Multiple cellular electrophysiological effects of azimilide in canine cardiac preparations

The cellular electrophysiological effect of azimilide (0.1–30 μM) was analyzed in canine ventricular preparations by applying the standard microelectrode and patch-clamp techniques at 37 °C. In papillary muscle, the drug prolonged the action potential duration (APD) in a concentration-dependent manner at a cycle length (CL) of 1000 ms. In Purkinje fibers, at the same CL, the concentration-dependent lengthening of the APD was observed in the presence of up to 3 μM azimilide (at 3.0 μM: 24.1±4.2%, n=9); at higher drug concentration, no further APD prolongation was observed. Azimilide lengthened APD in a reverse frequency-dependent manner in papillary muscle and Purkinje fibers alike. Azimilide (10 μM) caused a rate-dependent depression in the maximal upstroke velocity of the action potential (V_max) in papillary muscle. The time and rate constants of the offset and onset kinetics of this V_max block were 1754±267 ms (n=6) and 5.1±0.4 beats (n=6), respectively. Azimilide did not prevent the APD shortening effect of 10 μM pinacidil in papillary muscle, suggesting that the drug does not influence the ATP-sensitive K⁺ current. Azimilide inhibited the rapid (I_Kr) and slow component (I_Ks) of the delayed rectifier K⁺ current and the L-type Ca²⁺ current (I_Ca). The estimated EC₅₀ value of the drug was 0.59 μM for I_Ks, 0.39 μM for I_Kr and 7.5 μM for I_Ca. The transient outward (I_to) and the inward rectifier (I_k1) K⁺ currents were not influenced by the drug. It is concluded that the site of action of azimilide is multiple, it inhibits not only K⁺ (I_Kr, I_Ks) currents but, in higher concentrations, it also exerts calcium- and use-dependent sodium channel block.     

Modulation of the hyperpolarisation-activated current, Ih, in rat facial motoneurones in vitro by ZD-7288

ZD-7288 [4-(N-ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino) pyrimidinium chloride] and Cs⁺ have been used to distinguish the currents contributing to inward rectification in neonatal rat facial motoneurones (FMs). ZD-7288 (0.1–10 μM) inhibited a current that reversed at −43.7±3.7 mV in artificial cerebrospinal fluid (ACSF) containing 3 mM K⁺ (n=9), and displayed the time and voltage dependence of the hyperpolarisation-activated current, I_h. Depolarisation-activated transient (I_K(A)) and sustained outward currents were unaffected by ZD-7288. The IC₅₀ for block of I_h by ZD-7288 was around 0.2 μM. Onset of inhibition was slow and no recovery was seen after washing in ZD-7288-free ACSF for up to 4 h. In the presence of ZD-7288, Ba²⁺ and Rb⁺ blocked an inwardly rectifying potassium (K⁺) current, confirming both the presence of I_K(IR) and its insensitivity to ZD-7288. Cs⁺ rapidly and reversibly blocked both I_h and I_K(IR). Inhibition of I_h by ZD-7288 showed no use dependence, internally applied ZD-7288 also blocked I_h, and tail current analysis indicated inhibition to be voltage-independent. In the presence of internal guanosine 5′-O-(3-thiotriphosphate) (GTP-γ-S) and after previous exposure to ZD-7288, 5-hydroxytryptamine (5-HT), but not noradrenaline, promoted a recovery of I_h that was not observed if ZD-7288 was present throughout the recording period. This interaction between ZD-7288 and irreversible 5-HT-receptor activation may be related to the mechanism underlying ZD-7288-mediated block of these channels.     

Inhibition of cardiac inward-rectifier K current by terodiline

The antispasmodic agent terodiline has cardiotoxic effects that include QT lengthening. To determine whether inhibition of inwardly-rectifying K⁺ current (I_K1) might be a factor in the cardiotoxicity, we measured I_K1 in guinea pig ventricular myocytes. Terodiline reduced outward I_K1 with an IC₅₀ of 7 μM; maximal reduction was 60% with 100–300 μM concentration. Inhibition was independent of current direction, and persisted after removal of the drug. Terodiline (3–5 μM) lengthened action potentials in guinea pig papillary muscles by ca. 10%, primarily by slowing phase 3 repolarization; higher concentrations abbreviated the plateau and markedly slowed late repolarization. Terodiline washout provoked an extra lengthening, consistent with persistent inhibition of I_K1 and rapid recovery of net inward plateau current. The results suggest that inhibition of I_K1 is a likely factor in the cardiotoxicity of the drug.     

Inhibitory effect of bepridil on hKv1.5 channel current: comparison with amiodarone and E-4031

Effects of bepridil on the depolarization-activated outward K⁺ currents (I_out) in rat atrial myocytes and the human cardiac K⁺ (hKv1.5) channel current stably expressed in human embryonic kidney (HEK) 293 cells were examined, and compared with those of amiodarone and N-[4-[[1-[2-(6-methyl-2-pyridinyl)ethyl]-4-piperidinyl]carbonyl]phenyl] methanesulphonamide dihydrochloride dihydrate (E-4031). Membrane currents were recorded using patch-clamp techniques in enzymatically isolated rat atrial myocytes and HEK 293 cells expressing hKv1.5 channels. Bepridil potently inhibited I_out elicited by depolarization pulses and prolonged the action potential in rat atrial cells. Bepridil also inhibited the hKv1.5 channel current with the IC₅₀ value of 6.6 μM. The inhibitory effects of bepridil on the currents in HEK 293 cells were voltage-dependent. Amiodarone weakly inhibited rat atrial I_out and hKv1.5 channel current. In contrast, E-4031 at a concentration of 10 μM had little influence on these currents. Thus, bepridil inhibits hKv1.5 channel current and the inhibitory effect may be useful for the treatment of atrial fibrillation.     

Effects of ginkgo biloba extract and bilobalide,a main constituent,on the ionic currents in guinea pig ventricular cardiomyocytes

Effects of Ginkgo biloba extract (GBE, 0.01 to 1 mg/ml) and a main constituent, bilobalide (0.1 to 1 mumol/l), on the action potentials and the underlying ionic currents in guinea pig ventricular cardiomyocytes were investigated using a patch-clamp technique. Both GBE and bilobalide at high concentrations caused depressant actions on the action potential configuration. GBE (0.3 mg/ml) decreased the Vmax by 17.1 +/- 2.1% (n = 6, p < 0.05), and bilobalide (1 mumol/l) by 14.7 +/- 2.2% (n = 8, p < 0.05). GBE prolonged the action potential duration at 90% repolarization (APD90), by 70.6 +/- 2.8% (n = 6, p < 0.001) at 1 mg/ml; in contrast, bilobalide shortened APD, by 11.1 +/- 2.0% (n = 8, p < 0.05) at 3 mumol/l. In voltage-clamp experiments, GBE (1 mg/ml) markedly inhibited the Ca2+ current (ICa) at 10 mV by 90.1 +/- 3.0% (n = 6, p < 0.001), the delayed rectifier K+ current (IK) at 60 mV by 63.7 +/- 3.0% (n = 6, p < 0.01), and the inwardly rectifying K+ current (IK1) at -120 mV by 47.8 +/- 2.6% (n = 6, p < 0.01). On the other hand, bilobalide at 1 mumol/l enhanced the ICa by 40.0 +/- 2.3% (n = 6, p < 0.05), and the IK by 14.0 +/- 2.3% (n = 6, p < 0.05), concentration-dependently. The IK1 was unaffected. These responses were reversible (to approximately 50-80%) after 10- to 20-min washout. These results indicate that even after acute administrations, GBE and bilobalide produced active actions on the APD and the ionic currents in cardiomyocytes. Although each chemical exhibited the responses in opposite directions, GBE acts totally as a mixture.     

The actions of phenylglycine derived metabotropic glutamate receptor antagonists on multiple (1S,3R)-ACPD responses in the rat nucleus of the tractus solitarius

The effects of the metabotropic glutamate receptor (mGluR) agonist (1S,3R)-1-aminocy-clopentane-1,3-dicarboxylic acid [(1S,3R)-ACPD] and a series of phenylglycine-derived putative mGluR antagonists were examined on electrophysiological responses mediated by glutamate and GABA receptors in the nucleus of the tractus solitarius (NTS) in transverse brainsten slices of the rat. Monosynaptic excitatory currents (EPSC's) evoked by electrical stimulation in the region of the tractus solitarius (TS) were reduced in the presence of (1S,3R)-ACPD in > 90% of neurons recorded in the dorsomedial subdivision of the NTS adjacent to the area postrema (AP). Monosynaptic evoked inhibitory currents (IPSC's) were similarly inhibited by (1S,3R)-ACPD. The inward current evoked by pressure application of -amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (I_AMPS) was potentiated in the presence of (1S,3R)-ACPD, whereas the outward current evoked by the γ-amino-butyric acid-A (GABA-A) receptor agonist muscimol (I_MUSC) was inhibited. (1S,3R)-APCD also produced a postsynaptic inward current (I_K(ACPD)) associated with a decrease in membrane conductance in approximately 50% of cells. The novel mGluR antagonists (S)-4-carboxy-3-hydroxy-phenylglycine (4C3H-PG), (R,S)-4-carboxy-phenylglycine (4C-PG) and (R,S)--methyl-4-carboxy-phenylglycine (M4C-PG) reversibly antagonized the effects of (1S,3R)-ACPD on EPSC's IPSC's, I_AMPA and I_MUSC. The first two compounds also displayed weak agonist activity. However, none of the antagonists significantly inhibited I_K(ACPD) concentrations which blocked (1S,3R)-ACPD effects on synaptic transmission. These results suggest that pharmacologically distinct mGluR's may be present in the NTS.     

蛇床子素对乳鼠心肌细胞钠通道的影响

目的:研究蛇床子素对体外培养大鼠乳鼠心肌细胞钠通道电流的影响。方法:采用全细胞膜片钳技术,首先在乳鼠心肌细胞上诱导出钠电流,然后观察蛇床子素对钠电流的影响。结果:蛇床子素可以浓度依赖性抑制心肌细胞钠通道,抑制的半有效浓度为110.3±6.5μM。蛇床子素对钠电流的激活阈电位和峰电位没有影响,但是其对钠电流的抑制表现出电压依赖性。蛇床子素使钠通道稳态失活曲线向超极化方向漂移。此外,蛇床子素对钠电流的抑制是快速可逆的,这种抑制具有使用依赖性。结论:蛇床子素以浓度依赖和使用依赖的方式抑制乳鼠心肌细胞钠通道电流。     

Ionic basis for OPC-8212-induced increase in action potential duration in isolated rabbit, guinea pig and human ventricular myocytes

Changes in transmembrane ionic currents induced by OPC-8212 (3,4-dihydro-6-[4-(3,4-dimethoxybenzoyl)-1-piperazinyl]- 2(1H)-quinoline), a recently introduced positive inotropic agent which lengthens cardiac action potential duration, were examined using whole-cell voltage-clamp techniques in single rabbit, guinea pig and human ventricular myocytes. In rabbit, OPC-8212 (12 μmol/l) significantly increased membrane action potential duration measured at 90% of repolarization by an average of 88 ms (from 462 ± 25 to 550 ± 35 ms, n = 4; P < 0.05). In rabbit this increase in duration was not associated with significant changes in either the inward rectifier or transient outward K⁺ currents. The magnitude of the secondary inward current evoked from a holding potential of −50 mV was significantly increased by 97 ± 8% (n = 6; P < 0.01) while a demonstrable delayed rectifier outward current could not be identified in the rabbit myocytes examined at room temperature. In guinea pig ventricular myocytes, where the delayed rectifier was large, 12 μmol/l OPC-8212 significantly depressed the current by 58 ± 10% (n = 6; P < 0.01). The effects of OPC-8212 in human ventricular myocytes obtained from the explanted heart of a single patient having an idiopathic cardiomyopathy most closely resembled those observed in isolated rabbit ventricular myocytes. Thus, in rabbit and a few human ventricular myocytes examined at room temperature, OPC-8212 appeared to lengthen cardiac membrane action potential duration primarily by increasing the amplitude of the secondary inward current believed to primarily represent current through L-type Ca²⁺ channels. In guinea pig preparations, OPC-8212 also decreased the delayed rectifier outward K⁺ current which also would account for an increase in action potential duration. OPC-8212 could not be demonstrated to affect Na⁺ current inactivation in a manner similar to that produced by 1 mg/l veratrine, a recognized Na + channel agonist, which dramatically slowed this process.     

Effects of Fe on ion channels: Na channel, delayed rectified and transient outward K channels

The effects of Fe²⁺ on the properties of three types of ion channels were studied in acutely dissociated rat hippocampal pyramidal neurons from area CA1 at postnatal ages of 7–14 days using the whole cell patch clamp technique. The results indicated that: (1) in the existence of Fe²⁺, the activation voltage threshold of transient outward K⁺ currents (I_A) was decreased. The normalized current-voltage curves of activation were well fitted with a single Boltzmann function, and the V_1/2 was 2.44±1.14 mV (n=15) in control, whereas 1.79±1.53 (n=15), −2.96±0.92 (n=14), −5.11±1.31 (n=13), −9.05±1.64 mV (n=12) in 1, 10, 100 and 1000 μ Fe²⁺, respectively. Differences between two groups were significant (P<0.05, n=12–15), except for that between the control and 1 μ (P>0.05, n=15). (2) Fe²⁺ caused a left shift of the current–voltage curves of steady-state inactivation of I_A in a concentration-dependent manner. The curves were well fitted with a single Boltzmann function with similar slope (P>0.05, n=10–13). The V_1/2 were −70.71±1.23 (n=13), −71.14±1.37 (n=13), −78.21±1.17 (n=11), −84.61±1.34 (n=12), and −89.68±2.59 mV (n=10) in control, 1, 10, 100 and 1000 μ Fe²⁺, respectively. Fe²⁺ also shifted the current–voltage curves of Na⁺ channel steady-state inactivation to more negative depolarization potentials in parallel, with V_1/2, −67.37±1.33 mV (n=12) in control, and −67.52±1.28 mV (n=12), −68.24±1.61 mV (n=10), −71.58±1.45 mV (n=10), −76.65±1.76 mV (n=9) in 1, 10, 100 and 1000 μ Fe²⁺ solutions, respectively. (3) In Fe²⁺ solutions, the recovery from inactivation of I_A was slowed. (4) With application of different concentrations of Fe²⁺, the voltage threshold of activation of delayed rectified outward K⁺ currents (I_K) was decreased, while Fe²⁺ showed a little inhibition at more positive depolarization. Briefly, the results demonstrated that Fe²⁺ is a dose- and voltage-dependent, reversible modulator of I_A, I_K and Na⁺ channels. The results will be helpful to explain the mechanism of Fe²⁺ physiological function and Fe²⁺ intoxication in the central nervous system.     

Potentiation of inhibitory amino acid receptors-mediated responses by lanthanum in rat sacral dorsal commissural neurons

Lanthanum is one of rare earth cations with extremely active chemical property and has been reported to influence neuronal transmitter systems. To date, little attention has been directed towards the sacral dorsal commissural nucleus (SDCN), which serves as a relay of sensory information from the pelvic viscera in the spinal cord. Therefore, the effect of lanthanum on the inhibitory neurotransmitter γ-aminobutyric acid (GABA) and glycine (Gly) responses in neurons acutely dissociated from the rat SDCN was investigated using the nystatin-perforated patch-recording configuration under voltage-clamp conditions. At a holding potential of − 40 mV, La³⁺ reversibly potentiated GABA (3 μM)-activated currents (I_GABA) in a concentration-dependent manner over the concentration range of 10 μM to 30 mM, with the EC₅₀ value of 67.3 ± 16.4 μM. Similarly, La³⁺ reversibly potentiated glycine (10 μM)-activated currents (I_Gly) in a concentration-dependent manner over the concentration range of 1 μM to 1 mM, with the EC₅₀ value of 52.3 ± 10.9 μM. The effects of La³⁺ on I_GABA and I_Gly were voltage-independent. Moreover, both of the potentiations were not use-dependent and were overcome by increasing the concentration of agonist. Our results indicate that La³⁺ potentiates the inhibitory amino acid receptors-mediated responses in SDCN, which may reduce the transmission of the pelvic visceral information. The information provided by this work may help to elucidate the mechanisms and effects of lanthanum on brain functions.     

Effect of ramosetron on short-circuit current response in rat colonic mucosa

We investigated the effects of ramosetron (YM060, (−)-(R)-5-[(1-methyl-1H-indol-3-yl)carbonyl]-4,5,6,7-tetrahydro-1H-benzimidazole monohydrochloride) on the short-circuit current (I_sc) responses to 5-HT receptor agonists in the rat distal colon, and compared its potency to that of other 5-HT₃ receptor antagonists. 5-Hydroxytryptamine (5-HT) concentration-dependently increased I_sc. The I_sc response to 5-HT was partially reduced by tetrodotoxin and ramosetron, and strongly inhibited by GR113808 ([[1-[(2-methylsulphonyl)amino]ethyl]-4-piperidin-yl]methyl 1-methyl-1H-indole-3-carboxylate). 2-Methyl-5-HT and 5-methoxytryptamine also increased I_sc. The former response was inhibited by ramosetron, and the latter was abolished by GR113808. Ramosetron, YM114 (KAE-393, (−)-(R)-5-[(1-indolinyl)carbonyl]-4,5,6,7-tetrahydro-1H-benzimidazole monohydrochloride) and granisetron concentration-dependently antagonized the I_sc responses to 2-methyl-5-HT with reduction in the maximal response at higher concentrations. Apparent pA₂ values for these antagonists were 10.40, 10.37 and 8.99, respectively. Ondansetron produced clear rightward shifts of the concentration-response curves to 2-methyl-5-HT, with a pA₂ value of 8.53. These results suggest that 5-HT increases I_sc through the 5-HT₃ and 5-HT₄ receptors, and that ramosetron is a potent and selective 5-HT₃ receptor antagonist in rat colonic mucosa.     

Effects of thymol on calcium and potassium currents in canine and human ventricular cardiomyocytes

1. Concentration-dependent effects of thymol (1 - 1000 microM) was studied on action potential configuration and ionic currents in isolated canine ventricular cardiomyocytes using conventional microelectrode and patch clamp techniques. 2. Low concentration of thymol (10 microM) removed the notch of the action potential, whereas high concentrations (100 microM or higher) caused an additional shortening of action potential duration accompanied by progressive depression of plateau and reduction of V(max). 3. In the canine cells L-type Ca current (I(Ca)) was decreased by thymol in a concentration-dependent manner (EC(50): 158+/-7 microM, Hill coeff.: 2.96+/-0.43). In addition, thymol (50 - 250 microM) accelerated the inactivation of I(Ca), increased the time constant of recovery from inactivation, shifted the steady-state inactivation curve of I(Ca) leftwards, but voltage dependence of activation remained unaltered. Qualitatively similar results were obtained with thymol in ventricular myocytes isolated from healthy human hearts. 4. Thymol displayed concentration-dependent suppressive effects on potassium currents: the transient outward current, I(to) (EC(50): 60.6+/-11.4 microM, Hill coeff.: 1.03+/-0.11), the rapid component of the delayed rectifier, I(Kr) (EC(50): 63.4+/-6.1 microM, Hill coeff.: 1.29+/-0.15), and the slow component of the delayed rectifier, I(Ks) (EC(50): 202+/-11 microM, Hill coeff.: 0.72+/-0.14), however, K channel kinetics were not much altered by thymol. These effects on Ca and K currents developed rapidly (within 0.5 min) and were readily reversible. 5. In conclusion, thymol suppressed cardiac ionic channels in a concentration-dependent manner, however, both drug-sensitivities as well as the mechanism of action seems to be different when blocking calcium and potassium channels.     

Relaxant and Ca2+ channel blocking properties of norbormide on rat non-vascular smooth muscles

We have investigated the effects of the rat-specific vasoconstrictor agent norbormide on the mechanical and electrophysiological properties of rat non-vascular smooth muscles. Norbormide (50 μM) did not affect the resting tone of urinary bladder, tracheal, and duodenal rings. In all tissues, KCl-induced concentration–response curves were shifted downward by norbormide (5 and 50 μM). In urinary bladder and tracheal rings, norbormide inhibited contractile responses to carbachol only at the higher concentration (50 μM). In single gastric fundus myocytes, 50 μM norbormide inhibited L-type Ca²⁺ current (I_Ca(L)) by about 60%, neither affecting both activation and inactivation rates of the current nor the current–voltage curve along the voltage axis. Our results indicate that rat non-vascular smooth muscles are relaxed by norbormide with a mechanism likely involving a reduction of Ca²⁺ entry through L-type Ca²⁺ channels.     

Effect of ethanol on the electrophysiological characteristics of pulmonary vein cardiomyocytes

Ethanol consumption has been considered to contribute to the occurrences of paroxysmal atrial fibrillation. Pulmonary veins are known to initiate atrial fibrillation. This study investigated whether ethanol may induce atrial fibrillation through increasing arrhythmogenic activity of pulmonary vein cardiomyocytes. Using the whole-cell clamp technique, the action potential and ionic currents were investigated in rabbit single pulmonary vein beating cardiomyocytes with and without (control) incubation of ethanol. Compared with control cardiomyocytes, pulmonary vein cardiomyocytes receiving 0.3 mg/ml or 1 mg/ml ethanol had shorter action potential duration, but had similar beating rates (2.6+/-1.3, 2.7+/-1.2, 2.7+/-1.2 Hz) and incidences (45%, 41%, 32%) of delayed after depolarization. Pulmonary vein cardiomyocytes receiving ethanol had smaller L-type Ca(2+) currents and larger transient outward currents, but had similar transient inward, delayed rectified outward, inward rectified and pacemaker currents. These results suggest that ethanol has no direct effect on the arrhythmogenic potential of pulmonary vein cardiomyocytes.     

The effect of the venom of the yellow Iranian scorpion Odontobuthus doriae on skeletal muscle preparations in vitro

The yellow Iranian scorpion Odontobuthus doriae can cause fatal envenoming, but its mechanism of action is unclear. One of the reported manifestations of envenoming is moderate to severe involuntary tremor of skeletal muscle. In order to understand better the mechanism of action of this venom on skeletal muscle function, we examined the effects of the venom in vitro on chick biventer cervicis (CBC) and mouse hemidiaphragm (MHD) nerve muscle preparations. O. doriae venom (0.3–10 μg/ml) initially increased and then decreased twitch height. The venom also caused contracture in both preparations. In mouse triangularis sterni preparations, used for all intracellular recording techniques, the venom enhanced the release of acetylcholine and induced repetitive firing of nerve action potentials and endplate potentials in response to single-shock stimulation. With extracellular recording techniques, scorpion venom (1 μg/ml) was found to cause changes to the perineural waveform associated with nerve terminal action potentials consistent with effects on Na⁺ and K⁺ currents. The main facilitatory effects of O. doriae venom are likely to be due to toxins that affect Na⁺ channels in nerve–muscle preparations similar to most Old World scorpion venoms, but blocking effects on K⁺ channels are also possible. Such effects could lead to initial enhancement of transmitter release that could underlie the muscle tremors seen in victims. Toxins acting on Na⁺ and K+ currents have been isolated from the venom [Jalali, A., Bosmans, F., Amininasab, M., Clynen, E., Cuypers, E., Zaremirakabadi, A., Sarbolouki, M.N., Schoofs, L., Vatanpour, H., Tytgat, J., 2005. OD1, the first toxin isolated from the venom of the scorpion Odontobuthus doriae active on voltage-gated Na⁺ channels. FEBS Lett. 579, 4181–4186; Abdel-Mottaleb, Y., Clynen, E., Jalali, A., Bosmans, F., Vatanpour, H., Schoofs, L., Tytgat, J., 2006. The first potassium channel toxin from the venom of the Iranian scorpion Odontobuthus doriae. FEBS Lett. 580, 6254–6258]; however, the muscle paralysis seen at higher concentrations of venom may be due to additional, as yet uncharacterised, components of the venom.     

Effects of zatebradine on ouabain-, two-stage coronary ligation- and epinephrine-induced ventricular tachyarrhythmias

To determine whether a hyperpolarization-activated current (I_f) participates in ventricular tachyarrhythmias, we investigated the effects of zatebradine, an I_f inhibitor, on the ventricular tachyarrhythmias induced by ouabain, two-stage coronary ligation and epinephrine infusion in the dog heart. We determined atrial rate, ectopic ventricular rate, total heart rate and arrhythmic ratio (the number of ectopic ventricular beats divided by total heart beats). Zatebradine (0.15, 0.5 and 1.5 mg/kg, i.v.) dose dependently decreased the arrhythmic ratio, ectopic ventricular rate and atrial rate of the ouabain-induced ventricular tachyarrhythmias in pentobarbital-anesthetized dogs. The inhibition by zatebradine of the ventricular arrhythmias needed larger doses than the inhibition of the atrial rate. Zatebradine weakly depressed the ectopic ventricular rate but not the arrhythmic ratio of the ventricular arrhythmias induced by two-stage coronary ligation 24 h after the ligation in conscious dogs. Although neither the ectopic ventricular rate nor the arrhythmic ratio of the epinephrine-induced ventricular arrhythmias was affected by zatebradine, after treatment with zatebradine, the arrhythmias elicited by epinephrine developed more slowly. Together with the previously reported spectra of the effects of the antiarrhythmic agents in three ventricular tachyarrhythmia models, our results suggest that zatebradine may improve automaticity-related ventricular tachyarrhythmias due to I_f inhibition or to other undetermined mechanisms in the heart.     

Inhibitory effects of cilnidipine on peripheral and brain N-type Ca2+ channels expressed in BHK cells

We investigated differences in electrophysiological characteristics between peripheral and central N-type Ca²⁺ channels, containing _1B-a and a_1B-c, respectively. In addition, we examined the inhibitory effects of cilnidipine, a dihydropyridine (DHP) derivative, on both channels. Both _1B subunits were transiently expressed in BHK cells, and then analyzed using the whole-cell patch-clamp technique. The current–voltage relationship showed that _1B-c currents were activated at more negative potentials than _1B-a currents. The voltage-dependent steady-state inactivation and activation showed that the V_1/2 values for inactivation and activation of _1B-c (−88.5±1.3 and −33.2±1.3 mV) were both significantly more negative than those for _1B-a (−83.3±1.3 and −27.9±2.3 mV). Despite the different electrophysiological characteristics of these two N-type channels, cilnidipine blocked both with similar potency within the range 0.1 to 10 μM. Furthermore, cilnidipine had no effect on the I–V relationships or the steady-state inactivation curves. Our data indicate that the spliced positions of _1B-a and a_1B-c may affect not only their voltage-sensing abilities but also the kinetics of channel activation and inactivation. The data also suggest that cilnidipine binds to sites independent of those controlling voltage-sensing and channel kinetics in these _1B subunits.     

Response of IKr and hERG Currents to the Antipsychotics Tiapride and Sulpiride

The human ether-a-go-go-related gene (hERG) channel is important for repolarization in human myocardium and is a common target for drugs that prolong the QT interval. We studied the effects of two antipsychotics, tiapride and sulpiride, on hERG channels expressed in Xenopus oocytes and also on delayed rectifier K⁺ currents in guinea pig cardiomyocytes. Neither the amplitude of the hERG outward currents measured at the end of the voltage pulse, nor the amplitude of hERG tail currents, showed any concentration-dependent changes with either tiapride or sulpiride (3~300 µM). However, our findings did show that tiapride increased the potential for half-maximal activation (V_1/2) of HERG at 10~300 µM, whereas sulpiride increased the maximum conductance (G_max) at 3, 10 and 100 µM. In guinea pig ventricular myocytes, bath applications of 100 and 500 µM tiapride at 36℃ blocked rapidly activating delayed rectifier K⁺ current (I_Kr) by 40.3% and 70.0%, respectively. Also, sulpiride at 100 and 500 µM blocked I_Kr by 38.9% and 76.5%, respectively. However, neither tiapride nor sulpiride significantly affected the slowly activating delayed rectifier K⁺ current (I_Ks) at the same concentrations. Our findings suggest that the concentrations of the antipsychotics required to evoke a 50% inhibition of I_Kr are well above the reported therapeutic plasma concentrations of free and total compound.     

Electrophysiological effects of bimoclomol in canine ventricular myocytes

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 microM) 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 microM glibenclamide (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 Vmax-depressant effect of bimoclomol was not affected by charybdotoxin, anthracene-9-carboxylic acid, glibenclamide, or BAPTA load. In voltage clamped cardiomyocytes bimoclomol (100 microM) had no effect on the amplitude of I(Ca), but decreased significantly the inactivation time constant of I(Ca) (from 19.8+/-1.6 ms to 16.8+/-1.2 ms at 0 mV). Bimoclomol also decreased significantly the amplitude of I(K1) (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 BaCl2, when I(K1) current was blocked. The bimoclomol-induced current had a reversal potential close to -90 mV. Bimoclomol (100 microM) had no effect on the amplitude or kinetic properties of the transient outward K current (I(to)) and the delayed rectifier K current (I(K)). It is concluded that bimoclomol exerts both Ca-independent (inhibition of I(Na) and I(K1), activation of the ATP-sensitive K current) and Ca-dependent effects (mediated by Ca-activated Cl and probably K currents) in canine ventricular myocytes.     

Mechanistic model of acute autoinhibitory feedback action after administration of SSRIs in rats: Application to escitalopram-induced effects on brain serotonin levels

This study presents the development and evaluation of a feedback turnover model that mimics drug-induced effects on brain extracellular levels of serotonin (5-HT) after acute administration of the selective serotonin reuptake inhibitor (SSRI) escitalopram (S-citalopram) in rats. The extracellular 5-HT output in the hippocampus was continuously monitored by intracerebral microdialysis in conjunction with serial arterial blood sampling for evaluation of escitalopram pharmacokinetics. 5-HT levels were significantly increased following administration of 2.5, 5 and 10 mg/kg of escitalopram and the 5-HT levels gradually declined to its baseline value within 360 min. However, at 5 and 10 mg/kg, the response–time curves were almost identical. This might be explained by activation of serotonergic autoreceptors exerting negative feedback, leading to a reduced release of new 5-HT into the synapse. The dynamics of escitalopram-evoked changes of 5-HT response was characterized by a turnover model, which included an inhibitory feedback moderator component. Thus, the response acted linearly on the production of the moderator, which acted inversely on the production of response. The plasma kinetics served as input to an inhibitory function acting on the loss of response. Simultaneous fitting of the model after three constant rate infusions demonstrated the flexibility of the system. The efficacy (I_max) and potency (IC₅₀) of inhibition of reuptake were 0.9 ± 0.03 and 4.4 ± 1.4 ng/ml, respectively, corresponding to an EC₅₀ of escitalopram about 30 ng/ml. In conclusion, the model lends itself to ‘what–if’ predictions at different drug exposure scenarios, and has potential for extrapolation of the pharmacodynamics of SSRIs in man.