Guinea-pig Ventricular Cells Research Articles

Amiodarone inhibits sarcolemmal but not mitochondrial KATP channels in Guinea pig ventricular cells.

ATP-sensitive K(+) (KATP) channels are present on the sarcolemma (sarcKATP channels) and mitochondria (mitoKATP channels) of cardiac myocytes. Amiodarone, a class III antiarrhythmic drug, reduces sudden cardiac death in patients with organic heart disease. The objective of the present study was to investigate the effects of amiodarone on sarcKATP and mitoKATP channels. Single sarcKATP channel current and flavoprotein fluorescence were measured in guinea pig ventricular myocytes to assay sarcKATP and mitoKATP channel activity, respectively. Amiodarone inhibited the sarcKATP channel currents in a concentration-dependent manner without affecting its unitary amplitude. The IC50 values were 0.35 microM in the inside-out patch exposed to an ATP-free solution and 2.8 microM in the cell-attached patch under metabolic inhibition, respectively. Amiodarone (10 microM) alone did not oxidize the flavoprotein. In addition, the oxidative effect of the mitoKATP channel opener diazoxide (100 microM) was unaffected by amiodarone. Exposure to ouabain (1 mM) for 30 min produced mitochondrial Ca(2+) overload, and the intensity of rhod-2 fluorescence increased to 246 +/- 16% of baseline (n = 9). Amiodarone did not alter the ouabain-induced mitochondrial Ca(2+) overload (236 +/- 10% of baseline, n = 7). Treatment with diazoxide significantly reduced the ouabain-induced mitochondrial Ca(2+) overload (158 +/- 15% of baseline, n = 8, p < 0.05 versus ouabain); this effect was not abolished by amiodarone (154 +/- 10% of baseline, n = 8, p < 0.05 versus ouabain). These results suggest that amiodarone inhibits sarcKATP but not mitoKATP channels in cardiac myocytes. Such an action of amiodarone may effectively prevent ischemic arrhythmias without causing ischemic damage.

Mechanism of pacemaking in I(K1)-downregulated myocytes.

Biological pacemakers were recently created by genetic suppression of inward rectifier potassium current, I(K1), in guinea pig ventricular cells. We simulated these cells by adjusting I(K1) conductance in the Luo-Rudy model of the guinea pig ventricular myocyte. After 81% I(K1) suppression, the simulated cell reached steady state with pacemaker period of 594 ms. Pacemaking current is carried by the Na+-Ca2+ exchanger, I(NaCa), which depends on the intracellular calcium concentration [Ca2+]i. This [Ca2+]i dependence suggests responsiveness (increase in rate) to beta-adrenergic stimulation (betaAS), as observed experimentally. Simulations of betaAS demonstrate such responsiveness, which depends on I(NaCa) expression. However, a simultaneous betaAS-mediated increase in the slow delayed rectifier, I(Ks), limits betaAS sensitivity.

Molecular and functional diversity of ATP-sensitive K+ channels: the pathophysiological roles and potential drug targets

ATP-sensitive K(+) (K(ATP)) channels comprise the pore-forming subunit (Kir6.1 or Kir6.2) and the regulatory subunit sulfonylurea receptors (SUR1 or SUR2). K(ATP) channels with different combinations of these subunits are present in various tissues and regulate cellular functions. From the analysis of mouse models with targeted deletion of the gene encoding the pore-forming subunit Kir6.1 or Kir6.2, functional roles of K(ATP) channels in various organs have been clarified. Kir6.1(-/-) mice showed sudden death associated with ST elevation and atrioventricular block in ECG, a phenotype resembling Prinzmetal angina in humans. Kir6.2(-/-) mice were more susceptible to generalized seizure during hypoxia than wild-type (WT) mice, suggesting that neuronal K(ATP) channels, probably composed of Kir6.2 and SUR1, play a crucial role for the protection of the brain against lethal damage due to seizure. In Kir6.2(-/-) mice lacking the sarcolemmal K(ATP) channel activity in cardiac cells, ischemic preconditioning failed to reduce the infarct size, suggesting that sarcolemmal K(ATP) channels play an important role in cardioprotection against ischemia/reperfusion injuries in the heart. Mitochondrial K(ATP) channels have been also proposed to play a crucial role in cardioprotection, although the molecular identity of the channel has not been established. Nicorandil and minoxidil, K(+) channel openers activating mitochondrial K(ATP) channels, decreased the mitochondrial membrane potential, thereby preventing the Ca(2+) overload in the mitochondria of guinea-pig ventricular cells. SURs are the receptors for K(+) channel openers and the activating effects on sarcolemmal K(ATP) channels in cardiovascular tissues could be modulated by the interaction of nucleotides. Due to the molecular diversity of the accessory and pore subunits of K(ATP) channels, there would be considerable differences in the tissue selectivity of K(ATP) channel-acting drugs. Studies of Kir6.1 and Kir6.2 knockout mice indicate that K(ATP) channels are involved in the mechanisms of the protection against metabolic stress. Further clarification of physiological as well as pathophysiological roles of K(ATP) channels may lead to a new therapeutic strategy to improve the quality of life.

Cytoprotective role of Ca2+- activated K+ channels in the cardiac inner mitochondrial membrane.

Ion channels on the mitochondrial inner membrane influence cell function in specific ways that can be detrimental or beneficial to cell survival. At least one type of potassium (K+) channel, the mitochondrial adenosine triphosphate-sensitive K+ channel (mitoKATP), is an important effector of protection against necrotic and apoptotic cell injury after ischemia. Here another channel with properties similar to the surface membrane calcium-activated K+ channel was found on the mitochondrial inner membrane (mitoKCa) of guinea pig ventricular cells. MitoKCa significantly contributed to mitochondrial K+ uptake of the myocyte, and an opener of mitoKCa protected hearts against infarction.

Effects of acidic preconditioning on [formula omitted] exchange current in guinea pig ventricular cells

Effects of acidic preconditioning on [formula omitted] exchange current in guinea pig ventricular cells

Spatial heterogeneity of transmembrane potential responses of single guinea-pig cardiac cells during electric field stimulation.

Changes in transmembrane voltage (V(m)) of cardiac cells during electric field stimulation have a complex spatial- and time-dependent behaviour that differs significantly from electrical stimulation of space-clamped membranes by current pulses. A multisite optical mapping system was used to obtain 17 or 25 microm resolution maps of V(m) along the long axis of guinea-pig ventricular cells (n = 57) stained with voltage-sensitive dye (di-8-ANEPPS) and stimulated longitudinally with uniform electric field (2, 5 or 10 ms, 3-62 V cm(-1)) pulses (n = 201). The initial polarizations of V(m) responses (V(mr)) varied linearly along the cell length and reversed symmetrically upon field reversal. The remainder of the V(m) responses had parallel time courses among the recording sites, revealing a common time-varying signal component (V(ms)). V(ms) was depolarizing for pulses during rest and hyperpolarizing for pulses during the early plateau phase. V(ms) varied in amplitude and time course with increasing pulse amplitude. Four types of plateau response were observed, with transition points between the different responses occurring when the maximum polarization at the ends of the cell reached values estimated as 60, 110 and 220 mV. Among the cells that had a polarization change of > 200 mV at their ends (for fields > 45 V cm(-1)), some (n = 17/25) had non-parallel time courses among V(m) recordings of the various sites. This implied development of an intracellular field (E(i)) that was found to increase exponentially with time (tau = 7.2 +/- 3.2 ms). Theoretical considerations suggest that V(ms) represents the intracellular potential (phi(i)) as well as the average polarization of the cell, and that V(mr) is the manifestation of the extracellular potential gradient resulting from the field stimulus. For cells undergoing field stimulation, phi(i) acts as the cellular physiological state variable and substitutes for V(m), which is the customary variable for space-clamped membranes.

Regulation of K(ATP) channels by P(2Y) purinoceptors coupled to PIP(2) metabolism in guinea pig ventricular cells.

We used patch-clamp techniques to elucidate the regulatory mechanisms of ATP-sensitive K(+) (K(ATP)) channels by stimulation of P(2) purinoceptors in guinea pig ventricular myocytes. Extracellular ATP at 0.1 mM transiently inhibited by 90.5 +/- 5.0% the whole cell K(ATP) channel current evoked by a reduction in intracellular ATP concentration to 0.5 mM and exposure to 30 microM pinacidil. ADP and AMP (both 1 mM) also decreased the current by 42.8 +/- 9.3% and 9.4 +/- 4.8%, respectively, but adenosine did not, even at 10 mM. ATP-induced channel inhibition was hardly observed in the presence of 0.2 mM suramin, 0.2 mM guanosine 5'-O-(2-thiodiphosphate), or 0.1 mM compound 48/80, whereas it was not influenced by the presence of 0.1 microM staurosporine or 10 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid in the pipette. In the presence of 10 microM wortmannin or the absence of ATP in the cytosol, the ATP-induced channel inhibition was irreversible. Phosphatidylinositol 4,5-bisphosphate (PIP(2)) at 0.1 mM in the outside-out patch pipette prevented ATP-induced channel inhibition. The half-maximal internal ATP concentrations for inhibition of channel activity determined in inside-out membrane patches were 13.8 microM in the presence and 1.12 mM in the absence of 0.1 mM ATP at the external side. It is concluded that activity of K(ATP) channels is modulated by extracellular ATP by a mechanism involving P(2Y) purinoceptors coupled to GTP-binding proteins associated with reduction of the sarcolemmal PIP(2) concentration via stimulation of phospholipase C.

Caffeine-induced immobilization of gating charges in isolated guinea-pig ventricular heart cells.

The effects of 10 mM caffeine (CAF) on intramembrane charge movements (ICM) were studied in isolated guinea-pig ventricular heart cells with the whole-cell patch-clamp technique. In the presence of CAF, the properties (voltage dependence, maximum Q(ON) [Q(max)], availability with voltage) of Q(ON) charge activated from -110 mV were barely affected. Following a 100 ms prepulse to -50 mV to decrease the participation of charges originating from Na channels, the voltage dependence of Q(ON) was shifted by 5 mV (negative component) and by 10 mV (positive component) towards negative potentials, and Q(max) was depressed by 16.5%. CAF drastically reduced in a time- and voltage-dependent manner Q(OFF) on repolarization to -50 mV, the effects being greater at positive potentials. CAF-induced Q(OFF) immobilization could be almost entirely removed by repolarization to voltages as negative as -170 mV. In these conditions, the voltage-dependence of Q(OFF) (repolarization to +30 to -170 mV) was shifted by 17 mV (negative component) and 30 mV (positive component) towards negative potentials, suggesting an interconversion into charge 2. Most of CAF effects were suppressed when the sarcoplasmic reticulum (SR) was not functional or when the cells were loaded with BAPTA-AM. We conclude that CAF effects on ICM are likely due to Ca(2+) ions released from the SR, and which accumulate in the subsarcolemmal fuzzy spaces in the vicinity of the Ca channels. Because CAF effects were more pronounced on Q(OFF) than on Q(ON) the channels have likely to open before Ca(2+) ions could affect their gating properties. It is speculated that such an effect on gating charges might contribute to the Ca-induced inactivation of the Ca current.

Effects of benzyltetrahydropalmatine on two components of the delayed rectifier K+ current in Guinea pig ventricular myocytes.

The effects of benzyltetrahydropalmatine (BTHP), a new class III antiarrhythmic agent, on the action potential in guinea pig papillary muscle and the rapidly activating component (I(Kr)) and the slowly activating component (I(Ks)) of the delayed rectifier potassium current (I(K)) in isolated guinea pig ventricular myocytes were investigated. The action potentials of papillary muscles were studied using a standard microelectrode technique, while the K(+) currents were recorded using the whole-cell patch clamp technique. The results showed that BTHP prolonged the action potential duration (APD) without altering other variables of the action potential in guinea pig papillary muscles. The 2 components of I(K) were blocked by BTHP (1 approximately 100 micromol x L(-1)) in time-, concentration-, voltage-, and specifically frequency-dependent fashion. The IC(50) value for blockade of I(Kr) was 13.5 micromol x L(-1), while the IC(50) value for blockade of I(Ks) was 9.3 micromol x L(-1). BTHP 30.0 micromol x L(-1) reduced I(Kr) and I(Kr,tail) by 31 +/- 4.3% and 36 +/- 4.7% (n = 6, p < 0.01) and decreased I(Ks) and I(Ks,tail) by 40 +/- 6.3% and 39 +/- 4.6% (n = 7, p < 0.01) respectively. BTHP accelerated their deactivation course by reducing the time constants of deactivation of I(Kr) and I(Ks). The activation kinetics of I(Kr) or I(Ks) were not affected by BTHP. It is concluded that BTHP prolonged the action potential duration with respect to its non-selective action on I(Kr) and I(Ks) in single guinea pig ventricular cell in a frequency-dependent fashion.

Spiral-wave meandering in reaction–diffusion models of ventricular muscle

Reentrant arrhythmias in cardiac tissue can be idealised as spiral-wave solutions of reaction–diffusion equations of excitable media. The dynamical behaviour of such solutions depends on the properties of the excitable medium. Here we characterise the effects of model-parameter changes for ionic conductances and concentrations on the meandering patterns and dynamics of spiral waves in a model of mammalian ventricular tissue. A two-dimensional reaction–diffusion system with kinetics described by the Oxsoft ® Heart equations for the single guinea-pig ventricular cell was used. We examine the development and stability of reentrant spiral-wave solutions as well as the effects of ionic conductance and concentration changes on spiral-wave meandering. Spiral-wave meandering behaviour provides a significant validation test for detailed biophysical models.

Protein kinase C isoform-dependent modulation of ATP-sensitive K+ channels during reoxygenation in guinea-pig ventricular myocytes.

ATP-sensitive K+ (KATP) channels activated by glucose-free anoxia close immediately upon reoxygenation in single guinea-pig ventricular myocytes, while KATP channels open persistently during reperfusion in coronary-perfused guinea-pig ventricular myocardium. To investigate the reasons behind this discrepancy, we investigated whether protein kinase C (PKC) modulates the opening of KATP channels during anoxia-reoxygenation and ischaemia-reperfusion. Exposure of guinea-pig ventricular cells to glucose-free anoxia shortened the action potential duration at 90% repolarisation (APD90) and evoked the glibenclamide-sensitive robust outward current (IK,ATP). Subsequent reoxygenation caused an immediate prolongation of APD90 and a decrease in IK,ATP within approximately 20 s. When the novel (Ca2+-independent) PKC was activated by applying 1,2-dioctanoyl-sn-glycerol (1,2DOG, 20 M) with EGTA (20 mM) in the pipette, the APD90 restored gradually after reoxygenation and the extent of recovery was appoximately 80% of the pre-anoxic value. Moreover, IK,ATP decreased slowly and remained opened for up to approximately 4 min after reoxygenation. These results suggest persistent opening of KATP channels during reoxygenation. The persistent activation of KATP channels was augmented when both novel and conventional (Ca2+-dependent) isoforms of PKC were activated by applying 1,2DOG without EGTA in the pipette. In coronary-perfused right ventricular myocardium, APD90 remained shortened for up to approximately 30 min of reperfusion. The gradual restoration of APD90 after ischaemia-reperfusion was facilitated by the KATP channel blocker glibenclamide and by the potent PKC inhibitor chelerythrine. Our results provide the first evidence that PKC activation contributes to the persistent opening of KATP channels during reoxygenation and reperfusion. We also conclude that both novel and conventional PKC isoforms co-operatively modulate the opening of KATP channels during the early phase of reoxygenation.

Inhibitory effect of 2,3-butanedione monoxime (BDM) on Na(+)/Ca(2+) exchange current in guinea-pig cardiac ventricular myocytes.

1. The effect of 2,3-butanedione monoxime (BDM), a 'chemical phosphatase', on Na(+)/Ca(2+) exchange current (I(NCX)) was investigated using the whole-cell voltage-clamp technique in single guinea-pig cardiac ventricular myocytes and in CCL39 fibroblast cells expressing canine NCX1. 2. I(NCX) was identified as a current sensitive to KB-R7943, a relatively selective NCX inhibitor, at 140 mM Na(+) and 2 mM Ca(2+) in the external solution and 20 mM Na(+) and 433 nM free Ca(2+) in the pipette solution. 3. In guinea-pig ventricular cells, BDM inhibited I(NCX) in a concentration-dependent manner. The IC(50) value was 2.4 mM with a Hill coefficients of 1. The average time for 50% inhibition by 10 mM BDM was 124+/-31 s (n=5). 4. The effect of BDM was not affected by 1 microM okadaic acid in the pipette solution, indicating that the inhibition was not via activation of okadaic acid-sensitive protein phosphatases. 5. Intracellular trypsin treatment via the pipette solution significantly suppressed the inhibitory effect of BDM, implicating an intracellular site of action of BDM. 6. PAM (pralidoxime), another oxime compound, also inhibited I(NCX) in a manner similar to BDM. 7. Isoprenaline at 50 microM and phorbol 12-myristate 13-acetate (PMA) at 8 microM did not reverse the inhibition of I(NCX) by BDM. 8. BDM inhibited I(NCX) in CCL39 cells expressing NCX1 and in its mutant in which its three major phosphorylatable serine residues were replaced with alanines. 9. We conclude that BDM inhibits I(NCX) but the mechanism of inhibition is not by dephosphorylation of the Na(+)/Ca(2+) exchanger as a 'chemical phosphatase'.

Independent modulation of L-type Ca2+ channel in guinea pig ventricular cells by nitrendipine and isoproterenol.

Dihydropyridine (DHP) Ca2+ channel blockers decrease L-type Ca2+ channel current (I(CaL)) by enhancing steady-state inactivation, whereas beta-adrenergic stimulation increases I(CaL) with small changes in the kinetics. We studied the effects of DHP Ca2+ channel blockers on cardiac I(CaL) augmented by beta-adrenergic stimulation. We recorded I(CaL) as Ba2+ currents (I(Ba)) from guinea pig ventricular myocytes using the whole-cell patch clamp technique. and compared the effects of nitrendipine (NIT) in the absence and presence of isoproterenol (1 microM, ISO) or forskolin (10 microM, FSK). Maximal I(Ba) elicited from a holding potential of -80 mV were diminished to 69.4+/-13.5% (mean and SE, n=5) of control by NIT (100 nM) and the diminished I(Ba) were increased to 180.3+/-23.2% of control by ISO in the presence of NIT, which was similar to the enhancement seen in the absence of NIT. NIT shifted the V(1/2) of the I(Ba) inactivation curve from -34.6+/-1.9 mV (n=5) to -48.7+/-1.2 mV, enhancing I(Ba) decay with shortening T(1/2) at -10 mV from 164.6+/-24.2 ms (n=7) to 105.4+/-15.2 ms. ISO elicited a small additional shift in the V(1/2) of I(Ba) inactivation in the same direction. ISO and FSK each slowed I(Ba) decay in the absence of NIT, but not in its presence. Thus, beta-adrenergic agonists increase and DHP Ca2+ channel blockers decrease the amplitude of cardiac I(CaL) independently and the kinetics of I(CaL) is determined mainly by the latter when these drugs coexist.

Regulation of cardiac calcium current by NO and cGMP-modulating agents.

Several effects of nitric oxide (NO) on the control of L-type calcium current (ICa) and of calcium handling in cardiomyocytes have been described. Cardiomyocytes have been shown to express in different conditions all types of nitric oxide synthases (NOS), but the role of NO in the regulation of calcium current remains controversial. Previously, we have shown in guinea pig ventricular cells a stimulatory effect of NOS inhibitors on ICa. Here we investigate the intracellular mechanisms involved in the putative inhibitory role of NO on basal ICa in ventricular cells. The stimulatory effect of the NOS inhibitor NG-monomethyl-L-arginine (L-NMMA) (1 mM) was present also in calcium transient measurements, but only after a preincubation with L-arginine (L-arg, 0.1 mM). The nitric oxide scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO, 0.5 mM) increased peak ICa in a similar manner to NOS inhibitors in whole-cell voltage-clamp experiments. Also ODQ (1H-[1,2,4]oxidiazolo[4,3-a]quinoxaline-1-one, 0.1 mM), a specific inhibitor of a target of NO, the soluble guanylate cyclase, was able to stimulate ICa. The block of type II phosphodiesterase (cGMP-activated) by EHNA (erythro-9-[2-hydroxy-3-nonylladenine, 30 microM) exerted a similar effect on ICa as PTIO and ODQ. Carbachol (CCh, 1 microM) was able to revert the stimulatory effect on ICa observed with PTIO, ODQ, and EHNA. We propose that the increase of basal ICa in guinea pig cardiomyocytes previously observed with L-NMMA depends on the removal of a tonic NO inhibition. This increase of ICa is mimicked by blocking at different steps the cGMP-cascade activated by NO, suggesting a NO-guanylate cyclase mechanism in the basal control of ventricular calcium current.

Regulation kinetics of Na+‐Ca2+ exchange current in guinea‐pig ventricular myocytes

To investigate the regulation of native cardiac Na+-Ca2+ exchange by cytoplasmic Na+ (Na+i) and Ca2+ (Ca2+i), we recorded the Na+-Ca2+ exchange current (INa-Ca) from inside-out 'macro patches' excised from intact guinea-pig ventricular cells. The half-maximal concentration (Kh) of Ca2+i required to induce an inward INa-Ca was 7 microM. The Kh of Na+i required to induce an outward INa-Ca was 21 mM, and tended to decrease at the steady state of Na+-dependent inactivation. The time constant (tau) of Na+-dependent inactivation was approximately 1.5 s at 100 mM Na+i and 1 microM Ca2+i. The Kh for Na+i was 14 mM. Ca2+i augmented the peak outward INa-Ca (Kh = 0. 2 microM) and attenuated Na+-dependent inactivation (Kh = 2.2 microM). The outward INa-Ca was activated by 5 microM Ca2+i with a half-time to reach steady state (t1/2) of approximately 0.4 s. This activation was composed of two exponential processes. Deactivation of the current upon Ca2+i removal also consisted of two exponential processes and had a t1/2 of approximately 0.5 s. A Na+-Ca2+ exchange model, consisting of one consecutive 4Na+:1Ca2+ exchange cycle and two inactive states, well mimicked the experimental data with regard to ion dependencies and regulation kinetics. These data provide detailed information on the kinetics of the Na+i- and Ca2+i-dependent regulation of native Na+-Ca2+ exchange. They also indicate that the regulation kinetics operate faster in macro patches than in the giant membrane patch from cardiac 'blebs', or in Xenopus oocytes expressing a cloned exchanger (NCX1.1).

Effects of JTV-519, a novel anti-ischaemic drug, on the delayed rectifier K+ current in guinea-pig ventricular myocytes.

We studied the effects of a newly synthesized anti-ischaemic agent, 4-[3-(4-benzylpiperidin-1-yl) propionyl]-7-methoxy-2, 3, 4, 5-tetrahydro-1, 4-benzothiazepine monohydrochloride (JTV-519) on the delayed rectifier potassium current (IK), using guinea-pig ventricular myocytes and whole-cell voltage-clamp techniques, under blockade of the L-type calcium current (ICa,L) by D600 (1 microM) or nitrendipine (5 microM). The IK in guinea-pig ventricular cells consists of two different components; the rapidly activating, E4031-sensitive component (IKr) and the slowly activating E4031-resistant component (IKs). Under steady-state conditions, JTV-519 (1 and 5 microM) did not change the amplitude of IKs remaining after blockade of IKr with 5 microM E4031. The effect of JTV-519 on IKr was assessed using short (50 ms) pulses which evoked a tail current that was sensitive to E4031 but not to chromanol 293B, a specific blocker of IKs. JTV-519 suppressed the IKr with a half-maximal inhibitory concentration of 1.2 microM. Selective inhibition of IKr by this agent was confirmed by using the "envelope of tails" test. These results suggest that the blockade of IKr may underlie the prolongation of action potential duration in ventricular muscle and QT-intervals alleged to occur in animal as well as human hearts.

Time-dependent block of the slowly activating delayed rectifier K(+) current by chromanol 293B in guinea-pig ventricular cells.

The slowly activating delayed rectifier K(+) current (I(Ks)) was recorded in single myocytes dissociated from guinea-pig ventricles and the mechanism underlying the block of I(Ks) by a chromanol derivative, 293B, was investigated. In the presence of 1 - 100 microM 293B, activation phase of I(Ks) was followed by a slower decay during 10 s depolarizing pulses. Both the rate and extent of the decay were increased in a concentration-dependent manner. The relationship between the concentration of 293B and the block showed a Hill's coefficient of approximately 1. The half-inhibitory concentration was approximately 3.0 microM and did not differ significantly at various membrane potentials from +20 to +80 mV. A mathematical model for the 293B block was constructed on the basis of multiple closed and open states for the I(Ks) channels, and the blocking rate was calculated by fitting the model to the original current traces. The blocking rate constant showed a linear function with the 293B concentration, indicating 1 : 1 binding stoichiometry. At +80 mV the blocking rate was 4x10(4) M(-1) s(-1) and the unblocking rate was 0.2 s(-1). The results indicate that 293B is an open channel blocker with relatively smaller blocking rate than those reported so far for time-dependent blockade of various ionic channels.

Cardiotoxic effects of fenfluramine hydrochloride on isolated cardiac preparations and ventricular myocytes of guinea-pigs.

The cardiotoxic effects of fenfluramine hydrochloride on mechanical and electrical activity were studied in papillary muscles, Purkinje fibres, left atria and ventricular myocytes of guinea-pigs. Force of contraction (f(c)) was measured isometrically, action potentials and maximum rate of rise of the action potential (V(max)) were recorded by means of the intracellular microelectrode technique and the sodium current (I(Na)) with patch-clamp technique in the cell-attached mode. For kinetic analysis (S)-DPI-201-106-modified Na(+) channels from isolated guinea-pig ventricular heart cells were used. Fenfluramine (1 - 300 microM) produced negative chronotropic and inotropic effects; additional extracellular Ca(2+) competitively antagonized the negative inotropic effect. Fenfluramine concentration-dependently reduced V(max) and showed tonic blockade of sodium channels, shortened the action potential duration in papillary muscles and Purkinje fibres. In cell-attached patches, fenfluramine decreased I(Na) concentration-dependently (10 - 100 microM), frequency-independently (0.1 - 3 Hz; 30 microM). The h(infinity) curve was shifted towards hyperpolarizing direction. At 30 microM, fenfluramine blocked the sodium channel at all test potentials to the same degree, and neither changed the threshold and reversal potentials nor the peak of the curve. No effect on single channel availability, but a significant decrease in mean open times and increase in mean closed times was observed. Mean duration of the bursts decreased and number of openings per record increased with increasing drug concentration. It is concluded that the effect on I(Na) plays an important role in the cardiotoxicity of fenfluramine in addition to primary pulmonary hypertension and valvular disorders.

Stoichiometry of Na+‐Ca2+ exchange in inside‐out patches excised from guinea‐pig ventricular myocytes

1. The stoichiometry (nx) of cardiac Na+-Ca2+ exchange was examined by measuring the reversal potential of the Na+-Ca2+ exchange current (INa-Ca) in large inside-out patches, 'macro patches', excised from intact guinea-pig ventricular cells. 2. Cytoplasmic application of Na+ (Na+i) or Ca2+ (Ca2+i) induced INa-Ca which showed properties similar to INa-Ca in the giant membrane patch. The outward INa-Ca was depressed by an exchanger inhibitory peptide, XIP. 3. The reversal potential of the XIP-sensitive current indicated that nx was approximately 4 (3.6-4.2) at 9-40 mM Na+i, and nx tended to increase as Na+i was increased. Proteolysis by trypsin did not significantly affect the stoichiometry. Similar results were obtained from the reversal potential of INa-Ca that was induced by application of both Na+i and Ca2+i. 4. At 0.1 microM Ca2+i, nx was approximately 4 (3.7-4. 4) at 6-25 mM Na+i and tended to increase as Na+i was increased. When Ca2+i was changed from 0.1 to 1 and 1000 microM at constant 50 mM Na+i, the value was approximately 4 (3.6-4.4). 5. When the extracellular Na+ (Na+o) and Ca2+ (Ca2+o) concentrations were varied in the presence of 25 or 9 mM Na+i and 1 microM Ca2+i, nx was almost constant ( approximately 4) over the range 0.3-20 mM Ca2+o and 10-145 mM Na+o. 6. These results indicated that the stoichiometry of Na+-Ca2+ exchange is different from generally accepted 3Na+:1Ca2+, and suggested that the stoichiometry is either 4Na+:1Ca2+ or variable depending on Na+i and Ca2+i.

Analysis of the electrophysiologic effects of short-term oxybutynin on guinea pig and rabbit ventricular cells.

The objective of this study was to investigate the cardioactive properties of oxybutynin, a drug that is widely prescribed for management of voiding dysfunction. Membrane currents were recorded from whole-cell-configured guinea pig ventricular myocytes, and action potentials were recorded from guinea pig and rabbit papillary muscles. L-type Ca2+ current (I(Ca),L), inward-rectifier K+ current (I(K1)), and delayed-rectifier K+ current (I(K)) were unaffected by < or = 1 microM oxybutynin, and inhibited by higher concentrations. The concentrations that reduced the currents to one-half of predrug control amplitude (K0.5) were as follows: 1(Ca),L, 16.1 microM, I(K1), 18.2 microM, rapidly activating I(K)(I(Kr)), 11.4 microM, and slowly activating I(K)(I(Ks)), 28.7 microM. Action-potential durations at 20 and 90% repolarization (APD20, APD90) were unaffected by oxybutynin < or =3 microM in guinea pig papillary muscles driven at 1 Hz; higher concentrations selectively shortened the APD20 by as much as 25% (100 microM), and caused moderate reductions in maximal upstroke velocity. Changes in the action potentials of rabbit papillary muscles were even smaller than in the guinea pig muscles. Because the peak therapeutic plasma concentration of oxybutynin is in the 0.01-0.1 microM range, the results suggest that the drug is highly unlikely to have adverse effects on cardiac electrical activity.