Effects of potassium channel blockers on hyperinflation-induced rapidly adapting pulmonary stretch receptor stimulation in the rabbit.

The effects of K+ channel blockers, such as 4-aminoprydine (4-AP) and tetraethylammonium (TEA), on the excitatory responses of rapidly adapting pulmonary stretch receptor (RAR) activity to hyperinflation (inflation volume=3 tidal volumes) were investigated in anesthetized, artificially ventilated rabbits after vagus nerve section. The changes in the RAR adaptation index (AI) produced by constant-pressure (approximately 30 cmH2O, 29.7+/-0.2 cmH2O) inflation of the lungs were also examined before and after pretreatment with 4-AP and TEA. The administration of 4-AP (0.7 and 2.0 mg/kg) potentiated hyperinflation-induced RAR stimulation in a dose-dependent manner. During hyperinflation after 2.0 mg/kg 4-AP administration the discharge of RARs showed a relatively regular firing pattern in both inflation and deflation phases. The RAR AI values during constant-pressure inflation of the lungs were significantly reduced by 4-AP treatment (2.0 mg/kg). TEA treatment (2.0 and 7.0 mg/kg) did not significantly alter either the excitatory response of RAR activity to hyperinflation or the RAR AI values seen during constant-pressure inflation of the lungs. These results suggest that during hyperinflation in in vivo experiments on rabbits, RARs may be maintained at a lower activity by opening the 4-AP-sensitive K+ channels on the receptor endings, which can determine accommodation of the receptor discharge.     

The inhibitory effect of ouabain on the response of slowly adapting pulmonary stretch receptors to hyperinflation in the rabbit

The combined effects of ouabain (Na(+)-K(+) ATPase inhibitor) and hyperinflation (inflation volume=three tidal volumes) on slowly adapting pulmonary stretch receptors (SARs) were studied before and after administration of nifedipine (an L-type Ca(2+) channel blocker) and KB-R7943 (a reverse-mode Na(+)-Ca(2+) exchanger blocker) in anesthetized, artificially ventilated rabbits after bilateral vagotomy. Before ouabain administration, hyperinflation stimulated SAR activity. After 20 min of ouabain administration (30 microg/kg) the SARs increased discharge rates in normal inflation. Under these conditions, hyperinflation initially stimulated SAR activity but subsequently inhibited the activity at peak inflation. Additional administration of 60 microg/kg ouabain (total dose=90 microg/kg) caused a further stimulation of SAR activity, but 20 min later both normal inflation and hyperinflation resulted in a greater inhibition of the receptor activity. The hyperinflation-induced SAR inhibition in the presence of ouabain (30 microg/kg) was not significantly altered by administration of either nifedipine (2 and 4 mg/kg) or KB-R7943 (1 and 3 mg/kg). In another series of experiments, we further examined the combined effects of ouabain and hyperinflation in veratridine (a Na(+) channel opener, 40 microg/kg)-treated animals. After recovery from the veratridine effect on SAR activity, which vigorously stimulated the receptor activity, ouabain treatment (30 microg/kg) that silenced the receptor activity at peak inflation greatly inhibited hyperinflation-induced SAR stimulation. These results suggest that hyperinflation-induced SAR inhibition in the presence of ouabain may be related to a Na(+) overload, but not to a Ca(2+) influx via activation of L-type Ca(2+) channels, in the SAR endings.     

Effects of ouabain and flecainide on CO(2)-induced slowly adapting pulmonary stretch receptor inhibition in the rabbit

The inhibitory effect of CO2 on slowly adapting pulmonary stretch receptors (SARs) was examined before and after administration of ouabain, a Na+-K+ ATPase inhibitor, and flecainide, a Na+ channel blocker. The experiments were performed in anesthetized, artificially ventilated rabbits after vagus nerve section. CO2 inhalation (maximal tracheal CO2 concentration ranging from 9.2 % to 10.4%) for about 60 sec decreased the receptor activity during both inflation and deflation. The magnitude of decreased SAR activity during deflation was greater than that seen during inflation. Administration of ouabain (25 microg/kg) initially stimulated SAR activities during inflation and deflation, and after 20 min, the SAR response was still kept excitatory in both inflation and deflation phases. Under these conditions, CO2 inhalation inhibited SAR activities during inflation and deflation. Flecainide treatment (3 mg/kg) that abolished veratridine (30 microg/kg)-induced SAR excitation had no significant effect on the inhibitory responses of SAR activity to CO2. These results suggest that the inhibitory effect of CO2 occurs when ouabain results in intracellular Na+ concentration ([Na+]i) increases in the SAR endings, and that CO2-induced SAR inhibition may not be related to the reduction of influx of Na+ through voltage-gated Na+ channels.     

Inhibitory mechanism of slowly adapting pulmonary stretch receptors after release from hyperinflation in anesthetized rabbits

In anesthetized, artificially ventilated rabbits with vagus nerve section, release from 10 consecutive hyperinflations (inflation volume = 3 tidal volume) caused an inhibition of the slowly adapting pulmonary stretch receptor (SAR) activity for 16-22 sec. Intravenous administration of tetraethylammonium (TEA, 10 and 20 mg/kg), a K+ channel blocker, did not significantly alter either basal SAR discharge or tracheal pressure (PT). Although TEA treatment at 10.0 mg/kg had no significant effect on the magnitude and duration of inhibited SAR activity seen after release from hyperinflation, the increasing dose of this K+ channel blocker up to 20 mg/kg inhibited these effects of the receptor activity but this inhibition was small. The Na+ -K+ ATPase inhibitor ouabain (5 and 10 microg/kg) that had no significant effect on SAR activity and P(T) in the control abolished or attenuated the inhibitory action of SARs in a dose-dependent manner. Furthermore, the changes in dynamic lung compliance (Cdyn) and P(T) in response to post-hyperinflation were not significantly influenced by pretreatment with either TEA or ouabain. These results suggest that the inhibitory action of receptors seen during post-hyperinflation corresponded with the induction of slow afterhyperpolarization (sAHP), and that the mechanism of generating the sAHP of SARs is mainly mediated by the activation of Na+ -K+ pump activity.     

Effects of acetazolamide and 4-aminoprydine on the responses of deflationary slowly adapting pulmonary stretch receptors to CO2 inhalation in the rat

The inhibitory effect of CO(2) on deflationary slowly adapting pulmonary stretch receptors (deflationary SARs) was investigated before and after administration of acetazolamide, a carbonic anhydrase (CA) inhibitor, or 4-aminopyridine (4-AP), a K(+) channel blocker, in anesthetized, artificially ventilated rats after unilateral vagotomy. CO(2) inhalation (maximum tracheal CO(2) concentration ranging from 9 to 12%) for approximately 60 s decreased the impulse activity of deflationary SARs but had no significant effect on tracheal pressure (P(T)) as an index of bronchomotor tone. Acetazolamide treatment (20 mg/kg) diminished the inhibitory response of deflationary SARs to CO(2) inhalation. 4-AP (0.7 and 2.0 mg/kg) dose-dependently attenuated the decrease in deflationary SAR activity induced by CO(2) inhalation. When comparing the maximum attenuation due to 4-AP (2.0 mg/kg) and acetazolamide (20 mg/kg) in CO(2)-induced deflationary SAR inhibition, blockade of K(+) channels had a more pronounced effect. These results suggest that inhibition of deflationary SARs by CO(2) inhalation may be largely mediated by the stimulating action of 4-AP-sensitive K(+) currents in the nerve terminals of the receptors.     

哇巴因刺激肺慢适应感受器

钠钾泵抑制剂——哇巴因能引起气道内慢适应感受器异相发放,表现为冲动在正常时的吸气相发放,呼气相终止转变为在呼气相发放,吸气相终止。我们推测异相发放由过度兴奋所致,如果假设正确,那么降低气道压力从而减少对感受器刺激,将能防止异相发放。本工作在麻醉、开胸、机械通气（在呼气末附加3cm水柱的正压）的家兔中记录颈迷走神经中慢适应感受器的单位放电,向感受野注射微量哇巴因（1μmol／L,20μ1）,可观察到感受器活动发生变化。感受器放电经历紧张性发放、异相发放、以及不规则发放三个时期,随后放电终止,进入静息状态。在紧张期,感受器呈持续发放,冲动频率随肺部通气变化的波动幅度明显减小。在异相发放期,感受器活动出现突然发放（呼气相）与终止（吸气相）,其冲动快速转换于高频发放和静止之间。此时,若撤除呼气末正压而减少气道内压力,感受器活动恢复正常,即冲动频率于气管压峰值时为最高,在呼气相减少或终止。在不规则期,感受器通常处于静止状态,时而出现突发高频冲动,且与呼吸周期无关。可以设想：在吸气相,感受器受到牵拉,引起钠、钙等阳离子内流,产生感受器电位。正常时,由于激活钠泵,将钠离子泵出细胞,使感受器电位回复。当钠泵受到抑制后,钠外流受阻,感受器电位加大。在异相发放期,肺充气时牵拉感受器,进一步增加感受器电位,当它超越了产生动作电位的活动范围后,则感受器因过度去极化而失去兴奋性。     

Effects of acetazolamide and 4-aminopyridine on CO2-induced slowly adapting pulmonary stretch receptor inhibition in rats

Inhibitory responses of slowly adapting pulmonary stretch receptor (SAR) activity to CO(2) inhalation (maximal tracheal CO(2) concentration ranging from 9.5 to 12.5%) for approximately 60 s were examined before and after administration of acetazolamide (a carbonic anhydrase inhibitor) or 4-aminopyridine (4-AP, a K(+) channel blocker). The experiments were performed in 35 anesthetized, artificially ventilated rats after unilateral vagotomy. Sixty-eight of eighty-four SARs were inhibited by CO(2) inhalation. The SAR inhibition was attenuated by pretreatment with either acetazolamide (20 mg/kg, n = 10) or 4-AP (0.7 and 2.0 mg/kg, n = 10). In other series of experiments, stainings to show the existence of carbonic anhydrase (CA) enzymatic reaction were not found in the smooth muscle of either extrapulmonary or intrapulmonary bronchi. Protein gene product 9.5 (PGP 9.5)-immunoreactive SAR terminals to form leaflike extensions were found in the bronchioles at different diameters and were smooth-muscle-related receptors. But in the same sections, CA isozyme II-like (erythrocyte CA) immunoreactive SAR terminals were not identified. These results suggest that CO(2)-induced inhibition of SARs may be involved in the CA-dependent CO(2) hydration in addition to the activation of 4-AP sensitive K(+) currents.     

Common molecular determinants of flecainide and lidocaine block of heart Na+ channels: evidence from experiments with neutral and quaternary flecainide analogues

Flecainide (pKa 9.3, 99% charged at pH 7.4) and lidocaine (pKa 7.6-8.0, approximately 50% neutral at pH 7.4) have similar structures but markedly different effects on Na(+) channel activity. Both drugs cause well-characterized use-dependent block (UDB) of Na(+) channels due to stabilization of the inactivated state, but flecainide requires that channels first open before block develops, whereas lidocaine is believed to bind directly to the inactivated state. To test whether the charge on flecainide might determine its state specificity of Na(+) channel blockade, we developed two flecainide analogues, NU-FL (pKa 6.4), that is 90% neutral at pH 7.4, and a quaternary flecainide analogue, QX-FL, that is fully charged at physiological pH. We examined the effects of flecainide, NU-FL, QX-FL, and lidocaine on human cardiac Na(+) channels expressed in human embryonic kidney (HEK) 293 cells. At physiological pH, NU-FL, like lidocaine but not flecainide, interacts preferentially with inactivated channels without prerequisite channel opening, and causes minimal UDB. We find that UDB develops predominantly by the charged form of flecainide as evidenced by investigation of QX-FL at physiological pH and NU-FL investigated over a more acidic pH range where its charged fraction is increased. QX-FL is a potent blocker of channels when applied from inside the cell, but acts very weakly with external application. UDB by QX-FL, like flecainide, develops only after channels open. Once blocked, channels recover very slowly from QX-FL block, apparently without requisite channel opening. Our data strongly suggest that it is the difference in degree of ionization (pKa) between lidocaine and flecainide, rather than gross structural features, that determines distinction in block of cardiac Na(+) channels. The data also suggest that the two drugs share a common receptor but, consistent with the modulated receptor hypothesis, reach this receptor by distinct routes dictated by the degree of ionization of the drug molecules.     

Channel openings are necessary but not sufficient for use-dependent block of cardiac Na(+) channels by flecainide: evidence from the analysis of disease-linked mutations

Na(+) channel blockers such as flecainide have found renewed usefulness in the diagnosis and treatment of two clinical syndromes arising from inherited mutations in SCN5A, the gene encoding the alpha subunit of the cardiac voltage-gated Na(+) channel. The Brugada syndrome (BrS) and the LQT-3 variant of the Long QT syndrome are caused by disease-linked SCN5A mutations that act to change functional and pharmacological properties of the channel. Here we have explored a set of SCN5A mutations linked both to BrS and LQT-3 to determine what disease-modified channel properties underlie distinct responses to the Na(+) channel blocker flecainide. We focused on flecainide block that develops with repetitive channel activity, so-called use-dependent block (UDB). Our results indicate that mutation-induced changes in the voltage-dependence of channel availability (inactivation) may act as determinants of flecainide block. The data further indicate that UDB by flecainide requires channel opening, but is not likely due to open channel block. Rather, flecainide appears to interact with inactivation states that follow depolarization-induced channel opening, and mutation-induced changes in channel inactivation will alter flecainide block independent of the disease to which the mutation is linked. Analysis of flecainide block of mutant channels linked to these rare disorders has provided novel insight into the molecular determinants of drug action.     

Effect of the Anti-depressant Sertraline,the Novel Anti-seizure Drug Vinpocetine and Several Conventional Antiepileptic Drugs on the Epileptiform EEG Activity Induced by 4-Aminopyridine

Seizures are accompanied by an exacerbated activation of cerebral ion channels. 4-aminopyridine (4-AP) is a pro-convulsive agent which mechanism of action involves activation of Na⁺ and Ca²⁺ channels, and several antiepileptic drugs control seizures by reducing these channels permeability. The antidepressant, sertraline, and the anti-seizure drug vinpocetine are effective inhibitors of cerebral presynaptic Na⁺ channels. Here the effectiveness of these compounds to prevent the epileptiform EEG activity induced by 4-AP was compared with the effectiveness of seven conventional antiepileptic drugs. For this purpose, EEG recordings before and at three intervals within the next 30 min following 4-AP (2.5 mg/kg, i.p.) were taken in anesthetized animals; and the EEG-highest peak amplitude values (HPAV) calculated. In control animals, the marked increase in the EEG-HPAV observed near 20 min following 4-AP reached its maximum at 30 min. Results show that this epileptiform EEG activity induced by 4-AP is prevented by sertraline and vinpocetine at a dose of 2.5 mg/kg, and by carbamazepine, phenytoin, lamotrigine and oxcarbazepine at a higher dose (25 mg/kg). In contrast, topiramate (25 mg/kg), valproate (100 mg/kg) and levetiracetam (100 mg/kg) failed to prevent the epileptiform EEG activity induced by 4-AP. It is concluded that 4-AP is a useful tool to elicit the mechanism of action of anti-seizure drugs at clinical meaningful doses. The particular efficacy of sertraline and vinpocetine to prevent seizures induced by 4-AP is explained by their high effectiveness to reduce brain presynaptic Na⁺ and Ca²⁺ channels permeability.     

Vinpocetine blockade of sodium channels inhibits the rise in sodium and calcium induced by 4-aminopyridine in synaptosomes

The objective of this study was to get a more understandable picture of the mechanism underlying the anticonvulsant action of vinpocetine. The question of how the cerebral excitability is affected was investigated by determining the effect of vinpocetine on the changes on the internal concentrations of Na(+) (Na(i)) and Ca(2+) (Ca(i)) induced by different concentrations of the convulsing agent 4-aminopyridine (4-AP) in striatal isolated nerve endings. The cytosolic concentrations of Na(i) and Ca(i) were detected fluorimetrically with sodium-binding benzofuran isophthalate (SBFI) and fura-2, respectively. Vinpocetine, like the Na(+) channel blocker, tetrodotoxin, abolished the increase in Na(i) induced by 0.1 mM 4-AP and only inhibited in 30% the rise in Na(i) induced by 1mM 4-AP. In contrast with the different sensitivity of the rise in Na(i) induced by 0.1 and 1mM 4-AP to vinpocetine and tetrodotoxin, the rise in Ca(i) induced by the two concentrations of 4-AP was markedly inhibited by vinpocetine (and tetrodotoxin), indicating that only the voltage-sensitive sodium channels (VSSC)-mediated fraction of the rise in Na(i) induced by 4-AP is linked with the activation of pre-synaptic Ca(2+) channels. The elevation of Ca(2+) induced by high K(+) (30 mM) does not require a Na(+) gradient and is vinpocetine and tetrodotoxin insensitive. In contrast, the elevation of Ca(i) induced by 4-AP, requires a physiological (out/in) Na(+) gradient and is vinpocetine and tetrodotoxin-sensitive. It is concluded that by blocking the tetrodotoxin-sensitive fraction of the rise in Na(i) induced by 4-AP, vinpocetine inhibits the concomitant rise in Ca(i) induced by 4-AP. The inhibitory effect of vinpocetine on pre-synaptic voltage-sensitive sodium channels may underlie the in vivo anticonvulsant action of vinpocetine.     

Voltage-dependent K+ channels regulate the duration of reactive hyperemia in the canine coronary circulation

We previously demonstrated a role for voltage-dependent K(+) (K(V)) channels in coronary vasodilation elicited by myocardial metabolism and exogenous H(2)O(2), as responses were attenuated by the K(V) channel blocker 4-aminopyridine (4-AP). Here we tested the hypothesis that K(V) channels participate in coronary reactive hyperemia and examined the role of K(V) channels in responses to nitric oxide (NO) and adenosine, two putative mediators. Reactive hyperemia (30-s occlusion) was measured in open-chest dogs before and during 4-AP treatment [intracoronary (ic), plasma concentration 0.3 mM]. 4-AP reduced baseline flow 34 +/- 5% and inhibited hyperemic volume 32 +/- 5%. Administration of 8-phenyltheophylline (8-PT; 0.3 mM ic or 5 mg/kg iv) or N(G)-nitro-L-arginine methyl ester (L-NAME; 1 mg/min ic) inhibited early and late portions of hyperemic flow, supporting roles for adenosine and NO. 4-AP further inhibited hyperemia in the presence of 8-PT or L-NAME. Adenosine-induced blood flow responses were attenuated by 4-AP (52 +/- 6% block at 9 microg/min). Dilation of arterioles to adenosine was attenuated by 0.3 mM 4-AP and 1 microM correolide, a selective K(V)1 antagonist (76 +/- 7% and 47 +/- 2% block, respectively, at 1 microM). Dilation in response to sodium nitroprusside, an NO donor, was attenuated by 4-AP in vivo (41 +/- 6% block at 10 microg/min) and by correolide in vitro (29 +/- 4% block at 1 microM). K(V) current in smooth muscle cells was inhibited by 4-AP (IC(50) 1.1 +/- 0.1 mM) and virtually eliminated by correolide. Expression of mRNA for K(V)1 family members was detected in coronary arteries. Our data indicate that K(V) channels play an important role in regulating resting coronary blood flow, determining duration of reactive hyperemia, and mediating adenosine- and NO-induced vasodilation.     

Role of pertussis toxin-sensitive G-protein, K+ channels, and voltage-gated Ca2+ channels in the antinociceptive effect of inosine

Inosine is the first metabolite of adenosine. It exerts an antinociceptive effect by activating the adenosine A₁ and A_2A receptors. We have previously demonstrated that inosine exhibits antinociceptive properties in acute and chronic mice models of nociception. The aim of this study was to investigate the involvement of pertussis toxin-sensitive G-protein-coupled receptors, as well as K⁺ and Ca²⁺ channels, in the antinociception promoted by inosine in the formalin test. Mice were pretreated with pertussis toxin (2.5 μg/site, i.t., an inactivator of G_i/0 protein); after 7 days, they received inosine (10 mg/kg, i.p.) or morphine (2.5 mg/kg, s.c., used as positive control) immediately before the formalin test. Another group of animals received tetraethylammonium (TEA) or 4-aminopyridine (4-AP) (1 μg/site, i.t., a non-specific voltage-gated K⁺ channel blockers), apamin (50 ng/site, i.t., a small conductance Ca²⁺-activated K⁺ channel blocker), charybdotoxin (250 pg/site, i.t., a large-conductance Ca²⁺-activated K⁺ channel blocker), glibenclamide (100 μg/site, i.t., an ATP-sensitive K⁺ channel blocker) or CaCl₂ (200 nmol/site, i.t.). Afterwards, the mice received inosine (10 mg/kg, i.p.), diclofenac (10 mg/kg, i.p., a positive control), or morphine (2.5 mg/kg, s.c., a positive control) immediately before the formalin test. The antinociceptive effect of inosine was reversed by the pre-administration of pertussis toxin (2.5 μg/site, i.t.), TEA, 4-aminopyridine, charybdotoxin, glibenclamide, and CaCl₂, but not apamin. Further, all K⁺ channel blockers and CaCl₂ reversed the antinociception induced by diclofenac and morphine, respectively. Taken together, these data suggest that the antinociceptive effect of inosine is mediated, in part, by pertussis toxin-sensitive G-protein coupled receptors and the subsequent activation of voltage gated K⁺ channel, large conductance Ca²⁺-activated and ATP-sensitive K⁺ channels or inactivation of voltage-gated Ca²⁺ channels. Finally, small conductance Ca²⁺-activated K⁺ channels are not involved in the antinociceptive effect of inosine.     

Inhibition of Neuronal Degenerin/Epithelial Na+ Channels by the Multiple Sclerosis Drug 4-Aminopyridine

The voltage-gated K⁺ (Kv) channel blocker 4-aminopyridine (4-AP) is used to target symptoms of the neuroinflammatory disease multiple sclerosis (MS). By blocking Kv channels, 4-AP facilitates action potential conduction and neurotransmitter release in presynaptic neurons, lessening the effects of demyelination. Because they conduct inward Na⁺ and Ca²⁺ currents that contribute to axonal degeneration in response to inflammatory conditions, acid-sensing ion channels (ASICs) contribute to the pathology of MS. Consequently, ASICs are emerging as disease-modifying targets in MS. Surprisingly, as first demonstrated here, 4-AP inhibits neuronal degenerin/epithelial Na⁺ (Deg/ENaC) channels, including ASIC and BLINaC. This effect is specific for 4-AP compared with its heterocyclic base, pyridine, and the related derivative, 4-methylpyridine; and akin to the actions of 4-AP on the structurally unrelated Kv channels, dose- and voltage-dependent. 4-AP has differential actions on distinct ASICs, strongly inhibiting ASIC1a channels expressed in central neurons but being without effect on ASIC3, which is enriched in peripheral sensory neurons. The voltage dependence of the 4-AP block and the single binding site for this inhibitor are consistent with 4-AP binding in the pore of Deg/ENaC channels as it does Kv channels, suggesting a similar mechanism of inhibition in these two classes of channels. These findings argue that effects on both Kv and Deg/ENaC channels should be considered when evaluating the actions of 4-AP. Importantly, the current results are consistent with 4-AP influencing the symptoms of MS as well as the course of the disease because of inhibitory actions on Kv and ASIC channels, respectively.     

Is the sarcolemmal or mitochondrial KATP channel activation important in the antiarrhythmic and cardioprotective effects during acute ischemia/reperfusion in the intact anesthetized rabbit model?

The relative contributions of cardiomyocyte sarcolemmal ATP-sensitive K(+) (K(ATP)) and mitochondrial K(ATP) channels in the cardioprotection and antiarrhythmic activity induced by K(ATP) channel openers remain obscure, though the mitochondrial K(ATP) channels have been proposed to be involved as a subcellular mediator in cardioprotection afforded by ischemic preconditioning. In the present study, we sought to investigate the effects of administration of ATP-sensitive K(+) channel (K(ATP)) openers (nicorandil and minoxidil), a specific mitochondrial K(ATP) channel blocker (5-hydroxydecanoate (5-HD)) and a specific sarcolemmal K(ATP) channel blocker (HMR 1883; (1-[5-[2-(5-chloro-o-anisamido)ethyl]-2-methoxyphenyl]sulfonyl-3-methylthiourea) prior to coronary occlusion as well as prior to post-ischemic reperfusion on survival rate, ischemia-induced and reperfusion-induced arrhythmias and myocardial infarct size in anesthetized albino rabbits. The thorax was opened in the left 4th intercostal space and after pericardiotomy the heart was exposed. In Group I (n=88), occlusion of the left main coronary artery and hence, myocardial ischemia-induced arrhythmias was achieved by tightening a previously placed loose silk ligature for 30 min. In Group II (n=206), arrhythmias were induced by reperfusion following a 20-min ligation of the left main coronary artery. Both in Group I and Group II, intravenous (i.v.) administration of nicorandil (0.47 mg/kg), minoxidil (0.5 mg/kg), HMR 1883 (3 mg/kg)/nicorandil and HMR 1883 (3 mg/kg)/minoxidil before coronary artery occlusion increased survival rate (86%, 75%, 75% and 86% vs. 55% in the control subgroup in Group I; 75%, 67%, 67% and 75% vs. 46% in the control subgroup in Group II), significantly decreased the incidence and severity of life-threatening arrhythmias. In Group II, i.v. administration of nicorandil and minoxidil before coronary artery occlusion significantly decreased myocardial infarct size. However, i.v. administration of nicorandil or minoxidil before reperfusion did neither increase survival rate nor confer any antiarrhythmic or cardioprotective effects. The antiarrhythmic and cardioprotective effects of both nicorandil and minoxidil were abolished by pretreating the rabbits with 5-HD (5 mg/kg, i.v. bolus), a selective mitochondrial K(ATP) channel blocker but not by HMR 1883 (3 mg/kg). In the present study, higher levels of malondialdehyde (MDA) and lower levels of reduced glutathione (GSH) and superoxide dismutase (SOD) in necrotic zone of myocardium in all the 16 subgroups in Group II suggest little anti-free radical property of nicorandil and minoxidil. We conclude that intervention by intravenous administration of nicorandil and minoxidil (through the selective activation of mitochondrial K(ATP) channels) increased survival rate and exhibited antiarrhythmic and cardioprotective effects during coronary occlusion and reperfusion in anesthetized rabbits when administered prior to coronary occlusion. The cardiomyocyte mitochondrial K(ATP) channel may be a pharmacologically modulable target of cardioprotection and antiarrhythmic activity.     

Characterization of the Participation of Sodium Channels on the Rise in Na+ Induced by 4-Aminopyridine (4-AP) in Synaptosomes

The participation of voltage-sensitive Na+ channels (VSSC) on the changes on internal (i) Na+, K+, Ca2+, and on DA, Glu, and GABA release caused by different concentrations of 4-AP was investigated in striatum synaptosomes. TTX, which abolished the increase in Na(i) (as determined with SBFI), induced by 0.1 mM 4-AP only inhibited by 30% the rise in Na(i) induced by 1 mM 4-AP. One millimolar 4-AP markedly decreased the fluorescence of the K+ indicator dye PBFI but 0.1 mM 4-AP did not. Like 1 mM 4-AP, ouabain decreased PBFI fluorescence and increased a considerable fraction of Na(i) in a TTX-insensitive manner. In contrast with the different TTX sensitivity of the rise in Na(i) induced by 0.1 and 1 mM 4-AP, the rise in Ca(i) (as determined with fura-2) induced by the two concentrations of 4-AP was markedly inhibited by TTX, as well as by omega-agatoxin in combination with omega-conotoxin GVIA, indicating that only the TTX-sensitive fraction of the rise in Na(i) induced by 4-AP is linked with the activation of presynaptic Ca2+ channels. It is concluded that the TTX-sensitive fraction of neurotransmitter release evoked by 4-AP is released by exocytosis, and the TTX insensitive fraction involves reversal of the neurotransmitters transporters. This contrasts with the exocytosis evoked by high K+ that is unchanged by TTX and with the neurotransmitter-transporter-mediated release evoked by veratridine, which is highly TTX sensitive and does not require activation of Ca2+ channels.     

State-dependent block of wild-type and inactivation-deficient Na+ channels by flecainide

The antiarrhythmic agent flecainide appears beneficial for painful congenital myotonia and LQT-3/DeltaKPQ syndrome. Both diseases manifest small but persistent late Na+ currents in skeletal or cardiac myocytes. Flecainide may therefore block late Na+ currents for its efficacy. To investigate this possibility, we characterized state-dependent block of flecainide in wild-type and inactivation-deficient rNav1.4 muscle Na+ channels (L435W/L437C/A438W) expressed with beta1 subunits in Hek293t cells. The flecainide-resting block at -140 mV was weak for wild-type Na+ channels, with an estimated 50% inhibitory concentration (IC50) of 365 micro M when the cell was not stimulated for 1,000 s. At 100 micro M flecainide, brief monitoring pulses of +30 mV applied at frequencies as low as 1 per 60 s, however, produced an approximately 70% use-dependent block of peak Na+ currents. Recovery from this use-dependent block followed an exponential function, with a time constant over 225 s at -140 mV. Inactivated wild-type Na+ channels interacted with flecainide also slowly at -50 mV, with a time constant of 7.9 s. In contrast, flecainide blocked the open state of inactivation-deficient Na+ channels potently as revealed by its rapid time-dependent block of late Na+ currents. The IC50 for flecainide open-channel block at +30 mV was 0.61 micro M, right within the therapeutic plasma concentration range; on-rate and off-rate constants were 14.9 micro M-1s-1 and 12.2 s-1, respectively. Upon repolarization to -140 mV, flecainide block of inactivation-deficient Na+ channels recovered, with a time constant of 11.2 s, which was approximately 20-fold faster than that of wild-type counterparts. We conclude that flecainide directly blocks persistent late Na+ currents with a high affinity. The fast-inactivation gate, probably via its S6 docking site, may further stabilize the flecainide-receptor complex in wild-type Na+ channels.     

Butyl 2-(4-[1.1'-biphenyl]-4-yl-1H-imidazol-2-yl)ethylcarbamate, a potent sodium channel blocker for the treatment of neuropathic pain

A series of 4-arylimidazole carbamates was synthesized and their binding affinities to the site-2 sodium (Na+) channel were determined. SAR studies led to the identification of compound 10, a potent Na+ channel blocker which was efficacious in pain models in vivo.     

Redox properties of local anesthetics: A structural determinant of closed channel blockers in BTX-modified Na+ channels

Single channel analyses and macroscopic current measurements have shown that benzocaine is a predominantly closed channel blocker in BTX-modified Na+ channels; cocaine is an open channel blocker; and tetracaine, a dual channel blocker (Wang & Wang, 1994; Wang et al., 1994). The reason for such a selective state-dependent block by local anesthetics in BTX-modified Na+ channels is not clear. We assessed the redox properties of tetracaine, benzocaine, cocaine, and various derivatives by their ability to donate electrons to radical intermediates of eosin dye excited by visible light. Electron-donor properties of the drugs were previously proposed to be involved in Na+ channel blockade (Marinov, 1991). Our results provide evidence that redox properties of tetracaine, benzocaine, and their homologs correlate with their ability to enhance Na+ channel inactivation in BTX-modified Na+ channels. This correlation may be explained in terms of the previously proposed redox model of ion channels.     

Involvement of Na+ and Ca2+ channel activation and resultant nitric oxide synthesis in glutamate-mediated hypoxic injury in rat cerebrocortical slices

The role of Na+ and Ca2+ channels in glutamate-mediated hypoxic injury was investigated in slices of the rat cerebral cortex. Hypoxic injury was determined by mitochondrial reduction of 3-(4,5-dimethyl-2-thiazol)-2,5-diphenyltetrazolium bromide after exposure of brain slices to 30-min of hypoxia/glucose deprivation followed by 3-h of reoxygenation. Endogenous glutamate release was markedly elevated during hypoxia/glucose deprivation, but it returned almost to basal level during reoxygenation. Hypoxic injury was prevented by MK-801 or 6-cyano-7-nitroquinoxaline-2,3-dione. Combined treatment with omega-conotoxin GVIA, omega-agatoxin IVA, and tetrodotoxin reversed the hypoxic injury, although none of these agents alone or nifedipine was effective. Moreover, a novel Na+/Ca2+ channel blocker NS-7 [4-(4-fluorophenyl)-2-methyl-6-(5-piperidinopentyloxy) pyrimidine hydrochloride] significantly inhibited the hypoxic injury. Several inhibitors of nitric oxide synthase also blocked the hypoxic injury. Consistently, nitric oxide synthesis, as estimated from cyclic GMP formation in the extracellular fluids, was enhanced during hypoxia/glucose deprivation. NS-7 and other Na+ and Ca2+ channel blockers suppressed the enhancement of nitric oxide synthesis, although these compounds alone, or in combination, did not reduce hypoxic glutamate release. These findings suggest that hypoxic injury in rat cerebrocortical slices is triggered by glutamate and subsequent enhancement of nitric oxide synthesis through activation of both Na+ and Ca2+ channels. Thus, the simultaneous blockade of both Na+ channel as well as N-type and P/Q-type Ca2+ channels is required to sufficiently reverse the hypoxic injury.