Functional expression of the human cardiac Na+/Ca2+ exchanger in Sf9 cells: rapid and specific Ni2+ transport

Although inhibition of the Na+/Ca2+ exchanger normally increases [Ca2+]i in neonatal cardiac myocytes, application of the inhibitor Ni2+ appears to reduce [Ca2+] measured by fluo-3. To investigate how the apparent reduction in [Ca2+]i occurs we examined Ca2+ transport by the human Na+/Ca2+ exchanger expressed in Sf9 cells. Transport of Ca2+ by the Na+/Ca2+ exchanger was examined using a laser-scanning confocal microscope and the fluorescent Ca2+ indicator fluo-3, and the electrogenic function was determined by measuring the Na+/Ca2+ exchange current (INaCa) using patch clamp methods. INaCa was elicited with voltage-clamp steps or flash photolysis of caged Ca2+. We show significant expression of Na+/Ca2+ exchanger function in Sf9 cells infected with a recombinant Baculovirus carrying the Na+/Ca2+ exchanger. In addition to measurements of INaCa, characterization includes Ca2+ transport via the Na+/Ca2+ exchanger and the voltage dependence of Ca2+ transport. Application of Ni2+ blocked INaCa but, contrary to expectation, decreased fluo-3 fluorescence. Experiments with infected Sf9 cells suggested that Ni2+ was transported via the Na+/Ca2+ exchanger at a rate comparable to the Ca2+ transport. Once inside the cells, Ni2+ reduced fluorescence, presumably by quenching fluo-3. We conclude that Ni2+ does indeed block INaCa, but is also rapidly translocated across the cell membrane by the Na+/Ca2+ exchanger itself, most likely via an electroneutral partial reaction of the exchange cycle.     

Acidosis facilitates spontaneous sarcoplasmic reticulum Ca2+ release in rat myocardium

Previous studies have shown that acidosis increases myoplasmic [Ca2+] (Cai). We have investigated whether this facilitates spontaneous sarcoplasmic reticulum (SR) Ca2+ release and its functional sequelae. In unstimulated rat papillary muscles, exposure to an acid solution (produced by increasing the [CO2] of the perfusate from 5 to 20%) caused a rapid increase in the mean tissue Cai, as measured by the photoprotein aequorin. This was paralleled by an increase in spontaneous microscopic tissue motion caused by localized Ca2+ myofilament interactions, as monitored in fluctuations in the intensity of laser light scattered by the muscle. In regularly stimulated muscles, acidosis increased the size of the Ca2+ transient associated with each contraction and caused the appearance of Cai oscillations in the diastolic period. In unstimulated single myocytes, acidosis depolarized the resting membrane potential by approximately 5 mV and enhanced the frequency of spontaneous contractile waves. The small sarcolemmal depolarization associated with each contractile wave increased and occasionally initiated spontaneous action potentials. In regularly stimulated myocytes, acidosis caused de novo spontaneous contractile waves between twitches; these waves were associated with a decrease in the amplitude of the subsequent stimulated twitch. Ryanodine (2 microM) abolished all evidence of spontaneous Ca2+ release during acidosis, markedly reduced the acidosis-induced increase in aequorin light, and reduced resting tension. We conclude that acidosis increases the likelihood for the occurrence of spontaneous SR Ca2+ release, which can cause spontaneous action potentials, increase resting tension, and negatively affect twitch tension.     

Effects of impaired Ca2+ homeostasis on contraction in postinfarction myocytes.

The significance of altered Ca2+ influx and efflux pathways on contractile abnormalities of myocytes isolated from rat hearts 3 wk after myocardial infarction (MI) was investigated by varying extracellular Ca2+ concentration ([Ca2+]o, 0.6-5.0 mM) and pacing frequency (0.1-5.0 Hz). Myocytes isolated from 3-wk MI hearts were significantly longer than those from sham-treated (Sham) hearts (125 +/- 1 vs. 114 +/- 1 micrometer, P < 0.0001). At high [Ca2+]o and low pacing frequency, conditions that preferentially favored Ca2+ influx over efflux, Sham myocytes shortened to a greater extent than 3-wk MI myocytes. Conversely, under conditions that favored Ca2+ efflux (low [Ca2+]o and high pacing frequency), MI myocytes shortened more than Sham myocytes. At intermediate [Ca2+]o and pacing frequencies, differences in steady-state contraction amplitudes between Sham and MI myocytes were no longer significant. Collectively, the interpretation of these data was that Ca2+ influx and efflux pathways were subnormal in MI myocytes and that they contributed to abnormal cellular contractile behavior. Because Na+/Ca2+ exchange activity, but not whole cell Ca2+ current, was depressed in 3-wk MI rat myocytes, our results on steady-state contraction are consistent with, but not proof of, the hypothesis that depressed Na+/Ca2+ exchange accounted for abnormal contractility in MI myocytes. The effects of depressed Na+/Ca2+ exchange on MI myocyte mechanical activity were further evaluated in relaxation from caffeine-induced contractures. Because Ca2+ uptake by sarcoplasmic reticulum was inhibited by caffeine and with the assumption that intracellular Na+ and membrane potential were similar between Sham and MI myocytes, myocyte relaxation from caffeine-induced contracture can be taken as an estimate of Ca2+ extrusion by Na+/Ca2+ exchange. In MI myocytes, in which Na+/Ca2+ exchange activity was depressed, the half time of relaxation (1.54 +/- 0.14 s) was significantly (P < 0.02) prolonged compared with that measured in Sham myocytes (1.10 +/- 0.10 s).     

Antisense inhibition of Na+/Ca2+ exchange during anoxia/reoxygenation in ventricular myocytes

This study investigated the role of the Na+/Ca2+ exchanger (NCX) in regulating cytosolic intracellular Ca2+ concentration ([Ca2+]i) during anoxia/reoxygenation in guinea pig ventricular myocytes. The hypothesis that the NCX is the predominant mechanism mediating [Ca2+]i overload in this model was tested through inhibition of NCX expression by an antisense oligonucleotide. Immunocytochemistry revealed that this antisense oligonucleotide, directed at the area around the start site of the guinea pig NCX1, specifically reduced NCX expression in cultured adult myocytes by 90 +/- 4%. Antisense treatment inhibited evoked NCX activity by 94 +/- 3% and decreased the rise in [Ca2+]i during anoxia/reoxygenation by 95 +/- 3%. These data suggest that NCX is the predominant mechanism mediating Ca2+ overload during anoxia/reoxygenation in guinea-pig ventricular myocytes.     

Modulation of agonist-induced Ca2+ release by SR Ca2+ load: direct SR and cytosolic Ca2+ measurements in rat uterine myocytes

Release of Ca2+ from sarcoplasmic reticulum (SR) is one of the most important mechanisms of smooth muscle stimulation by a variety of physiologically active substances. Agonist-induced Ca2+ release is considered to be dependent on the Ca2+ content of the SR, although the mechanism underlying this dependence is unclear. In the present study, the effect of SR Ca2+ load on the amplitude of [Ca2+]i transients elicited by application of the purinergic agonist ATP was examined in uterine smooth muscle cells isolated from pregnant rats. Measurement of intraluminal Ca2+ level ([Ca2+]L) using a low affinity Ca indicator, mag-fluo-4, revealed that incubation of cells in a high-Ca2+ (10 mM) extracellular solution leads to a substantial increase in [Ca2+]L (SR overload). However, despite increased SR Ca2+ content this did not potentiate ATP-induced [Ca2+]i transients. Repetitive applications of ATP in the absence of extracellular Ca2+, as well as prolonged incubation in Ca2+-free solution without agonist, depleted the [Ca2+]L (SR overload). In contrast to overload, partial depletion of the SR substantially reduced the amplitude of Ca2+ release. ATP-induced [Ca2+]i transients were completely abolished when SR Ca2+ content was decreased below 80% of its normal value indicating a steep dependence of the IP3-mediated Ca2+ release on the Ca2+ load of the store. Our results suggest that in uterine smooth muscle cells decrease in the SR Ca2+ load below its normal resting level substantially reduces the IP3-mediated Ca2+ release, while Ca2+ overload of the SR has no impact on such release.     

Overexpression of Na+/Ca2+ exchanger alters contractility and SR Ca2+ content in adult rat myocytes

The functional consequences of overexpression of rat heart Na+/Ca2+ exchanger (NCX1) were investigated in adult rat myocytes in primary culture. When maintained under continued electrical field stimulation conditions, cultured adult rat myocytes retained normal contractile function compared with freshly isolated myocytes for at least 48 h. Infection of myocytes by adenovirus expressing green fluorescent protein (GFP) resulted in >95% infection as ascertained by GFP fluorescence, but contraction amplitude at 6-, 24-, and 48-h postinfection was not affected. When they were examined 48 h after infection, myocytes infected by adenovirus expressing both GFP and NCX1 had similar cell sizes but exhibited significantly altered contraction amplitudes and intracellular Ca2+ concentration ([Ca2+]i) transients, and lower resting and diastolic [Ca2+]i when compared with myocytes infected by the adenovirus expressing GFP alone. The effects of NCX1 overexpression on sarcoplasmic reticulum (SR) Ca2+ content depended on extracellular Ca2+ concentration ([Ca2+]o), with a decrease at low [Ca2+]o and an increase at high [Ca2+]o. The half-times for [Ca2+]i transient decline were similar, suggesting little to no changes in SR Ca2+-ATPase activity. Western blots demonstrated a significant (P < or = 0.02) threefold increase in NCX1 but no changes in SR Ca2+-ATPase and calsequestrin abundance in myocytes 48 h after infection by adenovirus expressing both GFP and NCX1 compared with those infected by adenovirus expressing GFP alone. We conclude that overexpression of NCX1 in adult rat myocytes incubated at high [Ca2+]o resulted in enhanced Ca2+ influx via reverse NCX1 function, as evidenced by greater SR Ca2+ content, larger twitch, and [Ca2+]i transient amplitudes. Forward NCX1 function was also increased, as indicated by lower resting and diastolic [Ca2+]i.     

Expression of active p21-activated kinase-1 induces Ca2+ flux modification with altered regulatory protein phosphorylation in cardiac myocytes

p21-Activated kinase-1 (Pak1) is a serine-threonine kinase that associates with and activates protein phosphatase 2A in adult ventricular myocytes and, thereby, induces increased Ca2+ sensitivity of skinned-fiber tension development mediated by dephosphorylation of myofilament proteins (Ke Y, Wang L, Pyle WG, de Tombe PP, Solaro RJ. Circ Res 94: 194-200, 2004). We test the hypothesis that activation of Pak1 also moderates cardiac contractility through regulation of intracellular Ca2+ fluxes. We found no difference in field-stimulated intracellular Ca2+ concentration ([Ca2+]i) transient amplitude and extent of cell shortening between myocytes expressing constitutively active Pak1 (CA-Pak1) and controls expressing LacZ; however, time to peak shortening was significantly faster and rate of [Ca2+]i decay and time of relengthening were slower. Neither caffeine-releasable sarcoplasmic reticulum (SR) Ca2+ content nor fractional release was different in CA-Pak1 myocytes compared with controls. Isoproterenol application revealed a significantly blunted increase in [Ca2+]i transient amplitude, as well as a slowed rate of [Ca2+]i decay, increased SR Ca2+ content, and increased cell shortening, in CA-Pak1 myocytes. We found no significant change in phospholamban phosphorylation at Ser16 or Thr17 in CA-Pak1 myocytes. Analysis of cardiac troponin I revealed a significant reduction in phosphorylated species that are primarily attributable to Ser(23/24) in CA-Pak1 myocytes. Nonstimulated, spontaneous SR Ca2+ release sparks were significantly smaller in amplitude in CA-Pak1 than LacZ myocytes. Propagation of spontaneous Ca2+ waves resulting from SR Ca2+ overload was significantly slower in CA-Pak1 myocytes. Our data indicate that CA-Pak1 expression has significant effects on ventricular myocyte contractility through altered myofilament Ca2+ sensitivity and modification of the [Ca2+]i transient.     

The mitochondrial Na+/Ca2+ exchanger plays a key role in the control of cytosolic Ca2+ oscillations

There is increasing evidence that mitochondria play an important role in the control of cytosolic Ca2+ signaling. We show here that the main mitochondrial Ca2+-exit pathway, the mitochondrial Na+/Ca2+ exchanger, controls the pattern of cytosolic Ca2+ oscillations in non-excitable cells. In HeLa cells, the inhibitor of the mitochondrial Na+/Ca2+ exchanger CGP37157 changed the pattern of the oscillations induced by histamine from a high-frequency irregular one to a lower frequency baseline spike type, surprisingly with little changes in the average Ca2+ values of a large cell population. In human fibroblasts, CGP37157 increased the frequency of the baseline oscillations in cells having spontaneous activity and induced the generation of oscillations in cells without spontaneous activity. This effect was dose-dependent, disappeared when the inhibitor was washed out and was not mimicked by mitochondrial depolarization. CGP37157 increased mitochondrial [Ca2+] and ATP production in histamine-stimulated HeLa cells, but the effect on ATP production was only transient. CGP37157 also activated histamine-induced Ca2+ release from the endoplasmic reticulum and increased the size of the cytosolic Ca2+ peak induced by histamine in HeLa cells. Our results suggest that the mitochondrial Na+/Ca2+ exchanger directly modulates inositol 1,4,5-trisphosphate-induced Ca2+ release and in that way controls cytosolic Ca2+ oscillations.     

Synaptosomal [Ca2+]i as influenced by Na+/Ca2+ exchange and K+ depolarization.

The modulation of the intrasynaptosomal concentration of Ca2+, [Ca2+]i, by Na+/Ca2+ exchange was studied using Indo-1 fluorescence. The electrochemical gradient of Na+ was manipulated by substituting Li+ or choline for Na+ in the external medium and, then, the influx of 45Ca2+ and the [Ca2+]i were measured. It was found that the increase in [Ca2+]i induced by K+ depolarization is lower if the value of [Ca2+]i has been previously raised by Na+/Ca2+ exchange, suggesting that Ca2+ entering by Na+/Ca2+ exchange reduces the Ca2+ entering by voltage-dependent calcium channels. Our results show that a value of [Ca2+]i of about 650 nM induced by Na+/Ca2+ exchange reduces by 50% the Ca2+ entering due to K+ depolarization and no Ca2+ enters through the channels if the [Ca2+]i is previously raised above about 800 nM. Furthermore, predepolarization of the synaptosomes in a Ca-free medium also inhibits by at least 40% the [Ca2+]i rise through Ca2+ channels. Thus, the results suggest that both predepolarization and [Ca2+]i rise due to Na+/Ca2+ exchange decrease the Ca2+ entering by voltage-sensitive Ca2+ channels. The Ca2+ entering by Na+/Ca2+ exchange might contribute to the regulation of neurotransmitter release. Our results also show that the presence of Li+ in the external medium decreases the buffering capacity of synaptosomes, probably by releasing Ca2+ from mitochondria by Li+/Ca2+ exchange.     

Effects of antisense oligonucleotides to the cardiac Na+/Ca2+ exchanger on calcium dynamics in cultured cardiac myocytes.

The present study was designed to explore the role of the Na+/Ca2+ exchanger on spontaneous beating of cultured cardiac myocytes. Antisense oligonucleotides (AS) based on the sequence of the cardiac Na+/Ca2+ exchanger were used to decrease expression of this Ca2+ transporting protein in cardiac myocytes. An application of AS (10 microM) caused an increase in beating rate of myocytes within 6-24 h. After 24 h of exposure, AS increased the beating rate from an average rate of 77 beats/min in control and sense-treated myocytes to 103 beats/min. Moreover, myocytes treated for 24 h with 10 microM AS exhibited an increase in diastolic [Ca2+]i levels. The antisense treatment also led to a approximately 20% decrease in expression of Na+/Ca2+ exchanger proteins within 6-24 h. Changes in mRNA levels following AS treatment could not be detected within 3- to 24-h periods. The results of these studies suggest that the Na+/Ca2+ exchanger plays a potentiating role in spontaneous the beating process by regulating [Ca2+]i dynamics and that even a small reduction in the levels of the exchanger protein has marked effects on the handling of [Ca2+]i during the cardiac cycle.     

Propagation of Ca2+ release in cardiac myocytes: role of mitochondria

Factors contributing to "local control" of Ca2+ release in cardiac myocytes are incompletely understood. We induced local release of Ca2+ by regional exposure of mouse atrial and ventricular myocytes to 10mM caffeine for 500 ms using a rapid solution switcher. Propagation of Ca2+ release was imaged by means of a Nipkow confocal microscope, and fluo-3. Under physiologic conditions, a local release of Ca2+ propagated in atrial myocytes, not in ventricular myocytes. Inhibition of SR Ca2+ uptake (500 nM thapsigargin), and of Ca2+ extrusion via Na/Ca exchange (5mM Ni2+), did not result in propagation in ventricular myocytes. The density of mitochondria was greater in ventricular than in atrial myocytes, although the abundance of ryanodine receptors and myofilaments was similar. Partial inhibition of Ca2+ uptake via the mitochondrial Ca2+ uniporter (5 microM Ru360) caused an increase in the [Ca2+]i transient in paced ventricular myocytes, and consistently resulted in propagation of Ca2+ release. This effect of Ru360 did not appear to be due to altered SR Ca2+ content. These data indicate that Ca2+ uptake via the mitochondrial uniporter occurs on a beat-to-beat basis, and may contribute to local control of Ca2+ release. Propagation of Ca2+ release in atrial myocytes may result in part from the relatively low density of mitochondria present.     

Ca2+ channel-sarcoplasmic reticulum coupling: a mechanism of arterial myocyte contraction without Ca2+ influx

Contraction of vascular smooth muscle cells (VSMCs) depends on the rise of cytosolic [Ca2+] owing to either Ca2+ influx through voltage-gated Ca2+ channels of the plasmalemma or receptor-mediated Ca2+ release from the sarcoplasmic reticulum (SR). We show that voltage-gated Ca2+ channels in arterial myocytes mediate fast Ca2+ release from the SR and contraction without the need of Ca2+ influx. After sensing membrane depolarization, Ca2+ channels activate G proteins and the phospholipase C-inositol 1,4,5-trisphosphate (InsP3) pathway. Ca2+ released through InsP3-dependent channels of the SR activates ryanodine receptors to amplify the cytosolic Ca2+ signal. These observations demonstrate a new mechanism of signaling SR Ca(2+)-release channels and reveal an unexpected function of voltage-gated Ca2+ channels in arterial myocytes. Our findings may have therapeutic implications as the calcium-channel-induced Ca2+ release from the SR can be suppressed by Ca(2+)-channel antagonists.     

Ca2+ release from the sarcoplasmic reticulum activated by the low affinity Ca2+ chelator TPEN in ventricular myocytes

The Ca2+ content of the sarcoplasmic reticulum (SR) of cardiac myocytes is thought to play a role in the regulation and termination of SR Ca2+ release through the ryanodine receptors (RyRs). Experimentally altering the amount of Ca2+ within the SR with the membrane-permeant low affinity Ca2+ chelator TPEN could improve our understanding of the mechanism(s) by which SR Ca2+ content and SR Ca2+ depletion can influence Ca2+ release sensitivity and termination. We applied laser-scanning confocal microscopy to examine SR Ca2+ release in freshly isolated ventricular myocytes loaded with fluo-3, while simultaneously recording membrane currents using the whole-cell patch-clamp technique. Following application of TPEN, local spontaneous Ca2+ releases increased in frequency and developed into cell-wide Ca2+ waves. SR Ca2+ load after TPEN application was found to be reduced to about 60% of control. Isolated cardiac RyRs reconstituted into lipid bilayers exhibited a two-fold increase of their open probability. At the low concentration used (20-40microTPEN did not significantly inhibit the SR-Ca2+-ATPase in SR vesicles. These results indicate that TPEN, traditionally used as a low affinity Ca2+ chelator in intracellular Ca2+ stores, may also act directly on the RyRs inducing an increase in their open probability. This in turn results in an increased Ca2+ leak from the SR leading to its Ca2+ depletion. Lowering of SR Ca2+ content may be a mechanism underlying the recently reported cardioprotective and antiarrhythmic features of TPEN.     

Differential segmental activation of Ca2+ -dependent CI-and K+ channels in pulmonary arterial myocytes

Differential segmental distribution of electrophysiologically distinct myocytes helps to explain the variability of the pulmonary arteries to vasoactive agents. We have studied whether Ca2+ -dependent CI- (CICa) and K+ (KCa) channels are activated differentially in enzymatically dispersed conduit and resistance myocytes. We measured cytosolic [Ca2+] and the changes of membrane current and potential elicited by spontaneous or agonist-induced Ca2+ oscillations. Conduit arteries contained a heterogeneous cell population with a variable mixture of KCa and CICa conductances. Resistance arteries contained a more homogeneous cell population with predominance of CICa channel activation. The relation between KCa and CICa conductances in a given conduit myocyte determines the size of the V(m)change in response to a rise of cytosolic [Ca2+]. Conduit myocytes tend to hyperpolarize towards the K+ equilibrium potential (approximately - 90 m V). In resistance myocytes, release of Ca2+ from stores activates CI Cachannels and brings Vm to a value close to the chloride equilibrium potential (approximately - 20 or - 30 m V) thus favouring opening of Ca2+ channels and Ca2+ influx. In resistance vessels CICachannels contribute to link agonist-induced Ca2+ release from stores and membrane depolarization, thus permitting protracted vasoconstriction.     

A mechanism of Ca2+ release from Ca2+ stores coupling to the Na+/Ca2+ exchanger in cultured smooth muscle cells.

We previously observed Ca2+ release from intracellular Ca2+ stores caused by reduction in extracellular Na+ concentration ([Na+]o). The purpose of this study was to determine whether lowering [Na+]o can elicit Ca2+ release from Ca2+ stores via the Na+/Ca2+ exchanger and to elucidate the mechanisms related to the Ca2+ release pathway in cultured longitudinal smooth muscle cells obtained from guinea pig ileum. Low [Na+]o-induced Ca2+ release was inhibited by antisense oligodeoxynucleotides for Na+/Ca2+ exchanger type 1 (anti-NCX). Application of anti-NCX to cells attenuated both the number of Ca2+ responding cells and the expression of the exchanger. Moreover, microinjection of heparin, a blocker of inositol 1,4,5-trisphosphate (IP3) receptors, into the cells inhibited low [Na+]o-induced Ca2+ release. These findings suggest that low [Na+]o-induced Ca2+ release occurs through an IP3-induced Ca2+ release mechanism due to changes in the Ca2+ flux regulated by the Na+/Ca2+ exchanger.     

Influence of age and run training on cardiac Na+/Ca2+ exchange.

Effects of age and training on myocardial Na+/Ca2+ exchange were examined in young sedentary (YS; 14-15 mo), aged sedentary (AS; 27-31 mo), and aged trained (AT; 8- to 11-wk treadmill run training) male Fischer Brown Norway rats. Whole heart performance and isolated cardiocyte Na+/Ca2+ exchange characteristics were measured. At the whole heart level, a small but significant slowing of late isovolumic left ventricular (LV) relaxation, which may be indicative of altered Na+/Ca2+ exchange activity, was seen in hearts from AS rats. This subtle impairment in relaxation was not observed in hearts from AT rats. At the single-cardiocyte level, late action potential duration was prolonged, resting membrane potential was more positive, and overshoot potential was greater in cardiocytes from AS rats than from YS rats (P < 0.05). Training did not influence any of these age-related action potential characteristics. In electrically paced cardiocytes, neither shortening nor intracellular Ca2+ concentration ([Ca2+]i) dynamics was influenced by age or training. Similarly, neither age nor training influenced the rate of [Ca2+]i clearance via forward (Nain+ /Caout2+) Na+/Ca2+ exchange after caffeine-induced Ca2+ release from the sarcoplasmic reticulum or cardiac Na+/Ca2+ exchanger protein (NCX1) expression. However, when whole cell patch-clamp techniques combined with fluorescence microscopy were used to evaluate the ability of Na+/Ca2+ exchange to alter cytosolic [Ca2+] ([Ca2+]c) under conditions where membrane potential (Vm) and internal and external [Na+] and [Ca2+] could be controlled, we observed age-associated increases in forward Na+/Ca2+ exchange-mediated [Ca2+]c clearance (P < 0.05) that were not influenced by training. The age-related increase in forward Na+/Ca2+ exchange activity provides a hypothetical explanation for the late action potential prolongation observed in this study.     

Axial stretch enhances sarcoplasmic reticulum Ca2+ leak and cellular Ca2+ reuptake in guinea pig ventricular myocytes: experiments and models

Cardiac cellular calcium (Ca2+) handling is the well-investigated mediator of excitation-contraction coupling, the process that translates cardiac electrical activation into mechanical events. The reverse--effects of mechanical stimulation on cardiomyocyte Ca2+ handling--are much less well understood, in particular during the inter-beat period, called 'diastole'. We have investigated the effects of diastolic length changes, applied axially using a pair of carbon fibres attached to opposite ends of Guinea pig isolated ventricular myocytes, on the availability of Ca2+ in the main cellular stores (the sarcoplasmic reticulum; SR), by studying the rest-decay of SR Ca2+ content [Ca2+]SR, and the reloading of the SR after prior depletion of Ca2+ from the cell. Cells were loaded with Fura-2 AM (an indicator of the cytosolic 'free' Ca2+ concentration, [Ca2+]i), and pre-conditioned by field-stimulation (2 Hz) at 37 degrees C, while [Ca2+]i transients and sarcomere length (SL) were recorded simultaneously. After reaching a steady state in the behaviour of observed parameters, stimulation was interrupted for between 5 and 60s, while cells were either held at resting length, or stretched (controlled to cause a 10% increase in SL, to aid inter-individual comparison). Thereafter, each cell was returned to its original resting length, followed by swift administration of 10mM of caffeine (in Na+/Ca2+-free solution), which causes the release of Ca2+ from the SR (caffeine), but largely prevents extrusion of Ca2+ from the cytosol to the cell exterior (Na+/Ca2+-free solution). By comparing the [Ca2+]i in cells exposed/not exposed to diastolic stretch of different duration, we assessed the rest-decay dynamics of [Ca2+]SR. To assess SR reloading after initial Ca2+ depletion, the same stretch protocol was implemented after prior emptying of the cell by application of 10mM of caffeine in normal Tyrode solution (which causes Ca2+ to be released from the SR and extruded from the cell via the Na+/Ca2+ exchanger; NCX). Axial stretch enhanced the rate of both rest-decay and reloading of [Ca2+]SR. Application of 40 microM streptomycin, a blocker of stretch-activated ion channels, did not affect the stretch-induced increase in SR reloading. This behaviour was reproduced in a computer simulation study, using a modified version of the 2006 Iribe-Kohl-Noble model of single cardiac myocyte Ca2+ handling, suggesting that stretch increases both Ca2+ leak from the SR and Ca2+ influx via the sarcolemma. This may have important implications for the mobilisation of Ca2+ in stretched cells, and could contribute to the regional 'matching' of individual cardiomyocyte contractility to dynamic, and regionally varying, changes in mechanical loads, such as diastolic pre-load, of cardiac tissue.     

Effects of ryanodine on intracellular Ca2+ transients in mammalian cardiac muscle

We observed the effects of ryanodine on the aequorin luminescence, membrane potential, and contraction of canine cardiac Purkinje fibers and ferret ventricular muscle. In canine Purkinje fibers, ryanodine (10 nM to 1 microM) abolished the spontaneous spatiotemporal fluctuations in [Ca2+] that occur as a result of Ca2+-induced Ca2+ release from the sarcoplasmic reticulum (SR) during exposure to low-Na+ solutions. Ryanodine strongly reduced the twitch and both components of the intracellular aequorin luminescence signal (L1 and L2), which normally accompanies contraction. The small luminescence signals that remained in ryanodine could be abolished by a Ca2+ channel blocker (nitrendipine, 10 microM). The plateau phase of the action potential was reduced by nitrendipine in the presence of ryanodine, which suggests that Ca2+ current was not blocked by ryanodine. In ferret ventricular tissue, ryanodine (1 microM) prolonged the action potential and reduced the peak amplitudes of both the aequorin transient and the twitch, while greatly prolonging the time-to-peak of both signals. Increases in extracellular [Ca2+] restored the peak amplitudes of the twitch and the aequorin luminescence, but did not restore the normal time-to-peak. The results show that in both tissues, the negative inotropic effect of ryanodine is due to the reduction of the intracellular [Ca2+] transient. Inasmuch as neither Ca2+ entry via surface membrane Ca2+ channels nor Na+-Ca2+ exchange appears to be blocked by ryanodine, the most probable cause of reduction of the [Ca2+] transient is an inhibition of Ca2+ release by the SR.     

Na+/Ca2+ exchange in plasma membrane vesicles from a glucose-responsive insulinoma.

Plasma membrane vesicles from a glucose-responsive insulinoma exhibited properties consistent with the presence of a membrane Na+/Ca2+ exchange. The exchange was rapid, reversible, and was dependent on the external Ca2+ concentration (Km = 4.1 +/- 1.1 microM). External Na+ inhibited the uptake in a dose-dependent manner (IC50 = 15 mM). Dissipation of the Na+ gradient by 10 microM monensin decreased Na+/Ca2+ exchange from 0.74 +/- 0.17 nmoles/mg protein/s to 0.11 +/- 0.05 nmoles/mg protein/s. Exchange was not influenced by veratridine, tetrodotoxin and ouabain, or by modifiers of cAMP. No effect was seen using the calcium channel blockers, nitrendipine or nifedipine. Glucose had no direct effect on Na+/Ca2+ exchange, while glyceraldehyde, glyceraldehyde-3-phosphate and dihydroxyacetone inhibited the exchange. Na+ induced efflux of calcium was seen in Ca2+ loaded vesicles and was half maximal at [Na+] of 11.1 +/- 0.75 mM. Ca2+ efflux was dependent on [Na+], with a Hill coefficient of 2.7 +/- 0.07 indicating that activation of Ca2+ release involves a minimum of three sites. The electrogenicity of this exchange was demonstrated using the lipophilic cation tetraphenylphosphonium [( 3H]-TPP), a membrane potential sensitive probe. [3H]-TPP uptake increased transiently during Na+/Ca2+ exchange indicating that the exchange generated a membrane potential. These results show that Na+/Ca2+ exchange operates in the beta cell and may be an important regulator of intracellular free Ca2+ concentrations.