Monensin-Induced Increase in Intracellular Na+ Induces Changes in Na+ and Ca2+ Currents and Regulates Na+-K+ and Na+-Ca2+ Transport in Cardiomyocytes.

BACKGROUND/AIMS: Monensin, an Na ionophore, increases intracellular Na ([Na]i). Alteration of [Na]i influences ion transport through the sarcolemmal membrane. So far, the effects of monensin on ventricular myocytes have not been examined in detail. The main objective of this study was to elucidate the mechanism via which monensin-evoked increases in [Na]i affect the membrane potential and currents in ventricular myocytes of guinea pigs. METHODS: Membrane potentials and currents were measured using the whole-cell patch-clamp technique in single myocytes. The concentration of intracellular Ca ([Ca]i) was evaluated by measuring fluorescence intensity of Fluo-4. RESULTS: Monensin (10-5M) shortened the action potential duration (APD) and reduced the amplitude of the plateau phase. In addition, monensin decreased the sodium current (INa) and shifted the inactivation curve to the hyperpolarized direction. Moreover, it decreased the L-type calcium current (ICa). However, this effect was attenuated by increasing the buffering capacity of [Ca]i. The Na-Ca exchange current (INa-Ca) was activated particularly in the reverse mode. Na-K pump current (INa-K) was also activated. Notably, the inward rectifying K current (IK1) was not affected, and the change in the delayed outward K current (IK) was not evident. CONCLUSION: These results suggest that the monensin-induced shortened APD and reduced amplitude of the plateau phase are primarily due to the decrease in the ICa, the activation of the reverse mode of INa-Ca, and the increased INa-K, and second due to the decreased INa. The IK and the IK1 may not be associated with the abovementioned changes induced by monensin. The elevation of [Na]i can exert multiple influences on electrophysiological phenomena in cardiac myocytes.

Methylene Blue Counteracts H2S-Induced Cardiac Ion Channel Dysfunction and ATP Reduction.

We have previously demonstrated that methylene blue (MB) counteracts the effects of hydrogen sulfide (H2S) cardiotoxicity by improving cardiomyocyte contractility and intracellular Ca2+ homeostasis disrupted by H2S poisoning. In vivo, MB restores cardiac contractility severely depressed by sulfide and protects against arrhythmias, ranging from bundle branch block to ventricular tachycardia or fibrillation. To dissect the cellular mechanisms by which MB reduces arrhythmogenesis and improves bioenergetics in myocytes intoxicated with H2S, we evaluated the effects of H2S on resting membrane potential (Em), action potential (AP), Na+/Ca2+ exchange current (INaCa), depolarization-activated K+ currents and ATP levels in adult mouse cardiac myocytes and determined whether MB could counteract the toxic effects of H2S on myocyte electrophysiology and ATP. Exposure to toxic concentrations of H2S (100 µM) significantly depolarized Em, reduced AP amplitude, prolonged AP duration at 90% repolarization (APD90), suppressed INaCa and depolarization-activated K+ currents, and reduced ATP levels in adult mouse cardiac myocytes. Treating cardiomyocytes with MB (20 µg/ml) 3 min after H2S exposure restored Em, APD90, INaCa, depolarization-activated K+ currents, and ATP levels toward normal. MB improved mitochondrial membrane potential (∆ψm) and oxygen consumption rate in myocytes in which Complex I was blocked by rotenone. We conclude that MB ameliorated H2S-induced cardiomyocyte toxicity at multiple levels: (1) reversing excitation-contraction coupling defects (Ca2+ homeostasis and L-type Ca2+ channels); (2) reducing risks of arrhythmias (Em, APD, INaCa and depolarization-activated K+ currents); and (3) improving cellular bioenergetics (ATP, ∆ψm).

Pro-death and pro-survival properties of ouabain in U937 lymphoma derived cells.

BACKGROUND: Epidemiological studies revealed significantly lower mortality rates in cancer patients receiving cardiac glycosides, which turned on interest in the anticancer properties of these drugs. However, cardiac glycosides have also been shown to stimulate cell growth in several cell types. In the present investigation we analyzed the pro-death and pro-survival properties of ouabain in the human lymphoma derived cell line U937. METHODS: ROS, intracellular Ca++, cell cycle were evaluated by loading the cells with fluorescent probes under cytofluorimetry. Cell counts and evaluation of trypan blue-excluding cells were performed under optic microscope. Protein detection was done by specific antibodies after protein separation from cellular lysates by SDS-PAGE and transfer blot. RESULTS: High doses of ouabain cause ROS generation, elevation of [Ca++]i and death of lymphoma derived U937 cells. Lower doses of OUA activate a survival pathway in which plays a role the Na+/Ca++-exchanger (NCX), active in the Ca++ influx mode rather than in the Ca++ efflux mode. Also p38 MAPK plays a pro-survival role. However, the activation of this MAPK does not appear to depend on NCX. CONCLUSION: This investigation shows that the cardiac glycoside OUA is cytotoxic also for the lymphoma derived cell line U937 and that can activate a survival pathway in which are involved NCX and p38 MAPK. These molecules can represent potential targets of combined therapy.

Nanomolar ouabain increases NCX1 expression and enhances Ca2+ signaling in human arterial myocytes: a mechanism that links salt to increased vascular resistance?

The mechanisms by which NaCl raises blood pressure (BP) in hypertension are unresolved, but much evidence indicates that endogenous ouabain is involved. In rodents, arterial smooth muscle cell (ASMC) Na(+) pumps with an α(2)-catalytic subunit (ouabain EC(50) ≤1.0 nM) are crucial for some hypertension models, even though ≈80% of ASMC Na(+) pumps have an α(1)-subunit (ouabain EC(50) ≈ 5 µM). Human α(1)-Na(+) pumps, however, have high ouabain affinity (EC(50) ≈ 10-20 nM). We used immunoblotting, immunocytochemistry, and Ca(2+) imaging (fura-2) to examine the expression, distribution, and function of Na(+) pump α-subunit isoforms in human arteries and primary cultured human ASMCs (hASMCs). hASMCs express α(1)- and α(2)-Na(+) pumps. Further, α(2)-, but not α(1)-, pumps are confined to plasma membrane microdomains adjacent to sarcoplasmic reticulum (SR), where they colocalize with Na/Ca exchanger-1 (NCX1) and C-type transient receptor potential-6 (receptor-operated channels, ROCs). Prolonged inhibition (72 h) with 100 nM ouabain (blocks nearly all α(1)- and α(2)-pumps) was toxic to most cultured hASMCs. Treatment with 10 nM ouabain (72 h), however, increased NCX1 and sarco(endo)plasmic reticulum Ca(2+)-ATPase expression and augmented ATP (10 µM)-induced SR Ca(2+) release in 0 Ca(2+), ouabain-free media, and Ca(2+) influx after external Ca(2+) restoration. The latter was likely mediated primarily by ROCs and store-operated Ca(2+) channels. These hASMC protein expression and Ca(2+) signaling changes are comparable with previous observations on myocytes isolated from arteries of many rat hypertension models. We conclude that the same structurally and functionally coupled mechanisms (α(2)-Na(+) pumps, NCX1, ROCs, and the SR) regulate Ca(2+) homeostasis and signaling in hASMCs and rodent ASMCs. These ouabain/endogenous ouabain-modulated mechanisms underlie the whole body autoregulation associated with increased vascular resistance and elevation of BP in human, salt-sensitive hypertension.

Role of calcium-activated potassium channels in the genesis of 3,4-diaminopyridine-induced periodic contractions in isolated canine coronary artery smooth muscles.

We found that 3,4-diaminopyridine (3,4-DAP), a voltage-gated potassium channel (K(V)) inhibitor, elicits pH-sensitive periodic contractions (PCs) of coronary smooth muscles. Underlying mechanisms of PCs, however, remained to be elucidated. The present study was performed to examine the roles of ion channels in the genesis of PCs. To determine the electromechanical changes of smooth muscles, isolated coronary arterial rings from beagles were suspended in organ chambers filled with Krebs-Henseleit solution, and 10(-2) M 3,4-DAP was added to elicit PCs. 3,4-DAP caused periodic spike-and-plateau depolarization accompanied by contraction. PCs were not produced when the CaCl(2) concentration in the chamber was ≤ 0.3 × 10(-3) or ≥ 10(-2) M. PCs were eliminated by a CaCl(2) concentration ≥ 5 × 10(-3) M or by lowering pH below 7.20 with HCl and recovered by the addition of iberiotoxin or charybdotoxin, which inhibit large-conductance calcium-activated potassium channels (K(Ca)), or by elevating pH above 7.35 with NaOH. PCs, as well as the spike-and-plateau depolarization, were eliminated by nifedipine, which inhibits L-type voltage-gated calcium channels (Ca(V)). Influx of Ca(2+) through L-type Ca(V), which was opened because closing of K(Ca), secondary to 3,4-DAP-induced closing of K(V), resulted in contraction; the intracellular Ca(2+) increased by this influx opened K(Ca), leading to closure of Ca(V) and consequent cessation of Ca(2+) influx with resultant relaxation. These processes were repeated spontaneously to cause PCs. H(+) and OH(-) were considered to act as the opener and closer of K(Ca), respectively.

In dialyzed squid axons oxidative stress inhibits the Na+/Ca2+ exchanger by impairing the Cai2+-regulatory site.

The Na(+)/Ca(2+) exchanger, a major mechanism by which cells extrude calcium, is involved in several physiological and physiopathological interactions. In this work we have used the dialyzed squid giant axon to study the effects of two oxidants, SIN-1-buffered peroxynitrite and hydrogen peroxide (H(2)O(2)), on the Na(+)/Ca(2+) exchanger in the absence and presence of MgATP upregulation. The results show that oxidative stress induced by peroxynitrite and hydrogen peroxide inhibits the Na(+)/Ca(2+) exchanger by impairing the intracellular Ca(2+) (Ca(i)(2+))-regulatory sites, leaving unharmed the intracellular Na(+)- and Ca(2+)-transporting sites. This effect is efficiently counteracted by the presence of MgATP and by intracellular alkalinization, conditions that also protect H(i)(+) and (H(i)(+) + Na(i)(+)) inhibition of Ca(i)(2+)-regulatory sites. In addition, 1 mM intracellular EGTA reduces oxidant inhibition. However, once the effects of oxidants are installed they cannot be reversed by either MgATP or EGTA. These results have significant implications regarding the role of the Na(+)/Ca(2+) exchanger in response to pathological conditions leading to tissue ischemia-reperfusion and anoxia/reoxygenation; they concur with a marked reduction in ATP concentration, an increase in oxidant production, and a rise in intracellular Ca(2+) concentration that seems to be the main factor responsible for cell damage.

Glycolytic inhibition causes spontaneous ventricular fibrillation in aged hearts.

Selective glycolytic inhibition (GI) promotes electromechanical alternans and triggered beats in isolated cardiac myocytes. We sought to determine whether GI promotes triggered activity by early afterdepolarization (EAD) or delayed afterdepolarizations in intact hearts isolated from adult and aged rats. Dual voltage and intracellular calcium ion (Ca(i)(2+)) fluorescent optical maps and single cell glass microelectrode recordings were made from the left ventricular (LV) epicardium of isolated Langendorff-perfused adult (∼4 mo) and aged (∼24 mo) rat hearts. GI was induced by replacing glucose with 10 mM pyruvate in oxygenated Tyrode's. Within 20 min, GI slowed Ca(i)(2+) transient decline rate and shortened action potential duration in both groups. These changes were associated with ventricular fibrillation (VF) in the aged hearts (64 out of 66) but not in adult hearts (0 out of 18; P < 0.001). VF was preceded by a transient period of focal ventricular tachycardia caused by EAD-mediated triggered activity leading to VF within seconds. The VF was suppressed by the ATP-sensitive K (K(ATP)) channel blocker glibenclamide (1 µM) but not (0 out of 7) by mitochondrial K(ATP) block. The Ca-calmodulin-dependent protein kinase II (CaMKII) blocker KN-93 (1 µM) prevented GI-mediated VF (P < 0.05). Block of Na-Ca exchanger (NCX) by SEA0400 (2 µM) prevented GI-mediated VF (3 out of 6), provided significant bradycardia did not occur. Aged hearts had significantly greater LV fibrosis and reduced connexin 43 than adult hearts (P < 0.05). We conclude that in aged fibrotic unlike in adult rat hearts, GI promotes EADs, triggered activity, and VF by activation of K(ATP) channels CaMKII and NCX.

Specific mitochondrial calcium overload induces mitochondrial fission in prostate cancer cells.

Mitochondria are structurally complex organelles that undergo fragmentation or fission in apoptotic cells. Mitochondrial fission requires the cytoplasmic dynamin-related protein, Drp1, which translocates to the mitochondria during apoptosis and interacts with the mitochondrial protein, Fis1. Finely tuned changes in cellular calcium modulate a variety of intracellular functions; in resting cells, the level of mitochondrial calcium is low, while it is higher during apoptosis. Mitochondria take up Ca(2+) via the Uniporter and extrude it to the cytoplasm through the mitochondrial Na+/Ca(2+) exchanger. Overload of Ca(2+) in the mitochondria leads to their damage, affecting cellular function and survival. The mitochondrial Na+/Ca2+ exchanger was blocked by benzodiazepine, CGP37157 (CGP) leading to increased mitochondrial calcium and enhancing the apoptotic effects of TRAIL, TNFalpha related apoptosis inducing ligand. In the present study, we observed that increasing mitochondrial calcium induced mitochondrial fragmentation, which correlated with the presence of Drp1 at the mitochondria in CGP treated cells. Under these conditions, we observed interactions between Drp1 and Fis1. The importance of Drp1 in fragmentation was confirmed by transfection of dominant negative Drp1 construct. However, fragmentation of the mitochondria was not sufficient to induce apoptosis, although it enhanced TRAIL-induced apoptosis. Furthermore, oligomerization of Bak was partially responsible for the increased apoptosis in cells treated with both CGP and TRAIL. Thus, our results show that combination of an apoptogenic agent and an appropriate calcium channel blocker provide therapeutic advantages.

Mitochondrial regulation of sarcoplasmic reticulum Ca2+ content in vascular smooth muscle cells.

Subplasmalemmal ion fluxes have global effects on Ca(2+) signaling in vascular smooth muscle. Measuring cytoplasmic and mitochondrial [Ca(2+)]and [Na(+)], we previously showed that mitochondria buffer both subplasmalemmal cytosolic [Ca(2+)] and [Na(+)] in vascular smooth muscle cells. We have now directly measured sarcoplasmic reticulum [Ca(2+)] in aortic smooth muscle cells, revealing that mitochondrial Na(+)/Ca(2+) exchanger inhibition with CGP-37157 impairs sarcoplasmic reticulum Ca(2+) refilling during purinergic stimulation. By overexpressing hFis1 to remove mitochondria from the subplasmalemmal space, we show that the rate and extent of sarcoplasmic reticulum refilling is augmented by a subpopulation of peripheral mitochondria. In ATP-stimulated cells, hFis-1-mediated relocalization of mitochondria impaired the sarcoplasmic reticulum refilling process and reduced mitochondrial [Ca(2+)] elevations, despite increased cytosolic [Ca(2+)] elevations. Reversal of plasmalemmal Na(+)/Ca(2+) exchange was the primary Ca(2+) entry mechanism following ATP stimulation, based on the effects of KB-R7943. We propose that subplasmalemmal mitochondria ensure efficient sarcoplasmic reticulum refilling by cooperating with the plasmalemmal Na(+)/Ca(2+) exchanger to funnel Ca(2+) into the sarcoplasmic reticulum and minimize cytosolic [Ca(2+)] elevations that might otherwise contribute to hypertensive or proliferative vasculopathies.

Epileptiform activity induces distance-dependent alterations of the Ca2+ extrusion mechanism in the apical dendrites of subicular pyramidal neurons.

The cellular and molecular mechanisms that underlie acquired changes in Ca(2+) dynamics of different neuronal compartments are important in the induction and maintenance of epileptiform activity. Simultaneous electrophysiology and Ca(2+) imaging techniques were used to understand the basic properties of dendritic Ca(2+) signaling in rat subicular pyramidal neurons during epileptiform activity. Distance-dependent changes in the Ca(2+) decay kinetics locked to spontaneous epileptiform discharges and back-propagating action potentials were observed in the apical dendrites. A decrement in the mean tau value of Ca(2+) decay was observed in distal parts (95-110 mum) of the apical dendrites compared with proximal segments (30-45 mum) in in-vitro epileptic conditions but not in control. Pharmacological agents that block Ca(2+) transporters, i.e. Na(+)/ Ca(2+) exchangers (Benzamil), plasma membrane Ca(2+)-ATPase pumps (Calmidazolium) and smooth endoplasmic reticulum Ca(2+)-ATPase pumps (Thapsigargin), were applied locally to the proximal and distal part of the apical dendrites in both experimental conditions to understand the molecular aspects of the Ca(2+) extrusion mechanisms. The relative contribution of Na(+)/Ca(2+) exchangers in Ca(2+) extrusion was higher in the distal apical dendrites in the in-vitro epileptic condition and this property modulated the excitability of the neuron in simulation. The Ca(2+) homeostatic mechanisms that restore normal Ca(2+) levels could play a major neuroprotective role in the distal dendrites that receive synaptic inputs.

Adenosine reduces the reverse mode of the Na+/Ca(2+) exchanger in ferret cardiac fibres.

The aim of this study was to investigate the effects of adenosine on reverse mode Na+/Ca(2+) exchange. In intact ferret cardiac trabeculae, Na+-free contractures were investigated after treating preparations with ryanodine, a sarcoplasmic reticulum Ca(2+) -channel inhibitor, and thapsigargin, a sarcoplasmic reticulum Ca(2+) -pump inhibitor added to suppress the sarcoplasmic reticulum function. The effects of adenosine (50-100 nmol/L), adenosine deaminase (ADA, 0.1-0.5 U/L), the A1 and A2A receptor agonists CCPA (3-100 nmol/L) and CGS 21680 (25-100 nmol/L), and the A1 and A2A receptor antagonists DPCPX (25 nmol/L) and ZM 241385 (25 nmol/L) were tested on Na+-free contractures. The application of adenosine (50-100 nmol/L) had no significant effect on the characteristics of the Na+-free contractures. However, the results show that treatment with ADA (0.3 U/L), adenosine (> or =50 nmol/L) and CCPA, a specific A1 receptor agonist (3-100 nmol/L), all reduced the Na+-free contracture amplitude. In the presence of ADA, the effects of adenosine and CCPA were also reduced by a specific antagonist of A1 receptors (DPCPX, 25 nmol/L). Furthermore, adenosine, ADA, and CCPA did not affect the properties of the contractile apparatus in Triton-skinned fibres. It is therefore proposed that endogenous adenosine reduced the reverse mode of the Na+/Ca(2+) exchanger by acting on A1 receptors present in the sarcolemmal membrane.

Plasma membrane Ca(2+)-ATPase in the cilia of olfactory receptor neurons: possible role in Ca(2+) clearance.

Olfactory sensory neurons respond to odorants increasing Ca(2+) concentrations in their chemosensory cilia. Calcium enters the cilia through cAMP-gated channels, activating Ca(2+)-dependent chloride or potassium channels. Calcium also has a fundamental role in odour adaptation, regulating cAMP turnover rate and the affinity of the cyclic nucleotide-gated channels for cAMP. It has been shown that a Na(+)/Ca(2+) exchanger (NCX) extrudes Ca(2+) from the cilia. Here we confirm previous evidence that olfactory cilia also express plasma membrane Ca(2+)-ATPase (PMCA), and show the first evidence supporting a role in Ca(2+) removal. Both transporters were detected by immunoblot of purified olfactory cilia membranes. The pump was also revealed by immunocytochemistry and immunohistochemistry. Inside-out cilia membrane vesicles transported Ca(2+) in an ATP-dependent fashion. PMCA activity was potentiated by luminal Ca(2+) (K(0.5) = 670 nm) and enhanced by calmodulin (CaM; K(0.5) = 31 nm). Both carboxyeosin (CE) and calmidazolium reduced Ca(2+) transport, as expected for a CaM-modulated PMCA. The relaxation time constant (tau) of the Ca(2+)-dependent Cl(-) current (272 +/- 78 ms), indicative of luminal Ca(2+) decline, was increased by CE (2181 +/- 437 ms), by omitting ATP (666 +/- 49 ms) and by raising pH (725 +/- 65 ms), suggesting a role of the pump on Ca(2+) clearance. Replacement of external Na(+) by Li(+) had a similar effect (tau = 442 +/- 8 ms), confirming the NCX involvement in Ca(2+) extrusion. The evidence suggests that both Ca(2+) transporters contribute to re-establish resting Ca(2+) levels in the cilia following olfactory responses.

A comparison of adenovirally delivered molecular methods to inhibit Na+/Ca2+ exchange.

The ability to use molecular biology tools to down-regulate Na+/Ca2+ exchanger (NCX) expression will allow us to better understand the regulation of Ca(i)2+ and contractility in heart. Three different techniques to deplete NCX expression were compared: short hairpin RNA (shRNA), antisense RNA and exchanger inhibitory peptide expression via adenoviral transfection. Our results demonstrate that the most efficient method to deplete NCX expression and activity from cardiomyocytes is shRNA. It is also possible to replace the endogenous NCX with alternative isoforms or mutant forms of the NCX. Adenovirally delivered shRNA is an efficient tool for the study of the NCX and could be adapted for many other cardiac proteins.

Potassium channel opener pinacidil induces relaxation of the isolated human radial artery.

Taking into consideration that the search for drugs capable of modifying blood flow through human radial artery (RA) is warranted, the present study was designed to examine the vasodilatatory effects of the potassium channel opener, pinacidil on the RA and to define the contribution of different K+ -channel subtypes in the endothelium-independent pinacidil action on this blood vessel. Pinacidil relaxed the RA rings with endothelium and without endothelium with comparable potency. N-nitro-L-arginine methyl ester (L-NAME) and methylene blue did not affect the pinacidil-induced vasorelaxation in rings with endothelium. In the rings without endothelium, the K+ -channel blockers glibenclamide and tetraethylammonium (TEA) moderately antagonized the pinacidil-induced relaxation, while charybdotoxin and 4-aminopiridine did not. In endothelium-denuded rings, precontracted with 100 mM K+, the relaxant responses to pinacidil were highly significantly shifted to the right compared to those obtained in RA precontracted with phenylephrine, but pinacidil-induced maximal relaxation was not affected. Addition of nifedipine did not but addition of nifedipine and nickel (Na+ -Ca2+ exchanger inhibitor) did cause a statistically significant rightward shift of the pinacidil concentration-relaxation curve, although the effect 0.1 mM pinacidil was preserved. Thus, pinacidil induces relaxation of the human RA in endothelium-independent manner, and glibenclamide- and TEA-sensitive vascular smooth muscle K+ channels are probably involved. Its ability to completely relax the RA precontracted with K+ -rich solution suggests that pinacidil has additional K+ channel-independent mechanism(s) of action. It seems that stimulation of the forward mode of the Na+ -Ca2+ exchanger plays a part in this K+ channel-independent effect of pinacidil.

Sarcolemmal cation channels and exchangers modify the increase in intracellular calcium in cardiomyocytes on inhibiting Na+-K+-ATPase.

Although inhibition of the sarcolemmal (SL) Na(+)-K(+)-ATPase is known to cause an increase in the intracellular concentration of Ca(2+) ([Ca(2+)](i)) by stimulating the SL Na(+)/Ca(2+) exchanger (NCX), the involvement of other SL sites in inducing this increase in [Ca(2+)](i) is not fully understood. Isolated rat cardiomyocytes were treated with or without different agents that modify Ca(2+) movements by affecting various SL sites and were then exposed to ouabain. Ouabain was observed to increase the basal levels of both [Ca(2+)](i) and intracellular Na(+) concentration ([Na(+)](i)) as well as to augment the KCl-induced increases in both [Ca(2+)](i) and [Na(+)](i) in a concentration-dependent manner. The ouabain-induced changes in [Na(+)](i) and [Ca(2+)](i) were attenuated by treatment with inhibitors of SL Na(+)/H(+) exchanger and SL Na(+) channels. Both the ouabain-induced increase in basal [Ca(2+)](i) and augmentation of the KCl response were markedly decreased when cardiomyocytes were exposed to 0-10 mM Na(+). Inhibitors of SL NCX depressed but decreasing extracellular Na(+) from 105-35 mM augmented the ouabain-induced increase in basal [Ca(2+)](i) and the KCl response. Not only was the increase in [Ca(2+)](i) by ouabain dependent on the extracellular Ca(2+) concentration, but it was also attenuated by inhibitors of SL L-type Ca(2+) channels and store-operated Ca(2+) channels (SOC). Unlike the SL L-type Ca(2+)-channel blocker, the blockers of SL Na(+) channel and SL SOC, when used in combination with SL NCX inhibitor, showed additive effects in reducing the ouabain-induced increase in basal [Ca(2+)](i). These results support the view that in addition to SL NCX, SL L-type Ca(2+) channels and SL SOC may be involved in raising [Ca(2+)](i) on inhibition of the SL Na(+)-K(+)-ATPase by ouabain. Furthermore, both SL Na(+)/H(+) exchanger and Na(+) channels play a critical role in the ouabain-induced Ca(2+) increase in cardiomyocytes.

Intracellular calcium handling in ventricular myocytes from mdx mice.

Duchenne muscular dystrophy (DMD) is a lethal degenerative disease of skeletal muscle, characterized by the absence of the cytoskeletal protein dystrophin. Some DMD patients show a dilated cardiomyopathy leading to heart failure. This study explores the possibility that dystrophin is involved in the regulation of a stretch-activated channel (SAC), which in the absence of dystrophin has increased activity and allows greater Ca(2+) into cardiomyocytes. Because cardiac failure only appears late in the progression of DMD, we examined age-related effects in the mdx mouse, an animal model of DMD. Ca(2+) measurements using a fluorescent Ca(2+)-sensitive dye fluo-4 were performed on single ventricular myocytes from mdx and wild-type mice. Immunoblotting and immunohistochemistry were performed on whole hearts to determine expression levels of key proteins involved in excitation-contraction coupling. Old mdx mice had raised resting intracellular Ca(2+) concentration ([Ca(2+)](i)). Isolated ventricular myocytes from young and old mdx mice displayed abnormal Ca(2+) transients, increased protein expression of the ryanodine receptor, and decreased protein expression of serine-16-phosphorylated phospholamban. Caffeine-induced Ca(2+) transients showed that the Na(+)/Ca(2+) exchanger function was increased in old mdx mice. Two SAC inhibitors streptomycin and GsMTx-4 both reduced resting [Ca(2+)](i) in old mdx mice, suggesting that SACs may be involved in the Ca(2+)-handling abnormalities in these animals. This finding was supported by immunoblotting data, which demonstrated that old mdx mice had increased protein expression of canonical transient receptor potential channel 1, a likely candidate protein for SACs. SACs may play a role in the pathogenesis of the heart failure associated with DMD. Early in the disease process and before the onset of clinical symptoms increased, SAC activity may underlie the abnormal Ca(2+) handling in young mdx mice.

The influence of sodium-calcium exchange inhibitors on rabbit lens ion balance and transparency.

Calcium regulation is essential to the maintenance of lens transparency. To maintain cytoplasmic calcium concentration at the required low level the lens must export calcium continuously. Here, studies were conducted to test whether sodium-calcium exchanger (NCX) inhibitors disturb calcium balance in the rabbit lens. Intact lenses were incubated up to 48 h in the presence or absence of the NCX inhibitor bepridil. Lens sodium, potassium and calcium content were determined by atomic absorption spectrophotometry. Fluo-4 was used to measure epithelial cell cytoplasmic calcium concentration in an intact lens preparation. NCX1 protein expression in lens epithelium was examined by western blot. NCX1 band density was similar in central and equatorial epithelium samples. Lenses exposed to bepridil (30 microM) lost transparency at the anterior and exhibited significant changes in electrolyte and water content. After 48 h, lens calcium content more than doubled, sodium increased four fold and potassium was significantly reduced. In contrast, lenses exposed to inhibitors of reverse mode calcium transport by NCX (KBR7943 or SN-6) remained transparent and the electrolyte and water content of the lens remained unchanged. The ability of bepridil to cause significant changes in lens transparency and electrolyte content points to an important role for NCX-meditated calcium export in the lens.

The sodium channel of human excitable cells is a target for gambierol.

BACKGROUND: Gambierol is a polycyclic ether toxin with the same biogenetic origin as ciguatoxins. Gambierol has been associated with neurological symptoms in humans even though its mechanism of action has not been fully characterized. METHODS: We studied the effect of gambierol in human neuroblastoma cells by using bis-oxonol to measure membrane potential and FURA-2 to monitor intracellular calcium. RESULTS: We found that this toxin: i) produced a membrane depolarization, ii) potentiated the effect of veratridine on membrane potential iii) decreased ciguatoxin-induced depolarization and iv) increased cytosolic calcium in neuroblastoma cells. CONCLUSION: These results indicate that gambierol modulate ion fluxes by acting as a partial agonist of sodium channels.

Modulation of calcium entry and glutamate release in cultured cerebellar granule cells by palytoxin.

A channel open on the membrane can be formed by palytoxin (PTX). Ten nanomolar PTX caused an irreversible increase in the cytosolic calcium concentration ([Ca(2+)](c)), which was abolished in the absence of external calcium. The increase was eliminated by saxitoxin (STX) and nifedipine (NIF). Calcium rise is secondary to the membrane depolarization. PTX effect on calcium was dependent on extracellular Na(+). Li(+) decreased the PTX-evoked rise in [Ca(2+)](c); replacement of Na(+) by N-methyl-D-glucamine (NMDG) abolished PTX-induced calcium increase. [Ca(2+)](c) increase by PTX was strongly reduced after inhibition of the reverse operation of the Na(+)/Ca(2+) exchanger, in the presence of antagonists of excitatory amino acid (EAA) receptors, and by inhibition of neurotransmitter release. PTX did not modify calcium extrusion by the plasma membrane Ca(2+)-ATPase (PMCA), because blockade of the calcium pump increased rather than decreased the PTX-induced calcium influx. Extracellular levels of glutamate and aspartate were measured by HPLC and exocytotic neurotransmitter release by determination of synaptic vesicle exocytosis using total internal reflection fluorescence microscopy (TIRFM). PTX caused a concentration-dependent increase in EAA release to the culture medium. Ten nanomolar PTX decreased cell viability by 30% within 5 min. PTX-induced calcium influx involves three pathways: Na(+)-dependent activation of voltage-dependent sodium channels (VDSC) and voltage-dependent calcium channels (VDCC), reverse operation of the Na(+)/Ca(2+) exchanger, and indirect activation of EAA receptors through glutamate release. The neuronal injury produced by the toxin could be partially mediated by the PTX-induced overactivation of EAA receptors, VDSC, VDCC and the glutamate efflux into the extracellular space.

Anesthetic-induced alteration in expression of myocardial calcium cycling proteins.

OBJECTIVE: The purpose of this study was to test the hypothesis that anesthesia alone or in combination with high F(I)(O)2 alters expression of the myocardial calcium cycling proteins, sarcoplasmic endoreticular calcium adenosine triphosphatase subtype 2a (SERCA2a), and the sarcolemmal sodium-calcium exchanger (NCX). DESIGN: Multigroup comparison of protein expression using analysis of variance. SETTING: University research laboratory. SUBJECTS: Twenty-seven New Zealand white rabbits. INTERVENTIONS: After sedation and the induction of anesthesia, animals underwent either tracheal intubation and ventilation for 5 hours with 1.0% end-tidal halothane in oxygen (HAL-O(2) , n = 5) or air (HAL-air, n = 5) or time-control recovery while spontaneously breathing oxygen (TC-O(2) , n = 5) or air (TC-air, n = 5) for 5 hours. Halothane dose was based on pilot data from 2 rabbits. Animals were then sacrificed, and the hearts were removed for Western blot analysis. Data were normalized to those from a group of rabbits immediately sacrificed (n = 5) without any prior treatment. MEASUREMENTS AND MAIN RESULTS: In comparison to their respective time controls, SERCA2a was decreased 23 % in both HAL-air and HAL-O(2) groups, whereas NCX was increased 34% and 122%, respectively. Expression was distinctly different between HAL-air and HAL-O(2) for both SERCA2a (p = 0.009) and NCX (p < 0.001), indicating an influence of high F(I)O(2). Similarly, SERCA2a in the TC-O(2) group was reduced 25% relative to the TC-air group. CONCLUSION: Halothane alters the expression of myocardial calcium cycling proteins, and this effect is potentiated by high F(I)O(2) . These data offer the broad conclusion that perioperative interventions may influence the study of myocardial molecular remodeling and suggest the possibility of anesthetic-induced myocardial molecular remodeling.