Connexin 43 hemichannels contribute to cytoplasmic Ca2+ oscillations by providing a bimodal Ca2+-dependent Ca2+ entry pathway

Many cellular functions are driven by changes in the intracellular Ca(2+) concentration ([Ca(2+)](i)) that are highly organized in time and space. Ca(2+) oscillations are particularly important in this respect and are based on positive and negative [Ca(2+)](i) feedback on inositol 1,4,5-trisphosphate receptors (InsP(3)Rs). Connexin hemichannels are Ca(2+)-permeable plasma membrane channels that are also controlled by [Ca(2+)](i). We aimed to investigate how hemichannels may contribute to Ca(2+) oscillations. Madin-Darby canine kidney cells expressing connexin-32 (Cx32) and Cx43 were exposed to bradykinin (BK) or ATP to induce Ca(2+) oscillations. BK-induced oscillations were rapidly (minutes) and reversibly inhibited by the connexin-mimetic peptides (32)Gap27/(43)Gap26, whereas ATP-induced oscillations were unaffected. Furthermore, these peptides inhibited the BK-triggered release of calcein, a hemichannel-permeable dye. BK-induced oscillations, but not those induced by ATP, were dependent on extracellular Ca(2+). Alleviating the negative feedback of [Ca(2+)](i) on InsP(3)Rs using cytochrome c inhibited BK- and ATP-induced oscillations. Cx32 and Cx43 hemichannels are activated by <500 nm [Ca(2+)](i) but inhibited by higher concentrations and CT9 peptide (last 9 amino acids of the Cx43 C terminus) removes this high [Ca(2+)](i) inhibition. Unlike interfering with the bell-shaped dependence of InsP(3)Rs to [Ca(2+)](i), CT9 peptide prevented BK-induced oscillations but not those triggered by ATP. Collectively, these data indicate that connexin hemichannels contribute to BK-induced oscillations by allowing Ca(2+) entry during the rising phase of the Ca(2+) spikes and by providing an OFF mechanism during the falling phase of the spikes. Hemichannels were not sufficient to ignite oscillations by themselves; however, their contribution was crucial as hemichannel inhibition stopped the oscillations.     

Opening hemichannels in nonjunctional membrane stimulates gap junction formation

We studied gap junction formation in pairs of Xenopus laevis oocytes expressing connexins that form functional hemichannels and found no correlation between junctional conductance (G(j)) and whole-cell hemichannel conductances (G(hemi)) within the first few hours of pairing. However, opening hemichannels to a threshold current stimulated a rapid G(j) increase. Moreover, cx46 hemichannel current stimulated cx40 G(j) even though cx40 and cx46 do not form heteromeric or heterotypic gap junctions. Initial growth rate and final steady-state level of stimulated G(j) were proportional to the product of hemichannel conductances. External calcium affected the growth rate of stimulated G(j) but not the final steady-state value. Time constants of formation were short in low [Ca(2+)](out) (3 min in 200 micro M Ca(2+)) and long in high [Ca(2+)](out) (15 min in 1 mM Ca(2+)), but in oocyte pairs pretreated with lectins to reduce steric hindrance imposed by large membrane glycoproteins the time constant was short and Ca(2+)-independent. We suggest that hemichannel activity stimulates G(j) by collapsing the extracellular volume between membranes to allow the end-to-end binding between hemichannels. These studies suggest the possibility that functional hemichannels could trigger or enhance junctional formation in vivo in response to appropriate stimuli.     

Hypoxia induces an increase in intracellular magnesium via transient receptor potential melastatin 7 (TRPM7) channels in rat hippocampal neurons in vitro

TRPM7, a divalent cation channel, plays an important role in neurons damaged from cerebral ischemia due to permitting intracellular calcium overload. This study aimed to explore whether magnesium was transported via a TRPM7 channel into the intracellular space of rat hippocampal neurons after 1 h of oxygen-glucose deprivation (OGD) and acute chemical ischemia (CI) by using methods of the Mg(2+) fluorescent probe Mag-Fura-2 to detect intracellular magnesium concentration ([Mg(2+)](i)) and flame atomic absorption spectrometry to measure extracellular magnesium concentration ([Mg(2+)](o)). The results showed that the neuronal [Mg(2+)](i) was 1.51-fold higher after 1 h of OGD at a basal level, and the increase of neuronal [Mg(2+)](i) reached a peak after 1 h of OGD and was kept for 60 min with re-oxygenation. Meanwhile, the [Mg(2+)](o) decreased after 1 h of OGD and recovered to the pre-ischemic level within 15 min after re-oxygenation. In the case of CI, the [Mg(2+)](i) peak immediately appeared in hippocampal neurons. This increase of [Mg(2+)](i) declined by removing extracellular magnesium in OGD or CI. Furthermore, by using Gd(3+) or 2-aminoethoxydiphenyl borate to inhibit TRPM7 channels, the [Mg(2+)](i) increase, which was induced by OGD or CI, was attenuated without altering the basal level of [Mg(2+)](i). By silencing TRPM7 with shRNA in hippocampal neurons, it was found that not only was the increase of [Mg(2+)](i) induced by OGD or CI but also the basal levels of [Mg(2+)](i) were attenuated. In contrast, overexpression of TRPM7 in HEK293 cells exaggerated both the basal levels and increased [Mg(2+)](i) after 1 h of OGD/CI. These results suggest that anoxia induced the increase of [Mg(2+)](i) via TRPM7 channels in rat hippocampal neurons.     

Elevated endogenous nitric oxide increases Ca2+ flux via L-type Ca2+ channels by S-nitrosylation in rat hippocampal neurons during severe hypoxia and in vitro ischemia

Nitric oxide (NO) mediates pathogenic changes in the brain subsequent to energy deprivation; yet the NO mechanism involved in the early events remains unclear. We examined the acute effects of severe hypoxia and oxygen-glucose deprivation (OGD) on the endogenous NO production and the NO-mediated pathways involved in the intracellular calcium ([Ca(2+)](i)) response in the rat hippocampal neurons. The levels of NO and [Ca(2+)](i) in the CA1 region of the slices rapidly elevated in hypoxia and were more prominent in OGD, measured by the electrochemical method and spectrofluorometry, respectively. The NO and [Ca(2+)](i) responses were enhanced by L-arginine and were reduced by NO synthase inhibitors, suggesting that the endogenous NO increases the [Ca(2+)](i) response to energy deprivation. Nickel and nifedipine significantly decreased the NO and [Ca(2+)](i) responses to hypoxia and OGD, indicating an involvement of L-type Ca(2+) channels in the NO-mediated mechanisms. In addition, the [Ca(2+)](i) responses were attenuated by ODQ or KT5823, inhibitors of the cGMP-PKG pathway, and by acivicin, an inhibitor of gamma-glutamyl transpeptidase for S-nitrosylation, and by the thiol-alkylating agent N-ethylmaleimide (NEM). Moreover, L-type Ca(2+) currents in cultured hippocampal neurons with whole-cell recording were significantly increased by L-arginine and were decreased by L-NAME. Pretreatment with NO synthase inhibitors or NEM but not ODQ abolished the effect of L-arginine on the Ca(2+) currents. Also, vitamin C, which decomposes nitrosothiol but not disulfide by reduction, reversed the change in the Ca(2+) current with L-arginine. Taken together, the results suggest that an elevated endogenous NO production enhances the influx of Ca(2+) via the hippocampal L-type Ca(2+) channel by S-nitrosylation during an initial phase of energy deprivation.     

Intracellular calcium changes trigger connexin 32 hemichannel opening

Connexin hemichannels have been proposed as a diffusion pathway for the release of extracellular messengers like ATP and others, based on connexin expression models and inhibition by gap junction blockers. Hemichannels are opened by various experimental stimuli, but the physiological intracellular triggers are currently not known. We investigated the hypothesis that an increase of cytoplasmic calcium concentration ([Ca2+]i) triggers hemichannel opening, making use of peptides that are identical to a short amino-acid sequence on the connexin subunit to specifically block hemichannels, but not gap junction channels. Our work performed on connexin 32 (Cx32)-expressing cells showed that an increase in [Ca2+]i triggers ATP release and dye uptake that is dependent on Cx32 expression, blocked by Cx32 (but not Cx43) mimetic peptides and a calmodulin antagonist, and critically dependent on [Ca2+]i elevation within a window situated around 500 nM. Our results indicate that [Ca2+]i elevation triggers hemichannel opening, and suggest that these channels are under physiological control.     

Separation of fluorescence signals from Ca2+ and NADH during cardioplegic arrest and cardiac ischemia

Determinations of intracellular [Ca(2+)](i) during ischemia using fluorescent indicators are hampered by overlapping cellular autofluorescence (AF), which largely depends on NADH. If Ca(2+) is to be determined under different kinds of ischemia, signal separation merits special attention. We used triple wavelength excitation fluorescence to separate autofluorescence from [Ca(2+)]-dependent fura-2 fluorescence. Excitation at 360 nm served as third, Ca(2+)-insensitive wavelength. Using an appropriate evaluation procedure, we separated Ca(2+)-dependent signals from autofluorescence which is semiquantitatively associated with NADH, an indicator of the cellular redox state. We compared changes of [Ca(2+)](i) in isolated hearts during ischemia following cardioplegic arrest with those after transient stop of nutritive perfusion. We observed [Ca(2+)] transients in spontaneously beating hearts, persisting during ischemic episodes, and an increase of mean [Ca(2+)](i). In contrast, cardioplegic arrest stopped periodical [Ca(2+)](i) transients and heart beats simultaneously. [Ca(2+)](i) remained at diastolic values, tended to decrease during the first minutes of cardioplegic arrest and then increased slowly. Autofluorescence increased under both conditions. During ischemia, this increase was faster than in cardioplegia experiments. It started after the last heart beat despite persisting perfusion. Our measurements demonstrate that rhythmical heart beat is essential for sufficient perfusion. Reduced [Ca(2+)](i) under cardioplegic arrest may influence metabolism.     

Time-correlation between membrane depolarization and intracellular calcium in insulin secreting BRIN-BD11 cells: studies using FLIPR

Cytoplasmic Ca(2+) ([Ca(2+)](i)) and membrane potential changes were measured in clonal pancreatic beta cells using a fluorimetric imaging plate reader (FLIPR). KCl (30 mM) produced a fast membrane depolarization immediately followed by increase of [Ca(2+)](i) in BRIN-BD11 cells. l-Alanine (10 mM) but not l-arginine (10 mM) mimicked the KCl profile and also produced a fast membrane depolarization and elevation of [Ca(2+)](i). Conversely, a rise in glucose from 5.6 mM to 11.1 or 16.7 mM induced rapid membrane depolarization, followed by a slower and delayed increase of [Ca(2+)](i). GLP-1 (20 nM) did not affect membrane potential or [Ca(2+)](i). In contrast, acetylcholine (ACh, 100 microM) induced fast membrane depolarization immediately followed by a modest [Ca(2+)](i) increase. When extracellular Ca(2+) was buffered with EGTA, ACh mobilized intracellular calcium stores and the [Ca(2+)](i) increase was reduced by 2-aminoethoxydiphenyl borate but not by dantrolene, indicating the involvement of inositol triphosphate receptors (InsP(3)R). It is concluded that membrane depolarization of beta cells by glucose stimulation is not immediately followed by elevation of [Ca(2+)](i) and other metabolic events are involved in glucose induced stimulus-secretion coupling. It is also suggested that ACh mobilizes intracellular Ca(2+) through store operated InsP(3)R.     

Endoplasmic reticulum Ca2+ dysregulation and endoplasmic reticulum stress following in vitro neuronal ischemia: role of Na+-K+-Cl- cotransporter

We investigated the role of Na(+)-K(+)-Cl(-) cotransporter (NKCC1) in conjunction with Na(+)/Ca(2+) exchanger (NCX) in disruption of endoplasmic reticulum (ER) Ca(2+) homeostasis and ER stress development in primary cortical neurons following in vitro ischemia. Oxygen-glucose deprivation (OGD) and reoxygenation (REOX) caused a rise in [Na(+)](cyt) which was accompanied by an elevation in [Ca(2+)](cyt). Inhibition of NKCC1 with its potent inhibitor bumetanide abolished the OGD/REOX-induced rise in [Na(+)](cyt) and [Ca(2+)](cyt). Moreover, OGD significantly increased Ca(2+)(ER) accumulation. Following REOX, a biphasic change in Ca(2+)(ER) occurred with an initial release of Ca(2+)(ER) which was sensitive to inositol 1,4,5-trisphosphate receptor (IP(3)R) inhibition and a subsequent refilling of Ca(2+)(ER) stores. Inhibition of NKCC1 activity with its inhibitor or genetic ablation prevented the release of Ca(2+)(ER). A similar result was obtained with inhibition of reversed mode operation of NCX (NCX(rev)). OGD/REOX also triggered a transient increase of glucose regulated protein 78 (GRP78), phospho-form of the alpha subunit of eukaryotic initiation factor 2 (p-eIF2alpha), and cleaved caspase 12 proteins. Pre-treatment of neurons with NKCC1 inhibitor bumetanide inhibited upregulation of GRP78 and attenuated the level of cleaved caspase 12 and p-eIF2alpha. Inhibition of NKCC1 reduced cytochrome C release and neuronal death. Taken together, these results suggest that NKCC1 and NCX(rev) may be involved in ischemic cell damage in part via disrupting ER Ca(2+) homeostasis and ER function.     

Negative-feedback regulation of ATP release: ATP release from cardiomyocytes is strictly regulated during ischemia

Extracellular ATP acts as a potent agonist on cardiomyocytes, inducing a broad range of physiological responses via P2 purinoceptors. Its concentration in the interstitial space within the heart is elevated during ischemia or hypoxia due to its release from a number of cell types, including cardiomyocytes. However, the exact mechanism responsible for the release of ATP from cardiomyocytes during ischemia is not known. In this study, we investigated whether and how the release of ATP was strictly regulated during ischemia in cultured neonatal rat cardiomyocytes. Ischemia was mimicked by oxygen-glucose deprivation (OGD). Exposure of cardiomyocytes to OGD resulted in an increase in the concentration of extracellular ATP shortly after the onset of OGD (15 min), and the increase was reversed by treatment with blockers of maxi-anion channels. Unexpectedly, at 1 and 2h after the onset of OGD, the blocking of maxi-anion channels increased the concentration of extracellular ATP, and the increase was significantly suppressed by co-treatment with blockers of hemichannels, suggesting that ATP release via maxi-anion channels was involved in the suppression of ATP release via hemichannels during persistent OGD. Here we show the possibility that the release of ATP from cardiomyocytes was strictly regulated during ischemia by negative-feedback mechanisms; that is, maxi-anion channel-derived ATP-induced suppression of ATP release via hemichannels in cardiomyocytes.     

Stimulated efflux of amino acids and glutathione from cultured hippocampal slices by omission of extracellular calcium: likely involvement of connexin hemichannels

Omission of extracellular Ca(2+) for 15 min from the incubation medium of cultured hippocampal slices stimulated the efflux of glutathione, phosphoethanolamine, hypotaurine, and taurine. The efflux was reduced by several blockers of gap junctions, i.e. carbenoxolone, flufenamic acid, and endothelin-1, and by the connexin43 hemichannel blocking peptide Gap26 but was unchanged by the P2X(7) receptor inhibitor oxidized ATP, a pannexin1 hemichannel blocking peptide and an inactive analogue of carbenoxolone. Pretreatment of the slices with the neurotoxin N-methyl-d -aspartate left the efflux by Ca(2+) omission unchanged, indicating that the stimulated efflux primarily originated from glia. Elevated glutamate efflux was detected when Ca(2+) omission was combined with the glutamate uptake blocker l-trans-pyrrolidine-2,4-dicarboxylate and when both Ca(2+) and Mg(2+) were omitted from the medium. Omission of Ca(2+) for 15 min alone did not induce delayed toxicity, but in combination with blocked glutamate uptake, significant cell death was observed 24 h later. Our results indicate that omission of extracellular Ca(2+) stimulates efflux of glutathione and specific amino acids including glutamate via opening of glial hemichannels. This type of efflux may have protective functions via glutathione efflux but can aggravate toxicity in situations when glutamate reuptake is impaired, such as following a stroke.     

Depressed tolerance to fluorocarbon-simulated ischemia in failing myocardium due to impaired [Ca(2+)](i) modulation

The aim of this study was to investigate the tolerance of failing myocardium from postinfarction rats to simulated ischemia. Myocardial infarction (MI) was induced by ligation of the left coronary artery in male Wistar rats. Isometric force and free intracellular Ca(2+) concentration ([Ca(2+)](i)) were measured in isolated left ventricular papillary muscles from sham-operated and post-MI animals 6 wk after surgery. Ischemia was simulated by using fluorocarbon immersion with hypoxia. Results showed that mechanical performance was depressed during the period of hypoxia in physiological salt solution (44 +/- 7% of baseline in sham vs. 30 +/- 6% of baseline in MI, P < 0.05) or ischemia (16 +/- 2% of baseline in sham vs. 9 +/- 1% of baseline in MI, P < 0.01) accompanied by no corresponding decrease of peak [Ca(2+)](i) (hypoxia: 51 +/- 8% of baseline in sham vs. 46 +/- 7% of baseline in MI, P = NS; ischemia: 47 +/- 5% of baseline in sham, 39 +/- 7% of baseline in MI, P = NS). After reoxygenation, [Ca(2+)](i) rapidly returned to near preischemic basal levels, whereas developed tension in fluorocarbon remained significantly lower. This dissociation between peak [Ca(2+)](i) and isometric contractility was more pronounced in the failing myocardium from postinfarction rats. In conclusion, more severe impairment of [Ca(2+)](i) homeostasis in the failing myocardium from postinfarction rats increases susceptibility to ischemia-reperfusion injury.     

The presenilin 1 deltaE9 mutation gives enhanced basal phospholipase C activity and a resultant increase in intracellular calcium concentrations

We studied effects of the familial Alzheimer's disease presenilin 1 (PS1) exon 9 deletion (PS1-DeltaE9) mutation on basal and carbachol-stimulated phosphoinositide (PI) hydrolysis and intracellular Ca(2+) concentrations ([Ca(2+)](i)) in human SH-SY5Y neuroblastoma cells. We demonstrate that PS1-DeltaE9 cells have an enhanced basal PI hydrolysis and [Ca(2+)](i) as compared with both wild type PS1 (PS1-WT) and nontransfected (NT) cells. Both were reversed by the phospholipase C (PLC) inhibitor neomycin. The PS1-DeltaE9-related high basal [Ca(2+)](i) was also reversed by xestospongin C confirming that this effect was inositol trisphosphate receptor-mediated. Carbachol gave a greater stimulation of [Ca(2+)](i) in PS1-DeltaE9 cells that took longer to return to basal as compared with responses seen in NT and PS1-WT cells. This long tail-off effect seen in PS1-DeltaE9 cells after carbachol stimulation was reversed by xestospongin C and dantrolene, suggesting that it was mediated by inositol trisphosphate receptor and ryanodine receptor amplification of Ca(2+). Ruthenium red only reduced carbachol peak elevations of [Ca(2+)](i) in NT and PS1-WT cells and not in PS1-DeltaE9 cells. No significant between cell type differences were seen for basal and carbachol-stimulated [Ca(2+)](i) with either ryanodine or the endoplasmic reticulum Ca(2+) ATPase inhibitor cyclopiazonic acid. Immunostaining experiments revealed that for all the cell types PS1 is present at the plasma membrane and co-localizes with N-cadherin, a component of the cell-cell adhesion complex. Immunoblotting of cell extracts for PLC-beta1 showed that, compared with NT and PS1-WT cells, the PS1-DeltaE9 transfectants gave a relative increase in levels of the calpain generated N-terminal fragment (100 kDa) over full-length (150 kDa) PLC-beta1. Our results suggest that the PS1-DeltaE9 mutation causes upstream changes in PI signaling with enhanced basal PLC activity as a primary effect that leads to a higher [Ca(2+)](i). This may provide a novel mechanism by which the PS1-DeltaE9 mutation sensitizes cells to apoptotic stimuli and enhanced amyloid beta generation.     

Functional significance of the negative-feedback regulation of ATP release via pannexin-1 hemichannels under ischemic stress in astrocytes

The opening of pannexin-1 (Px1) hemichannels is regulated by the activity of P2X(7) receptors (P2X(7)Rs). At present, however, little is known about how extracellular ATP-sensitive P2X(7)Rs regulates the opening and closure of Px1 hemichannels. Several lines of evidence suggest that P2X(7)Rs are activated under pathological conditions such as ischemia, resulting in the opening of Px1 hemichannels responsible for the massive influx of Ca(2+) from the extracellular space and the release of ATP from the cytoplasm, leading to cell death. Here we show in cultured astrocytes that the suppression of the activity of P2X(7)Rs during simulated ischemia (oxygen/glucose deprivation, OGD) resulted in the opening of Px1 hemichannels, leading to the enhanced release of ATP. In addition, the suppression of the activity of P2X(7)Rs during OGD resulted in a significant increase in astrocytic damage. Both the P2X(7)Rs suppression-induced enhancement of the release of ATP and cell damage were reversed by co-treatment with blockers of Px1 hemichannels, suggesting that suppression of the activity of PX(7)Rs resulted in the opening of Px1 hemichannels. All these findings suggested the existence of a negative-feedback loop regulating the release of ATP via Px1 hemichannels; ATP-induced suppression of ATP release. The present study indicates that ATP, released through Px1 hemichannels, activates P2X(7)Rs, resulting in the closure of Px1 hemichannels during ischemia. This negative-feedback mechanism, suppressing the loss of cellular ATP and Ca(2+) influx, might contribute to the survival of astrocytes under ischemic stress.     

Amiloride和Ni2+抑制缺血/复灌引起的大鼠心肌细胞内钙的增加

本文旨在研究Na+/H+交换以及Na+/Ca2 +交换对模拟缺血 /复灌引起的大鼠心肌细胞内游离钙水平变化的调节作用。分别利用模拟缺血液和正常台氏液对大鼠心肌细胞进行缺血 /复灌处理 ,在缺血期间分别应用Na+/H+交换抑制剂阿米洛利 (amiloride)、Na+/Ca2 +交换抑制剂NiCl2 以及无钙液 ,观察它们对细胞内游离Ca2 +浓度变化的影响。利用Zeiss LSM 5 10激光共聚焦显微镜检测、采集细胞内游离Ca2 +的指示剂Fluo 3 AM的荧光信号 ,计算出相对于正常(缺血前 )的相对荧光强度 ,以表示胞内游离Ca2 +浓度的变化。结果显示 ,模拟缺血引起大鼠心肌细胞内游离Ca2 +持续上升 ,缺血前的相对荧光强度值为 10 0 % ,模拟缺血 5min后为 140 3± 13 0 % (P <0 0 5 ) ,复灌 15min后为 142 8±15 5 % (P <0 0 5 )。经 10 0 μmol/Lamiloride、5mmol/LNiCl2 和无钙液分别预处理 ,模拟缺血 5min后的相对荧光强度分别为 10 1 4± 16 3 % (P <0 0 5 )、110 4± 11 1% (P <0 0 5 )和 10 7 1± 10 8(P <0 0 5 ) ;复灌 15min后则分别为 97 8±14 3 % (P <0 0 5 )、10 6 2± 14 5 % (P <0 0 5 )和 10 6 6± 15 7(P <0 0 5 )。另外 ,与对照组细胞相比 ,再灌注期间NiCl2和无钙液处理的细胞钙振荡的产生幅度明显减弱 ,amilorid     

Genomic and nongenomic stimulatory effect of aldosterone on H+-ATPase in proximal S3 segments

The genomic and nongenomic effects of aldosterone on the intracellular pH recovery rate (pHirr) via H(+)-ATPase and on cytosolic free calcium concentration ([Ca(2+)](i)) were investigated in isolated proximal S3 segments of rats during superfusion with an Na(+)-free solution, by using the fluorescent probes BCECF-AM and FLUO-4-AM, respectively. The pHirr, after cellular acidification with a NH(4)Cl pulse, was 0.064 ± 0.003 pH units/min (n = 17/74) and was abolished with concanamycin. Aldosterone (10(-12), 10(-10), 10(-8), or 10(-6) M with 1-h or 15- or 2-min preincubation) increased the pHirr. The baseline [Ca(2+)](i) was 103 ± 2 nM (n = 58). After 1 min of aldosterone preincubation, there was a transient and dose-dependent increase in [Ca(2+)](i) and after 6-min preincubation there was a new increase in [Ca(2+)](i) that persisted after 1 h. Spironolactone [mineralocorticoid (MR) antagonist], actinomycin D, or cycloheximide did not affect the effects of aldosterone (15- or 2-min preincubation) on pHirr and on [Ca(2+)](i) but inhibited the effects of aldosterone (1-h preincubation) on these parameters. RU 486 [glucocorticoid (GR) antagonist] and dimethyl-BAPTA (Ca(2+) chelator) prevented the effect of aldosterone on both parameters. The data indicate a genomic (1 h, via MR) and a nongenomic action (15 or 2 min, probably via GR) on the H(+)-ATPase and on [Ca(2+)](i). The results are compatible with stimulation of the H(+)-ATPase by increases in [Ca(2+)](i) (at 10(-12)-10(-6) M aldosterone) and inhibition of the H(+)-ATPase by decreases in [Ca(2+)](i) (at 10(-12) or 10(-6) M aldosterone plus RU 486).     

N(omega)-nitro-L-arginine inhibits inducible HSP-70 via Ca(2+), PKC, and PKA in human intestinal epithelial T84 cells

The nitric oxide (NO) synthase inhibitor N(omega)-nitro-L-arginine (L-NNA) inhibits heat stress (HS)-induced NO production and the inducible 70-kDa heat shock protein (HSP-70i) in many rodent organs. We used human intestinal epithelial T84 cells to characterize the inhibitory effect of L-NNA on HS-induced HSP-70i expression. Intracellular Ca(2+) concentration ([Ca(2+)](i)) was measured using fura-2, and protein kinase C (PKC), and PKA activities were determined. HS increased HSP-70i mRNA and protein in T84 cells exposed to 45 degrees C for 10 min and allowed to recover for 6 h. L-NNA treatment for 1 h before HS inhibited the induction of HSP-70i mRNA and protein, with an IC(50) of 0.0471 +/- 0.0007 microM. Because the HS-induced increase in HSP-70i mRNA and protein is Ca(2+) dependent, we measured [Ca(2+)](i) after treating cells with L-NNA. L-NNA at 100 microM significantly decreased resting [Ca(2+)](i). Likewise, treatment with 1 microM GF-109203X or H-89 (inhibitors of PKC and PKA, respectively) for 30 min also significantly decreased [Ca(2+)](i) and inhibited HS-induced increase in HSP-70i. GF-109203X- or H-89-treated cells failed to respond to L-NNA by further decreasing [Ca(2+)](i) and HSP-70i. L-NNA effectively blocked heat shock factor-1 (HSF1) translocation from the cytosol to the nucleus, a process requiring PKC phosphorylation. These results suggest that L-NNA inhibits HSP-70i by reducing [Ca(2+)](i) and decreasing PKC and PKA activity, thereby blocking HSF1 translocation from the cytosol to the nucleus.     

Preconditioning limits mitochondrial Ca(2+) during ischemia in rat hearts: role of K(ATP) channels

Prolonged myocardial ischemia results in an increase in intracellular calcium concentration ([Ca(2+)]i), which is thought to play a critical role in ischemia-reperfusion injury. Ischemic preconditioning (PC) improves myocardial function during ischemia-reperfusion, a process that may involve opening mitochondrial ATP-sensitive potassium (K(ATP)) channels. Because pharmacological limitation of mitochondrial calcium concentration ([Ca(2+)]m) overload during ischemia-reperfusion has been shown to improve myocardial function, we hypothesized that PC would reduce [Ca(2+)]m during ischemia-reperfusion and that this effect was mediated by opening mitochondrial K(ATP) channels. Isolated rat hearts were subjected to 25 min of global ischemia and 30 min of reperfusion with or without PC in the presence of mitochondrial K(ATP) channel opening (diazoxide, 100 microM) and blockade [5-hydroxydecanoic acid (5-HD), 100 microM]. Contracture during ischemia (end-diastolic pressure) and functional recovery on reperfusion (developed pressure) were assessed. Total [Ca(2+)]i and [Ca(2+)]m were measured using indo 1 fluorescence. Both PC and diazoxide limited the increase in end-diastolic pressure and resulted in greater functional recovery after 30 min of reperfusion, functional effects that were partially or completely abolished by 5-HD. PC and diazoxide also significantly limited the increase in [Ca(2+)]m during ischemia-reperfusion. In addition, PC lowered [Ca(2+)]i during reperfusion, whereas diazoxide paradoxically resulted in increased [Ca(2+)]i during reperfusion. There was an inverse linear relationship between [Ca(2+)]m and developed pressure during reperfusion. PC limits the ischemia-induced increase in mitochondrial, but not total, [Ca(2+)]i, an effect mediated by opening mitochondrial K(ATP) channels. These data suggest that the lowering of mitochondrial calcium overload is a mechanism of cardioprotection in PC.     

Rescue of contractile abnormalities by Na+/Ca2+ exchanger overexpression in postinfarction rat myocytes.

Previous studies on myocytes isolated from rat hearts 3 wk after myocardial infarction (MI) demonstrated increased cell length, reduced Na(+)/Ca(2+) exchange (NCX1) activity, altered contractility, and intracellular Ca(2+) concentration ([Ca(2+)](i)) transients. In the present study, we investigated whether NCX1 overexpression in MI myocytes would restore contraction and [Ca(2+)](i) transients to normal. When myocytes were placed in culture under continued electrical-field stimulation conditions, differences in contraction amplitudes and cell lengths between sham and MI myocytes were preserved for at least 48 h. Infection of both sham and MI myocytes by adenovirus expressing green fluorescent protein resulted in >95% infection, as evidenced by green fluorescent protein fluorescence, but contraction amplitudes at 6-, 24-, and 48-h postinfection were not affected. NCX1 overexpression in MI myocytes resulted in lower diastolic [Ca(2+)](i) levels at all extracellular Ca(2+) concentrations ([Ca(2+)](o)) examined, suggesting enhanced forward NCX1 activity. At 5 mM [Ca(2+)](o), subnormal contraction and [Ca(2+)](i) transient amplitudes in MI myocytes (compared with sham myocytes) were restored toward normal levels by overexpressing NCX1. At 0.6 mM [Ca(2+)](o), supranormal contraction and [Ca(2+)](i) transient amplitudes in MI myocytes (compared with sham myocytes) were lowered by NCX1 overexpression. We conclude that overexpression of NCX1 in MI myocytes was effective in improving contractile dysfunction, most likely because of enhancement of both Ca(2+) efflux and influx during a cardiac cycle. We suggest that decreased NCX1 activity may play an important role in contractile abnormalities in postinfarction myocytes.