Effect of calbindin-D28K on cyclosporine toxicity in cultured renal proximal tubular cells

Cyclosporine A (CsA) is known to have direct toxicity to renal tubular cells. Its toxicity may be mediated by intracellular calcium because CsA increases intracellular calcium concentration and enhances the activities of calcium-dependent calpains and caspases. Calbindin-D28k, a cytosolic calcium binding protein, has been used as an intracellular Ca2+ buffer to reduce calcium-mediated cytotoxicity in non-renal cells such as neuronal cells. We investigated the effects of gene transfer of calbindin-D28k cDNA on CsA cytotoxicity and intracellular calcium concentration ([Ca2+]i) in cultured murine proximal tubular (MCT) cells. A plasmid containing calbindin-D28k cDNA under the control of CMV promoter was transfected to MCT cells with liposomes. Cytotoxicity was assessed by LDH release and cell viability assay, and [Ca2+]i was measured ratiometrically with fura-2. Compared with MCT cells, cells transfected with calbindin-D28k cDNA showed a reduction in LDH release by 27, 30, 32, 33, and 19% (all P < 0.05), respectively, after 24 h exposure to 1, 2.5, 5, 10, and 25 microM CsA. Cell viability after CsA treatment was also significantly higher in CB cells. A mock transfection using plasmid without calbindin-D28k cDNA insert did not affect the LDH release or cell viability after CsA treatment. CsA treatment did not affect the protein and mRNA abundance of transfected calbindin-D28k cDNA. The expression of calbindin-D28k did not affect the baseline [Ca2+]i, but significantly suppressed CsA-induced elevation in [Ca2+]i. The expression of calbindin-D28k in renal tubular cells provides cytoprotective effects against CsA toxicity, probably through its buffering effects on [Ca2+]i.     

Cytosolic Free Calcium and Gene Expression During Chemical Hypoxia

Understanding the cellular response to hypoxia may help elucidate the role of altered oxidation in neuronal death or abnormal cell function. In PC12 cells, 30 min of chemical hypoxia (i.e., KCN) reduced ATP concentrations by 92%, but diminished viability by only 10%. Ten minutes of hypoxia increased cytosolic free calcium ([Ca2+]i) 2.5-fold above control, but after 30 min of hypoxia, [Ca2+]i was slightly below that of nonhypoxic cells. Short periods of hypoxia also exaggerated the K(+)-induced elevation of [Ca2+]i, but by 30 min these ATP-depleted cells reestablished a calcium gradient that was equal to nonhypoxic, K(+)-depolarized cells. Thus, 30 min of severe ATP depletion left [Ca2+]i and viability relatively unaffected. Nerve growth factor caused slight, but significant, improvements in ATP and viability of hypoxic cells, but had no effect on [Ca2+]i. Although [Ca2+]i was equivalent in control and hypoxic cells after 30 or 60 min, hypoxia abolished the K(+)-stimulated elevation of [Ca2+]i. The nerve growth factor induction of c-fos, an indicator of the genomic response, was diminished by approximately 80%. Thus, hypoxic PC12 cells with greatly reduced ATP stores maintained normal [Ca2+]i, but their ability to respond to external stimulation was impaired. Further, the reduced oxidation that occurs in the brain in a variety of pathological conditions may interfere with the cellular response to stimulation and growth factors.     

Graded response of K+ current,membrane potential,and [Ca2+]i to hypoxia in pulmonary arterial smooth muscle

Many studies indicate that hypoxic inhibition of some K+ channels in the membrane of the pulmonary arterial smooth muscle cells (PASMCs) plays a part in initiating hypoxic pulmonary vasoconstriction. The sensitivity of the K+ current (I(k)), resting membrane potential (E(m)), and intracellular Ca2+ concentration ([Ca2+]i) of PASMCs to different levels of hypoxia in these cells has not been explored fully. Reducing PO2 levels gradually inhibited steady-state I(k) of rat resistance PASMCs and depolarized the cell membrane. The block of I(k) by hypoxia was voltage dependent in that low O2 tensions (3 and 0% O2) inhibited I(k) more at 0 and -20 mV than at 50 mV. As expected, the hypoxia-sensitive I(k) was also 4-aminopyridine sensitive. Fura 2-loaded PASMCs showed a graded increase in [Ca2+]i as PO2 levels declined. This increase was reduced markedly by nifedipine and removal of extracellular Ca2+. We conclude that, as in the carotid body type I cells, PC-12 pheochromocytoma cells, and cortical neurons, increasing severity of hypoxia causes a proportional decrease in I(k) and E(m) and an increase of [Ca2+]i.     

Cell Swelling, Blebbing, and Death Are Dependent on ATP Depletion and Independent of Calcium During Chemical Hypoxia in a Glial Cell Line (ROC-1)

The morphological and biochemical changes that occur during chemical hypoxic injury in a neural cell line were studied in the presence and absence of calcium. Oligodendroglial-glioma hybrid cells (ROC-1) were subjected to inhibitors of glycolytic and oxidative ATP synthesis (chemical hypoxia). Complete respiratory inhibition depleted [ATP] to less than 5% of control by 4 min. Blebs appeared on the cell surfaces and cells began to swell within a few minutes of ATP depletion. A 200% increase in cell volume and bleb coalescence preceded irreversible cell injury (lactate dehydrogenase release) which began at approximately 20 min with 50% cell death by 40 min. In energized cells an equivalent degree of osmotic swelling induced by ouabain inhibition of the Na+, K(+)-ATPase pump did not produce blebbing or cell death. Partial inhibition of respiration decreased [ATP] to approximately 10% of control by 40 min. Blebbing and swelling began at 40 min and bleb coalescence preceded plasma membrane disruption which began at approximately 55 min. ATP depletion, blebbing, swelling, and death followed similar time courses in the presence or absence of extracellular calcium ([Ca2+]e). Intracellular calcium ([Ca2+]i) was measured using fura-2. In calcium-containing medium metabolic inhibition caused a transient increase in resting [Ca2+]i (100 +/- 17 nM) followed by a low steady-state level preceding plasma membrane disruption. Following deenergization in calcium-free medium, [Ca2+]i remained below 60 nM throughout injury and death. These data suggest that decreased ATP initiates a sequence of events including bleb formation and cell swelling that lead to irreversible cell injury in the absence of large increases in [Ca2+]i.     

Sodium nitroprusside prevents chemical hypoxia-induced cell death through iron ions stimulating the activity of the Na+-Ca2+ exchanger in C6 glioma cells

In C6 glioma cells exposed to chemical hypoxia, an increase of extracellular lactate dehydrogenase (LDH) activity, cell death, and intracellular Ca2+ concentration ([Ca2+]i) occurred. Sodium nitroprusside (SNP), a nitric oxide donor and an iron-containing molecule, reduced chemical hypoxia-induced LDH release and cell death. These effects were counteracted by bepridil and by 5-(N-4-chlorobenzyl)-2',4'-dimethylbenzamil (CB-DMB), two specific inhibitors of the Na+-Ca2+ exchanger. SNP also increased the activity of the Na+-Ca2+ exchanger as a Na+ efflux pathway, stimulated by Na+-free conditions and evaluated by monitoring [Ca2+]i in single cells. In addition, SNP produced a further increase of chemical hypoxia-elicited [Ca2+]i elevation, and this effect was blocked by bepridil. Chemical hypoxia-evoked cell death and LDH release were counteracted by the ferricyanide moiety of the SNP molecule, K3Fe(CN)6, and by ferric chloride (FeCl3), and this effect was counteracted by CB-DMB. In addition, the iron ion chelator deferoxamine reversed the protective effect exerted by SNP on cell injury. Collectively, these findings suggest that the protective effect of SNP on C6 glioma cells exposed to chemical hypoxia is due to the activation of the Na+-Ca2+ exchanger operating as a Na+ efflux-Ca2+ influx pathway induced by iron present in the SNP molecule.     

Preconditioning improves function and recovery of single muscle fibers during severe hypoxia and reoxygenation

Reperfusion following prolonged ischemia induces cellular damage in whole skeletal muscle models. Ischemic preconditioning attenuates the deleterious effects. We tested whether individual skeletal muscle fibers would be similarly affected by severe hypoxia and reoxygenation (H/R) in the absence of extracellular factors and whether cellular damage could be alleviated by hypoxic preconditioning. Force and free cytosolic Ca2+ ([Ca2+]c) were monitored in Xenopus single muscle fibers (n = 24) contracting tetanically at 0.2 Hz during 5 min of severe hypoxia and 5 min of reoxygenation. Twelve cells were preconditioned by a shorter bout of H/R 1 h before the experimental trial. In preconditioned cells, force relative to initial maximal values (P/P(o)) and relative peak [Ca2+]c fell (P < 0.05) during 5 min of hypoxia and recovered during reoxygenation. In contrast, P/P(o) and relative peak [Ca2+]c fell more during hypoxia (P < 0.05) and recovered less during reoxygenation (P < 0.05) in control cells. The ratio of force to [Ca2+]c was significantly higher in the preconditioned cells during severe hypoxia, suggesting that changes in [Ca2+]c were not solely responsible for the loss in force. We conclude that 1) isolated skeletal muscle fibers contracting in the absence of extracellular factors are susceptible to H/R injury associated with changes in Ca2+ handling; and 2) hypoxic preconditioning improves contractility, Ca2+ handling, and cell recovery during subsequent hypoxic insult.     

Role of mitochondrial reactive oxygen species in hypoxia-dependent increase in intracellular calcium in pulmonary artery myocytes

Previous studies examining the role of mitochondria-derived reactive oxygen species (ROS) in hypoxic responses have been mainly conducted in isolated lungs and cultured pulmonary artery smooth muscle cells (PASMCs) using mitochondrial inhibitors, and yielded largely conflicting results. Here we report that in freshly isolated mouse PASMCs, which are devoid of the mixed responses from multi-types of cells in lungs and significant changes in gene expression in cultured cells, the mitochondrial electron transport chain (ETC) complex I, II, or III inhibitors blocked hypoxia-induced increases in intracellular ROS and Ca2+ concentration ([ROS]i and [Ca2+]i) without effects on their resting levels. Inhibition of the complex I plus II and/or III did not produce an additive effect. Glutathione peroxidase-1 (Gpx1) or catalase gene overexpression to enhance H2O2 removal remarkably reduced hypoxic increases in [ROS]i and [Ca2+]i, whereas Gpx1 gene deletion had the opposite effect. None of these genetic modifications changed the resting [ROS]i and [Ca2+]i. H2O2 at 51 microM caused a similar increase in DCF fluorescence ([ROS]i) as that by hypoxia, but only induced 33% of hypoxic increase in [Ca2+]i. Moreover, H2O2 (5.1 microM) reversed the inhibition of the hypoxia-induced increase in [Ca2+]i by rotenone. Collectively, our study using various mitochondrial inhibitors and genetic approaches demonstrates that in response to acute hypoxia, the mitochondrial ETC molecules prior to the complex III ubisemiquinone site act as a functional unit to increase the generation of ROS, particularly H2O2, which is important for, but may not fully cause, the hypoxic increase in [Ca2+]i in freshly isolated PASMCs.     

Characterization of hypoxia-induced [Ca2+]i rise in rabbit pulmonary arterial smooth muscle cells

We have investigated the effects of hypoxia on the intracellular Ca2+ concentration ([Ca2+]i) in rabbit pulmonary (PASMCs) and coronary arterial smooth muscle cells with fura-2. Perfusion of a glucose-free and hypoxic (PO2<50 mmHg) external solution increased [Ca2+]i in cultured as well as freshly isolated PASMCs. However it had no effect on [Ca2+]i in freshly isolated coronary arterial myocytes. In the absence of extracellular Ca2+, hypoxic stimulation elicited a transient [Ca2+]i increase in cultured PASMCs which was abolished by the simultaneous application of cyclopiazonic acid and ryanodine, suggesting the involvement of sarcoplasmic reticulum (SR) Ca2+ store. Pretreatment with the mitochondrial protonophore, carbonyl cyanide m-chlorophenyl-hydrazone (CCCP) enhanced the [Ca2+]i rise in response to hypoxia. A short application of caffeine gave a transient [Ca2+]i rise which was prolonged by CCCP. Decay of the caffeine-induced [Ca2+]i transients was significantly slowed by treatment of CCCP or rotenone. After full development of the hypoxia-induced [Ca2+]i rise, nifedipine did not decrease [Ca2+]i. These data suggest that the [Ca2+]i increase in response to hypoxia may be ascribed to both Ca2+ release from the SR and the subsequent activation of nifedipine-insensitive capacitative Ca2+ entry. Mitochondria appear to modulate hypoxia induced Ca2+ release from the SR.     

缺氧时大鼠红细胞变形性损伤的机制研究

本实验通过测定平原和模拟高原减压缺氧３０天大鼠红细胞滤过指数（ＩＦ）、红细胞内［ｐＨ］ｉ、［Ｋ＋］ｉ／［Ｎａ＋］ｉ比值、［Ｃａ２＋］ｉ、［Ｍｇ２＋］ｉ、平均红细胞体积（ＭＣＶ）及平均红细胞血红蛋白浓度（ＭＣＨＣ）,从而探讨缺氧条件下大鼠红细胞变形性损害的机制。结果发现：１．缺氧组大鼠红细胞［Ｃａ２＋］ｉ明显升高,且与ＩＦ呈显著正相关,但［Ｍｇ２＋］ｉ无明显差异;２．缺氧组［Ｋ＋］ｉ／［Ｎａ＋］ｉ值较平原组明显降低,且与ＩＦ呈显著负相关;３．缺氧组ＭＣＨＣ与平原组无明显差异,但ＭＣＶ显著升高;４．缺氧组红细胞内［ｐＨ］ｉ较平原组明显升高。提示：缺氧时红细胞［Ｃａ２＋］ｉ升高,［Ｋ＋］ｉ／［Ｎａ＋］ｉ值降低,ＭＣＶ增大以及红细胞［ｐＨ］ｉ值的改变在其变形性损伤中起重要作用。     

Effects of Hypoxia on the Catecholamine Release, Ca2+ Uptake, and Cytosolic Free Ca2+ Concentration in Cultured Bovine Adrenal Chromaffin Cells

The purpose of the present study is to clarify the effects of hypoxia on catecholamine release and its mechanism of action. For this purpose, using cultured bovine adrenal chromaffin cells, we examined the effects of hypoxia on high (55 mM) K(+)-induced increases in catecholamine release, in cytosolic free Ca2+ concentration ([Ca2+]i), and in 45Ca2+ uptake. Experiments were carried out in media preequilibrated with a gas mixture of either 21% O2/79% N2 (control) or 100% N2 (hypoxia). High K(+)-induced catecholamine release was inhibited by hypoxia to approximately 40% of the control value, but on reoxygenation the release returned to control levels. Hypoxia had little effect on ATP concentrations in the cells. In the hypoxic medium, [Ca2+]i (measured using fura-2) gradually increased and reached a plateau of approximately 1.0 microM at 30 min, whereas the level was constant in the control medium (approximately 200 nM). High K(+)-induced increases in [Ca2+]i were inhibited by hypoxia to approximately 30% of the control value. In the cells permeabilized by digitonin, catecholamine release induced by Ca2+ was unaffected by hypoxia. Hypoxia had little effect on basal 45Ca2+ uptake into the cells, but high K(+)-induced 45Ca2+ uptake was inhibited by hypoxia. These results suggest that hypoxia inhibits high K(+)-induced catecholamine release and that this inhibition is mainly the result of the inhibition of high K(+)-induced increases in [Ca2+]i subsequent to the inhibition of Ca2+ influx through voltage-dependent Ca2+ channels.     

Calbindin-D(28k) controls [Ca(2+)](i) and insulin release. Evidence obtained from calbindin-d(28k) knockout mice and beta cell lines

The role of the calcium-binding protein, calbindin-D(28k) in potassium/depolarization-stimulated increases in the cytosolic free Ca(2+) concentration ([Ca(2+)](i)) and insulin release was investigated in pancreatic islets from calbindin-D(28k) nullmutant mice (knockouts; KO) or wild type mice and beta cell lines stably transfected and overexpressing calbindin. Using single islets from KO mice and stimulation with 45 mM KCl, the peak of [Ca(2+)](i) was 3.5-fold greater in islets from KO mice compared with wild type islets (p < 0.01) and [Ca(2+)](i) remained higher during the plateau phase. In addition to the increase in [Ca(2+)](i) in response to KCl there was also a significant increase in insulin release in islets isolated from KO mice. Evidence for modulation by calbindin of [Ca(2+)](i) and insulin release was also noted using beta cell lines. Rat calbindin was stably expressed in betaTC-3 and betaHC-13 cells. In response to depolarizing concentrations of K(+), insulin release was decreased by 45-47% in calbindin expressing betaTC cells and was decreased by 70-80% in calbindin expressing betaHC cells compared with insulin release from vector transfected betaTC or betaHC cells (p < 0.01). In addition, the K(+)-stimulated intracellular calcium peak was markedly inhibited in calbindin expressing betaHC cells compared with vector transfected cells (225 nM versus 1,100 nM, respectively). Buffering of the depolarization-induced rise in [Ca(2+)](i) was also observed in calbindin expressing betaTC cells. In summary, our findings, using both isolated islets from calbindin-D(28k) KO mice and beta cell lines, establish a role for calbindin in the modulation of depolarization-stimulated insulin release and suggest that calbindin can control the rate of insulin release via regulation of [Ca(2+)](i).     

A single cell model of myocardial reperfusion injury: Changes in intracellular Na+ and Ca2+ concentrations in guinea pig ventricular myocytes

To investigate the contribution of the changes in intracellular Na+ and Ca2+ concentrations ([Na+]i and [Ca2+]i) to myocardial reperfusion injury, we made an ischemia/reperfusion model in intact guinea pig myocytes. Myocardial ischemia was simulated by the perfusion of metabolic inhibitors (3.3 mM amobarbital and 5 M carbonyl cyanide m-chlorophenylhydrazone) with pH 6.6 and reperfusion was achieved by the washout of them with pH 7.4. [Na+]i increased from 7.9 ± 2.0 to 14.0 ± 3.4 mM (means ± S.E., p < 0.01) during 7.5 min of simulated ischemia (SI) and increased further to 18.8 ± 3.0 mM at 7.5 min after reperfusion. [Ca2+]i, expressed as the ratio of fluo 3 fluorescence intensity, increased to 133 ± 8% (p < 0.01) during SI and gradually returned to the control level after reperfusion. Intracellular pH decreased from 7.53 ± 0.04 to 6.31 ± 0.04 (p < 0.01) and recovered quickly after reperfusion. Reperfusion with the acidic solution or the continuous perfusion of hexamethylene amiloride (2 M) prevented the reperfusion-induced increase in [Na+]i. When the duration of SI was prolonged to 15 min, the cell response after reperfusion varied, 16 of 37 cells kept quiescent, 21 cells showed spontaneous Ca2+ waves, and 4 cells out of these 21 cells became hypercontracted. In quiescent cells, both [Na+]i and [Ca2+]i decreased immediately after reperfusion. In cells with Ca2+ waves, [Na+]i transiently increased further at the early phase of reperfusion, while [Ca+]i declined. In hypercontracted cells, [Na+]i increased as much as in Ca2+ wave cells, but [Ca2+]i increased extensively and both ion concentrations continued to increase. Reperfusion with the Ca2+-free solution prevented both the [Ca2+]i increase and morphological change. In the presence of ryanodine (10 M), the increase in [Ca2+]i after reperfusion was augmented and some cells became hypercontracted. We concluded that (1) Na+/H+ exchange is active both during SI and reperfusion, resulting in the additional [Na+]i elevation on reperfusion, (2) the [Na+]i level after reperfusion and the following Ca2+ influx via Na+/Ca2+ exchange are crucial for reperfusion cell injury, and (3) the Ca2+ buffering capacity of sarcoplasmic reticulum would also contribute to the Ca2+ regulation and cell injury after reperfusion.     

Stable transfection of calbindin-D28k into the GH3 cell line alters calcium currents and intracellular calcium homeostasis.

Previous work demonstrating the presence and differential distribution of Ca(2+)-binding proteins in the CNS has led to the proposal that cytosolic proteins, such as calbindin-D28k (CB), may play a pivotal role in neurons. We have used a retrovirus containing the full-length cDNA for CB to transfect the pituitary tumor cell line GH3, to generate CB-expressing GH3 cells and to investigate whether ionic channel activities as well as the concentration of intracellular free Ca2+ ([Ca2+]i) homeostasis could be altered by the presence of this Ca(2+)-binding protein. We show that CB-transfected GH3 cells exhibited lower Ca2+ entry through voltage-dependent Ca2+ channels and were better able to reduce [Ca2+]i transients evoked by voltage depolarizations than the wild-type parent cell line. These observations provide a mechanism by which CB may protect tissues against Ca(2+)-mediated excitotoxicity.     

Characterization of the calcium response to thyrotropin-releasing hormone (TRH) in cells transfected with TRH receptor complementary DNA: importance of voltage-sensitive calcium channels.

TRH stimulates a biphasic increase in intracellular free calcium ion, [Ca2+]i. Cells stably transfected with TRH receptor cDNA were used to compare the response in lines with and without L type voltage-gated calcium channels. Rat pituitary GH-Y cells that do not normally express TRH receptors, rat glial C6 cells, and human epithelial Hela cells were transfected with mouse TRH receptor cDNA. All lines bound similar amounts of [3H][N3-Me-His2]TRH with identical affinities (dissociation constant = 1.5 nM). Both pituitary lines expressed L type voltage-gated calcium channels; depolarization with high K+ increased 45Ca2+ uptake 20- to 25-fold and [Ca2+]i 12- to 14-fold. C6 and Hela cells, in contrast, appeared to have no L channel activity. GH4C1 cells responded to TRH with a calcium spike (6-fold) followed by a sustained second phase. When TRH was added after 100 nM nimodipine, an L channel blocker, the initial calcium burst was unaffected but the second phase was abolished. GH-Y cells transfected with TRH receptor cDNA responded to TRH with a 6-fold [Ca2+]i spike followed by a plateau phase (>8 min) in which [Ca2+]i remained elevated or increased. Nimodipine did not alter the peak TRH response or resting [Ca2+]i but reduced the sustained phase, which was eliminated by chelation of extracellular Ca2+. In the transfected glial C6 and Hela cells without calcium channels, TRH evoked transient, monophasic 7- to 9-fold increases in [Ca2+]i, and [Ca2+]i returned to resting levels within 3 min. Thapsigargin stimulated a gradual, large increase in [Ca2+]i in transfected C6 cells, and subsequent addition of TRH caused no further rise. Removal of extracellular Ca2+ from transfected C6 cells shortened the [Ca2+]i responses to TRH, to endothelin 1, and to thapsigargin. The TRH responses were pertussis toxin-insensitive. In summary, TRH can generate a calcium spike in pituitary, C6, and Hela cells transfected with TRH receptor cDNA, but the plateau phase of the [Ca2+]i response is not observed when the receptor is expressed in a cell line without L channel activity.     

Calbindin-D28k decreases L-type calcium channel activity and modulates intracellular calcium homeostasis in response to K+ depolarization in a rat beta cell line RINr1046-38

Calbindin-D(28k), acts as a modulator of depolarization induced calcium transients in the pancreatic beta cell. However, specific mechanisms have not been defined. Here we show for the first time that the calcium binding protein calbindin-D(28k) acts by affecting calcium influx through voltage-dependent calcium channels in RIN pancreatic beta cells. Whole-cell patch-clamp recordings revealed that Ca(2+) current amplitudes of calbindin-D(28k) expressing RINr1046-38 beta cells were smaller than the Ca(2+) current amplitudes in control cells in response to depolarizing pulses. The peak current was observed at +20mV and the average amplitude was approximately 50pA in the calbindin expressing cells compared to approximately 250pA in control cells. In calbindin-D(28k) expressing cells, the channels had enhanced sensitivity to Ca(2+) dependent inactivation and currents decayed much more rapidly than in control cells. The Ca(2+) channels affected by calbindin were found to have biophysical properties consistent with dihydropyridine-sensitive L-type calcium channels. In response to depolarizing concentrations of K(+), calbindin expression caused a five-fold decrease in the rate of rise of [Ca(2+)](i) and decay was slower in the calbindin expressing cells. Application of verapamil resulted in a drop in the [Ca(2+)](i) signal to pre-stimulation levels indicating that the Ca(2+) channel responsible for the depolarization evoked Ca(2+) entry, modulated by calbindin, is the L-type. Co-immunoprecipitation and GST pull-down assays indicate that calbindin-D(28k) can interact with the alpha(1) subunit of Ca(v)1.2. We thus conclude that calbindin-D(28k) can regulate calcium influx via L-type calcium channels. Our findings suggest a role for calbindin-D(28k) in the beta cell in modulating Ca(2+) influx via L-type voltage-dependent calcium channels.     

Hypoxic remodelling of Ca2+ mobilization in type I cortical astrocytes: involvement of ROS and pro-amyloidogenic APP processing

Chronic hypoxia (CH) alters Ca2+ homeostasis in various cells and may contribute to disturbed Ca2+ homeostasis of Alzheimer's disease. Here, we have employed microfluorimetric measurements of [Ca2+]i to investigate the mechanism underlying augmentation of Ca2+ signalling by chronic hypoxia in type I cortical astrocytes. Application of bradykinin evoked significantly larger rises of [Ca2+]i in hypoxic cells as compared with control cells. This augmentation was prevented fully by either melatonin (150 micro m) or ascorbic acid (200 micro m), indicating the involvement of reactive oxygen species. Given the association between hypoxia and increased production of amyloid beta peptides (AbetaPs) of Alzheimer's disease, we performed immunofluorescence studies to show that hypoxia caused a marked and consistent increased staining for AbetaPs and presenilin-1 (PS-1). Western blot experiments also confirmed that hypoxia increased PS-1 protein levels. Hypoxic increases of AbetaP production was prevented with inhibitors of either gamma- or beta-secretase. These inhibitors also partially prevented the augmentation of Ca2+ signalling in astrocytes. Our results indicate that chronic hypoxia enhances agonist-evoked rises of [Ca2+]i in cortical astrocytes, and that this can be prevented by antioxidants and appears to be associated with increased AbetaP formation.     

BHK cells transfected with NCX3 are more resistant to hypoxia followed by reoxygenation than those transfected with NCX1 and NCX2: Possible relationship with mitochondrial membrane potential

The specific role played by NCX1, NCX2, and NCX3, the three isoforms of the Na+/Ca2+ exchanger (NCX), has been explored during hypoxic conditions in BHK cells stably transfected with each of these isoforms. Six major findings emerged from the present study: (1) all the three isoforms were highly expressed on the plasma membranes of BHK cells; (2) under physiological conditions, the three NCX isoforms showed similar functional activity; (3) hypoxia plus reoxygenation induced a lower increase of [Ca2+]i in BHK-NCX3-transfected cells than in BHK-NCX1- and BHK-NCX2-transfected cells; (4) NCX3-transfected cells were more resistant to chemical hypoxia plus reoxygenation than NCX1- and NCX2-transfected cells. Interestingly, such augmented resistance was eliminated by CBDMD (10 microM), an inhibitor of NCX and by the specific silencing of the NCX3 isoform; (5) chemical hypoxia plus reoxygenation produced a loss of mitochondrial membrane potential in NCX1- and NCX2-transfected cells, but not in NCX3-transfected cells; (6) the forward mode of operation in NCX3-transfected cells was not affected by ATP depletion, as it occurred in NCX1- and NCX2-transfected cells. Altogether, these results indicate that the brain specifically expressed NCX3 isoform more significantly contributes to the maintenance of [Ca2+]i homeostasis during experimental conditions mimicking ischemia, thereby preventing mitochondrial delta psi collapses and cell death.     

Increased [Mg2+]o reduces Ca2+ influx and disruption of mitochondrial membrane potential during reoxygenation

Increase in extracellular Mg2+ concentration ([Mg2+]o) reduces Ca2+ accumulation during reoxygenation of hypoxic cardiomyocytes and exerts protective effects. The aims of the present study were to investigate the effect of increased [Mg(2+)](o) on Ca2+ influx and efflux, free cytosolic Ca2+ ([Ca2+]i) and Mg2+ concentrations ([Mg2+]i), Ca2+ accumulation in the presence of inhibitors of mitochondrial or sarcoplasmatic reticulum Ca2+ transport, and finally mitochondrial membrane potential (Delta(psi)m). Isolated adult rat cardiomyocytes were exposed to 1 h of hypoxia and subsequent reoxygenation. Cell Ca2+ was determined by 45Ca2+ uptake, and the levels of [Mg2+]i and [Ca2+]i were determined by flow cytometry as the fluorescence of magnesium green and fluo 3, respectively. Ca2+ influx rate was significantly reduced by approximately 40%, whereas Ca2+ efflux was not affected by increased [Mg2+]o (5 mM) during reoxygenation. [Ca2+]i and [Mg2+]i were increased at the end of hypoxia, fell after reoxygenation, and were unaffected by increased [Mg2+]o. Clonazepam, a selective mitochondrial Na+/Ca2+ exchange inhibitor (100 microM), significantly reduced Ca2+ accumulation by 70% and in combination with increased [Mg2+]o by 90%. Increased [Mg2+]o, clonazepam, and the combination of both attenuated the hypoxia-reoxygenation-induced reduction in Delta(psi)m, determined with the cationic dye JC-1 by flow cytometry. A significant inverse correlation was observed between Delta(psi)m and cell Ca2+ in reoxygenated cells treated with increased [Mg2+]o and clonazepam. In conclusion, increased [Mg2+]o (5 mM) inhibits Ca2+ accumulation by reducing Ca2+ influx and preserves Delta(psi)m without affecting [Ca2+]i and [Mg2+]i during reoxygenation. Preservation of mitochondria may be an important effect whereby increased [Mg2+]o protects the postischemic heart.     

Changes in smooth muscle cell pH during hypoxic pulmonary vasoconstriction: a possible role for ion transporters

Hypoxic pulmonary vasoconstriction (HPV) occurs in smooth muscle cells (SMC) from small pulmonary arteries (SPA) and is accompanied by increases in free cytoplasmic calcium ([Ca2+]i) and cytoplasmic pH (pHi). SMC from large pulmonary arteries (LPA) relax during hypoxia, and [Ca2+]i and pHi decrease. Increases in pHi and [Ca2+]i in cat SPA SMC during hypoxia and the augmentation of hypoxic pulmonary vasoconstriction by alkalosis seen in isolated arteries and lungs suggest that cellular mechanisms, which regulate inward and outward movement of Ca2+ and H+, may participate in the generation of HPV. SMC transport systems that regulate pHi include the Na+ - H+ transporter which regulates intracellular Na+ and H+ and aids in recovery from acid loads, and the Na+ -dependent and Na+ -independent Cl-/HCO3- transporters which regulate intracellular chloride. The Na+ -dependent Cl-/HCO3- transporter also aids in recovery from acidosis in the presence of CO2 and HCO3-. The Na+ -independent Cl-/HCO3- transporter aids in recovery from cellular alkalosis. The Na+ - H+ transporter was present in SMC from SPA and LPA of the cat, but it seemed to have little if any role in regulating pHi in the presence of CO2 and HCO3-. Inhibiting the Cl-/HCO3- transporters reversed the normal direction of pHi change during hypoxia, suggesting a role for these transporters in the hypoxic response. Future studies to determine the interaction between pHi, [Ca2+]i and HPV should ascertain whether pHi and [Ca2+]i changes are linked and how they may interact to promote or inhibit SMC contraction.