Attenuation of pain behaviour by local administration of alpha-2 adrenoceptor antagonists to dorsal root ganglia in a rat radiculopathy model.

BACKGROUND: There were several reports suggesting α-adrenoceptor antagonists are effective to treat neuropathic pain. The aims of this study were as follows: (1) to introduce drug delivery system for dorsal root ganglion (DRG) neurons; (2) to elucidate the effects of α-adrenoceptor antagonists in acute, subacute or chronic phase and (3) to determine which subtype of adrenoceptor was mainly involved. METHOD: We used 130 male Sprague-Dawley rats. After root constriction, rats received three local injections of α-adrenoceptor antagonists around DRG. We administered the non-selective α-adrenoceptor antagonist phentolamine for 3 consecutive days from day 0, 4 or 11 after the surgery, and the α1-adrenoceptor antagonist prazosin, the α1-adrenoceptor antagonist silodosin, the more preferred α1-adrenoceptor than prazosin and the α2-adrenoceptor antagonist yohimbine for 3 consecutive days from day 0 after the surgery. RESULTS: Phentolamine and yohimbine continually attenuated pain behaviour. Prazosin at high dose attenuated pain behaviour, however, prazosin at low dose did not attenuate pain behaviour every experimental day. Silodosin had no analgesic effect. Phentolamine injections from day 4 after surgery attenuated pain behaviour that had been established on the 3rd experimental day until the 28th post-operative day, although effect of phentolamine wore off. Phentolamine injections from day 11 after surgery temporarily attenuated pain behaviour that had been established on the 3rd, 7th and 10th experimental days. CONCLUSIONS: This study showed α-adrenoceptor antagonists could suppress pain behaviour via α2-adrenoceptor in acute phase and temporary attenuate pain behaviour in chronic phase. These findings presented potentials sympathetic nerve blockade contributed to treat neuropathic pain.

Sympathectomy attenuates excitability of dorsal root ganglion neurons and pain behaviour in a lumbar radiculopathy model.

OBJECTIVES: In order to elucidate the influence of sympathetic nerves on lumbar radiculopathy, we investigated whether sympathectomy attenuated pain behaviour and altered the electrical properties of the dorsal root ganglion (DRG) neurons in a rat model of lumbar root constriction. METHODS: Sprague-Dawley rats were divided into three experimental groups. In the root constriction group, the left L5 spinal nerve root was ligated proximal to the DRG as a lumbar radiculopathy model. In the root constriction + sympathectomy group, sympathectomy was performed after the root constriction procedure. In the control group, no procedures were performed. In order to evaluate the pain relief effect of sympathectomy, behavioural analysis using mechanical and thermal stimulation was performed. In order to evaluate the excitability of the DRG neurons, we recorded action potentials of the isolated single DRG neuron by the whole-cell patch-clamp method. RESULTS: In behavioural analysis, sympathectomy attenuated the mechanical allodynia and thermal hyperalgesia caused by lumbar root constriction. In electrophysiological analysis, single isolated DRG neurons with root constriction exhibited lower threshold current, more depolarised resting membrane potential, prolonged action potential duration, and more depolarisation frequency. These hyperexcitable alterations caused by root constriction were significantly attenuated in rats treated with surgical sympathectomy. CONCLUSION: The present results suggest that sympathectomy attenuates lumbar radicular pain resulting from root constriction by altering the electrical property of the DRG neuron itself. Thus, the sympathetic nervous system was closely associated with lumbar radicular pain, and suppressing the activity of the sympathetic nervous system may therefore lead to pain relief.

Desflurane but not sevoflurane can increase lung resistance via tachykinin pathways.

BACKGROUND: Although there is evidence that the volatile anaesthetic desflurane directly relaxes preconstricted airway smooth muscle in vitro, the anaesthetic increases the lung resistance in vivo. The constrictive mechanisms of desflurane are, however, still unknown. This study was conducted to clarify the increasing mechanisms of desflurane on lung resistance by examining the vagal nerve reflexes in guinea pigs. METHODS: The effects of desflurane and sevoflurane on total lung resistance (R(L)) and dynamic lung compliance (C(Dyn)) were investigated in animals that were either untreated, pretreated with atropine or vagotomy, pretreated with the tachykinin receptor antagonists sendide or MEN-10376, or given chronic pretreatment with capsaicin. RESULTS: Desflurane biphasically and dose-dependently increased R(L) (by 180% and 230% at the first and second peaks, respectively, at 2 minimum alveolar concentration) concomitant with a decrease in C(Dyn). However, sevoflurane had little effect on either R(L) or C(Dyn). Although vagotomy partially inhibited the first peak of R(L) by 30%, neither atropine nor vagotomy had any effect on the other respiratory responses to desflurane. Antagonization of tachykinin receptors of airway smooth muscles completely diminished the increase in R(L) induced by desflurane. Desflurane also had little effect on respiratory parameters after the capsaicin pretreatment, in which tachykinin containing afferent C-fibres was desensitized. CONCLUSIONS: Desflurane but not sevoflurane increased R(L) concomitant with a decrease in C(Dyn) in guinea pigs. The increase in lung resistance by desflurane might be due to antidromic tachykinin release from afferent C-fibres but not acetylcholine release from parasympathetic efferent nerves.

The effects of the sympathetic nerves on lumbar radicular pain: a behavioural and immunohistochemical study.

A rat model of lumbar root constriction with an additional sympathectomy in some animals was used to assess whether the sympathetic nerves influenced radicular pain. Behavioural tests were undertaken before and after the operation. On the 28th post-operative day, both dorsal root ganglia and the spinal roots of L4 and L5 were removed, frozen and sectioned on a cryostat (8 microm to 10 microm). Immunostaining was then performed with antibodies to tyrosine hydroxylase (TH) according to the Avidin Biotin Complex method. In order to quantify the presence of sympathetic nerve fibres, we counted TH-immunoreactive fibres in the dorsal root ganglia using a light microscope equipped with a micrometer graticule (10 x 10 squares, 500 mm x 500 mm). We counted the squares of the graticule which contained TH-immunoreactive fibres for each of five randomly-selected sections of the dorsal root ganglia. The root constriction group showed mechanical allodynia and thermal hyperalgesia. In this group, TH-immunoreactive fibres were abundant in the ipsilateral dorsal root ganglia at L5 and L4 compared with the opposite side. In the sympathectomy group, mechanical hypersensitivity was attenuated significantly. We consider that the sympathetic nervous system plays an important role in the generation of radicular pain.

Chloride conductance determining membrane potential of rabbit articular chondrocytes.

Membrane conductance of cultured rabbit articular chondrocytes was characterized by means of the patch-clamp technique. The resting membrane potential of the articular chondrocytes was about -42 mV. The membrane potential shifted in accordance with the prediction by the Nernst equation for Cl- when intracellular and extracellular concentrations of Cl- were changed. On the other hand, change in extracellular concentration of K+ produced no shift in the membrane potential of chondrocytes. The Cl- channel blocker 4-acetamido-4'-isothiocyanatostilbene-2'2-disulfonic acid (SITS) depolarized the membrane potential. These findings suggest that the membrane potential of the chondrocytes is determined mainly by Cl- conductance. Using the cell-attached patch-clamp method, a large unitary conductance of 217 pS was observed in the articular chondrocytes. The unitary current was reversibly blocked by SITS. Therefore, the unitary current was carried by Cl-. The Cl- channel showed voltage-dependent activation and the channels exhibited long-lasting openings. Therefore, the membrane potential of rabbit cultured articular chondrocytes was mainly determined by the activities of the large-conductance and voltage-dependent Cl- channels.

Cloning of a functional splice variant of L-type calcium channel beta 2 subunit from rat heart.

L-type Ca(2+) channels are heteromultimeric and finely tuned by auxiliary subunits in different tissues and regions. Among auxiliary subunits, beta subunit has been shown to play important roles in many functional aspects of Ca(2+) channel. Rat heart was reported to specifically express beta(2a) subunit. However, the slow inactivation rates of Ca(2+) currents recorded from recombinant Ca(2+) channels with the beta(2a) subunit, and the reported inability to detect beta(2a) subunit in rabbit heart by reverse transcription-PCR analysis raise the possibility of the existence of other beta subunits. We cloned a splice variant of beta(2) subunit from rat heart, using rapid amplification of cDNA 5' ends. The splice variant is highly similar to human beta(2c) subunit that was cloned from human ventricle. Northern blot analysis detected the rat beta(2c) subunit abundantly in rat heart and brain. The deduced amino acid sequence of the beta(2c) subunit was different from that of the beta(2a) subunit only in the N-terminal region. When the beta(2c) subunit was expressed along with alpha(1c) and alpha(2)delta subunits in baby hamster kidney cells, the inactivation rates were comparable with those from native cardiac myocytes, although those with the beta(2a) subunit were slow. Taken together, these observations suggest that the beta(2c) subunit is a functional beta(2) subunit expressed in heart and that the short N-terminal region plays a major role in modifying inactivation kinetics.

Decrease in the Ca2+-activated K+ current of pulmonary arterial smooth muscle in pulmonary hypertension rats.

Pulmonary hypertension exhibits acute elevation of vascular tone and hyperreactivity of pulmonary vasculature, which are closely related to patient mortality. In the present study, we investigated the characteristics of membrane currents of isolated pulmonary artery smooth muscle cells taken from rats with monocrotaline-induced pulmonary hypertension. Male Wistar rats were given a single subcutaneous injection of monocrotaline or saline, and then sacrificed between 18 to 21 days after the injection. The membrane currents in the smooth muscle cells from both groups of rats were compared using the whole-cell patch clamp technique. With 0.1 mM EGTA in the pipette, the densities of outward currents in monocrotaline-injected rats were smaller than those in control rats. When EGTA in patch pipettes was increased to 10 mM, the densities of the outward currents in monocrotaline-injected rats were equal to those of control rats. The Ca2+-activated K+ channel blockers (TEA, iberiotoxin) and nisoldipine were less effective on the outward currents of monocrotaline-injected rats. In the current clamp mode, a depolarization of membrane potential induced by 4-aminopyridine was greater in monocrotaline-injected rats than in control rats because of the reduced activity of the Ca2+-activated K+ channels. The Ca2+-activated K+ channels were decreased in pulmonary hypertension. The reduced activity of the currents may be related to the vascular hyperreactivity in pulmonary hypertension.

[Alternation of regulation system for pulmonary arterial tone in pulmonary hypertension].

In pulmonary artery smooth muscle cells(PASMC), the Ca-influx required for myogenic tone is mainly supplied through the L-type Ca channels, which sustained open around resting membrane potential. Therefore, the voltage-gated K channels, which determine membrane potential in PASMC, are critical for regulation of the Ca-influx and myogenic tone. The voltage-gated K channels are inhibited in hypoxic condition. The inhibition may be related to the hypoxic pulmonary vasoconstriction and the chronic hypoxia-induced pulmonary hypertension. In subjects with primary pulmonary hypertension, the voltage-gated K channels are also inhibited. On the other hand, the Ca-activated K channels of PASMC are also inhibited in chronic exposure with pulmonary hypertension. The reduction of the K channels in pulmonary hypertension may enhance proliferation of PASMC.

Alternation of inwardly rectifying background K+ channel during development of rat fetal cardiomyocytes.

The resting membrane potential of rat ventricular myocytes dramatically hyperpolarizes in the late phase of the fetal period. In order to investigate the mechanisms of this hyperpolarization, we examined the electrophysiological properties and molecular structure of the inwardly rectifying background K+ channels of rat fetal ventricular myocytes. In a patch-clamp experiment the whole-cell current of the inwardly rectifying background K+ channel increased 12-fold from between 12 and 18 days after impregnation. In the single channel recording, the large-conductance (35 pS) channels were mainly observed in the 18-day fetal ventricular myocytes. In the 12-day cells, the large-conductance channel was not observed although the low-conductance channels (11 and 16 pS) were infrequently observed. These data of single channel recordings suggested that channel proteins conducting the inwardly rectifying background K+ current were altered during the fetal development. Therefore, we compared the expression of Kir 2.1 mRNA and Kir 2.2 mRNA between 12 days and 18 days using the RT-PCR method, in order to investigate the possible molecular regulation which contributes to the electrophysiological changes. During the fetal period, the expression of Kir 2.2 mRNA increased tremendously (17 times), whereas the increase in the expression of Kir 2.1 mRNA (two times) was not so great. These results show that hyperpolarization in the late fetal period seems to be mainly due to the dramatic increase in expression of Kir 2.2 mRNA rather than expression of Kir 2.1 mRNA.

Characteristics of current fluctuations originating from activities of inward-rectifier K+ channels in guinea-pig heart cells.

Although outward current through inward-rectifier K+ channels has been observed in the whole-cell mode of the patch-clamp technique, no outward unitary current in single-channel studies has been recorded with the physiological ionic conditions. Hence, the relationship between single-channel activities and the inward rectification of the whole-cell current has been poorly understood. Therefore, characteristics of inward-rectifier K+ channels in guinea-pig ventricular myocytes were assessed by the noise analysis of the K+ current using the whole-cell patch clamp method. Partial blockade of the inward-rectifier K+ current by Ba2+ was used to obtain different levels of mean current and current fluctuation as needed for variance-to-mean analysis. The plot of variance of current fluctuation against mean currents was well fitted by theoretical parabolic curves, and the unitary conductance, the open probability, and the density of functional channels were deduced. The unitary conductance of the inward-rectifier K+ channel exhibited an inward-rectification, although the channel open probability and the density of functional channels were not much different at various holding potentials used. The unitary conductance was not changed when the intrapipette concentration of Mg2+ was reduced, but tended to be smaller when the pipette contained high Mg2+ concentration. Spermine also tended to reduce the outward unitary conductances, although the reduction was not statistically significant. These results suggest that the inward rectification in the whole-cell current was due to the inward-rectifying property of the unitary conductance of the K+ channels. Inward rectification of the unitary conductance may be caused by blocking of the channels by both Mg2+ and polyamines.

Inhibition of the delayed rectifier K current in guinea-pig cardiomyocytes by thiamine tetrahydrofurfuryl disulfide.

We examined effect of thiamine tetrahydrofurfuryl disulfide on electrophysiological characteristics of single atrial myocytes, obtained by digestion of guinea-pig heart, using collagenase. Membrane potential and ion channel current in the atrial myocytes were recorded by the patch clamp method. Thiamine tetrahydrofurfuryl disulfide prolonged action potentials at cycle lengths from 250 to 10,000 ms. The degree of thiamine tetrahydrofurfuryl disulfide-induced prolongation was similar among these cycle lengths. Thiamine tetrahydrofurfuryl disulfide inhibited the delayed rectifier K+ current, without affecting Ca2+ current and inward-rectifier K+ current. Thiamine tetrahydrofurfuryl disulfide blocked the delayed rectifier K+ current in voltage- and time-independent manner, indicating that thiamine tetrahydrofurfuryl disulfide blocked both subtypes of the delayed rectifier K+ current (rapid and slow components). Thiamine, the parent molecule of thiamine tetrahydrofurfuryl disulfide, blocked the delayed rectifier K+ current only when thiamine was applied intracellularly. Thiamine tetrahydrofurfuryl disulfide may be converted to thiamine in the cytoplasm, and then may block the the delayed rectifier K+ channel from the intracellular side. Although thiamine tetrahydrofurfuryl disulfide (or thiamine) has some of the properties of class III antiarrhythmics agents, thiamine tetrahydrofurfuryl disulfide did not exhibit reverse use-dependent prolongation of action potential.

A de novo missense mutation of human cardiac Na+ channel exhibiting novel molecular mechanisms of long QT syndrome.

Mutations in a human cardiac Na+ channel gene (SCN5A) are responsible for chromosome 3-linked congenital long QT syndrome (LQT3). Here we characterized a de novo missense mutation (R1623Q, S4 segment of domain 4) identified in an infant Japanese girl with a severe form of LQT3. When expressed in oocytes, mutant Na+ channels exhibited only minor abnormalities in channel activation, but in contrast to three previously characterized LQT3 mutations, had significantly delayed macroscopic inactivation. Single channel analysis revealed that R1623Q channels have significantly prolonged open times with bursting behavior, suggesting a novel mechanism of pathophysiology in Na+ channel-linked long QT syndrome.

Vascular reactivity to endothelium-derived relaxing factor in human umbilical artery at term pregnancy.

It has been reported that human umbilical artery (HUA) at term pregnancy released endothelium-derived relaxing factor (EDRF), using a superfusion bioassay system. However, other reports showed that endothelium-dependent relaxation was not observed in isometric tension studies using HUA ring with intact endothelium. Thus, we intended to clarify whether vascular smooth muscle of HUA at term is sensitive to EDRF. HUA was obtained after normal vaginal delivery or cesarean section at term. Isometric tension studies were performed in normal Krebs solution, using HUA rings or strips, which were prepared in calcium-free Krebs solution. Sodium nitroprusside (SNP), a nitric oxide (NO) donor drug, relaxed HUA rings precontracted with 0.1 microM 5-hydroxytryptamine (5HT) in a dose-dependent manner (1 nM-10 microM). Histamine, substance P, carbachol, or the calcium ionophore A23187, which are considered to be EDRF-releasing agents, did not relax the HUA rings. By immunohistochemical study, it was confirmed that endothelial cells were present in the luminal surface of the HUA rings after the isometric tension recording. In a co-axial bioassay system involving HUA strips denuded of endothelium and rabbit aorta with intact endothelium, HUA strips precontracted with 0.1 microM 5HT were relaxed in response to 1 microM SNP but not 1 microM carbachol, which released EDRF from the endothelium of rabbit aorta. These findings suggest that HUA at term is sensitive to NO but not EDRF.

Alpha 1-adrenoceptor subtype involved in the positive and negative inotropic responses to phenylephrine in rat papillary muscle.

1. In rat papillary muscle, stimulation of alpha 1-adrenoceptors results in a biphasic inotropic response: a transient negative inotropic phase and a subsequent sustained positive inotropic phase. This study was designed to determine whether the positive and negative inotropic effects in this tissue are mediated by different alpha 1-adrenoceptor subtypes. 2. After treatment with the tumor-promoting compound, phorbol 12, 13-dibutyrate, phenylephrine (in the presence of propranolol) produced only a positive inotropic effect. The selective alpha 1A-adrenoceptor antagonist, WB4101, significantly inhibited the positive inotropic effect. In contrast, inactivation of alpha 1B-adrenoceptors with chloroethylclonidine (CEC) did not alter the positive effect. 3. In the presence of the Ca2+ channel antagonist, nifedipine, phenylephrine induced only a sustained negative inotropic effect. The negative inotropic effect was significantly attenuated by WB4101, but was not affected by CEC. 4. We conclude that both the positive and negative inotropic responses of rat papillary muscle to phenylephrine are mediated exclusively by the WB4101-sensitive but CEC-resistant alpha 1-adrenoceptor subtype. The alpha 1-adrenoceptor subtype with such a property may correspond to the alpha 1A-subtype.

Restoration of action potential duration and transient outward current by regression of left ventricular hypertrophy.

The presence of left ventricular hypertrophy (LVH) is associated with an increased incidence of arrhythmias. Our previous study on hypertrophied rat hearts has demonstrated that regression of LVH prevents ischemia-induced lethal arrhythmias. To elucidate the underlying mechanism of the reduced incidence of arrhythmias in regression of LVH, we examined electrophysiological properties of both hypertrophied and regressed left ventricular cells. Hearts from spontaneously hypertensive rats (SHR) were used as LVH, and those from Wistar-Kyoto rats (WKY) served as control. SHR with regression of LVH (REG) was produced by captopril treatment. Action potentials and membrane currents of subendocardial left ventricular cells were compared by the whole-cell patch-clamp techniques. Although the membrane capacitance of SHR cells was significantly greater than that of WKY cells, that of REG cells was normalized to the control level. Prolonged action potential duration (APD) and reduced density of transient outward current (ito) in SHR cells was normalized by LVH regression (APD at 75% repolarization (ms) and ito density at +60 mV (pA/pF): WKY 36.1 +/- 4.2, 11.9 +/- 1.3, SHR 73.1 +/- 12.9, 5.2 +/- 0.7, REG 29.5 +/- 3.9, 10.4 +/- 2.0, P = 0.015, P = 0.001 v WKY). No significant differences were observed in the densities of steady-state outward current, inward rectifier current, and L-type Ca2+ current. The restoration of ito density by regression of LVH could normalize the prolonged APD in hypertensive LVH, which may be causally related to the reduced incidence of arrhythmias in LVH regression.

Alpha 1-adrenoceptor subtypes mediating inotropic and electrophysiological effects in mammalian myocardium.

Stimulation of alpha 1-adrenoceptors produces a positive inotropic effect in rat and rabbit ventricular myocardium via different mechanisms, the prolongation of action potential duration (APD) exclusively in the former and an increase in myofibrillar Ca2+ sensitivity in large part in the latter. This study was designed to determine whether the two inotropic mechanisms are mediated by different alpha 1-adrenoceptor subtypes. In rat papillary muscles, the positive inotropic effect and APD prolongation induced by phenylephrine (in the presence of propranolol) were inhibited by WB-4101, but not affected by chlorethylclonidine (CEC). WB-4101, but not CEC, blocked the phenylephrine-induced inhibition of the transient outward current (Ito) in rat ventricular cells. On the other hand, WB-4101 and CEC each antagonized the positive inotropic effect of phenylephrine in rabbit papillary muscles. However, the phenylephrine-induced APD prolongation observed in rabbit papillary muscles was blocked only by WB-4101. These results indicate that the WB-4101 sensitive alpha 1-adrenoceptor subtype mediates the positive inotropism that is correlated with the APD prolongation resulting from Ito reduction, whereas the CEC-sensitive subtype mediates the positive inotropism that is probably associated with increased myofibrillar Ca2+ sensitivity. Radioligand binding studies with [3H] prazosin showed a similar ratio of alpha 1A-to alpha 1B-adrenoceptor subtypes in rat and rabbit ventricular myocardium, implying that the different degree of contribution of each action mechanism to the overall inotropic effect in the two species cannot be explained by distribution of the alpha 1-adrenoceptor subtypes.

Unitary current through the inward rectifier K+ channel cloned from rabbit heart--comparison with the native K+ channel.

Recently, we have cloned a cDNA for a putative cardiac inward rectifier K+ channel (RBHIK1) from rabbit cardiac muscles. However, it's single channel characteristics have remained unknown. Therefore, we investigated the single channel characteristics of the RBHIK1 channel expressed in Xenopus oocytes by the cell attached patch clamp configuration, and compared them with those of the native Ik1 channel of the freshly-isolated ventricular myocytes under similar temperature conditions. In patch clamp experiments with 145 mmol/l K+ in the pipette at room temperature (20-22 degrees C), both the RBHIK1 currents and the native Ik1 showed a strong inward rectifying property. The single channel conductance of the RBHIK1 channel was 17.8 +/- 0.47 pS (n = 4), and that of the native Ik1 channel was 23.5 +/- 0.29 pS (n = 5). The activities of the cloned channel were sensitive to the putative K+ channel blockers (TEA, Cs+ and Ba2+). The open and closed time histograms at -140 mV could be fitted by a single exponential both in the RBHIK1 channel and the native Ik1 channel. Although the closed-time histogram of the native Ik1 channel was fitted by a sum of two exponential curves, that of the RBHIK1 channel was fitted by a single exponential curve. The sublevel corresponding to two-thirds of the unitary current was observed both in the RBHIK1 channel and the native Ik1, but it was more frequently detected in the RBHIK1 channel. Amplitude histogram constructed at -140mV in the RBHIK1 channel exhibited three peaks, which indicated closed, full-open, and 2/3 sublevel state, respectively. Unitary current was calculated to be 2.5 pA and sublevel of the unitary current was 1.68 pA. These characterization in the single channel activities of the RBHIK1 channel will help to study the molecular regulation of the Ik1 channel in cardiac cells.

[Developmental aspects of electrophysiology in cardiac muscle].

Electrical properties of the cardiac muscles drastically change with development. The changes in the current density of ionic currents of cardiomyocytes are inconsistent among species. In cultured embryonic chick ventricular myocytes, the developmental changes in the fast Na+ channel properties (3- to 17-day-old) are reviewed. The sensitivity to TTX, with a KD as high as 2 nM, remains unchanged. The limiting conductance (GNa) increased by 8-10-fold. The activation kinetics such as the steady-state activation (m infinity) and time constant of activation (tau m) remain unchanged. The voltage-dependence of inactivation kinetics such as the steady state inactivation (h infinity) and time constant (tau h) shift in the hyperpolarizing direction. The window conductance tends to be reduced. On the other hand, the L-type Ca2+ channel is important during the development of rat heart, and also the fe-type current (dihydropyridine-resistant) is important in the fetal stage. In chick embryo cardiomyocytes, the L-type channel exhibits long-lasting opening behavior. The behavior is gradually abolished during development, cAMP-dependent protein kinase enhances the Ca2+ channel current on and after the late fetal/embryonic stage. cGMP-dependent protein kinase markedly inhibits the Ca2+ channel current in the fetal/embryonic stage, compared with adult heart. These changes would play an important role for cardiac functions during development.

Effects of dofetilide on membrane currents in sinoatrial node cells of rabbit.

The effects of dofetilide (UK 68798) on membrane currents were examined in isolated sinoatrial node cells of rabbits by using patch clamping. At a concentration of 1 microM, dofetilide decreased the delayed rectifier K+ current (IK) (50.2 +/- 10.2%, mean +/- S.E.). The Ca2+ current was slightly decreased during the application of dofetilide. However, the decrease in the current may be attributed to the "run down" phenomenon. The drug did not affect the hyperpolarization-activated inward current. Therefore, dofetilide exhibited class III antiarrhythmic activity in rabbit sinoatrial node cells. Similarly, E-4031 (1-[2- (6-methyl-2-pyridyl)ethyl]-4-(4-methylsulfonylaminobenzoyl)pire ridine) (1 microM), a standard class III agent, also showed specific inhibition of IK. Furthermore, dofetilide depolarized the maximum diastolic potentials, reduced the slope of the pacemaker potential and then abolished spontaneously firing action potentials in the nodal cells. The results demonstrate that dofetilide may produce negative chronotropic effects as a result of its class III activity.

Voltage-dependence of Ca2+ agonist effect of YC-170 on cardiac L-type Ca2+ channels.

1. We investigated the voltage-dependence of the agonist actions of YC-170, a dihydropyridine (DHP) derivative, on cardiac L-type Ca2+ channels in rabbit ventricular cells, using the patch clamp technique. The characteristics of YC-170 were compared with those of other DHP Ca2+ agonists (Bay K 8644, CGP 28392). Ca2+ channel activities were elicited by depolarizing pulses to 0 mV from a holding potential (HP) of either -80 mV or -40 mV. 2. YC-170 (10 microM) increased Ca2+ channel activities when HP was set at -80 mV. However, decreasing HP to -40 mV abolished the agonist action. The agonist effect of Bay K 8644 (1 microM) on Ca2+ channels was elicited to the same extent at either HP. CGP 28392 (10 microM) also increased Ca2+ channel activities at both HPs, but its agonist effect was significantly larger at an HP of -80 mV than at -40 mV. 3. All of the three DHP Ca2+agonists prolonged open times of Ca2+ channels, but the prolongation did not correspond to the voltage-dependence of Ca2+ agonist effects of the three DHPs. 4. YC-170 markedly reduced the closed time of the Ca2+ channel when the HP was at -80 mV, but prolonged it at HP of -40 mV. Bay K 8644 reduced closed times at an HP of -80 mV. At an HP of -40 mV, Bay K 8644 slightly reduced closed times. CGP 28392 reduced closed times at an HP of -80 mV and prolonged those at an HP of -40 mV. Thus the voltage-dependence of the agonist effects of these agents was in good agreement with the voltage-dependence of changes in closed times of Ca2+ channel. 5. Two mechanisms may be involved in the agonist action of YC-170; a prolongation of open times, and a reduction of closed times of Ca2+ channels, i.e. an increase in reopening. The former mechanism is not dependent on Hp and the latter mechanism is highly dependent on HP. Thus, the voltage-dependence of the agonist action may be attributed to the voltage-dependence of their enhancing effect on reopening of Ca2+ channels.