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Current density, the membrane current value divided by membrane capacitance (Cm), is widely used in cellular electrophysiology. Comparing current densities obtained in different cell populations assume that Cm and ion current magnitudes are linearly related, however data is scarce about this in cardiomyocytes. Therefore, we statistically analyzed the distributions, and the relationship between parameters of canine cardiac ion currents and Cm, and tested if dividing original parameters with Cm had any effect. Under conventional voltage clamp conditions, correlations were high for IK1, moderate for IKr and ICa,L, while negligible for IKs. Correlation between Ito1 peak amplitude and Cm was negligible when analyzing all cells together, however, the analysis showed high correlations when cells of subepicardial, subendocardial or midmyocardial origin were analyzed separately. In action potential voltage clamp experiments IK1, IKr and ICa,L parameters showed high correlations with Cm. For INCX, INa,late and IKs there were low-to-moderate correlations between Cm and these current parameters. Dividing the original current parameters with Cm reduced both the coefficient of variation, and the deviation from normal distribution. The level of correlation between ion currents and Cm varies depending on the ion current studied. This must be considered when evaluating ion current densities in cardiac cells. [ABSTRACT FROM AUTHOR]

Enhancement of the late Na+ current (INaL) increases arrhythmia propensity in the heart, while suppression of the current is antiarrhythmic. GS967 is an agent considered as a selective blocker of INaL. In the present study, effects of GS967 on INaL and action potential (AP) morphology were studied in canine ventricular myocytes by using conventional voltage clamp, action potential voltage clamp and sharp microelectrode techniques. The effects of GS967 (1 µM) were compared to those of the class I/B antiarrhythmic compound mexiletine (40 µM). Under conventional voltage clamp conditions, INaL was significantly suppressed by GS967 and mexiletine, causing 80.4 ± 2.2% and 59.1 ± 1.8% reduction of the densities of INaL measured at 50 ms of depolarization, and 79.0 ± 3.1% and 63.3 ± 2.7% reduction of the corresponding current integrals, respectively. Both drugs shifted the voltage dependence of the steady-state inactivation curve of INaL towards negative potentials. GS967 and mexiletine dissected inward INaL profiles under AP voltage clamp conditions having densities, measured at 50% of AP duration (APD), of −0.37 ± 0.07 and −0.28 ± 0.03 A/F, and current integrals of −56.7 ± 9.1 and −46.6 ± 5.5 mC/F, respectively. Drug effects on peak Na+ current (INaP) were assessed by recording the maximum velocity of AP upstroke (V+max) in multicellular preparations. The offset time constant was threefold faster for GS967 than mexiletine (110 ms versus 289 ms), while the onset of the rate-dependent block was slower in the case of GS967. Effects on beat-to-beat variability of APD was studied in isolated myocytes. Beat-to-beat variability was significantly decreased by both GS967 and mexiletine (reduction of 42.1 ± 6.5% and 24.6 ± 12.8%, respectively) while their shortening effect on APD was comparable. It is concluded that the electrophysiological effects of GS967 are similar to those of mexiletine, but with somewhat faster offset kinetics of V+max block. However, since GS967 depressed V+max and INaL at the same concentration, the current view that GS967 represents a new class of drugs that selectively block INaL has to be questioned and it is suggested that GS967 should be classified as a class I/B antiarrhythmic agent. [ABSTRACT FROM AUTHOR]

Omecamtiv mecarbil (OM) is a myosin activator agent developed for the treatment of heart failure. OM was reported to increase left ventricular ejection fraction and systolic ejection time, but little is known about the effect of heart rate on the action of OM. The present study, therefore, was designed to investigate the effects of OM on unloaded cell shortening and intracellular Ca ([Ca]) transients as a function of the pacing frequency. Isolated cardiomyocytes were stimulated at various frequencies under steady-state conditions. Cell length was monitored by an optical edge detector and changes in [Ca] were followed using the Ca-sensitive dye Fura-2. At the pacing frequency of 1 Hz, OM (1-10 μM) significantly decreased both diastolic and systolic cell length, however, fractional shortening was augmented only by 1 μM OM. Time to peak tension and time of 90% relaxation were progressively increased by OM. At the frequency of 2 Hz, diastolic cell length was reduced by 10 μM OM to a larger extent than systolic cell length, resulting in a significantly decreased fractional shortening under these conditions. OM had no effect on the parameters of the [Ca] transient at any pacing frequency. The results suggest that supratherapeutic concentrations of OM may decrease rather than increase the force of cardiac contraction especially in tachycardic patients. [ABSTRACT FROM AUTHOR]

Although beat-to-beat variability (short-term variability, SV) of action potential duration (APD) is considered as a predictor of imminent cardiac arrhythmias, the underlying mechanisms are still not clear. In the present study, therefore, we aimed to determine the role of the major cardiac ion currents, APD, stimulation frequency, and changes in the intracellular Ca concentration ([Ca]) on the magnitude of SV. Action potentials were recorded from isolated canine ventricular cardiomyocytes using conventional microelectrode techniques. SV was an exponential function of APD, when APD was modified by current injections. Drug effects were characterized as relative SV changes by comparing the drug-induced changes in SV to those in APD according to the exponential function obtained with current pulses. Relative SV was increased by dofetilide, HMR 1556, nisoldipine, and veratridine, while it was reduced by BAY K8644, tetrodotoxin, lidocaine, and isoproterenol. Relative SV was also increased by increasing the stimulation frequency and [Ca]. In summary, relative SV is decreased by ion currents involved in the negative feedback regulation of APD ( I, I, and I), while it is increased by I and I. We conclude that drug-induced effects on SV should be evaluated in relation with the concomitant changes in APD. Since relative SV was decreased by ion currents playing critical role in the negative feedback regulation of APD, blockade of these currents, or the beta-adrenergic pathway, may carry also some additional proarrhythmic risk in addition to their well-known antiarrhythmic action. [ABSTRACT FROM AUTHOR]

Understanding the role of ionic currents in shaping the cardiac action potential (AP) has great importance as channel malfunctions can lead to sudden cardiac death by inducing arrhythmias. Therefore, researchers frequently use inhibitors to selectively block a certain ion channel like 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS) and 9-anthracene carboxylic acid (9-AC) for calcium-activated chloride current (I). This study aims to explore which blocker is preferable to study I. Whole-cell voltage-clamp technique was used to record I I I and I, while action potentials were measured using sharp microelectrodes. DIDS- (0.2 mM) and 9-AC-sensitive (0.5 mM) currents were identical in voltage-clamp conditions, regardless of intracellular Ca buffering. DIDS-sensitive current amplitude was larger with the increase of stimulation rate and correlated well with the rate-induced increase of calcium transients. Both drugs increased action potential duration (APD) to the same extent, but the elevation of the plateau potential was more pronounced with 9-AC at fast stimulation rates. On the contrary, 9-AC did not influence either the AP amplitude or the maximal rate of depolarization ( V), but DIDS caused marked reduction of V. Both inhibitors reduced the magnitude of phase-1, but, at slow stimulation rates, this effect of DIDS was larger. All of these actions on APs were reversible upon washout of the drugs. Increasing concentrations of 9-AC between 0.1 and 0.5 mM in a cumulative manner gradually reduced phase-1 and increased APD. 9-AC at 1 mM had no additional actions upon perfusion after 0.5 mM. The half-effective concentration of 9-AC was approximately 160 μM with a Hill coefficient of 2. The amplitudes of I I I and I were not changed by 0.5 mM 9-AC. These results suggest that DIDS is equally useful to study I during voltage-clamp but 9-AC is superior in AP measurements for studying the physiological role of I due to the lack of sodium channel inhibition. 9-AC has also no action on other ion currents (I, I, I, I); however, I tracings can be contaminated with I when measured in voltage-clamp condition. [ABSTRACT FROM AUTHOR]

Adrenergic activation of L-type Ca and various K currents is a crucial mechanism of cardiac adaptation; however, it may carry a substantial proarrhythmic risk as well. The aim of the present work was to study the timing of activation of Ca and K currents in isolated canine ventricular cells in response to exposure to isoproterenol (ISO). Whole cell configuration of the patch-clamp technique in either conventional voltage clamp or action potential voltage clamp modes were used to monitor I, I, and I, while action potentials were recorded using sharp microelectrodes. ISO (10 nM) elevated the plateau potential and shortened action potential duration (APD) in subepicardial and mid-myocardial cells, which effects were associated with multifold enhancement of I and I and moderate stimulation of I. The ISO-induced plateau shift and I increase developed faster than the shortening of APD and stimulation of I and I. Blockade of β-adrenoceptors (using 300 nM CGP-20712A) converted the ISO-induced shortening of APD to lengthening, decreased its latency, and reduced the plateau shift. In contrast, blockade of β-adrenoceptors (by 50 nM ICI 118,551) augmented the APD-shortening effect and increased the latency of plateau shift without altering its magnitude. All effects of ISO were prevented by simultaneous blockade of both receptor types. Inhibition of phosphodiesterases decreased the differences observed in the turn on of the ISO-induced plateau shift and APD shortening. ISO-induced activation of I is turned on faster than the stimulation of I and I in canine ventricular cells due to the involvement of different adrenergic pathways and compartmentalization. [ABSTRACT FROM AUTHOR]

Tacrolimus is a commonly used immunosuppressive agent which causes cardiovascular complications, e.g., hypertension and hypertrophic cardiomyopathy. In spite of it, there is little information on the cellular cardiac effects of the immunosuppressive agent tacrolimus in larger mammals. In the present study, therefore, the concentration-dependent effects of tacrolimus on action potential morphology and the underlying ion currents were studied in canine ventricular cardiomyocytes. Standard microelectrode, conventional whole cell patch clamp, and action potential voltage clamp techniques were applied in myocytes enzymatically dispersed from canine ventricular myocardium. Tacrolimus (3-30 μM) caused a concentration-dependent reduction of maximum velocity of depolarization and repolarization, action potential amplitude, phase-1 repolarization, action potential duration, and plateau potential, while no significant change in the resting membrane potential was observed. Conventional voltage clamp experiments revealed that tacrolimus concentrations ≥3 μM blocked a variety of ion currents, including I, I, I, I, and I. Similar results were obtained under action potential voltage clamp conditions. These effects of tacrolimus developed rapidly and were fully reversible upon washout. The blockade of inward currents with the concomitant shortening of action potential duration in canine myocytes is the opposite of those observed previously with tacrolimus in small rodents. It is concluded that although tacrolimus blocks several ion channels at higher concentrations, there is no risk of direct interaction with cardiac ion channels when applying tacrolimus in therapeutic concentrations. [ABSTRACT FROM AUTHOR]

Tetrodotoxin (TTX) is believed to be the most selective inhibitor of voltage-gated fast Na channels in excitable tissues, including nerve, skeletal muscle, and heart, although TTX sensitivity of the latter is lower than the former by at least three orders of magnitude. In the present study, the TTX sensitivity of L-type Ca current ( I) was studied in isolated canine ventricular cells using conventional voltage clamp and action potential voltage clamp techniques. TTX was found to block I in a reversible manner without altering inactivation kinetics of I. Fitting results to the Hill equation, an IC value of 55 ± 2 μM was obtained with a Hill coefficient of unity (1.0 ± s0.04). The current was fully abolished by 1 μM nisoldipine, indicating that it was really I. Under action potential voltage clamp conditions, the TTX-sensitive current displayed the typical fingerprint of I, which was absent in the presence of nisoldipine. Stick-and-ball models for Cav1.2 and Nav1.5 channel proteins were constructed to explain the differences observed between action of TTX on cardiac I and I. This is the first report demonstrating TTX to interact with L-type calcium current in the heart. [ABSTRACT FROM AUTHOR]

Protein kinase C (PKC) inhibitors are useful tools for studying PKC-dependent regulation of ion channels. For this purpose, high PKC specificity is a basic requirement excluding any direct interaction between the PKC inhibitor and the ion channel. In the present study, the effects of two frequently applied PKC inhibitors, chelerythine and bisindolylmaleimide I, were studied on the rapid and slow components of the delayed rectifier K current ( I and I) in canine ventricular cardiomyocytes and on the human ether-à-go-go-related gene (hERG) channels expressed in human embryonic kidney (HEK) cells. The whole cell version of the patch clamp technique was used in all experiments. Chelerythrine and bisindolylmaleimide I (both 1 μM) suppressed I in canine ventricular cells. This inhibition developed rapidly, suggesting a direct drug-channel interaction. In HEK cells heterologously expressing hERG channels, chelerythrine and bisindolylmaleimide I blocked hERG current in a concentration-dependent manner, having EC values of 0.11 ± 0.01 and 0.76 ± 0.04 μM, respectively. Both chelerythrine and bisindolylmaleimide I strongly modified gating kinetics of hERG-voltage dependence of activation was shifted towards more negative voltages and activation was accelerated. Deactivation was slowed by bisindolylmaleimide I but not by chelerythrine. I was not significantly altered by bisindolylmaleimide I and chelerythrine. No significant effect of 0.1 μM bisindolylmaleimide I or 0.1 μM PMA (PKC activator) was observed on I arguing against significant contribution of PKC to regulation of I. It is concluded that neither chelerythrine nor bisindolylmaleimide I is suitable for selective PKC blockade due to their direct blocking actions on the hERG channel. [ABSTRACT FROM AUTHOR]

In spite of its widespread clinical application, there is little information on the cellular cardiac effects of the dopamine receptor agonist ropinirole. In the present study, therefore, the concentration-dependent effects of ropinirole on action potential morphology and the underlying ion currents were studied in enzymatically dispersed canine ventricular cardiomyocytes using standard microelectrode, conventional whole-cell patch clamp, and action potential voltage clamp techniques. At concentrations ≥ 1 μM, ropinirole increased action potential duration (APD) and suppressed the rapid delayed rectifier K current ( I) with an IC value of 2.7 ± 0.25 μM and Hill coefficient of 0.92 ± 0.09. The block increased with increasing depolarizations to more positive voltages, but paradoxically, the activation of I was accelerated by 3 μM ropinirole (time constant decreased from 34 ± 4 to 14 ± 1 ms). No significant changes in the fast and slow deactivation time constants were observed with ropinirole. At higher concentrations, ropinirole decreased the amplitude of early repolarization (at concentrations ≥ 10 μM), reduced the maximum rate of depolarization and caused depression of the plateau (at concentrations ≥ 30 μM), and shortened APD measured at 50% repolarization (at 300 μM) indicating a concentration-dependent inhibition of I, I, and I. Suppression of I, I, and I has been confirmed under conventional patch clamp and action potential voltage clamp conditions. I and I were not influenced significantly by ropinirole at concentrations less than 300 μM. All these effects of ropinirole were fully reversible upon washout. The results indicate that ropinirole treatment may carry proarrhythmic risk for patients with inherited or acquired long QT syndrome due to inhibition of I—especially in cases of accidental overdose or intoxication. [ABSTRACT FROM AUTHOR]

Class III antiarrhythmic agents exhibit reverse rate-dependent lengthening of the action potential duration (APD). In spite of the several theories developed so far to explain this reverse rate-dependency (RRD), its mechanism has not yet been clarified. The aim of the present work was to further elucidate the mechanisms responsible for RRD in mammalian ventricular myocardium. Action potentials were recorded using conventional sharp microelectrodes from human, canine, rabbit and guinea pig ventricular myocardium in a rate-dependent manner varying the cycle length (CL) between 0.3 and 5 s. Rate-dependent drug effects were studied using agents known to lengthen or shorten action potentials, and these drug-induced changes in APD were correlated with baseline APD values. Both drug-induced lengthening (by dofetilide, sotalol, E-4031, BaCl2, veratrine, BAY K 8644) and shortening (by mexiletine, tetrodotoxin, lemakalim) of action potentials displayed RRD, i.e., changes in APD were greater at longer than at shorter CLs. In rabbit, where APD is a biphasic function of CL, the drug-induced APD changes were proportional to baseline APD values but not to CL. Similar results were obtained when repolarization was modified by injection of inward or outward current pulses in isolated canine cardiomyocytes. In each case the change in APD was proportional to baseline APD (i.e., that measured before the superfusion of drug or injection of current). Also, the net membrane current ( Inet), determined from the action potential waveform at the middle of the plateau, was inversely proportional to APD and consequently with to CL. The results indicate that RRD is a common characteristic of all the drugs tested regardless of the modified ion current species. Thus, drug-induced RRD can be considered as an intrinsic property of cardiac membranes based on the inverse relationship between Inet and APD. [ABSTRACT FROM AUTHOR]

The aim of the present work was to study the profile of the rapid delayed rectifier potassium current ( I Kr) and the inward rectifier potassium current ( I K1) during ventricular repolarization as a function of action potential duration and rate of repolarization. Whole cell configuration of the patch clamp technique was used to monitor I Kr and I K1 during the action potential plateau and terminal repolarization. Action potentials recorded at various cycle lengths (0.4–5 s) and repolarizing voltage ramps having various slopes (0.5–3 V/s) were used as command signals. I Kr and I K1 were identified as difference currents dissected by E-4031 and BaCl2, respectively. Neither peak amplitudes nor mean values of I Kr and I K1 recorded during the plateau of canine action potentials were influenced by action potential duration. The membrane potential where I Kr and I K1 peaked during the terminal repolarization was also independent of action potential duration. Similar results were obtained in undiseased human ventricular myocytes, and also in canine cells when I Kr and I K1 were evoked using repolarizing voltage ramps of various slopes. Action potential voltage clamp experiments revealed that the peak values of I Kr, I K1, and net outward current during the terminal repolarization were independent of the pacing cycle length within the range of 0.4 and 5 s. The results indicate that action potential configuration fails to influence the amplitude of I Kr and I K1 during the ventricular action potential in dogs and humans, suggesting that rate-dependent changes in action potential duration are not likely related to rate-dependent alterations in I Kr or I K1 kinetics in these species. [ABSTRACT FROM AUTHOR]

The role of the slow delayed rectifier K+ current ( I Ks) in cardiac repolarization seems to be largely influenced by the experimental conditions including the species and tissue studied. The aim of this study was to determine the contribution of I Ks to repolarization in canine ventricular myocytes by measuring the frequency dependent action potential lengthening effect of 10 μM chromanol 293B using sharp microelectrodes. Pretreatment with isoproterenol (2 nM), E-4031 (1 μM), and injection of inward current pulses were applied to modify action potential configuration. Chromanol alone caused moderate but statistically significant lengthening of action potentials at cycle lengths longer than 500 ms. The lengthening effect of chromanol, which was strongly enhanced in the presence of either isoproterenol or E-4031, was proportional to the amplitude of plateau, whereas poor correlation was found with action potential duration. Similar results were obtained when action potential configuration was modified by injection of depolarizing current pulses. Computer simulations revealed that activation of I Ks is a sharp function of the plateau amplitude within the physiological range, while elongation of repolarization may enhance I Ks only when it is excessive. It was concluded that the effect of I Ks on ventricular repolarization critically depends on the level of action potential plateau; however, other factors, like action potential duration, cycle length, or suppression of other K+ currents can also influence its contribution. [ABSTRACT FROM AUTHOR]

Activators of the slow delayed rectifier K+ current (IKs) are promising tools to suppress ventricular arrhythmias originating from prolongation of action potentials. A recently synthesized compound, L-364,373, was shown to activate IKs in ventricular cells isolated from guinea pigs and rabbits. Due to the interspecies differences known to exist in the properties of the delayed rectifier K+ currents, the effect of L-364,373 on IKs was studied and compared with that of another IKs activator mefenamic acid in canine ventricular myocytes. Mefenamic acid (100 μM) significantly increased the amplitude of the fully activated IKs current, as well as the IKs current tails, by shifting the voltage dependence of its activation towards negative voltages and increased the time constant for deactivation. In contrast, L-364,373, up to concentrations of 3 μM, failed to augment IKs at any membrane potential studied, but slightly increased the time constant of deactivation. It is concluded that human studies are required to evaluate the therapeutically beneficial effects of IKs activators. Rodent cardiac tissues are not suitable for this purpose. [ABSTRACT FROM AUTHOR]

The aim of the present study was to compare the distribution of ion currents and the major underlying ion channel proteins in canine and human subepicardial (EPI) and midmyocardial (MID) left-ventricular muscle. Ion currents and action potentials were recorded from canine cardiomyocytes derived from the very superficial EPI and central MID regions of the left ventricle. Amplitude, duration and the maximum velocity of depolarization of the action potential were significantly greater in MID than EPI myocytes, whereas phase-1 repolarization was more pronounced in the EPI cells. Amplitudes of the transient outwards K+ current (29.5±1.5 vs. 19.0±2.3 pA/pF at +50 mV) and the slow component of the delayed rectifier K+ current (10.3±2.3 vs. 6.5±1.0 pA/pF at +50 mV) were significantly larger in EPI than in MID myocytes under whole-cell voltage-clamp conditions. The densities of the inwards rectifier K+ current, rapid delayed rectifier K+ current and L-type Ca2+ current were similar in both cell types. Expression of channel proteins in both canine and human ventricular myocardium was determined by Western blotting. In the canine heart, the expression of Kv4.3, Kv1.4, KChIP2 and KvLQT1 was significantly higher, and that of Nav1.5 and MinK much lower, in EPI than in MID. No significant EPI-MID differences were observed in the expression of the other channel proteins studied (Kir2.1, α1C, HERG and MiRP1). Similar results were obtained in human hearts, although the HERG was more abundant in the EPI than in the MID layer. In the canine heart, the EPI-MID differences in ion current densities were proportional to differences in channel protein expression. Except for the density of HERG, the pattern of EPI-MID distribution of ion-channel proteins was identical in canine and human ventricles. [ABSTRACT FROM AUTHOR]

SEA0400 and KB-R7943 are compounds synthesised to block transsarcolemmal Na+/Ca2+ exchange current (INa/Ca); however, they Have also been shown to inhibit L-type Ca2+ current (ICa). The potential value of these compounds depends critically on their relative selectivity for INa/Ca over ICa. In the present work, therefore, the concentration-dependent effects of SEA0400 and KB-R7943 on INa/Ca and ICa were studied and compared in canine ventricular cardiomyocytes using the whole-cell configuration of the patch clamp technique. SEA0400 and KB-R7943 decreased INa/Ca in a concentration-dependent manner, having EC50 values of 111±43 nM and 3.35±0.82 μM, when suppressing inward currents, while the respective EC50 values were estimated at 108±18 nM and 4.74±0.69 μM in the case of outward current block. SEA0400 and KB-R7943 also blocked ICa, having comparable EC50 values (3.6 μM and 3.2 μM, respectively). At higher concentrations (10 μM) both drugs accelerated inactivation of ICa, retarded recovery from inactivation and shifted the voltage dependence of inactivation towards more negative voltages. The voltage dependence of activation was slightly modified by SEA0400, but not by KB-R7943. Based on the relatively good selectivity of submicromolar concentrations of SEA0400—but not KB-R7943—for INa/Ca over ICa, SEA0400 appears to be a suitable tool to study the role of INa/Ca in Ca2+ handling in canine cardiac cells. At concentrations higher than 1 μM, however, ICa is progressively suppressed by the compound. [ABSTRACT FROM AUTHOR]

Norfluoxetine is the most important active metabolite of the widely used antidepressant compound fluoxetine. Although the cellular electrophysiological actions of fluoxetine are well characterized in cardiac cells, little is known about the effects of its metabolite. In this study, therefore, the effects of norfluoxetine on action potential (AP) configuration and transmembrane ion currents were studied in isolated canine cardiomyocytes using the whole cell configuration of patch clamp techniques. Micromolar concentrations of norfluoxetine (1-10 µM) modified AP configuration: amplitude and duration of the AP and maximum velocity of depolarization were decreased in addition to depression of the plateau and elimination of the incisura of AP. Voltage clamp experiments revealed a concentration-dependent suppression of both L-type Ca2+ current,ICa (EC50=1.13±0.08 µM) and transient outward K+ current,Ito (EC50=1.19±0.17 µM) having Hill coefficients close to unity. The midpoint potential of the steady-state inactivation ofICa was shifted from -20.9±0.75 mV to -27.7±1.35 mV by 3 µM norfluoxetine (P<0.05,n=7). No such shift in the steady-state inactivation curve was observed in the case ofIto. Similarly, norfluoxetine caused no change in the steady-state current-voltage relationship of the membrane or in the density of the inward rectifier K+ current,IK1. All these effects of norfluoxetine developed rapidly and were fully reversible. Comparing present results with those obtained previously with fluoxetine, it can be concluded that norfluoxetine displays stronger suppression of cardiac ion channels than fluoxetine. Consequently, the majority of the cardiac side effects observed during fluoxetine treatment are likely to be attributed to its metabolite norfluoxetine. [ABSTRACT FROM AUTHOR]

In this study, the effects of risperidone, the widely used antipsychotic drug, on isolated canine ventricular myocytes and guinea-pig papillary muscles were analyzed using conventional microelectrode and whole cell voltage-clamp techniques. Risperidone concentration-dependently lengthened action potential duration in guinea-pig papillary muscles (EC50=0.29±0.02 µM) and single canine ventricular myocytes (EC50=0.48±0.14 µM). This effect was reversible, showed reverse rate dependence, and it was most prominent on the terminal portion of repolarization. No significant effect of risperidone on the resting membrane potential, action potential amplitude or maximum rate of depolarization was observed. In voltage-clamped canine ventricular myocytes risperidone caused concentration-dependent block of the rapid component of the delayed rectifier K+ current (IKr), measured as outward current tails at –40 mV, with an IC50 of 0.92±0.26 µM. Suppression of IKr was not associated with changes in activation or deactivation kinetics. High concentration of risperidone (10 µM) suppressed also the slow component of the delayed rectifier K+ current (IKs) by 9.6±1.5% at +50 mV. These effects of risperidone developed rapidly and were readily reversible. Risperidone had no significant effect on the amplitude of other K+ currents (IK1 and Ito). The inhibition of cardiac IKr current by risperidone may explain the cardiac side-effects observed occasionally with the drug. Our results suggest that risperidone displays class III antiarrhythmic properties, and as such, may produce QTc prolongation, especially in patients with long QT syndrome. Therefore, in psychotic patients having also cardiac disorders, ECG control may be suggested during risperidone therapy. [ABSTRACT FROM AUTHOR]

Based on earlier pharmacological studies performed using conventional microelectrodes EGIS-7229 (S 21407), the novel antiarrhythmic candidate, was suggested to have a combined mode of action in cardiac tissues isolated from various mammalian species. In order to characterize the electrophysiological effects of the compound, its effects on calcium and potassium currents of isolated canine ventricular cardiomyocytes were studied in the present work using the whole cell configuration of the patch clamp technique. L-type Ca current (ICa) was significantly depressed by EGIS-7229 at concentrations of 3 µM or higher with no concomitant changes in the voltage-dependence of activation and time course of inactivation of ICa. The drug reversibly suppressed the rapid component of the delayed rectifier K current (IKr) in a concentration-dependent manner, having a K0.5 value of 1.1±0.1 µM and a slope factor of close to unity (1.23±0.16), indicating that probably one single binding site of high affinity may be involved in binding of EGIS-7229 to the IKr channel. In contrast, no changes in the slow component of the delayed rectifier K current (IKs) was observed with the compound up to the concentration of 100 µM, even if the current was fully activated by 8-bromo-cAMP. At a concentration of 10 µM or higher, EGIS-7229 caused also a moderate but significant reduction in the inward rectifier K current (IK1) and the transient outward K current (Ito) with no change in the voltage-dependence of activation and steady-state inactivation of Ito. Present results indicate that EGIS-7229 can be considered as a selective IKr blocker at low (1 µM) concentration; however, its combined (class III + IV) mechanism of action is evident at concentrations of 3 µM or higher. Suppression of ICa may explain the lack of development of early afterdepolarizations in the presence of EGIS-7229, predicting a relatively safe clinical application in contrast to pure class III compounds. [ABSTRACT FROM AUTHOR]

Effects of endothelin-1 (ET-1) on the L-type calcium current (ICa) and delayed rectifier potassium current (IK) were studied in isolated canine ventricular cardiomyocytes using the whole-cell configuration of the patch-clamp technique. ET-1 (8 nM) was applied in three experimental arrangements: untreated cells, in the presence of 50 nM isoproterenol, and in the presence of 250 µM 8-bromo-cAMP. In untreated cells, ET-1 significantly decreased the peak amplitude of ICa by 32.3±4.8% at +5 mV (P<0.05) without changing activation or inactivation characteristics of ICa. ET-1 had no effect on the amplitude of IK, Ito (transient outward current) or IK1 (inward rectifier K current) in untreated cells; however, the time course of recovery from inactivation of Ito was significantly increased by ET-1 (from 26.5±4.6 ms to 59.5±1.8 ms, P<0.05). Amplitude and time course of intracellular calcium transients, recorded in voltage-clamped cells previously loaded with the fluorescent calcium indicator dye Fura-2, were not affected by ET-1. ET-1 had no effect on force of contraction in canine ventricular trabeculae. Isoproterenol increased the amplitude of ICa to 263±29% of control. ET-1 reduced ICa also in isoproterenol-treated cells by 17.8±2% (P<0.05); this inhibition was significantly less than obtained in untreated cells. IK was increased by isoproterenol to 213±18% of control. This effect of isoproterenol on IK was reduced by 31.8±4.8% if the cells were pretreated with ET-1. Similarly, in isoproterenol-treated cells ET-1 decreased IK by 16.2±1.5% (P<0.05). Maximal activation of protein kinase A (PKA) was achieved by application of 8-bromo-cAMP in the pipette solution. In the presence of 8-bromo-cAMP ET-1 failed to alter ICa or IK. It was concluded that differences in effects of ET-1 on ICa and IK may be related to differences in cAMP sensitivity of the currents. [ABSTRACT FROM AUTHOR]

Concentration-dependent effects of bimoclomol, a novel heat shock protein (HSP) coinducer, were studied on the parameters of action potential and transmembrane ionic currents in enzymatically dispersed canine ventricular cardiomyocytes using conventional microelectrode and whole cell voltage clamp techniques. Bimoclomol (10-100 μ,M) decreased the maximum velocity of depolarization (Vmax) and amplitude of action potentials in a concentration-dependent manner. These effects were fully reversible after a 5-min period of washout in drug-free medium. Action potential duration measured at 50% or 90% level of repolarization (APD-50 and APD-90, respectively) was markedly shortened by bimoclomol. Both APD-50 and APD-90 were decreased, but the reduction in APD-50 was more pronounced. The APD-shortening effect of bimoclomol was significantly reduced in the presence of 20 nM charybdotoxin (inhibitor of the Ca-dependent K current) or 0.5 mM anthracene-9-carboxylic acid (inhibitor of the Ca-dependent Cl current) or 1 μM glibenclaniide (inhibitor of the ATP-sensitive K current). In the presence of anthracene-9-carboxylic acid, APD-90 was lengthened by bimoclomol. The APD-shortening effect of bimoclomol was also partially antagonized by chelation of intracellular Ca2+ by application of the cell permeant form of BAPTA, or when using 10 mM EGTA-containing patch pipettes to record action potentials. The &Vdot;maxdepressant effect of bimoclomol was not affected by charybdotoxin, anthracene-9-carboxylic acid, glibenclamide, or BAPTA load. In voltage clamped cardiomyocytes bimoclomol (100 μM) had no effect on the amplitude of ICa but decreased significantly the inactivation time constant of ICa (from 19.8±1.6 ms to 16.8±1.2 ms at 0 mV). Bimoclomol also decreased significantly the amplitude of IK1 (from -20.5±1.1 pA/pF to -16.6±0.8 pA/pF at -135 mV), causing reduction in slope of the negative branch of the I-V curve. At positive potentials, however, bimoclomol increased outward current. The bimoclomol -induced current, therefore, was studied in the presence of BaC12, when IK1 current was blocked. The bimoclomol-induced current had a reversal potential close to -90 mV. Bimoclomol (100 μM) had no effect on the amplitude or kinetic properties of the transient outward K current (Ito) and the delayed rectifier K current (IK). It is concluded that bimoclomol exerts both Ca-independent (inhibition of INa and IK1 activation of the ATP-sensitive K current) and Ca-dependent effects (mediated by Ca-activated CI and probably K currents) in canine ventricular myocytes. [ABSTRACT FROM AUTHOR]

Cell proliferation and apoptosis in renal cysts induced by streptozotocin, alloxan and ferric-nitrilotriacetate were investigated in rats. In the kidneys of all treated animals dilated tubules at the cortico-medullary region, large cysts, glomerular cysts and tubular dilation in the medullary area were found. Both cell proliferation and apoptosis were increased in the epithelium of the non-dilated tubules, in the mesangial and interstitial cells. Cells lining the dilated tubules or cysts demonstrated apoptosis but their proliferating activity was low. By calculating the proliferation–apoptosis ratio we found that alloxan did not change the balance between the two mechanisms. Meanwhile streptozotocin resulted in an increased apoptosis and ferric-nitrilotriacetate in an increased cell proliferation. p53 expression might be responsible for the uncontrolled proliferation in rats treated with ferric-nitrilotriacetate as this oncoprotein was diffusely present in tubular cell nuclei. The observed apoptosis seemed to be independent of bcl-2 oncoprotein expression. We assume that the initial factor in such cystogenesis should be a cellular injury due to direct toxic or to the diabetogenic effect of the drugs. The latter is more likely as all the animals were hyperglycemic and insulin treatment following administration of streptozotocin prevented the morphologic changes. [ABSTRACT FROM AUTHOR]

The influence of experimental hyperthyroidism on the catecholamine induced stimulation of rubidium ion transport in the soleus (SOL), a slow-twitch muscle and the extensor digitorum longus (EDL), a fast-twitch skeletal muscle of rats was studied. Thyroxine administration (800 μg/kg/day), for ten days induced a rise of ouabainsensitiveRb uptake in SOL muscle, without affecting the ouabain-insensitive uptake, whereas both fractions ofRb uptake were increased in EDL muscle from hyperthyroid rats. Isoproterenol (5 μmol/l) caused a two-fold rise in ouabain-sensitive Rb uptake of euthyroid SOL muscle, while in hyperthyroid SOL it could stimulate only the ouabain-insensitive fraction ofRb influx. On the other hand, the stimulating action of isoproterenol on euthyroid EDL muscle was due to an enhancement of ouabain-insensitive Rb uptake, but in hyperthyroid EDL it failed to stimulate the ouabain-insensitive transport and caused a marked rise in ouabain-sensitive Rb uptake. The changes in catecholamine mediated transport properties in SOL muscle may be related to fibre type trans-formation induced by thyroid hormone, although in EDL the changes of catecholamine stimulation are unlikely due to fibre type conversion. Basal and isoproterenol stimulated cAMP levels were significantly reduced in both EDL and SOL muscles from hyperthyroid rats, in contrast with an insignificant decrease in net rubidium uptake caused by isoproterenol at the same concentration. [ABSTRACT FROM AUTHOR]

The cellular electrophysiological effects of EGIS-7229 (5-chlor-4-[N-(3,4-dimethoxy-phenyl-ethyl)-amino-propylamino]-3(2H)-pyridazinone fumarate), a novel antiarrhythmic agent, was studied using conventional microelectrode techniques in canine cardiac Purkinje fibers and papillary muscle preparations obtained from man, rabbits and guinea pigs. Low concentration of EGIS-7229 (3 μmol/l) selectively lengthened action potential duration (both APD50 and APD90) in all preparations. The effect of higher concentrations (30–100 μmol/l) of EGIS-7229 on action potential duration was variable depending on the preparation studied: in rabbit and human papillary muscles both APD50 and APD90 were lengthened, in canine Purkinje fibers APD90 was lengthened but APD50 was shortened, while in guinea pig papillary muscles both APD50 and APD90 were shortened by high concentrations of the drug. At these higher concentrations EGIS-7229 also decreased the maximum velocity of action potential upstroke ( V max) and depressed the plateau of action potentials without affecting the resting membrane potential or action potential amplitude. Both reduction of V max and lengthening of APD were frequency dependent. The former effect was more prominent at higher pacing frequencies, while the latter was more pronounced at lower driving rates. In guinea pig papillary muscle, the time constant of recovery from V max-block was 719 ± 33 ms ( n = 18) and the rate of onset of the block was 1.81 ± 0.06 AP–1 ( n = 16) in the presence of 100 μmol/l EGIS-7229. EGIS-7229 had a complex action on refractoriness in guinea pig papillary muscles: ERP was lengthened at low concentrations (3 to 10 μmol/l), unchanged at 30 μmol/l and shortened at 100 μmol/l. The ratio of ERP/APD90, however, was significantly increased at concentrations higher than 3 μmol/l. In canine Purkinje fiber, when the delayed rectifier K current (IK) was blocked by d-sotalol (60 μmol/l) and APD was shortened back to its control value by additional application of nicorandil (15 μmol/l), APD was not affected by 3 μmol/l but was shortened by 30 μmol/l of EGIS-7229. 100 μmol/l EGIS-7229 shortened APD in guinea pig papillary muscle. This effect of EGIS-7229 was effectively prevented by nifedipine pretreatment (10 μmol/l). In this preparation, EGIS-7229 also decreased the V max of the slow action potential, evoked in the presence of 20 mmol/l external K+ plus 0.5 mmol/l Ba2+. It is likely that EGIS-7229 at low concentrations blocks IK in human, canine, rabbit and guinea pig cardiac preparations, but at higher concentrations also inhibits Ca and Na currents. Therefore, EGIS-7229 appears to carry mixed class III, IV and IB antiarrhythmic properties. [ABSTRACT FROM AUTHOR]