Endothelin-1 induced hypertrophic effect in neonatal rat cardiomyocytes: involvement of Na+/H+ and Na+/Ca2+ exchangers

Endothelin-1 (ET-1) is a potent agonist of cell growth that also stimulates Na(+)/H(+) exchanger isoform 1 (NHE-1) activity. It was hypothesized that the increase in intracellular Na(+) ([Na(+)](i)) mediated by NHE-1 activity may induce the reverse mode of Na(+)/Ca(2+) exchanger (NCX(rev)) increasing intracellular Ca(2+) ([Ca(2+)](i)) which in turn will induce hypertrophy. The objective of this work was to test whether the inhibition of NHE-1 or NCX(rev) prevents ET-1 induced hypertrophy in neonatal rat cardiomyocytes (NRVMs). NRVMs were cultured (24 h) in the absence (control) and presence of 5 nmol/L ET-1 alone, or combined with 1 mumol/L HOE 642 or 5 mumol/L KB-R7943. Cell surface area, (3)H-phenylalanine incorporation and atrial natriuretic factor (ANF) mRNA expression were increased to 131 +/- 3, 220 +/- 12 and 190 +/- 25% of control, respectively (P < 0.05) by ET-1. [Na(+)](i) and total [Ca(2+)](i) were higher (8.1 +/- 1.2 mmol/L and 636 +/- 117 nmol/L, respectively) in ET-1-treated than in control NRVMs (4.2 +/- 1.3 and 346 +/- 85, respectively, P < 0.05), effects that were cancelled by NHE-1 inhibition with HOE 642. The rise in [Ca(2+)](i) induced by extracellular Na(+) removal (NCX(rev)) was higher in ET-1-treated than in control NRVMs and the effect was prevented by co-treatment with HOE 642 or KB-R7943 (NCX(rev) inhibitor). The ET-1-induced increase in cell area, ANF mRNA expression and (3)H-phenylalanine incorporation in ET-1-treated NRVM were decreased by NHE-1 or NCX(rev) inhibition. Our results provide the first evidence that NCX(rev) is, secondarily to NHE-1 activation, involved in ET-1-induced hypertrophy in NRVMs.     

A low dose of angiotensin II increases inotropism through activation of reverse Na(+)/Ca(2+) exchange by endothelin release

OBJECTIVE: This work was aimed to prove that release/formation of endogenous endothelin acting in an autocrine/paracrine fashion contributes to the increase in contractility promoted by a low dose of angiotensin II. METHODS: Isolated cat papillary muscles were used for force, pH(i), [Na(+)](i) and [Ca(2+)](i) measurements and isolated cat myocytes for patch-clamp experiments. RESULTS: In papillary muscles, 1.0 nmol/l angiotensin II increased force by 23+/-2% (n=4, P<0.05), [Na(+)](i) by 2.2+/-0.2 mmol/l (n=4, P<0.05), and peak (but not diastolic) Ca(2+) from 0.674+/-0.11 to 0.768+/-0.13 micromol/l (n=4, P<0.05), without affecting pH(i). Force and [Na(+)](i) increase were abolished by inhibition of the Na(+)/H(+) exchanger (NHE) with the inhibitor HOE642, blockade of endothelin receptors with the nonselective antagonist TAK044 and by inhibition of the endothelin-converting enzyme with phosphoramidon. Force but not [Na(+)](i) increase was abolished by inhibition of reverse Na(+)/Ca(2+) exchange (NCX) with the inhibitor KB-R7943. Similar increase in force (21+/-2%, n=4, P<0.05) and in [Na(+)](i) (2.4+/-0.4 mmol/l, n=4, P<0.05) that were also suppressed by TAK044 and HOE642 were induced by exogenous 5.0 nmol/l endothelin-1. KB-R7943 reverted the endothelin-1 effect on force but not on [Na(+)](i). In isolated myocytes, exogenous endothelin-1 dose-dependently increased the NCX current and shifted the NCX reversal potential (E(NCX)) to a more negative value (DeltaE(NCX): -10+/-3 and -17+/-5 mV, with 1 and 10 nmol/l endothelin-1, respectively, n=12). The latter effect was prevented by HOE642. CONCLUSION: Taken together, the results indicate that a low dose of angiotensin II induces release of endothelin, which, in autocrine/paracrine fashion activates the Na(+)/H(+) exchanger, increases [Na(+)](i) and changes E(NCX), promoting the influx of Ca(2+) that leads to a positive inotropic effect (PIE).     

Na(+)-Ca(2+) exchanger overexpression predisposes to reactive oxygen species-induced injury

OBJECTIVE: In heart failure (HF), the generation of reactive oxygen species (ROS) is enhanced. It was shown that failing cardiac myocytes are more susceptible to ROS-induced damage, possibly due to increased expression of the sarcolemmal Na-Ca exchanger (NCX). METHODS: We investigated the consequences of increased expression levels of NCX in adult rabbit ventricular cardiomyocytes (via adenovirus-mediated gene transfer, Ad-NCX1-GFP) with respect to tolerance towards ROS. After 48-h incubation, cells were monitored for morphological changes on an inverted microscope. ROS were generated via hydrogen peroxide (H(2)O(2)) (100 micromol/l) and Fe(3+)/nitrilotriacetate (Fe(3+)/NTA, 100/200 micromol/l) for 4 min and cell morphology was followed over 30 min. [Na(+)](i) and [Ca(2+)](i) in native cells were measured using SBFI-AM and Indo1-AM, respectively. RESULTS: In native myocytes, exposure to ROS induced hypercontracture. This was accompanied by a 1.3-fold increase in diastolic Indo1 fluorescence ratio (P<0.05). Overexpression of NCX significantly enhanced development of hypercontracture. After 15 min, the percentage of cells that had undergone hypercontracture (F(hyper)) was 85+/-4% vs. only 44+/-10% in control cells (P<0.05). Inhibition of NCX-mediated Ca(2+) entry with KB-R7943 (5 micromol/l) reduced F(hyper) to 33+/-11% (P<0.05). [Na(+)](i) was increased 2.9-fold 1 min prior to hypercontracture (P<0.05). CONCLUSIONS: ROS-induced hypercontracture is due to Ca(2+) entry via NCX which could be triggered by a concomitant substantial increase in [Na(+)](i). Elevated NCX levels predispose to ROS-induced injury, a mechanism likely contributing to myocyte dysfunction and death in heart failure.     

Effects of Na(+)/Ca(2+)-exchanger overexpression on excitation-contraction coupling in adult rabbit ventricular myocytes

The Na(+)/Ca(2+)-exchanger (NCX) is the main mechanism by which Ca(2+) is transported out of the ventricular myocyte. NCX levels are raised in failing human heart, and the consequences of this for excitation-contraction coupling are still debated. We have increased NCX levels in adult rabbit myocytes by adenovirally-mediated gene transfer and examined the effects on excitation-contraction coupling after 24 and 48 h. Infected myocytes were identified through expression of green fluorescent protein (GFP), transfected under a separate promoter on the same viral construct. Control experiments were done with both non-infected myocytes and those infected with adenovirus expressing GFP only. Contraction amplitude was markedly reduced in NCX-overexpressing myocytes at either time point, and neither increasing frequency nor raising extracellular Ca(2+) could reverse this depression. Resting membrane potential and action potential duration were largely unaffected by NCX overexpression, as was peak Ca(2+) entry via the L-type Ca(2+) channel. Systolic and diastolic Ca(2+) levels were significantly reduced, with peak systolic Ca(2+) in NCX-overexpressing myocytes lower than diastolic levels in control cells at 2 m m extracellular Ca(2+). Both cell relengthening and the decay of the Ca(2+) transient were significantly slowed. Sarcoplasmic reticulum (SR) Ca(2+) stores were completely depleted in a majority of myocytes, and remained so despite increasingly vigorous loading protocols. Depressed contractility following NCX overexpression is therefore related to decreased SR Ca(2+) stores and low diastolic Ca(2+) levels rather than reduced Ca(2+) entry.     

The role of p38 in the regulation of Na+-Ca2+ exchanger expression in adult cardiomyocytes

The Na(+)-Ca(2+) exchanger is crucial in the regulation of [Ca(2+)](i) in the cardiac myocyte. The exchanger is upregulated in cardiac hypertrophy and failure. This upregulation can have an effect on calcium transients and possibly contribute to diastolic dysfunction and an increased risk of arrhythmias. Here we use adenovirus mediated gene expression to examine the role of p38 MAP kinase in upregulation of the exchanger in adult cardiac myocytes. We demonstrate that p38 mediates a part of the alpha-adrenergic stimulated upregulation of the Na(+)-Ca(2+) exchanger gene. Overexpression of dominant-negative p38 isoforms and activated MKK3 and MKK6 in isolated adult cardiac myocytes demonstrates that p38 activation is sufficient for NCX1 promoter upregulation and that this is mediated primarily by the p38alpha isoform. Lastly, this work demonstrates that the p38alpha stimulated upregulation of the NCX1 promoter is mediated via the -80 CArG box element. This is the first time that a specific role for p38alpha in gene regulation has been demonstrated in isolated adult cardiomyocytes and provides an important clue to our understanding some of the factors regulating exchanger gene expression in the hypertrophic and failing heart.     

Three Na+/Ca2+ exchanger (NCX) variants are expressed in mouse osteoclasts and mediate calcium transport during bone resorption

The plasma membrane Na(+)/Ca(2+) exchanger (NCX) is a bidirectional transporter that mediates the exchange of Na(+) for Ca(2+) depending on the electrochemical gradients. Mammalian NCXs form a multigene family comprising NCX1, NCX2, and NCX3 isoforms. Although it has been known that NCX1 in rat osteoclasts is coupled with the Na(+)/ H(+) exchanger for regulation of intracellular Ca(2+) concentration ([Ca(2+)](i)), it is unclear what kind of NCX1 variants are expressed and whether the other two NCX isoforms are also present in mouse osteoclasts. To clarify the role of NCXs during bone resorption, we investigated the expression of NCXs, the ion transport via NCXs, and the effects of NCX inhibitors on bone-resorbing activity in mouse osteoclasts. Using RT-PCR, immunocytochemical, and Western blot methods, we detected three splice variants of NCX1 and NCX3, namely NCX1.3, NCX1.41, and NCX3.2. Of these, NCX1.41 is a newly identified splice variant. Low extracellular sodium ([Na(+)](o)) solution increases the intracellular Ca(2+) concentration via NCX transporter in fura-2-loaded osteoclasts. The [Na(+)](o)-free solution-induced [Ca(2+)](i) increase was suppressed by benzyloxyphenyl NCX inhibitors. Bidirectional NCX currents in mouse osteoclasts were recorded using the patch clamp method and could be suppressed with NCX inhibitors. NCX inhibitors also decreased the resorption pit area surrounding osteoclasts in a dose-dependent manner. Furthermore, small interference RNAs targeted against NCX1.3, NCX1.41, and NCX3.2 expressed in mouse osteoclasts suppressed osteoclastic pit formation. These results show that three NCX variants are expressed in mouse osteoclasts and play an important role for Ca(2+) transport and regulation during osteoclastic bone resorption.     

Reperfusion-induced Ins(1,4,5)P(3) generation and arrhythmogenesis require activation of the Na(+)/Ca(2+) exchanger

Reperfusion of globally ischemic rat hearts causes rapid generation of inositol(1,4,5) trisphosphate [Ins(1,4,5)P(3)] and the development of arrhythmias, following stimulation of alpha(1)-adrenergic receptors by norepinephrine released from the cardiac sympathetic nerves. The heightened inositol phosphate response in reperfusion depends on the activation of the Na(+)/H(+) exchanger, which might reflect a central role for increased Ca(2+)following reverse mode activation of the Na(+)/Ca(2+) exchanger (NCX). Isolated, perfused rat hearts were subjected to 20 min ischemia followed by 2 min reperfusion and the content of Ins(1,4,5)P(3) measured by mass analysis or by anion-exchange high performance liquid chromatography (HPLC) following [(3)H]inositol labeling. Reperfusion caused generation of Ins(1,4,5)P(3) (1266+/-401 to 3387+/-256 cpm/g tissue, mean+/-s.e.m., n=6, P<0.01) and the development of arrhythmias. Inhibition of NCX either by reperfusion at low Ca(2+) (1133+/-173 cpm/g tissue, mean+/-s.e.m., n=6, P<0.01 relative to reperfusion control) or by adding 10 microm KB-R7943, an inhibitor of reverse mode Na(+)/Ca(2+) exchange, prevented the Ins(1,4,5)P(3) response (1151+/-243 cpm/g tissue, mean+/-s.e.m., n=6, P<0.01 relative to reperfusion control) and the development of ventricular fibrillation. Lower concentrations of KB-R7943 were less effective. Reverse mode activation of NCX is therefore required for the enhanced Ins(1,4,5)P(3) response in early reperfusion, and inhibitors of this transporter may be useful in the prevention of arrhythmias under such conditions.     

Association of annexin A5 with Na+/Ca2+ exchanger and caveolin-3 in non-failing and failing human heart

Annexin A5 is a Ca2+ dependent phosphatidylserine binding protein mainly located in the T-tubules and sarcolemma of cardiomyocytes. Our objectives were to determine whether annexin A5 was associated with various protein(s) and whether such an association was modified in failing (F) hearts. The association between annexin A5 and the cardiac Na+/Ca2+ exchanger (NCX) was demonstrated by immunohistofluorescence, annexin A5-biotin overlay and co-immunoprecipitations (IPs) performed with microsomal preparations (MPs) from non-failing (NF) (n = 8) and F (dilated cardiomyopathy, n = 7) human hearts. We moreover found caveolin-3 in the immunoprecipitates, indicating the presence of multimolecular subsarcolemmal complexes. Surface plasmon resonance assays in NF MPs allowed us to demonstrate direct interaction between the NCX and caveolin-3 and immobilized annexin A5. Interaction was Ca2+-dependent and inhibited by the specific antibody. In addition, dissociation by zwittergent 3-14 (ZW 3-14) of the complexes from MPs increased specific interactions. In F hearts, specific interactions were blunted in native MPs but were fully recovered after treatment with ZW 3-14. In conclusion, we demonstrated that a direct interaction between annexin A5 and the cardiac NCX occurs in complexes including caveolin-3. In F hearts, despite the increase in the exchanger level, almost all of the NCX was involved in complexes. These interactions probably occurred in the intracytoplasmic regulatory loop of the exchanger, suggesting a different regulation of the exchanger in heart failure, consistent with a role in altered Ca2+ handling.     

Sinoatrial nodal cell ryanodine receptor and Na(+)-Ca(2+) exchanger: molecular partners in pacemaker regulation

The rate of spontaneous diastolic depolarization (DD) of sinoatrial nodal cells (SANCs) that triggers recurrent action potentials (APs) is a fundamental aspect of the heart's pacemaker. Here, in experiments on isolated SANCs, using confocal microscopy combined with a patch clamp technique, we show that ryanodine receptor Ca(2+) release during the DD produces a localized subsarcolemmal Ca(2+) increase that spreads in a wavelike manner by Ca(2+)-induced Ca(2+) release and produces an inward current via the Na(+)-Ca(2+) exchanger (NCX). Ryanodine, a blocker of the sarcoplasmic reticulum Ca(2+) release channel, in a dose-dependent manner reduces the SANC beating rate with an IC(50) of 2.6 micromol/L and abolishes the local Ca(2+) transients that precede the AP upstroke. In voltage-clamped cells in which the DD was simulated by voltage ramp, 3 micromol/L ryanodine decreased an inward current during the voltage ramp by 1.6+/-0.3 pA/pF (SEM, n=4) leaving the peak of L-type Ca(2+) current unchanged. Likewise, acute blockade of the NCX (via rapid substitution of bath Na(+) by Li(+)) abolished SANC beating and reduced the inward current to a similar extent (1.7+/-0.4 pA/pF, n=4), as did ryanodine. Thus, in addition to activation/inactivation of multiple ion channels, Ca(2+) activation of the NCX, because of localized sarcoplasmic reticulum Ca(2+) release, is a critical element in a chain of molecular interactions that permits the heartbeat to occur and determines its beating rate.     

Loss of beta-adrenoceptor response in myocytes overexpressing the Na+/Ca(2+)-exchanger

Increased Na+/Ca(2+)-exchanger (NCX) and altered beta-adrenoceptor (betaAR) responses are observed in failing human heart. To determine the possible interaction between these changes, we investigated the effect of NCX overexpression on responses to isoproterenol in adult rat ventricular myocytes. Responses to isoproterenol were largely mediated through the beta1AR in control myocytes. Adenovirally-mediated overexpression of NCX, at levels, which did not alter basal contraction of myocytes, markedly depressed the isoproterenol concentration-response curve. Responses to isoproterenol could be restored to normal by beta2AR blockade, suggesting a beta2AR-mediated inhibition of beta1AR signalling. Pertussis toxin normalised isoproterenol responses in NCX cells, indicating that beta2AR effects were mediated by Gi. Negative-inotropic effects of high concentrations of ICI 118,551, previously shown to be due to beta2AR-Gi coupling, were increased in NCX cells. We conclude that NCX upregulation can markedly alter the consequences of betaAR stimulation and that this may contribute to the alterations in betaAR response seen in failing human heart.     

Enhanced Ca(2+)-activated Na(+)-Ca(2+) exchange activity in canine pacing-induced heart failure

Defective excitation-contraction coupling in heart failure is generally associated with both a reduction in sarcoplasmic reticulum (SR) Ca(2+) uptake and a greater dependence on transsarcolemmal Na(+)-Ca(2+) exchange (NCX) for Ca(2+) removal. Although a relative increase in NCX is expected when SR function is impaired, few and contradictory studies have addressed whether there is an absolute increase in NCX activity. The present study examines in detail NCX density and function in left ventricular midmyocardial myocytes isolated from normal or tachycardic pacing-induced failing canine hearts. No change of NCX current density was evident in myocytes from failing hearts when intracellular Ca(2+) ([Ca(2+)](i)) was buffered to 200 nmol/L. However, when [Ca(2+)](i) was minimally buffered with 50 micromol/L indo-1, Ca(2+) extrusion via NCX during caffeine application was doubled in failing versus normal cells. In other voltage-clamp experiments in which SR uptake was blocked with thapsigargin, both reverse-mode and forward-mode NCX currents and Ca(2+) transport were increased >2-fold in failing cells. These results suggest that, in addition to a relative increase in NCX function as a consequence of defective SR Ca(2+) uptake, there is an absolute increase in NCX function that depends on [Ca(2+)](i) in the failing heart.     

Functional expression of the Na+/Ca2+ exchanger in the embryonic mouse heart

The Na(+)/Ca(2+) exchanger (NCX) is one of the earliest functional genes and is currently assumed to compensate at least in part for the rudimentary sarcoplasmic reticulum in the developing mouse heart. However, to date little is known about the functional expression of NCX during development. This prompted us to investigate the NCX current (I(NCX)) in very early (embryonic day E8.5-E9.5 post coitum), early (E10.5-E11.5), middle (E13.5) and late (E16.5) stage mouse embryonic cardiomyocytes. For standard I(NCX) measurements, [Ca(2+)](i) was buffered to 150 nmol/l and voltage ramps were applied from +60 mV to -120 mV. At very early stages of development, we observed a prominent role of the I(NCX) Ca(2+) inward mode in elevating the cytosolic Ca(2+) concentration ([Ca(2+)](i)). Accordingly, a high I(NCX) density was observed (+60 mV: 4.6+/-0.7 pA/pF, n=14). Likewise, we found a strong Ca(2+) outward mode of I(NCX) (-120 mV: -3.9+/-0.7 pA/pF, n=14). At later stages, however, I(NCX) Ca(2+) inward mode was reduced by 54+/-6% (n=15, p<0.0001) in ventricular and 68+/-10% (n=9, p<0.0006) in atrial cells. For the outward mode, a reduction by 43+/-10% (n=15, p<0.01) in ventricular and 62+/-11% (n=9, p<0.004) in atrial cardiomyocytes was observed. By contrast, NCX isoform expression and the reversal potential did not significantly change during development. Thus, NCX displays a prominent Ca(2+) inward and outward mode during early embryonic heart development pointing to its important contribution to maintain [Ca(2+)](i) homeostasis. The functional and protein expression of NCX declines during further development.     

Impaired contractile performance of cultured rabbit ventricular myocytes after adenoviral gene transfer of Na(+)-Ca(2+) exchanger

Na(+)-Ca(2+) exchanger (NCX) gene expression is increased in the failing human heart. We investigated the hypothesis that upregulation of NCX can induce depressed contractile performance. Overexpression of NCX was achieved in isolated rabbit ventricular myocytes through adenoviral gene transfer (Ad-NCX). After 48 hours, immunoblots revealed a virus dose-dependent increase in NCX protein. Adenoviral beta-galactosidase transfection served as a control. The fractional shortening (FS) of electrically stimulated myocytes was analyzed. At 60 min(-1), FS was depressed by 15.6% in the Ad-NCX group (n=143) versus the control group (n=163, P:<0.05). Analysis of the shortening-frequency relationship showed a steady increase in FS in the control myocytes (n=26) from 0.027+/-0.002 at 30 min(-1) to 0. 037+/-0.002 at 120 min(-1) (P:<0.05 versus 30 min(-1)) and to 0. 040+/-0.002 at 180 min(-1) (P:<0.05 versus 30 min(-1)). Frequency potentiation of shortening was blunted in NCX-transfected myocytes (n=27). The FS was 0.024+/-0.002 at 30 min(-1), 0.029+/-0.002 at 120 min(-1) (P:<0.05 versus 30 min(-1), P:<0.05 versus control), and 0. 026+/-0.002 at 180 min(-1) (NS versus 30 min(-1), P:<0.05 versus control). Caffeine contractures, which indicate sarcoplasmic reticulum Ca(2+) load, were significantly reduced at 120 min(-1) in NCX-transfected cells. An analysis of postrest behavior showed a decay of FS with longer rest intervals in control cells. Rest decay was significantly higher in the Ad-NCX group; after 120 seconds of rest, FS was 78+/-4% in control and 65+/-3% in the Ad-NCX group (P:<0.05) relative to steady-state FS before rest (100%). In conclusion, the overexpression of NCX in rabbit cardiomyocytes results in the depression of contractile function. This supports the hypothesis that upregulation of NCX can result in systolic myocardial failure.     

The Na+/K+-ATPase alpha2-isoform regulates cardiac contractility in rat cardiomyocytes

OBJECTIVE: The presence of both alpha1- and alpha2-isoforms of the Na+/K+-ATPase (NKA) in cardiomyocytes indicates different functions. We hypothesized that preferential localization of the alpha2-isoform to the t-tubules, locally controlling the Na+/Ca2+-exchanger (NCX), underlies a specific role in Ca2+ handling. METHODS: We studied NKA isoform distribution in isolated cardiomyocytes from Wistar rats using immunocytochemistry. NKA pump and NCX currents (I(pump) and I(NCX)) were measured in control and detubulated cardiomyocytes. Intracellular Na+ concentration [Na+]i was assessed with the fluorescent dye SBFI. RESULTS: The alpha2-isoform abundance was higher in the t-tubules than in the surface sarcolemma. We established that 0.3 microM ouabain specifically blocked the alpha2-isoform in isolated rat cardiomyocytes. This low concentration blocked 10.7+/-0.6% of I(pump) in control, but only 6.0+/-0.5% in detubulated cardiomyocytes. Moreover, measured and calculated alpha1-specific and alpha2-specific I(pump) in control (547+/-29 pA and 66 pA, respectively) and in detubulated cells (495+/-30 pA and 31 pA, respectively) showed that 53% of the alpha2-isoform, but only 9.5% of the alpha1-isoform, were localized to the t-tubules. Despite the small abundance of the alpha2-isoform (approximately 11% of total NKA), selective inhibition of this isoform induced a 40% increase in contractility in field stimulated cardiomyocytes, but no increase in global [Na+]i. However, inhibition of the alpha2-isoform increased I(NCX) indicating local subsarcolemmal accumulation of Na+ near NCX. CONCLUSIONS: The alpha2-isoform of the NKA is functionally coupled to the NCX and can regulate Ca2+ handling without changing global [Na+]i.     

Autocrine stimulation of cardiac Na(+)-Ca(2+) exchanger currents by endogenous endothelin released by angiotensin II

The goal of the present study was to evaluate the effects of Ang II on the current produced by the Na(+)-Ca(2+) exchanger (I(NCX)) working in the reverse mode and the possible autocrine role played by the release of endothelin (ET) in these actions. I(NCX) was studied in isolation in cat cardiac myocytes. Angiotensin II (Ang II) (100 nmol/L) increased I(NCX) at potentials higher than 0 mV (at +60 mV: 2.07 +/- 0.22 pA/pF in control versus 2.73 +/- 0.22 pA/pF in Ang II, n=9; P<0.05). The increase in I(NCX) induced by Ang II was prevented by the treatment of the cells with the unspecific blocker of the ET receptors, TAK 044 (1 micromol/L) (at +60 mV: 2.15 +/- 0.27 pA/pF in control versus 2.01+/- 0.26 pA/pF in Ang II, n=5, NS). These results show, for the first time, that the effect of Ang II on I(NCX) is the result of the autocrine actions of ET released by the octapeptide.     

Stretch-dependent modulation of [Na+]i, [Ca2+]i, and pHi in rabbit myocardium--a mechanism for the slow force response

OBJECTIVE: Rabbit ventricular myocardium is characterized by a biphasic response to stretch with an initial, rapid increase in force followed by a delayed, slow increase in force (slow force response, SFR). The initial phase is attributed to increased myofilament Ca(2+) sensitivity, but the mechanisms of the delayed phase are only incompletely understood. We tested whether stretch-dependent stimulation of Na(+)/H(+) exchange (NHE1) and consecutive changes in pH(i) and/or [Na(+)](i) may underlie the SFR. METHODS: Isometric contractions of rabbit ventricular muscles were recorded in bicarbonate-containing Tyrode's (Tyrode) or bicarbonate-free HEPES-buffered solution (HEPES). Muscles were loaded with the Ca(2+) indicator aequorin, the pH indicator BCECF, or the Na(+) indicator SBFI and rapidly stretched from 88% (L(88)) to 98% (L(98)) of optimal length. The resulting immediate and slow increases in twitch force (1st phase and SFR) as well as changes in [Ca(2+)](i), [Na(+)](i), or pH(i) were quantified before and after inhibition of NHE1 by HOE 642 (3 microM) or reverse-mode Na(+)/Ca(2+) exchange (NCX) by KB-R 7943 (5 microM). RESULTS: In both Tyrode (n=21) and HEPES (n=22), developed force increased to approximately 160% during the 1st phase followed by a further increase to approximately 205% during the SFR. The SFR was accompanied by a 21% increase of the aequorin light transient (n=4; normalized to the 1st phase) and a approximately 3 mM increase in [Na(+)](i) (n=4-7). The SFR was also associated with an increase in pH(i). However, this increase was delayed and was significant only after the SFR had reached its maximum. The delayed pH(i) increase was larger in HEPES than in Tyrode. HOE 642 and/or KB-R 7943 reduced the SFR by approximately 30-40%. In addition, HOE 642 diminished the stretch-mediated elevation of [Na(+)](i) by 72% and the delayed alkalinization. CONCLUSIONS: The data are consistent with the hypothesis that SFR results from increases in [Ca(2+)](i) secondary to altered flux via NCX in part resulting from increases in [Na(+)](i) mediated by NHE1.     

Bimodal regulation of Na(+)--Ca(2+) exchanger by beta-adrenergic signaling pathway in shark ventricular myocytes

In shark heart, the Na(+)--Ca(2+) exchanger serves as a major pathway for both Ca(2+) influx and efflux, as there is only rudimentary sarcoplasmic reticulum in these hearts. The modulation of the exchanger by a beta-adrenergic agonist in whole-cell clamped ventricular myocytes was compared with that of the Na(+)--Ca(2+) exchanger blocker KB-R7943. Application of 5 microM isoproterenol and 10 microM KB-R7943 suppressed both the inward and the outward Na(+)--Ca(2+) exchanger current (I(Na--Ca)). The isoproterenol effect was mimicked by 10 microM forskolin. Isoproterenol and forskolin shifted the reversal potential (E(rev)) of I(Na--Ca) by approximately -23 mV and -30 mV, respectively. An equivalent suppression of outward I(Na--Ca) by KB-R7943 to that by isoproterenol produced a significantly smaller shift in E(rev) of about --4 mV. The ratio of inward to outward exchanger currents was also significantly larger in isoproterenol- than in control- and KB-R7943-treated myocytes. Our data suggest that the larger ratio of inward to outward exchanger currents as well as the larger shift in E(rev) with isoproterenol results from the enhanced efficacy of Ca(2+) efflux via the exchanger. The protein kinase A-mediated bimodal regulation of the exchanger in parallel with phosphorylation of the Ca(2+) channel and enhancement of its current may have evolved to satisfy the evolutionary needs for accelerated contraction and relaxation in hearts of animals with vestigial sarcoplasmic Ca(2+) release stores.     

Na(+)-Ca(2+) exchange current and submembrane [Ca(2+)] during the cardiac action potential

Na(+)-Ca(2+) exchange (NCX) is crucial in the regulation of [Ca(2+)](i) and cardiac contractility, but key details of its dynamic function during the heartbeat are not known. In the present study, we assess how NCX current (I(NCX)) varies during a rabbit ventricular action potential (AP). First, we measured the steady-state voltage and [Ca(2+)](i) dependence of I(NCX) under conditions when [Ca(2+)](i) was heavily buffered. We then used this relationship to infer the submembrane [Ca(2+)](i) ([Ca(2+)](sm)) sensed by NCX during a normal AP and [Ca(2+)](i) transient (when the AP was interrupted to produce an I(NCX) tail current). The [Ca(2+)](i) dependence of I(NCX) at -90 mV allowed us to convert the peak inward I(NCX) tail currents to [Ca(2+)](sm). Peak [Ca(2+)](sm) measured via this technique was >3.2 micromol/L within < 32 ms of the AP upstroke (versus peak [Ca(2+)](i) of 1.1 micromol/L at 81 ms measured with the global Ca(2+) indicator indo-1). The voltage and [Ca(2+)](sm) dependence of I(NCX) allowed us to infer I(NCX) during the normal AP and Ca(2+) transient. The early rise in [Ca(2+)](sm) causes I(NCX) to be inward for the majority of the AP. Thus, little Ca(2+) influx via NCX is expected under physiological conditions, but this can differ among species and in pathophysiological conditions.     

Cardiac Na(+)-Ca(2+) exchange: molecular and pharmacological aspects

The Na(+)-Ca(2+) exchanger (NCX) is one of the essential regulators of Ca(2+) homeostasis in cardiomyocytes and thus an important modulator of the cardiac contractile function. The purpose of this review is to survey recent advances in cardiac NCX research, with particular emphasis on molecular and pharmacological aspects. The NCX function is thought to be regulated by a variety of cellular factors. However, data obtained by use of different experimental systems often appear to be in conflict. Where possible, we endeavor to provide a rational interpretation of such data. We also provide a summary of current work relating to the structure and function of the cardiac NCX. Recent molecular studies of the NCX protein are beginning to shed light on structural features of the ion translocation pathway in the NCX membrane domain, which seems likely to be formed, at least partly, by the phylogenetically conserved alpha-1 and alpha-2 repeat structures and their neighboring membrane-spanning segments. Finally, we discuss new classes of NCX inhibitors with improved selectivity. One of these, 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulfonate (KB-R7943), appears to exhibit unique selectivity for Ca(2+)-influx-mode NCX activity. Data obtained with these inhibitors should provide a basis for designing more selective and clinically useful drugs targeting NCX.