium in electropermeabilized rod photoreceptors.

This study examines the regulation of cGMP by illumination and by calcium during signal transduction in vertebrate retinal photoreceptor cells. We employed an electropermeabilized rod outer segment (EP-ROS) preparation which permits perfusion of low molecular weight compounds into the cytosol while retaining many of the features of physiologically competent, intact rod outer segments (ROS). When nucleotide-depleted EP-ROS were incubated with MgGTP, time- and dose-dependent increases in intracellular cGMP levels were observed. The steady state cGMP concentration in EP-ROS (0.007 mol cGMP per mol rhodopsin) approached the cGMP concentration in intact ROS. Flash illumination of EP-ROS in a 250-nM free calcium medium resulted in a transient decrease in cGMP levels; this occurred in the absence of changes in calcium concentration. The kinetics of the cGMP response to flash illumination of EP-ROS were similar to that of intact ROS. To further examine the effects of calcium on cGMP metabolism, dark-adapted EP-ROS were incubated with MgGTP containing various concentrations of calcium. We observed a twofold increase in cGMP steady state levels as the free calcium was lowered from 1 microM to 20 nM; this increase was comparable to the behavior of intact ROS. Measurements of guanylate cyclase activity in EP-ROS showed a 3.5-fold increase in activity over this range of calcium concentrations, indicating a retention of calcium regulation of guanylate cyclase in EP-ROS preparations. Flash illumination of EP-ROS in either a 50- or 250-nM free calcium medium revealed a slowing of the recovery time course at the lower calcium concentration. This observation conflicts with any hypothesis whereby a reduction in free calcium concentration hastens the recovery of cytoplasmic cGMP levels, either by stimulating guanylate cyclase activity or by inhibiting phosphodiesterase activity. We conclude that changes in the intracellular calcium concentration during visual transduction may have more complex effects on the recovery of the photoresponse than can be accounted for solely by guanylate cyclase activation.

Calcium Levels Affect the Ability to Immunolocalize Calmodulin to Cortical Microtubules.

Calcium affects the stability of cortical microtubules (MTs) in lysed protoplasts. This calmodulin (CaM)-mediated interaction may provide a mechanism that serves to integrate cellular behavior with MT function. To test the hypothesis that CaM associates with these MTs, monoclonal antibodies were produced against CaM, and one (designated mAb1D10) was selected for its suitability as an immunocytochemical reagent. It is shown that CaM associates with the cortical MTs of cultured carrot (Daucus carota L.) and tobacco (Nicotiana tabacum L.) cells. Inasmuch as CaM interacts with calcium and affects the behavior of these MTs, we hypothesized that calcium would alter this association. To test this, protoplasts containing taxol-stabilized MTs were lysed in the presence of various concentrations of calcium and examined for the association of CaM with cortical MTs. At 1 [mu]M calcium, many protoplasts did not have CaM in association with the cortical MTs, whereas at 3.6 [mu]M calcium, this association was completely abolished. Control experiments were performed to eliminate alternate explanations including differential antibody binding in the presence of calcium and/or taxol, detergent-induced redistribution of antigen, and epitope masking. The results are discussed in terms of a model in which CaM associates with MTs via two types of interactions, one that occurs in the presence of calcium and another that occurs only in its absence.

Transient hypocalcemic reperfusion does not improve postischemic recovery in the rat heart after preservation with St. Thomas’ Hospital cardioplegic solution

We used the isolated perfused working rat heart to investigate the effects of transient hypocalcemic reperfusion after cardioplegic arrest with the St. Thomas' Hospital cardioplegic solution and 25 minutes of global normothermic (37 degrees C) ischemia. Hearts were reperfused (Langendorff mode) transiently (20 minutes) with solutions containing various concentrations of calcium; this was followed by 30 minutes of reperfusion with standard (1.4 mmol/L, the physiologic concentration) calcium buffer (10 minutes in the Langendorff mode and 20 minutes in the working mode). Recovery of cardiac output in control hearts (calcium concentration 1.4 mmol/L throughout) was 51.7% +/- 4.6%; in hearts transiently reperfused with hypocalcemic buffer (0.25, 0.5, 0.75, or 1.0 mmol/L) the recoveries of cardiac output were 49.3% +/- 6.4%, 52.2% +/- 7.2%, 58.7% +/- 3.2%, and 47.2 +/- 4.7%, respectively (all not significant), whereas recovery was only 14.7% +/- 2.8% (p less than 0.05) in hearts transiently reperfused with calcium 0.1 mmol/L. Creatine kinase leakage was significantly (p less than 0.05) greater in the group reperfused with calcium 0.1 mmol/L, but it did not vary significantly between the other groups. Tissue high-energy phosphate content was similar and in the normal range in all groups except for the group reperfused with calcium 0.1 mmol/L. In further experiments, the duration of hypocalcemic (0.5 mmol/L) reperfusion was varied (0, 5, 10, 15, 20, or 30 minutes). No significant differences in recovery of cardiac output were observed (58.2% +/- 5.0%, 52.3% +/- 5.7%, 52.0% +/- 8.2%, 61.2% +/- 5.0%, 62.2% +/- 4.3%, and 66.2% +/- 3.2%, respectively). In additional studies, the standard calcium concentration (1.4 mmol/L) used before and after ischemia was replaced by hypercalcemic solution (2.5 mmol/L). Despite this, transient (10 minutes) hypocalcemic (0.5 mmol/L) reperfusion did not improve recovery. Finally, studies were undertaken with a longer duration of ischemia (40 minutes), and although recovery of cardiac output in the hypocalcemic group (0.5 mmol/L for 10 minutes) tended to be higher than in the control group (29.7% +/- 4.8% versus 18.5% +/- 4.9%, respectively), statistical significance was not achieved. We conclude that in these studies transient hypocalcemic reperfusion did not afford any additional protection over and above that afforded by cardioplegia alone.

Effect of hypoxia upon intracellular calcium concentration of human endothelial cells.

Ischemia is a situation occurring in several diseases including myocardial infarction and organ transplantation in which oxygenated blood supply is impaired. Ischemia leads to many cellular and tissue modifications, the most important one being cell death. Several explanations have been proposed to account for these modifications and cell death; among them is calcium overload. However, the influence of calcium concentration on the alteration of endothelial cell functions or viability during ischemia are still unknown. We developed here an in vitro model where human endothelial cell monolayers were submitted to hypoxia with or without reoxygenation and variation in calcium concentration was followed using a specific intracellular probe Fura 2. We observed a significant increase of [Ca2+]i during 2 h hypoxia reaching values similar to those observed during agonist stimulation of endothelial cells but far lower than values toxic for the cells. This increase was constant during the hypoxic incubation and was due mainly to an influx of extracellular calcium. Viability was also followed during hypoxia and using calcium channel blockers, we could show that there was no correlation between viability and the rise in calcium concentration. During the reoxygenation period, [Ca2+]i decreased to reach the normal value of resting cells after 45 min, suggesting that cells were still able to recover their calcium homeostasis. The use of a ketone body (beta-hydroxybutyrate) indicated that an energy deficiency was responsible for the hypoxia-induced increase in [Ca2+]i. We actually observed a 43% decrease in ATP concentration after 2 h hypoxia. This decrease was already significant after 30 min which thus precedes the changes in [Ca2+]i. These results show that during hypoxia, energy deficiency led to an increase in [Ca2+]i which is, however, too low to account for the loss of viability but which is within the range of concentrations observed during stimulation of endothelial cells. We propose that such increased intracellular calcium concentrations could play a role in the synthesis of mediators leading to the development of local inflammation.

Lung protection against paraquat is calcium dependent.

Isolated, perfused, and ventilated rat lungs were challenged by paraquat (0.01 M) in the presence of 2.5 mM Ca2+, 2.5 mM Ca2+ with trifluoperazine (100 microM), 0.025 mM Ca2+, or 0.025 mM Ca2+ with sodium metavanadate (10 microM) to establish the effect of varying calcium concentration or calcium-dependent enzyme activities on injury induced by paraquat. Segmental vascular resistances, microvascular permeability (as assessed by the capillary filtration coefficient), lung tissue oxidized glutathione, and lung paraquat accumulation were measured. Exposure to paraquat for 2.5 h did not increase microvascular permeability or pulmonary vascular resistance in the presence of either normal extracellular calcium or low extracellular calcium and sodium metavanadate. Lungs exposed to paraquat were injured (as assessed by increased filtration coefficient) only in the presence of low extracellular calcium or after trifluoperazine was added. This injury was associated with decreased levels of oxidized glutathione and increased paraquat accumulation, suggesting that calcium's protective effect was both by inhibition of paraquat accumulation and maintenance of NADPH. Pulmonary vascular resistance was not increased with paraquat challenge.

Mechanisms of calcium relaxation of vascular smooth muscle

We examined mechanisms of relaxation in intact and endothelium-denuded thoracic aortic rings of rat in response to various concentrations of calcium (from 1.6 to 10.1 mM) after contraction induced by 30 mM KCl. The relaxation to calcium was concentration dependent in intact and denuded rings. This effect was greater in intact preparations than in denuded preparations or in intact preparations treated with methylene blue (10(-5) M). This indicates that the relaxation by calcium is partly mediated by releasing endothelium-derived relaxing factors (EDRFs). In the presence of A23187, the relaxation to calcium was attenuated in intact rings but not in denuded rings, whereas calcium relaxation was not significantly altered by sodium nitroprusside. These observations suggest that the calcium-induced relaxation is reduced if EDRF has already been released by A23187. We conclude that, paradoxically, the same increase in extracellular Ca2+ concentration that causes an increase in endothelial intracellular calcium concentration ([Ca2+]i) to produce EDRF also results in membrane stabilization of the vascular smooth muscle cell to decrease [Ca2+]i and cause relaxation. These two mechanisms are additive in producing calcium relaxation in the intact artery.