H2O2-induced left ventricular dysfunction in isolated working rat hearts is independent of calcium accumulation

Reactive oxygen species (ROS) and intracellular Ca²⁺ overload play key roles in myocardial ischemia–reperfusion (IR) injury but the relationships among ROS, Ca²⁺ overload and LV mechanical dysfunction remain unclear. We tested the hypothesis that H₂O₂ impairs LV function by causing Ca²⁺ overload by increasing late sodium current (I_Na), similar to Sea Anemone Toxin II (ATX-II). Diastolic and systolic Ca²⁺ concentrations (d[Ca²⁺]_i and s[Ca²⁺]_i) were measured by indo-1 fluorescence simultaneously with LV work in isolated working rat hearts. H₂O₂ (100 μM, 30 min) increased d[Ca²⁺]_i and s[Ca²⁺]_i. LV work increased transiently then declined to 32% of baseline before recovering to 70%. ATX-II (12 nM, 30 min) caused greater increases in d[Ca²⁺]_i and s[Ca²⁺]_i. LV work increased transiently before declining gradually to 17%. Ouabain (80 μM) exerted similar effects to ATX-II. Late I_Na inhibitors, lidocaine (10 μM) or R56865 (2 μM), reduced effects of ATX-II on [Ca²⁺]_i and LV function, but did not alter effects of H₂O₂. The antioxidant, N-(2-mercaptopropionyl)glycine (MPG, 1 mM) prevented H₂O₂-induced LV dysfunction, but did not alter [Ca²⁺]_i. Paradoxically, further increases in [Ca²⁺]_i by ATX-II or ouabain, given 10 min after H₂O₂, improved function. The failure of late I_Na inhibitors to prevent H₂O₂-induced LV dysfunction, and the ability of MPG to prevent H₂O₂-induced LV dysfunction independent of changes in [Ca²⁺]_i indicate that impaired contractility is not due to Ca²⁺ overload. The ability of further increases in [Ca²⁺]_i to reverse H₂O₂-induced LV dysfunction suggests that Ca²⁺ desensitization is the predominant mechanism of ROS-induced contractile dysfunction.     

Ranolazine improves diastolic dysfunction in isolated myocardium from failing human hearts--role of late sodium current and intracellular ion accumulation

The goal of this study was to test the hypothesis that the novel anti-ischemic drug ranolazine, which is known to inhibit late I_Na, could reduce intracellular [Na⁺]_i and diastolic [Ca²⁺]_i overload and improve diastolic function. Contractile dysfunction in human heart failure (HF) is associated with increased [Na⁺]_i and elevated diastolic [Ca²⁺]_i. Increased Na⁺ influx through voltage-gated Na⁺ channels (late I_Na) has been suggested to contribute to elevated [Na⁺]_i in HF. In isometrically contracting ventricular muscle strips from end-stage failing human hearts, ranolazine (10 µmol/L) did not exert negative inotropic effects on twitch force amplitude. However, ranolazine significantly reduced frequency-dependent increase in diastolic tension (i.e., diastolic dysfunction) by ~ 30% without significantly affecting sarcoplasmic reticulum (SR) Ca²⁺ loading. To investigate the mechanism of action of this beneficial effect of ranolazine on diastolic tension, Anemonia sulcata toxin II (ATX-II, 40 nmol/L) was used to increase intracellular Na⁺ loading in ventricular rabbit myocytes. ATX-II caused a significant rise in [Na⁺]_i typically seen in heart failure via increased late I_Na. In parallel, ATX-II significantly increased diastolic [Ca²⁺]_i. In the presence of ranolazine the increases in late I_Na, as well as [Na⁺]_i and diastolic [Ca²⁺]_i were significantly blunted at all stimulation rates without significantly decreasing Ca²⁺ transient amplitudes or SR Ca²⁺ content. In summary, ranolazine reduced the frequency-dependent increase in diastolic tension without having negative inotropic effects on contractility of muscles from end-stage failing human hearts. Moreover, in rabbit myocytes the increases in late I_Na, [Na⁺]_i and [Ca²⁺]_i caused by ATX-II, were significantly blunted by ranolazine. These results suggest that ranolazine may be of therapeutic benefit in conditions of diastolic dysfunction due to elevated [Na⁺]_i and diastolic [Ca²⁺]_i.     

Intracellular Ca- and PKC-dependent upregulation of T-type Ca channels in LPC-stimulated cardiomyocytes

Lysophosphatidylcholine (LPC) accumulation in intracellular and/or interstitial space in cardiomyocytes may underlie as a mechanism for tachycardia and various arrhythmias during cardiac ischemia, which is usually accompanied by elevation of intracellular Ca²⁺ concentration ([Ca²⁺]_i). The present study was therefore designed to investigate possible mechanisms responsible for [Ca²⁺]_i elevation by LPC focusing on T-type Ca²⁺ channel current (I_Ca.T). LPC as well as phorbol 12-myristate 13-acetate (PMA) significantly accelerated the beating rates of neonatal rat cardiomyocytes. Augmentation of I_Ca.T by LPC was dependent on the intracellular Ca²⁺ concentration: an increase of I_Ca.T was significantly larger in high [Ca²⁺]_i condition (pCa = 7) than those in low [Ca²⁺]_i condition (pCa = 11). In heterologous expression system by use of human cardiac Ca_V3.1 and Ca_V3.2 channels expressed in HEK293 cells, LPC augmented Ca_V3.2 channel current (I_Cav3.2) in a concentration-dependent manner but not Ca_V3.1 channel current (I_Cav3.1). Augmentation of I_Cav3.2 by LPC was highly [Ca²⁺]_i dependent: I_Cav3.2 was unchanged when pCa was 11 but was markedly increased when [Ca²⁺]_i was higher than 10⁻¹⁰ M (pCa ≤ 10) by LPC application (10-50 μM). A specific inhibitor of protein kinase Cα (Ro-32-0432) attenuated the increase of I_Cav3.2 by LPC. LPC stimulates I_Ca.T in a [Ca²⁺]_i-dependent manner via PKCα activation, which may play a role in triggering arrhythmias in pathophysiological conditions of the heart.     

Mitochondrial Ca2+ flux and respiratory enzyme activity decline are early events in cardiomyocyte response to H2O2

Oxidative stress is involved in mitochondrial apoptosis, and plays a critical role in ischemic heart disease and cardiac failure. Exposure of cardiomyocytes to H₂O₂ leads to oxidative stress and mitochondrial dysfunction. In this study, we investigated the temporal order of mitochondrial-related events in the neonatal rat cardiomyocyte response to H₂O₂ treatment. At times ranging from 10 to 90 min after H₂O₂ treatment, levels were determined for respiratory complexes I, II, IV and V, and citrate synthase activities, mitochondrial Ca²⁺ flux, intracellular oxidation, mitochondrial membrane potential and apoptotic progression. Complexes II and IV activity levels were significantly reduced within 20 min of H₂O₂ exposure while complexes I and V, and citrate synthase were unaffected. Mitochondrial membrane potential declined after 20 and 60 min of H₂O₂ exposure while intracellular oxidation, declining complex I activity and apoptotic progression were detectable only after 60 min. Measurement of mitochondrial Ca²⁺ ([Ca²⁺]_m) using rhodamine 2 detected an early accumulation of [Ca²⁺]_m occurring between 5 and 10 min. Pretreatment of cardiomyocytes with either ruthenium red or cyclosporin A abrogated the H₂O₂-induced decline in complexes II and IV activities, indicating that [Ca²⁺]_m flux and onset of mitochondrial permeability transition pore opening likely precede the observed early enzymatic decline. Our findings suggest that [Ca²⁺]_m flux represents an early pivotal event in H₂O₂-induced cardiomyocyte damage, preceding and presumably leading to reduced mitochondrial respiratory activity levels followed by accumulation of intracellular oxidation, mitochondrial membrane depolarization and apoptotic progression concomitant with declining complex I activity.     

Activation of Connexin-43 Hemichannels Can Elevate [Ca]iand [Na]iin Rabbit Ventricular Myocytes During Metabolic Inhibition

ATP depletion due to ischemia or metabolic inhibition (MI) causes Na⁺and Ca²⁺accumulation in myocytes, which may be in part due to opening of connexin-43 hemichannels. Halothane (H) has been shown to reduce conductance of connexin-43 hemichannels and to protect the heart against ischemic injury. We therefore investigated the effect of halothane on [Ca²⁺]_iand [Na⁺]_iin myocytes during MI. Isolated rabbit left ventricular myocytes were loaded with 4μ m fluo-3 AM for 30 min, or with 5 μ m sodium green AM for 60 min at 37°C. After washing, the myocytes were exposed to: (1) Normal HEPES solution; (2) MI solution (2 m NaCN, 20 m 2-deoxy- -glucose and 0-glucose); or (3) MI+H (0.95 m , 4.7 m ) for 60 min. Propidium iodide (PI, 25 μ m) was added to all samples before data acquisition. The fluorescence intensity was measured by flow cytometry with 488 nm excitation and 530 nm emission for fluo-3 or sodium green, and 670 nm for PI. The [Ca²⁺]_iand [Na⁺]_iwere then calculated by calibration. In some experiments, the effect of 10 μ m tetrodotoxin (TTX) and 20 μ m nifedipine (NIF) were studied. Metabolic inhibition for 60 min caused a significant increase in [Ca²⁺]_iand [Na⁺]_iin myocytes when compared to controls, which was significantly reduced by halothane in a dose-dependent fashion. In the presence of TTX and NIF, halothane also significantly reduced the rise in the [Ca²⁺]_iand [Na⁺]_iin myocytes subjected to MI. 1-heptanol, another gap junction blocker, had similar effects. Thus, halothane reduced [Ca²⁺]_iand [Na⁺]_ioverload produced by MI in myocytes. This effect is not solely due to block of voltage-gated Na⁺and Ca²⁺channels, and is likely mediated by inhibiting the opening of connexin-43 hemichannels.     

Hyperpolarization induced by K+ channel openers inhibits Ca2+ influx and Ca2+ release in coronary artery

The vasodilating mechanisms of the K⁺ channel openers—cromakalim, pinacidil, nicorandil, KRN2391, and Ki4032—were examined by measurement of the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) using the fura-2 method in canine or porcine coronary arterial smooth muscle. The five K⁺ channel openers all produced a reduction of [Ca²⁺]_i in 5 and 30 mM KCl physiological salt solution (PSS), the effects of which were antagonized by tetrabutylammonium (TBA) or glibenclamide, but failed to affect [Ca²⁺]_i in 45 and 90 mM MCl-PSS. Cromakalim and Ki4032 only partially inhibited the 30 mM KCl-induced contractures, whereas pinacidil, nicorandil, and KRN2391 nearly abolished contractions produced by high KCl-PSS. The increased [Ca²⁺]_i and force produced by a thromboxane A₂ analogue, U46619, were inhibited by K⁺ channel openers and verapamil. In the absence of extracellular Ca²⁺, U46619 induced a transient increase in [Ca²⁺]_i with a contraction, which is effectively inhibited by cromakalim and Ki4032. Their inhibitory effects were blocked by TBA and counteracted by 20 mM KCl-induced depolarization. Cromakalim and Ki4032 did not affect caffeine-induced Ca²⁺ release. Cromakalim reduced U46619-induced IP₃ production and TBA blocked this inhibitory effect. Thus, cromakalim and Ki4032 are more specific K⁺ channel openers than pinacidil, nicorandil, and KRN2391. The vasodilation related with a reduction of [Ca²⁺]_i produced by K⁺ channel openers is due to the hyperpolarization of the plasma membrane resulting in not only the closure of voltage-dependent Ca²⁺ channels but also inhibition of the production of IP₃ and Ca²⁺ release from intracellular stores related to stimulation of the thromboxane A₂ receptor.     

Mechanisms of Ca2+ overload induced by extracellular H2O2 in quiescent isolated rat cardiomyocytes

Rat cardiomyocytes were exposed to H₂O₂ (1–100 μmol/L) for 10 min with washout for 10 min. Intracellular Ca²⁺ concentration ([Ca²⁺]_i) was measured using fluo-3. [Ca²⁺]_i increased with 100 μmol/L H₂O₂ and further increased during washout, causing irreversible contracture in one-half of the cells. The increase in [Ca²⁺]_i with 10 μmol/L H₂O₂ was modest with few cells showing irreversible contracture and attenuated by caffeine, and [Ca²⁺]_i gradually decreased during washout and this decrease was accelerated by a calcium-free solution, while 1 μmol/L H₂O₂ did not have any effects on [Ca²⁺]_i or cell viability. Ca²⁺ overload caused during exposure to 100 μmol/L H₂O₂ was attenuated by caffeine with improved cellular viability but not by chelerythrine, KB-R7943 or nifedipine. With 100 μmol/L H₂O₂ calcium-free solution attenuated the increase during exposure and washout while KB-R7943 or chelerythrine partly attenuated further increase during washout but not improved cell viability, but chelerythrine did not have additional effect on calcium-free treatment. Catalase abolished the effects of H₂O₂. We concluded that the increased [Ca²⁺]_i during exposure to 100 μmol/L H₂O₂ was caused both by release of Ca²⁺ from the intracellular store sites including the sarcoplasmic reticulum and by influx through route(s) other than the voltage-dependent Ca²⁺ channels or Na⁺/Ca²⁺ exchanger, although the Na⁺/Ca²⁺ exchanger or protein kinase C-mediated mechanism was partly responsible for a further increase during washout. Received: 1 February 2001, Returned for revision: 19 February 2001, Revision received: 11 April 2001, Accepted: 11 April 2001     

Sarcoplasmic Reticulum Function in Murine Ventricular Myocytes Overexpressing SR CaATPase

To examine the effects of the overexpression of sarcoplasmic reticulum (SR) CaATPase on function of the SR and Ca²⁺homeostasis, we measured [Ca²⁺]_itransients (fluo-3), and L-type Ca²⁺currents (I_Ca,L), Na/Ca exchanger currents (I_Na/Ca), and SR Ca²⁺content with voltage clamp in ventricular myocytes isolated from wild type (WT) mice and transgenic (SRTG) mice. The amplitude of [Ca²⁺]_itransients was insignificantly increased in SRTG myocytes, while the diastolic [Ca²⁺]_itended to be lower. The initial and terminal declines of [Ca²⁺]_itransients were significantly accelerated in SRTG myocytes, implying a functional upregulation of the SR CaATPase. We examined the functional contribution of only the SR CaATPase to the initial and the terminal phase of the decline of [Ca²⁺]_i, by abruptly inhibiting Na/Ca exchange with a rapid switcher device. The rate of [Ca²⁺] decline mediated by the SR CaATPase was increased by 40% in SRTG compared with WT myocytes. The function of the L-type Ca²⁺channel was unchanged in SRTG myocytes, while INa/Ca density was slightly (10%) decreased. Measured SR Ca²⁺content was significantly increased by 29% in SRTG myocytes. Thus, overexpression of SR CaATPase markedly accelerates the decline of [Ca²⁺]_itransients, and induces an increase in SR Ca²⁺content, with some downregulation of the Na/Ca exchanger.     

Theoretical investigation of action potential duration dependence on extracellular Ca in human cardiomyocytes

Reduction in [Ca²⁺]_o prolongs the AP in ventricular cardiomyocytes and the QT_c interval in patients. Although this phenomenon is relevant to arrhythmogenesis in the clinical setting, its mechanisms are counterintuitive and incompletely understood. To evaluate in silico the mechanisms of APD modulation by [Ca²⁺]_o in human cardiomyocytes. We implemented the Ten Tusscher-Noble-Noble-Panfilov model of the human ventricular myocyte and modified the formulations of the rapidly and slowly activating delayed rectifier K⁺ currents (I_Kr and I_Ks) and L-type Ca²⁺ current (I_CaL) to incorporate their known sensitivity to intra- or extracellular Ca²⁺. Simulations were run with the original and modified models at variable [Ca²⁺]_o in the clinically relevant 1 to 3 mM range. The original model responds with APD shortening to decrease in [Ca²⁺]_o, i.e. opposite to the experimental observations. Incorporation of Ca²⁺ dependency of K⁺ currents cannot reproduce the inverse relation between APD and [Ca²⁺]_o. Only when I_CaL inactivation process was modified, by enhancing its dependency on Ca²⁺, simulations predict APD prolongation at lower [Ca²⁺]_o. Although Ca²⁺-dependent I_CaL inactivation is the primary mechanism, secondary changes in electrogenic Ca²⁺ transport (by Na⁺/Ca²⁺ exchanger and plasmalemmal Ca²⁺-ATPase) contribute to the reversal of APD dependency on [Ca²⁺]_o. This theoretical investigation points to Ca²⁺-dependent inactivation of I_CaL as a mechanism primarily responsible for the dependency of APD on [Ca²⁺]_o. The modifications implemented here make the model more suitable to analyze repolarization mechanisms when Ca²⁺ levels are altered.     

Role of mitochondrial dysfunction in cardiac glycoside toxicity

Cardiac glycosides, which inhibit the plasma membrane Na⁺ pump, are one of the four categories of drug recommended for routine use to treat heart failure, yet their therapeutic window is limited by toxic effects. Elevated cytoplasmic Na⁺ ([Na⁺]_i) compromises mitochondrial energetics and redox balance by blunting mitochondrial Ca²⁺ ([Ca²⁺]_m) accumulation, and this impairment can be prevented by enhancing [Ca²⁺]_m. Here, we investigate whether this effect underlies the toxicity and arrhythmogenic effects of cardiac glycosides and if these effects can be prevented by suppressing mitochondrial Ca²⁺ efflux, via inhibition of the mitochondrial Na⁺/Ca²⁺ exchanger (mNCE). In isolated cardiomyocytes, ouabain elevated [Na⁺]_i in a dose-dependent way, blunted [Ca²⁺]_m accumulation, decreased the NADH/NAD + redox potential, and increased reactive oxygen species (ROS). Concomitant treatment with the mNCE inhibitor CGP-37157 ameliorated these effects. CGP-37157 also attenuated ouabain-induced cellular Ca²⁺ overload and prevented delayed afterdepolarizations (DADs). In isolated perfused hearts, ouabain's positive effects on contractility and respiration were markedly potentiated by CGP-37157, as were those mediated by β-adrenergic stimulation. Furthermore, CGP-37157 inhibited the arrhythmogenic effects of ouabain in both isolated perfused hearts and in vivo. The findings reveal the mechanism behind cardiac glycoside toxicity and show that improving mitochondrial Ca²⁺ retention by mNCE inhibition can mitigate these effects, particularly with respect to the suppression of Ca²⁺-triggered arrhythmias, while enhancing positive inotropic actions. These results suggest a novel strategy for the treatment of heart failure.     

Influence of Infrasound Exposure on the Whole L-type Calcium Currents in Rat Ventricular Myocytes

This study was designed to examine the effect of infrasound exposure (5 Hz at 130 dB) on whole-cell L-type Ca²⁺ currents (WLCC) in rat ventricular myocytes and the underlying mechanism(s) involved. Thirty-two adult Sprague-Dawley rats were randomly assigned to infrasound exposure and control groups. [Ca²⁺]_i, WLCC, mRNA expression of the a_1c subunit of L-type Ca²⁺ channels (LCC), and SERCA2 protein were examined on day 1, 7, and 14 after initiation of infrasound exposure. Fluo-3/AM fluorescence and the laser scanning confocal microscope techniques were used to measure [Ca²⁺]_i in freshly isolated ventricular myocytes. The Ca²⁺ fluorescence intensity (FI), denoting [Ca²⁺]_i in cardiomyocytes, was significantly elevated in a time-dependent manner in the exposure groups. There was a significant increase in WLCC in the 1-day group and a further significant increase in the 7- and 14-day groups. LCC mRNA expression measured by RT-PCR revealed a significant rise in the 1-day group and a significant additional rise in the 7- and 14-day groups compared with control group. SERCA2 expression was significantly upregulated in the 1-day group followed by an overt decrease in the 7- and 14-day groups. Prolonged exposure of infrasound altered WLCC in rat cardiomyocytes by shifting the steady-state inactivation curves to the right (more depolarized direction) without altering the slope and biophysical properties of I _Ca,L. Taken together, our data suggest that changes in [Ca²⁺]_I levels as well as expression of LCC and SERCA2 may contribute to the infrasound exposure-elicited cardiac response. Zhaohui Pei and Zhiqiang Zhuang contributed equally to this work.     

4-Hydroxy-2-nonenal Induces Calcium Overload via the Generation of Reactive Oxygen Species in Isolated Rat Cardiac Myocytes

BackgroundIt has been reported that that the amount of 4-hydroxy-2-nonenal (HNE), which is a major lipid peroxidation product and a cytotoxic aldehyde, is increased in the human failing myocardium. This study was designed to determine whether HNE has a pro-oxidant effect in cardiac myocytes and whether HNE causes Ca²⁺ overload.Methods and ResultsExposure to HNE for 10 minutes in the presence of ferric nitrilotriacetate induced the production of hydroxyl radical (·OH) in the rat myocardium as assessed by electron spin resonance spectroscopy, and HNE induced the generation of reactive oxygen species (ROS) in a dose-dependent manner as assessed by 2′, 7′-dichlorofluorescein diacetate fluorescence. HNE increased intracellular Ca²⁺ concentration ([Ca²⁺]_i) as assessed by fura-2 ratio in a dose- and time-dependent manner. After 20 minutes of HNE (400 μmol/L) exposure, hypercontracture was induced in 67% of the cells. Catalase, an antioxidative enzyme that can decompose hydrogen peroxide (H₂O₂), significantly attenuated the increase in [Ca²⁺]_i and completely inhibited hypercontracture. Carvedilol, a β-blocker with potent antioxidant activity, also significantly attenuated the increase in [Ca²⁺]_i and completely inhibited hypercontracture, but propranolol had no effect on either [Ca²⁺]_i increase or hypercontracture.ConclusionsHNE induces the formation of ROS, especially H₂O₂ and ·OH, in cardiomyocytes and subsequently ROS cause intracellular Ca²⁺ overload. HNE formation may play an important role as a mediator of oxidative stress in heart failure.     

Ca2+ release-activated Ca2+ channel blockade as a potential tool in antipancreatitis therapy

Alcohol-related acute pancreatitis can be mediated by a combination of alcohol and fatty acids (fatty acid ethyl esters) and is initiated by a sustained elevation of the Ca²⁺ concentration inside pancreatic acinar cells ([Ca²⁺]_i), due to excessive release of Ca²⁺ stored inside the cells followed by Ca²⁺ entry from the interstitial fluid. The sustained [Ca²⁺]_i elevation activates intracellular digestive proenzymes resulting in necrosis and inflammation. We tested the hypothesis that pharmacological blockade of store-operated or Ca²⁺ release-activated Ca²⁺ channels (CRAC) would prevent sustained elevation of [Ca²⁺]_i and therefore protease activation and necrosis. In isolated mouse pancreatic acinar cells, CRAC channels were activated by blocking Ca²⁺ ATPase pumps in the endoplasmic reticulum with thapsigargin in the absence of external Ca²⁺. Ca²⁺ entry then occurred upon admission of Ca²⁺ to the extracellular solution. The CRAC channel blocker developed by GlaxoSmithKline, GSK-7975A, inhibited store-operated Ca²⁺ entry in a concentration-dependent manner within the range of 1 to 50 μM (IC₅₀ = 3.4 μM), but had little or no effect on the physiological Ca²⁺ spiking evoked by acetylcholine or cholecystokinin. Palmitoleic acid ethyl ester (100 μM), an important mediator of alcohol-related pancreatitis, evoked a sustained elevation of [Ca²⁺]_i, which was markedly reduced by CRAC blockade. Importantly, the palmitoleic acid ethyl ester-induced trypsin and protease activity as well as necrosis were almost abolished by blocking CRAC channels. There is currently no specific treatment of pancreatitis, but our data show that pharmacological CRAC blockade is highly effective against toxic [Ca²⁺]_i elevation, necrosis, and trypsin/protease activity and therefore has potential to effectively treat pancreatitis.     

Intracellular [Na<Superscript>+</Superscript>] modulates synergy between Na<Superscript>+</Superscript>/Ca<Superscript>2+</Superscript> exchanger and L-type Ca<Superscript>2+</Superscript> current in cardiac excitation–contraction coupling during action potentials

Excitation–contraction coupling (ECC) in cardiac myocytes involves triggering of Ca²⁺ release from the sarcoplasmic reticulum (SR) by L-type Ca channels, whose activity is strongly influenced by action potential (AP) profile. The contribution of Ca²⁺ entry via the Na⁺/Ca²⁺ exchanger (NCX) to trigger SR Ca²⁺ release during ECC in response to an AP remains uncertain. To isolate the contribution of NCX to SR Ca²⁺ release, independent of effects on SR Ca²⁺ load, Ca²⁺ release was determined by recording Ca²⁺ spikes using confocal microscopy on patch-clamped rat ventricular myocytes with [Ca²⁺]_i fixed at 150 nmol/L. In response to AP clamps, normalized Ca²⁺ spike amplitudes (ΔF/F ₀) increased sigmoidally and doubled as [Na⁺]_i was elevated from 0 to 20 mmol/L with an EC₅₀ of ~10 mmol/L. This [Na⁺]_i-dependence was independent of I _Na as well as SR Ca²⁺ load, which was unchanged under our experimental conditions. However, NCX inhibition using either KB-R7943 or XIP reduced ΔF/F ₀ amplitude in myocytes with 20 mmol/L [Na⁺]_i, but not with 5 mmol/L [Na⁺]_i. SR Ca²⁺ release was complete before the membrane repolarized to −15 mV, indicating Ca²⁺ entry into the dyad (not reduced extrusion) underlies [Na⁺]_i-dependent enhancement of ECC. Because I _Ca,L inhibition with 50 mmol/L Cd²⁺ abolished Ca²⁺ spikes, our results demonstrate that during cardiac APs, NCX enhances SR Ca²⁺ release by synergistically increasing the efficiency of I _Ca,L-mediated ECC.     

Relationships between dopamine-induced changes in cytosolic free calcium concentration ([Ca2+]i) and rate of prolactin secretion

This study was undertaken to investigate the relationship between dopamine (DA) induced changes in the cytosolic calcium concentration ([Ca²⁺]_i) and the rate of prolactin secretion using GH₄ZR₇, a rat pituitary cell line, which express only one subtype of D₂ receptor. GH₄ZR₇ cells were loaded with Fluo-3, a fluorescent Ca²⁺ indicator, and then perifused with two different doses of DA (10⁻⁷ mol/L and 5×10⁻⁴ mol/L). We monitored changes in [Ca²⁺]_i and rate of prolactin release simultaneously by attaching a spectrofluorometer to a dynamic perifusion system. DA has stimulatory and inhibitory effect on prolactin secretion in GH₄ZR₇ cells; 10⁻⁷ mol/L DA slightly increased [Ca²⁺]_i and stimulated prolactin release, whereas 5×10⁻⁴ mol/L DA decreased [Ca²⁺]_i and inhibited prolactin secretion. When the cells were pretreated with pertussis toxin (PTX), 10⁻⁷ mol/L DA had no significant change in [Ca²⁺]_i while stimulating prolactin release, and 5×10⁻⁴ mol/L DA reduced [Ca²⁺]_i without having any significant effect on the rate of prolactin secretion. The results of this study demonstrate that changes in [Ca²⁺]_i do not always correlate with the rate of prolactin release from lactotrophs. The dissociation between [Ca²⁺]_i and prolactin release is somewhat expected considering the diverse role of [Ca²⁺]_i and post-[Ca²⁺]_i events, which can change the rate of prolactin release.     

L-type Ca channel facilitation mediated by H2O2-induced activation of CaMKII in rat ventricular myocytes

The Ca²⁺-dependent facilitation (CDF) of L-type Ca²⁺ channels, a major mechanism for force-frequency relationship of cardiac contraction, is mediated by Ca²⁺/CaM-dependent kinase II (CaMKII). Recently, CaMKII was shown to be activated by methionine oxidation. We investigated whether oxidation-dependent CaMKII activation is involved in the regulation of L-type Ca²⁺ currents (I_Ca,L) by H₂O₂ and whether Ca²⁺ is required in this process. Using patch clamp, I_Ca,L was measured in rat ventricular myocytes. H₂O₂ induced an increase in I_Ca,L amplitude and slowed inactivation of I_Ca,L. This oxidation-dependent facilitation (ODF) of I_Ca,L was abolished by a CaMKII blocker KN-93, but not by its inactive analog KN-92, indicating that CaMKII is involved in ODF. ODF was not affected by replacement of external Ca²⁺ with Ba²⁺ or presence of EGTA in the internal solutions. However, ODF was abolished by adding BAPTA to the internal solution or by depleting sarcoplasmic reticulum (SR) Ca²⁺ stores using caffeine and thapsigargin. Alkaline phosphatase, β-iminoadenosine 5′-triphosphate (AMP-PNP), an autophosphorylation inhibitor autocamtide-2-related inhibitory peptide (AIP), or a catalytic domain blocker (CaM-KIINtide) did not affect ODF. In conclusion, oxidation-dependent facilitation of L-type Ca²⁺ channels is mediated by oxidation-dependent CaMKII activation, in which local Ca²⁺ increases induced by SR Ca²⁺ release is required.     

Oscillations in cytoplasmic free calcium concentration in human pancreatic islets from subjects with normal and impaired glucose tolerance

Summary Plasma insulin levels in healthy subjects oscillate and non-insulin-dependent diabetic patients display an irregular pattern of such oscillations. Since an increase in cytoplasmic free Ca²⁺ concentration ([Ca²⁺]_i) in the pancreatic beta cell is the major stimulus for insulin release, this study was undertaken to investigate the dynamics of electrical activity, [Ca²⁺]_i-changes and insulin release, in stimulated islets from subjects of varying glucose tolerance. In four patients it was possible to investigate more than one of these three parameters. Stimulation of pancreatic islets with glucose and tolbutamide sometimes resulted in the appearance of oscillations in [Ca²⁺]_i, lasting 2–3 min. Such oscillations were observed even in some islets from patients with impaired glucose tolerance. In one islet from a diabetic patient there was no response to glucose, whereas that islet displayed [Ca²⁺]_i-oscillations in response to tolbutamide, suggesting that sulphonylurea treatment can mimic the complex pattern of glucose-induced [Ca²⁺]_i-oscillations. We also, for the first time, made patch-clamp recordings of membrane currents in beta-cells in situ in the islet. Stimulation with glucose and tolbutamide resulted in depolarization and appearance of action potentials. The islet preparations responded to stimulation with a number of different secretagogues with release of insulin. The present study shows that human islets can respond to stimulation with glucose and sulphonylurea with oscillations in [Ca²⁺]_i, which is the signal probably underlying the oscillations in plasma insulin levels observed in healthy subjects. Interestingly, even subjects with impaired glucose tolerance had islets that responded with oscillations in [Ca²⁺]_i upon glucose stimulation, although it is not known to what extent the response of these islets was representative of most islets in these patients.Abbreviations [Ca²⁺]_i Cytoplasmic free Ca²⁺ - NIDDM non-insulin-dependent diabetes mellitus - DMSO dimethylsulphoxide - PC pancreatic cancer     

Experimental diabetes enhances Ca2+ mobilization and glutamate exocytosis in cerebral synaptosomes from mice

The present study was conducted to investigate the effects of the diabetic condition on the Ca²⁺ mobilization and glutamate release in cerebral nerve terminals (synaptosomes). Diabetes was induced in male mice by intraperitoneal injection of streptozotocin. Cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) and glutamate release in synaptosomes were determined using fura-2 and enzyme-linked fluorometric assay, respectively. Diabetes significantly enhanced the ability of the depolarizing agents K⁺ and 4-aminopyridine (4-AP) to increase [Ca²⁺]_i. In addition, diabetes significantly enhanced K⁺- and 4-AP-evoked Ca²⁺-dependent glutamate release. The pretreatment of synaptosomes with a combination of ω-agatoxin IVA (a P-type Ca²⁺ channel blocker) and ω-conotoxin GVIA (an N-type Ca²⁺ channel blocker) inhibited K⁺- or 4-AP-induced increases in [Ca²⁺]_i and Ca²⁺-dependent glutamate release in synaptosomes from the control and diabetic mice to a similar extent, respectively. These results indicate that diabetes enhances a K⁺- or 4-AP-evoked Ca²⁺-dependent glutamate release by increasing [Ca²⁺]_i via stimulation of Ca²⁺ entry through both P- and N-type Ca²⁺ channels.     

Intracellular calcium ion response to glucose in beta-cells of calbindin-D28k nullmutant mice and in betaHC13 cells overexpressing calbindin-D28k

This article describes studies on the glucose-induced responses of intracellular Ca²⁺ concentration ([Ca²⁺]_i), insulin release, and redistribution of calbindin-D_28k, a calcium-binding regulatory protein, in β-cells of pancreatic islets of calbindin-D_28k knockout (KO) and wild-type mice (C57BL6) as well as in βHC-13 control cells and βHC-13 CaBP40 cells (β-cell line overexpressing calbindin-D_28k). Upon increasing the glucose concentration from 2.8 to 30 mM, islets of KO mice showed a significantly greater increase in [Ca²⁺]_i (mean increase in [Ca²⁺]_i, i.e., Δ[Ca²⁺], was 296 nM) compared with wild-type mice (Δ[Ca²⁺]_i=97 nM). βHC-13 CaBP40 cells showed little change in [Ca²⁺]_i upon elevation of glucose from 5.5 to 32.7 mM, whereas βHC-13 control cells exhibited significant increases in [Ca²⁺]_i (Δ[Ca²⁺]_i=510 nM). Similarly, upon addition of 30 mM glucose, the rate of insulin release increased from 25.2 (basal rate) to 145.2 pg/mL/min in βHC-13 control cells, whereas in βHC-13 CaBP40 cells the rate of insulin release was only 27.5 pg/mL/min in high glucose. Thus, levels of calbindin-D_28k in β-cells affect both [Ca²⁺]_i and insulin secretion in response to glucose. The three-dimensional reconstruct of confocal immunofluorescent images showed that glucose caused redistribution of calbindin-D_28k resulting in co-localization in the region of L-type voltage-dependent calcium channels (VDCC). This colocalization may be an important regulatory function concerning Ca²⁺ influx via L-type VDCC and exocytosis of insulin granules.