Inhibition of Na+ channel currents in rat myoblasts by 4-aminopyridine

Our previous study revealed that 4-aminopyridine (4-AP), a specific blocker of A-type current, could also inhibit inward Na+ currents (I(Na)) with a state-independent mechanism in rat cerebellar granule cells. In the present study, we report an inhibitory effect of 4-AP on voltage-gated and tetrodotoxin (TTX)-sensitive I(Na) recorded from cultured rat myoblasts. 4-AP inhibited I(Na) amplitude in a dose-dependent manner between the concentrations of 0.5 and 10 mM without significant alteration in the activation or inactivation kinetics of the channel. By comparison to the 4-AP-induced inhibitory effect on cerebellum neurons, the inhibitory effect on myoblasts was enhanced through repetitive pulse and inflected by changing frequency. Specifically, the lower the frequency of pulse, the higher the inhibition observed, suggesting that block manner is inversely use-dependent. Moreover, experiments adding 4-AP to the intracellular solution indicate that the inhibitory effects are localized inside the cell. Additionally, 4-AP significantly modifies the properties of steady-state activation and inactivation kinetics of the channel. Our data suggest that the K+ channel blocker 4-AP inhibits both neuron and myoblast Na+ channels via different mechanisms. These findings may also provide information regarding 4-AP-induced pharmacological and toxicological effects in clinical use and experimental research.     

Heterologous expression and functional analysis of rat Nav1.8 (SNS) voltage-gated sodium channels in the dorsal root ganglion neuroblastoma cell line ND7-23

The voltage-gated sodium channel NaV1.8 (SNS, PN3) is thought to be a molecular correlate of the dorsal root ganglion (DRG) tetrodotoxin resistant (TTX-R) Na+ current. TTX-R/NaV1.8 is an attractive therapeutic drug target for inflammatory and neuropathic pain on the basis of its specific distribution in sensory neurones and its modulation by inflammatory mediators. However, detailed analysis of recombinant NaV1.8 has been hampered by difficulties in stably expressing the functional protein in mammalian cells. Here, we show stable expression and functional analysis of rat NaV1.8 (rNaV1.8) in the rat DRG/mouse N18Tg2 neuroblastoma hybridoma cell line ND7-23. Rat NaV1.8 Na+ currents were recorded (789 +/- 89 pA, n=62, over 20-cell passages) that qualitatively resembled DRG TTX-R in terms of gating kinetics and voltage-dependence of activation and inactivation. The local anaesthetic drug tetracaine produced tonic inhibition of rNaV1.8 (mean IC50 value 12.5 microM) and in repeated gating paradigms (2-10 Hz) also showed frequency-dependent block. There was a correlation between the ability of several analogues of the anticonvulsant/analgesic compound lamotrigine to inhibit TTX-R and rNaV1.8 (r=0.72, P<0.001). RT-PCR analysis of wild type ND7-23 cells revealed endogenous expression of the beta1 and beta3 accessory Na+ channel subunits-the possibility that the presence of these subunits assists and stabilises expression of rNaV1.8 is discussed. We conclude that the neuroblastoma ND7-23 cell line is a suitable heterologous expression system for rNaV1.8 Na+ channels in that it allows stable expression of a channel with biophysical properties that closely resemble the native TTX-R currents in DRG neurones. This reagent will prove useful in the search for pharmacological inhibitors of rNaV1.8 as novel analgesics.     

Vasopressin V(1) receptor-mediated activation of central sympatho-adrenomedullary outflow in rats

High-threshold Ca(2+) channels and tetrodotoxin-resistant Na(+) channels are highly expressed in small dorsal root ganglion neurons. In acutely isolated rat dorsal root ganglion neurons, the effects of neomycin, one of the aminoglycoside antibiotics, on high-threshold Ca(2+) currents and tetrodotoxin-resistant Na(+) currents were examined using whole-cell patch recording. We showed for the first time that neomycin dose-dependently inhibited peak high-threshold Ca(2+) currents and peak tetrodotoxin-resistant Na(+) currents with half-maximal inhibitory concentrations at 3.69 microM (n=20) and 1213.44 microM (n=25), respectively. Inactivation properties of high-threshold Ca(2+) currents and activation properties of tetrodotoxin-resistant Na(+) currents were also affected by neomycin with reduction of excitability of small dorsal root ganglion neurons. Half-maximal inactivation voltage of high-threshold Ca(2+) currents was -45.56 mV before and -50.46 mV after application of neomycin (n=10). Half-maximal activation voltage of tetrodotoxin-resistant Na(+) currents was -19.93 mV before and -11.19 mV after administration of neomycin (n=15). These results suggest that neomycin can inhibit high-threshold Ca(2+) currents and tetrodotoxin-resistant Na(+) currents in small dorsal root ganglion neurons, which may contribute to neomycin-induced peripheral and central analgesia.     

The gasotransmitter hydrogen sulphide decreases Na⁺ transport across pulmonary epithelial cells

BACKGROUND AND PURPOSE The transepithelial absorption of Na(+) in the lungs is crucial for the maintenance of the volume and composition of epithelial lining fluid. The regulation of Na(+) transport is essential, because hypo- or hyperabsorption of Na(+) is associated with lung diseases such as pulmonary oedema or cystic fibrosis. This study investigated the effects of the gaseous signalling molecule hydrogen sulphide (H(2) S) on Na(+) absorption across pulmonary epithelial cells. EXPERIMENTAL APPROACH Ion transport processes were electrophysiologically assessed in Ussing chambers on H441 cells grown on permeable supports at air/liquid interface and on native tracheal preparations of pigs and mice. The effects of H(2)S were further investigated on Na(+) channels expressed in Xenopus oocytes and Na(+) /K(+)-ATPase activity in vitro. Membrane abundance of Na(+) /K(+)-ATPase was determined by surface biotinylation and Western blot. Cellular ATP concentrations were measured colorimetrically, and cytosolic Ca(2+) concentrations were measured with Fura-2. KEY RESULTS H(2)S rapidly and reversibly inhibited Na(+) transport in all the models employed. H(2)S had no effect on Na(+) channels, whereas it decreased Na(+) /K(+)-ATPase currents. H(2)S did not affect the membrane abundance of Na(+) /K(+)-ATPase, its metabolic or calcium-dependent regulation, or its direct activity. However, H(2)S inhibited basolateral calcium-dependent K(+) channels, which consequently decreased Na(+) absorption by H441 monolayers. CONCLUSIONS AND IMPLICATIONS H(2) S impairs pulmonary transepithelial Na(+) absorption, mainly by inhibiting basolateral Ca(2+)-dependent K(+) channels. These data suggest that the H(2)S signalling system might represent a novel pharmacological target for modifying pulmonary transepithelial Na(+) transport.     

Potassium and potassium clouds in endothelium-dependent hyperpolarizations

A small increase in extracellular K(+) acts as a local, physiological regulator of blood flow to certain vascular beds. The K(+) derives from active tissues such as contracting skeletal muscle and brain and increases blood supply to these organs by the activation of Na(+)/K(+)-ATPases and/or inwardly-rectifying K(+) channels on the vascular myocytes. K(+) liberated from the vascular endothelium also acts as an endothelium-derived hyperpolarizing and relaxing factor within blood vessels. The K(+) effluxes from endothelial cell intermediate- and small-conductance, Ca(2+)-sensitive K(+) channels which open in response to stretch and local hormones. In many vessels, endothelium-derived hyperpolarizing factor (EDHF) seems identical to the K(+) derived from endothelial cells; it activates Na(+)/K(+)-ATPases (particularly those containing alpha2 and alpha3 subunits) and inward rectifiers (particularly Kir2.1) located on the vascular myocytes. Vasospastic agents generate "potassium clouds" around vascular smooth muscle cells via the efflux of this ion through large conductance, Ca(2+)-sensitive K(+) channels on the myocytes. These potassium clouds can reduce the hyperpolarizing actions of endothelium-derived K(+) by effectively saturating the Na(+)/K(+)-ATPases and inward rectifiers on the muscle cells and they may be of clinical significance in vasospastic conditions.     

Imipramine inhibits A-type delayed rectifier and ATP-sensitive K+ currents independent of G-protein and protein kinase C in murine proximal colonic myocytes

The effects of imipramine on A-type delayed rectifier K+ currents and ATP-sensitive K+ (KATP) currents were studied in isolated murine proximal colonic myocytes using the whole-cell patch-clamp technique. Depolarizing test pulses between -80 mV and +30 mV with 10 mV increments from the holding potential of -80 mV activated voltage-dependent outward K+ currents that peaked within 50 ms followed by slow decreasing sustained currents. Early peak currents were inhibited by the application of 4-aminopyridine, whereas sustained currents were inhibited by the application of TEA. The peak amplitude of A-type delayed rectifier K+ currents was reduced by external application of imipramine. The half-inactivation potential and the half-recovery time of A-type delayed rectifier K+ currents were not changed by imipramine. With 0.1 mM ATP and 140 mM K+ in the pipette and 90 mM K+ in the bath solution and a holding potential of -80 mV, pinacidil activated inward currents; this effect was blocked by glibenclamide. Imipramine also inhibited KATP currents. The inhibitory effects of imipramine in A-type delayed rectifier K+ currents and KATP currents were not changed by guanosine 5-O-(2-thiodiphosphate) (GDPbetaS) and chelerythrine, a protein kinase C inhibitor. These results suggest that imipramine inhibits A-type delayed rectifier K+ currents and KATP currents in a manner independent of G-protein and protein kinase C.     

Comparison of the effects of antiarrhythmic drugs flecainide and verapamil on fKv1.4ΔN channel currents in Xenopus oocytes

Aim:

To study the effects of Na⁺ channel blocker flecainide and L-type Ca²⁺ channel antagonist verapamil on the voltage-gated fKv1.4ΔN channel, an N-terminal-deleted mutant of the ferret Kv1.4 K⁺ channel.

Methods:

fKv1.4ΔN channels were stably expressed in Xenopus oocytes. The K⁺ currents were recorded using a two-electrode voltage-clamp technique. The drugs were administered through superfusion.

Results:

fKv1.4ΔN currents displayed slow inactivation, with a half-inactivation potential of −41.74 mV and a slow recovery from inactivation (τ=1.90 s at −90 mV). Flecainide and verapamil blocked the currents with IC₅₀ values of 512.29±56.92 and 260.71±18.50 μmol/L, respectively. The blocking action of the drugs showed opposite voltage-dependence: it was enhanced with depolarization for flecainide, and was attenuated with depolarization for verapamil. Both the drugs exerted state-dependent blockade on fKv1.4ΔN currents, but verapamil showed a stronger use-dependent blockage compared with flecainide. Flecainide accelerated the C-type inactivation rate without affecting the recovery kinetics and the steady-state activation. Verapamil also accelerated the inactivation kinetics of the currents, but unlike flecainide, it affected both the recovery and the steady-state activation, causing slower recovery of fKv1.4ΔN channel and a depolarizing shift of the steady-state activation curve.

Conclusion:

The results demonstrate that widely used antiarrhythmic drugs flecainide and verapamil substantially inhibit fKv1.4ΔN channels expressed in Xenopus oocytes by binding to the open state of the channels. Therefore, caution should be taken when these drugs are administered in combination with K⁺ channel blockers to treat arrhythmia.     

Voltage-dependent inhibition of brain Na(+) channels by American ginseng

American ginseng (Panax quinquefolius) is a major species of ginseng that has many pharmacological effects. Studies have demonstrated that constituents of ginseng have neuroprotective effects during ischemia. Neuronal damage during ischemic episodes has been associated with abnormal Na(+) fluxes. Drugs that block voltage-dependent Na(+) channels provide cytoprotection during cerebral ischemia. We thus hypothesized that American ginseng may block Na(+) channels. In this study, effects of an American ginseng aqueous extract was evaluated in tsA201 cells transfected with cDNA expressing alpha subunits of the Brain(2a) Na(+) channel using the whole-cell patch clamp technique. We found that American ginseng extract tonically and reversibly blocked the channel in a concentration- and voltage-dependent manner. It shifted the voltage-dependence of inactivation by 14 mV (3 mg/ml) in the hyperpolarizing direction and delayed recovery from inactivation, whereas activation of the channel was unaffected. Ginsenoside Rb(1), a major constituent of the American ginseng extract, produced similar effects. The data were compared with the actions of lidocaine, a Na(+) channel blocker. Our results suggest that Na(+) channel block by American ginseng extract and Rb(1) was primarily due to interaction with the inactive state of the channel. Inhibition of the Na(+) channel activity by American ginseng extract may contribute to its neuroprotective effect during ischemia.     

Evaluation of endomorphin-1 on the activity of Na(+),K(+)-ATPase using in vitro and in vivo studies

The goal of this study was to investigate the effects of endomorphin-1 on Na(+),K(+)-ATPase activity in mouse brain synaptosome in vitro, and its antinociceptive interaction with the Na(+),K(+)-ATPase inhibitor ouabain. Endomorphin-1 (0.1 nM-10 microM) produced a concentration-dependent (EC(50): 43.19 nM, CI: 23.38-65.71 nM, E(max): 25.86%, CI: 24.53-27.20%), naloxone-reversible increase of the synaptosomal Na(+),K(+)-ATPase activity. The intrathecally (i.t.) administered endomorphin-1 (2-20 microg) produced a dose-dependent short-lasting increase in the tail-flick latency. Ouabain itself (1-1000 ng, i.t.) did not cause antinociception. Treatment with 10 ng ouabain significantly decreased the antinociceptive effect of 2 microg endomorphin-1, but none of the other combinations did significantly differ from the endomorhin-1-treated groups. These data indicate that endomorphin-1 increases the activity of Na(+),K(+)-ATPase in vitro but this effect may play a weak role in the antinociception induced by intrathecal endomorphin-1.     

Halothane attenuates the cerebroprotective action of several Na+ and Ca2+ channel blockers via reversal of their ion channel blockade

We have previously shown the involvement of Na(+) channel as well as N-type and P/Q-type Ca(2+) channels in the oxygen and glucose deprivation-induced injury in rat cerebrocortical slices. In the present study, we investigated the influence of halothane on the cerebroprotective effects of a variety of Na(+) and Ca(2+) channel blockers in rat cerebrocortical slices. The hypoxic injury was attenuated by Na(+) channel blockers including tetrodotoxin, lidocaine and dibucaine, and Ca(2+) channel blockers, such as verapamil, omega-agatoxin IVA and omega-conotoxin GVIA. Halothane abolished the protective effects of lidocaine, dibucaine and verapamil, all of which block the respective cation channels in a voltage-dependent manner, without affecting the actions of tetrodotoxin, omega-agatoxin IVA and omega-conotoxin GVIA, which reveal voltage-independent blockade. On the other hand, the nitric oxide synthesis estimated from the extracellular cyclic GMP formation was elevated during exposure to hypoxia. All channel blockers tested here attenuated hypoxia-evoked nitric oxide synthesis. Halothane blocked almost completely these actions of lidocaine and verapamil. Moreover, the Na(+) and Ca(2+) channel blockade by these compounds, as determined by veratridine- and KCl-stimulated nitric oxide synthesis, respectively, was also reversed by halothane. These findings suggest that an anesthetic agent halothane reversed the Na(+) and Ca(2+) channel blockade of several voltage-dependent ion channel blockers, leading to the attenuation of their cerebroprotective actions. Therefore, the influence of halothane anesthesia should be taken into consideration for the evaluation of neuroprotective action of Na(+) and Ca(2+) channel blockers.     

Molecular and functional characterization of the human platelet Na(+) /Ca(2+) exchangers

BACKGROUND AND PURPOSE The Na(+) /Ca(2+) exchanger is a bi-directional transporter that plays an important role in maintaining the concentration of cytosolic Ca(2+) ([Ca(2+) ](i) ) of quiescent platelets and increasing it during activation with some, but not all, agonists. There are two classes of Na(+) /Ca(2+) exchangers: K(+) -independent Na(+) /Ca(2+) exchanger (NCX) and K(+) -dependent Na(+) /Ca(2+) exchanger (NCKX). Platelets have previously been shown to express NCKX1. However, initial studies from our laboratory suggest that NCX may also play a role in platelet activation. The objective of this study was to determine if the human platelet expresses functional NCXs. EXPERIMENTAL APPROACH RT-PCR, DNA sequencing and Western blot analysis were utilized to characterize the human platelet Na(+) /Ca(2+) exchangers. Their function during quiescence and collagen-induced activation was determined by measuring [Ca(2+) ](i) with calcium-green/fura-red in response to: changes in the Na(+) and K(+) gradient, NCX pharmacological inhibitors (CBDMB, KB-R7943 and SEA0400) and antibodies specific to extracellular epitopes of the exchangers. KEY RESULTS Human platelets express NCX1.3, NCX3.2 and NCX3.4. The NCXs operate in the Ca(2+) efflux mode in resting platelets and also during their activation with thrombin but not collagen. Collagen-induced increase in [Ca(2+) ](i) was reduced with the pharmacological inhibitors of NCX (CBDMB, KB-R7943 or SEA0400), anti-NCX1 and anti-NCX3. In contrast, anti-NCKX1 enhanced the collagen-induced increase in [Ca(2+) ](i) . CONCLUSIONS AND IMPLICATIONS Human platelets express K(+) -independent Na(+) /Ca(2+) exchangers NCX1.3, NCX3.2 and NCX3.4. During collagen activation, NCX1 and NCX3 transiently reverse to promote Ca(2+) influx, whereas NCKX1 continues to operate in the Ca(2+) efflux mode to reduce [Ca(2+) ](i) .     

Amitriptyline modulation of Na(+) channels in rat dorsal root ganglion neurons

The effects of amitriptyline, a tricyclic antidepressant, on tetrodotoxin-sensitive and tetrodotoxin-resistant Na(+) currents in rat dorsal root ganglion neurons were studied using the whole-cell patch clamp method. Amitriptyline blocked both types of Na(+)currents in a dose-and holding potential-dependent manner. At the holding potential of -80 mV, the apparent dissociation constants (K(d)) for amitriptyline to block tetrodotoxin-sensitive and tetrodotoxin-resistant Na(+) channels were 4.7 and 105 microM, respectively. These values increased to 181 and 193 microM, respectively, when the membrane was held at a potential negative enough to remove the steady-state inactivation. Amitriptyline dose-dependently shifted the steady-state inactivation curves in the hyperpolarizing direction and increased the values of the slope factors for both types of Na(+) channels. The voltage dependence of the activation of both types of Na(+) channels was shifted in the depolarizing direction. It was concluded that amitriptyline blocked the two types of Na(+) channels in rat sensory neurons by modulating the activation and the inactivation kinetics.     

Phenol derivatives accelerate inactivation kinetics in one inactivation-deficient mutant human skeletal muscle Na(+) channel

Altered inactivation kinetics in skeletal muscle Na(+) channels due to mutations in the encoding gene are causal for the alterations in muscle excitability in nondystrophic myotonia. Na(+) channel blockers like lidocaine and mexiletine, suggested for therapy of myotonia, do not reconstitute inactivation in channels with defective inactivation in vitro. We examined the effects of four methylated and/or halogenated phenol derivatives on one heterologously expressed inactivation-deficient Paramyotonia congenita-mutant (R1448H) muscle Na(+) channel in vitro. All these compounds accelerated delayed inactivation of R1448H-whole-cell currents during a depolarization and delayed accelerated recovery from inactivation. The potency of these effects paralleled the potency of the drugs to block the peak current amplitude. We conclude that the investigated phenol derivatives affect inactivation-deficient Na(+) channels more specifically than lidocaine and mexiletine. However, for all compounds, the effect on inactivation was accompanied by a substantial block of the peak current amplitude.     

Lithium inhibits function of voltage-dependent sodium channels and catecholamine secretion independent of glycogen synthase kinase-3 in adrenal chromaffin cells

Lithium has been proven to be effective in the therapy of bipolar disorder, but its mechanism of pharmacological action is not clearly defined. We examined the effects of lithium on voltage-dependent Na(+) channels, nicotinic acetylcholine receptors, and voltage-dependent Ca(2+) channels, as well as catecholamine secretion in cultured bovine adrenal chromaffin cells. Lithium chloride (LiCl) reduced veratridine-induced (22)Na(+) influx in a concentration-dependent manner, even in the presence of ouabain, an inhibitor of Na(+), K(+)-ATPase. Glycogen synthase kinase-3 (GSK-3) inhibitors (SB216763, SB415286 or the GSK-3 inhibitor IX) did not affect veratridine-induced (22)Na(+) influx, as well as inhibitory effect of LiCl on veratridine-induced (22)Na(+) influx. Enhancement of veratridine (site 2 toxin)-induced (22)Na(+) influx caused by alpha-scorpion venom (site 3 toxin), beta-scorpion venom (site 4 toxin), or Ptychodiscus brevis toxin-3 (site 5 toxin), still occurred in the presence of LiCl in the same manner as in the control cells. LiCl also reduced veratridine-induced (45)Ca(2+) influx and catecholamine secretion. In contrast, LiCl (< or = 30 mM) had no effect on nicotine-induced (22)Na(+) influx, (45)Ca(2+) influx and catecholamine secretion, as well as on high K(+)-induced (45)Ca(2+) influx and catecholamine secretion. Chronic treatment with LiCl at 100mM (but not at < or = 30 mM) significantly reduced cell viability in a time-dependent manner. These results suggest that lithium selectively inhibits Na(+) influx thorough Na(+) channels and subsequent Ca(2+) influx and catecholamine secretion, independent of GSK-3 inhibition.     

Characterization of aconitine-induced block of delayed rectifier K+ current in differentiated NG108-15 neuronal cells

The effects of aconitine (ACO), a highly toxic alkaloid, on ion currents in differentiated NG108-15 neuronal cells were investigated in this study. ACO (0.3-30 microM) suppressed the amplitude of delayed rectifier K+ current (I K(DR)) in a concentration-dependent manner with an IC50 value of 3.1 microM. The presence of ACO enhanced the rate and extent of I K(DR) inactivation, although it had no effect on the initial activation phase of I K(DR). It could shift the inactivation curve of I K(DR) to a hyperpolarized potential with no change in the slope factor. Cumulative inactivation for I K(DR) was also enhanced by ACO. Orphenadrine (30 microM) or methyllycaconitine (30 microM) slightly suppressed I K(DR) without modifying current decay. ACO (10 microM) had an inhibitory effect on voltage-dependent Na+ current (I Na). Under current-clamp recordings, ACO increased the firing and widening of action potentials in these cells. With the aid of the minimal binding scheme, the ACO actions on I K(DR) was quantitatively provided with a dissociation constant of 0.6 microM. A modeled cell was designed to duplicate its inhibitory effect on spontaneous pacemaking. ACO also blocked I K(DR) in neuroblastoma SH-SY5Y cells. Taken together, the experimental data and simulations show that ACO can block delayed rectifier K+ channels of neurons in a concentration- and state-dependent manner. Changes in action potentials induced by ACO in neurons in vivo can be explained mainly by its blocking actions on I K(DR) and I Na.     

Effects of fluoroquinolones on insulin secretion and beta-cell ATP-sensitive K+ channels

Although fluoroquinolones are used widely in the treatment of various infectious diseases, some of the drugs are known to cause hypoglycemia as a side-effect. We have investigated the effects of three fluoroquinolone derivatives, levofloxacin, gatifloxacin, and temafloxacin, on insulin secretion and pancreatic beta-cell ATP-sensitive K(+) channel (K(ATP) channel) activity. While levofloxacin had only a small effect on insulin secretion and K(ATP) currents, gatifloxacin and temafloxacin stimulated insulin secretion and inhibited K(ATP) channel currents in a dose-dependent manner. We also determined the site of action of gatifloxacin and temafloxacin on the K(ATP) channel. In a reconstituted system, gatifloxacin and temafloxacin inhibited Kir6.2 Delta C26 channels, which function in the absence of the SUR subunit, indicating direct action of the drugs on the Kir6.2 subunits. These results suggest that stimulation of insulin secretion by inhibition of pancreatic beta-cell K(ATP) channels underlies the hypoglycemia caused by certain fluoroquinolones.     

Cromakalim activates the KATP and enhances spontaneous transient outward potassium currents in rat saphenous arterial myocytes

Potassium channel openers, e.g. cromakalim are held to relax smooth muscle by hyperpolarizing the cell membrane via activation of ATP-sensitive K(+) (K(ATP)) channels. A recent report indicates that members of this group dilate cerebral arteries also by enhancing the K(Ca)-based spontaneous transient outward currents (STOCs) through the activation of mitochondrial K(ATP) channels. We extended the study to rat saphenous arterial myocytes, a model for peripheral resistance vessels, to investigate the effects of cromakalim on K(ATP) and STOCs, and the underlaying mechanisms. Smooth muscle myocytes were enzymatically dissociated from the saphenous branch of the femoral artery. Macroscopic currents were recorded from acutely isolated cells using the perforated-patch and whole-cell variants of the patch-clamp technique. Predictably metabolic inhibitors and cromakalim activated a background K(+) current blocked by glibenclamide, identified as the K(ATP) channel. However, in addition, cromakalim markedly increased the amplitude and frequency of STOCs. The latter action was not sensitive to the specific K(ATP) channel blocker glibenclamide, excluding the participation of mitocondrial K(ATP) channels in this action. In conclusion, this study suggests that, in addition to the opening of K(ATP) channels, the increased STOC activity may have an important role in the vasorelaxing action of cromakalim, but through a mechanism different from that reported on cerebral artery.     

The peripheral antinociceptive effect of resveratrol is associated with activation of potassium channels

The possible participation of K(+) channels in the antinociceptive action induced by resveratrol was assessed in the 1% formalin test. Local administration of resveratrol produced a dose-dependent antinociception in the second phase of the test. The antinociception produced by resveratrol was due to a local action as its administration in the contralateral paw was not active. Local pretreatment of the injured paw with glibenclamide, tolbutamide or glipizide (ATP-sensitive K(+) channel inhibitors) did not modify resveratrol-induced antinociception. In contrast, charybdotoxin and apamin (large and small conductance Ca(2+) activated-K(+) channel blockers, respectively), 4-aminopyridine or tetraethylammonium (voltage-dependent K(+) channel inhibitors) dose-dependently prevented resveratrol-induced antinociception. Local peripheral administration of glibenclamide, but not charybdotoxin or apamin, significantly reduced the antinociceptive effect produced by peripheral morphine (positive control). At the highest effective doses, none of the drugs used induced behavioral side effects as revealed by the evaluation of stepping, righting, corneal and pinna reflexes. In addition, when given alone, none of the inhibitors modified the nociceptive behavior induced by 1% formalin. The results suggest that resveratrol opens large and small conductance Ca(2+)-activated K(+) channels, but not ATP-sensitive K(+) channels, in order to produce its peripheral antinociceptive effect in the formalin test. The participation of voltage-dependent K(+) channels was also suggested, but since non-selective inhibitors were used the data awaits further confirmation.     

Inhibition of Na(+) current by imipramine and related compounds: different binding kinetics as an inactivation stabilizer and as an open channel blocker

Use-dependent block of Na(+) channels plays an important role in the action of many medications, including the anticonvulsants phenytoin, carbamazepine, and lamotrigine. These anticonvulsants all slowly yet selectively bind to a common receptor site in inactivated but not resting Na(+) channels, constituting the molecular basis of the use-dependent block. However, it remains unclear what channel gating process "makes" the receptor, where the receptor is located, and how the slow drug binding rate (to the inactivated channels) is contrived. Imipramine has a diphenyl structural motif almost identical to that in carbamazepine (a dibenzazepine tricyclic compound), as well as a tertiary amine chain similar to that in many prototypical local anesthetics, and has also been reported to inhibit Na(+) channels in a use-dependent fashion. We found that imipramine selectively binds to the inactivated (dissociation constant approximately 1.3 microM) rather than the resting Na(+) channels (dissociation constant >130 microM). Moreover, imipramine rapidly blocks open Na(+) channels, with a binding rate approximately 70-fold faster than its binding to the inactivated channels. Similarly, carbamazepine and diphenhydramine are open Na(+) channel blockers with faster binding rates to the open than to the inactivated channels. These findings indicate that the anticonvulsant receptor responsible for the use-dependent block of Na(+) channels is located in or near the pore (most likely in the pore mouth) and is made suitable for drug binding during channel activation. The receptor, however, continually changes its conformation in the subsequent gating process, causing the slower drug binding rates to the inactivated Na(+) channels.     

Diclofenac, a nonsteroidal anti-inflammatory drug, activates the transient outward K+ current in rat cerebellar granule cells

Diclofenac, a nonsteroidal anti-inflammatory drug (NSAID), has been widely investigated in terms of its pharmacological action, but less is known about its direct effect on ion channels. Here, the effect of diclofenac on voltage-dependent transient outward K+ currents (I(A)) in cultured rat cerebellar granule cells was investigated using the whole-cell voltage-clamp technique. At concentrations of 10(-5)-10(-3) M, diclofenac reversibly increased the I(A) amplitude in a dose-dependent manner and significantly modulated the steady-state inactivation properties of the I(A) channels, but did not alter the steady-state activation properties. Furthermore, diclofenac treatment resulted in a slightly accelerated recovery from I(A) channel inactivation. Intracellular application of diclofenac could mimic the effects induced by extracellular application, although once the intracellular response reached a plateau, extracellular application of diclofenac could induce further increases in the current. These observations indicate that diclofenac might exert its effects on the channel protein at both the inner and outer sides of the cell membrane. Our data provide the first evidence that diclofenac is able to activate transient outward potassium channels in neurons. Although further work will be necessary to define the exact mechanism of diclofenac-induced I(A) channel activation, this study provides evidence that the nonsteroidal anti-inflammatory drug, diclofenac, may play a novel neuronal role that is worthy of future study.