Exchanger In Reverse Mode Research Articles

The reverse mode of the Na+/Ca2+ exchanger contributes to the pacemaker mechanism in rabbit sinus node cells

Sinus node (SN) pacemaking is based on a coupling between surface membrane ion-channels and intracellular Ca2+-handling. The fundamental role of the inward Na+/Ca2+ exchanger (NCX) is firmly established. However, little is known about the reverse mode exchange. A simulation study attributed important role to reverse NCX activity, however experimental evidence is still missing. Whole-cell and perforated patch-clamp experiments were performed on rabbit SN cells supplemented with fluorescent Ca2+-tracking. We established 2 and 8 mM pipette NaCl groups to suppress and enable reverse NCX. NCX was assessed by specific block with 1 μM ORM-10962. Mechanistic simulations were performed by Maltsev–Lakatta minimal computational SN model. Active reverse NCX resulted in larger Ca2+-transient amplitude with larger SR Ca2+-content. Spontaneous action potential (AP) frequency increased with 8 mM NaCl. When reverse NCX was facilitated by 1 μM strophantin the Ca2+i and spontaneous rate increased. ORM-10962 applied prior to strophantin prevented Ca2+i and AP cycle change. Computational simulations indicated gradually increasing reverse NCX current, Ca2+i and heart rate with increasing Na+i. Our results provide further evidence for the role of reverse NCX in SN pacemaking. The reverse NCX activity may provide additional Ca2+-influx that could increase SR Ca2+-content, which consequently leads to enhanced pacemaking activity.

Cyclic AMP-dependent signaling system is a primary metabolic target for non-thermal effect of microwaves on heart muscle hydration

ABSTRACTPreviously, we have suggested that cell hydration is a universal and extra-sensitive sensor for the structural changes of cell aqua medium caused by the impact of weak chemical and physical factors. The aim of present work is to elucidate the nature of the metabolic messenger through which physiological solution (PS) treated by non-thermal (NT) microwaves (MW) could modulate heart muscle hydration of rats. For this purpose, the effects of NT MW–treated PS on heart muscle hydration, [3H]-ouabain binding with cell membrane, 45Ca2+ uptake and intracellular cyclic nucleotides contents in vivo and in vitro experiments were studied. It is shown that intraperitoneal injections of both Sham-treated PS and NT MW–treated PS elevate heart muscle hydration. However, the effect of NT MW–treated PS on muscle hydration is more pronounced than the effect of Sham-treated PS. In vitro experiments NT MW–treated PS has dehydration effect on muscle, which is not changed by decreasing Na+ gradients on membrane. Intraperitoneal injection of Sham- and NT MW–treated PS containing 45Ca2+ have similar dehydration effect on muscle, while NT MW–treated PS has activation effect on Na+/Ca2+ exchange in reverse mode. The intraperitoneal injection of NT MW–treated PS depresses [3H]-ouabain binding with its high-affinity membrane receptors, elevates intracellular cAMP and decreases cGMP contents. Based on the obtained data, it is suggested that cAMP-dependent signaling system serves as a primary metabolic target for NT MW effect on heart muscle hydration.

Non-thermal microwave radiation-induced brain tissue dehydration as a potential factor for brain functional impairment

Based on our previous finding that metabolically controlled cell hydration is sensitive to the changes of physicochemical properties of cell aqua medium, which take place upon the effect of weak physical signals, including microwaves with non-thermal intensity (NT MW), it has been hypothesized that cell aqua medium serves as a primary target for NT MW effect on brain tissue. To elucidate the nature of the metabolic pathway through which the effect of NT MW-induced changes of physicochemical properties of cell aqua medium modulate cell hydration, the effects of intraperitoneal (IP) injection of PS treated by NT MW on brain tissue hydration, 45Ca2+ uptake, [3H]-ouabain binding with cell membrane and intracellular cyclic nucleotides have been studied. The obtained data indicate that PS treated by NT MW through the activation of high affinity ouabain receptors in membrane stimulates cAMP-dependent Na+/Ca2+ exchange in reverse mode (R), which leads to brain tissue hydration in healthy (young) and dehydration in non-healthy (old) rats.As NT MW radiation-induced activation of R Na+/Ca2+ exchange leads to the increase of intracellular Ca2+ ([Ca2+]i), it is considered as a potential factor leading to the brain functional impairment, especially when brain metabolic activity is depressed (e.g. during aging).

Primary mechanism responsible for age-dependent neuronal dehydration

<p>Neuronal dehydration and high [Ca<sup>2+</sup>]<sub>i</sub> are essential hallmarks for age-dependent memory impairment. Na<sup>+</sup>/K<sup>+</sup>-ATPase, having membrane transporting and intracellular signaling functions, has age-induced dysfunctional character. Therefore, it could have a key role in age-dependent neuronal dehydration and increase of [Ca<sup>2+</sup>]<sub>i</sub>. However, it is not clear the dysfunction of which above mentioned functions of Na<sup>+</sup>/K<sup>+</sup>-ATPase serves as a primary mechanism for generation of age-dependent neuronal dehydration and increase of [Ca<sup>2+</sup>]<sub>i</sub>. In present work age-dependent effects of ≤10<sup>-9</sup>M (agonist for α<sub>3</sub> isoform which has only signaling function) and 10<sup>-4</sup>M ouabain (agonist for α<sub>1</sub> isoform which has ion-transporting function) on brain cortex tissue hydration, <sup>45</sup>Ca<sup>2+</sup> uptake and <sup>45</sup>Ca<sup>2+</sup> efflux through plasma membrane were studied. It was shown that ≤10<sup>-9</sup>M and 10<sup>-4</sup>M ouabain concentrations have stimulation effects on cortex tissue hydration and Na<sup>+</sup>/Ca<sup>2+</sup> exchange in reverse mode. However, these effects have age-dependent weakening and increasing characters, respectively. It is suggested that ≤10<sup>-9</sup>M ouabain-induced tissue hydration is due to cAMP-activated Ca<sup>2+</sup>-ATPase in endoplasmic reticulum membrane leading to reversion of Na<sup>+</sup>/Ca<sup>2+</sup> exchange and elevation of endogenous H<sub>2</sub>O release in cytoplasm. This effect has age-dependent depressing character. Thus, the dysfunction of α<sub>3</sub> isoform-dependent intracellular signaling system could be considered as a primary mechanism for age-dependent neuronal dehydration.</p>

Voltage-gated sodium channels contribute to action potentials and spontaneous contractility in isolated human lymphatic vessels.

Voltage-gated sodium channels (VGSC) play a key role for initiating action potentials (AP) in excitable cells. VGSC in human lymphatic vessels have not been investigated. In the present study, we report the electrical activity and APs of small human lymphatic collecting vessels, as well as mRNA expression and function of VGSC in small and large human lymphatic vessels. The VGSC blocker TTX inhibited spontaneous contractions in six of 10 spontaneously active vessels, whereas ranolazine, which has a narrower VGSC blocking profile, had no influence on spontaneous activity. TTX did not affect noradrenaline-induced contractions. The VGSC opener veratridine induced contractions in a concentration-dependent manner (0.1-30 μm) eliciting a stable tonic contraction and membrane depolarization to -18 ± 0.6 mV. Veratridine-induced depolarizations and contractions were reversed ∼80% by TTX, and were dependent on Ca(2+) influx via L-type calcium channels and the sodium-calcium exchanger in reverse mode. Molecular analysis determined NaV 1.3 to be the predominantly expressed VGSC isoform. Electrophysiology of mesenteric lymphatics determined the resting membrane potential to be -45 ± 1.7 mV. Spontaneous APs were preceded by a slow depolarization of 5.3 ± 0.6 mV after which a spike was elicited that almost completely repolarized before immediately depolarizing again to plateau. Vessels transiently hyperpolarized prior to returning to the resting membrane potential. TTX application blocked APs. We have shown that VGSC are necessary for initiating and maintaining APs and spontaneous contractions in human lymphatic vessels and our data suggest the main contribution from comes NaV 1.3. We have also shown that activation of these channels augments the contractile activity of the vessels.

The Dysfunction of Camp-Dependent Na+/Ca2+ Exchange in Reverse Mode as a Primary Mechanism for Age-Dependent Cardio-Muscle Failure

The Dysfunction of Camp-Dependent Na+/Ca2+ Exchange in Reverse Mode as a Primary Mechanism for Age-Dependent Cardio-Muscle Failure

Early signals after stretch leading to cardiac hypertrophy. Key role of NHE-1

The enhanced activity of the cardiac Na+/H+ exchanger (NHE-1) after myocardial stretch is considered a key step of the intracellular signaling pathway leading to the slow force response to stretch as well as an early signal for the development of cardiac hypertrophy. We propose that the chain of events triggered by stretch begins with the release of small amounts of Angiotensin II (Ang II)/endothelin (ET) and ends with the increase in intracellular Ca2+ concentration ([Ca2+]i) through the Na+/Ca2+ exchanger in reverse mode (NCX(rev)), which triggers cardiac hypertrophy by activation of widely recognized Ca2+-dependent intracellular signaling pathways.

CDNA Cloning and Expression of the Cardiac Na+/Ca2+ Exchanger from Mozambique Tilapia (Oreochromis mossambicus) Reveal a Teleost Membrane Transporter with Mammalian Temperature Dependence

The complete cDNA sequence of the tilapia cardiac Na(+)/Ca2+ exchanger (NCX-TL1.0) was determined. The 3.1-kb transcript encodes a protein 957 amino acids in length, with a predicted signal peptide cleaved at residue 31 and two potential N-glycosylation sites in the extracellular N terminus. Hydropathy analysis and sequence comparison predicted a mature protein with nine transmembrane-spanning segments, consistent with the structural topologies of other known mammalian and teleost NCX isoforms. Overall sequence comparison shows high identity to both trout NCX-TR1.0 ( approximately 81%) and mammalian NCX1.1 ( approximately 73%), and phylogenetic analyses confirmed its identity as a member of the NCX1 gene family, expressing exons A, C, D, and F in the alternative splice site. Sequence identity is even higher in the alpha-repeats, the exchanger inhibitory peptide (XIP) site, and Ca(2+)-binding domains, which is reflected in the functional and regulatory properties of tilapia NCX-TL1.0. When NCX-TL1.0 was expressed in Xenopus oocytes and the currents were measured in giant excised patches, they displayed both positive regulation by Ca2+ and Na(+)-dependent inactivation in a manner similar to trout NCX-TR1.0. However, tilapia NCX-TL1.0 exhibited a relatively high sensitivity to temperature compared with trout NCX-TR1.0. Whereas trout NCX-TR1.0 currents displayed activation energies of approximately 7 kJ/mol, tilapia NCX-TL1.0 currents showed mammal-like temperature dependence, with peak and steady-state current activation energies of 53 +/- 9 and 67 +/- 21 kJ/mol, respectively. Using comparative sequence analysis, we highlighted 10 residue positions in the N-terminal domain of the NCX that, in combination, may confer exchanger temperature dependence through subtle changes in protein flexibility. Tilapia NCX-TL1.0 represents the first non-mammalian NCX to exhibit a mammalian temperature dependence phenotype and will prove to be a useful model in defining the interplay between molecular flexibility and stability in NCX function.

Localization and function of the Na +/Ca 2+-exchanger in normal and detubulated rat cardiomyocytes

It is controversial whether the Na +/Ca 2+-exchanger (NCX) can induce cardiomyocyte contraction through reverse-mode exchange and Ca 2+-induced Ca 2+ release (CICR). Information about the spatial distribution and functional activity within different sarcolemmal (SL) regions could shed light on this potential role. We raised a new antibody to the NCX and showed by confocal laser scanning microscopy (CLSM) that immunoreactivity is strongly expressed throughout the surface SL and intercalated disk regions with punctate labeling of the vertical transverse (T)-tubules but not the longitudinal T-tubules. Immuno-electron microscopy confirmed CLSM observations. Gold particles associated with the exchanger were within nanometer range of particles signaling ryanodine receptors. A similar close association was found between the L-type Ca 2+ channel (known to be concentrated in the dyad) and ryanodine receptors. In whole-cell patch-clamped cardiomyocytes, peak I NCX (measured at 90 mV) decreased by ~40% (497 ± 32 vs. 304 ± 12 pA, P < 0.001) after detubulation, while membrane capacitance decreased by 27% (204 ± 11 vs. 150 ± 7 pF, P < 0.01) thus giving a small but significant 16% reduction in current density. Thus, the density and/or functional activity of the NCX is greater in the vertical T-tubules than in the longitudinal T-tubules, surface SL or disk regions, pointing to important functional differences between these plasma membrane domains. Our combined co-immunolocalization and physiological data suggest that the NCX has multiple functions depending upon membrane location. We suggest the possibility that NCX modulates CICR, sarcoplasmic reticulum Ca 2+ load, and that it also serves to regulate Ca 2+ handling in neighboring cells.

Mode-dependent inhibition by quinidine of Na+-Ca2+ exchanger current from guinea-pig isolated ventricular myocytes

1. The aim of the present study was to determine whether or not the class Ia anti-arrhythmic agent quinidine modulates ionic current (INaCa) generated by the sarcolemmal Na+-Ca2+ exchanger of adult ventricular cardiac myocytes. 2. Selective whole-cell voltage-clamp recordings of INaCa were made from guinea-pig ventricular myocytes, with major interfering currents blocked. The INaCa was measured as the ionic current sensitive to 10 mmol/L external Ni2+ during a descending voltage ramp protocol. 3. The effects of quinidine concentrations in the range 10-100 micromol/L were studied. Quinidine produced a concentration-dependent partial blockade of outward INaCa, generated by reverse-mode exchange. At +60 mV, 100 micromol/L quinidine blocked INaCa by 33.0 +/- 4.1% (mean+/-SEM; n = 4). This was the maximal concentration that we were able to test, because concentrations of quinidine higher than 100 micromol/L were found to be toxic to cells under our conditions. The drug did not produce any significant inhibition of inward INaCa (generated by forward-mode exchange). 4. The Ni2+-insensitive residual current was not significantly altered by quinidine at any membrane potential, confirming that the inhibitory effects of quinidine we observed could be attributed to an action on the Na+-Ca2+ exchanger. 5. For the purpose of comparison, quinidine was tested against L-type Ca current (ICa,L). It blocked peak ICa,L at 0 mV, with an IC50 of 14.9 +/- 1.5 micromol/L. Thus, quinidine was less potent against the exchanger than against ICa,L. 6. Our data suggest that quinidine preferentially inhibits the Naout/Cain mode of exchanger function. We conclude that this drug is a weak inhibitor of ventricular INaCa and that the inhibitory effect is mode dependent.

Inhibition by KB-r7943 of the reverse mode of the Na+/Ca2+ exchanger reduces Ca2+ overload in ischemic-reperfused rat hearts.

Ca2+ influx via the Na+/Ca2+ exchanger (NCX) may lead to Ca2+ overload and myocardial injury in ischemia-reperfusion. Direct evidence that increased cytoplasmic Ca2+ concentration ([Ca2+]i) is mediated by the reverse mode of the NCX is limited, so in the present study the [Ca2+]i dynamics and left ventricular pressure were monitored in perfused beating hearts. The effects of KB-R7943 (KBR), a selective inhibitor of the NCX in the reverse mode, were analyzed during low-Na+ exposure and ischemia-reperfusion. Hearts from Sprague-Dawley rats were retrogradely perfused and loaded with 4 micromol/L fura-2 to measure the fluorescence ratio as an index of [Ca2+]i. To evaluate KBR effects on the reverse mode exchanger, the increase in [Ca2+]i induced by low-Na+ exposure (Na+: 30 mmol/L, 10 mmol/L caffeine pre-treatment) was measured with and without 10 micromol/L KBR (n=5). In another series, the hearts were subjected to 10 min of low-flow ischemia with pacing, followed by reperfusion in the absence (n=6) or in the presence of 10 micromol/L KBR (n=6). Background autofluorescence was subtracted to estimate the ratio in the ischemia-reperfusion protocol. KBR significantly suppressed the increase in [Ca2+]i induced by low-Na+ (40.2 +/- 11.2% of control condition, p=0.014), as well as on increase in diastolic [Ca2+]i during ischemia (% increase from pre-ischemia in [Ca2+]i at 10 min: KBR, 17.9 +/- 6.4%; no KBR, 44.4 +/- 7.7%; p=0.024). After reperfusion, diastolic [Ca2+]i normalized more rapidly in KBR-treated hearts (% increase at 1 min: KBR, 4.5 +/- 7.0%; no KBR, 39.8 +/- 12.2%; p=0.03). Treatment with KBR also accelerated recovery of the rate-pressure product on reperfusion (1 min: KBR, 8,944 +/- 1,554 min(-1) mmHg; no KBR, 4,970 +/- 1,325; p<0.05). Thus, inhibition of the reverse mode exchanger by KBR reduced ischemic Ca2+ overload and possibly improved functional myocardial recovery during reperfusion in a whole heart model.

Sodium/calcium exchange contributes to contraction and relaxation in failed human ventricular myocytes.

Defects in myocyte contraction and relaxation are key features of human heart failure. Sodium/calcium exchanger-mediated contribution to contraction and relaxation were separated from other mechanisms [L-type calcium current, sarco(endo)plasmic reticulum (SR) Ca(2+)-ATPase] based on voltage, temperature, and selective blockers. Rod-shaped left ventricular myocytes were isolated from failed human explants (n = 29) via perfusion with collagenase-containing Krebs solution. Action potentials using perforated patch and contractions using an edge detector were recorded at 0.5-1.5 Hz in Tyrode solution at 25 degrees C and 37 degrees C. Contraction duration was dependent on action potential (AP) duration at 37 degrees C but not at 25 degrees C, suggesting the role of the exchanger in relaxation and linking myocyte relaxation to the repolarization phase of the AP. Voltage-clamp experiments from -50 to +10 mV for 1,500 ms in Tyrode or Na(+)- and K(+)-free solutions after conditioning pulses triggered biphasic contractions that included a rapid SR-mediated component and a slower voltage-dependent exchanger-mediated component. We used thapsigargin to block the SR, which eliminated the rapid component, and we used an exchanger blocker, Kanebo 7943, which eliminated the slow component. The exchanger was shown to contribute to contraction through reverse-mode exchange, as well as to play a key role in relaxation of human ventricular myocytes.

K+-dependence of Na+-Ca2+ exchange in type I vestibular sensory cells of guinea-pig.

The properties of the vestibular Na+-Ca2+ exchanger in mammalian type I vestibular sensory cells were studied using fura-2 fluorescence and immunocytochemical techniques. In the absence of external Na+, the activation of Na+-Ca2+ exchange in reverse mode required the presence of external K+ (K+o) and depended on K+o concentration. Alkali cations Rb+ and NH4+ but not Li+ or Cs+ substituted for K+o to activate the exchange. For pressure applications of 10 mm K+, the contribution of voltage-sensitive calcium channels to the increase in [Ca2+]i was < 15%. The dependence of the exchange on [K+]o was also recorded when the membrane potential was clamped using carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP) and monensin ionophores. In these conditions, where there was no intracellular Na+, the increase in [Ca2+]i was completely blocked. These physiological results suggest that in reverse mode, Ca2+ entry is driven by both an outward transport of Na+ and an inward transport of K+. The dependence of the vestibular Na+-Ca2+ exchanger on K+ is more reminiscent of the properties of the retinal type Na+-Ca2+ exchanger than those of the more widely distributed cardiac type exchanger. Moreover, the immunocytochemical localization of both types of exchange proteins in the vestibular sensory epithelium confirmed the presence in the vestibular sensory cells of a Na+-Ca2+ exchanger which is recognized by an antibody raised against retinal type and not by an antibody raised against the cardiac type.