Effects of potassium channel blockers on CO2-induced slowly adapting pulmonary stretch receptor inhibition.

In anesthetized, artificially ventilated rabbits with vagus nerve section, inhalation of CO(2) gas mixtures (tracheal CO(2) concentration ranging from 8.0 to 10.2%) for 60 s decreased slowly adapting pulmonary stretch receptor (SAR) activity during both inflation and deflation. The magnitude of decreased receptor activity during deflation had a more pronounced effect than that seen during inflation. CO(2) inhalation did not cause any significant change in tracheal pressure (P(T)) as an index of bronchomotor tone. Intravenous administration of 4-aminopyridine (0. 7 and 2.0 mg/kg i.v.), a K(+) channel blocker, which dose-dependently increased SAR activity during deflation and had no effect on P(T), abolished or attenuated the decrease in SAR activities induced by CO(2) inhalation in a dose-dependent manner. The K(+) channel blocker tetraethylammonium (2.0 and 6.0 mg/kg i.v.) that did not significantly alter either basal SAR discharge or P(T) had no effect on the inhibitory responses of receptor activity to CO(2) inhalation. These results suggest that the inhibitory mechanism of CO(2) inhalation on SARs may be involved in the activation of 4-aminopyridine-sensitive K(+) channels in the nerve terminals of SARs.     

Nitric oxide from enteric nerves acts by a different mechanism from myogenic nitric oxide in canine lower esophageal sphincter

In canine lower esophageal sphincter, myogenic constitutive nitric-oxide (NO) synthase (NOS) in plasma membrane limits tone by opening large conductance Ca(2+)-dependent K(+) channels (BK(Ca) channels) and hyperpolarizing the membrane. We examined whether K(V) channels were involved and whether NO from enteric nerves and from NO donors used the same mechanisms. With nerves inactive, 100 nM iberiotoxin, like N-nitro-L-arginine (L-NOARG), increased tone but less. 4-Aminopyridine (4-AP) at 5 mM behaved similarly. Tetraethyl ammonium (TEA) at 20 mM equaled the effect of L-NOARG and occluded any tone increase from any combination of these agents. More than iberiotoxin or 4-AP, TEA decreased relaxations in response to sodium nitroprusside (SNP) or 3-morpholino-sydnonimine (Sin-1) by approximately 50%. In whole-cell patch-clamp recordings, TEA and 4-AP reduced outward K(+) currents additively by >90% at depolarization of +90 mV. Thus, K(+) channels in addition to BK(Ca) channels are opened by myogenic NO, and exogenous NO had relaxing effects both related and unrelated to K(+) channel openings. TEA (20 mM) increased tone but did not inhibit relaxations to electrical field stimulation (EFS) of enteric nerves. 4-AP relaxed tone, an effect that was abolished and reversed by L-NOARG. 4-AP apparently released NO and acetylcholine from nerves. The putative Cl(-) channel blocker niflumic acid (NFA; 30-100 microM) dose dependently reduced tone, but tone, restored by 10(-6) M carbachol or 20 mM TEA, was still relaxed by EFS and by SNP. 4,4'-Diisothiocyanatostilbene-2, 2'-disulfonic acid (DIDS) at 500 to 1000 microM did not inhibit relaxation to EFS or SNP. The addition of TEA (20 mM) to DIDS (1000 microM) induced tonic and phasic activity and markedly inhibited relaxations to EFS. DIDS plus TEA reduced the relaxations to SNP like TEA alone. Reduction in extracellular ?Cl(-) by isethionate substitution reduced tone but did not reduce relaxations when tone was restored. The combination of reduced extracellular ?Cl(-) and TEA did not abolish relaxation to EFS until DIDS was added. Thus, multiple K(+) channels are opened by myogenic NO, and openings of these channels, as well as DIDS-sensitive, undefined mechanisms, are induced when NO is released from nerves or SNP.     

Block of Na+,K+-ATPase and induction of hybrid death by 4-aminopyridine in cultured cortical neurons

K(+) channel blockers such as 4-aminopyridine (4-AP) can be toxic to neurons; the cellular mechanism underlying the toxicity, however, is obscure. In cultured mouse cortical neurons, we tested the hypothesis that the toxic effect of 4-AP might result from inhibiting the Na(+),K(+)-ATPase (Na(+),K(+)-pump) and thereafter induction of a hybrid death of concomitant apoptosis and necrosis. The Na(+),K(+)-pump activity, monitored as whole-cell membrane currents, was markedly blocked by 4-AP in concentration- and voltage-dependent manners in low millimolar ranges. At similar concentrations, 4-AP induced a neuronal death sensitive to attenuation by the caspase inhibitor Z-VAD-FMK (Z-Val-Ala-Asp(OMe)-fluoromethyl ketone) or Ca(2+) chelator BAPTA-AM (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester). Electron microscopy confirmed hybrid ultrastructural features of coexisting apoptotic and necrotic components in same cells. We suggest that 4-AP is a potent antagonist of the Na(+),K(+)-ATPase and an inducer of the hybrid death of central neurons.     

Frequency-independent blockade of cardiac Na+ channels by riluzole: comparison with established anticonvulsants and class I anti-arrhythmics

The Na+ channel blocking activity and the antiarrhythmic effects of riluzole, and established anticonvulsants (lamotrigine and lifarizine) and class I antiarrhythmics (lidocaine, flecainide and disopyramide) were studied under in vitro and in vivo conditions. Guinea-pig cardiac Purkinje fibres were superfused with Tyrode solution and electrically driven for recording action potentials with intracellular microelectrodes. In these preparations paced at 1 Hz, all compounds tested produced concentration-dependent (0.3-100 microM) reductions in the maximum rate of depolarization of the action potential (Vmax). For riluzole, phenytoin and carbamazepine this effect was frequency-independent (0.5-6 Hz) but for lamotrigine, lifarizine, lidocaine, flecainide and disopyramide it was frequency-dependent. In anaesthetized rats, riluzole, in contrast to flecainide, did not delay the appearance of aconitine-induced arrhythmias. Riluzole (0.3-3.9 mg/kg, i.v.) also lacked notable cardiac electrophysiological effects in anaesthetized dogs. At an i.v. dose of 3.0 mg/kg riluzole failed to restore a normal sinus rhythm in conscious dogs with polymorphic arrhythmias produced by ligation of the left anterior descending coronary artery 24 h earlier. These results indicate that riluzole, phenytoin and carbamazepine, unlike lamotrigine, lifarizine and flecainide, block cardiac Na+ channels in a frequency-independent manner. This property may account for the lack of antiarrhythmic activity of riluzole, phenytoin and carbamazepine in animal models of arrhythmias that respond to class I antiarrhythmic drugs. It may also account for the clinical observation that riluzole does not seem to cause the unfavourable electrocardiographic changes characteristic of drugs that block cardiac Na+ channels in a frequency-dependent manner.     

Effects of K+-channel blockers on transmitter release in bullfrog sympathetic ganglia

Effects of K+-channel blockers, tetraethylammonium (TEA), 4-aminopyridine (4-AP) and Cs+ on synaptic transmission were studied with an intracellular electrode in bullfrog sympathetic ganglia. TEA (25-500 microM), 4-AP (0.6-5 microM) and Cs+ (50 microM-10 mM) all increased the quantal content of the fast excitatory postsynaptic potential in a dose-dependent manner. The effects of TEA were rapid in onset and recovery, whereas those of 4-AP and Cs+ appeared with a notable delay and reversed slowly. All blockers lengthened synaptic delay. When compared at approximately equipotent concentrations for potentiation of transmitter release, TEA and 4-AP were found to produce a similar lengthening of the synaptic delay whereas Cs+ caused a much greater prolongation. The quantal size of the fast excitatory postsynaptic potential and the amplitude of the acetylcholine potential were not affected by 4-AP or Cs+, but were depressed by TEA. These results indicate that TEA, 4-AP and Cs+ enhance evoked transmitter release in bullfrog sympathetic ganglia. It is suggested that the potentiation is caused, at least in part, by a mechanism that increases Ca++ influx in the nerve terminal. The enhanced influx is presumed to be mediated by a broadening of the presynaptic action potential after K+-channel blockade.     

Anti-allodynic effect of NW-1029, a novel Na(+) channel blocker,in experimental animal models of inflammatory and neuropathic pain

NW-1029, a benzylamino propanamide derivative, was selected among several molecules of this chemical class on the basis of its affinity for the [(3)H]batracotoxin ligand displacement of the Na(+) channel complex and also on the basis of its voltage and use-dependent inhibitory action on the Na(+) currents of the rat DRG (dorsal root ganglia) sensory neuron. This study evaluated the analgesic activity of NW-1029 in animal models of inflammatory and neuropathic pain (formalin test in mice, complete Freund's adjuvant and chronic constriction injury in rats) as well as in acute pain test (hot-plate and tail-flick in rats). Orally administered NW-1029 dose-dependently reduced cumulative licking time in the early and late phase of the formalin test (ED(50)=10.1 mg/kg in the late phase). In the CFA model, NW-1029 reversed mechanical allodynia (von Frey test) after both i.p. and p.o. administration (ED(50)=0.57 and 0.53 mg/kg), respectively. Similarly, NW-1029 reversed mechanical allodynia in the CCI model after both i.p. and p.o. administration yielding an ED(50) of 0.89 and 0.67 mg/kg, respectively. No effects were observed in the hot-plate and tail-flick tests up to 30 mg/kg p.o. The compound orally administered (0.1-10 mg/kg) was well tolerated, without signs of neurological impairment up to high doses (ED(50)=470 and 245 mg/kg in rat and mice Rotarod test, respectively). These results indicate that NW-1029 has anti-nociceptive properties in models of inflammatory and neuropathic pain.     

Effects of 4-aminopyridine and tetraethylammonium chloride on the electrical activity and cable properties of canine tracheal smooth muscle

The actions of 4-aminopyridine (4-AP) on passive membrane properties of canine tracheal smooth muscle were compared to those of tetraethylammonium chloride (TEA). Transmembrane potential was studied with intracellular electrodes in a partitioned bath by extracellular current injection. The cells were electrically quiescent and showed rectification to depolarizing pulses. TEA (30 mM) depolarized the muscle and increased membrane resistance, time constant (282-421 msec), space constant (2.16-2.67 mm), decreased rectification and generated action potentials. D600, with TEA, abolished the spikes but further increased the membrane resistance, time constant (690 msec) and space constant (3.79 mm). TEA apparently blocked potassium conductance and initiated Ca++-dependent spikes owing to depolarization. 4-AP (5 mM) depolarized the membrane, but caused slow oscillations, decreased membrane resistance and space constant (1.94 mm), without affecting the time constant (265 msec) or rectification. These membrane effects of 4-AP were reversed to a large extent by atropine (10(-7) to 10(-6) M). D600 blocked the 4-AP-induced oscillations but not the decreased membrane resistance or space constant. 4-AP produced characteristic electrical and mechanical responses in a sucrose gap chamber when the muscle was functionally denervated by tetrodotoxin or scorpion venom. 4-AP appeared to activate the muscarinic receptor of canine trachealis to increase conductance, possibly to Na+ and K+. An effect on potassium conductance blockade might have been masked by the muscarinic receptor gated conductance changes, but is unlikely as it was not evident after exposure to atropine. We conclude that 4-AP actions in canine trachealis reflect chiefly activation of muscarinic receptors and not interference with potassium conductance.     

Effects of K+-channel blockers on A1-adenosine receptor-mediated negative inotropy and chronotropy of guinea-pig isolated left and right atria

Adenosine has previously been shown to stimulate K(+)-efflux and to block L-type calcium channels in atrial myocytes. The aim of the present study was to evaluate the contribution of K(+)-channels in the development of the negative inotropic and chronotropic responses to adenosine agonists in guinea-pig left and right atria, respectively. Tetraethylammonium (TEA) potentiated the negative inotropic and chronotropic responses to R-(-)-N6-(2-phenyl-isopropyl)-adenosine (R-PIA), seen as leftward shifts of the concentration-response curves. Glibenclamide had no effect on the negative inotropic response to R-PIA but increased the rate of onset of the negative chronotropic response in right atria. 4-Aminopyridine (4-AP, 10 mM), potentiated the left atrial inotropic responses to R-PIA, seen as a leftward shift of the concentration-response curve, but slowed the speed of onset of the response to a single concentration (10(-6) M) of R-PIA. This reduction in speed of onset of the response can explain the differences in effects of 4-AP on concentration-response curves reported here and previously. In the right atria, 4-AP (10 mM) inhibited the negative chronotropic responses to R-PIA, seen as a rightward shift of the concentration-response curve and reduction of the maximum response. 4-AP also slowed the onset of the right atrial rate response to R-PIA. These results support the theory that K(+)-efflux plays only a minor role in the negative inotropic responses of guinea-pig left atria to R-PIA. This apparently controls the speed of onset of the response. The negative chronotropic response of guinea-pig right atria to R-PIA appears to be much more dependent upon K(+)-efflux than for the negative inotropic response of the left atria.     

Pharmacology of 2-[4-(4-chloro-2-fluorophenoxy)phenyl]-pyrimidine-4-carboxamide: a potent, broad-spectrum state-dependent sodium channel blocker for treating pain states

Voltage-gated Na(+) channels may play important roles in establishing pathological neuronal hyperexcitability associated with chronic pain in humans. Na(+) channel blockers, such as carbamazepine (CBZ) and lamotrigine (LTG), are efficacious in treating neuropathic pain; however, their therapeutic utility is compromised by central nervous system side effects. We reasoned that it may be possible to gain superior control over pain states and, in particular, a better therapeutic index, by designing broad-spectrum Na(+) channel blockers with higher potency, faster onset kinetics, and greater levels of state dependence than existing drugs. 2-[4-(4-Chloro-2-fluorophenoxy)phenyl]-pyrimidine-4-carboxamide (PPPA) is a novel structural analog of the state-dependent Na(+) channel blocker V102862 [4-(4-fluorophenoxy)benzaldehyde semicarbazone]. Tested on recombinant rat Na(v)1.2 channels and native Na(+) currents in cultured rat dorsal root ganglion neurons, PPPA was approximately 1000 times more potent, had 2000-fold faster binding kinetics, and > or =10-fold higher levels of state dependence than CBZ and LTG. Tested in rat pain models against mechanical endpoints, PPPA had minimal effective doses of 1 to 3 mg/kg p.o. in partial sciatic nerve ligation, Freund's complete adjuvant, and postincisional pain. In all cases, efficacy was similar to clinically relevant comparators. Importantly, PPPA did not produce motor deficits in the accelerating Rotarod assay of ataxia at doses up to 30 mg/kg p.o., indicating a therapeutic index >10, which was superior to CBZ and LTG. Our experiments suggest that high-potency, broad-spectrum, state-dependent Na(+) channel blockers will have clinical utility for treating neuropathic, inflammatory, and postsurgical pain. Optimizing the biophysical parameters of broad-spectrum voltage-gated Na(+) channel blockers may lead to improved pain therapeutics.     

Neutralization of α2-antiplasmin by microplasmin: A randomized,double-blind,placebo-controlled,ascending-dose study in healthy male volunteers

Background: Microplasmin is the isolated proteinase domain of plasmin. Administration of microplasmin has been found to neutralize α₂-antiplasmin (α₂-AP) activity, which has been associated with reduced infarct size in various preclinical models of stroke.Objectives: The goals of this first-in-man study were to investigate the ability of microplasmin to neutralize α₂-AP activity and to monitor its tolerability in healthy male volunteers.Methods: This Phase I, double-blind, placebo-controlled, ascending-dose study included 10 groups, each containing 6 subjects who were randomized to receive microplasmin or placebo in a 2:1 ratio. The study had 3 parts. In part 1, subjects received a single intravenous bolus of microplasmin 0.1, 0.5, 1, 1.5, or 2 mg/kg or placebo over 15 minutes. In part 2, subjects received a bolus of microplasmin 1 mg/kg or placebo, followed by an infusion of 1, 2, 3, or 4 mg/kg or placebo over 60 minutes. In part 3, subjects, all of whom were aged >55 years, received a bolus of mi-croplasmin 1 mg/kg or placebo, followed by an infusion of 1 mg/kg or placebo. The primary pharmaco-dynamic end point was the change in α₂-AP activity, measured at different time points up to 4 days after dosing using a chromogenic assay. All adverse events were monitored based on spontaneous reports and nondirected questioning at study visits up to the post-study visit 21 days after administration of study drug.Results: The mean (SD) age of subjects in parts 1, 2, and 3 was 30 (8), 30 (8), and 64 (8) years, respectively. All groups receiving microplasmin had a dose-dependent decrease in α₂-AP activity. In part 1, the mean maximal inhibition of α₂-AP was 11.8% (6.0%), 27.7% (4.3%), 53.0% (4.8%), 65.3% (4.3%), and 84.0% (6.0%) after bolus administration of microplasmin 0.1, 0.5, 1, 1.5, and 2 mg/kg, respectively, and 7.4% (6.9%) after administration of placebo. In part 2, the mean maximal inhibition of α₂-AP was 74.6% (11.2%), 95.5% (3.3%), 99.0% (1.0%), and 88.0% (12.5%) after bolus administration of microplasmin 1 mg/kg followed by an infusion of 1, 2, 3, and 4 mg/kg, respectively, compared with 12.9% (6.8%) after administration of placebo. In part 3, the mean maximal inhibition was 69.7% (3.4%) after bolus administration of microplasmin 1 mg/kg followed by an infusion of 1 mg/kg, compared with 8.8% (4.1%) with placebo. One subject in the highest dose group in part 1 (2 mg/kg) and 2 subjects in the highest dose group in part 2 (1 + 4 mg/kg) had an urticarial reaction. All 3 subjects also had a decrease in total hemolytic complement and an increase in complement 5a.Conclusions: Neutralization of α₂-AP activity by microplasmin was feasible in these healthy male volunteers. The urticarial reactions observed in the highest dose groups were considered dose-limiting adverse events. Further trials are needed to investigate the tolerability of this therapy and whether it is neuroprotective in patients with acute ischemic stroke.     

PD 122860: a novel dihydropyridine with sodium channel stimulating and calcium channel blocking properties

The cardiac and vascular activities of ethyl 5-cyano-1,4-dihydro-6-methyl-2-[(4-pyridinyl-sulfonyl)methyl]-4-[2- (trifluoromethyl)phenyl]-3-pyridine carboxylase (PD 122860), a novel dihydropyridine, were investigated in vitro using rat heart and rabbit aorta, and compared with reference inotropic and vasodilator agents. In the rat heart, PD 122860 increased left ventricular contractility, decreased coronary resistance and altered the shape of the electrocardiogram T-wave. All three effects were observed at comparable concentrations of PD 122860. The inotropic response to PD 122860 was reversed by the Na+ channel blocker tetrodotoxin and blunted by the Na+-Ca++ exchange inhibitor dichlorobenzamil. The effects of tetrodotoxin and dichlorobenzamil on the inotropic response to the reference Na+ channel stimulant veratridine were comparable to PD 122860, whereas tetrodotoxin and dichlorobenzamil had no inhibitory effect on the inotropic responses to the adenylate cyclase stimulator forskolin or the Ca++ channel stimulant BAY K 8644. PD 122860 selectively relaxed potassium-contracted aortic rings and inhibited [3H]nitrendipine binding to rat brain membranes, suggesting that the vasodilator activity of PD 122860 is due to Ca++ channel blockade. In contrast to BAY K 8644, PD 122860 did not contract partially depolarized aortic rings, suggesting an absence of Ca++ channel stimulant activity. PD 122860 is a racemic mixture and both the vasorelaxant and [3H]nitrendipine binding inhibitory activities selectively reside in the (+)-enantiomer [(+)-PD 122860]. In contrast, the inotropic response resides with both enantiomers of PD 122860. It is therefore concluded that PD 122860 represents a unique dihydropyridine derivative which possesses both Na+ channel stimulating and Ca++ channel blocking activities.     

Up-regulation of cell surface sodium channels by cyclosporin A, FK506, and rapamycin in adrenal chromaffin cells

Treatment of cultured bovine adrenal chromaffin cells with cyclosporin A (CsA) increased cell surface [(3)H]saxitoxin ([(3)H]STX) binding by 56% in a time (t(1/2) = 15.2 h)- and concentration (EC(50) = 2.9 microM)-dependent manner but did not change the K(d) value. In CsA-treated cells, veratridine-induced (22)Na(+) influx was augmented with no change in the EC(50) of veratridine; also, alpha- and beta-scorpion venom and Ptychodiscus brevis toxin-3 enhanced veratridine-induced (22)Na(+) influx in a more than additive manner, as in nontreated cells. CsA treatment for 1 to 24 h inhibited calcineurin activity, measured by the in vitro assay, with the IC(50) of 0.6 microM but did not alter cellular level of calcineurin. FK506 or rapamycin elevated [(3)H]STX binding by 36 or 25%, whereas GPI-1046, an immunophilin ligand incapable to inhibit calcineurin, or okadaic acid, an inhibitor of protein phosphatases 1 and 2A, had no increasing effect. The rise of [(3)H]STX binding by CsA was attenuated by the coincident treatment with brefeldin A (BFA), an inhibitor of vesicular exit from the trans-Golgi network. The internalization rate of cell surface Na(+) channels, as determined in the presence of BFA, was decreased in CsA (but not rapamycin)-treated cells (t(1/2) = 20.3 h), compared with nontreated cells (t(1/2) = 13.7 h). CsA treatment, however, did not elevate cellular levels of Na(+) channel alpha-subunit and Na(+) channel alpha- and beta(1)-subunit mRNAs. In CsA-treated cells, veratridine-induced (45)Ca(2+) influx via voltage-dependent Ca(2+) channels and catecholamine secretion were enhanced, whereas high K(+)-induced (45)Ca(+) influx was not. Thus, the inhibition of calcineurin or rapamycin-binding protein causes up-regulation of cell surface functional Na(+) channels via modulating externalization and internalization of Na(+) channels, thus enhancing Ca(2+) channel gating and catecholamine secretion.     

Depolarizing agents induce oscillations in canine bronchial smooth muscle membrane potential: possible mechanisms.

Tetraethylammonium (TEA) 925 mM), 4-aminopyridine (4-AP) (5 mM) and carbachol (3 X 10(-7) M) elicited membrane depolarization (approximately 20 mV) and oscillation (0.5-1.0 Hz; up to 25 mV in amplitude) in canine bronchi (3rd to 5th order). BaCl2 (1 mM) also elicited large depolarizations but not oscillations. The oscillations were antagonized by nitrendipine (NT) (10(-8) M) or atropine (10(-6) M) and were unaffected by phentolamine, propranolol or apamin (all 10(-7) M). TEA- or 4-AP-induced membrane depolarizations were partially reversed by atropine. After replacement of extracellular Ca++ with Sr++, oscillations elicited by carbachol or TEA were considerably slower (although amplitudes were similar to those seen in Ca++). Excitatory junction potentials were not altered by NT, were inhibited by 4-AP or replacement of extracellular Ca++ with Sr++, were potentiated by Ba++ and were variably affected by TEA. Contractile responses to acetylcholine were supported by Ca++ or Sr++ (with reduced efficacy), and only the latter were sensitive to NT. Our data suggest that, in canine bronchi (3rd to 5th order), 1) there are K+ channels and Ca++ channels (not L-type) on the cholinergic nerve endings, which modulate neurotransmitter release, 2) there are voltage-dependent K+ channels on the smooth muscle, which regulate membrane potential and suppress excitatory activity, and 3) membrane depolarization leads to alternating opening and closing of voltage-dependent (L-type) Ca++ channels on the smooth muscle, producing oscillations in membrane potential.(ABSTRACT TRUNCATED AT 250 WORDS)     

Competitive and noncompetitive antagonists at N-methyl-D-aspartate receptors protect against methamphetamine-induced dopaminergic damage in mice.

The administration of methamphetamine (METH) to experimental animals results in damage to nigrostriatal dopaminergic neurons. We have demonstrated previously that the excitatory amino acids may be involved in this neurotoxicity. For example, several compounds which bind to the phenyclidine site within the ion channel linked to the N-methyl-D-aspartate (NMDA) receptor protected mice from the METH-induced loss of neostriatal tyrosine hydroxylase activity and dopamine content. The present study was conducted to characterize further the role of the excitatory amino acids in mediating the neurotoxic effects of METH. The administration of three or four injections of METH (10 mg/kg) every 2 hr to mice produced large decrements in neostriatal dopamine content (80-84%) and in tyrosine hydroxylase activity (65-74%). A dose-dependent protection against these METH-induced decreases was seen with two noncompetitive NMDA antagonists, ifenprodil and SL 82.0715 (25-50 mg/kg/injection), both of which are thought to bind to a polyamine or sigma site associated with the NMDA receptor complex, and with two competitive NMDA antagonists, CGS 19755 (25-50 mg/kg/injection) and NPC 12626 (150-300 mg/kg/injection). Moreover, an intrastriatal infusion of NMDA (0.1 mumol) produced a slight but significant loss of neostriatal dopamine which was potentiated in mice that also received a systemic injection of METH. The results of these studies strengthen the hypothesis that the excitatory amino acids play a critical role in the nigrostriatal dopaminergic damage induced by METH.     

Cooperative modulation of voltage-dependent sodium channels by brevetoxin and classical neurotoxins in cultured bovine adrenal medullary cells.

The effects of Ptychodiscus brevis toxin (PbTx-3) on 22Na influx, 45Ca influx and catecholamine secretion were examined in cultured bovine adrenal medullary cells and compared with the effects of classical neurotoxins. PbTx-3 alone had no effects, but greatly enhanced veratridine (30 microM)-induced Na influx, Ca influx and secretion, with a EC50 of 30, 25 and 23 nM, respectively. PbTx-3 (1 microM) reduced EC50 values of veratridine approximately 3-fold and increased the maximal responses caused by saturating concentration (300 microM) of veratridine approximately 1.3 fold. alpha- and beta-Scorpion venom shifted the concentration-response curves of veratridine to the left without altering maximal responses. PbTx-3 in combination with either alpha- or beta-scorpion venom showed only additive effects on Na influx, but augmented veratridine (30 microM)-induced Na influx to a greater extent than PbTx-3, alpha- or beta-scorpion venom alone. Na influx due to these toxins was abolished by 1 microM saxitoxin. Our results suggest that Na channels in adrenal medullary cells have neurotoxin receptors for brevetoxin that allosterically stimulate Na influx initiated by veratridine, leading to increased Ca influx and catecholamine secretion. Allosteric interactions do not exist between brevetoxin and alpha-scorpion venom, or between brevetoxin and beta-scorpion venom, but once Na channels are gated by veratridine, these toxins cooperatively augment Na influx.     

Plasma concentrations of flecainide acetate, a new antiarrhythmic agent, in humans

Flecainide, a new antiarrhythmic agent, was given to eight healthy men to ascertain plasma drug levels and to assess tolerance for the drug. Each subject received a single intravenous (IV) dose (0.5, 1, 1.5, or 2 mg/kg) of flecainide, and plasma levels of unchanged flecainide were measured by gas-liquid chromatography. After an initial rapid distribution phase, the drug's plasma half-life (terminal elimination phase) ranged from seven to 15 hours (mean, 11 hours); half-life was apparently independent of dose level. Immediately after IV administration, the relatively high plasma drug levels seen during the short distribution phase were associated with minor and transient side effects in some subjects receiving the two higher doses; overall, the drug was well tolerated. Plasma flecainide levels 60 minutes after administration (after distribution) ranged from 52 to 300 ng/ml and were reasonably proportional to dose level. The drug's relatively long plasma half-life in humans indicates that plasma levels will be maintained for prolonged periods; thus flecainide should provide sustained therapeutic activity in patients with cardiac arrhythmias.     

The Na(+)-dependent release of endogenous dopamine and noradrenaline from rat brain synaptosomes

The involvement of intrasynaptosomal-free Ca++ concentration [( Ca++]i) in Na(+)-dependent release of endogenous dopamine and noradrenaline from rat brain synaptosomes was studied. The release of endogenous dopamine and noradrenaline from rat whole brain synaptosomes were measured using high-performance liquid chromatography with electrochemical detector. The change of [Ca++]i was measured fluorometrically using a Ca++ indicator, Quin-2. Whether extracellular Ca++ was present or not, 30 microM veratridine, a Na(+)-ionophore, increased the release of endogenous dopamine and noradrenaline. In the presence of 1.25 mM Ca++, 30 microM veratridine increased [Ca++]i. In contrast, in the absence of extracellular Ca++, veratridine did not affect [Ca++]i. Ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) increased the release of dopamine and noradrenaline in Ca-Mg-free medium. This stimulatory effect of EGTA seemed to be the result of an increase in the influx of Na+ through Ca++ channels in the absence of divalent cation. In Ca-Mg-free medium, EGTA caused a slight decrease in [Ca++]i. The EGTA-stimulated release of dopamine and noradrenaline was blocked by La which also significantly blocked the decrease in [Ca++]i observed after the addition of EGTA. These results suggest that the Na(+)-dependent release of dopamine and noradrenaline may not depend on a change in [Ca++]i. Veratridine (30 microM)-induced release of dopamine and noradrenaline were detected simultaneously. However, the time needed to induce the maximal stimulatory effect of veratridine on the release of dopamine was apparently shorter than that of noradrenaline. This delay might suggest that the Na(+)-dependent release process of dopamine is not similar to that of noradrenaline.     

The effects of the potassium channel opener minoxidil on renal electrolytes transport in the loop of henle

ATP-sensitive potassium channels (K(ATP)) in the thick ascending limb of the loop of Henle play an important role in apical K(+) recycling, a mechanism essential for maintaining the activity of the Na/2Cl/K-cotransporter. We have previously demonstrated that inhibition of K(ATP) decreases Na(+) and K(+) absorption in the loop of Henle and induces diuretic and natriuretic effects. In the present study, we used renal clearance and in vivo microperfusion techniques to evaluate the effects of the K(ATP) opener minoxidil on the urinary excretion and absorption in the loop of Henle of Na(+), K(+), Ca(2+), and Mg(2+). Intravenous injection of minoxidil (1.5 mg/kg) significantly decreased fractional Na(+) (FENa) and Mg(2+) (FEMg) excretion and urine volume with a moderate decrease in blood pressure (12%) and glomerular filtration rate (15%). Urine volume decreased 63%, and FENa and FEMg decreased 58 and 37%, respectively. In contrast, K(+) and Ca(2+) excretion did not change significantly. In the microperfusion of the loop of Henle, addition of minoxidil to the perfusion fluid significantly increased fluid (J(v)), Na(+) (J(Na)), Cl(-) (J(Cl)), and K(+) (J(K)) absorption. J(v) increased 44% (from 8.32 to 11.95 nl/min), J(Na) increased 14% (from 1.96 to 2.34 nmol/min), J(Cl) increased 21% (from 1.72 to 2.08 nmol/min), and J(K) increased 57% (from 35.8 to 56.4 pmol/min). We conclude that the activation of K(ATP) leads to stimulation of Na/2Cl/K-cotransporter activity and increases the rates of Na(+), Cl(-), and K(+) absorption in the loop of Henle, an effect contributing to the antidiuretic and antinatriuretic action of this K channel opener.     

Effect of alpha-asarone and a derivative on lipids, bile flow and Na+/K+-ATPase in ethinyl estradiol-induced cholestasis in the rat

Administration of ethinyl estradiol (EE), a widely used component of oral contraceptives, has been associated with impairment of bile flow and the capacity to excrete organic anions in man and experimental animals. alpha-Asarone (2,4,5-trimethoxypropenylbenzene) and 2-methoxy-4-(2-propenyl) phenoxyacetic acid (MPPA) have shown hypolipidemic effects. In addition to these effects, we decided to evaluate the properties of these compounds on EE-induced cholestasis. Wistar male rats were injected subcutaneously with 10 mg/kg of EE for 5 days; simultaneously, alpha-asarone or MPPA were also administered and appropriate controls were performed. alpha-asarone and MPPA decreased plasma and bile cholesterol. EE diminished triglycerides total, low-density lipoprotein, high-density lipoprotein and bile cholesterol. MPPA further decreased these lipid parameters. Alkaline phosphatase (an enzyme marker of cholestasis) was increased after administration of EE, but this effect was prevented significantly by alpha-asarone or MPPA administration. Bile flow was importantly decreased by EE and increased by alpha-asarone alone. Furthermore, alpha-asarone or MPPA preserved the normal bile flow in EE-treated rats. EE inhibited the activity of the Na(+)/K(+)-ATPase, while both alpha-asarone and MPPA preserved this enzyme activity. Na(+)/K(+)-ATPase is involved in Na(+)-coupled uptake of bile acids into hepatocytes and, therefore, ultimately is the driving force for the generation of bile flow. Therefore, the anticholestatic effects of alpha-asarone and MPPA, described herein by the first time, may be due to its ability to preserve ATPase activity. This enzyme is negatively regulated by membrane cholesterol, thus the hypolipidemic effects of the compounds tested may be responsible for Na(+)/K(+)-ATPase activity and bile flow maintenance.     

Studies on functional sites of organic cation/carnitine transporter OCTN2 (SLC22A5) using a Ser467Cys mutant protein

The organic cation/carnitine transporter OCTN2 mediates transport of carnitine and organic cations in Na(+)-dependent and Na(+)-independent manners, respectively. However, the mechanism of molecular recognition of different substrates has not been clarified yet. We previously found a single amino acid change in OCTN2, Ser467Cys (S467C), in the Japanese population and observed a decreased carnitine transport but unchanged organic cation transport compared with wild type. Therefore, we conducted detailed kinetic and functional analyses of the substrate recognition sites of wild-type and S467C-mutant OCTN2. The K(m) value for carnitine of S467C-mutant was increased about 15-fold over that of the wild type. Mutual inhibition kinetics of carnitine and tetraethylammonium (TEA) were not completely competitive, suggesting that the binding sites are very close to each other, but not identical. Several organic anions such as valproate, as well as organic cations, significantly inhibited carnitine and TEA uptake by OCTN2, and valproate showed Na(+)-dependent inhibition of OCTN2-mediated TEA uptake. The Na(+)-activation kinetics of the S467C mutant was similar to that of the wild type. Furthermore, a significant decrease of the TEA uptake-inhibitory potency of valproate was observed in S467C-mutant OCTN2. These observations suggest that the decrease in affinity of S467C-mutant OCTN2 for carnitine was caused by functional alteration of the anion (carboxyl moiety of carnitine) recognition site located in trans-membrane domain 11, which is closely related to the Na(+)-binding site, on OCTN2 protein. These results demonstrate that OCTN2 has functional sites for carnitine and Na(+) and that the carnitine-binding site is involved, in part, in the recognition of organic cations.