Effects of mitochondria-associated Ca2+ transporters suppression on oocyte activation.

Oocyte activation deficiency leads to female infertility. [Ca2+ ]i oscillations are required for mitochondrial energy supplement transition from the resting to the excited state, but the underlying mechanisms are still very little known. Three mitochondrial Ca2+ channels, Mitochondria Calcium Uniporter (MCU), Na+ /Ca2+ Exchanger (NCLX) and Voltage-dependent Ca2+ Channel (VDAC), were deactivated by inhibitors RU360, CGP37157 and Erastin, respectively. Both Erastin and CGP37157 inhibited mitochondrial activity significantly while attenuating [Ca2+ ]i and [Ca2+ ]m oscillations, which caused developmental block of pronuclear formation. Thus, NCLX and VDAC are two mitochondria-associated Ca2+ transporter proteins regulating oocyte activation, which may be used as potential targets to treat female infertility. SIGNIFICANCE OF THE STUDY: NCLX and VDAC are two mitochondria-associated Ca2+ transporter proteins regulating oocyte activation.

Roles Played by the Na+/Ca2+ Exchanger and Hypothermia in the Prevention of Ischemia-Induced Carrier-Mediated Efflux of Catecholamines into the Extracellular Space: Implications for Stroke Therapy.

The release of [3H]dopamine ([3H]DA) and [3H]noradrenaline ([3H]NA) in acutely perfused rat striatal and cortical slice preparations was measured at 37 °C and 17 °C under ischemic conditions. The ischemia was simulated by the removal of oxygen and glucose from the Krebs solution. At 37 °C, resting release rates in response to ischemia were increased; in contrast, at 17 °C, resting release rates were significantly reduced, or resting release was completely prevented. The removal of extracellular Ca2+ further increased the release rates of [3H]DA and [3H]NA induced by ischemic conditions. This finding indicated that the Na+/Ca2+ exchanger (NCX), working in reverse in the absence of extracellular Ca2+, fails to trigger the influx of Ca2+ in exchange for Na+ and fails to counteract ischemia by further increasing the intracellular Na+ concentration ([Na+]i). KB-R7943, an inhibitor of NCX, significantly reduced the cytoplasmic resting release rate of catecholamines under ischemic conditions and under conditions where Ca2+ was removed. Hypothermia inhibited the excessive release of [3H]DA in response to ischemia, even in the absence of Ca2+. These findings further indicate that the NCX plays an important role in maintaining a high [Na+]i, a condition that may lead to the reversal of monoamine transporter functions; this effect consequently leads to the excessive cytoplasmic tonic release of monoamines and the reversal of the NCX. Using HPLC combined with scintillation spectrometry, hypothermia, which enhances the stimulation-evoked release of DA, was found to inhibit the efflux of toxic DA metabolites, such as 3,4-dihydroxyphenylacetaldehyde (DOPAL). In slices prepared from human cortical brain tissue removed during elective neurosurgery, the uptake and release values for [3H]NA did not differ from those measured at 37 °C in slices that were previously maintained under hypoxic conditions at 8 °C for 20 h. This result indicates that hypothermia preserves the functions of the transport and release mechanisms, even under hypoxic conditions. Oxidative stress (H2O2), a mediator of ischemic brain injury enhanced the striatal resting release of [3H]DA and its toxic metabolites (DOPAL, quinone). The study supports our earlier findings that during ischemia transmitters are released from the cytoplasm. In addition, the major findings of this study that hypothermia of brain slice preparations prevents the extracellular calcium concentration ([Ca2+]o)-independent non-vesicular transmitter release induced by ischemic insults, inhibiting Na+/Cl--dependent membrane transport of monoamines and their toxic metabolites into the extracellular space, where they can exert toxic effects.

Benzothiazepine CGP37157 Analogues Exert Cytoprotection in Various in Vitro Models of Neurodegeneration.

Mitochondria regulate cellular Ca(2+) oscillations, taking up Ca(2+) through its uniporter and releasing it through the mitochondrial sodium/calcium exchanger. The role of mitochondria in the regulation of Ca(2+) cycle has received much attention recently, as it is a central stage in neuronal survival and death processes. Over the last decades, the 4,1-benzothiazepine CGP37157 has been the only available blocker of the mitochondrial sodium/calcium exchanger, although it targets several other calcium transporters. We report the synthesis of 4,1-benzothiazepine derivatives with the goal of enhancing mitochondrial sodium/calcium exchanger blockade and selectivity, and the evaluation of their cytoprotective effect. The compound 4c presented an interesting neuroprotective profile in addition to an important blockade of the mitochondrial sodium/calcium exchanger. The use of this benzothiazepine could help to understand the physiological functions of the mitochondrial sodium/calcium exchanger. In addition, we hypothesize that a moderate blockade of the mitochondrial sodium/calcium exchanger would provide enhanced neuroprotection in neurons.

Trafficking of Na+/Ca2+ exchanger to the site of persistent inflammation in nociceptive afferents.

Persistent inflammation results in an increase in the amplitude and duration of depolarization-evoked Ca(2+) transients in putative nociceptive afferents. Previous data indicated that these changes were the result of neither increased neuronal excitability nor an increase in the amplitude of depolarization. Subsequent data also ruled out an increase in voltage-gated Ca(2+) currents and recruitment of Ca(2+)-induced Ca(2+) release. Parametric studies indicated that the inflammation-induced increase in the duration of the evoked Ca(2+) transient required a relatively large and long-lasting increase in the concentration of intracellular Ca(2+) implicating the Na(+)/Ca(2+) exchanger (NCX), a major Ca(2+) extrusion mechanism activated with high intracellular Ca(2+) loads. The contribution of NCX to the inflammation-induced increase in the evoked Ca(2+) transient in rat sensory neurons was tested using fura-2 AM imaging and electrophysiological recordings. Changes in NCX expression and protein were assessed with real-time PCR and Western blot analysis, respectively. An inflammation-induced decrease in NCX activity was observed in a subpopulation of putative nociceptive neurons innervating the site of inflammation. The time course of the decrease in NCX activity paralleled that of the inflammation-induced changes in nociceptive behavior. The change in NCX3 in the cell body was associated with a decrease in NCX3 protein in the ganglia, an increase in the peripheral nerve (sciatic) yet no change in the central root. This single response to inflammation is associated with changes in at least three different segments of the primary afferent, all of which are likely to contribute to the dynamic response to persistent inflammation.

Pharmacological characterization of the newly synthesized 5-amino-N-butyl-2-(4-ethoxyphenoxy)-benzamide hydrochloride (BED) as a potent NCX3 inhibitor that worsens anoxic injury in cortical neurons, organotypic hippocampal cultures, and ischemic brain.

The Na(+)/Ca(2+) exchanger (NCX), a 10-transmembrane domain protein mainly involved in the regulation of intracellular Ca(2+) homeostasis, plays a crucial role in cerebral ischemia. In the present paper, we characterized the effect of the newly synthesized compound 5-amino-N-butyl-2-(4-ethoxyphenoxy)-benzamide hydrochloride (BED) on the activity of the three NCX isoforms and on the evolution of cerebral ischemia. BED inhibited NCX isoform 3 (NCX3) activity (IC50 = 1.9 nM) recorded with the help of single-cell microflorimetry, (45)Ca(2+) radiotracer fluxes, and patch-clamp in whole-cell configuration. Furthermore, this drug displayed negligible effect on NCX2, the other isoform expressed within the CNS, and it failed to modulate the ubiquitously expressed NCX1 isoform. Concerning the molecular site of action, the use of chimera strategy and deletion mutagenesis showed that α1 and α2 repeats of NCX3 represented relevant molecular determinants for BED inhibitory action, whereas the intracellular regulatory f-loop was not involved. At 10 nM, BED worsened the damage induced by oxygen/glucose deprivation (OGD) followed by reoxygenation in cortical neurons through a dysregulation of [Ca(2+)]i. Furthermore, at the same concentration, BED significantly enhanced cell death in CA3 subregion of hippocampal organotypic slices exposed to OGD and aggravated infarct injury after transient middle cerebral artery occlusion in mice. These results showed that the newly synthesized 5-amino-N-butyl-2-(4-ethoxyphenoxy)-benzamide hydrochloride is one of the most potent inhibitor of NCX3 so far identified, representing an useful tool to dissect the role played by NCX3 in the control of Ca(2+) homeostasis under physiological and pathological conditions.

KB-R7943, an inhibitor of the reverse Na(+)/Ca(2+) exchanger, does not modify secondary pathology in the thalamus following focal cerebral stroke in rats.

Remote areas connected to cortical infarcts, such as the thalamus, are affected by stroke due to delayed retrograde degeneration of afferent connections. This is temporally associated with the accumulation of ß-amyloid (Aß) and calcium. Here we tested a hypothesis that prevention of excessive Ca(2+) influx into the axoplasm via the reverse Na(+)/Ca(2+) exchanger (NCX) would provide axonal protection and eventually lessen the Aß and calcium load in the thalamus. We found that chronic treatment with a specific inhibitor of the reverse NCX, KB-R7943 (30mg/kg once daily, 27 days) after middle cerebral artery occlusion did not prevent atypical secondary pathology in the thalamus or improve functional outcome. The present data do not support a role for reverse NCX activity in the complex pathology within the thalamus after cerebral ischemia.

[Effects of selective inhibition of reverse mode of Na(+)/Ca(2+) exchanger on rats with contrast-induced acute kidney injury].

OBJECTIVE: Intracellular Ca(2+) overload is a key factor in contrast-induced renal tubular toxicity. Na(+)/Ca(2+) exchanger (NCX) system is one of main pathways of intracellular Ca(2+) overload. We explore the effects of KB-R7943, an inhibitor of reverse mode of NCX, on contrast-induced acute kidney injury (CI-AKI). METHODS: Rats were divided into control, CI-AKI and pre-treatment groups with KB-R7943 (5, 10 mg/kg). CI-AKI was induced by diatrizoate administration in rats with cholesterol-supplemented diet for 8 weeks. Renal function and hemodynamics were determined at Day 1 post-administration. Renal histopathology was observed under light microscope. Renal tubular apoptosis was examined by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). Renal endothelin-1 (ET-1) was measured by radioimmunoassay. The oxidative markers of renal malondialdehyde (MDA) and catalase (CAT) were measured. The expression of NCX was evaluated by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Levels of serum creatinine (Scr, µmol/L ) in CI-AKI rats ((149 ± 35) µmol/L) were significantly higher than those of normal rats ((55 ± 4) µmol/L, P < 0.01). Renal ET-1, MDA and CAT, resistance index (RI) of renal blood vessels increased significantly in CI-AKI rats. The contrast-induced increases in Scr and RI of renal blood vessels were suppressed significantly and dose-dependently by pretreatment with KB-R7943. Histopathological and TUNEL results showed that contrast-induced severe renal tubular necrosis and apoptosis were significantly and dose-dependently attenuated by KB-R7943. KB-R7943 significantly suppressed the contrast-induced increments of ET-1, MDA and CAT. No significant changes in NCX1 mRNA expression were observed following contrast administration. CONCLUSION: Renal oxidative stress and ET-1 overproduction via the activation of reverse mode of NCX play an important role in the pathogenesis of CI-AKI. And inhibition of reverse mode of NCX expressed in renal tubular epithelial cell has protective effects on CI-AKI.

Simultaneous measurement of cytosolic and mitochondrial Ca(2+) during ischemia in mice whole-brain slice preparation and its application to drug evaluation.

We developed a conventional imaging method to measure Ca(2+) concentration in cytosol (using FuraRed as an indicator) and mitochondria (using Rhod-2 as an indicator), simultaneously, by alternative excitation with specific wave length. After confirming the availability of the method in Hela cells, we applied it to mouse whole-brain slice -preparation, which was exposed to oxygen- and glucose-deprived artificial cerebrospinal fluid (ischemic ACSF) for 12 min. The fluorescence (>570 nm) at the cerebral cortex and hippocampus due to FuraRed (excited by 480 ± 10 nm) decreased (indicating the increase in cytosolic Ca(2+)-concentration), while the fluorescence due to Rhod-2 (excited by 560 ± 10 nm) increased (indicating the increase in mitochondrial Ca(2+) concentration) during exposure to ischemic conditions. We found the characteristic protective effects of cyclosporine A (10(-6) M), a known blocker for mitochondrial permeability transition, and SEA0400 (10(-6) M), a blocker for Na(+)/Ca(2+) exchanger, on the abnormal Ca(2+) increase in cytosol. We confirmed that the present method will be useful for future pathological and pharmacological studies on ischemia-induced brain damage.

Na+/Ca2+ exchange inhibitor, KB-R7943, attenuates contrast-induced acute kidney injury.

BACKGROUND: Intracellular Ca2+ overload is considered to be a key factor in contrast-induced acute kidney injury (CI-AKI). The Na+/Ca2+ exchanger (NCX) system is one of the main pathways of intracellular Ca2+ overload. We investigated the effects of KB-R7943, an inhibitor of the reverse mode of NCX, on CI-AKI in a rat model. METHOD: Rats were divided into control group, CI-AKI group and pretreatment groups (with KB-R7943 dose of 5 or 10 mg/kg). CI-AKI was induced by diatrizoate administration in rats with cholesterol-supplemented diet for 8 weeks. Renal function and renal hemodynamics were determined 1 day following contrast medium administration. Renal histopathology was observed by light microscope. Renal tubular apoptosis was examined by TUNEL. Renal endothelin-1 (ET-1) was measured by radioimmunoassay. Renal malondialdehyde (MDA) and catalase (CAT) were measured as oxidative markers. RESULTS: Levels of serum creatinine (Scr), renal ET-1, MDA and CAT, and resistance index (RI) of renal blood vessels increased significantly in CI-AKI rats. The increases in Scr and RI of renal blood vessels induced by diatrizoate were suppressed significantly and dose-dependently by pretreatment with KB-R7943. Histopathological and TUNEL results showed that the contrast medium-induced severe renal tubular necrosis and apoptosis were significantly and dose-dependently attenuated by KB-R7943. KB-R7943 significantly suppressed the increment of renal ET-1 content and MDA and CAT level induced by contrast medium administration. CONCLUSION: Activation of the reverse mode of NCX, followed by ET-1 overproduction and increased oxidative stress, seems to play an important role in the pathogenesis of CI-AKI. The inhibitor of the reverse mode of NCX, KB-R7943, has renoprotective effects on CI-AKI.

Vasopressin and oxytocin excite MCH neurons, but not other lateral hypothalamic GABA neurons.

Neurons that synthesize melanin-concentrating hormone (MCH) colocalize GABA, regulate energy homeostasis, modulate water intake, and influence anxiety, stress, and social interaction. Similarly, vasopressin and oxytocin can influence the same behaviors and states, suggesting that these neuropeptides may exert part of their effect by modulating MCH neurons. Using whole cell recording in MCH-green fluorescent protein (GFP) transgenic mouse hypothalamic brain slices, we found that both vasopressin and oxytocin evoked a substantial excitatory effect. Both peptides reversibly increased spike frequency and depolarized the membrane potential in a concentration-dependent and tetrodotoxin-resistant manner, indicating a direct effect. Substitution of lithium for extracellular sodium, Na(+)/Ca(2+) exchanger blockers KB-R7943 and SN-6, and intracellular calcium chelator BAPTA, all substantially reduced the vasopressin-mediated depolarization, suggesting activation of the Na(+)/Ca(2+) exchanger. Vasopressin reduced input resistance, and the vasopressin-mediated depolarization was attenuated by SKF-96265, suggesting a second mechanism based on opening nonselective cation channels. Neither vasopressin nor oxytocin showed substantial excitatory actions on lateral hypothalamic inhibitory neurons identified in a glutamate decarboxylase 67 (GAD67)-GFP mouse. The primary vasopressin receptor was vasopressin receptor 1a (V1aR), as suggested by the excitation by V1aR agonist [Arg(8)]vasotocin, the selective V1aR agonist [Phe(2)]OVT and by the presence of V1aR mRNA in MCH cells, but not in other nearby GABA cells, as detected with single-cell RT-PCR. Oxytocin receptor mRNA was also detected in MCH neurons. Together, these data suggest that vasopressin or oxytocin exert a minimal effect on most GABA neurons in the lateral hypothalamus but exert a robust excitatory effect on presumptive GABA cells that contain MCH. Thus, some of the central actions of vasopressin and oxytocin may be mediated through MCH cells.

Conus magus vs. Irukandji syndrome: a computational approach of a possible new therapy.

The Irukandji syndrome is caused by the sting of some small jellyfish species. The syndrome has severe life-threatening consequences. The exacerbating pain and cardiovascular symptoms (tachycardia and hypertension) are hard to control in many cases. We suggest a way to experiment a new possible therapy with an FDA approved analgesic, ziconotide, a synthetic derivative from a marine cone snail (Conus magus) venom component, which is administrated intravenously. The proposed experimental plasma concentration of ziconotide for rats is in the range of 0-6µgml(-1). Based on a molecular biological scenario of the venom action mechanism at cellular level, we suggest that the proposed method should be functional in re-establishing the normal cardiovascular parameters of the experimental animals and concomitantly it should abolish the severe pain caused by envenomation. We expect that positive experimental results in agreement with our theory will lead to the possibility of a new therapy for the Irukandji syndrome and possibly for other envenomations with similar ethyology.

Dual effects of nobiletin, a citrus polymethoxy flavone, on catecholamine secretion in cultured bovine adrenal medullary cells.

Nobiletin, a compound of polymethoxy flavones found in citrus fruits, possesses a wide range of pharmacological activities. Here we report the effects of nobiletin on catecholamine secretion in cultured bovine adrenal medullary cells. Nobiletin (1.0-100 microM) concentration-dependently stimulated catecholamine secretion and (45)Ca(2+) influx. Its stimulatory effect of nobiletin on catecholamine secretion was abolished by deprivation of extracellular Ca(2+) and partially inhibited by specific inhibitors of voltage-dependent Ca(2+) channels and Na(+)/Ca(2+) exchangers. On the other hand, nobiletin suppressed catecholamine secretion and (22)Na(+) and (45)Ca(2+) influx induced by acetylcholine, an agonist of nicotinic acetylcholine receptors, in a concentration-dependent manner. It also inhibited catecholamine secretion, (22)Na(+) influx and/or (45)Ca(2+) influx induced by veratridine, an activator of voltage-dependent Na(+) channels, and 56 mM K(+), an activator of voltage-dependent Ca(2+) channels. In Xenopus oocytes expressing alpha3beta4 neuronal acetylcholine receptors, nobiletin directly inhibited the current evoked by acetylcholine in a concentration-dependent manner similar to that observed in catecholamine secretion. The present findings suggest that nobiletin, by itself, stimulates catecholamine secretion via activation of voltage-dependent Ca(2+) channels or Na(+)/Ca(2+) exchangers, whereas it inhibits catecholamine secretion induced by acetylcholine through the suppression of Na(+) influx and Ca(2+) influx in cultured bovine adrenal medullary cells.

Pharmacological modifications of the stretch-induced effects on ventricular fibrillation in perfused rabbit hearts.

Stretch induces modifications in myocardial electrical and mechanical activity. Besides the effects of substances that block the stretch-activated channels, other substances could modulate the effects of stretch through different mechanisms that affect Ca(2+) handling by myocytes. Thirty-six Langendorff-perfused rabbit hearts were used to analyze the effects of the Na(+)/Ca(2+) exchanger blocker KB-R7943, propranolol, and the adenosine A(2) receptor antagonist SCH-58261 on the acceleration of ventricular fibrillation (VF) produced by acute myocardial stretching. VF recordings were obtained with two epicardial multiple electrodes before, during, and after local stretching in four experimental series: control (n = 9), KB-R7943 (1 microM, n = 9), propranolol (1 microM, n = 9), and SCH-58261 (1 microM, n = 9). Both the Na(+)/Ca(2+) exchanger blocker KB-R7943 and propranolol induced a significant reduction (P < 0.001 and P < 0.05, respectively) in the dominant frequency increments produced by stretching with respect to the control and SCH-58261 series (control = 49.9%, SCH-58261 = 52.1%, KB-R7943 = 9.5%, and propranolol = 12.5%). The median of the activation intervals, the functional refractory period, and the wavelength of the activation process during VF decreased significantly under stretch in the control and SCH-58261 series, whereas no significant variations were observed in the propranolol and KB-R7943 series, with the exception of a slight but significant decrease in the median of the fibrillation intervals in the KB-R7943 series. KB-R7943 and propranolol induced a significant reduction in the activation maps complexity increment produced by stretch with respect to the control and SCH-58261 series. In conclusion, the electrophysiological effects responsible for stretch-induced VF acceleration in the rabbit heart are reduced by the Na(+)/Ca(2+) exchanger blocker KB-R7943 and by propranolol but not by the adenosine A(2) receptor antagonist SCH-58261.

Involvement of Na(+)-Ca (2+) exchanger in the endothelium-independent vasorelaxation induced by Curcuma longa L. in isolated rat superior mesenteric arteries.

Previous studies confirmed that the methanolic extract from Curcuma longa L. (CLME) lowers arterial blood pressure and heart rate in rats due to the blockade of extracellular Ca(2+) influx. The aim of this study was to investigate the involvement of Na(+)-Ca (2+) exchanger in the vasorelaxant effects elicited by CLME in isolated rat superior mesenteric arteries. CLME (1-1,000 microg/ml) concentration-dependently relaxed phenylephrine (PHE) (10 microM) pre-contracted arterial rings with intact-endothelium (pD(2) and E(max) = 2.04 +/- 0.06 and 88.3 +/- 3.2%) or denuded-endothelium (pD(2) and E(max) = 2.06 +/- 0.03 and 91.4 +/- 1.0%), respectively, suggesting that the removal of endothelium has no significant effect (P>0.05) on the vasorelaxation induced by CLME. Furthermore, CLME (30, 100 and 300 microg/ml) inhibited the cumulative concentration-response curves to PHE (10(-8)-10(-5) M) in a concentration-dependent manner, whereas, treatment with ouabain 100 microM (selective blocker of Na(+)-K(+) ATPase) has no effect on the relaxant responses of CLME. However, treatment with nickel chloride (NiCl(2)) (100, 300 and 400 microM), a putative Na(+)-Ca(2+) exchanger inhibitor, concentration-dependently reduced the vasorelaxant responses of CLME. Precisely, NiCl(2) at 100, 300 and 400 microM significantly (P<0.05) decreased the pD(2) and E(max) values of CLME (1.86 +/- 0.03 and 81.3 +/- 1.2%, 1.77 +/- 0.03 and 60.2 +/- 0.8%, 1.69 +/- 0.04 and 55.3 +/- 1.6%, respectively). Also, CLME (100 microg/ml) produced less relaxant effect with decreasing extracellular Na(+) concentration. CLME-induced vasorelaxation was completely abolished in a Na(+)-free Tyrode's solution, a condition that eliminates the influence of the forward mode of the exchanger. The results provide indirect evidence that the stimulation of the forward mode of Na(+)-Ca (2+) exchanger may probably contribute to the vasorelaxation induced by CLME in endothelium-denuded arterial rings.

Cardioprotective effects of KB-R7943, a novel inhibitor of Na+/Ca2+ exchanger, on stunned myocardium in anesthetized dogs.

PURPOSE: The present study was carried out to determine the cardioprotective effects of KB-R7943 (KBR), a selective inhibitor of the reverse mode of Na+/Ca2+ exchanger (NCX), on stunned myocardium in anesthetized dogs. METHODS: The dogs were allocated to one of three groups (n = 7 for each group), and received drug vehicle (group C), low-dose KBR (5 mg x kg(-1) i.v.) (group L) or high-dose KBR (10 mg x kg(-1) i.v.) (group H) at 15 min before left anterior descending coronary artery (LAD) occlusion. Stunned myocardium was produced by 15-min occlusion of LAD and 90-min reperfusion in all dogs. Regional myocardial contractility was evaluated with segment shortening (%SS). RESULTS: Recovery of %SS at 90 min after reperfusion was significantly improved in group H (70.8% +/- 3.9% of baseline), whereas the recovery was poor in groups C and L (34.3% +/- 2.8% and 36.4% +/- 5.4% of baseline, respectively). Regional myocardial blood flow showed no significant difference among groups. KBR had no effect on coronary or systemic hemodynamics. CONCLUSION: The results show that preischemic administration of high-dose KBR markedly improves myocardial contractile dysfunction after ischemia-reperfusion in anesthetized dogs, indicating that KBR protects myocardium against the ischemia-reperfusion injury in vivo.

External bioenergy-induced increases in intracellular free calcium concentrations are mediated by Na+/Ca2+ exchanger and L-type calcium channel.

External bioenergy (EBE, energy emitted from a human body) has been shown to increase intracellular calcium concentration ([Ca2+]i, an important factor in signal transduction) and regulate the cellular response to heat stress in cultured human lymphoid Jurkat T cells. In this study, we wanted to elucidate the underlying mechanisms. A bioenergy specialist emitted bioenergy sequentially toward tubes of cultured Jurkat T cells for one 15-minute period in buffers containing different ion compositions or different concentrations of inhibitors. [Ca2+], was measured spectrofluorometrically using the fluorescent probe fura-2. The resting [Ca2+]i in Jurkat T cells was 70 +/- 3 nM (n = 130) in the normal buffer. Removal of external calcium decreased the resting [Ca2+]i to 52 +/- 2 nM (n = 23), indicating that Ca2+ entry from the external source is important for maintaining the basal level of [Ca2+]i. Treatment of Jurkat T cells with EBE for 15 min increased [Ca2+]i by 30 +/- 5% (P < 0.05, Student t-test). The distance between the bioenergy specialist and Jurkat T cells and repetitive treatments of EBE did not attenuate [Ca2+]i responsiveness to EBE. Removal of external Ca2+ or Na+, but not Mg2+, inhibited the EBE-induced increase in [Ca2+]i. Dichlorobenzamil, an inhibitor of Na+/Ca2+ exchangers, also inhibited the EBE-induced increase in [Ca2+]i in a concentration-dependent manner with an IC50 of 0.11 +/- 0.02 nM. When external [K+] was increased from 4.5 mM to 25 mM, EBE decreased [Ca2+]i. The EBE-induced increase was also blocked by verapamil, an L-type voltage-gated Ca2+ channel blocker. These results suggest that the EBE-induced [Ca2+]i increase may serve as an objective means for assessing and validating bioenergy effects and those specialists claiming bioenergy capability. The increase in [Ca2+]i is mediated by activation of Na+/Ca2+ exchangers and opening of L-type voltage-gated Ca2+ channels.

Plasma membrane KATP channel-mediated cardioprotection involves posthypoxic reductions in calcium overload and contractile dysfunction: mechanistic insights into cardioplegia.

Our recent data demonstrate that activation of pmKATP channels polarizes the membrane of cardiomyocytes and reduces Na+/Ca2+ exchange-mediated Ca2+ overload. However, it is important that these findings be extended into contractile models of hypoxia/reoxygenation injury to further test the notion that pmKATP channel activation affords protection against contractile dysfunction and calcium overload. Single rat heart right ventricular myocytes were enzymatically isolated, and cell contractility and Ca2+ transients in field-stimulated myocytes were measured in a cellular model of metabolic inhibition and reoxygenation. Activation of pmKATP with P-1075 (5 microM) or inhibition of the Na+/Ca2+ exchanger with KB-R7943 (5 microM)reduced reoxygenation-induced diastolic Ca2+ overload and improved the rate and magnitude of posthypoxic contractile recovery during the first few minutes of reoxygenation. Moreover,diastolic Ca2+ overload and posthypoxic contractile dysfunction were aggravated in ventricular myocytes either subjected to specific blockade of pmKATP with HMR1098 (20 microM) or expressing the dominant-negative pmKATP construct Kir6.2(AAA) in the presence of P-1075. Our results suggest that a common mechanism, involving resting membrane potential-modulated increases in diastolic [Ca2+]i, is responsible for the development of contractile dysfunction during reoxygenation following metabolic inhibition. This novel and highly plausible cellular mechanism for pmKATP-mediated cardioprotection may have direct clinical relevance as evidenced by the following findings: a hypokalemic polarizing cardioplegia solution supplemented with the pmKATP opener P-1075 improved Ca2+ homeostasis and recovery of function compared with hyperkalemic depolarizing St. Thomas' cardioplegia following contractile arrest in single ventricular myocytes and working rat hearts. We therefore propose that activation of pmKATP channels improves posthypoxic cardiac function via reductions in abnormal diastolic Ca2+ homeostasis mediated by reverse-mode Na+/Ca2+ exchange.

Cross-talk between native plasmalemmal Na+/Ca2+ exchanger and inositol 1,4,5-trisphosphate-sensitive ca2+ internal store in Xenopus oocytes.

Because the presence of a native plasmalemmal Na+/Ca2+ exchange (NCX) activity in Xenopus laevis oocytes remains controversial, its possible functional role in these cells is poorly understood. Here, in experiments on control oocytes and oocytes overexpressing a cloned NCX1 cardiac protein, confocal microscopy combined with electrophysiological techniques reveal that these cells express an endogenous NCX protein forming a functional microdomain with inositol 1,4,5-trisphosphate receptors (InsP3R) that controls intracellular Ca2+ in a restricted subplasmalemmal space. The following data obtained in control denuded oocytes are consistent with this view: (i) reverse transcription-PCR revealed that the oocyte expresses two transcripts for the NCX1 and NCX3 isoforms; (ii) immunofluorescence experiments showed that native NCX1 and InsP3Rs are largely codistributed in discrete areas of the plasma membrane in close apposition to the cortical endoplasmic reticulum shell; (iii) when stimulated by rabbit serum, which elevates intracellular Ca2+ mediated by InsP3, voltage-clamped oocytes display a large and transient inward Ca2+ -activated chloride current, IClCa, as a result of the Ca2+ rise at the inner surface membrane; (iv) this current is significantly enhanced by KB-R7943 and by an extracellular sodium-depleted medium, two maneuvers that prevent "Ca2+ extrusion" via NCX; and (v) blocking NCX enhanced the IClCa elicited by InsP3 but not by Ca2+ photolysis in oocytes injected with the respective caged compounds. Moreover, overexpression of cardiac NCX1, confirmed by confocal microscopy, has functional consequences for the "Ca2+ influx" but not for the serum-elicited "Ca2+ efflux" mode of basal exchange activity and does not alter the number of endogenous NCX/InsP3Rs colocalization sites. Our results suggest that native NCX, because of its strategic position, may regulate InsP3-mediated Ca2+ signaling during the early phases of oocyte maturation and/or fertilization, and furthermore foreign cardiac protein is excluded from the Ca2+ microdomains surrounding the native NCX/InsP3Rs complex in the oocyte.

Inhibitory effect of n-3 fish oil fatty acids on cardiac Na+/Ca2+ exchange currents in HEK293t cells.

Abnormal activity of the cardiac Na+/Ca2+ exchanger (NCX1) can affect intracellular Ca2+ homeostasis and cause arrhythmias. The n-3 polyunsaturated fatty acids (PUFAs), however, may prevent arrhythmias. To test the effect of PUFAs on the cardiac NCX1 current (I(NCX1)), the canine NCX1 cDNA was expressed in human embryonic kidney (HEK293t) cells. The average density of I(NCX1) was 10.9+/-2.6 pA/pF (n=44) in NCX1-transfected cells and eicosapentaenoic acid (EPA, C20:5n-3) significantly inhibited I(NCX1) The suppression of I(NCX1) by EPA was concentration-dependent with an IC50 of 0.82+/-0.27 microM. EPA had a similar effect on outward or inward I(NCX1). Docosahexaenoic acid (DHA, C22:6n-3) and arachidonic acid (AA, C20:4n-6) also significantly inhibited I(NCX1), whereas the saturated fatty acid, stearic acid (SA, C18:0), did not. Our data demonstrate that the n-3 PUFAs significantly suppress cardiac I(NCX1), which is probably one of their protective effects against lethal arrhythmias.

Electrophysiological effects of ranolazine, a novel antianginal agent with antiarrhythmic properties.

BACKGROUND: Ranolazine is a novel antianginal agent capable of producing antiischemic effects at plasma concentrations of 2 to 6 micromol/L without reducing heart rate or blood pressure. The present study examines its electrophysiological effects in isolated canine ventricular myocytes, tissues, and arterially perfused left ventricular wedge preparations. METHODS AND RESULTS: Transmembrane action potentials (APs) from epicardial and midmyocardial (M) regions and a pseudo-ECG were recorded simultaneously from wedge preparations. APs were also recorded from epicardial and M tissues. Whole-cell currents were recorded from epicardial and M myocytes. Ranolazine inhibited I(Kr) (IC50=11.5 micromol/L), late I(Na), late I(Ca), peak I(Ca), and I(Na-Ca) (IC50=5.9, 50, 296, and 91 micromol/L, respectively) and I(Ks) (17% at 30 micromol/L), but caused little or no inhibition of I(to) or I(K1). In tissues and wedge preparations, ranolazine produced a concentration-dependent prolongation of AP duration of epicardial but abbreviation of that of M cells, leading to reduction or no change in transmural dispersion of repolarization (TDR). At [K+]o=4 mmol/L, 10 micromol/L ranolazine prolonged QT interval by 20 ms but did not increase TDR. Extrasystolic activity and spontaneous torsade de pointes (TdP) were never observed, and stimulation-induced TdP could not be induced at any concentration of ranolazine, either in normal or low [K+]o. Ranolazine (5 to 20 micromol/L) suppressed early afterdepolarizations (EADs) and reduced the increase in TDR induced by the selective I(Kr) blocker d-sotalol. CONCLUSIONS: Ranolazine produces ion channel effects similar to those observed after chronic amiodarone (reduced I(Kr), I(Ks), late I(Na), and I(Ca)). The actions of ranolazine to suppress EADs and reduce TDR suggest that, in addition to its antianginal actions, the drug may possess antiarrhythmic activity.