Increase In Extracellular Calcium Research Articles

Differential effects of Fe2+ and Fe3+ on osteoblasts and the effects of 1,25(OH)2D3, deferiprone and extracellular calcium on osteoblast viability under iron-overloaded conditions.

One of the potential contributing factors for iron overload-induced osteoporosis is the iron toxicity on bone forming cells, osteoblasts. In this study, the comparative effects of Fe3+ and Fe2+ on osteoblast differentiation and mineralization were studied in UMR-106 osteoblast cells by using ferric ammonium citrate and ferrous ammonium sulfate as Fe3+ and Fe2+ donors, respectively. Effects of 1,25 dihydroxyvitamin D3 [1,25(OH)2D3] and iron chelator deferiprone on iron uptake ability of osteoblasts were examined, and the potential protective ability of 1,25(OH)2D3, deferiprone and extracellular calcium treatment in osteoblast cell survival under iron overload was also elucidated. The differential effects of Fe3+ and Fe2+ on reactive oxygen species (ROS) production in osteoblasts were also compared. Our results showed that both iron species suppressed alkaline phosphatase gene expression and mineralization with the stronger effects from Fe3+ than Fe2+. 1,25(OH)2D3 significantly increased the intracellular iron but minimally affected osteoblast cell survival under iron overload. Deferiprone markedly decreased intracellular iron in osteoblasts, but it could not recover iron-induced osteoblast cell death. Interestingly, extracellular calcium was able to rescue osteoblasts from iron-induced osteoblast cell death. Additionally, both iron species could induce ROS production and G0/G1 cell cycle arrest in osteoblasts with the stronger effects from Fe3+. In conclusions, Fe3+ and Fe2+ differentially compromised the osteoblast functions and viability, which can be alleviated by an increase in extracellular ionized calcium, but not 1,25(OH)2D3 or iron chelator deferiprone. This study has provided the invaluable information for therapeutic design targeting specific iron specie(s) in iron overload-induced osteoporosis. Moreover, an increase in extracellular calcium could be beneficial for this group of patients.

Length-Dependent Prolongation of Force Relaxation Is Unaltered by Delay of Intracellular Calcium Decline in Early-Stage Rabbit Right Ventricular Hypertrophy.

Chronic pressure overload can result in ventricular hypertrophy and eventually diastolic dysfunction. In normal myocardium, the time from peak tension to 50% relaxation of isolated cardiac myocardium is not directly determined by the time for calcium decline. This study aims to determine whether the time for calcium decline is altered with a change in preload in early-stage hypertrophied myocardium, and whether this change in time for calcium decline alters the rate of relaxation of the myocardium. Young New Zealand white rabbits underwent a pulmonary artery banding procedure and were euthanized 10 weeks later. Twitch contractions and calibrated bis-fura-2 calcium transients were measured in isolated thin right ventricular trabeculae at optimal length and with the muscle taut. Systolic calcium, calcium transient amplitude, and time from peak tension to 50% relaxation all increased with an increase in preload for both hypertrophied and sham groups. Time for intracellular calcium decline increased both with an increase in preload and an increase in extracellular calcium concentration in hypertrophied myocardium but not in sham, while time from peak tension to 50% relaxation did not significantly change between groups under either condition. Also, time for intracellular calcium decline generally decreased with an increase in extracellular calcium for both hypertrophied and sham groups, while time from peak tension to 50% relaxation generally did not significantly change in either group. Combined, these results indicate that the mild hypertrophy significantly changes calcium handling, but does not impact on the rate of force relaxation. This implies that the rate-limiting step in force relaxation is not directly related to calcium transient decline.

The increase of extracellular calcium and calcium ion channel activity may affect the inflammatory action of lipopolysaccharide‐induced RAW 264.7 murine macrophage cells

Calcium is known to be a ubiquitous secondary signaling molecule that has very important roles in practically all cell types. Calcium is known to be involved in various processes such as proliferation, differentiation, apoptosis and transcriptional programs. In the current study, we determined the effects of alterations in the extracellular calcium as well as calcium ion channel activity on the immune modulative function of LPS‐induced RAW 264.7 murine macrophage cells. Several assays such as cell viability analysis, nitric oxide production analysis and reverse transcriptase–polymerase chain reaction were done in order to investigate the effects of extracellular calcium on immunomodulation. Results have shown that increasing extracellular calcium up to 6mM leads to an increase in the proliferation rate of the cells after stimulation with lipopolysaccharide. This increase in the proliferation rate had no corresponding increase in the rate of nitric oxide production and basal expression of inflammation‐related genes (NF‐KB, IL‐1β, TNFα, IL‐6 and IFNγ). In order to assess the effect of an altered calcium ion movement in the immune cells, ionomycin was used to increase the cellular entry of calcium. Treatment with ionomycin resulted in an increase in cellular proliferation as well as nitric oxide production but not to a significant extent which could imply a tight regulation for the functions of the mitogen‐activated cell involving calcium as a secondary signaling molecule. Also, RT‐PCR assay have shown only slight changes in the inflammation‐related genes in the study. In conclusion, the study was able to show that changes in calcium ion concentration across macrophage cell population results in minimal changes in immune function probably due to the highly regulated nature of the ion in such a way as to preserve its function as an immune cell.

Calcium Modulates Herg Function by Acting at Two Distinct Binding Sites

Reduction of the current carried by the cardiac potassium channel HERG can lead to Long QT syndrome, an arrhythmia characterized by a rapid heart rate and reduced cardiac output, which can, in certain situations, be fatal. The effect of extracellular electrolytes on the biophysical properties of the HERG channel have been studied in some detail. In particular, increases in extracellular calcium, magnesium and hydrogen have been shown to slow channel activation, increase channel deactivation, and shift the G-V curve to more positive voltages. A number of reports have also shown that hydrogen can reduce HERG current by a mechanism that does not involve an effect on channel deactivation and which likely involves pore block. We have shown previously that reducing extracellular potassium results in a significant increase in HERG current reduction by a number of extracellular divalent ions, including calcium, magnesium, cobalt, manganese, and hydrogen. We have also shown that current reduction of the HERG mutant G628CS631A is significantly reduced compared to WT at all voltages tested. We show here that the positive shift in the G-V curve of the WT HERG due to an increase in extracellular calcium is much less sensitive to potassium than the effect of extracellular potassium on current reduction. In addition, the G-V curve of the HERG double mutant G682CS631 (which shows reduced current reduction with an increase in extracellular calcium) is shifted to the right with an increase in extracellular calcium in a similar manner to the positive shift with increased extracellular calcium seen with the WT channel. Both of these findings are consistent two separate calcium binding sites: one site that interacts with the voltage sensor and one site that may interact with the outer pore of the HERG channel.

T-type calcium channels contribute to calcium disturbances in brain during hyponatremia

Disturbance of calcium homeostasis is implicated in the normal process of aging and brain pathology prevalent in the elderly such as Alzheimer's, Parkinson's, and amyotrophic lateral sclerosis. Previous studies demonstrated that applying a hyponatremic iso-osmotic (low-NaCl) artificial cerebrospinal fluid (ACSF) to rodent hippocampus causes extracellular calcium to rapidly decrease. Restoring normonatremia after low-NaCl treatment causes a rapid increase in extracellular calcium that overshoots baseline. This study examined the amplitude, timing, and mechanism of these surprising calcium changes. We also tested whether hyponatremia increased calcium entry into brain cells or calcium binding to chondroitin sulfate (CS), a negatively charged constituent of the extracellular matrix (ECM) that may be occupied by sodium during normonatremia. We report three major findings. First we show that CS does not contribute to extracellular calcium changes during low-NaCl treatments. Second, we show that the time to minimum extracellular calcium during low-NaCl treatment is significantly shorter than the time to maximum extracellular calcium in recovery from low-NaCl treatment. Third, we show that the decrease in extracellular calcium observed during hyponatremia is attenuated by ML 218, a highly selective T-type calcium channel blocker. Together these data suggest that calcium rapidly enters cells at the onset of low-NaCl treatment and is extruded from cells when normonatremia is restored. Calcium binding to CS does not significantly contribute to calcium changes in brain during hyponatremia. Differences in timing suggest that extracellular calcium changes during and in recovery from hyponatremia occur by distinct mechanisms or by a multistep process. Finally, partial block of extracellular calcium influx by ML 218 suggests that T-type channels are involved in calcium entering cells during hyponatremia. Given the high prevalence of hyponatremia among elderly patients and the growing understanding of calcium's role in multiple neurologic pathologies, this study promotes a novel approach for studying and potentially preventing the effects of hyponatremia on calcium dysregulation in brain tissue.

The cardiac potassium channel HERG is blocked by calcium at a site near the outer mouth of the channel

A reduction in the number of HERG channels has been implicated in Long QT Syndrome, which can degenerate into the lethal arrhythmia Torsades de Pointes. Patients can present with abnormal serum electrolyte levels due to a variety of conditions including gastrointestinal dysfunction, renal and endocrine disorders, diuretic use, alcoholism, and aging. The aim of this study is to determine if extracellular calcium blocks the pore of the HERG channel by acting at a site in the outer vestibule of the channel.Divalent block was measured using two‐electrode voltage clamping of Xenopus oocytes expressing either wild‐type HERG or the HERG mutants S631A, and G628CS631C. S631 is thought to be located in the outer pore of the HERG channel.Changing extracellular potassium from 0 mM to 20 mM resulted in a greater decrease in WT HERG current due to an increase in extracellular calcium, as well as a number of other divalent ions (Mg2+, Co2+ and Mn2+). In addition, block of WT HERG by calcium was voltage dependent, with increased block at negative voltages. Finally, there was little or no block of the HERG double mutant G628CS631C by calcium at all voltages tested, whereas there was increased block of the HERG mutant S631A by calcium at negative voltages.3 results suggest that calcium blocks HERG by physically occluding the pore of the channel: 1) the dependence of block on extracellular potassium. 2) the lack of block of G628CS631C 3) the altered voltage dependence of block of S631A. This study has implications for an increased risk of cardiac arrhythmias with hypokalemia as well as for patients with acute promyelocitic leukemia treated with arsenic, a trivalent ion that might block HERG at the same site as calcium.Funded by Touro University and NIHR 15‐1R15HL079971

The role of calcium and nicotinic acid adenine dinucleotide phosphate (NAADP) in human osteoclast formation and resorption.

Osteoclasts are specialised bone resorbing cells which form by fusion of circulating mononuclear phagocyte precursors. Bone resorption results in the release of large amounts of calcium into the extracellular fluid (ECF), but it is not certain whether changes in extracellular calcium concentration [Ca(2+)]e influence osteoclast formation and resorption. In this study, we sought to determine the effect of [Ca(2+)]e and NAADP, a potent calcium mobilising messenger that induces calcium uptake, on human osteoclast formation and resorption. CD14+ human monocytes were cultured with M-CSF and RANKL in the presence of different concentrations of calcium and NAADP and the effect on osteoclast formation and resorption evaluated. We found that the number of TRAP+ multinucleated cells and the extent of lacunar resorption were reduced when there was an increase in extracellular calcium and NAADP. This was associated with a decrease in RANK mRNA expression by CD14+ cells. At high concentrations (20mM) of [Ca(2+)]e mature osteoclast resorption activity remained unaltered relative to control cultures. Our findings indicate that osteoclast formation is inhibited by a rise in [Ca(2+)]e and that RANK expression by mononuclear phagocyte osteoclast precursors is also [Ca(2+)]e dependent. Changes in NAADP also influence osteoclast formation, suggesting a role for this molecule in calcium handling. Osteoclasts remained capable of lacunar resorption, even at high ECF [Ca(2+)]e, in keeping with their role in physiological and pathological bone resorption.

Ruling in a Suspect: The Role ofAP2S1Mutations in Familial Hypocalciuric Hypercalcemia Type 3

Ruling in a Suspect: The Role of<i>AP2S1</i>Mutations in Familial Hypocalciuric Hypercalcemia Type 3

The influence of pH on the cardiac depressant action of tobramycin

Tobramycin, like other aminoglycoside antibiotics, is a basic compound which causes a reduction of cardiac contractile force and inhibits the inotropic action of calcium. Its antagonistic action towards calcium had been analysed at different pH values of the solution bathing the rat left atrium. The isometric tension of the electrically driven atrium (4 Hz, 2 ms, supramaximal voltage) in response to cumulative increase in the bath calcium concentration (2.0 to 32.0 mM) was measured in the absence and in the presence of 2.0 x 10(-5) M tobramycin. The increase in pH from 6.6 to 8.2, which decreased the ionization of tobramycin, reduced the cardiac depression. Similar experiments were carried with other cardiac depressants. Thus, the effect of methoxyverapamil (3.5 x 10(-7) M), also a basic compound, was increased at higher values of pH; the acidic pentobarbitone (4 x 10(-5)M) was less effective at more alkaline pH; the cardiac depression of the non-electrolyte ethanol (2 x 10(-4)M) was practically the same over the pH range studied. The time for equilibrium of the antagonism was small for tobramycin, moreover, its antagonism could only be fully reversed by an increase in extracellular calcium. These results suggest a competitive type of antagonism, between the ionized fraction of tobramycin and calcium, for superficial sites of the sarcolemma. The effect of methoxyverapamil, pentobarbitone and ethanol seems to occur at a more internal site of the cardiac cell since the unionized form of the drug is the most active.

TOLL-LIKE RECEPTOR 4 MODULATES CALCIUM SENSITIZATION IN RESISTANCE ARTERIES: 3D.07

Objectives: Inflammation is associated with hypertension and cardiovascular disease. The role of the innate immune system on microvascular smooth muscle function remains unclear. We tested whether Toll-like receptors (TLRs), key players of the innate immune system, sensitize microvascular myosin light chain phosphatase (MLCP), thus calcium sensitization, and thereby the contractile apparatus. Methods: Mesenteric resistance arteries (RA) of TLR4-/−, TLR2−/−, MyD88−/− and wildtype mice (C57/Bl6) were investigated by wire myography. After viability testing calcium sensitivity was assessed in depolarized RA (120 mol/L K+) exposed to stepwise increases in extracellular calcium (0 to 3 mmol/L). Calcium sensitivity was determined before and after application of 10 μmol/L sodium nitroprusside (SNP), the soluble guanylate cyclase (sGC) inhibitor ODQ (1 μmol/L), the MLCP inhibitor Calyculin A (120 nmol/L) or in presence of the Rho kinase inhibitor Y27632 (1 μmol/L). Wall-to-Lumen ratio (W/L) was assessed by histology. Blood pressure was assessed by radiotelemetry. Results: TLR4−/−, but not TLR2−/− or MyD88−/− mice were characterized by a reduced calcium sensitivity as compared to wildtype. Calyculin A and ODQ, but not Y27632 or the supplementation of NO abolished this difference in TLR4−/−. W/L was reduced in TLR4−/−, whereas blood pressure was similar between TLR4−/− and wildtype mice. Conclusions: The MyD88-independent TLR4 signaling pathway is characterized by attenuated vascular contractility under resting conditions. This involves attenuated calcium desensitization based on a cGMP-dependent activation of the MLCP in TLR4−/−.

Dinámica in vitro de la secreción de hormona paratiroidea regulada por calcio y efecto sobre el ciclo celular: adenoma frente a hiperplasia paratiroidea

To compare the dynamics of calcium-regulated PTH secretion in vitro from adenomatous versus hyperplastic glands and to investigate the relationship between the parathyroid cell cycle and the calcium-regulated PTH secretion in these glands. A total of 31 parathyroid glands (8 adenomatous and 23 hyperplastic) from 8 patients with primary hyperparathyroidism and 7 with secondary hyperparathyroidism respectively were studied. For the evaluation of calcium-regulated PTH secretion, small parathyroid pieces of 1 mm were sequentially transferred to wells with varying Ca concentrations: 0.4, 0.6, 0.8, 1, 1.25 and 1.35 or 1.5 mM. PTH concentrations were determined in the medium. For the parathyroid cell cycle studies, parathyroid cells were isolated without the use of enzymes and cell cycle was analyzed using the method described by Vindelov. The nuclei were acquired by flow cytometer and analyzed using the CELLFIT software. In parathyroid tissues from hyperplastic glands, the increase in extracellular calcium produced a decrease in PTH secretion which was apparent with a calcium level as low as 0.8 mM and the maximal inhibition of PTH secretion was obtained with a calcium of 1.25 mM, by the contrary, adenomatous glands required a calcium of 1.2 mM to produce a minimal decrease in PTH secretion. In hyperplastic parathyroid glands but not in parathyroid adenomas there was a significant correlation between the percentage of cells in G0/G1 phase with the set point (r = 0.914; P < 0.005) and the basal serum Ca (r = 0.862; P < 0.02). The control of the extracellular calcium-PTH release in vitro is less sensitive in parathyroid adenomas than hyperplasic parathyroid glands. In parathyroid hyperplasia the cell proliferation may be regulated by the extracellular calcium concentration (higher calcemia less proliferation).

The calcium-sensing receptor changes cell shape via a β-arrestin-1–ARNO–ARF6–ELMO protein network

G-protein-coupled receptors (GPCRs) transduce the binding of extracellular stimuli into intracellular signalling cascades that can lead to morphological changes. Here, we demonstrate that stimulation of the calcium-sensing receptor (CaSR), a GPCR that promotes chemotaxis by detecting increases in extracellular calcium, triggers plasma membrane (PM) ruffling via a pathway that involves beta-arrestin 1, Arf nucleotide binding site opener (ARNO), ADP-ribosylating factor 6 (ARF6) and engulfment and cell motility protein (ELMO). Expression of dominant negative beta-arrestin 1 or its knockdown with siRNA impaired the CaSR-induced PM ruffling response. Expression of a catalytically inactive ARNO also reduced CaSR-induced PM ruffling. Furthermore, beta-arrestin 1 co-immunoprecipitated with the CaSR and ARNO under resting conditions. Agonist treatment did not markedly alter beta-arrestin 1 binding to the CaSR or to ARNO but it did elicit the translocation and colocalisation of the CaSR, beta-arrestin 1 and ARNO to membrane protrusions. Furthermore, ARF6 and ELMO, two proteins known to couple ARNO to the cytoskeleton, were required for CaSR-dependent morphological changes and translocated to the PM ruffles. These data suggest that cells ruffle upon CaSR stimulation via a mechanism that involves translocation of beta-arrestin 1 pre-assembled with the CaSR or ARNO, and that ELMO plays an essential role in this CaSR-signalling-induced cytoskeletal reorganisation.

Hypertrophy and transcriptional regulation induced in myogenic cell line L6-C5 by an increase of extracellular calcium.

Calcium plays a pivotal role in the establishment of the differentiated phenotype in myogenic cells but the involved molecular mechanisms are still matter of debate. Here we studied the effects of exposing L6-C5 myogenic cells to high extracellular Ca2+ concentration ([Ca2+]o), which induces an increase of intracellular calcium ([Ca2+]i) without involving Ca2+ release from the intracellular stores but exclusively due to plasma membrane influx (Naro et al., 2003). Exposure of L6-C5 cells to [Ca2+]o up to 20 mM for 30 min, before shifting them into a differentiative medium, induced the appearance of multinucleated, myosin-positive myotubes, much larger than in control cells with an increased protein/DNA ratio. These large myotubes showed nuclear accumulation of the hypertrophy marker GATA-2. The hypertrophic growth of these cells was blocked by cyclosporin A (CsA), FK506, or overexpression of a calcineurin-dominant negative protein, suggesting the involvement in this process of the Ca2+ responsive phosphatase calcineurin. Furthermore, transient exposure of L6-C5 cells to high [Ca2+]o increased the expression of luciferase reporter driven by myoglobin (Mb) and beta-MHC promoters but not IIB-MHC and MCK promoters. Luciferase transcription driven by CK promoter was, instead, enhanced by mobilizing Ca2+ from the intracellular stores. These data indicate that a transient increase of [Ca2+]i due to plasma-membrane influx is sufficient to induce a hypertrophic phenotype and an increased expression of slow-fiber genes but not fast-fiber genes.

Role of acetylated human AP-endonuclease (APE1/Ref-1) in regulation of the parathyroid hormone gene.

The human AP-endonuclease (APE1/Ref-1), a multifunctional protein central to repairing abasic sites and single-strand breaks in DNA, also plays a role in transcriptional regulation. Besides activating some transcription factors, APE1 is directly involved in Ca2+-dependent downregulation of parathyroid hormone (PTH) expression by binding to negative calcium response elements (nCaREs) present in the PTH promoter. Here we show that APE1 is acetylated both in vivo and in vitro by the transcriptional co-activator p300 which is activated by Ca2+. Acetylation at Lys6 or Lys7 enhances binding of APE1 to nCaRE. APE1 stably interacts with class I histone deacetylases (HDACs) in vivo. An increase in extracellular calcium enhances the level of acetylated APE1 which acts as a repressor for the PTH promoter. Moreover, chromatin immunoprecipitation (ChIP) assay revealed that acetylation of APE1 enhanced binding of the APE1-HDACs complex to the PTH promoter. These results indicate that acetylation of APE1 plays an important role in this key repair protein's action in transcriptional regulation.

Calcium and polyamine regulated calcium-sensing receptors in cardiac tissues.

Activation of a calcium-sensing receptor (Ca-SR) leads to increased intracellular calcium concentration and altered cellular activities. The expression of Ca-SR has been identified in both nonexcitable and excitable cells, including neurons and smooth muscle cells. Whether Ca-SR was expressed and functioning in cardiac myocytes remained unclear. In the present study, the transcripts of Ca-SR were identified in rat heart tissues using RT-PCR that was further confirmed by sequence analysis. Ca-SR proteins were detected in rat ventricular and atrial tissues as well as in isolated cardiac myocytes. Anti-(Ca-SR) Ig did not detect any specific bands after preadsorption with standard Ca-SR antigens. An immunohistochemistry study revealed the presence of Ca-SR in rat cardiac as well as other tissues. An increase in extracellular calcium or gadolinium induced a concentration-dependent sustained increase in [Ca2+]i in isolated ventricular myocytes from adult rats. Spermine (1-10 mm) also increased [Ca2+]i. Pre-treatment of cardiac myocytes with thapsigargin or U73122 abolished the extracellular calcium, gadolinium or spermine-induced increase in [Ca2+]i. The blockade of Na+/Ca2+ exchanger or voltage-dependent calcium channels did not alter the extracellular calcium-induced increase in [Ca2+]i. Finally, extracellular calcium, gadolinium and spermine all increased intracellular inositol 1,4,5-triphosphate (IP3) levels. Our results demonstrated that Ca-SR was expressed in cardiac tissue and cardiomyocytes and its function was regulated by extracellular calcium and spermine.

Calcium Modulates Promoter Occupancy by the Entamoeba histolytica Ca2+-binding Transcription Factor URE3-BP

The Entamoeba histolytica upstream regulatory element 3-binding protein (URE3-BP) binds to the URE3 sequence of the Gal/GalNAc-inhibitable lectin hgl5 and ferredoxin 1 (fdx) gene promoters. This binding can be inhibited in vitro by addition of calcium. Two EF-hand motifs, which are associated with the ability to bind calcium, are present in the amino acid sequence of URE3-BP. Mutation of the second EF-hand motif in URE3-BP resulted in the loss of calcium inhibition of DNA binding as monitored by electrophoretic mobility shift assay. Chromatin immunoprecipitation assays revealed that URE3-BP was physically bound to the hgl5 and fdx promoters in vivo. Parasite intracellular calcium concentrations were altered by changes in extracellular calcium. Promoter occupancy was lost when intracellular calcium levels were increased by coordinate increases in extracellular calcium. Increased intracellular calcium also resulted in decreased levels of URE3-BP mRNA. Together these results demonstrate that changes in extracellular calcium result in changes in URE3-BP mRNA and in the ability of URE3-BP to bind to URE3-containing promoters. Modulation of URE3-BP by calcium may represent an important mechanism of control of gene expression in E. histolytica.

Is cytotoxic cellular edema real? The effect of calcium ion on water homeostasis in the rat heart.

The objective of this study was to determine the effect of calcium on water homeostasis in cardiac muscle and whether cytotoxic edema is involved in ischemic-reperfusion injury. Cellular water was quantified by multinuclear nuclear magnetic resonance (NMR) spectroscopy in isolated rat hearts during 30 min of ischemia and reperfusion. An increase in extracellular calcium from 0.75 to 2.25 mM increased ischemic swelling twofold, with a marginal effect on functional recovery at reperfusion. Inhibition of Ca2+ channels with verapamil and bepridil reduced ischemic swelling by 58-66% and improved cardiac viability and functional recovery. Inhibition of the Na/Ca exchange with amiloride resulted in reperfusion swelling resulting from the inhibition of calcium efflux via the Na/Ca exchange. This was confirmed by inhibition with amiloride of 63% of cellular swelling induced by calcium overload via the Na/Ca exchange after calcium-free perfusion (calcium paradox). The volume-related activity of amiloride was attributed to the inhibition of Na/Ca exchange because the inhibition of the Na/H antiport resulted in low cellular volumes. The consistent changes in cellular volumes induced by the various agents used to alter calcium fluxes provide evidence of a pro-edematous effect of intracellular calcium accumulation during myocardial ischemia. The administration of toxic doses of bepridil to the perfused and ischemic heart resulted in the shrinkage of cellular volumes and in functional failure. The toxicity of bepridil was mediated by unrestricted release of calcium from the sarcoplasmic reticulum because magnesium cardioplegia, which limits calcium release, lowered ischemic swelling without cellular shrinkage and improved functional recovery. In all experimental groups, cellular edema subsided during the early stages of reperfusion, indicating efficient dissipation of intracellular hyperosmolarity. There was no evidence that cytotoxic edema develops during ischemia or reperfusion, or both. On the contrary, calcium-mediated cardiac toxicity was associated with cellular volume shrinkage as a result of the efflux of osmolytes through permeabilized sarcolemmal membranes. It is concluded that calcium ion is involved in the regulation of cellular volumes, and its intracellular accumulation promotes swelling in ischemic myocytes with intact membranes.

Calcium regulates estrogen increase in permeability of cultured CaSki epithelium by eNOS-dependent mechanism.

Estrogen increases baseline transepithelial permeability across CaSki cultures and augments the increase in permeability in response to hypertonic gradients. In estrogen-treated cells, lowering cytosolic calcium abrogated the hypertonicity-induced augmented increase in permeability and decreased baseline permeability to a greater degree than in estrogen-deprived cells. Steady-state levels of cytosolic calcium in estrogen-deprived cells were higher than in estrogen-treated cells. Increases in extracellular calcium increased cytosolic calcium more in estrogen-deprived cells than in estrogen-treated cells. However, in estrogen-treated cells, increasing cytosolic calcium was associated with greater increases in permeability in response to hypertonic gradients than in estrogen-deprived cells. Lowering cytosolic calcium blocked the estrogen-induced increase in nitric oxide (NO) release and in the in vitro conversion of L-[(3)H]arginine to L-[(3)H]citrulline. Treatment with estrogen upregulated mRNA of the NO synthase isoform endothelial nitric oxide synthase (eNOS). These results indicate that cytosolic calcium mediates the responses to estrogen and suggest that the estrogen increase in permeability and the augmented increase in permeability in response to hypertonicity involve an increase in NO synthesis by upregulation of the calcium-dependent eNOS.

Signal Transduction Mechanisms Linking Increased Extracellular Calcium to Proliferation in Ovarian Surface Epithelial Cells

Although ovarian surface epithelial (OSE) cells are the cell type responsible for malignant ovarian carcinoma, relatively little is known about either the extracellular stimuli or the intracellular signaling mechanisms responsible for regulating proliferation in these cells. We have demonstrated that OSE cells proliferate in response to elevation of extracellular calcium and that OSE cells express functional calcium-sensing receptors (CaR). Here we show that agonists of the CaR increase the kinase activity of Src and ERKs (extracellular signal-regulated kinases) in rat OSE cells and promote association between tyrosine-phosphorylated Shc and p120rasGAP. Expression of an interfering mutant CaR inhibited the proliferative response to elevated extracellular calcium, as well as CaR agonist-induced tyrosine phosphorylation and ERK activation. Transfection with dominant negative mutants of Ras, Raf, and MKK1 also inhibited the increase in ERK activity in response to calcium, as did treatment with herbimycin, a selective inhibitor for Src family kinases. These results indicate that the ability of OSE cells to proliferate in response to increases in extracellular calcium involves cross-talk between the G-protein-coupled CaR and the activation of a tyrosine kinase-dependent Ras–Raf–ERK signaling pathway.

Glowing with Calcium

Proteins that luminesce when they bind calcium are useful tools for studying calcium signaling and homeostasis. Torrecilla et al. generated aequorin-expressing cyanobacteria to determine whether these bacteria used calcium as a signal in response to temperature shock. First, the authors characterized the calcium homeostasis mechanism and found a biphasic response to increases in extracellular calcium with the slower decay phase dependent on both the reduction of [Ca2+]i by calcium binding proteins and calcium transporters. Calcium transients were generated in response to both heat and cold shock. The mechanisms of increased [Ca2+]i were different: Increased [Ca2+]i produced by heat shock arises from calcium released from both intracellular stores and by influx from the extracellular space, and increased [Ca2+]i produced by cold shock arises from predominantly extracellular sources.Torrecilla, I., Leganés, F., Bonilla, I., and Fernández-Piñas, F. (2000) Use of recombinant aequorin to study calcium homeostasis and monitor calcium transients in response to heat and cold shock in cyanobacteria. Plant Physiol. 123: 161-175. [Abstract] [Full Text]