Effects of Paroxetine on a Human Ether-a-go-go-related Gene (hERG) K⁺ Channel Expressed in Xenopus Oocytes and on Cardiac Action Potential

K⁺ channels are key components of the primary and secondary basolateral Cl- pump systems, which are important for secretion from the salivary glands. Paroxetine is a selective serotonin reuptake inhibitor (SSRI) for psychiatric disorders that can induce QT prolongation, which may lead to torsades de pointes. We studied the effects of paroxetine on a human K⁺ channel, human ether-a-go-go-related gene (hERG), expressed in Xenopus oocytes and on action potential in guinea pig ventricular myocytes. The hERG encodes the pore-forming subunits of the rapidly-activating delayed rectifier K⁺ channel (I(Kr)) in the heart. Mutations in hERG reduce I(Kr) and cause type 2 long QT syndrome (LQT2), a disorder that predisposes individuals to life-threatening arrhythmias. Paroxetine induced concentration-dependent decreases in the current amplitude at the end of the voltage steps and hERG tail currents. The inhibition was concentration-dependent and time-dependent, but voltage-independent during each voltage pulse. In guinea pig ventricular myocytes held at 36℃, treatment with 0.4 µM paroxetine for 5 min decreased the action potential duration at 90% of repolarization (APD₉₀) by 4.3%. Our results suggest that paroxetine is a blocker of the hERG channels, providing a molecular mechanism for the arrhythmogenic side effects of clinical administration of paroxetine.

Inhibition of K⁺ outward currents by linopirdine in the cochlear outer hair cells of circling mice within the first postnatal week

Inhibition of K⁺ outward currents by linopirdine in the outer hair cells (OHCs) of circling mice (homozygous (cir/cir) mice), an animal model for human deafness (DFNB6 type), was investigated using a whole cell patch clamp technique. Littermate heterozygous (+/cir) and ICR mice of the same age (postnatal day (P) 0 –P6) were used as controls. Voltage steps from –100 mV to 40 mV elicited small inward currents (–100 mV~–70 mV) and slow rising K⁺ outward currents (–60 mV ~40 mV) which activated near –50 mV in all OHCs tested. Linopirdine, a known blocker of K⁺ currents activated at negative potentials (I(K,n)), did cause inhibition at varying degree (severe, moderate, mild) in K⁺ outward currents of heterozygous (+/cir) or homozygous (cir/cir) mice OHCs in the concentration range between 1 and 100 µM, while it was apparent only in one ICR mice OHC out of nine OHCs at 100 µM. Although the half inhibition concentrations in heterozygous (+/cir) or homozygous (cir/cir) mice OHCs were close to those reported in I(K,n), biophysical and pharmacological properties of K⁺ outward currents, such as the activation close to –50 mV, small inward currents evoked by hyperpolarizing steps and TEA sensitivity, were not in line with I(K,n) reported in other tissues. Our results show that the delayed rectifier type K⁺ outward currents, which are not similar to I(K,n) with respect to biophysical and pharmacological properties, are inhibited by linopirdine in the developing (P0~P6) homozygous (cir/cir) or heterozygous (+/cir) mice OHCs.

DAMGO modulates two-pore domain K⁺ channels in the substantia gelatinosa neurons of rat spinal cord

The analgesic mechanism of opioids is known to decrease the excitability of substantia gelatinosa (SG) neurons receiving the synaptic inputs from primary nociceptive afferent fiber by increasing inwardly rectifying K⁺ current. In this study, we examined whether a µ-opioid agonist, [D-Ala2,N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO), affects the two-pore domain K⁺ channel (K2P) current in rat SG neurons using a slice whole-cell patch clamp technique. Also we confirmed which subtypes of K2P channels were associated with DAMGO-induced currents, measuring the expression of K2P channel in whole spinal cord and SG region. DAMGO caused a robust hyperpolarization and outward current in the SG neurons, which developed almost instantaneously and did not show any time-dependent inactivation. Half of the SG neurons exhibited a linear I~V relationship of the DAMGO-induced current, whereas rest of the neurons displayed inward rectification. In SG neurons with a linear I~V relationship of DAMGO-induced current, the reversal potential was close to the K⁺ equilibrium potentials. The mRNA expression of TWIK (tandem of pore domains in a weak inwardly rectifying K⁺ channel) related acid-sensitive K⁺ channel (TASK) 1 and 3 was found in the SG region and a low pH (6.4) significantly blocked the DAMGO-induced K⁺ current. Taken together, the DAMGO-induced hyperpolarization at resting membrane potential and subsequent decrease in excitability of SG neurons can be carried by the two-pore domain K⁺ channel (TASK1 and 3) in addition to inwardly rectifying K⁺ channel.

Characteristics of K+ Outward Currents in the Cochlear Outer Hair Cells of Circling Mice within the First Postnatal Week

K+ outward currents in the outer hair cells (OHCs) of circling mice (homozygous (cir/cir) mice), an animal model for human deafness (DFNB6 type), were investigated using a whole cell patch clamp technique. Littermate heterozygous (+/cir) mice of the same age (postnatal day (P) 0 -P6) were used as controls. Similar slow rising K+ currents were observed in both genotypes, but their biophysical and pharmacological properties were quite different. The values of V(half) for activation were significantly different in the heterozygous (+/cir) and homozygous (cir/cir) mice (-8.1+/-2.2 mV, heterozygous (+/cir) mice (n=7) and -17.2+/-4.2 mV, homozygous (cir/cir) mice (n=5)). The inactivation curve was expressed by a single first order Boltzmann equation in the homozygous (cir/cir) mice, while it was expressed by a sum of two first order Boltzmann equations in the heterozygous (+/cir) mice. The K+ current of homozygous (cir/cir) mice was more sensitive to TEA in the 1 to 10 mM range, while the 4-AP sensitivities were not different between the two genotypes. Removal of external Ca2+ did not affect the K+ currents in either genotype, indicating that the higher sensitivity of K+ current to TEA in the homozygous (cir/cir) mice was not due to an early expression of Ca2+ activated K+ channels. Our results suggest that the K+ outward current of developing homozygous (cir/cir) mice OHCs is different in both biophysical and pharmacological aspects than that of heterozygous (+/cir) mice.

Effect of trimetazidine treatment on the transient outward potassium current of the left ventricular myocytes of rats with streptozotocin-induced type 1 diabetes mellitus

Cardiovascular complications are a leading cause of mortality in patients with diabetes mellitus (DM). The present study was designed to investigate the effects of trimetazidine (TMZ), an anti-angina drug, on transient outward potassium current (Ito) remodeling in ventricular myocytes and the plasma contents of free fatty acid (FFA) and glucose in DM. Sprague-Dawley rats, 8 weeks old and weighing 200-250 g, were randomly divided into three groups of 20 animals each. The control group was injected with vehicle (1 mM citrate buffer), the DM group was injected with 65 mg/kg streptozotocin (STZ) for induction of type 1 DM, and the DM + TMZ group was injected with the same dose of STZ followed by a 4-week treatment with TMZ (60 mg·kg-1·day-1). All animals were then euthanized and their hearts excised and subjected to electrophysiological measurements or gene expression analyses. TMZ exposure significantly reversed the increased plasma FFA level in diabetic rats, but failed to change the plasma glucose level. The amplitude of Ito was significantly decreased in left ventricular myocytes from diabetic rats relative to control animals (6.25 ± 1.45 vs 20.72 ± 2.93 pA/pF at +40 mV). The DM-associated Ito reduction was attenuated by TMZ. Moreover, TMZ treatment reversed the increased expression of the channel-forming alpha subunit Kv1.4 and the decreased expression of Kv4.2 and Kv4.3 in diabetic rat hearts. These data demonstrate that TMZ can normalize, or partially normalize, the increased plasma FFA content, the reduced Ito of ventricular myocytes, and the altered expression Kv1.4, Kv4.2, and Kv4.3 in type 1 DM.

Sustained K+ Outward Currents are Sensitive to Intracellular Heteropodatoxin2 in CA1 Neurons of Organotypic Cultured Hippocampi of Rats

Blocking or regulating K+ channels is important for investigating neuronal functions in mammalian brains, because voltage-dependent K+ channels (Kv channels) play roles to regulate membrane excitabilities for synaptic and somatic processings in neurons. Although a number of toxins and chemicals are useful to change gating properties of Kv channels, specific effects of each toxin on a particular Kv subunit have not been sufficiently demonstrated in neurons yet. In this study, we tested electrophysiologically if heteropodatoxin2 (HpTX2), known as one of Kv4-specific toxins, might be effective on various K+ outward currents in CA1 neurons of organotypic hippocampal slices of rats. Using a nucleated-patch technique and a pre-pulse protocol in voltage-clamp mode, total K+ outward currents recorded in the soma of CA1 neurons were separated into two components, transient and sustained currents. The extracellular application of HpTX2 weakly but significantly reduced transient currents. However, when HpTX2 was added to internal solution, the significant reduction of amplitudes were observed in sustained currents but not in transient currents. This indicates the non-specificity of HpTX2 effects on Kv4 family. Compared with the effect of cytosolic 4-AP to block transient currents, it is possible that cytosolic HpTX2 is pharmacologically specific to sustained currents in CA1 neurons. These results suggest that distinctive actions of HpTX2 inside and outside of neurons are very efficient to selectively reduce specific K+ outward currents.

Ionic Mechanisms of Desflurane on Prolongation of Action Potential Duration in Rat Ventricular Myocytes

PURPOSE: Despite the fact that desflurane prolongs the QTC interval in humans, little is known about the mechanisms that underlie these actions. We investigated the effects of desflurane on action potential (AP) duration and underlying electrophysiological mechanisms in rat ventricular myocytes. MATERIALS AND METHODS: Rat ventricular myocytes were enzymatically isolated and studied at room temperature. AP was measured using a current clamp technique. The effects of 6% (0.78 mM) and 12% (1.23 mM) desflurane on transient outward K+ current (I(to)), sustained outward current (I(sus)), inward rectifier K+ current (I(KI)), and L-type Ca2+ current were determined using a whole cell voltage clamp. RESULTS: Desflurane prolonged AP duration, while the amplitude and resting membrane potential remained unchanged. Desflurane at 0.78 mM and 1.23 mM significantly reduced the peak I(to) by 20+/-8% and 32+/-7%, respectively, at +60 mV. Desflurane (1.23 mM) shifted the steady-state inactivation curve in a hyperpolarizing direction and accelerated inactivation of the current. While desflurane (1.23 mM) had no effects on I(sus) and I(KI), it reduced the L-type Ca2+ current by 40+/-6% (p<0.05). CONCLUSION: Clinically relevant concentrations of desflurane appear to prolong AP duration by suppressing Ito in rat ventricular myocytes.

The underlying proantiarrhythmic mechanism of 5-HT_4 receptor agonist and 5-HT_3 receptor antagonist 2-[1-(4-piperonyl)piperazinyl]benzothiazole / 中国药理学通报

Aim To investigate the effects of 5-HT_4 receptor agonist and 5-HT_3 receptor antagonist 2-[1-(4-piperonyl)piperazinyl]benzothiazole on rat heart rhythm and the involved ionic mechanisms.Methods Langendorff-perfused rat hearts were subjected to 0.1～10 μmol·L~(-1) 2-[1-(4-piperonyl)-piperazinyl]benzothiazole for 15 minutes with simultaneous ECGs recording.The whole-cell patch-clamp electrophysiology was used to record effects of 2-[1-(4-piperonyl)piperazinyl]benzothiazole on inward rectifier K~+ current(I_(K1)),transient outward K~+ current(I_(to)),resting membrane potential(RMP)and action potential(AP)in enzymatic dissociated rat ventricular myocytes.Results In ex vivo Langendorff-perfused hearts,0.1～10 μmol·L~(-1) 2-[1-(4-piperonyl)piperazinyl]benzothiazole elicited singnificant rhythm disturbances.In the presence of 10 μmol·L~(-1) agent,the total of PVB were 236±37,87.5%(7/8)hearts exhibited VT,and 62.5%(5/8)hearts exhibited VF(P<0.01).At the concentration of 0.1～10 μmol·L~(-1),2-[1-(4-piperonyl)piperazinyl]benzothiazole could inhibit I_(K1)(EC50=0.74 μmol·L~(-1))and I_(to)(EC50=2.16 μmol·L~(-1)),decrease RMP and prolong action potential duration(APD)in concentration-dependent manners(n=6,P<0.01).Conclusion Inhibition of IK1,Ito and resultant prolongation of APD,depolarization of RMP might be the critical causes for induction of arrhythmias by 2-[1-(4-piperonyl)piperazinyl]benzothiazole in rat.

A Carbohydrate Fraction, AIP1, from Artemisia Iwayomogi Reduces the Action Potential Duration by Activation of Rapidly Activating Delayed Rectifier K+ Channels in Rabbit Ventricular Myocytes

We investigated the effects of a hot-water extract of Artemisia iwayomogi, a plant belonging to family Compositae, on cardiac ventricular delayed rectifier K+ current (I(K)) using the patch clamp technique. The carbohydrate fraction AIP1 dose-dependently increased the heart rate with an apparent EC(50) value of 56.1+/-5.5 microgram/ml. Application of AIP1 reduced the action potential duration (APD) in concentration-dependent fashion by activating I(K) without significantly altering the resting membrane potential (IC(50) value of APD(50): 54.80+/-2.24, IC(50) value of APD(90): 57.45+/-3.47 microgram/ml). Based on the results, all experiments were performed with 50 microgram/ml of AIP1. Pre-treatment with the rapidly activating delayed rectifier K+ current (I(Kr)) inhibitor, E-4031 prolonged APD. However, additional application of AIP1 did not reduce APD. The inhibition of slowly activating delayed rectifier K+ current (I(Ks)) by chromanol 293B did not change the effect of AIP1. AIP1 did not significantly affect coronary arterial tone or ion channels, even at the highest concentration of AIP1. In summary, AIP1 reduces APD by activating I(Kr) but not I(Ks). These results suggest that the natural product AIP1 may provide an adjunctive therapy of long QT syndrome.

Sodium-activated Potassium Current in Guinea pig Gastric Myocytes

This study was designed to identify and characterize Na+ -activated K+ current (I(K(Na))) in guinea pig gastric myocytes under whole-cell patch clamp. After whole-cell configuration was established under 110 mM intracellular Na+ concentration ([Na+]i) at holding potential of -60 mV, a large inward current was produced by external 60 mM K+([K+] degree). This inward current was not affected by removal of external Ca2+. K+ channel blockers had little effects on the current (p>0.05). Only TEA (5 mM) inhibited steady-state current to 68+/-2.7% of the control (p<0.05). In the presence of K+ channel blocker cocktail (mixture of Ba2+, glibenclamide, 4-AP, apamin, quinidine and TEA), a large inward current was activated. However, the amplitude of the steadystate current produced under [K+]degree (140 mM) was significantly smaller when Na+ in pipette solution was replaced with K+ - and Li+ in the presence of K+ channel blocker cocktail than under 110 mM [Na+]i. In the presence of K+ channel blocker cocktail under low Cl- pipette solution, this current was still activated and seemed K+ -selective, since reversal potentials (E(rev)) of various concentrations of [K+]degree-induced current in current/voltage (I/V) relationship were nearly identical to expected values. R-56865 (10-20 microgram), a blocker of IK(Na), completely and reversibly inhibited this current. The characteristics of the current coincide with those of IK(Na) of other cells. Our results indicate the presence of IK(Na) in guinea pig gastric myocytes.

5-Azacytidine actives three types of K~+ currents during MSCs proliferating and differentiating processes / 基础医学与临床

Objective To study the changes of the transient outward K+ current (Ito),delayed rectifier K+ current(IkDR) and the inward rectifier K+ current(Ik1) of the rat bone mesenchymal stem cells (MSCs) induced by 5-Azacytidine(5-Aza) during proliferating and differentiating process in vitro. Methods MSCs were cultured according to related articles for two weeks and then some of the cells were induced by 5-Azacytidine.The experiment was divided into uninduced(cultured for 6w) and induced(1,2,3 and 4w) cell groups. Each week had twenty cells random tested by the whole-cell patch clamp technology and the K+ currents were identified by corresponding ionic blockers.Results The detection rate of the same type of K+ current during test weeks had no significant deviation;The three types of K+ current intensity were gradually augmented after being inducing cultured for 1,2,3 and 4 weeks(P

Electrophysiologic Mechanisms of Sevoflurane on Prolongation of the QT Interval: K+ Currents in Rat Ventricular Myocytes / 대한마취과학회지

BACKGROUND: Whereas sevoflurane (SEVO) has been reported to prolong the QT interval, little has been known on the electrophysiologic effects of SEVO which contributes to the prolongation of action potential (AP) duration. METHODS: The ventricular myocytes were obtained from enzymatically treated rat hearts. The standard whole cell voltage-clamp methods were used. The AP was measured using current clamp technique. As a repolarizing K+ current, the transient outward K+ current (I(to)), the sustained outward K+ current (I(sus)), and the inwardly rectifying K+ current (I(kI)) were measured. The L-type Ca2+ current (I(Ca), L) was also obtained. After the baseline measurements, the myocytes were exposed to 1.7 and 3.4% SEVO. SEVO concentrations in Tyrode superfusate at room temperature were 0.35 and 0.7 mM for 1.7 and 3.4% SEVO, respectively. Results are mean +/- SEM. RESULTS: SEVO prolonged the AP duration, while the amplitude and the resting membrane potential remained unchanged. At membrane potential of +60 mV, peak I(to) was significantly reduced by 18 +/- 2 and 24 +/- 2% by 0.35 and 0.7 mM SEVO, respectively. 0.7 mM SEVO did not shift the steady-state inactivation curve. Isus was unaffected by 0.7 mM SEVO. The I(kI) at -130 mV was little altered by 0.7 mM SEVO. I(Ca), L was significantly reduced by 28 +/- 3 and 33 +/- 1% by 0.35 and 0.7 mM SEVO, respectively. CONCLUSIONS: Prolongation of AP duration by SEVO in rat ventricular myocytes is likely to be caused by a reduction of I(to). Resting membrane potential was unaffected by SEVO, which seems to be related to no alteration of I(kI).

Electrophysiologic Effect of Desflurane on the Prolongation of Action Potential Duration in Ventricular Myocytes / 대한마취과학회지

BACKGROUND: Desflurane has been reported to prolong the QTc. Several ionic currents that contribute to the prolongation of the action potential (AP) duration were investigated using guinea pig (GP) and rat ventricular myocytes. METHODS: The normal APs were measured in isolated GP papillary muscles at 37 degrees C. Ventricular myocytes were obtained from GP and rat hearts. Both the delayed outward K+ current (I(K)) and the inward rectifier K+ current (I(KI)) were assessed using a voltage ramp protocol. A more detailed study on the I(K) was performed. The ICa, L was measured. In the rat ventricular myocytes, the transient outward K+ current (I(to)) was obtained. All the patch clamp experiments were carried out at room temperature. The values are presented as mean +/- SD. RESULTS: 0.91 mM desflurane significantly prolonged the APD in the GP ventricular myocytes. Using a linear voltage ramp protocol, the I(KI) at -130 mV and the peak outward I(KI) at -60 to -50 mV were not found to be significantly reduced by 0.78 and 1.23 mM desflurane, respectively. However, the peak outward I(K) at +60 mV was significantly reduced to 63 +/- 19% and 58 +/- 12% of the baseline by 0.78 and 1.23 mM desflurane, respectively. At a membrane potential of +60 mV, 0.78 and 1.23 mM desflurane reduced the Ito to 80 +/- 8% and 68 +/- 7%, respectively. A concentration-dependent reduction in the ICa, L was observed. CONCLUSIONS: The prolongation of the APD induced by clinically relevant concentrations of desflurane in GP and rat ventricular myocytes is most likely the result of I(K) and I(to) suppression.

Changes in Excitability of Neurons in Rat Medial Vestibular Nucleus Following Vestibular Neurectomy

Intrinsic excitabilities of acutely isolated medial vestibular nucleus (MVN) neurons of rats with normal labyrinth and with undergoing vestibular compensation from 30 min to 24 h after unilateral vestibular deafferentation (UVD) were compared. In control rats, proportions of type A and B cells were 30 and 70%, respectively, however, the proportion of type A cells increased following UVD. Bursting discharge and irregular firing patterns were recorded from 2 to 12 h post UVD. The spontaneous discharge rate of neurons in the ipsilesional MVN increased significantly at 2 h post-UVD and remained high until 12 h post-UVD in both type A and type B cells. After-hyperpolarization (AHP) of the MVN neurons decreased significantly from 2 h post-UVD in both types of cells. These results suggest that the early stage of vestibular compensation after peripheral neurectomy is associated with an increase in intrinsic excitability due to reduction of AHP in MVN neurons.

Effects of Erythromycin on Voltage-dependent Potassium Current in Hippocampal Neurons of Mice / 中国药房

OBJECTIVE:To study the effect of erythromycin on the voltage-dependent K~+ current in hippocampal neurons of mice.METHODS:Isolation of mice's hippocammal neurons cells and whole-cell patch-clamp techniques were applied to study the change of voltage-dependent K~+ current of hippocampal neurons and the current-voltage curves in mice before and after treatment with erythromycin.RESULTS:Erythromycin inhibited the voltage-dependent K~+ current of mice's hippocampal neurons,and it down-regulated the peak current from(810.67?250.86)pA to(529.96?171.83)pA(P

Stationary outward and transient Ca2+-dependent currents in hamster oocytes

The outward currents elicited in hamster eggs by depolarizing pulses were studied. The currents appeared to comprise at least two components, a transient outward component (Ito) and a steady-state outward component (Iinfin). Ito was transiently followed by the cessation of inward Ca2+ current (ICa), and its current-voltage (I-V) relation was a mirror image of that of ICa. Either blockade of ICa by Co2+ or replacement of Ca2+ with Sr2+ abolished Ito without change in Iinfin. Intracellular EGTA (10 mM) inhibited Ito but not Iinfin. suggesting strongly that generation of Ito requires intracellular Ca2+. Apamin (1 nM) abolished selectively Ito, indicating that Ito is Ca2+-dependent K+ current. On the other hand, Iinfin was Ca2+-independent. Both Ito and Iinfin were completely inhibited by internal Cs+ and external TEA. The estimated reversal potential of Ito was close to the theoretical EK. Taken together, both outward currents were carried by K+ channels. From these results, Ito is likely to be a current responsible for the hyperpolarizing responses seen in hamster eggs at fertilization.

Effect of capsaicin on delayed rectifier K+ current in adult rat dorsal root ganglion neurons

K+ currents play multiple roles in the excitability of dorsal root ganglion (DRG) neurons. Influences on these currents change the shape of the action potential, its firing threshold and the resting membrane potential. In this study, whole cell configuration of patch clamp technique had been applied to record the blocking effect of capsaicin, a lipophilic alkaloid, on the delayed rectifier K+ current in cultured small diameter DRG neurons of adult rat. Capsaicin reduced the amplitude of K+ current in dose dependent manner, and the concentration-dependence curve was well described by the Hill equation with KD value of 19.1 micrometer. The blocking effect of capsaicin was reversible. Capsaicin (10 micrometer) shifted the steady-state inactivation curve in the hyperpolarizing direction by about 15 mV and increased the rate of inactivation. The voltage dependence of activation was not affected by capsaicin. These multiple effects of capsaicin may suggest that capsaicin bind to the region of K+ channel, participating in inactivation process.

Sulfhydryl modification affects coronary artery tension by changing activity of delayed rectifier K+ current

It has been reported that a change in the cellular redox state may be involved in the regulation of vascular tone, but the underlying mechanism is not fully understood. The present study was designed to investigate the cellular effect of sulfhydryl modifying agents in the coronary artery of rabbit using the tension measurement and whole cell clamping method. The application of diamide, a sulfhydryl oxidizing agent, relaxed the endothelium denuded coronary arteries in a dose dependent manner. The fact that this diamide-induced relaxation was significantly attenuated by a pretreatment of 4-AP, and the coronary arteries precontracted with 100 mM K+ instead of histamine, suggests the involvement of 4-AP sensitive K+ channels in the diamide-induced relaxation of coronary arteries. Whole cell patch clamp studies revealed that the 4-AP sensitive IdK was significantly enhanced by the membrane permeant oxidizing agents, diamide and DTDP, and were reversed by subsequent exposure to the reducing agent, DTT. Neither the membrane impermeant oxidizing or reducing agents, GSSG or GSH, had any effect on the activity of IdK, indicating that intracellular sulfhydryl modification is critical for modulating IdK activity. The Diamide failed to significantly alter the voltage dependence of the activation and inactivation parameters, and did not change the inactivation process, suggesting that diamide increases the number of functional channels without altering their gating properties. Since IdK has been believed to play an important role in regulating membrane potential and arterial tone, our results about the effect of sulfhydryl modifying agents on coronary arterial tone and IdK activity should help understand the pathophysiology of the diseases, where oxidative damage has been implicated.

Depression of L-type Ca2+ and transient outward K+ currents in endotoxin-treated rat cardiac myocytes

Decreased cardiac contractility occurs in endotoxicosis, but little is known about the ionic mechanism responsible for myocardial dysfunction. In this study, we examined the changes in Ca2+ and K+ currents in cardiac myocytes from endotoxin-treated rat. Ventricular myocytes were isolated from normal and endotoxemic rats (ex vivo), that were treated for 10 hours with Salmonella enteritidis lipopolysaccharides (LPS; 1.5 mg/kg) intravenously. Normal cardiac myocytes were also incubated for 6 hours with 200 ng/ml LPS (in vitro). L-type Ca2+ current (ICa,L) and transient outward K+ current (Ito) were measured using whole cell patch clamp techniques. Peak ICa,L was reduced in endotoxemic myocytes (ex vivo; 6.00.4 pA/pF, P<0.01) compared to normal myocytes (control; 10.90.6 pA/pF). Exposure to endotoxin in vitro also attenuated ICa,L (8.40.4 pA/pF, P<0.01). The amplitude of Ito on depolarization to 60 mV was reduced in endotoxin treated myocytes (16.51.5 pA/pF, P<0.01, ex vivo; 20.00.9 pA/pF, P<0.01, in vitro) compared to normal myocytes (control; 24.71.0 pA/pF). There was no voltage shift in steady-state inactivation of ICa,L and Ito between groups. These results suggest that endotoxin reduces Ca2+ and K+ currents of rat cardiac myocytes, which may lead to cardiac dysfunction.

Effect of PKC-dependent Change of K+ Current Activity on Histamine-induced Contraction of Rabbit Coronary Artery

BACKGROUND: Histamine, released from mast cells in atheromatous plaque, has been known to cause cardiac ischemia or sudden cardiac death in atherosclerosis patient. Previous reports have suggested that histamine induced coronary vasoconstriction was due to increase in IP(3) and DAG, which induce release of Ca2+ from SR and increase the Ca2+ sensitivity of contractile element via activation of PKC. Recently, it was reported that application of histamine cause depolarization of intestinal smooth muscle, which may contribute to histamine-induced contraction via augmenting Ca2+ influx through activation of Ca2+ channels. However, the underyling mechanism of histamine-induced depolarization and its contribution to the magnitude of coronary vasoconstriction are still uncertain. METHOD: To elucidate the underlying mechanism of Ca2+ influx change during histamine-induced vasoconstriction, we examined the effect of Ca2+ channel antagonist and PKC blocker on histamine-induced contractions, and then measured the effect of PKC antagonist on whole cell K+ current using patch clamping method in rabbit coronary smooth muscle cells. RESULTS: Application of histamine induced phasic and tonic constraction of coronary rings via activation of H(1) receptors. Pretreatment of Ca2+ channel antagonist (nifedipine, 1 microM) or PKC blockers (10 nM staurosporine and 10 microM Go6976) markedly inhibited histamine-induced tonic contraction, which suggest that the magnitude of tonic contraction depend on the Ca2+ influx. Application of 4-AP, a blocker of voltage-dependent K+ channels, increased resting tone of coronary rings, and combined treatment of nifedipine blocked this 4-AP induced increase of resting tone. Application of active analoge of DAG (1,2-DiC(8)) significantly inhibited the activity of voltage-dependent K+ current in single smooth muscle cell, meanwhile the inactive analogue of DAG (1,3-DiC(8)) has no apparent effect on the activity of voltage-dependent K+ current. Furthermore, pretreatment of calphostin C (1 microM), a blocker of PKC, diminished the 1,2-DiC(8)-induced inhibition of K+ current. CONCLUSION: PKC dependent inhibition of voltage-dependent K+ current may be responsible for the maintaining of histamine-induced tonic contraction in rabbit coronary artery.