Polyaromatic hydrocarbons in pollution: a heart-breaking matter.

Air pollution is associated with detrimental effects on human health, including decreased cardiovascular function. However, the causative mechanisms behind these effects have yet to be fully elucidated. Here we review the current epidemiological, clinical and experimental evidence linking pollution with cardiovascular dysfunction. Our focus is on particulate matter (PM) and the associated low molecular weight polycyclic aromatic hydrocarbons (PAHs) as key mediators of cardiotoxicity. We begin by reviewing the growing epidemiological evidence linking air pollution to cardiovascular dysfunction in humans. We next address the pollution-based cardiotoxic mechanisms first identified in fish following the release of large quantities of PAHs into the marine environment from point oil spills (e.g. Deepwater Horizon). We finish by discussing the current state of mechanistic knowledge linking PM and PAH exposure to mammalian cardiovascular patho-physiologies such as atherosclerosis, cardiac hypertrophy, arrhythmias, contractile dysfunction and the underlying alterations in gene regulation. Our aim is to show conservation of toxicant pathways and cellular targets across vertebrate hearts to allow a broad framework of the global problem of cardiotoxic pollution to be established. AhR; Aryl hydrocarbon receptor. Dark lines indicate topics discussed in this review. Grey lines indicate topics reviewed elsewhere.

In silico investigation of a KCNQ1 mutation associated with familial atrial fibrillation.

Mutations in transmembrane domains of the KCNQ1 subunit of the I(Ks) potassium channel have been associated with familial atrial fibrillation. We have investigated mechanisms by which the S1 domain S140G KCNQ1 mutation influences atrial arrhythmia risk and, additionally, whether it can affect ventricular electrophysiology. In perforated-patch recordings, S140G-KCNQ1+KCNE1 exhibited leftward-shifted activation, slowed deactivation and marked residual current. In human atrial action potential (AP) simulations, AP duration and refractoriness were shortened and rate-dependence flattened. Simulated I(Ks) but not I(Kr) block offset AP shortening produced by the mutation. In atrial tissue simulations, temporal vulnerability to re-entry was little affected by the S140G mutation. Spatial vulnerability was markedly increased, leading to more stable and stationary spiral wave re-entry in 2D stimulations, which was offset by I(Ks) block, and to scroll waves in 3D simulations. These changes account for vulnerability to AF with this mutation. Ventricular AP clamp experiments indicate a propensity for increased ventricular I(Ks) with the S140G KCNQ1 mutation and ventricular AP simulations showed model-dependent ventricular AP abbreviation.

The key characteristics of cardiotoxicity for the pervasive pollutant phenanthrene.

The key characteristic (KCs) framework has been used previously to assess the carcinogenicity and cardiotoxicity of various chemical and pharmacological agents. Here, the 12 KCs of cardiotoxicity are used to evaluate the previously reported cardiotoxicity of phenanthrene (Phe), a tricyclic polycyclic aromatic hydrocarbon (PAH), and major component of fossil fuel-derived air pollution. Phe is a semi-volatile pollutant existing in both the gas phase and particle phase through adsorption onto or into particulate matter (PM). Phe can translocate across the airways and gastrointestinal tract into the systemic circulation, enabling body-wide effects. Our evaluation based on a comprehensive literature review, indicates Phe exhibits 11 of the 12 KCs for cardiotoxicity. These include adverse effects on cardiac electromechanical performance, the vasculature and endothelium, immunomodulation and oxidative stress, and neuronal and endocrine control. Environmental agents that have similarly damaging effects on the cardiovascular system are heavily regulated and monitored, yet globally there is no air quality regulation specific for PAHs like Phe. Environmental monitoring of Phe is not the international standard with benzo[a]pyrene being frequently used as a proxy despite the two PAH species exhibiting significant differences in sources, concentration variations and toxic effects. The evidence summarised in this evaluation highlights the need to move away from proxied PAH measurements and develop a monitoring network capable of measuring Phe concentration. It also stresses the need to raise awareness amongst the medical community of the potential cardiovascular impact of PAH exposure. This will allow the production of mitigation strategies and possibly the development of new policies for the protection of the societal groups most vulnerable to cardiovascular disease.

Pro-arrhythmic effects of gain-of-function potassium channel mutations in the short QT syndrome.

The congenital short QT syndrome (SQTS) is a rare condition characterized by abbreviated rate-corrected QT (QTc) intervals on the electrocardiogram and by increased susceptibility to both atrial and ventricular arrhythmias and sudden death. Although mutations to multiple genes have been implicated in the SQTS, evidence of causality is particularly strong for the first three (SQT1-3) variants: these result from gain-of-function mutations in genes that encode K+ channel subunits responsible, respectively, for the IKr, IKs and IK1 cardiac potassium currents. This article reviews evidence for the impact of SQT1-3 missense potassium channel gene mutations on the electrophysiological properties of IKr, IKs and IK1 and of the links between these changes and arrhythmia susceptibility. Data from experimental and simulation studies and future directions for research in this field are considered. This article is part of the theme issue 'The heartbeat: its molecular basis and physiological mechanisms'.

Enhanced inhibitory effect of acidosis on hERG potassium channels that incorporate the hERG1b isoform.

Extracellular acidosis occurs in the heart during myocardial ischemia and can lead to dangerous arrhythmias. Potassium channels encoded by hERG (human ether-à-go-go-related gene) mediate the cardiac rapid delayed rectifier K+ current (IKr), and impaired hERG function can exacerbate arrhythmia risk. Nearly all electrophysiological investigations of hERG have centred on the hERG1a isoform, although native IKr channels may be comprised of hERG1a and hERG1b, which has a unique shorter N-terminus. This study has characterised for the first time the effects of extracellular acidosis (an extracellular pH decrease from 7.4 to 6.3) on hERG channels incorporating the hERG1b isoform. Acidosis inhibited hERG1b current amplitude to a significantly greater extent than that of hERG1a, with intermediate effects on coexpressed hERG1a/1b. IhERG tail deactivation was accelerated by acidosis for both isoforms. hERG1a/1b activation was positively voltage-shifted by acidosis, and the fully-activated current-voltage relation was reduced in amplitude and right-shifted (by ∼10 mV). Peak IhERG1a/1b during both ventricular and atrial action potentials was both suppressed and positively voltage-shifted by acidosis. Differential expression of hERG isoforms may contribute to regional differences in IKr in the heart. Therefore inhibitory effects of acidosis on IKr could also differ regionally, depending on the relative expression levels of hERG1a and 1b, thereby increasing dispersion of repolarization and arrhythmia risk.

Pharmacological inhibition of the hERG potassium channel is modulated by extracellular but not intracellular acidosis.

INTRODUCTION: Human ether-à-go-go related gene (hERG) is responsible for channels that mediate the rapid delayed rectifier K(+) channel current (I(Kr) ), which participates in repolarization of the ventricles and is a target for some antiarrhythmic drugs. Acidosis occurs in the heart in some pathological situations and can modify the function and responses to drugs of ion channels. The aim of this study was to determine the effects of extracellular and intracellular acidosis on the potency of hERG channel current (I(hERG)) inhibition by the antiarrhythmic agents dofetilide, flecainide, and amiodarone at 37 °C. METHODS AND RESULTS: Whole-cell patch-clamp recordings of I(hERG) were made at 37 °C from hERG-expressing Human Embryonic Kidney (HEK293) cells. Half-maximal inhibitory concentration (IC(50)) values for I(hERG) tail inhibition at -40 mV following depolarizing commands to +20 mV were significantly higher at external pH 6.3 than at pH 7.4 for both flecainide and dofetilide, but not for amiodarone. Lowering pipette pH from 7.2 to 6.3 altered neither I(hERG) kinetics nor the extent of observed I(hERG) blockade by any of these drugs. CONCLUSION: Conditions leading to localized extracellular acidosis may facilitate heterogeneity of action of dofetilide and flecainide, but not amiodarone via modification of hERG channel blockade. Such effects depend on the external pH change rather than intracellular acidification.

One-dimensional mathematical model of the atrioventricular node including atrio-nodal, nodal, and nodal-his cells.

Mathematical models are a repository of knowledge as well as research and teaching tools. Although action potential models have been developed for most regions of the heart, there is no model for the atrioventricular node (AVN). We have developed action potential models for single atrio-nodal, nodal, and nodal-His cells. The models have the same action potential shapes and refractoriness as observed in experiments. Using these models, together with models for the sinoatrial node (SAN) and atrial muscle, we have developed a one-dimensional (1D) multicellular model including the SAN and AVN. The multicellular model has slow and fast pathways into the AVN and using it we have analyzed the rich behavior of the AVN. Under normal conditions, action potentials were initiated in the SAN center and then propagated through the atrium and AVN. The relationship between the AVN conduction time and the timing of a premature stimulus (conduction curve) is consistent with experimental data. After premature stimulation, atrioventricular nodal reentry could occur. After slow pathway ablation or block of the L-type Ca(2+) current, atrioventricular nodal reentry was abolished. During atrial fibrillation, the AVN limited the number of action potentials transmitted to the ventricle. In the absence of SAN pacemaking, the inferior nodal extension acted as the pacemaker. In conclusion, we have developed what we believe is the first detailed mathematical model of the AVN and it shows the typical physiological and pathophysiological characteristics of the tissue. The model can be used as a tool to analyze the complex structure and behavior of the AVN.

hERG1a/1b heteromeric currents exhibit amplified attenuation of inactivation in variant 1 short QT syndrome.

Potassium channels encoded by hERG (human ether-à-go-go-related gene) underlie the cardiac rapid delayed rectifier K+ current (IKr) and hERG mutations underpin clinically important repolarization disorders. Virtually all electrophysiological investigations of hERG mutations have studied exclusively the hERG1a isoform; however, recent evidence indicates that native IKr channels may be comprised of hERG1a together with the hERG1b variant, which has a shorter N-terminus. Here, for the first time, electrophysiological effects were studied of a gain-of-function hERG mutation (N588K; responsible for the 'SQT1' variant of the short QT syndrome) on current (I(hERG1a/1b)) carried by co-expressed hERG1a/1b channels. There were no significant effects of N588K on I(hERG1a/1b) activation or deactivation, but N588K I(hERG1a/1b) showed little inactivation up to highly positive voltages (< or = +80 mV), a more marked effect than seen for hERG1a expressed alone. I(hERG1a/1b) under action potential voltage-clamp, and the effects on this of the N588K mutation, also showed differences from those previously reported for hERG1a. The amplified attenuation of I(hERG) inactivation for the N588K mutation reported here indicates that the study of co-expressed hERG1a/1b channels should be considered when investigating clinically relevant hERG channel mutations, even if these reside outside of the N-terminus region.

More types than one: multiple muscarinic receptor coupled K+ currents undergo remodelling in an experimental model of atrial fibrillation.

The common cardiac arrhythmia atrial fibrillation (AF) tends to show progression in its severity, which is associated with 'remodelling': structural and electrophysiological changes that facilitate arrhythmia induction and maintenance. In this issue of the BJP, Yeh and colleagues demonstrate for the first time, down-regulation of three types of muscarinic cholinergic receptor (mAChR) coupled K+ currents (IKM2, IKM3 and IKM4) and of M2, M3 and M4 mAChR subtype proteins, in a canine model of atrial tachycardia (AT) induced remodelling. The IKMs and their extent of AT-induced remodelling were similar in left-atrial and pulmonary vein (PV) myocytes, so remodelling of M2-M4 receptor-linked currents appears not to underlie the unique contribution of PVs to AF. Parasympathetic stimulation can increase susceptibility to AF; thus remodelling of M2-M4 receptors and K+ currents could be adaptive in AT. Further work is warranted to determine whether or not remodelling of multiple mAChRs and currents also contributes to human AF.

Inhibition of the HERG potassium channel by the tricyclic antidepressant doxepin.

HERG (human ether-à-go-go-related gene) encodes channels responsible for the cardiac rapid delayed rectifier potassium current, I(Kr). This study investigated the effects on HERG channels of doxepin, a tricyclic antidepressant linked to QT interval prolongation and cardiac arrhythmia. Whole-cell patch-clamp recordings were made at 37 degrees C of recombinant HERG channel current (I(HERG)), and of native I(Kr) 'tails' from rabbit ventricular myocytes. Doxepin inhibited I(HERG) with an IC(50) value of 6.5+/-1.4 microM and native I(Kr) with an IC(50) of 4.4+/-0.6 microM. The inhibitory effect on I(HERG) developed rapidly upon membrane depolarization, but with no significant dependence on voltage and with little alteration to the voltage-dependent kinetics of I(HERG). Neither the S631A nor N588K inactivation-attenuating mutations (of residues located in the channel pore and external S5-Pore linker, respectively) significantly reduced the potency of inhibition. The S6 point mutation Y652A increased the IC(50) for I(HERG) blockade by approximately 4.2-fold; the F656A mutant also attenuated doxepin's action at some concentrations. HERG channel blockade is likely to underpin reported cases of QT interval prolongation with doxepin. Notably, this study also establishes doxepin as an effective inhibitor of mutant (N588K) HERG channels responsible for variant 1 of the short QT syndrome.

VEGF activates receptor-operated cation channels in human microvascular endothelial cells.

OBJECTIVE: Vascular endothelial growth factor (VEGF) exerts many of its effects by stimulating endothelial calcium influx, but little is known about channels mediating VEGF-induced cation entry. The aim of this study was to measure and characterize for the first time the VEGF-activated cation current in human microvascular endothelial cells (HMVECs). METHODS AND RESULTS: Whole-cell patch-clamp recordings were made from HMVECs. During applied voltage ramps, VEGF activated a current that reversed at 0 mV, was sensitive to gadolinium, and required extracellular cations. Noise analysis yielded a single-channel conductance of 27 pS. The current was not dependent on intracellular calcium stores, and was not blocked by inositol triphosphate (IP3) receptor or serine/threonine kinase inhibition but was partially inhibited by flufenamic acid. A similar current was activated by 1-oleoyl-2-acetyl-sn-glycerol (OAG), a membrane-permeant analog of diacylglycerol (DAG). To determine whether VEGF could activate recombinant ion channels with similar properties, we investigated the effect of VEGF on Chinese hamster ovary cells cotransfected with VEGFR2 and the canonical transient receptor potential (TRPC) channels, TRPC3 or TRPC6. VEGF induced a similar current to that described above in VEGFR2-TRPC3 and VEGFR2-TRPC6 cells but not in cells transfected with either cDNA alone. CONCLUSIONS: VEGF activates a receptor-operated cation current in HMVECs and OAG can activate directly a similar current in these cells. VEGF is also able to activate heterologously expressed TRPC3/6 channels through VEGFR2.

Combined hERG channel inhibition and disruption of trafficking in drug-induced long QT syndrome by fluoxetine: a case-study in cardiac safety pharmacology.

Drug-induced prolongation of the rate-corrected QT interval (QTCI) on the electrocardiogram occurs as an unwanted effect of diverse clinical and investigational drugs and carries a risk of potentially fatal cardiac arrhythmias. hERG (human ether-à-go-go-related gene) is the gene encoding the alpha-subunit of channels mediating the rapid delayed rectifier K+ current, which plays a vital role in repolarising the ventricles of the heart. Most QTCI prolonging drugs can inhibit the function of recombinant hERG K+ channels, consequently in vitro hERG assays are used widely as front-line screens in cardiac safety-testing of novel chemical entities. In this issue, Rajamani and colleagues report a case of QTCI prolongation with the antidepressant fluoxetine and correlate this with a dual effect of the drug and of its major metabolite norfluoxetine on hERG channels. Both compounds were found to produce an acute inhibition of the hERG channel by pharmacological blockade, but in addition they also were able to disrupt the normal trafficking of hERG protein to the cell membrane. Mutations to a key component of the drug binding site in the S6 region of the channel greatly attenuated channel block, but did not impair disruption of trafficking; this suggests that channel block and drug effects on trafficking were mediated by different mechanisms. These findings add to growing evidence for disruption of hERG channel trafficking as a mechanism for drug-induced long QT syndrome and raise questions as to possible limitations of acute screening methods in the assessment of QTcI prolonging liability of drugs in development.

A role for depolarisation induced calcium entry on the Na-Ca exchange in triggering intracellular calcium release and contraction in rat ventricular myocytes.

OBJECTIVE: The aim was to test whether depolarisation-induced calcium entry on the Na-Ca exchange is able to trigger calcium release from the sarcoplasmic reticulum in rat ventricular myocytes. METHODS: Myocytes were isolated enzymatically from the left ventricle of the rat heart. Cells were impaled with narrow tipped microelectrodes to minimise intracellular dialysis and maintain normal internal ionic conditions. Cells were voltage clamped, contraction was measured optically, and in some experiments intracellular calcium was measured with Fura-2. RESULTS: When the fast Na current was inactivated by using a holding potential of -40 mV, Ca entry via L-type Ca channels was expected to be the only mechanism capable of triggering sarcoplasmic reticular Ca release. In this situation, blocking L-type Ca channels should have abolished sarcoplasmic reticular release and the phasic twitch. However, after 2 min exposure to 20 microM nifedipine, which abolished the Ca current (ICa) completely, voltage clamp depolarisation from -40 mV to 0 mV still elicited 41(SEM 8.9)% of the control phasic twitch (n = 22 cells). This shows that there must be another mechanism, besides Ca entry via Ca channels, by which membrane depolarisation can trigger sarcoplasmic reticular release and the phasic twitch. The phasic twitch that remained in the presence of nifedipine increased progressively with the magnitude of step depolarisation, required a functional sarcoplasmic reticulum, was abolished by 5 mM external nickel, and was sensitive to both the Na and Ca transmembrane gradients. CONCLUSIONS: The voltage dependent sarcolemmal Na-Ca exchange is predicted theoretically to generate a transient Ca entry at the start of a step membrane depolarisation, when membrane potential suddenly becomes more positive than the reversal potential of the Na-Ca exchange. The results of this study indicate that in rat myocytes with normal internal ions, physiological levels of membrane depolarisation generate a sufficient Ca entry on the exchange to trigger sarcoplasmic reticular calcium release and contraction. In the absence of ICa, this mechanism is capable of triggering a calcium release which leads to about 40% of the phasic contraction in cells depolarised from -40 mV to 0 mV. The existence of this sarcoplasmic triggering mechanism may have significance for the normal control of cardiac muscle contraction.

One hump or two? The triggering of calcium release from the sarcoplasmic reticulum and the voltage dependence of contraction in mammalian cardiac muscle.

We have examined the hypothesis that the sarcolemmal Na/Ca exchanger is able to trigger calcium release from the sarcoplasmic reticulum in a direct fashion. We propose that when the cardiac muscle membrane is depolarised, for instance during the upstroke of the action potential or a square voltage clamp pulse, the voltage dependent Na/Ca exchanger generates an initial "spike" of calcium entry which is sufficient to trigger a fraction of the normal sarcoplasmic reticular calcium release, via calcium induced calcium release. For the last 20 years, it has been widely considered that calcium entry through L-type calcium channels is the only trigger for calcium release from sarcoplasmic reticulum in cardiac muscle. In the first section of this review, we examined some of the earlier studies of excitation-contraction coupling which used multicellular preparations of cardiac muscle. We suggested that these earlier studies do not support the idea that calcium entry via the calcium current (ICa) is the only trigger for sarcoplasmic reticular release. In contrast, more recent studies using isolated myocytes have supported ICa as the only trigger. However, these were performed mostly with a low or absent sodium inside the cell, or with an increased intracellular calcium buffering, or with other altered internal ions (eg, high magnesium or caesium in the pipette) or at a relatively low temperature. All these factors may have reduced or abolished the initial spike of calcium entry which the Na/Ca exchanger is expected to generate at the start of depolarisation. New studies on myocytes are presented, using conditions where cells are dialysed minimally, or where a normal level of internal sodium is preserved deliberately.(ABSTRACT TRUNCATED AT 250 WORDS)

Cultured adult cardiac myocytes: future applications, culture methods, morphological and electrophysiological properties.

Isolated adult cardiac myocytes maintained in primary culture have been used as a model of the adult myocardium for 20 years. With the recent advances and current interest in using molecular biological techniques to investigate cardiac physiology, culturing myocytes is becoming an increasingly important technique. Acutely isolated myocytes do not remain viable for the time needed for the changes in gene expression to occur, and therefore it is necessary to maintain myocytes in culture. The aims of this review are: (1) To describe a method for isolating and culturing myocytes in serum-free medium. This section is targeted at new researchers in the field, with particular emphasis on aspects of the isolation procedure which are important for optimising myocyte culture. (2) To review current knowledge of how contractile, electrophysiological and morphological properties of adult myocytes are preserved in culture. Over the past 5 to 10 years significant advances have been made in developing novel techniques which help maintain the in-vivo properties of myocytes in culture. Efficient methods for transporting exogenous genes and anti-sense oligonucleotides into adult myocytes are now available. We anticipate that in future these advances will make cultured myocytes more attractive for use in biophysical and molecular investigations of cardiac physiology.

Evidence for a modulatory effect of external potassium ions on ionic current mediated by the cardiac Na+-Ca2+ exchanger.

The aim of this study was to investigate whether or not the activity of the cardiac Na(+)-Ca(2+) exchanger might be directly sensitive to external K(+) concentration ([K(+)](e)). Measurements of whole-cell exchanger current (I(NaCa)) were made at 37 degrees C from guinea-pig isolated ventricular myocytes, using whole-cell patch clamp recording with major interfering conductances blocked. Changing [K(+)](e) from 0 to 5mM significantly reduced both outward and inward exchange currents in a time-dependent manner. Various [K(+)](e) between 1 and 15 mM were tested and the inhibitory effect was observed to be concentration-dependent. At steady-state, 5mM [K(+)](e) decreased the density of Ni(2+)-sensitive current by 52.8+/-4.3% (mean+/-S.E.M., n=6) and of 0Na0Ca-sensitive current by 39.0+/-4.4% (n=5). The possibility that the inhibitory effect of external K(+) on I(NaCa) might wholly or in part be secondary to activation of the sarcolemmal Na(+)-K(+) pump was investigated by testing the effect of K(+) addition in the presence of a high concentration of strophanthidin (500 microM). Ni(2+)-sensitive I(NaCa) was still observed to be sensitive to external K(+) (I(NaCa) decreased by 39.4+/-9.4%, n=4), suggesting that the inhibitory effect could occur independently of activation of the Na(+)-K(+) pump. The effect of external K(+) on I(NaCa) was verified using a baby hamster kidney (BHK) cell line stably expressing the cardiac Na(+)-Ca(2+) exchanger isoform, NCX1. Similar to native I(NaCa), NCX1 current was also suppressed by [K(+)](e). However, [K(+)](e) did not alter current amplitude in untransfected BHK cells. The effect of [K(+)](e) on I(NaCa) could not be attributed to simply adding any monovalent cation back to the external solution, since it was not reproduced by application of equimolar Li(+), Cs(+) and TEA(+). Rb(+), however, could mimic the effect of K(+). Collectively, these data suggest that external K(+) at physiologically and pathologically relevant concentrations might be able to modulate directly the activity of the cardiac Na(+)-Ca(2+) exchanger.

Anti-adrenergic effect of adenosine on Na(+)-Ca(2+) exchange current recorded from guinea-pig ventricular myocytes.

The Na(+)-Ca(2+) exchanger is a protein present in the cell membrane of many cell types. In heart it plays important roles in Ca homeostasis and ionic current generation. Recently, it has been reported that the beta-adrenergic agonist isoprenaline (ISO) can increase directly Na(+)-Ca(2+) exchanger activity in guinea-pig ventricular myocytes. Adenosine (ADO) exerts anti-adrenergic properties that make it effective against some arrhythmias and the aim of the present study was to determine whether or not ADO can antagonize the direct modulatory effect of ISO on the exchanger.Whole-cell patch clamp measurements of Na(+)-Ca(2+) exchanger current (I(NaCa)) were made from guinea-pig ventricular myocytes, with major interfering currents inhibited. I(NaCa) was measured at 378 degrees C as current sensitive to external nickel (Ni(2+), 10 mM) during an applied descending voltage ramp. ISO (1 microM) significantly increased both inward and outward I(NaCa). This effect was abolished in the presence of ADO (200 microM). ADO alone did not significantly alter the amplitude of I(NaCa). The effect of ADO on the response of I(NaCa) to ISO was mimicked by the A(1)ADO receptor agonist N(6)-cyclopentyladenosine (CPA, 10 microM), whereas the effect of ADO on the response of I(NaCa) to ISO was inhibited by the A(1)ADO receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 2 microM). These data suggest that the A(1)ADO receptor mediated the response. The anti-adrenergic effects on I(NaCa) of ADO were not affected by the protein kinase C (PKC) inhibitor, chelerythrine (CLT, 1 microM), nor by the nitric oxide (NO) synthase inhibitor, N (G)-nitro-L-arginine methyl ester((L)-NAME, 0.5 mM). Moreover, in the presence of PKC activator phorbol 12-myristate 13-acetate (PMA, 1 microM) or exogenous NO donor sodium nitroprusside (SNP, 100 microM), ISO preserved its stimulatory effect on I(NaCa). However, prior incubation of myocytes with pertussis toxin (PTX, 5 microg ml(-1) did prevent the effect of ADO. The anti-adrenergic effect of ADO on I(NaCa) was mimicked by externally applied carbachol (CCh, 10 microM), a muscarinic receptor agonist. We conclude that ADO antagonized the effect of beta-adrenergic stimulation of I(NaCa) by directly activating inhibitory G-protein (G(i))-linked A(1) receptors in guinea-pig ventricular myocytes. These findings may suggest a novel mechanism by which adenosine exerts some of its antiarrhythmic effects.

Regulation by endothelin-1 of Na+-Ca2+ exchange current (I(NaCa)) from guinea-pig isolated ventricular myocytes.

The cardiac Na+-Ca2+ exchanger participates in Ca homeostasis, and Na+-Ca2+ exchanger-mediated ionic current (I(NaCa)) also contributes to the regulation of cardiac action potential duration. Moreover, I(NaCa) can contribute to arrhythmogenesis under conditions of cellular Ca overload. Although it has been shown that the peptide hormone endothelin-1 (ET-1) can phosphorylate the cardiac Na+-Ca2+ exchanger via protein kinase C (PKC), little is known about the effect of ET-1 on I(NaCa). In order to examine the effects of ET-1 on I(NaCa), whole-cell patch clamp measurements were made at 378C from guinea-pig isolated ventricular myocytes. With major interfering currents inhibited, I(NaCa) was measured as the current sensitive to nickel (Ni; 10mM) during a descending voltage ramp. ET-1 (10 nM) significantly increased I(NaCa) ( approximately 2-fold at -100 mV). Application of a PKC activator (PMA; 1mM: phorbol 12-myristate 13-acetate), mimicked the effect of ET-1. In contrast, the PKC inhibitor chelerythrine (CLT, 1mM) abolished the stimulatory effect of ET-1. An inactive phorbol ester, 4-alpha-phorbol-12,13-didecanoate (4a-PDD, 1mM) had no effect on I(NaCa). Collectively, these data indicate that ET-1 activated I(NaCa) through a PKC-dependent pathway. In additional experiments, isoprenaline (ISO; which has also been reported to activate I(NaCa) ) was applied. The increase in I(NaCa) density with ISO (1mM) was similar to that induced by ET-1 (10nM). When I(NaCa) was pre-stimulated by ET-1, application of ISO elicited no further increase in current and vice versa. ISO also had no additional effect on I(NaCa) when the cells were pretreated with PMA. Application of CLT did not alter the response of I(NaCa) to ISO. We conclude that ET-1 stimulated ventricular I(NaCa) via a PKC-dependent mechanism under our recording conditions. Concentrations of ET-1 and ISO that stimulated I(NaCa) to similar extents when applied separately were not additive when co-applied. The lack of synergy between the stimulatory effects of ET-1 and ISO may be important in protecting the heart from the potentially deleterious consequences of excessive stimulation of I(NaCa).

Inhibition of Na/Ca exchange by external Ni in guinea-pig ventricular myocytes at 37 degrees C, dialysed internally with cAMP-free and cAMP-containing solutions.

In many mammalian tissue types an integral membrane protein--the sodium/calcium (Na/Ca) exchanger--plays a key role in intracellular Ca homeostasis, and evidence suggests that Na/Ca exchange function can be modulated by cAMP-dependent phosphorylation. External Nickel (Ni) ions are used widely to inhibit the exchange but little is known about the mode of Ni action. In guinea-pig ventricular myocytes, we investigated inhibition of Na/Ca exchange by external Ni under phosphorylated (cells dialysed with cAMP) and non-phosphorylated conditions. Ventricular myocytes were isolated from adult guinea-pig hearts, recordings were made at 37 degrees C using the whole-cell patch clamp technique. Internal and external solutions were used which allowed Na/Ca exchange current (INaCa) to be measured during a descending voltage ramp protocol (+80 to -120 mV) applied from a holding potential of -40 mV. The application of 10 mM Ni caused a maximal block of INaCa since inhibition was identical to that when a Na- and Ca-free (0Na/0Ca) solution was superfused externally. Kinetics of Ni-block of INaCa were assessed using applications of different external [Ni] to cells dialysed internally with cAMP-free and 100 microM cAMP-containing solutions. At +60 mV, Ni inhibited INaCa in cells dialysed with a cAMP-free solution with a dissociation constant (KD) of 0.29 +/- 0.03 mM and the data were fitted with a Hill coefficient of 0.89 +/- 0.07 (n = 9 cells). In cells dialysed with 100 microM cAMP the exchange was inhibited by Ni with a KD of 0.16 +/- 0.05 mM, the Hill coefficient was 0.82 +/- 0.16 (n = 6-7 cells). The KD and Hill coefficient values obtained in cells dialysed with cAMP-free and cAMP-containing solutions were not significantly different. Inhibition of INaCa by Ni did not appear to be voltage-dependent, was maximal within 3-4 s of application and was rapidly reversible. With cAMP-free internal dialysate, inhibition was 'mixed' showing competition with external Ca and a degree of non-competitive block. With 100 microM cAMP the inhibition appeared to be more non-competitive. We conclude that, under these experimental conditions, a concentration of external Ni of 10 mM is sufficient to produce maximal inhibition of INaCa in guinea-pig cardiac cells.

Actions of myristyl-FRCRCFa, a cell-permeant blocker of the cardiac sarcolemmal Na-Ca exchanger, tested in rabbit ventricular myocytes.

In cardiac muscle, the electrogenic Na-Ca exchanger plays important roles in determining action potential shape and in the beat-to-beat homeostasis of intracellular calcium. In this study we tested the actions of a putative cell-permeant blocker of the cardiac sarcolemmal Na-Ca exchange, "Myristyl- (Myr-) FRCRCFa". Experiments were performed using isolated rabbit right ventricular myocytes and whole-cell patch-clamp at 35-37 degreesC. The Na-Ca exchange current (INa-Ca), L-type calcium current (ICa,L), inward rectifier potassium current (IK1) and delayed rectifier potassium current (IK) were compared in untreated cells and cells incubated in a solution containing N-myristylated FRCRCFa. With other major currents blocked, INa-Ca was measured as the Ni-sensitive component of current during a voltage ramp applied from the holding potential of -40 mV, between +80 and -120 mV (ramp velocity 0.1 V s-1). In untreated cells, INa-Ca at +60 mV was 7.1+/-0.6 pA/pF and at -100 mV was -2.7+/-0.3 pA/pF (n=9). After a 15-min pre-incubation with 20 microM Myr-FRCRCFa, INa-Ca was reduced to 4.2+/-0.3 pA/pF at +60 mV and -1. 5+/-0.2 pA/pF at -100 mV (P<0.02; n=7). After incubation with 20 microM Myr-FRCRCFa for 1 h, INa-Ca at both potentials was further reduced (2.3+/-0.8 pA/pF at +60 mV; -0.9+/-0.3 pA/pF at -100 mV; P<0. 008 compared with control; n=4). Under selective recording conditions for ICa,L, there was little difference in ICa,L density between untreated and cells incubated with Myr-FRCRCFa. A Boltzmann fit to the ICa,L/V relation showed no significant alteration of half-maximal activation potential or slope factor of activation. IK1 was also largely unaffected by pre-incubation of cells with Myr-FRCRCFa. IK, measured as deactivating tail current following 1-s test depolarisations to a range of test potentials, was also not significantly altered by Myr-FRCRCFa. The suppression of INa-Ca in cells incubated in Myr-FRCRCFa suggests that addition of the myristyl group to FRCRCFa peptide conveys cell permeancy to the peptide and that Myr-FRCRCFa applied externally to rabbit ventricular myocytes is moderately effective as an INa-Ca blocker. ICa,L, IK1 and IK were largely unaffected by Myr-FRCRCFa. N-Myristylation of such conformationally constrained hexapeptides may, therefore, provide a means of producing cell-permeant inhibitors of the cardiac Na-Ca exchanger.