Glucose-6-phosphate dehydrogenase increases Ca2+ currents by interacting with Cav1.2 and reducing intrinsic inactivation of the L-type calcium channel.

Pyridine nucleotides, such as NADPH and NADH, are emerging as critical players in the regulation of heart and vascular function. Glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme in the pentose phosphate pathway, is the primary source and regulator of cellular NADPH. In the current study, we have identified two isoforms of G6PD (slow and fast migrating) and functionally characterized the slow migrating isoform of G6PD (G6PD545) in bovine and human arteries. We found that G6PD545 is eluted in the caveolae fraction of vascular smooth muscle (VSM) and has a higher maximum rate of reaction (Vmax: 1.65-fold) than its fast migrating isoform (G6PD515). Interestingly, caveolae G6PD forms a complex with the pore-forming α1C-subunit of the L-type Ca2+ channel, Cav1.2, as demonstrated by a proximity ligation assay in fixed VSMCs. Additionally, Förster resonance energy transfer (FRET) analysis of HEK293-17T cells cotransfected with red fluorescent protein (RFP)-tagged G6PD545 (C-G6PD545) and green fluorescent protein (GFP)-tagged Cav1.2-(Cav1.2-GFP) demonstrated strong FRET signals as compared with cells cotransfected with Cav1.2-GFP and C-G6PD515. Furthermore, L-type Ca2+ channel conductance was larger and the voltage-independent component of availability (c1) was augmented in C-G6PD545 and Cav1.2-GFP cotransfectants compared with those expressing Cav1.2-GFP alone. Surprisingly, epiandrosterone, a G6PD inhibitor, disrupted the G6PD-Cav1.2 complex, also decreasing the amplitude of L-type Ca2+ currents and window currents, thereby reducing the availability of the c1 component. Moreover, overexpression of adeno-G6PD545-GFP augmented the KCl-induced contraction in coronary arteries compared with control. To determine whether overexpression of G6PD had any clinical implication, we investigated its activity in arteries from patients and rats with metabolic syndrome and found that G6PD activity was high in this disease condition. Interestingly, epiandrosterone treatment reduced elevated mean arterial blood pressure and peripheral vascular resistance in metabolic syndrome rats, suggesting that the increased activity of G6PD augmented vascular contraction and blood pressure in the metabolic syndrome. These data suggest that the novel G6PD-Cav1.2 interaction, in the caveolae fraction, reduces intrinsic voltage-dependent inactivation of the channel and contributes to regulate VSM L-type Ca2+ channel function and Ca2+ signaling, thereby playing a significant role in modulating vascular function in physiological/pathophysiological conditions.NEW & NOTEWORTHY In this study we have identified a novel isozyme of glucose-6-phosphate dehydrogenase (G6PD), a metabolic enzyme, that interacts with and contributes to regulate smooth muscle cell l-type Ca2+ ion channel function, which plays a crucial role in vascular function in physiology and pathophysiology. Furthermore, we demonstrate that expression and activity of this novel G6PD isoform are increased in arteries of individuals with metabolic syndrome and in inhibition of G6PD activity in rats of metabolic syndrome reduced blood pressure.

Dehydroepiandrosterone inhibits ICa,L and its window current in voltage-dependent and -independent mechanisms in arterial smooth muscle cells.

Dehydroepiandrosterone (DHEA) is an adrenal steroid hormone, which has the highest serum concentration among steroid hormones with DHEA sulfate (DHEAS). DHEA possesses an inhibitory action on glucose-6-phosphate dehydrogenase (G6PD), the first pentose-phosphate pathway enzyme that reduces NADP+ to NADPH. DHEA induced relaxation of high K+-induced contraction in rat arterial strips, whereas DHEAS barely induced it. We studied the effects of DHEA on L-type Ca2+ current ( ICa,L) of A7r5 arterial smooth muscle cells and compared the mechanism of inhibition with that produced by the 6-aminonicotinamide (6-AN) competitive inhibitor of G6PD. DHEA moderately inhibited ICa,L that was elicited from a holding potential (HP) of -80 mV [voltage-independent inhibition (VIDI)] and accelerated decay of ICa,L during the depolarization pulse [voltage-dependent inhibition (VDI)]. DHEA-induced VDI decreased peak ICa,L at depolarized HPs. By applying repetitive depolarization pulses from multiple HPs, novel HP-dependent steady-state inactivation curves ( f∞-HP) were constructed. DHEA shifted f∞-HP to the left and inhibited the window current, which was recorded at depolarized HPs and obtained as a product of current-voltage relationship and f∞-HP. The IC50 value of ICa,L inhibition was much higher than serum concentration. DHEA-induced VDI was downregulated by the dialysis of guanosine 5'- O-(2-thiodiphosphate), which shifted f∞-voltage to the right before the application of DHEA. 6-AN gradually and irreversibly inhibited ICa,L by VIDI, suggesting that the inhibition of G6PD is involved in DHEA-induced VIDI. In 6-AN-pretreated cells, DHEA induced additional inhibition by increasing VIDI and generating VDI. The inhibition of G6PD underlies DHEA-induced VIDI, and DHEA additionally induces VDI as described for Ca2+ channel blockers. NEW & NOTEWORTHY Dehydroepiandrosterone, the most abundantly released adrenal steroid hormone with dehydroepiandrosterone sulfate, inhibited L-type Ca2+ current and its window current in aortic smooth muscle cells. The IC50 value of inhibition decreased with the depolarization of holding potential to 15 µM at -20 mV. The inhibition occurred in a voltage-dependent manner as described for Ca2+ channel blockers and in a voltage-independent manner because of the inhibition of glucose-6-phosphate dehydrogenase.

Rotenone-stimulated superoxide release from mitochondrial complex I acutely augments L-type Ca2+ current in A7r5 aortic smooth muscle cells.

Voltage-gated L-type Ca(2+) current (ICa,L) induces contraction of arterial smooth muscle cells (ASMCs), and ICa,L is increased by H2O2 in ASMCs. Superoxide released from the mitochondrial respiratory chain (MRC) is dismutated to H2O2 We studied whether superoxide per se acutely modulates ICa,L in ASMCs using cultured A7r5 cells derived from rat aorta. Rotenone is a toxin that inhibits complex I of the MRC and increases mitochondrial superoxide release. The superoxide content of mitochondria was estimated using mitochondrial-specific MitoSOX and HPLC methods, and was shown to be increased by a brief exposure to 10 µM rotenone. ICa,L was recorded with 5 mM BAPTA in the pipette solution. Rotenone administration (10 nM to 10 µM) resulted in a greater ICa,L increase in a dose-dependent manner to a maximum of 22.1% at 10 µM for 1 min, which gradually decreased to 9% after 5 min. The rotenone-induced ICa,L increase was associated with a shift in the current-voltage relationship (I-V) to a hyperpolarizing direction. DTT administration resulted in a 17.9% increase in ICa,L without a negative shift in I-V, and rotenone produced an additional increase with a shift. H2O2 (0.3 mM) inhibited ICa,L by 13%, and additional rotenone induced an increase with a negative shift. Sustained treatment with Tempol (4-hydroxy tempo) led to a significant ICa,L increase but it inhibited the rotenone-induced increase. Staurosporine, a broad-spectrum protein kinase inhibitor, partially inhibited ICa,L and completely suppressed the rotenone-induced increase. Superoxide released from mitochondria affected protein kinases and resulted in stronger ICa,L preceding its dismutation to H2O2 The removal of nitric oxide is a likely mechanism for the increase in ICa,L.

Poly(ethylene glycol)-cholesterol inhibits L-type Ca2+ channel currents and augments voltage-dependent inactivation in A7r5 cells.

Cholesterol distributes at a high density in the membrane lipid raft and modulates ion channel currents. Poly(ethylene glycol) cholesteryl ether (PEG-cholesterol) is a nonionic amphipathic lipid consisting of lipophilic cholesterol and covalently bound hydrophilic PEG. PEG-cholesterol is used to formulate lipoplexes to transfect cultured cells, and liposomes for encapsulated drug delivery. PEG-cholesterol is dissolved in the external leaflet of the lipid bilayer, and expands it to flatten the caveolae and widen the gap between the two leaflets. We studied the effect of PEG-cholesterol on whole cell L-type Ca(2+) channel currents (I(Ca),L) recorded from cultured A7r5 arterial smooth muscle cells. The pretreatment of cells with PEG-cholesterol decreased the density of ICa,L and augmented the voltage-dependent inactivation with acceleration of time course of inactivation and negative shift of steady-state inactivation curve. Methyl-ß-cyclodextrin (MßCD) is a cholesterol-binding oligosaccharide. The enrichment of cholesterol by the MßCD:cholesterol complex (cholesterol (MßCD)) caused inhibition of I(Ca),L but did not augment voltage-dependent inactivation. Incubation with MßCD increased I(Ca),L, slowed the time course of inactivation and shifted the inactivation curve to a positive direction. Additional pretreatment by a high concentration of MßCD of the cells initially pretreated with PEG-cholesterol, increased I(Ca),L to a greater level than the control, and removed the augmented voltage-dependent inactivation. Due to the enhancement of the voltage-dependent inactivation, PEG-cholesterol inhibited window I(Ca),L more strongly as compared with cholesterol (MßCD). Poly(ethylene glycol) conferred to cholesterol the efficacy to induce sustained augmentation of voltage-dependent inactivation of I(Ca),L.

Down-regulation of replication factor C-40 (RFC40) causes chromosomal missegregation in neonatal and hypertrophic adult rat cardiac myocytes.

BACKGROUND: Adult mammalian cardiac myocytes are generally assumed to be terminally differentiated; nonetheless, a small fraction of cardiac myocytes have been shown to replicate during ventricular remodeling. However, the expression of Replication Factor C (RFC; RFC140/40/38/37/36) and DNA polymerase δ (Pol δ) proteins, which are required for DNA synthesis and cell proliferation, in the adult normal and hypertrophied hearts has been rarely studied. METHODS: We performed qRT-PCR and Western blot analysis to determine the levels of RFC and Pol δ message and proteins in the adult normal cardiac myocytes and cardiac fibroblasts, as well as in adult normal and pulmonary arterial hypertension induced right ventricular hypertrophied hearts. Immunohistochemical analyses were performed to determine the localization of the re-expressed DNA replication and cell cycle proteins in adult normal (control) and hypertrophied right ventricle. We determined right ventricular cardiac myocyte polyploidy and chromosomal missegregation/aneuploidy using Fluorescent in situ hybridization (FISH) for rat chromosome 12. RESULTS: RFC40-mRNA and protein was undetectable, whereas Pol δ message was detectable in the cardiac myocytes isolated from control adult hearts. Although RFC40 and Pol δ message and protein significantly increased in hypertrophied hearts as compared to the control hearts; however, this increase was marginal as compared to the fetal hearts. Immunohistochemical analyses revealed that in addition to RFC40, proliferative and mitotic markers such as cyclin A, phospho-Aurora A/B/C kinase and phospho-histone 3 were also re-expressed/up-regulated simultaneously in the cardiac myocytes. Interestingly, FISH analyses demonstrated cardiac myocytes polyploidy and chromosomal missegregation/aneuploidy in these hearts. Knock-down of endogenous RFC40 caused chromosomal missegregation/aneuploidy and decrease in the rat neonatal cardiac myocyte numbers. CONCLUSION: Our novel findings suggest that transcription of RFC40 is suppressed in the normal adult cardiac myocytes and its insufficient re-expression may be responsible for causing chromosomal missegregation/aneuploidy and in cardiac myocytes during right ventricular hypertrophy.

Glucose-6-phosphate dehydrogenase is a regulator of vascular smooth muscle contraction.

Glucose-6-phosphate dehydrogenase (G6PD) is the rate-limiting enzyme in the pentose phosphate pathway and a major source of nicotinamide adenine dinucleotide phosphate reduced (NADPH), which regulates numerous enzymatic (including glutathione reductase and NADPH oxidase that, respectively, generates reduced glutathione and reactive oxygen species) reactions involved in various cellular actions, yet its physiological function is seldom investigated. We, however, recently showed that inhibiting G6PD causes precontracted coronary artery (CA) to relax in an endothelium-derived relaxing factor- and second messenger-independent manner. Here we assessed the role of G6PD in regulating CA contractility. Treating bovine CAs for 20 min with potassium chloride (KCl; 30 mM), amphotericin B (50 µM), or U46619 (100 nM) significantly (p < 0.05) increased both G6PD activity and glucose flux through the pentose phosphate pathway. The effect was Ca(2+) independent, and there was a corresponding increase in protein kinase C (PKC) activity. Activation of G6PD by KCl was blocked by the PKCδ inhibitor rottlerin (10 µM) or by knocking down PKCδ expression using siRNA. Phorbol 12, 13-dibutyrate (10 µM), a PKC activator, significantly increased G6PD phosphorylation and activity, whereas single (S210A, T266A) and double (S210A/T266A) mutations at sites flanking the G6PD active site significantly inhibited phosphorylation, shifted the isoelectric point, and reduced enzyme activity. Knocking down G6PD decreased NADPH and reactive oxygen species generation, and reduced KCl-evoked increases in [Ca(2+)](i) and myosin light chain phosphorylation, thereby reducing CA contractility. Similarly, aortas from G6PD-deficient mice developed less KCl/phorbol 12, 13-dibutyrate-evoked force than those from their wild-type littermates. Conversely, overexpression of G6PD augmented KCl-evoked increases in [Ca(2+)](i), thereby augmenting CA contraction. Our findings demonstrate that G6PD activity and NADPH is increased in activated CA in a PKCδ-dependent manner and that G6PD modulates Ca(2+) entry and CA contractions evoked by membrane depolarization.

Cholesterol depletion modulates basal L-type Ca2+ current and abolishes its -adrenergic enhancement in ventricular myocytes.

Cholesterol is a primary constituent of the plasmalemma, including the lipid rafts/caveolae, where various G protein-coupled receptors colocalize with signaling proteins and channels. By manipulating cholesterol in rabbit and rat ventricular myocytes using methyl-beta-cyclodextrin (MbetaCD), we studied the role of cholesterol in the modulation of L-type Ca(2+) currents (I(Ca,L)). MbetaCD was mainly dialyzed from BAPTA-containing pipette solution during whole cell clamp. In rabbit myocytes dialyzed with 30 mM MbetaCD for 10 min, a positive shift in membrane potential at half-maximal activation (V(0.5)) from -8 to -2 mV developed and was associated with an increase in current density at positive potentials (42% at +20 mV vs. time-matched controls). Isoproterenol (ISO) increased I(Ca,L) approximately threefold and caused a negative shift in V(0.5) in control cells, but it did not increase I(Ca,L) in MbetaCD-treated myocytes, nor did it shift V(0.5). The effect of MbetaCD (10 or 30 mM) was concentration dependent: 30 mM MbetaCD suppressed the ISO-induced increase in I(Ca,L) more effectively than 10 mM MbetaCD. MbetaCD dialysis also abolished the increase in I(Ca,L) elicited by forskolin or dibutyryl cAMP, but not that elicited by (-)BAY K 8644. External application of MbetaCD-cholesterol complex to rat myocytes attenuated the MbetaCD-mediated inhibition of the ISO-induced increase of I(Ca,L). Biochemical analysis confirmed that the myocytes' cholesterol content was diminished by MbetaCD and increased by MbetaCD-cholesterol complex. Cholesterol thus appears to contribute to the regulation of basal I(Ca,L) and beta-adrenergic cAMP/PKA-mediated increases in I(Ca,L). We suggest that cholesterol affects the structural coupling between L-type Ca(2+) channels and adjacent regulatory proteins.

Ryanodine receptor: a novel therapeutic target in heart disease.

In excitable cells such as skeletal and cardiac myocytes excitation-contraction coupling is an important intermediate step between initiation of the action potential and induction of contraction. This process is predominantly controlled by Ca(2+) release from the sarcoplasmic reticulum via the ryanodine receptor. This very large protein (MW 560 kDa) exists as a homotetramer (~2.2 MDa) and is expressed in three isoforms: RyR1, expressed in skeletal muscle; RyR2, expressed in cardiac muscle; and RyR3, expressed in various cells at lower levels than the other isoforms. Release of Ca(2+) via RyR2 is induced by Ca(2+) influx through L-type Ca(2+) channels and is modulated by multiple factors, including phosphorylation of RyR2 protein by protein kinase A, calmodulin kinase II and FKBP12.6, and stimulation via the beta-adrenergic receptor signaling pathway. Hyperphosphorylation of RyR2 induces Ca(2+) leak during diastole, which can cause fatal arrhythmias and lead to heart failure. This makes RyR2 an important therapeutic target. Although there are few commercially available drugs that inhibit Ca(2+) leak from RyR2, K201 (JTV-519), a benzothiazepine derivative, has emerged as a new ryanodine receptor-selective agent that prevents atrial fibrillation, ventricular arrhythmias, heart failure and exercise-induced sudden cardiac death. In this review, we discuss recent advances in our understanding of the basic structure and function of ryanodine receptors, their involvement in heart disease, and the development of drugs to prevent ryanodine receptor malfunction and recent patents.

Sustained beta-adrenergic stimulation increased L-type Ca2+ channel expression in cultured quiescent ventricular myocytes.

The abundance of voltage-gated L-type Ca2+ channels is altered by beta-adrenergic receptor (beta-AR) stimulation and by an elevation of the intracellular Ca2+ concentration in cardiac myocytes. In whole animal, chronic beta-AR stimulation or pacing heart results in various changes in the abundance of the channel, but it reduces the beta-AR responsiveness of the L-type channel. Because beta-AR stimulation facilitates the L-type calcium channels, it is difficult in the whole animal to study the effects of beta-AR and Ca2+ influx on the upregulation of the L-type channel independently of each other, which makes the culture of nonbeating adult myocytes an attractive model. We found that culturing quiescent adult rabbit ventricular myocytes with isoproterenol (ISO, 2 microM) for 72 h or more caused a significant increase in the expression of mRNA coding for the L-type channel alpha(1C) subunit by approximately twofold as compared to time-matched controls, and it was followed by a 1.8-fold increase in the Ca2+ current density at 96 h. Somewhat surprisingly, an acute application of 1 microM ISO increased the current amplitude even in ISO-treated cells. The increase in the current density, induced by sustained beta-AR stimulation, was blocked by a beta-AR antagonist, propranolol (10 microM), but not by a Ca2+ antagonist, nitrendipine (10 microM). In addition, the effects were reproduced by forskolin (10 microM), but not by a Ca2+ agonist, Bay-K 8644 (2 microM). Taken together, these results suggest that sustained beta-AR stimulation upregulates L-type channel expression, but does not alter the beta-AR responsiveness of the channel in quiescent myocytes.

Sphingosine-1-phosphate effects on guinea pig atrial myocytes: Alterations in action potentials and K+ currents.

OBJECTIVE: Sphingosine-1-phosphate (S-1-P), a potent lysophospholipid mediator which is released from platelets during clotting, activates a G-protein-gated inwardly rectifying K+ current (GIRK) in atrial and sino-atrial node myocytes. We denote this current I(K(S-1-P).) A similar GIRK, which is activated by acetylcholine (ACh) and denoted I(K(ACh)), is expressed in atrium. It shortens the action potential duration (APD) and reduces the effective refractory period (ERP). We have examined the effect of S-1-P on APD in guinea pig atrial myocytes by characterizing the rectification properties of I(K(S-1-P)) and evaluating whether I(K(S-1-P)) and I(K(ACh)) exhibit convergence/occlusion. METHODS: Membrane potential and K+ currents were recorded from guinea pig atrial myocytes. Inwardly rectifying K+ currents were recorded using a ramp voltage clamp waveform between +30 to -130 mV from a holding potential of -7 mV. Agonist-induced current changes were obtained by subtracting the control current. RESULTS: S-1-P (1 and 10 nM) altered both passive and active properties of atrial myocytes. S-1-P increased the threshold current for excitation and decreased the time constant of the subthreshold electrotonic potentials. In addition, both APD50 and APD90 were decreased substantially. Voltage clamp analysis showed that the outward conductance of I(K(IR)) (G(K(IR)out)) was 134.8+/-11.3 pS pF(-1) (n = 19) in S-1-P (100 nM), and 207.0+/-19.6 pS pF(-1) (n = 18) in ACh (10 microM). The ratio of G(K(IR)out):G(K(IR)in) was about 0.7 for both S-1-P and ACh. The EC50 values for the activation of G(K(IR)out) and G(K(IR)in) by S-1-P were 1.6 and 1.3 nM, respectively. Addition of S-1-P (100 nM) after the effect of ACh (10 microM) had developed fully caused very little additional change. CONCLUSION: I(K(S-1-P)) is carried by weakly inwardly-rectifying K+ channels that are the same as those activated by ACh. This K+ current can markedly shorten APD in guinea pig atrial myocytes. This effect would be expected to increase the incidence of atrial rhythm disturbances.

Hypoxia and hypercapnia affect contractile and histological properties of rat diaphragm and hind limb muscles.

The effects of hypoxia and hypercapnia on contractile and histological properties of the diaphragm and skeletal muscles of the hind limb were examined. Eight-week-old male Sprague-Dawley rats ( [Formula: see text] ) were kept in hypobaric hypoxic ( [Formula: see text] ) or hypercapnic ( [Formula: see text] ) chambers for 6 weeks, and compared with the control rats (room air, [Formula: see text] ). Contractile properties were evaluated with twitch kinetics, force-frequency curve and fatigue tolerance. After the experiments on contractile activities, muscles were fixed for histological examination with ATPase staining. It was demonstrated that peak twitch tension of diaphragm decreased with no significant histological changes under hypoxic conditions while significant contractile and histological changes were observed under hypercapnic conditions. Skeletal muscles of the hind limbs were affected also under hypoxic and hypercapnic conditions but the profiles of the changes in contraction and histology were different from those of the diaphragm. These results suggest that hypoxia and hypercapnia affect differently on contractile and histological properties of respiratory and hind limb muscles. Furthermore, when we consider the conditions involved in chronic obstructive respiratory disease (COPD; both hypoxia and hypercapnia are deeply involved), our results indicate that COPD should be regarded as a systemic disorder rather than a respiratory disease.

Modulation of manganese currents by 1, 4-dihydropyridines, isoproterenol and forskolin in rabbit ventricular cells.

Although often used as a Ca(2+) channel blocker, Mn(2+), in fact, permeates through Ca(2+) channels. Under Na(+)-free conditions, depolarizing pulses evoked slowly-decaying Mn(2+) currents ( I(Mn)). Maximal I(Mn) densities in the presence of 5 and 20 mM Mn(2+) were 0.42+/-0.12 pA/pF (mean+/-SEM, n=17) and 1.23+/-0.10 pA/pF ( n=40), respectively. At 5 mM, the ratio of maximal amplitude of I(Mn) to that of the Ca(2+) current ( I(Ca)) was 0.079+/-0.009 ( n=8). I(Mn) elicited from a holding potential of -50 mV was depressed by nitrendipine (1 microM) by approximately 70%. Nitrendipine (0.3 microM) shifted the steady-state inactivation curve to more negative potentials and shifted the potential for half-maximal inactivation ( E(0.5)) from 1.3 to -8.8 mV and also decreased the time constant of decay of I(Mn) at 20 mV from 986.2 to 167.9 ms. BAY K 8644 (1 microM), isoproterenol (10 microM) and forskolin (10 microM) all increased I(Mn) and shifted the current/voltage ( I/ V) relationship to more negative potentials. The small, slowly-inactivating I(Mn) is thus modulated by dihydropyridine Ca(2+) channel modulators and cyclic AMP-mediated phosphorylation in a manner similar to other L-type Ca(2+) channel currents. L-type Ca(2+) channels are involved in the regulation of intracellular [Mn] in ventricular myocytes.

Diltiazem facilitates inactivation of single L-type calcium channels in guinea pig ventricular myocytes.

Diltiazem is a benzothiazepine Ca2+ channel blocker used clinically for its antihypertensive and antiarrhythmic effects. We studied the mechanism of diltiazem blockade by recording L-type Ca2+ channel currents from cell-attached patches in isolated guinea pig ventricular myocytes using Ba2+ as the charge carrier. With diltiazem (200 microM) in the superfusate, multichannel currents showed a use-dependent decline in amplitude reflecting reductions in the numbers of superpositions of channel openings. Analysis of single-channel currents revealed that both open and closed times were little affected by diltiazem (50 and 100 microM). However, the rate of decay of the averaged current during 150-ms depolarization steps was significantly accelerated and the open state probability in current containing-sweeps was significantly decreased by diltiazem, suggesting that the drug accelerates transition from the activated state to the inactivated state. The effect of diltiazem on the slow gating process was studied by repetitively applying 500-1000 step pulses at selected holding potentials. Decreased channel availability by diltiazem was reflected by the increasing number of blank sweeps per run at depolarized holding potentials. These results suggest that diltiazem reduces Ca2+ influx by accelerating inactivation during action potentials, and that the use-dependent blockade is due to increases in the number of channels in a sustained closed state.

Epiandrosterone, a metabolite of testosterone precursor, blocks L-type calcium channels of ventricular myocytes and inhibits myocardial contractility.

The dehydroepiandrosterone metabolite epiandrosterone (EPI) inhibits the pentose phosphate pathway (PPP) and dilates isolated blood vessels pre-contracted by partial depolarization. We found that EPI (10-100 microM) also dose-dependently decreases left-ventricular developed pressure (LVDP), the rate of myocardial contraction (+d p /d t), and the pressure rate product (PRP); at 100 microM EPI, LVDP (131+/-9 vs 34+/-7 mmHg), +d p /dt (1515+/-94 vs 542+/-185 mmHg/s), and PRP (37870+/-2471 vs 9498+/-2375 HR x mmHg/min) were all significantly (P<0.05) reduced. EPI also elevated CPP in isolated hearts, decreased levels of myocardial NADPH and nitrite, and dose-dependently relaxed rat aortic rings pre-contracted with KCl. Electrophysiological analysis of single ventricular myocytes using whole cell clamp showed EPI to dose-dependently (100 n M-100 microM) and reversibly inhibit L-type channel currents carried by Ba2+ (IBa) (IC50=42+/-6 microM) by as much as 50%. At 30 microM, EPI shifted the steady-state inactivation curve to more negative potentials (V50=-26.6 mV vs -38.0 mV), thereby accelerating the decay of IBa during depolarization. These results suggest that EPI may act as a L-type Ca2+ channel antagonist with properties similar to those of 1,4-dihydropyridine (DHP) Ca2+ channel blockers.

KV2.1 K+ channels underlie major voltage-gated K+ outward current in H9c2 myoblasts.

The H9c2 clonal cell line derived from embryonic rat ventricle is an in vitro model system for cardiac and skeletal myocytes. We used the whole-cell patch clamp technique to characterize the electrophysiological and pharmacological properties of an outward K+ current (IK(V)) and determined its molecular correlate in H9c2 myoblasts. IK(V) was activated by threshold depolarization to -30 mV, and its current amplitude and rate of activation increased with further depolarizations. IK(V) inactivated slowly with a time constant of 1-2 s, and the V(0.5) for steady-state inactivation was -37.9 +/- 4.6 mV (n = 10). Tetraethylammonium and quinidine suppressed IK(V) with IC(50)'s of 3.7 mM and 11.6 microM, respectively. Using RT-PCR analysis we found that the K(V )2.1 gene is the most abundantly expressed among genes for K(V)1.2, 1.4, 1.5, 2.1, 4.2, and 4.3, and by Western blotting we confirmed the synthesis of the K(V)2.1 alpha-subunit protein. We conclude that IK(V), the predominant voltage-gated outward current in H9c2 myoblasts, flows through the channels comprised of the K(V)2.1-subunit gene product.