Sodium-dependent inactivation of sodium/calcium exchange in transfected Chinese hamster ovary cells

High concentrations of cytosolic Na⁺ ions induce the time-dependent formation of an inactive state of the Na⁺/Ca²⁺ exchanger (NCX), a process known as Na⁺-dependent inactivation. NCX activity was measured as Ca²⁺ uptake in fura 2-loaded Chinese hamster ovary (CHO) cells expressing the wild-type (WT) NCX or mutants that are hypersensitive (F223E) or resistant (K229Q) to Na⁺-dependent inactivation. As expected, 1) Na⁺-dependent inactivation was promoted by high cytosolic Na⁺ concentration, 2) the F223E mutant was more susceptible than the WT exchanger to inactivation, whereas the K229Q mutant was resistant, and 3) inactivation was enhanced by cytosolic acidification. However, in contrast to expectations from excised patch studies, 1) the WT exchanger was resistant to Na⁺-dependent inactivation unless cytosolic pH was reduced, 2) reducing cellular phosphatidylinositol-4,5-bisphosphate levels did not induce Na⁺-dependent inactivation in the WT exchanger, 3) Na⁺-dependent inactivation did not increase the half-maximal cytosolic Ca²⁺ concentration for allosteric Ca²⁺ activation, 4) Na⁺-dependent inactivation was not reversed by high cytosolic Ca²⁺ concentrations, and 5) Na⁺-dependent inactivation was partially, but transiently, reversed by an increase in extracellular Ca²⁺ concentration. Thus Na⁺-dependent inactivation of NCX expressed in CHO cells differs in several respects from the inactivation process measured in excised patches. The refractoriness of the WT exchanger to Na⁺-dependent inactivation suggests that this type of inactivation is unlikely to be a strong regulator of exchange activity under physiological conditions but would probably act to inhibit NCX-mediated Ca²⁺ influx during ischemia. ischemia; cytosolic calcium concentration; cytosolic sodium concentration; cellular phosphatidylinositol-4,5-bisphosphate     

Regulation of Na+/Ca2+ exchange activity by cytosolic Ca2+ in transfected Chinese hamster ovary cells

Transfected Chinese hamster ovary cells stably expressing thebovine cardiacNa⁺/Ca²⁺exchanger (CK1.4 cells) were used to determine the range of cytosolic Ca²⁺ concentrations([Ca²⁺]_i)that activateNa⁺/Ca²⁺exchange activity. Ba²⁺ influx wasmeasured in fura 2-loaded, ionomycin-treated cells under conditions inwhich the intracellular Na⁺concentration was clamped with gramicidin at ~20 mM.[Ca²⁺]_iwas varied by preincubating ionomycin-treated cells with either theacetoxymethyl ester of EGTA or medium containing 0-1 mM added CaCl₂. The rate ofBa²⁺ influx increased in asaturable manner with[Ca²⁺]_i,with the half-maximal activation value of 44 nM and a Hill coefficientof 1.6. When identical experiments were carried out with cellsexpressing a Ca²⁺-insensitivemutant of the exchanger, Ba²⁺influx did not vary with[Ca²⁺]_i.The concentration for activation of exchange activity was similar tothat reported for whole cardiac myocytes but approximately an order ofmagnitude lower than that reported for excised, giant patches. Thereason for the difference in Ca²⁺regulation between whole cells and membrane patches is unknown.

     

Calcium-dependent regulation of calcium efflux by the cardiac sodium/calcium exchanger

Allosteric regulation by cytosolic Ca²⁺ of Na⁺/Ca²⁺ exchange activity in the Ca²⁺ efflux mode has received little attention because it has been technically difficult to distinguish between the roles of Ca²⁺ as allosteric activator and transport substrate. In this study, we used transfected Chinese hamster ovary cells to compare the Ca²⁺ efflux activities in nontransfected cells and in cells expressing either the wild-type exchanger or a mutant, (241–680), that operates constitutively; i.e., its activity does not require allosteric Ca²⁺ activation. Expression of the wild-type exchanger did not significantly lower the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) compared with nontransfected cells. During Ca²⁺ entry through store-operated Ca²⁺ channels, Ca²⁺ efflux by the wild-type exchanger became evident only after [Ca²⁺]_i approached 100–200 nM. A subsequent decline in [Ca²⁺]_i was observed, suggesting that the activation process was time dependent. In contrast, Ca²⁺ efflux activity was evident under all experimental conditions in cells expressing the constitutive exchanger mutant. After transient exposure to elevated [Ca²⁺]_i, the wild-type exchanger behaved similarly to the constitutive mutant for tens of seconds after [Ca²⁺]_i had returned to resting levels. We conclude that Ca²⁺ efflux activity by the wild-type exchanger is allosterically activated by Ca²⁺, perhaps in a time-dependent manner, and that the activated state is briefly retained after the return of [Ca²⁺]_i to resting levels. persistent calcium activation; store-operated channels; calcium transient     

Role of Na+/Ca2+ exchange in regulating cytosolic Ca2+ in cultured human pulmonary artery smooth muscle cells

A rise in cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) in pulmonary artery smooth muscle cells (PASMC) is an important stimulus for cell contraction, migration, and proliferation. Depletion of intracellular Ca²⁺ stores opens store-operated Ca²⁺ channels (SOC) and causes Ca²⁺ entry. Transient receptor potential (TRP) cation channels that are permeable to Na⁺ and Ca²⁺ are believed to form functional SOC. Because sarcolemmal Na⁺/Ca²⁺ exchanger has also been implicated in regulating [Ca²⁺]_cyt, this study was designed to test the hypothesis that the Na⁺/Ca²⁺ exchanger (NCX) in cultured human PASMC is functionally involved in regulating [Ca²⁺]_cyt by contributing to store depletion-mediated Ca²⁺ entry. RT-PCR and Western blot analyses revealed mRNA and protein expression for NCX1 and NCKX3 in cultured human PASMC. Removal of extracellular Na⁺, which switches the Na⁺/Ca²⁺ exchanger from the forward (Ca²⁺ exit) to reverse (Ca²⁺ entry) mode, significantly increased [Ca²⁺]_cyt, whereas inhibition of the Na⁺/Ca²⁺ exchanger with KB-R7943 (10 µM) markedly attenuated the increase in [Ca²⁺]_cyt via the reverse mode of Na⁺/Ca²⁺ exchange. Store depletion also induced a rise in [Ca²⁺]_cyt via the reverse mode of Na⁺/Ca²⁺ exchange. Removal of extracellular Na⁺ or inhibition of the Na⁺/Ca²⁺ exchanger with KB-R7943 attenuated the store depletion-mediated Ca²⁺ entry. Furthermore, treatment of human PASMC with KB-R7943 also inhibited cell proliferation in the presence of serum and growth factors. These results suggest that NCX is functionally expressed in cultured human PASMC, that Ca²⁺ entry via the reverse mode of Na⁺/Ca²⁺ exchange contributes to store depletion-mediated increase in [Ca²⁺]_cyt, and that blockade of the Na⁺/Ca²⁺ exchanger in its reverse mode may serve as a potential therapeutic approach for treatment of pulmonary hypertension. sodium-calcium exchange; calcium homeostasis; vascular smooth muscle     

Upregulation of Na+/Ca2+ exchanger contributes to the enhanced Ca2+ entry in pulmonary artery smooth muscle cells from patients with idiopathic pulmonary arterial hypertension

A rise in cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) in pulmonary artery smooth muscle cells (PASMC) is a trigger for pulmonary vasoconstriction and a stimulus for PASMC proliferation and migration. Multiple mechanisms are involved in regulating [Ca²⁺]_cyt in human PASMC. The resting [Ca²⁺]_cyt and Ca²⁺ entry are both increased in PASMC from patients with idiopathic pulmonary arterial hypertension (IPAH), which is believed to be a critical mechanism for sustained pulmonary vasoconstriction and excessive pulmonary vascular remodeling in these patients. Here we report that protein expression of NCX1, an NCX family member of Na⁺/Ca²⁺ exchanger proteins is upregulated in PASMC from IPAH patients compared with PASMC from normal subjects and patients with other cardiopulmonary diseases. The Na⁺/Ca²⁺ exchanger operates in a forward (Ca²⁺ exit) and reverse (Ca²⁺ entry) mode. By activating the reverse mode of Na⁺/Ca²⁺ exchange, removal of extracellular Na⁺ caused a rapid increase in [Ca²⁺]_cyt, which was significantly enhanced in IPAH PASMC compared with normal PASMC. Furthermore, passive depletion of intracellular Ca²⁺ stores using cyclopiazonic acid (10 µM) not only caused a rise in [Ca²⁺]_cyt due to Ca²⁺ influx through store-operated Ca²⁺ channels but also mediated a rise in [Ca²⁺]_cyt via the reverse mode of Na⁺/Ca²⁺ exchange. The upregulated NCX1 in IPAH PASMC led to an enhanced Ca²⁺ entry via the reverse mode of Na⁺/Ca²⁺ exchange, but did not accelerate Ca²⁺ extrusion via the forward mode of Na⁺/Ca²⁺ exchange. These observations indicate that the upregulated NCX1 and enhanced Ca²⁺ entry via the reverse mode of Na⁺/Ca²⁺ exchange are an additional mechanism responsible for the elevated [Ca²⁺]_cyt in PASMC from IPAH patients. transient receptor potential channel; reverse and forward mode; proliferation     

Actin-dependent regulation of the cardiac Na(+)/Ca(2+) exchanger

In the present study, the bovine cardiac Na⁺/Ca²⁺ exchanger (NCX1.1) was expressed in Chinese hamster ovary cells. The surface distribution of the exchanger protein, externally tagged with the hemagglutinin (HA) epitope, was associated with underlying actin filaments in regions of cell-to-cell contact and also along stress fibers. After we treated cells with cytochalasin D, NCX1.1 protein colocalized with patches of fragmented filamentous actin (F-actin). In contrast, an HA-tagged deletion mutant of NCX1.1 that was missing much of the exchanger's central hydrophilic domain (241–680) did not associate with F-actin. In cells expressing the wild-type exchanger, cytochalasin D inhibited allosteric Ca²⁺ activation of NCX activity as shown by prolongation of the lag phase of low Ca²⁺ uptake after initiation of the reverse (i.e., Ca²⁺ influx) mode of NCX activity. Other agents that perturbed F-actin structure (methyl--cyclodextrin, latrunculin B, and jasplakinolide) also increased the duration of the lag phase. In contrast, when reverse-mode activity was initiated after allosteric Ca²⁺ activation, both cytochalasin D and methyl--cyclodextrin (Me--CD) stimulated NCX activity by 70%. The activity of the (241–680) mutant, which does not require allosteric Ca²⁺ activation, was also stimulated by cytochalasin D and Me--CD. The increased activity after these treatments appeared to reflect an increased amount of exchanger protein at the cell surface. We conclude that wild-type NCX1.1 associates with the F-actin cytoskeleton, probably through interactions involving the exchanger's central hydrophilic domain, and that this association interferes with allosteric Ca²⁺ activation. cytochalasin; methyl--cyclodextrin; allosteric calcium activation     

Na+/Ca2+ exchange activity in neonatal rabbit ventricular myocytes

Much less is known about the contributions of the Na⁺/Ca²⁺ exchanger (NCX) and sarcoplasmic reticulum (SR) Ca²⁺ pump to cell relaxation in neonatal compared with adult mammalian ventricular myocytes. Based on both biochemical and molecular studies, there is evidence of a much higher density of NCX at birth that subsequently decreases during the next 2 wk of development. It has been hypothesized, therefore, that NCX plays a relatively more important role for cytosolic Ca²⁺ decline in neonates as well as, perhaps, a role in excitation-contraction coupling in reverse mode. We isolated neonatal ventricular myocytes from rabbits in four different age groups: 3, 6, 10, and 20 days of age. Using an amphotericin-perforated patch-clamp technique in fluo-3-loaded myocytes, we measured the caffeine-induced inward NCX current (I_NCX) and the Ca²⁺ transient. We found that the integral of I_NCX, an indicator of SR Ca²⁺ content, was greatest in myocytes from younger age groups when normalized by cell surface area and that it decreased with age. The velocity of Ca²⁺ extrusion by NCX (V_NCX) was linear with [Ca²⁺] and did not indicate saturation kinetics until [Ca²⁺] reached 1–3 µM for each age group. There was a significantly greater time delay between the peaks of I_NCX and the Ca²⁺ transient in myocytes from the youngest age groups. This observation could be related to structural differences in the subsarcolemmal microdomains as a function of age. ontogeny of cardiac excitation-contraction coupling; sodium/calcium exchanger; cytosolic calcium concentration; subsarcolemmal calcium concentration; sarcoplasmic reticulum calcium content     

Functional comparison of the three isoforms of the Na+/Ca2+ exchanger (NCX1, NCX2, NCX3)

Three distinctmammalianNa⁺/Ca²⁺exchangers have been cloned: NCX1, NCX2, and NCX3. We have undertaken adetailed functional comparison of these three exchangers. Eachexchanger was stably expressed at high levels in the plasma membranesof BHK cells. Na⁺/Ca²⁺exchange activity was assessed using three different complementary techniques: Na⁺ gradient-dependent⁴⁵Ca²⁺uptake into intact cells, Na⁺gradient-dependent⁴⁵Ca²⁺uptake into membrane vesicles isolated from the transfected cells, andexchange currents measured using giant patches of excised cellmembrane. Apparent affinities for the transported ionsNa⁺ andCa²⁺ were markedly similar for thethree exchangers at both membrane surfaces. Likewise, generally similarresponses to changes in pH, chymotrypsin treatment, and application ofvarious inhibitors were obtained. Depletion of cellular ATP inhibitedNCX1 and NCX2 but did not affect the activity of NCX3. Exchangeactivities of NCX1 and NCX3 were modestly increased by agents thatactivate protein kinases A and C. All exchangers were regulated byintracellular Ca²⁺. NCX1-inducedexchange currents were especially large in excised patches and, likethe native myocardial exchanger, were stimulated by ATP. Results may beinfluenced by our choice of expression system and specific splicevariants, but, overall, the three exchangers appear to have verysimilar properties.

     

Physiological effects of Na+/Ca2+ exchanger knockdown by antisense oligodeoxynucleotides in arterial myocytes

Antisense oligodeoxynucleotides (AS-oligos) targeted to theNa⁺/Ca²⁺exchanger (NCX) inhibit NCX-mediatedCa²⁺ influx in mesenteric artery(MA) myocytes [Am. J. Physiol.269 (Cell Physiol. 38):C1340-C1345, 1995]. Here, we show AS-oligo knockdown ofNCX-mediated Ca²⁺ efflux. Ininitial experiments, the cytosolic freeCa²⁺ concentration([Ca²⁺]_cyt)was raised, and sarcoplasmic reticulum (SR)Ca²⁺ sequestration was blockedwith caffeine and cyclopiazonic acid; the extracellularNa⁺-dependent (NCX) component ofCa²⁺ efflux was then selectivelyinhibited in AS-oligo-treated cells but not in controls (no oligos ornonsense oligos). In contrast, theLa³⁺-sensitive (plasmalemmaCa²⁺ pump) component ofCa²⁺ efflux was unaffected inAS-oligo-treated cells. Knockdown of NCX activity was reversed byincubating AS-oligo-treated cells in normal media for 5 days. Transient[Ca²⁺]_cytelevations evoked by serotonin (5-HT) at 15-min intervals inAS-oligo-treated cells were indistinguishable from those in controls.When cells were stimulated every 3 min, however, the peak amplitudes ofthe second and third responses were larger, and[Ca²⁺]_cytreturned to baseline more slowly, in AS-oligo-treated cells than incontrols. Peak 5-HT-evoked responses in the controls, but notAS-oligo-treated cells, were augmented more than twofold inNa⁺-free media. This implies thatNCX is involved in Na⁺ gradientmodulation of SR Ca²⁺ stores andcell responsiveness. The repetitive stimulation data suggest that theNCX may be important during tonic activation of arterial myocytes.

     

Differential inhibition of Na+/Ca2+ exchanger isoforms by divalent cations and isothiourea derivative

We compared the properties of three mammalianNa⁺/Ca²⁺exchanger isoforms, NCX1, NCX2, and NCX3, by analyzing the effects of Ni²⁺ and other cations as well asthe recently identified inhibitor isothiourea derivatives onintracellular Na⁺-dependent⁴⁵Ca²⁺uptake into CCL-39 (Dede) fibroblasts stably expressingeach isoform. All these NCX isoforms had similar affinities for the extracellular transport substratesCa²⁺ andNa⁺.Ni²⁺ inhibited⁴⁵Ca²⁺uptake by competing with Ca²⁺ forthe external transport site, with 10-fold less affinity in NCX3 than inNCX1 or NCX2. Ni²⁺ andCo²⁺ were most efficient in suchdiscrimination of NCX isoforms, although their inhibitory potencieswere less than those of La³⁺ andCd²⁺. The monovalent cationLi⁺ stimulated⁴⁵Ca²⁺uptake rate by all NCX isoforms similarly with low affinity, althoughthe extent of stimulation was somewhat smaller in NCX1. On the otherhand, the isothiourea derivative KB-R7943 was threefold more inhibitoryto NCX3 than to NCX1 or NCX2. Thus distinct differences in the kineticand pharmacological properties were detected between NCX3 and the othertwo isoforms.

     

Changes in regulation of sodium/calcium exchanger of avian ventricular heart cells during embryonic development

It has been suggested that the sodium/calcium exchanger NCX1 may have a more important physiological role in embryonic and neonatal hearts than in adult hearts. However, in chick heart sarcolemmal vesicles, sodium-dependent calcium transport is reported to be small and, moreover, to be 3–12 times smaller in hearts at embryonic day (ED) 4–5 than at ED18, the opposite of what would be expected of a transporter that is more important in early development. To better assess the role of NCX1 in calcium regulation in the chick embryonic heart, we measured the activity of NCX1 in chick embryonic hearts as extracellular calcium-activated exchanger current (I_NCX) under controlled ionic conditions. With intracellular calcium concentration ([Ca²⁺]_i) = 47 nM, I_NCX density increased from 1.34 ± 0.28 pA/pF at ED2 to 3.22 ± 0.55 pA/pF at ED11 (P = 0.006); however, with [Ca²⁺]_i = 481 nM, the increase was small and statistically insignificant, from 4.54 ± 0.77 to 5.88 ± 0.73 pA/pF (P = 0.20, membrane potential = 0 mV, extracellular calcium concentration = 2 mM). Plots of I_NCX density against [Ca²⁺]_i were well fitted by the Michaelis-Menton equation and extrapolated to identical maximal currents for ED2 and ED11 cells (extracellular calcium concentration = 1, 2, or 4 mM). Thus the increase in I_NCX at low [Ca²⁺]_i appeared to reflect a developmental change in allosteric regulation of the exchanger by intracellular calcium rather than an increase in the membrane density of NCX1. Supporting this conclusion, RT-PCR demonstrated little change in the amount of mRNA encoding NCX1 expression from ED2 through ED18. NCX1; chick embryo; allosteric regulation; sodium/calcium exchange current     

Intermittent hypoxia protects cardiomyocytes against ischemia-reperfusion injury-induced alterations in Ca2+ homeostasis and contraction via the sarcoplasmic reticulum and Na+/Ca2+ exchange mechanisms

We have previously demonstrated that intermittent high-altitude (IHA) hypoxia significantly attenuates ischemia-reperfusion (I/R) injury-induced excessive increase in resting intracellular Ca²⁺ concentrations ([Ca²⁺]_i). Because the sarcoplasmic reticulum (SR) and Na⁺/Ca²⁺ exchanger (NCX) play crucial roles in regulating [Ca²⁺]_i and both are dysfunctional during I/R, we tested the hypothesis that IHA hypoxia may prevent I/R-induced Ca²⁺ overload by maintaining Ca²⁺ homeostasis via SR and NCX mechanisms. We thus determined the dynamics of Ca²⁺ transients and cell shortening during preischemia and I/R injury in ventricular cardiomyocytes from normoxic and IHA hypoxic rats. IHA hypoxia did not affect the preischemic dynamics of Ca²⁺ transients and cell shortening, but it significantly suppressed the I/R-induced increase in resting [Ca²⁺]_i levels and attenuated the depression of the Ca²⁺ transients and cell shortening during reperfusion. Moreover, IHA hypoxia significantly attenuated I/R-induced depression of the protein contents of SR Ca²⁺ release channels and/or ryanodine receptors (RyRs) and SR Ca²⁺ pump ATPase (SERCA2) and SR Ca²⁺ release and uptake. In addition, a delayed decay rate time constant of Ca²⁺ transients and cell shortening of Ca²⁺ transients observed during ischemia was accompanied by markedly inhibited NCX currents, which were prevented by IHA hypoxia. These findings indicate that IHA hypoxia may preserve Ca²⁺ homeostasis and contraction by preserving RyRs and SERCA2 proteins as well as NCX activity during I/R. intracellular Ca²⁺ concentration; Ca²⁺ transients; Ca²⁺ transporters; myofilament Ca²⁺ sensitivity     

Adenylyl cyclase expression and modulation of cAMP in rat taste cells

The present study determined Ca²⁺ handling in the hearts of rats subjected to chronic hypoxia (CH). Spectrofluorometry was used to measure intracellular Ca²⁺ concentration ([Ca²⁺]_i) and its responses to electrical stimulation, caffeine, and isoproterenol in myocytes from the right ventricle of rats breathing 10% oxygen for 1, 3, 7, 14, 21, 28, and 56 days and age-matched controls. The protein expression of sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA) and its ryanodine receptor (RyR) were measured. The uptake of ⁴⁵Ca²⁺ by SERCA, release by RyR, and extrusion by Na⁺/Ca²⁺ exchange (NCX) were determined. It was found that Ca²⁺ homeostasis and Ca²⁺ responses to -adrenoceptor stimulation reached a new equilibrium after 4 wk of CH. Ca²⁺ content in the sarcoplasmic reticulum (SR) was reduced, but cytosolic Ca²⁺ remained unchanged after CH. Expression of SERCA and its Ca²⁺ uptake, Ca²⁺ release via RyR, and NCX activity were suppressed by CH. The results indicate impaired Ca²⁺ handling, which may be responsible for the attenuated Ca²⁺ responses to -adrenoceptor stimulation in CH. intracellular calcium ion concentration; calcium-adenosinetriphosphatase; ryanodine receptor; sodium/calcium exchange; sarcoplasmic reticulum; -adrenoceptor; chronic hypoxia     

Novel role of the Ca2+-ATPase in NMDA-induced intracellular acidification

The mechanism involved inN-methyl-D-glucamine(NMDA)-induced Ca²⁺-dependentintracellular acidosis is not clear. In this study, we investigated indetail several possible mechanisms using cultured rat cerebellargranule cells and microfluorometry [fura 2-AM or 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-AM].When 100 µM NMDA or 40 mM KCl was added, a marked increase in theintracellular Ca²⁺ concentration([Ca²⁺]_i)and a decrease in the intracellular pH were seen. Acidosis wascompletely prevented by the use ofCa²⁺-free medium or1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM, suggesting that it resulted from an influx of extracellular Ca²⁺. The following fourmechanisms that could conceivably have been involved were excluded:1)Ca²⁺ displacement of intracellularH⁺ from common binding sites;2) activation of an acid loader or inhibition of acid extruders; 3)overproduction of CO₂ or lactate; and 4) collapse of the mitochondrialmembrane potential due to Ca²⁺uptake, resulting in inhibition of cytosolicH⁺ uptake. However,NMDA/KCl-induced acidosis was largely prevented by glycolyticinhibitors (iodoacetate or deoxyglucose in glucose-free medium) or byinhibitors of the Ca²⁺-ATPase(i.e.,Ca²⁺/H⁺exchanger), including La³⁺,orthovanadate, eosin B, or an extracellular pH of 8.5. Our results therefore suggest that Ca²⁺-ATPaseis involved in NMDA-induced intracellular acidosis in granule cells. Wealso provide new evidence that NMDA-evoked intracellular acidosisprobably serves as a negative feedback signal, probably with theacidification itself inhibiting the NMDA-induced[Ca²⁺]_i increase.

     

Temperature dependence of cloned mammalian and salmonid cardiac Na(+)/Ca(2+) exchanger isoforms

The cardiacNa⁺/Ca²⁺ exchanger (NCX), an importantregulator of cytosolic Ca²⁺ concentration in contractionand relaxation, has been shown in trout heart sarcolemmal vesicles tohave high activity at 7°C relative to its mammalian isoform. Thisunique property is likely due to differences in protein structure. Inthis study, outward NCX currents (I_NCX) of thewild-type trout (NCX-TR1.0) and canine (NCX 1.1) exchangers expressedin oocytes were measured to explore the potential contributions ofregulatory vs. transport mechanisms to this observation. cRNA wastranscribed in vitro from both wild-type cDNA and was injected intoXenopus oocytes. I_NCX of NCX-TR1.0 and NCX1.1 were measured after 3-4 days over a temperature range of 7-30°C using the giant excised patch technique. TheI_NCX for both isoforms exhibitedNa⁺-dependent inactivation and Ca²⁺-dependentpositive regulation. The I_NCX of NCX1.1exhibited typical mammalian temperature sensitivities withQ₁₀ values of 2.4 and 2.6 for peak and steady-statecurrents, respectively. However, the I_NCX ofNCX-TR1.0 was relatively temperature insensitive with Q₁₀values of 1.2 and 1.1 for peak and steady-state currents, respectively.I_NCX current decay was fit with a singleexponential, and the resultant rate constant of inactivation () wasdetermined as a function of temperature. As expected,  decreasedmonotonically with temperature for both isoforms. Although  wassignificantly greater in NCX1.1 compared with NCX-TR1.0 at alltemperatures, the effect of temperature on  was not differentbetween the two isoforms. These data suggest that thedisparities in I_NCX temperature dependencebetween these two exchanger isoforms are unlikely due to differences intheir inactivation kinetics. In addition, similar differences intemperature dependence were observed in both isoforms after-chymotrypsin treatment that renders the exchanger in a deregulatedstate. These data suggest that the differences in I_NCX temperature dependence between the twoisoforms are not due to potential disparities in either theI_NCX regulatory mechanisms or structuraldifferences in the cytoplasmic loop but are likely predicated ondifferences within the transmembrane segments.

     

Heterogeneity of mitochondrial matrix free Ca2+: resolution of Ca2+ dynamics in individual mitochondria in situ

The role of mitochondria inCa²⁺ homeostasis is controversial.We employed the Ca²⁺-sensitive dyerhod 2 with novel, high temporal and spatial resolution imaging toevaluate changes in the matrix freeCa²⁺ concentration of individualmitochondria([Ca²⁺]_m)in agonist-stimulated, primary cultured aortic myocytes. Stimulation with 10 µM serotonin (5-HT) evoked modest cytosolicCa²⁺ transients[cytosolic freeCa²⁺ concentration([Ca²⁺]_cyt)<500 nM; measured with fura 2] and triggered contractions inshort-term cultured myocytes. However, 5-HT triggered a large mitochondrial rhod 2 signal (indicating pronounced elevation of [Ca²⁺]_m)in only 4% of cells. This revealed heterogeneity in the responses ofindividual mitochondria, all of which stained with MitoTracker GreenFM. In contrast, stimulation with 100 µM ATP evoked large cytosolicCa²⁺ transients (>1,000 nM) andinduced pronounced, reversible elevation of[Ca²⁺]_m(measured as rhod 2 fluorescence) in 60% of cells. This mitochondrial Ca²⁺ uptake usually lagged behindthe cytosolic Ca²⁺ transient peakby 3-5 s, and[Ca²⁺]_mdeclined more slowly than did bulk[Ca²⁺]_cyt.The uptake delay may prevent mitochondria from interfering with rapidsignaling events while enhancing the mitochondrial response to large,long-duration elevations of[Ca²⁺]_cyt.The responses of arterial myocytes to modest physiological stimulationdo not, however, depend on such marked changes in [Ca²⁺]_m.     

Altered Ca(2+) handling by ryanodine receptor and Na(+)-Ca(2+) exchange in the heart from ovariectomized rats: role of protein kinase A

Our previous study has demonstrated that ovariectomy (Ovx) significantly increased the left ventricular developed pressure (LVDP) and the maximal rate of developed pressure over time (±dP/dt_max) in the isolated perfused rat heart and the effects were reversed by female sex hormone replacement. In the present investigation, we studied the effects of Ovx for 6 wk on Ca²⁺ homeostasis that determines the contractile function. Particular emphasis was given to Ca²⁺ handling by ryanodine receptor (RyR) and Na⁺-Ca²⁺ exchange (NCX). ⁴⁵Ca²⁺ fluxes via the RyR, NCX, and Ca²⁺-ATPase (SERCA) were compared with their expression in myocytes from Ovx rats with and without estrogen replacement. Furthermore, we correlated the handling of Ca²⁺ by these Ca²⁺ handling proteins with the overall Ca²⁺ homeostasis by determining the Ca²⁺ transients induced by electrical stimulation and caffeine, which reveals the dynamic changes of cytosolic Ca²⁺ concentration ([Ca²⁺]_i) in the heart. In addition, we determined the expression and contribution of protein kinase A (PKA) to the regulation of the aforementioned Ca²⁺ handling proteins in Ovx rats. It was found that after Ovx there were 1) increased Ca²⁺ fluxes via RyR and NCX, which were reversed not only by estrogen replacement, but more importantly by blockade of PKA; 2) an increased expression of PKA; and 3) no increase in expression of NCX and SERCA. We suggest that hyperactivities of RyR and NCX are a result of upregulation of PKA. The increased release of Ca²⁺ through RyR and removal of Ca²⁺ by NCX are believed to be responsible for the greater contractility and faster relaxation after Ovx. ovariectomy     

Glucagon-mediated Ca2+ signaling in BHK cells expressing cloned human glucagon receptors

From video imaging of fura 2-loaded baby hamster kidney (BHK)cells stably expressing the cloned human glucagon receptor, we foundthe Ca²⁺ response to glucagon tobe specific, dose dependent, synchronous, sensitive to pertussis toxin,and independent of Ca²⁺ influx.Forskolin did not elicit a Ca²⁺response, but treatment with a protein kinase A inhibitor, the Rp diastereomer of 8-bromoadenosine-3',5'-cyclicmonophosphothioate, resulted in a reduced glucagon-mediatedCa²⁺ response as well asCa²⁺ oscillations. The specificphospholipase C inhibitor U-73122 abolished theCa²⁺ response to glucagon, and amodest twofold increase in inositol trisphosphate(IP₃) production could beobserved after stimulation with glucagon. In BHK cells coexpressingglucagon and muscarinic (M₁)acetylcholine receptors, carbachol blocked the rise in intracellular free Ca²⁺ concentrations inresponse to glucagon, whereas glucagon did not affect thecarbachol-induced increase inCa²⁺. Furthermore, carbachol, butnot glucagon, could block thapsigargin-activated increases inintracellular free Ca²⁺concentration. These results indicate that, in BHK cells, glucagon receptors can activate not only adenylate cyclase but also a second independent G protein-coupled pathway that leads to the stimulation ofphospholipase C and the release ofCa²⁺ fromIP₃-sensitive intracellularCa²⁺ stores. Finally, we provideevidence to suggest that cAMP potentiates theIP₃-mediated effects onintracellular Ca²⁺ handling.

     

New insights into the molecular and cellular workings of the cardiac Na+/Ca2+ exchanger

The cardiac Na⁺/Ca²⁺ exchanger (NCX1) is almost certainly the major Ca²⁺ extrusion mechanism in cardiac myocytes, although the driving force for Ca²⁺ extrusion is quite small. To explain multiple recent results, it is useful to think of the exchanger as a slow Ca²⁺ buffer that can reverse its function multiple times during the excitation-contraction cycle (ECC). An article by the group of John Reeves brings new insights to this function by analyzing the role of regulatory domains of NCX1 that mediate its activation by a rise of cytoplasmic Ca²⁺. It was demonstrated that the gating reactions are operative just in the physiological range of Ca²⁺ changes, a few fold above resting Ca²⁺ level, and that they prevent the exchanger from damping out the influence of mechanisms that transiently increase Ca²⁺ levels. Furthermore, exchangers with deleted regulatory domains are shown to reduce resting Ca²⁺ to lower levels than achieved by wild-type exchangers. A study by the group of Kenneth Philipson demonstrated that the NCX1 regulatory domain can bind and respond to Ca²⁺ changes on the time scale of the ECC in rat myocytes. At the same time, studies of transgenic mice and NCX1 knockout mice generated by the Philipson group revealed that large changes of NCX1 activity have rather modest effects on ECC. Simple simulations predict these results very well: murine cardiac ECC is very sensitive to small changes of the Na⁺ gradient, very sensitive to changes of the sarcoplasmic reticulum Ca²⁺ pump activity, and very insensitive to changes of NCX1 activity. It is speculated that the NCX1 gating reactions not only regulate coupled 3Na⁺:1Ca²⁺ exchange but also control the exchanger’s Na⁺ leak function that generates background Na⁺ influx and depolarizing current in cardiac myocytes. excitation-contraction cycle     

Effects of chronic run training on Na+-dependent Ca2+ efflux from rat left ventricular myocytes

The effects ofendurance run training onNa⁺-dependentCa²⁺ regulation in rat leftventricular myocytes were examined. Myocytes were isolated fromsedentary and trained rats and loaded with fura 2. Contractile dynamicsand fluorescence ratio transients were recorded during electricalpacing at 0.5 Hz, 2 mM extracellular Ca²⁺ concentration, and 29°C.Resting and peak cytosolic Ca²⁺concentration([Ca²⁺]_c)did not change with exercise training. However, resting and peak[Ca²⁺]_cincreased significantly in both groups during 5 min of continuous pacing, although diastolic[Ca²⁺]_cin the trained group was less susceptible to this elevation ofintracellular Ca²⁺. Run trainingalso significantly reduced the rate of[Ca²⁺]_cdecay during relaxation. Myocytes were then exposed to 10 mM caffeinein the absence of external Na⁺ orCa²⁺ to trigger sarcoplasmicreticular Ca²⁺ release and tosuppress cellular Ca²⁺ efflux.This maneuver elicited an elevated steady-state[Ca²⁺]_c.External Na⁺ was then added, andthe rate of[Ca²⁺]_cclearance was determined. Run training significantly reduced the rateof Na⁺-dependent clearance of[Ca²⁺]_cduring the caffeine-induced contractures. These data demonstrate thatthe removal of cytosolic Ca²⁺ wasdepressed with exercise training under these experimental conditionsand may be specifically reflective of a training-induced decrease inthe rate of cytosolic Ca²⁺ removalviaNa⁺/Ca²⁺exchange and/or in the amount ofCa²⁺ moved across the sarcolemmaduring a contraction.