Mechanisms of Beat-to-Beat Regulation of Cardiac Pacemaker Cell Function by Ca2+ Cycling Dynamics

Whether intracellular Ca²⁺ cycling dynamics regulate cardiac pacemaker cell function on a beat-to-beat basis remains unknown. Here we show that under physiological conditions, application of low concentrations of caffeine (2–4 mM) to isolated single rabbit sinoatrial node cells acutely reduces their spontaneous action potential cycle length (CL) and increases Ca²⁺ transient amplitude for several cycles. Numerical simulations, using a modified Maltsev-Lakatta coupled-clock model, faithfully reproduced these effects, and also the effects of CL prolongation and dysrhythmic spontaneous beating (produced by cytosolic Ca²⁺ buffering) and an acute CL reduction (produced by flash-induced Ca²⁺ release from a caged Ca²⁺ buffer), which we had reported previously. Three contemporary numerical models (including the original Maltsev-Lakatta model) failed to reproduce the experimental results. In our proposed new model, Ca²⁺ releases acutely change the CL via activation of the Na⁺/Ca²⁺ exchanger current. Time-dependent CL reductions after flash-induced Ca²⁺ releases (the memory effect) are linked to changes in Ca²⁺ available for pumping into sarcoplasmic reticulum which, in turn, changes the sarcoplasmic reticulum Ca²⁺ load, diastolic Ca²⁺ releases, and Na⁺/Ca²⁺ exchanger current. These results support the idea that Ca²⁺ regulates CL in cardiac pacemaker cells on a beat-to-beat basis, and suggest a more realistic numerical mechanism of this regulation.     

[Na] in the subsarcolemmal ‘fuzzy’ space and modulation of [Ca]i and contraction in cardiac myocytes

The strength of the heart beat depends on the amplitude and time course of the transient increase in [Ca²⁺] in the myocytes with each cycle. [Na⁺]_i modulates cardiac contraction through its effect on the Ca²⁺ flux through the Na/Ca exchanger. Cardiac excitation–contraction coupling has been postulated to occur in a microdomain or ‘fuzzy’ space at the junction of the T-tubules and the sarcoplasmic reticulum. This ‘fuzzy’ space is well described for the Ca²⁺ fluxes and the interaction between the L-type Ca²⁺ channel, the Ca²⁺ release channel of the sarcoplasmic reticulum and the Na/Ca exchanger. Co-localization of the Na⁺ transporters, in particular the Na/K pump and the Na⁺ channel, within this ‘fuzzy’ space is not as well established. The functional and morphological characteristics of the ‘fuzzy’ space for Na⁺ and its interaction with the Ca²⁺ handling suggest that this space is not strictly co-inciding with the Ca²⁺ microdomain. In this space [Na⁺] can be several-fold higher or lower than [Na⁺] in the bulk cytosol. This has implications for modulation of [Ca²⁺]_i during a single beat as well as during alterations in Na⁺ fluxes seen in pathological conditions.     

Influence of monovalent cations on the Ca2+-ATPase of sarcoplasmic reticulum isolated from rabbit skeletal and dog cardiac muscles. An interpretation of transient-state kinetic data

Transient-state kinetics of phosphorylation and dephosphorylation of the Ca²⁺-ATPase of sarcoplasmic reticulum vesicles from rabbit skeletal and dog cardiac muscles were studied in the presence of varying concentrations of monovalent and divalent cations. Monovalent cations affect the two types of sarcoplasmic reticulum differently. When the rabbit skeletal sarcoplasmic reticulum was Ca²⁺ deficient, preincubation with K⁺ (as compared with preincubation with choline chloride) did not affect initial phosphorylation at various concentrations of Ca²⁺, added with ATP to phosphorylate the enzyme. This is in contrast to preincubation with K⁺ of the Ca²⁺-deficient dog cardiac sarcoplasmic reticulum, which resulted in an increase in the phosphoenzyme level. When Ca²⁺ was bound to the rabbit skeletal sarcoplasmic reticulum, K⁺ inhibited E ~ P formation; but under the same conditions, E ~ P formation of dog cardiac sarcoplasmic reticulum was activated by K⁺ at 12 μM Ca²⁺ and inhibited at 0.33 and 1.3 μM Ca²⁺. Li⁺, Na⁺ and K⁺ also have different effects on E ~ P decomposition of skeletal and cardiac sarcoplasmic reticulum. The latter responded less to these cations than the former. Studies with ADP revealed differences between the two types of sarcoplasmic reticulum. For rabbit skeletal sarcoplasmic reticulum, 40% of the phosphoenzyme formed was ‘ADP sensitive’, and the decay of the remaining E ~ P was enhanced by K⁺ and ADP. Dog cardiac sarcoplasmic reticulum yielded about 40–48% ADP-sensitive E ~ P, but the decomposition rate of the remaining E ~ P was close to the rate measured in the absence of ADP. Thus, these studies showed certain qualitative differences in the transformation and decomposition of phosphoenzymes between skeletal and cardiac muscle which may have bearing on physiological differences between the two muscle types.     

Role of myocardial viscoelasticity in disturbances of electrical and mechanical activity in calcium overloaded cardiomyocytes: mathematical modeling

Cardiomyocyte Ca²⁺ overload is closely linked to cardiac arrhythmias. We have earlier shown in a mathematical model that myocardium mechanical activity may contribute to rhythm disturbances induced by Ca²⁺ overload in cardiomyocytes with reduced Na⁺-K⁺ pump work (Sulman et al., 2008). The same model is used here to address possible contribution of the passive mechanical properties of cardiac muscle (i.e. myocardial viscous and elastic properties) to the arrhythmogenesis. In a series of contractions at regular pacing rate of 75 beats/min a model with higher viscosity demonstrated essentially earlier appearance of extrasystoles due to a faster cardiomyocyte Ca²⁺ loading up to a level triggering spontaneous Ca²⁺ releases from the sarcoplasmic reticulum. The model predicts that myocardial elasticity also may affect arrhythmogenesis in cardiomyocytes overloaded with Ca²⁺. Contribution of the mechanical properties of the myocardial tissue to the arrhythmia has been analyzed for wide ranges of both viscosity and elasticity coefficients. The results suggest that myocardial viscoelastic properties may be a factor affecting Ca²⁺ handling in cardiomyocytes and contributing to cardiac mechano-electric feedback in arrhythmogenesis.     

Ca-dependent inhibitory effects of Na and K on Ca transport in sarcoplasmic reticulum vesicles

Effects of Na⁺ and K⁺ on Ca²⁺ transport by sarcoplasmic reticulum vesicles were studied in a medium containing high Mg²⁺ and ATP (2mM) and low Ca²⁺ (0.44μM) concentrations. Under these conditions, Na⁺ and K⁺ inhibit Ca²⁺ uptake. ATPase activity and membrane phosphorylation by ATP. Since the concentrations of ATP and Ca²⁺ used are consistent with relaxation in vivo, the results suggest that under physiological resting conditions the Ca²⁺ pump of the sarcoplasmic reticulum operates below its maximal capacity.     

Ca2+-dependent inhibitory effects of Na+ and K− on Ca2+ transport in sarcoplasmic reticulum vesicles

Effects of Na⁺ and K⁺ on Ca²⁺ transport by sarcoplasmic reticulum vesicles were studied in a medium containing high Mg²⁺ and ATP (2mM) and low Ca²⁺ (0.44μM) concentrations. Under these conditions, Na⁺ and K⁺ inhibit Ca²⁺ uptake. ATPase activity and membrane phosphorylation by ATP. Since the concentrations of ATP and Ca²⁺ used are consistent with relaxation in vivo, the results suggest that under physiological resting conditions the Ca²⁺ pump of the sarcoplasmic reticulum operates below its maximal capacity.     

The effect of Sirt1 deficiency on Ca2+ and Na+ regulation in mouse ventricular myocytes

This study addressed the hypothesis that cardiac Sirtuin 1 (Sirt1) deficiency alters cardiomyocyte Ca²⁺ and Na⁺ regulation, leading to cardiac dysfunction and arrhythmogenesis. We used mice with cardiac‐specific Sirt1 knockout (Sirt1^?/?). Sirt1^flox/flox mice were served as control. Sirt1^?/? mice showed impaired cardiac ejection fraction with increased ventricular spontaneous activity and burst firing compared with those in control mice. The arrhythmic events were suppressed by KN93 and ranolazine. Reduction in Ca²⁺ transient amplitudes and sarcoplasmic reticulum (SR) Ca²⁺ stores, and increased SR Ca²⁺ leak were shown in the Sirt1^?/? mice. Electrophysiological measurements were performed using patch‐clamp method. While L‐type Ca²⁺ current (I_{Ca, L}) was smaller in Sirt1^?/? myocytes, reverse‐mode Na⁺/Ca²⁺ exchanger (NCX) current was larger compared with those in control myocytes. Late Na⁺ current (I_{Na, L}) was enhanced in the Sirt1^?/? mice, alongside with elevated cytosolic Na⁺ level. Increased cytosolic and mitochondrial reactive oxygen species (ROS) were shown in Sirt1^?/? mice. Sirt1^?/? cardiomyocytes showed down‐regulation of L‐type Ca²⁺ channel α1c subunit (Cav1.2) and sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase 2a (SERCA2a), but up‐regulation of Ca²⁺/calmodulin‐dependent protein kinase II and NCX. In conclusions, these findings suggest that deficiency of Sirt1 impairs the regulation of intracellular Ca²⁺ and Na⁺ in cardiomyocytes, thereby provoking cardiac dysfunction and arrhythmogenesis.     

Modulation of the cardiac Na+-Ca2+ exchanger by cytoplasmic protons: Molecular mechanisms and physiological implications

A precise temporal and spatial control of intracellular Ca²⁺ concentration is essential for a coordinated contraction of the heart. Following contraction, cardiac cells need to rapidly remove intracellular Ca²⁺ to allow for relaxation. This task is performed by two transporters: the plasma membrane Na⁺-Ca²⁺ exchanger (NCX) and the sarcoplasmic reticulum (SR) Ca²⁺‐ATPase (SERCA). NCX extrudes Ca²⁺ from the cell, balancing the Ca²⁺entering the cytoplasm during systole through L-type Ca²⁺ channels. In parallel, following SR Ca²⁺ release, SERCA activity replenishes the SR, reuptaking Ca²⁺ from the cytoplasm.The activity of the mammalian exchanger is fine-tuned by numerous ionic allosteric regulatory mechanisms. Micromolar concentrations of cytoplasmic Ca²⁺ potentiate NCX activity, while an increase in intracellular Na⁺ levels inhibits NCX via a mechanism known as Na⁺-dependent inactivation. Protons are also powerful inhibitors of NCX activity. By regulating NCX activity, Ca²⁺, Na⁺ and H⁺ couple cell metabolism to Ca²⁺ homeostasis and therefore cardiac contractility. This review summarizes the recent progress towards the understanding of the molecular mechanisms underlying the ionic regulation of the cardiac NCX with special emphasis on pH modulation and its physiological impact on the heart.     

A model for the generation of localized transient [Na] elevations in vascular smooth muscle

We present a stochastic computational model to study the mechanism of signaling between a source and a target ionic transporter, both localized on the plasma membrane (PM). In general this requires a nanometer-scale cytoplasmic space, or nanodomain, between the PM and a peripheral organelle to reflect ions back towards the PM. Specifically we investigate the coupling between Na⁺ entry via the transient receptor potential canonical channel 6 (TRPC6) and the Na⁺/Ca²⁺ exchanger (NCX), a process which is essential for reloading the sarcoplasmic reticulum (SR) via the sarco/endoplasmic reticulum Ca²⁺ATPase (SERCA) and maintaining Ca²⁺ oscillations in activated vascular smooth muscle. Having previously modeled the flow of Ca²⁺ between reverse NCX and SERCA during SR refilling, this quantitative approach now allows us to model the upstream linkage of Na⁺ entry through TRPC6 to reversal of NCX. We have implemented a random walk (RW) Monte Carlo (MC) model with simulations mimicking a diffusion process originating at the TRPC6 within PM-SR junctions. The model calculates the average Na⁺ in the nanospace and also produces profiles as a function of distance from the source. Our results highlight the necessity of a strategic juxtaposition of the relevant ion translocators as well as other physical structures within the nanospaces to permit adequate Na⁺ build-up to initiate NCX reversal and Ca²⁺ influx to refill the SR.     

Analysis of the Na+/Ca2+ Exchanger Gene Family within the Phylum Nematoda

Na⁺/Ca²⁺ exchangers are low affinity, high capacity transporters that rapidly transport calcium at the plasma membrane, mitochondrion, endoplasmic (and sarcoplasmic) reticulum, and the nucleus. Na⁺/Ca²⁺ exchangers are widely expressed in diverse cell types where they contribute homeostatic balance to calcium levels. In animals, Na⁺/Ca²⁺ exchangers are divided into three groups based upon stoichiometry: Na⁺/Ca²⁺ exchangers (NCX), Na⁺/Ca²⁺/K⁺ exchangers (NCKX), and Ca²⁺/Cation exchangers (CCX). In mammals there are three NCX genes, five NCKX genes and one CCX (NCLX) gene. The genome of the nematode Caenorhabditis elegans contains ten Na⁺/Ca²⁺ exchanger genes: three NCX; five CCX; and two NCKX genes. Here we set out to characterize structural and taxonomic specializations within the family of Na⁺/Ca²⁺ exchangers across the phylum Nematoda. In this analysis we identify Na⁺/Ca²⁺ exchanger genes from twelve species of nematodes and reconstruct their phylogenetic and evolutionary relationships. The most notable feature of the resulting phylogenies was the heterogeneous evolution observed within exchanger subtypes. Specifically, in the case of the CCX exchangers we did not detect members of this class in three Clade III nematodes. Within the Caenorhabditis and Pristionchus lineages we identify between three and five CCX representatives, whereas in other Clade V and also Clade IV nematode taxa we only observed a single CCX gene in each species, and in the Clade III nematode taxa that we sampled we identify NCX and NCKX encoding genes but no evidence of CCX representatives using our mining approach. We also provided re-annotation for predicted CCX gene structures from Heterorhabditis bacteriophora and Caenorhabditis japonica by RT-PCR and sequencing. Together, these findings reveal a complex picture of Na⁺/Ca²⁺ transporters in nematodes that suggest an incongruent evolutionary history of proteins that provide central control of calcium dynamics.     

Gel electrophoretic and density gradient analysis of the (K + Ca)-ATPase and the (Na + K)-ATPase activities of cardiac membrane vesicles

The two major ATPase activities of intact and leaky cardiac membrane vesicles (microsomes) were characterized with respect to ionic activation requirements. The predominant ATPase activity of intact vesicles was (K⁺ + Ca²⁺)-ATPase, an enzymic activity localized to sarcoplasmic reticulum, whereas the predominant ATPase activity of leaky, sodium dodecyl sulfate-pretreated vesicles was (Na⁺ + K⁺)-ATPase, an enzymic activity localized to sarcolemma. The (K⁺ + Ca²⁺)-ATPase activity was stimulated 4- to 5-fold by 100 mM K⁺ in the presence of 50 μM Ca²⁺. Phosphorylation of the (K⁺ + Ca²⁺)-ATPase of intact vesicles with [γ-³²P]ATP was Ca²⁺ dependent, and monovalent cations including K⁺ increased the level of [³²P]phosphoprotein by up to 50% when phosphorylation was measured at 5°C. After the intact vesicles were treated with SDS (0.30 mg/ml), (K⁺ + Ca²⁺)-ATPase was inactivated, as was Ca²⁺-dependent ³²P incorporation. The monovalent cation-stimulated ATPase activity of the particulate residue (SDS-extracted membrane vesicles) displayed the usual characteristics of ouabain-sensitive (Na⁺ + K⁺)-ATPase and the activity was increased 9- to 14-fold over the small amount of patent (Na⁺ + K⁺)-ATPase activity of intact membrane vesicles. ³²P incorporation by the (Na⁺ + K⁺)-ATPase of SDS-extracted vesicles was Na⁺ dependent, and Na⁺-stimulated incorporation was increased 7- to 9-fold over that of intact vesicles.Slab gel polyacrylamide electrophoresis of both intact and SDS-extracted crude vesicle preparations revealed at least 40 distinct Coomassie Blue-positive protein bands and provided evidence for a possible heterogeneous membrane origin of the vesicles. Periodic acid-Schiff staining of the gels revealed at least two major glycoproteins. Simultaneous electrophoresis of the ³²P-intermediates of the (K⁺ + Ca²⁺)-ATPase and the (Na⁺ + K⁺)-ATPase in the same gels did not resolve the two enzymes clearly. With sucrose gradient centrifugation of intact membrane vesicles, it was possible to physically resolve the two ATPase activities. Latent (Na⁺ + K⁺)-ATPase activity (unmasked by exposing the various fractions to SDS) was found in the higher regions of the gradient, whereas (K⁺ + Ca²⁺)-ATPase activity was primarily in the denser regions. A reasonable interpretation of the data is that cardiac microsomes consist of membrane vesicles derived both from sarcolemma and sarcoplasmic reticulum. (Na⁺ + K⁺)-ATPase is localized to intact vesicles of sarcolemma but is mainly latent, whereas (K⁺ + Ca²⁺)-ATPase is mostly patent and is localized to vesicles of sarcoplasmic reticulum.     

Oxygen free radicals and calcium homeostasis in the heart

Many experiments have been done to clarify the effects of oxygen free radicals on Ca²⁺ homeostasis in the hearts. A burst of oxygen free radicals occurs immediately after reperfusion, but we have to be reminded that the exact levels of oxygen free radicals in the hearts are yet unknown in both physiological and pathophysiological conditions. Therefore, we should give careful consideration to this point when we perform the experiments and analyze the results. It is, however, evident that Ca²⁺ overload occurs when the hearts are exposed to an excess amount of oxygen free radicals. Though ATP-independent Ca²⁺ binding is increased, Ca²⁺ influx through Ca²⁺ channel does not increase in the presence of oxygen free radicals. Another possible pathway through which Ca²⁺ can enter the myocytes is Na⁺?Ca²⁺ exchanger. Although, the activities of Na⁺?K⁺ ATPase and Na⁺?H⁺ exchange are inhibited by oxygen free radicals, it is not known whether intracellular Na⁺ level increases under oxidative stress or not. The question has to be solved for the understanding of the importance of Na⁺?Ca²⁺ exchange in Ca²⁺ influx process from extracellular space. Another question is ‘which way does Na⁺?Ca²⁺ exchange work under oxidative stress? Net influx or efflux of Ca^2+?’ Membrane permeability for Ca²⁺ may be maintained in a relatively early phase of free radical injury. Since sarcolemmal Ca²⁺-pump ATPase activity is depressed by oxygen free radicals, Ca²⁺ extrusion from cytosol to extracellular space is considered to be reduced. It has also been shown that oxygen free radicals promote Ca²⁺ release from sarcoplasmic reticulum and inhibit Ca²⁺ sequestration to sarcoplasmic reticulum. Thus, these changes in Ca²⁺ handling systems could cause the Ca²⁺ overload due to oxygen free radicals.     

The effects of KB-R7943, an inhibitor of reverse Na<Superscript>+</Superscript>/Ca<Superscript>2+</Superscript> exchange,on the force of contraction of papillary muscles in the heart of the ground squirrel <Emphasis Type="Italic">Spermophilus undulatus</Emphasis>

We investigated the effect of KB-R7943, an inhibitor of the reverse mode of Na⁺/Ca²⁺ exchanger, on the force of isometric contractions, the contractile force–frequency relationship and post-rest potentiation (a qualitative parameter of Ca²⁺ levels in sarcoplasmic reticulum) in the right ventricle papillary muscles isolated from ground squirrel hearts during summer (June, n = 4) and autumn (October, n = 4) activities. In the presence of 1.8 mM Ca2+at 36°C, 1–1.5 hours-long treatment of the summer papillary muscles with KB-R7943 produced no significant effects on the contractile indices at the majority of stimulation frequencies. In the autumn papillary muscles KB-R7943 induced a 40–50% decrease in the force of contraction (negative inotropic effect) at low stimulation frequencies (0.1–0.3 Hz) without any significant effect at higher stimulation frequencies (0.4–3.0 Hz). Furthermore, in this group, KB-R7943 suppressed the post-rest potentiation of contractility by 50 ± 21% at pause durations exceeding 120 s. These observations indicate that KB-R7943 can affect Ca²⁺ levels in sarcoplasmic reticulum and that Na⁺/Ca2⁺ exchange may contribute to the physiological remodeling of intracellular Ca²⁺ homeostasis in myocardium of hibernating animals prior their transition to a hypometabolic torpid state.     

Life after the birth of the mitochondrial Na<Superscript>+</Superscript>/Ca<Superscript>2+</Superscript> exchanger,NCLX

Powered by the mitochondrial membrane potential, Ca²⁺ permeates the mitochondria via a Ca²⁺ channel termed Ca²⁺ uniporter and is pumped out by a Na⁺/Ca²⁺ exchanger, both of which are located on the inner mitochondrial membrane. Mitochondrial Ca²⁺ transients are critical for metabolic activity and regulating global Ca²⁺ responses. On the other hand, failure to control mitochondrial Ca²⁺ is a hallmark of ischemic and neurodegenerative diseases. Despite their importance, identifying the uniporter and exchanger remains elusive and their inhibitors are non-specific. This review will focus on the mitochondrial exchanger, initially describing how it was molecularly identified and linked to a novel member of the Na⁺/Ca²⁺ exchanger superfamily termed NCLX. Molecular control of NCLX expression provides a selective tool to determine its physiological role in a variety of cell types. In lymphocytes, NCLX is essential for refilling the endoplasmic reticulum Ca²⁺ stores required for antigendependent signaling. Communication of NCLX with the store-operated channel in astroglia controls Ca²⁺ influx and thereby neuro-transmitter release and cell proliferation. The refilling of the Ca²⁺ stores in the sarcoplasmic reticulum, which is controlled by NCLX, determines the frequency of action potential and Ca²⁺ transients in cardiomyocytes. NCLX is emerging as a hub for integrating glucose-dependent Na⁺ and Ca²⁺ signaling in pancreatic β cells, and the specific molecular control of NCLX expression resolved the controversy regarding its role in neurons and β cells. Future studies on an NCLX knockdown mouse model and identification of human NCLX mutations are expected to determine the role of mitochondrial Ca²⁺ efflux in organ activity and whether NCLX inactivation is linked to ischemic and/or neurodegenerative syndromes. Structure-function analysis and protein analysis will identify the NCLX mode of regulation and its partners in the inner membrane of the mitochondria.     

Allosteric Activation of Na-Ca Exchange by L-Type Ca Current Augments the Trigger Flux for SR Ca Release in Ventricular Myocytes

The possible contribution of Na⁺-Ca²⁺ exchange to the triggering of Ca²⁺ release from the sarcoplasmic reticulum in ventricular cells remains unresolved. To gain insight into this issue, we measured the “trigger flux” of Ca²⁺ crossing the cell membrane in rabbit ventricular myocytes with Ca²⁺ release disabled pharmacologically. Under conditions that promote Ca²⁺ entry via Na⁺-Ca²⁺ exchange, internal [Na⁺] (10 mM), and positive membrane potential, the Ca²⁺ trigger flux (measured using a fluorescent Ca²⁺ indicator) was much greater than the Ca²⁺ flux through the L-type Ca²⁺ channel, indicating a significant contribution from Na⁺-Ca²⁺ exchange to the trigger flux. The difference between total trigger flux and flux through L-type Ca²⁺ channels was assessed by whole-cell patch-clamp recordings of Ca²⁺ current and complementary experiments in which internal [Na⁺] was reduced. However, Ca²⁺ entry via Na⁺-Ca²⁺ exchange measured in the absence of L-type Ca²⁺ current was considerably smaller than the amount inferred from the trigger flux measurements. From these results, we surmise that openings of L-type Ca²⁺ channels increase [Ca²⁺] near Na⁺-Ca²⁺ exchanger molecules and activate this protein. These results help to resolve seemingly contradictory results obtained previously and have implications for our understanding of the triggering of Ca²⁺ release in heart cells under various conditions.     

Monovalent ion and calcium ion fluxes in sarcoplasmic reticulum

Summary The ion permeability of sarcoplasmic reticulum vesicles from skeletal and heart muscle has been characterized by radioisotope flux, osmotic and membrane potential measurements, and by incorporating vesicles into planar phospholipid bilayers. The sarcoplasmic reticulum membrane is uniquely permeable to various biologically relevant monovalent ions. At least two and possibly three separate passive permeation systems for monovalent ions have been identified: 1) a K⁺, Na⁺ channel, 2) an anion channel, and 3) a H⁺ (OH^–) permeable pathway which may or may not be synonymous with the anion channel. A possible physiological function of these monovalent ion permeation systems is to permit rapid movement of K⁺, Na⁺, H⁺ and Cl across the membrane to counter electrogenic Ca²⁺ fluxes during Ca²⁺ release and uptake by sacroplasmic reticulum.     

Sodium-calcium ion exchange in skeletal muscle sarcolemmal vesicles

Summary The Ca²⁺ permeability of rabbit skeletal muscle sarcolemmal vesicles was investigated by means of radioisotope flux measurements. A membrane vesicle fraction highly enriched in sarcolemma, as revealed by enzymatic markers, was obtained from the 22–27% region of sucrose gradients after isopycnic centrifugation. The ability of sarcolemmal vesicles to exchange Na⁺ for Ca²⁺ was investigated by measuring Ca²⁺ influx into and efflux from sarcolemmal vesicles in the presence and absence of a Na⁺ gradient. It was found that Ca²⁺ movements were enhanced in the direction of the higher Na⁺ concentration. When intra- and extravesicular Na⁺ concentrations were high, Na⁺–Na⁺ exchange predominated and Na⁺–Ca²⁺ exchange was low or absent. The presence of the Ca²⁺ ionophore A23187 in the dilution medium resulted in the rapid release of Ca²⁺ and the elimination of the Na⁺-enhanced efflux of Ca²⁺, suggesting that internal rather than bound external Ca²⁺ was exchanged with Na⁺. La³⁺ abolished Na⁺–Ca²⁺ exchange and decreased overall membrane permeability. Na⁺–Ca²⁺ exchange was not due to sarcoplasmic reticulum or mitochondrial contaminants. This investigation suggests that skeletal muscle, like cardiac muscle and neurons, is capable of a transmembranous Na⁺–Ca²⁺ exchange.     

Extracellular calcium depletion transiently elevates oxygen consumption in neurosecretory PC12 cells through activation of mitochondrial Na/Ca exchange

Fluctuating extracellular Ca²⁺ regulates many aspects of neuronal (patho)physiology including cell metabolism and respiration. Using fluorescence-based intracellular oxygen sensing technique, we demonstrate that depletion of extracellular Ca²⁺ from 1.8 to ≤ 0.6 mM by chelation with EGTA induces a marked spike in O₂ consumption in differentiated PC12 cells. This respiratory response is associated with the reduction in cytosolic and mitochondrial Ca²⁺, minor depolarization on the mitochondrial membrane, moderate depolarization of plasma membrane, and no changes in NAD(P)H and ATP. The response is linked to the influx of extracellular Na⁺ and the subsequent activation of mitochondrial Na⁺/Ca²⁺ and Na⁺/H⁺ exchange. The mitochondrial Na⁺/Ca²⁺ exchanger (_mNCX) activated by Na⁺ influx reduces Ca²⁺ and increases Na⁺ levels in the mitochondrial matrix. The excess of Na⁺ activates the mitochondrial Na⁺/H⁺ exchanger (NHE) increasing the outward pumping of protons, electron transport and O₂ consumption. Reduction in extracellular Na⁺ and inhibition of Na⁺ influx through the receptor operated calcium channels and plasmalemmal NHE reduce the respiratory response. Inhibition of the _mNCX, L-type voltage gated Ca²⁺ channels or the release of Ca²⁺ from the endoplasmic reticulum also reduces the respiratory spike, indicating that unimpaired intercompartmental Ca²⁺ exchange is critical for response development.     

An electrogenic Na+/Ca2+ antiporter in addition to the Ca2+ pump in cardiac sarcolemma

Vesicles isolated from rat heart, particularly enriched in sarcolemma markers, were examined for their sidedness by investigation of side-specific interactions of modulators with the asymmetric (Na⁺ + K⁺)-ATPase and adenylate cyclase complex. The membrane preparation with the properties expected for inside-out vesicles showed the highest rate of ATP-driven Ca²⁺ transport. The Ca²⁺ pump was stimulated 1.7- and 2.1-fold by external Na⁺ and K⁺, respectively, the half-maximal activation occurring at 35 mM monovalent cation concentration. In vesicles loaded with Ca²⁺ by pump action in a medium containing 160 mM KCl, a slow spontaneous release of Ca²⁺ started after 2 min. The rate of this release could be dramatically increased by the addition of 40 mM NaCl to the external medium. In contrast, 40 mM KCl exerted no appreciable effect on vesicles loaded with Ca²⁺ in a medium containing 160 mM NaCl. Ca²⁺ movements were also studied in the absence of ATP and Mg²⁺. Vesicles containing an outwardly directed Na⁺ gradient showed the highest Ca²⁺ uptake activity. These findings suggested the operation of a Ca²⁺/Na⁺ antiporter in addition to the active Ca²⁺ pump in these sarcolemmal vesicles. A valinomycin-induced inward K⁺-diffusion potential stimulated the Na⁺- Ca²⁺ exchange, suggesting its electrogenic nature. If in the absence of ATP and Mg²⁺ the transmembrane Na_i⁺/Na_o⁺ gradient exceeded 160/15 mM concentrations, Ca²⁺ uptake could be stimulated by the addition of 5 mM oxalate, indicating Na⁺ gradient-induced Ca²⁺ uptake to be a translocation of Ca²⁺ to the lumen of the vesicle. A sarcoplasmic reticulum contamination, removed by further sucrose gradient fractionation, contained rather low Na⁺-Ca²⁺ exchange activity. This result suggests that the activity can be entirely accounted for by the sarcolemmal content of the cardiac membrane preparation.