Triggered activity in atrial myocytes is influenced by Na+/Ca2+ exchanger activity in genetically altered mice.

AIMS: In atrial fibrillation, increased function of the Na+/Ca2+-exchanger (NCX) is one among several electrical remodeling mechanisms. METHODS/RESULTS: Using the patch-clamp- and Ca2+ imaging-methods, we investigated atrial myocytes from NCX-homozygous-overexpressor (OE)- and heterozygous-knockout (KO)-mice and their corresponding wildtypes (WTOE; WTKO). NCX mediated Ca2+ extrusion capacity was reduced in KO and increased in OE. There was no evidence for structural or molecular remodeling. During a proarrhythmic pacing-protocol, the number of low amplitude delayed afterdepolarizations (DADs) was unaltered in OE vs. WTOE and KO vs. WTKO. However, DADs triggered full spontaneous action potentials (sAP) significantly more often in OE vs. WTOE (ratio sAP/DAD: OE:0.18±0.05; WTOE:0.02±0.02; p<0.001). Using the same protocol, a DAD triggered an sAP by tendency less often in KO vs. WTKO (p=0.06) and significantly less often under a more aggressive proarrhythmic protocol (ratio sAP/DAD: KO:0.01±0.003; WT KO: 0.12±0.05; p=0.007). The DAD amplitude was increased in OE vs. WTOE and decreased in KO vs. WTKO. There were no differences in SR-Ca2+-load, the number of spontaneous Ca2+-release-events or IKACh/IK1. CONCLUSIONS: Atrial myocytes with increased NCX expression exhibited increased vulnerability towards sAPs while atriomyocytes with reduced NCX expression were protected. The underlying mechanism consists of a modification of the DAD-amplitude by the level of NCX-activity. Thus, although the number of spontaneous Ca2+-releases and therefore DADs is unaltered, the higher DAD-amplitude in OE made a transgression of the voltage-threshold of an sAP more likely. These findings indicate that the level of NCX activity could influence triggered activity in atrial myocytes independent of possible remodeling processes.

Phenylarsine oxide induces mitochondrial permeability transition, hypercontracture, and cardiac cell death.

The mitochondrial permeability transition (MPT) is implicated in cardiac reperfusion/reoxygenation injury. In isolated ventricular myocytes, the sulfhydryl (SH) group modifier and MPT inducer phenylarsine oxide (PAO) caused MPT, severe hypercontracture, and irreversible membrane injury associated with increased cytoplasmic free [Ca(2+)]. Removal of extracellular Ca(2+) or depletion of nonmitochondrial Ca(2+) pools did not prevent these effects, whereas the MPT inhibitor cyclosporin A was partially protective and the SH-reducing agent dithiothreitol fully protective. In permeabilized myocytes, PAO caused hypercontracture at much lower free [Ca(2+)] than in its absence. Thus PAO induced hypercontracture by both increasing myofibrillar Ca(2+) sensitivity and promoting mitochondrial Ca(2+) efflux during MPT. Hypercontracture did not directly cause irreversible membrane injury because lactate dehydrogenase (LDH) release was not prevented by abolishing hypercontracture with 2,3-butanedione monoxime. However, loading myocytes with the membrane-permeable Ca(2+) chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM) prevented PAO-induced LDH release, thus implicating the PAO-induced rise in cytoplasmic [Ca(2+)] as obligatory for irreversible membrane injury. In conclusion, PAO induces MPT and enhanced susceptibility to hypercontracture in isolated cardiac myocytes, both key features also implicated in cardiac reperfusion and reoxygenation injury.

Metabolic inhibition activates a non-selective current through connexin hemichannels in isolated ventricular myocytes.

Intracellular Na(+)accumulation and K(+)loss play important roles in the pathogenesis of arrhythmias and injury in the ischemic heart. We investigated the role of metabolically sensitive connexin hemichannels as a potential route for Na(+)influx and K(+)efflux during ischemia, using dye uptake and electrophysiological measurements to assay hemichannel activity in isolated rabbit ventricular myocytes. Consistent with the known size selectivity of connexin hemichannels,;50% of myocytes exposed to either low extracellular Ca(2+)(an established method for opening connexin hemichannels) or to metabolic inhibitors (a recently described method for opening hemichannels) accumulated fluorescent dyes with <1000 MW (propidium iodide and calcein), but excluded a larger dye with 1500-3000 MW (dextran-rhodamine). Using the whole cell patch clamp technique, we found that metabolic inhibitors activated a non-selective current permeant to both small and large cations, and blocked by La(3+), similar to the properties of connexin 43 when overexpressed in human embryonic kidney (HEK) cells. These findings indicate that isolated cardiac myocytes endogenously express metabolically-sensitive connexin hemichannels. If activated during ischemia, these hemichannels could contribute significantly to altered ionic fluxes promoting arrhythmias and myocardial injury.

Putative ryanodine receptors in the sarcolemma of ventricular myocytes.

The activity of caffeine-activated large conductance channels was recorded in whole-cell, patch-clamped, isolated ventricular myocytes from rabbit heart. The channels were permeable to monovalent and divalent cations and had a unitary monovalent cation conductance of 300-400 pS. Extracellular ruthenium red reduced the unitary conductance of the caffeine-activated channel in a concentration- and voltage-dependent manner. Ryanodine locked the caffeine-activated channels into a subconductance state. Elevating intracellular Ca2+ by photolysis of "caged calcium" increased the number of channel openings. The properties of this caffeine-activated channel were remarkably similar to those of cardiac ryanodine receptors (RyR) and support the novel finding that these channels may also be found on the sarcolemmal membrane.

Oxygen free radicals and excitation-contraction coupling.

Oxygen free radicals (OFR) contribute to contractile failure, rigor, and calcium (Ca2+) overload in ischemic/reperfused myocardium. Using both multicellular and isolated single-cell preparations, our laboratory has identified two fundamental mechanisms contributing to the deleterious effects of OFR: (i) impaired myocardial metabolism, and (ii) altered myocardial calcium handling. Impaired metabolism leads to activation of metabolically sensitive K+ currents, which shorten the action potential, thereby decreasing the duration of systole. Ultimately, high-energy phosphate depletion secondary to metabolic failure results in rigor. Altered myocardial Ca2+ handling is evidenced by a decrease in Ca2+ entry via L-type Ca2+ channels [another cause of decreased action potential duration (APD)], a reduction in sarcoplasmic reticulum (SR) Ca2+ content, slowed Ca2+ uptake in diastole, and increased sodium-calcium exchange (NaCaX) activity. The increase in NaCaX activity may contribute to the early increase in developed tension frequently observed in multicellular preparations exposed to free radicals, as well as the SR depletion occurring early on in voltage-clamped isolated cell preparations. Increased NaCaX activity is likely to be a critical factor underlying the late Ca2+ overload that occurs in the setting of increased intracellular Na+, and which leads to irreversible injury. The extent to which free radical-mediated metabolic inhibition participates in the dysfunction of the L-type Ca2+ channel is uncertain. The altered activity of the SR Ca2+ pump and NaCaX are more likely caused by direct actions of OFR on these proteins.

Local regulation of the threshold for calcium sparks in rat ventricular myocytes: role of sodium-calcium exchange.

1. To determine whether Na+-Ca2+ exchange modulates Ca2+ sparks, we studied enzymatically isolated patch clamped rat ventricular myocytes loaded with the Ca2+-sensitive indicator fluo-3, using confocal microscopy at 20-22 C. Two-dimensional images of Ca2+ sparks were recorded at 240 Hz using a laser scanning confocal microscope, allowing observation of a large area of the cell (820 microm2) at one time. 2. At a holding potential of -75 mV, spontaneous sparks were infrequent. Removal of extracellular Na+ for 520 ms, which in the absence of pipette Na+ should block Na+-Ca2+ exchange bidirectionally, was associated with a fourfold increase in spark frequency, without a significant change in cytoplasmic [Ca2+], sarcoplasmic reticulum (SR) Ca2+ content, or spark intensity, size or time course. 3. These findings are consistent with a model of excitation-contraction coupling in which Na+-Ca2+ exchange locally regulates the resting Ca2+ concentration in the diadic cleft (T-tubule-SR junction), thereby modulating the threshold for triggering Ca2+ sparks.

High-density lipoprotein increases intracellular calcium levels by releasing calcium from internal stores in human endothelial cells.

Elevated levels of high-density lipoproteins (HDL) appear to delay or prevent the development of atherosclerosis. The intracellular signaling mechanisms activated by HDL in vascular cells are currently under active investigation. In this study the effects of HDL on endothelial intracellular Ca levels (EC Ca(i)) are investigated. We show that HDL, like low density lipoproteins (LDL), increases EC Ca(i) in a dose-dependent fashion by releasing Ca from internal stores. Neither apolipoprotein A-I (apo A-I) nor apolipoprotein A-II (apo A-II) was responsible for the increase in EC Ca(i). HDL appeared to release Ca from the same internal stores as did LDL, since preincubation of EC with LDL prevented subsequent responses to HDL but not to the vasodilator ATP. In addition, preincubation of EC with pertussis toxin, an inhibitor of specific G proteins, as well as U73122, an inhibitor of phospholipase C, prevented a rise in EC Ca(i) in response to HDL. These findings suggest that HDL, like LDL, can modulate EC Ca(i) and that this occurs via a pertussis toxin-sensitive G protein-mediated pathway which involves phospholipase C.

Induction of monocyte binding to endothelial cells by MM-LDL: role of lipoxygenase metabolites.

Treatment of human aortic endothelial cells (EC) with minimally oxidized LDL (or minimally modified LDL, MM-LDL) produces a specific pattern of endothelial cell activation distinct from that produced by LPS, tumor necrosis factor-alpha, and interleukin-1, but similar to other agents that elevate cAMP. The current studies focus on the signal transduction pathways by which MM-LDL activates EC to bind monocytes. We now demonstrate that, in addition to an elevation of cAMP, lipoxygenase products are necessary for the MM-LDL response. Treatment of EC with inhibitors of the lipoxygenase pathway, 5,8,11, 14-eicosatetraynoic acid (ETYA) or cinnamyl-3, 4-dihydroxy-alpha-cyanocinnamate (CDC), blocked monocyte binding in MM-LDL-treated EC (MM-LDL=118+/-13%; MM-LDL+ETYA=33+/-4%; MM-LDL+CDC=23+/-4% increase in monocyte binding) without reducing cAMP levels. To further investigate the role of the lipoxygenase pathway, cellular phospholipids were labeled with arachidonic acid. Treatment of cells for 4 hours with 50 to 100 microg/mL MM-LDL, but not native LDL, caused a 60% increase in arachidonate release into the medium and increased the intracellular formation of 12(S)-HETE (approximately 100% increase). There was little 15(S)-HETE present, and no increase in its levels was observed. We demonstrated that 12(S)-HETE reversed the inhibitory effect of CDC. We also observed a 70% increase in the formation of 11,12-epoxyeicosatrienoic acid (11, 12-EET) in cells treated with MM-LDL. To determine the mechanism of arachidonate release induced by MM-LDL, we examined the effects of MM-LDL on intracellular calcium levels. Treatment of EC with both native LDL and MM-LDL caused a rapid release of intracellular calcium from internal stores. However, several pieces of evidence suggest that calcium release alone does not explain the increased arachidonate release in MM-LDL-treated cells. The present studies suggest that products of 12-lipoxygenase play an important role in MM-LDL action on the induction of monocyte binding to EC.

Connexin-43 hemichannels opened by metabolic inhibition.

The cause of altered ionic homeostasis leading to cell death during ischemia and metabolic inhibition is unclear. Hemichannels, which are precursors to gap junctions, are nonselective ion channels that are permeable to molecules of less than Mr 1000. We show that hemichannels open upon exposure to calcium-free solutions when they are either heterologously overexpressed in HEK293 cells or endogenously expressed in cardiac ventricular myocytes. In the presence of normal extracellular calcium, hemichannels open during metabolic inhibition. During ischemia and other forms of metabolic inhibition, activation of relatively few hemichannels will seriously compromise the cell's ability to maintain ionic homeostasis, which is an essential step promoting cell death.

Beta1 integrins participate in the hypertrophic response of rat ventricular myocytes.

Multiple signaling pathways have been implicated in the hypertrophic response of ventricular myocytes, yet the importance of cell-matrix interactions has not been extensively examined. Integrins are cell-surface molecules that link the extracellular matrix to the cellular cytoskeleton. They can function as cell signaling molecules and transducers of mechanical information in noncardiac cells. Given these properties and their abundance in cardiac cells, we evaluated the hypothesis that beta1 integrin function is involved in the alpha1-adrenergic mediated hypertrophic response of neonatal rat ventricular myocytes. The hypertrophic response of this model required interaction with extracellular matrix proteins. Specificity of these results was confirmed by demonstrating that ventricular myocytes plated onto an anti-beta1 integrin antibody supported the hypertrophic gene response. Adenovirus-mediated overexpression of beta1 integrin augmented the myocyte hypertrophic response when assessed by protein synthesis and atrial natriuretic factor production, a marker gene of hypertrophic induction. DNA synthesis was not altered by integrin overexpression. Transfection of cultured cardiac myocytes with either the ubiquitously expressed beta1A integrin or the cardiac/skeletal muscle-specific beta1 isoform (beta1D) activated reporter expression from both the atrial natriuretic factor and myosin light chain-2 ventricular promoters, genetic markers of ventricular cell hypertrophy. Finally, suppression of integrin signaling by overexpression of free beta1 integrin cytoplasmic domains inhibited the adrenergically mediated atrial natriuretic factor response. These findings show that integrin ligation and signaling are involved in the cardiac hypertrophic response pathway.

Safety and hemodynamic effects of intravenous triiodothyronine in advanced congestive heart failure.

Most patients with advanced congestive heart failure have altered thyroid hormone metabolism. A low triiodothyronine level is associated with impaired hemodynamics and is an independent predictor of poor survival. This study sought to evaluate safety and hemodynamic effects of short-term intravenous administration of triiodothyronine in patients with advanced heart failure. An intravenous bolus dose of triiodothyronine, with or without a 6- to 12-hour infusion (cumulative dose 0. 1 5 to 2.7 microg/kg), was administered to 23 patients with advanced heart failure (mean left ventricular ejection fraction 0.22 +/- 0.01). Cardiac rhythm and hemodynamic status were monitored for 12 hours, and basal metabolic rate by indirect calorimetry, echocardiographic parameters of systolic function and valvular regurgitation, thyroid hormone, and catecholamine levels were measured at baseline and at 4 to 6 hours. Triiodothyronine was well tolerated without episodes of ischemia or clinical arrhythmia. There was no significant change in heart rate or metabolic rate and there was minimal increase in core temperature. Cardiac output increased with a reduction in systemic vascular resistance in patients receiving the largest dose, consistent with a peripheral vasodilatory effect. Acute intravenous administration of triiodothyronine is well tolerated in patients with advanced heart failure, establishing the basis for further investigation into the safety and potential hemodynamic benefits of longer infusions, combined infusion with inotropic agents, oral triiodothyronine replacement therapy, and new triiodothyronine analogs.

Localization and functional effects of adenosine A1 receptors on cardiac vagal afferents in adult rats.

There is evidence to suggest that during ischemia adenosine acts on cardiac vagal afferent neurons to activate systemic reflexes and to modulate cardiac nociception. The purpose of this study was to determine whether adenosine receptors are present and have direct cellular electrophysiological actions on cardiac vagal afferent neurons. In radioreceptor assays of nodose ganglion tissue from rats, binding was detectable for A1 (39.6 +/- 1.2 fmol/mg protein) but not A2a adenosine receptors. These findings were confirmed using the complementary approach of receptor-labeling autoradiography. Using in situ hybridization, we saw specific labeling over approximately 50% of neurons in the nodose ganglia, but not over nonneuronal cells. In colabeling studies, cardiac vagal afferent neurons were identified by retroneuronal labeling with fluororuby. Of cardiac vagal afferents approximately one-half were strongly positive for A1 adenosine receptors (immunocytochemistry). In patch-clamping experiments, adenosine inhibited peak inward calcium current in 7 of 11 cells by 48 +/- 13%. In conclusion, adenosine A1 receptors reside on a subset of vagal afferent neurons, including cardiac vagal afferents, and have electrophysiological effects that modulate neuroexcitability in cultured nodose ganglion neurons.

Mechanism of hypoxic K loss in rabbit ventricle.

Although a critical factor causing lethal ischemic ventricular arrhythmias, net cellular K loss during myocardial ischemia and hypoxia is poorly understood. We investigated whether selective activation of ATP-sensitive K (KATP) channels causes net cellular K loss by examining the effects of the KATP channel agonist cromakalim on unidirectional K efflux, total tissue K content, and action potential duration (APD) in isolated arterially perfused rabbit interventricular septa. Despite increasing unidirectional K efflux and shortening APD to a comparable degree as hypoxia, cromakalim failed to induce net tissue K loss, ruling out activation of KATP channels as the primary cause of hypoxic K loss. Next, we evaluated a novel hypothesis about the mechanism of hypoxic K loss, namely that net K loss is a passive reflection of intracellular Na gain during hypoxia or ischemia. When the major pathways promoting Na influx were inhibited, net K loss during hypoxia was almost completely eliminated. These findings show that altered Na fluxes are the primary cause of net K loss during hypoxia, and presumably also in ischemia. Given its previously defined role during hypoxia and ischemia in promoting intracellular Ca overload and reperfusion injury, this newly defined role of intracellular Na accumulation as a primary cause of cellular K loss identifies it as a central pathogenetic factor in these settings.

Effects of TNF-alpha on [Ca2+]i and contractility in isolated adult rabbit ventricular myocytes.

The mechanism of the acute negative inotropic effect of tumor necrosis factor-alpha (TNF-alpha) was studied in enzymatically isolated adult rabbit ventricular myocytes. In cells loaded with fura 2 acetoxymethyl ester (AM) and paced intermittently at 0.2 Hz, TNF-alpha at doses < or = 10,000 U/ml caused a significant reduction in active cell shortening at 20 min, without reducing the amplitude of the accompanying intracellular Ca2+ concentration ([Ca2+]i) transient. Similar results were obtained in cells loaded with indo 1-AM and paced continuously at 0.2 Hz during exposure to TNF-alpha (10,000 U/ml). The effect of TNF-alpha on cell shortening could be prevented by the nitric oxide (NO) synthase blocker NG-nitro-L-arginine methyl ester (L-NAME) but not its inactive enantiomer NG-nitro-D-arginine methyl ester (D-NAME). The NO scavenger hemoglobin also attenuated the effects of TNF-alpha. TNF-alpha also caused a significant increase in diastolic cell length without any change in diastolic [Ca2+]i. The effect on cell length was prevented by L-NAME but not D-NAME. In cells loaded with the pH indicator seminaphthorhodafluor-AM, TNF-alpha did not alter pH sufficiently to account for the negative inotropic effect. These data suggest that high doses of TNF-alpha can acutely induce NO synthesis in isolated myocytes and reduce contractility by decreasing myofilament [Ca2+]i responsiveness. The mechanism of this altered myofilament [Ca2+]i response is unknown but does not appear to be pH mediated.

Free radicals enhance Na+/Ca2+ exchange in ventricular myocytes.

Oxygen-derived free radicals (OFR) have been implicated in the pathogenesis of intracellular Ca2+ overload and the arrhythmias that characterize cardiac reperfusion. These arrhythmias may in large part be due to activation of the pathological transient inward current (ITI). However, the identity of the ITI generated by OFR is uncertain. We previously found that H2O2, an OFR-generating compound, markedly stimulated the ITI elicited by brief caffeine pulses in patch-clamped guinea pig ventricular myocytes. In the present study, using patch-clamped rabbit ventricular myocytes loaded with the Ca(2+)-sensitive indicator fura 2, we have further characterized this ITI and have identified its major component to be Na+/Ca2+ exchange based on its dependence on extracellular Na+ and sarcoplasmic reticulum Ca2+ release, its sensitivity to Ni2+, and the effects of its inhibition on relaxation. The effect on ITI was not unique to H2O2, because another free radical-generating system, xanthine + xanthine oxidase, produced a similar response. We hypothesize that enhancement of Na+/Ca2+ exchange by OFR during reperfusion, when intracellular Na+ is elevated, may promote intracellular Ca2+ overload and triggered arrhythmias.

Endothelium-dependent vasodilators do not cause propagated intercellular Ca2+ waves in vascular endothelial monolayers.

Local application of a number of vasoactive agents affects vasomotor tone not only downstream to the point of application but also upstream. The mechanism(s) of upstream propagation is unknown. In endothelial cell monolayers, mechanical stimulation of one cell leads to intercellular propagation of increases in endothelial cell (EC) [Ca2+]i. In this study, we tested whether increases in EC [Ca2+]i induced by the local application of the endothelium-dependent vasodilators ATP, bradykinin and acetylcholine could spread across the monolayer. We demonstrate that unlike the response seen to a mechanical stimulus, there was no significant propagation of increases in EC [Ca2+]i levels in response to localized application of these agents. These findings suggest that upstream vasodilation in response to endothelium-dependent vasodilators is not mediated by propagation of EC [Ca2+]i waves and suggest that other electrical or chemical signals are responsible.

Excitation-contraction coupling in single guinea-pig ventricular myocytes exposed to hydrogen peroxide.

1. The effects of hydrogen peroxide (H2O2), an in vitro free radical generating system, on excitation-contraction (E-C) coupling were studied in isolated adult guinea-pig ventricular myocytes using Ca(2+)-sensitive dyes and the patch-clamp technique. 2. In paced myocytes loaded with indo-1 AM, 1 mM H2O2 briefly increased, then decreased the amplitude of intracellular Ca2+ ([Ca2+]i) transients and cell contractions. Diastolic [Ca2+]i increased in association with cell shortening. Automaticity also developed, followed shortly by inexcitability. In contrast, paced myocytes exposed to the metabolic inhibitors carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) and 2-deoxyglucose (DG), rapidly became inexcitable and exhibited marked diastolic shortening prior to increases in diastolic [Ca2+]i. 3. In patch-clamped myocytes loaded with fura-2, H2O2 reduced the amplitude of the Ca2+ current (ICa), the [Ca2+]i transient, and active cell shortening. H2O2 prolonged the relaxation phase of the [Ca2+]i transient, and activated an outward membrane current consistent with the ATP-sensitive K+ current (IK,ATP), but did not change the voltage dependence of ICa, the peak [Ca2+]i transient or active cell shortening. These responses were qualitatively similar to patch-clamped myocytes exposed to FCCP and DG. 4. Following exposure to H2O2, ICa elicited smaller [Ca2+]i transients than under control conditions. This was consistent with the observation that H2O2 reduced sarcoplasmic reticulum (SR) stores of Ca2+ by 42%, when assessed by observing the [Ca2+]i transients elicited by rapid extracellular application of 5 mM caffeine. In contrast FCCP-DG tended to increase SR Ca2+ stores. 5. Despite the decrease in the caffeine-induced Ca2+i release after H2O2, there was an increase in the Na(+)-Ca2+ exchange current associated with the caffeine-induced [Ca2+]i transient. 6. We conclude, therefore, that as with metabolic inhibitors, H2O2 interferes with E-C coupling in guinea-pig myocytes by impairing ICa and activating IK,ATP. However, unlike metabolic inhibitors, H2O2 stimulates Na(+)-Ca2+ exchange and depletes SR Ca2+ stores. Furthermore, diastolic [Ca2+]i becomes elevated while the myocyte is still excitable. These observations suggest that free radicals have primary effects on cardiac E-C coupling independent of their depressant effects on metabolism.

Lactate transport in mammalian ventricle. General properties and relation to K+ fluxes.

Net cellular L-lactate efflux associated with accelerated anaerobic glycolysis has been implicated as a potential cause of the marked cellular K+ loss contributing to lethal cardiac arrhythmias in ischemic heart and to impaired function of fatigued skeletal muscle. To examine the mechanisms of transsarcolemmal L-lactate movement in the heart, isolated guinea pig ventricular myocytes were loaded with the fluorescent H+ or K+ indicators, carboxy SNARF-1 or PBFI, respectively, under whole-cell patch-clamp conditions. With H+ as the only permeable monovalent cation, a rapid increase in extracellular L-lactate concentration ([L-]o) from 0 to 30 mmol/L at constant pHo (7.35) caused an intracellular acidification averaging 0.18 +/- 0.02 pH units in 60 seconds (n = 7), reflecting L-lactate influx in association with H+ influx (or OH- efflux). Under voltage-clamp conditions, no significant electrogenic current was associated with H(+)-coupled L-lactate influx, and membrane potential (-75 to +75 mV) had no effect on the degree of acidification produced by 30 mmol/L [L-]o, indicating that L-lactate influx was predominantly nonelectrogenic. Acidification in response to increased [L-]o was saturable (Km, approximately 5 mmol/L), partially stereospecific for L-lactate over D-lactate, and inhibited by 55 +/- 7% and 82 +/- 7% by the monocarboxylate carrier inhibitors alpha-cyano-4-hydroxycinnamate and mersalyl acid, respectively, consistent with a carrier-mediated transport mechanism. Extracellular K+ inhibited H(+)-coupled L-lactate influx by 36 +/- 2%, suggesting that K+ either inhibited or substituted for H+ in cotransport with L-lactate. However, in myocytes loaded with PBFI, no significant increase in [K+]i was detected during exposure to 30 mmol/L [L-]o, suggesting that only a minor component, if any, of L-lactate influx was cotransported or codiffused with K+.

Oxygen free radicals and cardiac reperfusion abnormalities.

Oxygen free radicals are highly reactive compounds causing peroxidation of lipids and proteins and are thought to play an important role in the pathogenesis of reperfusion abnormalities including myocardial stunning, irreversible injury, and reperfusion arrhythmias. Free radical accumulation has been measured in ischemic and reperfused myocardium directly using techniques such as electron paramagnetic resonance spectroscopy and tissue chemiluminescence and indirectly using biochemical assays of lipid peroxidation products. Potential sources of free radicals during ischemia and reperfusion have been identified in myocytes, vascular endothelium, and leukocytes. In several different experimental models exogenous free radical-generating systems have been shown to produce alterations in cardiac function that resemble the various reperfusion abnormalities described above. Injury to processes involved in regulation of the intracellular Ca2+ concentration may be a common mechanism underlying both free radical-induced and reperfusion abnormalities. Direct effects of free radicals on each of the known Ca(2+)-regulating mechanisms of the cell as well as the contractile proteins and various ionic membrane currents have been described. Free radicals also inhibit critical enzymes in anaerobic and aerobic metabolic pathways, which may limit the metabolic reserve of reperfused myocardium and contribute to intracellular Ca2+ overload. Inhibiting free radical accumulation during myocardial ischemia/reperfusion with free radical scavengers and inhibitors has been demonstrated to reduce the severity of myocardial stunning, irreversible injury, and reperfusion arrhythmias in many, but not all, studies. This evidence strongly implicates free radical accumulation during myocardial ischemia/reperfusion as an important pathophysiological mechanism of reperfusion abnormalities, although many issues remain unresolved.