Channels In Cardiac Myocytes Research Articles

Transient receptor potential channels meet phosphoinositides

Transient receptor potential (TRP) cation channels are unique cellular sensors that are involved in multiple cellular functions, ranging from transduction of sensory signals to the regulation of Ca2+ and Mg2+ homoeostasis. Malfunctioning of TRP channels is now recognized as the cause of several hereditary and acquired human diseases. At the time of cloning of the first Drosophila TRP channel, a close connection between gating and phosphatidylinositol phosphates (PIPs) was already recognized. In this review, we summarize current knowledge about the mechanisms of interaction between TRP channels and PIPs, and discuss the possible functional implications of TRP–PIP interactions to human physiology and pathophysiology.

Monte Carlo simulation of Ca2+ influx through L-type calcium channel in Cardiac Myocytes

Monte Carlo simulation of Ca2+ influx through L-type calcium channel in Cardiac Myocytes

The anchoring protein SAP97 retains Kv1.5 channels in the plasma membrane of cardiac myocytes

Membrane- associated guanylate kinase proteins (MAGUKs) are important determinants of localization and organization of ion channels into specific plasma membrane domains. However, their exact role in channel function and cardiac excitability is not known. We examined the effect of synapse-associated protein 97 (SAP97), a MAGUK abundantly expressed in the heart, on the function and localization of Kv1.5 subunits in cardiac myocytes. Recombinant SAP97 or Kv1.5 subunits tagged with green fluorescent protein (GFP) were overexpressed in rat neonatal cardiac myocytes and in Chinese hamster ovary (CHO) cells from adenoviral or plasmidic vectors. Immunocytochemistry, fluorescence recovery after photobleaching, and patch-clamp techniques were used to study the effects of SAP97 on the localization, mobility, and function of Kv1.5 subunits. Adenovirus-mediated SAP97 overexpression in cardiac myocytes resulted in the clustering of endogenous Kv1.5 subunits at myocyte-myocyte contacts and an increase in both the maintained component of the outward K(+) current, I(Kur) (5.64 +/- 0.57 pA/pF in SAP97 myocytes vs. 3.23 +/- 0.43 pA/pF in controls) and the number of 4-aminopyridine-sensitive potassium channels in cell-attached membrane patches. In live myocytes, GFP-Kv1.5 subunits were mobile and organized in clusters at the basal plasma membrane, whereas SAP97 overexpression reduced their mobility. In CHO cells, Kv1.5 channels were diffusely distributed throughout the cell body and freely mobile. When coexpressed with SAP97, Kv subunits were organized in plaquelike clusters and poorly mobile. In conclusion, SAP97 regulates the K(+) current in cardiac myocytes by retaining and immobilizing Kv1.5 subunits in the plasma membrane. This new regulatory mechanism may contribute to the targeting of Kv channels in cardiac myocytes.

Activation of Mitochondrial Large-Conductance Calcium-Activated K+ Channels via Protein Kinase A Mediates Desflurane-Induced Preconditioning

ATP-regulated K+ channels are involved in anesthetic-induced preconditioning (APC). The role of other K+ channels in APC is unclear. We tested the hypothesis that APC is mediated by large-conductance calcium-activated K+ channels (K(Ca)). Pentobarbital-anesthetized male C57BL/6 mice were subjected to 45 min of coronary artery occlusion and 3 h reperfusion. Thirty minutes before coronary artery occlusion, 1.0 MAC desflurane was administered for 15 min alone or in combination with the large-conductance K(Ca) channel activator NS1619 (1 microg/g i.p.), its respective vehicle dimethylsulfoxide (10 microL/g i.p.), the large-conductance K(Ca) channel blocker iberiotoxin (0.05 microg/g i.p.), or the protein kinase A (PKA) inhibitor H-89 (0.5 microg/g intraventricular). Infarct size was determined with triphenyltetrazolium chloride and area at risk with Evans blue. Mitochondrial and sarcolemmal localization of large-conductance K(Ca) channels in cardiac myocytes was investigated with immunocytochemical staining of isolated cardiac myocytes. Desflurane significantly reduced infarct size compared with control animals (7.4% +/- 0.8% vs 51.3% +/- 6.1%; P < 0.05). Activation of large-conductance K(Ca) channels by NS1619 (7.5% +/- 1.8%; P < 0.05) mimicked and blockade of large-conductance K(Ca) channels by iberiotoxin (49.1% +/- 7.5%) abrogated desflurane-induced preconditioning. PKA blockade by H-89 abolished desflurane-induced (45.1% +/- 4.0%) but not NS1619-induced (9.0% +/- 2.4%, P < 0.05) preconditioning. Immunocytochemical staining revealed that large-conductance K(Ca) channels were localized in the mitochondria but not in the sarcolemma of cardiac myocytes. These data suggest that desflurane-induced APC is mediated in part by activation of mitochondrial large-conductance K(Ca) channels, and that activation of these channels by desflurane is mediated by PKA.

Aberration of L-type Calcium Channel in Cardiac Myocytes is One of the Mechanisms of Arrhythmia Induced by Cerebral Ischemia

We have examined whether there is a close link between cerebral ischemia and arrhythmogenesis in rats as well as its possible electrophysiological mechanisms. Cerebral ischemic model was made by middle cerebral artery occlusion (MCAO). The incidence of electrocardiographic (ECG) abnormality was as high as 75.71% and the myocardium was severely damaged after MCAO 2 h. The normalized peak currents of I_Ca,L in ventricular myocytes investigated by whole-cell patch clamp were larger in cerebral ischemic rats with arrhythmias than those in sham-operated rats. The steady-state inactivation curve of I_Ca,L was shifted to more positive potentials and recovery of I_Ca,L from inactivation was accelerated significantly, but the activation of I_Ca,L was not influenced in cerebral ischemic rats with arrhythmias compared with control. Meanwhile the action potential duration (APD) of ventricular myocytes was prolonged obviously. The [Ca²⁺]_i induced by KCL in ventricular myocytes was also significantly increased by scanning confocal microscopy. Additionally, the mRNA and protein expression of α_1C/Ca_V1.2 detected by RT-PCR and immunohistochemistry analysis was also increased in myocardium of cerebral ischemic rats with arrhythmias. We conclude that the up-regulation in function and expression of L-type Ca²⁺ channel as well as [Ca²⁺]_i increasing in cardiac myocytes of cerebral ischemic rats may provide mechanisms of arrhythmogenesis.

Caffeine-activated large-conductance plasma membrane cation channels in cardiac myocytes: characteristics and significance

Caffeine-activated, large-conductance, nonselective cation channels (LCCs) have been found in the plasma membrane of isolated cardiac myocytes in several species. However, little is known about the effects of opening these channels. To examine such effects and to further understand the caffeine-activation mechanism, we carried out studies using whole-cell patch-clamp techniques with freshly isolated cardiac myocytes from rats and mice. Unlike previous studies, thapsigargin was used so that both the effect of opening LCCs and the action of caffeine were independent of Ca(2+) release from intracellular stores. These Ca(2+)-permeable LCCs were found in a majority of the cells from atria and ventricles, with a conductance of approximately 370 pS in rat atria. Caffeine and all its direct metabolic products (theophylline, theobromine, and paraxanthine) activated the channel, while isocaffeine did not. Although they share some similarities with ryanodine receptors (RyRs, the openings of which give rise to Ca(2+) sparks), LCCs also showed some different characteristics. With simultaneous Ca(2+) imaging and current recording, the localized fluorescence increase due to Ca(2+) entry through a single opening of an LCC (SCCaFT) was detected. When membrane potential, instead of current, was recorded, SCCaFT-like fluorescence transients (indicating single LCC openings) were found to accompany membrane depolarizations. To our knowledge, this is the first report directly linking membrane potential changes to a single opening of an ion channel. Moreover, these events in cardiac cells suggest a possible additional mechanism by which caffeine and theophylline contribute to the generation of cardiac arrhythmias.

Verrucotoxin inhibits K ATP channels in cardiac myocytes through a muscarinic M 3 receptor-PKC pathway

Verrucotoxin is the major component of venom from the stonefish ( Synanceia verrucosa). Stings from the dorsal spines of the stonefish produce intensive pain, convulsions, hypotension, paralysis, respiratory weakness and collapse of the cardiovascular system, occasionally leading to death. It has been reported that verrucotoxin might modulate ATP-sensitive K + (K ATP) current in frog atrial fibers. However, the mechanism by which verrucotoxin acts on K ATP current remains unclear. In this study, we examined whether verrucotoxin inhibited K ATP current in guinea pig ventricular myocytes, using the patch clamp method. Verrucotoxin suppressed K ATP current induced by pinacidil (K ATP channel opener) in a concentration-dependent manner, with a half maximum concentration of 16.3 μg/ml. The effect of verrucotoxin on K ATP current was suppressed by atropine (1 μM), a muscarinic receptor antagonist, or by 4-diphenylacetoxy- N-methylpiperidine (100 nM), a muscarinic M 3 receptor antagonist. Furthermore, the effect of verrucotoxin on K ATP current was attenuated by the protein kinase C (PKC) inhibitor chelerythrine (10 μM) and calphostin C (10 μM), yet not by the cAMP-dependent protein kinase (PKA) inhibitor H-89 (0.5 μM). These results suggest that verrucotoxin inhibits K ATP current through the muscarinic M 3 receptor-PKC pathway. These findings enhance our understanding of the toxic effects of verrucotoxin from the stonefish.

Hypericin Activates L-Type Ca2+ Channels in Cardiac Myocytes

The effects and the mode of action of hypericin (1) were studied, in the dark, on the action potential (AP) and the L-type Ca2+ channel of frog atrial heart muscle, using intracellular microelectrode and patch-clamp techniques, respectively. In the presence of Ca2+ in Ringer solution, hypericin (1 to 4 microM) did not markedly modify the AP. Total replacement of Ca2+ by Sr2+ in the solution (Ringer Sr2+) revealed that hypericin (4 microM) prolonged the AP duration (APD). Hypericin dose-dependently increased the magnitude of the Sr2+current, which develops through L-type Ca2+ channels in the Ringer solution containing tetrodotoxin (0.7 microM) and tetraethylammonium (10 mM), but did not modify the kinetics of activation and inactivation. This revealed that hypericin increased L-type Ca2+ channel conductance, which accounted for the APD lengthening. The hypericin-induced APD lengthening recorded in the Ringer Sr2+ was not prevented by (i) a blockade of alpha- and beta-adrenoceptors by yohimbine (1 microM), urapidil (1 microM), and propanolol (50 microM), respectively, and (ii) PKC blockade by staurosporine (1 microM). The hypericin-induced APD lengthening recorded in the Ringer Sr2+ was prevented by blocking soluble guanylate cyclase (sGC) activity by 1H-[1,2,4]-oxadiazolo[4,3-a]quinoxalin-1-one (13 microM), which mimicked the effects of hypericin. Hypericin decreased the cellular cGMP level by 69% in atrial myocytes. The compound also decreased the cellular cGMP level by inhibiting sGC, thus cancelling the nucleotide inhibitory effect on the cardiac L-type Ca2+ channel.

Protecting endothelial function: A novel therapeutic target of ATP-sensitive potassium channel openers

See article by Wang et al. [7] (pages 497–503) in this issue . Endothelial dysfunction is commonly observed in patients with hypertension, type II diabetes mellitus, and hypercholesteremia and is considered to be an early event in the changes accompanying atherosclerosis [1,2]. Disruption of normal endothelial function leads to loss of vasomotor control, reduced production of nitric oxide (NO), formation of a procoagulant surface, and promotion of inflammation [1,2]. These events may lead to destabilization of atherosclerotic plaques and could cause acute coronary syndrome. Indeed, recent clinical studies have demonstrated that endothelial dysfunction is a predictor for the development of cardiovascular events [3,4]. Endothelial cells express a diverse array of ion channels which play important roles in modulating cell function [5]. Since Noma first identified ATP-sensitive potassium (KATP) channels in cardiac myocytes, the physiological roles of KATP channels have been extensively studied in cardiac muscle and other tissues [5,6]. Endothelial KATP channels contribute to maintaining the resting membrane potential. In addition, they also regulate intracellular Ca2+ levels that affect the production and release of endothelial autacoids, … *Corresponding author. Tel.: +81 6 6879 3635; fax: +81 6 6879 3634. Email address: mhori{at}medone.med.osaka-u.ac.jp

Calcium influx through If channels in rat ventricular myocytes

The hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels, or cardiac (I(f))/neuronal (I(h)) time- and voltage-dependent inward cation current channels, are conventionally considered as monovalent-selective channels. Recently we discovered that calcium ions can permeate through HCN4 and I(h) channels in neurons. This raises the possibility of Ca(2+) permeation in I(f), the I(h) counterpart in cardiac myocytes, because of their structural homology. We performed simultaneous measurement of fura-2 Ca(2+) signals and whole cell currents produced by HCN2 and HCN4 channels (the 2 cardiac isoforms present in ventricles) expressed in HEK293 cells and by I(f) in rat ventricular myocytes. We observed Ca(2+) influx when HCN/I(f) channels were activated. Ca(2+) influx was increased with stronger hyperpolarization or longer pulse duration. Cesium, an I(f) channel blocker, inhibited I(f) and Ca(2+) influx at the same time. Quantitative analysis revealed that Ca(2+) flux contributed to approximately 0.5% of current produced by the HCN2 channel or I(f). The associated increase in Ca(2+) influx was also observed in spontaneously hypertensive rat (SHR) myocytes in which I(f) current density is higher than that of normotensive rat ventricle. In the absence of EGTA (a Ca(2+) chelator), preactivation of I(f) channels significantly reduced the action potential duration, and the effect was blocked by another selective I(f) channel blocker, ZD-7288. In the presence of EGTA, however, preactivation of I(f) channels had no effects on action potential duration. Our data extend our previous discovery of Ca(2+) influx in I(h) channels in neurons to I(f) channels in cardiac myocytes.

Cardiac and vascular KATP channels in rats are activated by endogenous epoxyeicosatrienoic acids through different mechanisms

We have reported that epoxyeicosatrienoic acids (EETs), the cytochrome P450 (CYP) epoxygenase metabolites of arachidonic acid (AA), are potent sarcolemmal ATP-sensitive K+ (KATP) channel activators. However, activation of cardiac and vascular KATP channels by endogenously produced EETs under physiological intracellular conditions has not been demonstrated and direct comparison of the mechanisms whereby EETs activate the KATP channels in cardiac myocytes versus vascular smooth muscle cells has not been made. In this study, we examined the effects of AA on KATP channels in freshly isolated cardiac myocytes from rats, wild-type (WT) and transgenic mice overexpressing CYP2J2 cDNA, and mesenteric arterial smooth muscle cells from rats. We also compared the activation of cardiac and vascular KATP channels by extracellularly and intracellularly applied 11,12-EET. We found that 1 microm AA enhanced KATP channel activities in both cardiac and vascular smooth muscle cells, and the AA effects were inhibited by preincubation with CYP epoxygenase inhibitors. Baseline cardiac KATP current densities in CYP2J2 transgenic mice were 190% higher than those of WT mice, and both were reduced to similar levels by CYP epoxygenase inhibition. Western blot analysis showed that expression of Kir6.2 and SUR2A was similar between WT and CYP2J2 transgenic hearts. 11,12-EET (5 microm) applied intracellularly enhanced the KATP currents by 850% in cardiac myocytes, but had no effect in vascular smooth muscle cells. In contrast, 11,12-EET (5 microm) applied extracellularly increased KATP currents by 520% in mesenteric arterial smooth muscle cells, but by only 209% in cardiac myocytes. Preincubation with 100 microm m-iodobenzylguanidine or 5 microm myristoylated PKI amide did not alter the activation of cardiac KATP channels by 5 microm 11,12-EET, but significantly inhibited activation of vascular KATP channels. Moreover, EET only enhanced the inward component of cardiac KATP currents, but activated both the inward and outward components of vascular KATP currents. Our results indicate that endogenously derived CYP metabolites of AA potently activate cardiac and vascular KATP channels. EETs regulate cardiac electrophysiology and vascular tone by KATP channel activation, albeit through different mechanisms: the cardiac KATP channels are directly activated by EETs, whereas activation of the vascular KATP channels by EETs is protein kinase A dependent.

ATP and PIP2 dependence of the magnesium-inhibited, TRPM7-like cation channel in cardiac myocytes

The Mg(2+)-inhibited cation (MIC) current (I(MIC)) in cardiac myocytes biophysically resembles currents of heterologously expressed transient receptor potential (TRP) channels, particularly TRPM6 and TRPM7, known to be important in Mg(2+) homeostasis. To understand the regulation of MIC channels in cardiac cells, we used the whole cell voltage-clamp technique to investigate the role of intracellular ATP in pig, rat, and guinea pig isolated ventricular myocytes. I(MIC), studied in the presence or absence of extracellular divalent cations, was sustained for >or=50 min after patch rupture in ATP-dialyzed cells, whereas in ATP-depleted cells I(MIC) exhibited complete rundown. Equimolar substitution of internal ATP by its nonhydrolyzable analog adenosine 5'-(beta,gamma-imido)triphosphate failed to prevent rundown. In ATP-depleted cells, inhibition of lipid phosphatases by fluoride + vanadate + pyrophosphate prevented I(MIC) rundown. In contrast, under similar conditions neither the inhibition of protein phosphatases 1, 2A, 2B or of protein tyrosine phosphatase nor the activation of protein kinase A (forskolin, 20 microM) or protein kinase C (phorbol myristate acetate, 100 nM) could prevent rundown. In ATP-loaded cells, depletion of phosphatidylinositol 4,5-bisphosphate (PIP(2)) by prevention of its resynthesis (10 microM wortmannin or 15 microM phenylarsine oxide) induced rundown of I(MIC). Finally, loading ATP-depleted cells with exogenous PIP(2) (10 microM) prevented rundown. These results suggest that PIP(2), likely generated by ATP-utilizing lipid kinases, is necessary for maintaining cardiac MIC channel activity.

Cocaine‐Induced Inhibition of ATP‐Sensitive K+ Channels in Rat Ventricular Myocytes and in Heart‐Derived H9c2 Cells

Cocaine use may cause coronary artery spasm and acute myocardial ischaemia/infarction. However, its effects on ATP-sensitive K+ (KATP) channel, an ion channel responsible for ischaemic preconditioning, remain unknown. In isolated rat ventricular myocytes with whole-cell experiments, cocaine can reverse action potential shortening and increased K+ current caused by the openers of ATP-sensitive K+ (KATP) channels. In inside-out patches, cocaine applied to intracellular surface suppressed KATP-channel activity in a concentration-dependent manner with an IC50 value of 9.2 microM; however, it did not modify the single-channel conductance of this channel. The change in the kinetic behaviour of KATP channels caused by cocaine is primarily the result of an increase in mean closed time and a decrease in mean open time. Cocaine-induced inhibition of KATP channels is independent of change in intracellular ATP concentrations. In heart-derived H9c2 cells, cocaine is also capable of suppressing KATP-channel activity. The present study provides evidence that cocaine can produce a depressant action on KATP channels in cardiac myocytes, and thus disturb ischaemic preconditioning in clinical settings.

Augmented Activity of Adenosine Triphosphate-Sensitive K+ Channels Induced by Droperidol in the Rat Aorta

Droperidol produces the inhibition of K+ channels in cardiac myocytes. However, the effects of droperidol on K+ channels have not been studied in blood vessels. Therefore, we designed the present study to determine whether droperidol modulates the activity of adenosine triphosphate (ATP)-sensitive K+ channels in vascular smooth muscle cells. Rat aortic rings without endothelium were suspended or used for isometric force and membrane potential recordings, respectively. Vasorelaxation and hyperpolarization induced by levcromakalim (10(-8) to 10(-5) M or 10(-5) M, respectively) were completely abolished by the ATP-sensitive K+ channel antagonist glibenclamide (10(-5) M). Droperidol (10(-7) M) and an alpha-adrenergic receptor antagonist phentolamine (3 x 10(-9) M) caused a similar vasodilator effect (approximately 20% of vasorelaxation compared with maximal vasorelaxation induced by papaverine [3 x 10(-4) M]), whereas glibenclamide did not alter vasorelaxation induced by droperidol. Droperidol (3 x 10(-8) M to 10(-7) M) augmented vasorelaxation and hyperpolarization produced by levcromakalim, whereas phentolamine (3 x 10(-9) M) did not alter this vasorelaxation. Glibenclamide (10(-5) M) abolished the vasodilating and hyperpolarizing effects of levcromakalim in the aorta treated with droperidol (10(-7) M). These results suggest that droperidol augments vasodilator activity via ATP-sensitive K+ channels. However, it is unlikely that this augmentation is mediated by the inhibition of alpha-adrenergic receptors in vascular smooth muscles.

Effects of sulfonylureas on mitochondrial ATP‐sensitive K+ channels in cardiac myocytes: implications for sulfonylurea controversy

Mitochondrial ATP-sensitive K(+) (mitoK(ATP)) channel plays a key role in cardioprotection. Hence, a sulfonylurea that does not block mitoK(ATP) channels would be desirable to avoid damage to the heart. Accordingly, we examined the effects of sulfonylureas on the mitoK(ATP) channel and mitochondrial Ca(2+) overload. Flavoprotein fluorescence in rabbit ventricular myocytes was measured to assay mitoK(ATP) channel activity. The mitochondrial Ca(2+) concentration was measured by loading cells with rhod-2. The mitoK(ATP) channel opener diazoxide (100 microM) reversibly increased flavoprotein oxidation to 31.8 +/- 4.3% (n = 5) of the maximum value induced by 2,4-dinitrophenol. Glimepiride (10 microM) alone did not oxidize the flavoprotein, and the oxidative effect of diazoxide was unaffected by glimepiride (35.4 +/- 3.2%, n = 5). Similarly, the diazoxide-induced flavoprotein oxidation was unaffected both by gliclazide (10 microM) and by tolbutamide (100 microM). Exposure to ouabain (1 mM) for 30 min produced mitochondrial Ca(2+) overload, and the intensity of rhod-2 fluorescence increased to 197.4 +/- 7.2% of baseline (n = 11). Treatment with diazoxide significantly reduced the ouabain-induced mitochondrial Ca(2+) overload (149.6 +/- 5.1%, n = 11, p < 0.05 versus ouabain alone), and the effect was antagonized by the mitoK(ATP) channel blocker 5-hydroxydecanoate (189.8 +/- 27.8%, n = 5) and glibenclamide (193.1 +/- 7.7%, n = 8). On the contrary, cardioprotective effect of diazoxide was not abolished by glimepiride (141.8 +/- 7.8%, n = 6), gliclazide (139.0 +/- 9.4%, n = 5), and tolbutamide (141.1 +/- 4.5%, n = 7). Our results indicate that glimepiride, gliclazide, and tolbutamide have no effect on mitoK(ATP) channel, and do not abolish the cardioprotective effects of diazoxide. Therefore, these sulfonylureas, unlike glibenclamide, do not interfere with the cellular pathways that confer cardioprotection.

Modulation of CaV1.2 channels by Mg2+ acting at an EF-hand motif in the COOH-terminal domain.

Magnesium levels in cardiac myocytes change in cardiovascular diseases. Intracellular free magnesium (Mgi) inhibits L-type Ca2+ currents through CaV1.2 channels in cardiac myocytes, but the mechanism of this effect is unknown. We hypothesized that Mgi acts through the COOH-terminal EF-hand of CaV1.2. EF-hand mutants were engineered to have either decreased (D1546A/N/S/K) or increased (K1543D and K1539D) Mg2+ affinity. In whole-cell patch clamp experiments, increased Mgi reduced both Ba2+ and Ca2+ currents conducted by wild type (WT) CaV1.2 channels expressed in tsA-201 cells with similar affinity. Exposure of WT CaV1.2 to lower Mgi (0.26 mM) increased the amplitudes of Ba2+ currents 2.6 ± 0.4–fold without effects on the voltage dependence of activation and inactivation. In contrast, increasing Mgi to 2.4 or 7.2 mM reduced current amplitude to 0.5 ± 0.1 and 0.26 ± 0.05 of the control level at 0.8 mM Mgi. The effects of Mgi on peak Ba2+ currents were approximately fit by a single binding site model with an apparent Kd of 0.65 mM. The apparent Kd for this effect of Mgi was shifted ∼3.3- to 16.5-fold to higher concentration in D1546A/N/S mutants, with only small effects on the voltage dependence of activation and inactivation. Moreover, mutant D1546K was insensitive to Mgi up to 7.2 mM. In contrast to these results, peak Ba2+ currents through the K1543D mutant were inhibited by lower concentrations of Mgi compared with WT, consistent with approximately fourfold reduction in apparent Kd for Mgi, and inhibition of mutant K1539D by Mgi was also increased comparably. In addition to these effects, voltage-dependent inactivation of K1543D and K1539D was incomplete at positive membrane potentials when Mgi was reduced to 0.26 or 0.1 mM, respectively. These results support a novel mechanism linking the COOH-terminal EF-hand with modulation of CaV1.2 channels by Mgi. Our findings expand the repertoire of modulatory interactions taking place at the COOH terminus of CaV1.2 channels, and reveal a potentially important role of Mgi binding to the COOH-terminal EF-hand in regulating Ca2+ influx in physiological and pathophysiological states.

Cardioprotection: A New Paradigm in the Management of Acute Heart Failure Syndromes

The management of acute heart failure syndromes (AHFS) focuses primarily on improving hemodynamic function and alleviating symptoms. Emerging evidence has raised the possibility that patients with AHFS may be susceptible to progressive myocardial failure because of the accelerated loss of cardiac myocytes. Although there are circumstantial data to suggest that the choice of therapeutic agent may affect long-term outcomes in such patients, the responsible mechanism is not known. Activation of mitochondrial adenosine triphosphate-dependent potassium (K(ATP)) channels in cardiac myocytes is a potent cardioprotective mechanism. We studied cardiac myocytes in culture to determine whether levosimendan can protect against apoptotic cell death in response to oxidative stress, a stimulus that appears to mediate myocyte loss in response to hemodynamic overload and beta-adrenergic stimulation, conditions commonly encountered in acute HF. Levosimendan, at concentrations below the therapeutic range in humans, protected myocytes from hydrogen peroxide-induced apoptosis. This effect was prevented by K(ATP) channel inhibitors. The demonstration that levosimendan can oppose myocyte apoptosis via the activation of mitochondrial K(ATP) channels provides a potential mechanism by which this agent might protect cardiac myocytes during episodes of acute HF. Although the alleviation of symptoms should remain an important goal of therapy in acute HF, a therapeutic approach that includes a cardioprotective strategy may be able to exert a clinically meaningful benefit on disease progression. This speculation, if proved true, would mandate a fundamental paradigm shift in the acute management of acute HF.

Phosphatidylinositol 3,4,5-trisphosphate and Ca2+/calmodulin competitively bind to the regulators of G-protein-signalling (RGS) domain of RGS4 and reciprocally regulate its action.

RGS (regulators of G-protein signalling) are a diverse group of proteins, which accelerate intrinsic GTP hydrolysis on heterotrimeric G-protein a subunits. They are involved in the control of a physiological behaviour known as 'relaxation' of G-protein-gated K+ channels in cardiac myocytes. The GTPase-accelerating activity of cardiac RGS proteins, such as RGS4, is inhibited by PtdIns(3,4,5)P3 (phosphatidylinositol 3,4,5-trisphosphate) and this inhibition is cancelled by Ca2+/calmodulin (CaM) formed during membrane depolarization. G-protein-gated K+ channel activity decreases on depolarization owing to the facilitation of GTPase-activating protein activity by RGS proteins and vice versa on hyperpolarization. The molecular mechanism responsible for this reciprocal control of RGS action by PtdIns(3,4,5)P3 and Ca2+/CaM, however, has not been fully elucidated. Using lipid-protein co-sedimentation assay and surface plasmon resonance measurements, we show in the present study that the control of the GTPase-accelerating activity of the RGS4 protein is achieved through the competitive binding of PtdIns(3,4,5)P3 and Ca2+/CaM within its RGS domain. Competitive binding occurs exclusively within the RGS domain and involves a cluster of positively charged residues located on the surface opposite to the Ga interaction site. In the RGS proteins conserving these residues, the reciprocal regulation by PtdIns(3,4,5)P3 and Ca2+/CaM may be important for their physiological regulation of G-protein signalling.

Redox modulation of basal and beta-adrenergically stimulated cardiac L-type Ca(2+) channel activity by phenylarsine oxide.

1. Phenylarsine oxide (PAO) is commonly used to inhibit tyrosine phosphatase activity. However, PAO can affect a variety of different processes because of its ability to promote sulfhydryl oxidation. In the present study, we investigated the effects that PAO has on basal and beta-adrenergically stimulated L-type Ca(2+) channel activity in isolated cardiac myocytes. 2. Extracellular application of PAO transiently stimulated the basal L-type Ca(2+) channel activity, whereas it irreversibly inhibited protein kinase A (PKA)-dependent regulation of channel activity by isoproterenol, forskolin and 8-CPT-cAMP (8-p-chlorophenylthioadenosine 3',5'-cyclic monophosphate). PAO also inhibited channel activity irreversibly stimulated in the presence of adenosine 5'-(3-thiotriphosphate) tetralithium salt. 3. Neither the stimulatory nor the inhibitory effects of PAO were affected by the tyrosine kinase inhibitor lavendustin A, suggesting that tyrosine phosphorylation is not involved. 4. Extracellular application of the sulfhydryl-reducing agent dithiothreitol (DTT) antagonized both the stimulatory and inhibitory effects of PAO. Yet, following intracellular dialysis with DTT, only the inhibitory effect of PAO was antagonized. 5. The inhibitory effect of PAO was mimicked by intracellular, but not extracellular application of the membrane impermeant thiol oxidant 5,5'-dithio-bis(2-nitrobenzoic acid). 6. These results suggest that the stimulatory effect of PAO results from oxidation of sulfhydryl residues at an extracellular site and the inhibitory effect is due to redox regulation of an intracellular site that affects the response of the channel to PKA-dependent phosphorylation. It is concluded that the redox state of the cell may play a critical role in modulating beta-adrenergic responsiveness of the L-type Ca(2+) channel in cardiac myocytes.

Pro-nucleotide Inhibitors of Adenylyl Cyclases in Intact Cells

9-substituted adenine derivatives with protected phosphoryl groups were synthesized and tested as inhibitors of adenylyl cyclase in isolated enzyme and intact cell systems. Protected 3'-phosphoryl derivatives of 2',5'-dideoxyadenosine (2',5'-dd-Ado) and beta-l-2',5'-dd-Ado, protected 5'-phosphoryl derivatives of beta-l-2',3'-dd-Ado, and protected phosphoryl derivatives of two 9-(2-phosphonomethoxy-acyl)-adenines were synthesized. Protection was afforded by two cyclosaligenyl- or three S-acyl-2-thioethyl-substituents. These pro-nucleotides were tested for their capacity to block forskolin-induced increases in [(3)H]cAMP in OB1771 and F442A preadipocytes and human macrophages prelabeled with [(3)H]adenine. A striking selectivity for 2',5'-dd-Ado-3'-phosphoryl derivatives was observed. Cyclosaligenyl-derivatives (IC(50) approximately 2 microm) were much less potent than S-acyl-2-thioethyl-derivatives. Best studied of these was 2',5'-dd-Ado-3'-O-bis(S-pivaloyl-2-thioethyl)-phosphate, which blocked [(3)H]cAMP formation in preadipocytes (IC(50) approximately 30 nm) and suppressed opening of cAMP-dependent Cl(-) channels in cardiac myocytes (IC(50) approximately 800 nm). None of the pro-nucleotides inhibited adenylyl cyclase per se, whether isolated from rat brain or OB1771 cells. These compounds exhibit the hallmarks of prodrugs. Data suggest they are taken up, are deprotected, and are converted to a potent inhibitory form to inhibit adenylyl cyclase, but only by intact cells. The availability and characteristics of these prodrugs should make them useful for blocking cAMP-mediated pathways in intact cell systems, in biochemical, pharmacological, and potentially therapeutic contexts.