Regulatory effect of sulphatides on BKCa channels

BACKGROUND AND PURPOSE: Sulphatides are sulphated glycosphingolipids expressed on the surface of many cell types, particularly neurones. Changes in sulphatide species or content have been associated with epilepsy and Alzheimer's disease. As the large conductance, calcium sensitive K(+) channel (BK(Ca)) are modulated by membrane lipids, the aim of the study was to explore possible effects of sulphatides on BK(Ca) channels. EXPERIMENTAL APPROACH: Using patch-clamp techniques, we studied effects of exogenous sulphatides on BK(Ca) channels expressed in Chinese hamster ovary cells. KEY RESULTS: Sulphatides reversibly increased the whole-cell current and the single channel open probability of BK(Ca) channels dose-dependently. The EC(50) value on the channel at +10 mV was 1.6 microM and the Hill coefficient was 2.5. In inside-out patches, sulphatides increased the single channel open probability from both intra- and extra-cellular faces of the membrane, but more effectively with external application. Furthermore, activation of the channels by sulphatides was independent of intracellular Ca(2+) concentration. Sulphatides also shifted the activation curve of the channels to less positive membrane potentials. Mutant BK(Ca) channels lacking a 59 aminoacid region important for amphipath activation (STREX) were less activated by the sulphatides. CONCLUSIONS AND IMPLICATIONS: Sulphatides are novel activators of BK(Ca) channels, independent of intracellular Ca(2+) or other signalling molecules but partly dependent on the STREX sequence of the channel protein. As changes of sulphatide content are associated with neuronal dysfunction, as in epilepsy and Alzheimer's disease, our results imply that these effects of sulphatides may play important pathophysiological roles in regulation of BK(Ca) channels.     

Evidence that BKCa channel activation contributes to K+ channel opener induced relaxation of the porcine coronary artery

The rank order of potency of a series of benzopyran and cyanoguanidine K⁺ channel openers (KCOs) for causing relaxation of the PGF₂-precontracted porcine coronary artery was determined. Glyburide, an inhibitor of K_ATP channels, showed an apparent competitive inhibition of the vasorelaxant activity of the KCOs. The pA₂ values of glyburide when cromakalim and CGP 14877 (P1060) were used as vasorelaxants were 7.66 and 7.83, respectively. Charybdotoxin (40 nM), an inhibitor of BK_Ca channels, also caused a significant inhibition of the cromakalim mediated relaxation of the porcine coronary artery. In order to clarify the site of action of these KCOs, we identified a K⁺ channel current in single porcine coronary arterial cells and measured channel activity in the presence of these compounds. The prominent K⁺ ion current in these cells had characteristics typical of the conventional large Ca²⁺-activated K⁺ channel BK_Ca present in other smooth muscle cells. Using symmetrical K⁺ concentrations, the channel had a conductance of 214 pS and was found to be sensitive to [Ca²⁺]_i and membrane potential. The KCOs were found to reversibly increase the open probability (P_o) of the channel without changing channel conductance. The potency of the KCOs to increase K⁺ channel opening was similar to the potency of these compounds to cause coronary artery relaxation. These results indicate that the porcine coronary artery contains the BK_Ca channel and that this channel, along with other types of K⁺ channels (K_ATP), mediate the vasorelaxant effects of K⁺ channel openers.     

Activation of BKCa channels via cyclic AMP- and cyclic GMP-dependent protein kinases by eugenosedin-A in rat basilar artery myocytes

BACKGROUND AND PURPOSE: The study investigated whether eugenosedin-A, a 5-hydroxytryptamine and alpha/beta adrenoceptor antagonist, enhanced delayed-rectifier potassium (K(DR))- or large-conductance Ca(2+)-activated potassium (BK(Ca))-channel activity in basilar artery myocytes through cyclic AMP/GMP-dependent and -independent protein kinases. EXPERIMENTAL APPROACH: Cerebral smooth muscle cells (SMCs) were enzymatically dissociated from rat basilar arteries. Conventional whole cell, perforated and inside-out patch-clamp electrophysiology was used to monitor K(+)- and Ca(2+)-channel activities. KEY RESULTS: Eugenosedin-A (1 microM) did not affect the K(DR) current but dramatically augmented BK(Ca) channel activity in a concentration-dependent manner. Increased BK(Ca) current was abolished by charybdotoxin (ChTX, 0.1 microM) or iberiotoxin (IbTX, 0.1 microM), but not affected by a small-conductance K(Ca) blocker (apamin, 100 microM). BK(Ca) current activation by eugenosedin-A was significantly inhibited by an adenylate cyclase inhibitor (SQ 22536, 10 microM), a soluble guanylate cyclase inhibitor (ODQ, 10 microM), competitive antagonists of cAMP and cGMP (Rp-cAMP, 100 microM and Rp-cGMP, 100 microM), and cAMP- and cGMP-dependent protein kinase inhibitors (KT5720, 0.3 microM and KT5823, 0.3 microM). Eugenosedin-A reversed the inhibition of BK(Ca) current induced by the protein kinase C activator, phorbol myristyl acetate (PMA, 0.1 microM). Eugenosedin-A also prevented BK(Ca) current inhibition induced by adding PMA, KT5720 and KT5823. Moreover, eugenosedin-A reduced the amplitude of voltage-dependent L-type Ca(2+) current (I(Ca,L)), but without modifying the voltage-dependence of the current. CONCLUSIONS AND IMPLICATIONS: Eugenosedin-A enhanced BK(Ca) currents by stimulating the activity of cyclic nucleotide-dependent protein kinases. Physiologically, this activation would result in the closure of voltage-dependent calcium channels and thereby relax cerebral SMCs.     

Amplified NO/cGMP-mediated relaxation and ryanodine receptor-to-BKCa channel signalling in corpus cavernosum smooth muscle from phospholamban knockout mice

BACKGROUND AND PURPOSE

Relaxation of corpus cavernosum smooth muscle (CCSM) is induced by NO. NO promotes the formation of cGMP, which activates cGMP-dependent protein kinase I (PKGI). The large conductance calcium-activated potassium (BK_Ca) channel is regarded as a major target of NO/cGMP signalling; however, the mechanism of BK_Ca activation remains unclear. The aim of the present study was to determine whether sarcoplasmic reticulum (SR) Ca²⁺ load and Ca²⁺ release from the SR via ryanodine receptors (RyRs) is important for BK_Ca channel activation in response to NO/cGMP.

EXPERIMENTAL APPROACH

In vitro myography was performed on CCSM strips from wild-type and PLB knockout (PLB^−/−) mice to evaluate contraction and relaxation in response to pharmacological agents and electrical field stimulation (EFS).

KEY RESULTS

In CCSM strips from PLB^−/− mice, a model of increased SR Ca²⁺ load, contractile force in response to EFS or phenylephrine (PE) was increased by nearly 100%. EFS of strips precontracted with PE induced transient relaxation in CCSM, an effect that was significantly larger in PLB^−/− strips. Likewise, the relaxation of PE-induced contraction in response to SNP and cGMP was greater in PLB^−/−, as demonstrated by a shift in the concentration–response curve towards lower concentrations. Blocking RyRs and BK_Ca channels diminished the induced relaxations and eliminated the difference between wild-type and PLB^−/−.

CONCLUSIONS AND IMPLICATIONS

NO/cGMP activates BK_Ca channels through RyR-mediated Ca²⁺ release. This signalling pathway is responsible for approximately 40% of the NO/cGMP effects and is amplified by increased SR Ca²⁺ concentrations.     

Inhibitory effect of protopine on K(ATP) channel subunits expressed in HEK-293 cells

Protopine is an isoquinoline alkaloid purified from Corydalis tubers and other families of medicinal plants. The purpose of the present study was to investigate the effects of protopine on K(ATP) channels and big conductance (BKCa) channels. Protopine concentration-dependently inhibited K(ATP) channel currents in human embryonic kidney cells (HEK-293) which were cotransfected with Kir6.1 and sulfonylurea receptor 1 (SUR1) subunits, but not that with Kir6.1 cDNA transfection alone. At 25 muM, protopine reversibly decreased Kir6.1/SUR1 currents densities from -17.4+/-3 to -13.2+/-2.4 pA/pF at -60 mV (n=5, P<0.05). The heterologously expressed mSlo-encoded BK(Ca) channel currents in HEK-293 cells were not affected by protopine (25 muM), although iberiotoxin (100 nM) significantly inhibited the expressed BK(Ca) currents (n=5, P<0.05). In summary, protopine selectively inhibited K(ATP) channels by targeting on SUR1 subunit. This discovery may help design specific agents to selectively modulate the function of Kir6.1/SUR1 channel complex and facilitate the understanding of the structure-function relationship of specific subtype of K(ATP) channels.     

Inhalational anaesthetics and n-alcohols share a site of action in the neuronal Shaw2 Kv channel

Background and purpose:

Neuronal ion channels are key targets of general anaesthetics and alcohol, and binding of these drugs to pre-existing and relatively specific sites is thought to alter channel gating. However, the underlying molecular mechanisms of this action are still poorly understood. Here, we investigated the neuronal Shaw2 voltage-gated K⁺ (K_v) channel to ask whether the inhalational anaesthetic halothane and n-alcohols share a binding site near the activation gate of the channel.

Experimental approach:

Focusing on activation gate mutations that affect channel modulation by n-alcohols, we investigated n-alcohol-sensitive and n-alcohol-resistant K_v channels heterologously expressed in Xenopus oocytes to probe the functional modulation by externally applied halothane using two-electrode voltage clamping and a gas-tight perfusion system.

Key results:

Shaw2 K_v channels are reversibly inhibited by halothane in a dose-dependent and saturable manner (K_0.5= 400 µM; n_H= 1.2). Also, discrete mutations in the channel''s S4S5 linker are sufficient to reduce or confer inhibition by halothane (Shaw2-T330L and K_v3.4-G371I/T378A respectively). Furthermore, a point mutation in the S6 segment of Shaw2 (P410A) converted the halothane-induced inhibition into halothane-induced potentiation. Lastly, the inhibition resulting from the co-application of n-butanol and halothane is consistent with the presence of overlapping binding sites for these drugs and weak binding cooperativity.

Conclusions and implications:

These observations strongly support a molecular model of a general anaesthetic binding site in the Shaw2 K_v channel. This site may involve the amphiphilic interface between the S4S5 linker and the S6 segment, which plays a pivotal role in K_v channel activation.     

N-arachidonoyl glycine suppresses Na+/Ca2+ exchanger-mediated Ca2+ entry into endothelial cells and activates BKCa channels independently of GPCRs

Background and Purpose

N-arachidonoyl glycine (NAGly) is a lipoamino acid with vasorelaxant properties. We aimed to explore the mechanisms of NAGly''s action on unstimulated and agonist-stimulated endothelial cells.

Experimental Approach

The effects of NAGly on endothelial electrical signalling were studied in combination with vascular reactivity.

Key Results

In EA.hy926 cells, the sustained hyperpolarization to histamine was inhibited by the non-selective Na⁺/Ca²⁺ exchanger (NCX) inhibitor bepridil and by an inhibitor of reversed mode NCX, KB-R7943. In cells dialysed with Cs⁺-based Na⁺-containing solution, the outwardly rectifying current with typical characteristics of NCX was augmented following histamine exposure, further increased upon external Na⁺ withdrawal and inhibited by bepridil. NAGly (0.3–30 μM) suppressed NCX currents in a URB597- and guanosine 5′-O-(2-thiodiphosphate) (GDPβS)-insensitive manner, [Ca²⁺]_i elevation evoked by Na⁺ removal and the hyperpolarization to histamine. In rat aorta, NAGly opposed the endothelial hyperpolarization and relaxation response to ACh. In unstimulated EA.hy926 cells, NAGly potentiated the whole-cell current attributable to large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels in a GDPβS-insensitive, paxilline-sensitive manner and produced a sustained hyperpolarization. In cell-free inside-out patches, NAGly stimulated single BK_Ca channel activity.

Conclusion and Implications

Our data showed that NCX is a Ca²⁺ entry pathway in endothelial cells and that NAGly is a potent G-protein-independent modulator of endothelial electrical signalling and has a dual effect on endothelial electrical responses. In agonist pre-stimulated cells, NAGly opposes hyperpolarization and relaxation via inhibition of NCX-mediated Ca²⁺ entry, while in unstimulated cells, it promotes hyperpolarization via receptor-independent activation of BK_Ca channels.     

NS11021, a novel opener of large-conductance Ca2+-activated K+ channels,enhances erectile responses in rats

Background and purpose:

Large-conductance Ca²⁺-activated K⁺ channels (BK_Ca), located on the arterial and corporal smooth muscle, are potential targets for treatment of erectile dysfunction (ED). This study investigated whether NS11021 (1-(3,5-Bis-trifluoromethyl-phenyl)-3-[4-bromo-2-(1H-tetrazol-5-yl)-phenyl]-thiourea), a novel opener of BK_Ca channels, relaxes erectile tissue in vitro and enhances erectile responses in intact rats. The effects were compared with sildenafil, an inhibitor of phosphodiesterase type 5.

Experimental approach:

Patch clamp was used to record whole cell current in rat isolated corpus cavernosum smooth muscle cells (SMCs) and human umbilical vein endothelial cells (HUVECs). Isometric tension was measured in intracavernous arterial rings and corpus cavernosum strips isolated from rats and men, and simultaneous measurements of intracellular Ca²⁺ concentration ([Ca²⁺]_i) and tension were performed in intracavernous arteries. Erectile response was measured in anaesthetized rats.

Key results:

In patch clamp recordings, NS11021 increased currents sensitive to the selective BK_Ca channel blocker, iberiotoxin (IbTX) in SMCs, but did not modulate K⁺ current in HUVECs. NS11021 reduced [Ca²⁺]_i and tension in penile arteries. IbTX inhibited the vasorelaxation induced by NS11021 and sildenafil in human erectile tissue. NS11021 and sildenafil but not vehicle increased erectile responses in anaesthetized rats, an effect which was abolished after pretreatment with tetraethylammonium.

Conclusions and implications:

NS11021 leads to relaxation of both intracavernous arteries and corpus cavernosum strips primarily through opening of BK_Ca channels. It is also effective in facilitating erectile responses in anaesthetized rats. These results suggest a potential for use of BK_Ca openers in the treatment of ED.     

The TRPM4 channel inhibitor 9-phenanthrol

The phenanthrene-derivative 9-phenanthrol is a recently identified inhibitor of the transient receptor potential melastatin (TRPM) 4 channel, a Ca²⁺-activated non-selective cation channel whose mechanism of action remains to be determined. Subsequent studies performed on other ion channels confirm the specificity of the drug for TRPM4. In addition, 9-phenanthrol modulates a variety of physiological processes through TRPM4 current inhibition and thus exerts beneficial effects in several pathological conditions. 9-Phenanthrol modulates smooth muscle contraction in bladder and cerebral arteries, affects spontaneous activity in neurons and in the heart, and reduces lipopolysaccharide-induced cell death. Among promising potential applications, 9-phenanthrol exerts cardioprotective effects against ischaemia-reperfusion injuries and reduces ischaemic stroke injuries. In addition to reviewing the biophysical effects of 9-phenanthrol, here we present information about its appropriate use in physiological studies and possible clinical applications.     

Resistance to myocardial infarction induced by heat stress and the effect of ATP-sensitive potassium channel blockade in the rat isolated heart

1. Heat stress (HS) is known to protect against myocardial ischaemia-reperfusion injury by improving mechanical dysfunction and decreasing necrosis. However, the mechanisms responsible for this form of cardioprotection remain to be elucidated. ATP-sensitive potassium (K_ATP) channels have been shown to be involved in the delayed phase of protection following ischaemic preconditioning, a phenomenon closely resembling the HS-induced cardioprotection. The aim of this study was thus to investigate the role of K_ATP channels in HS-induced protection of the isolated rat heart.
2. Twenty four hours after whole body heat stress (at 42°C for 15 min) or sham anaesthesia, isolated perfused hearts were subjected to a 15 min stabilization period followed by a 15 min infusion of either 10 μM glibenclamide (Glib), 100 μM sodium 5-hydroxydecanoate (5HD) or vehicle (0.04% DMSO). Regional ischaemia (35 min) and reperfusion (120 min) were then performed.
3. Prior heat stress significantly reduced infarct-to-risk ratio (from 42.4±2.4% to 19.4±2.9, P<0.001). This resistance to myocardial infarction was abolished in both Glib-treated (40.1±1.8% vs 42.3±1.8%) and 5HD-treated (41.2±1.8% vs 41.8±1.2%) groups.
4. The results of this study suggest that K_ATP channel activation contributes to the cytoprotective response induced by heat stress.

     

Effect of phenothiazine neuroleptic drugs and tricyclic antidepressants on phosphodiesterase activity in rat cerebral cortex

The activity of phosphodiesterase (PDE) of rat cerebral cortex following the administration in vitro and in vivo of various concentrations of neuroleptic phenothiazine drugs and tricyclic antidepressive drugs has been investigated. It has been shown that PDE activity is inhibited by phenothiazine neuroleptic drugs (fluphenazine > trifluperazine > thioproperazine > chlorpromazine = thioridazine). Tricyclic antidepressants nortriptyline, chlorimipramine, protiptyline, imipramine and desipramine at a concentration of 10^–3 M caused 60–80% inhibition of PDE activity. It has also been found that the investigated phenothiazine compounds inhibit the high affinity PDE activity more than the PDE activity of low affinity to the substrate.The results obtained suggest that the mechanism of the neuroleptic action of phenothiazine drugs is partially connected with their influence on cyclic 3,5-AMP metabolism.Supported by Polish Academy of Sciences, 09.4.1.5.     

Functional characterization of large conductance calcium-activated K<Superscript>+</Superscript> channel openers in bladder and vascular smooth muscle

Calcium activated K⁺ channels (K_Ca channels) are found in a variety of smooth muscle tissues, the most characterized of which are the large conductance K_Ca channels (BK_Ca or maxi-K⁺ channels). Recent medicinal chemistry efforts have identified novel BK_Ca openers including 2-amino-5-(2-fluoro-phenyl)-4-methyl-1H-pyrrole-3-carbonitrile (NS-8), BMS-204352 and its analog 3-(5-chloro-2-hydroxy-phenyl)-3-hydroxy-6-trifluoromethyl-1,3-dihydro-indol-2-one (compound 1), and 5,7-dichloro-4-(5-chloro-2-hydroxy-phenyl)-3-hydroxy-1H-quinolin-2-one (compound 2). Although these compounds are effective BK_Ca openers as shown by electrophysiological methods, little is known about their effects on smooth muscle contractility. In this study, the responsiveness of structurally diverse BK_Ca openers—NS-8, compounds 1 and 2 and the well characterized nonselective NS-1619—was assessed using segments of endothelium denuded rat aorta, rat and guinea pig detrusor precontracted with extracellular K⁺, and Landrace pig detrusor stimulated by electrical field. In all preparations, the compounds tested inhibited or completely abolished contractions with similar potencies (–logIC₅₀ values: 3.8 to 5.1). In rat aorta, in the presence of 80 mM K⁺, the compounds significantly shifted the concentration-response curve to the right compared with those obtained in 30 mM K⁺. These data are consistent with K⁺ channel (BK_Ca channel) activation as the underlying mechanism of relaxation by compounds that share the electrophysiological property of BK_Ca current activation. The similar potencies at detrusor and vascular smooth muscle suggest that the achievement of smooth muscle selectivity in vitro with the representative compounds examined in this study may prove to be a challenge when targeting BK_Ca channels for smooth muscle indications such as overactive bladder.Abbreviations KCO Potassium channel opener - BK_Ca Large conductance calcium-activated K⁺ channel - IC₅₀ Molar concentration of test compound for 50% inhibition of response     

Stimulated release of a hyperpolarizing factor (ADHF) from mesenteric artery perivascular adipose tissue: involvement of myocyte BKCa channels and adiponectin

Background and Purpose

Perivascular adipose tissue (PVAT) releases adipocyte-derived hyperpolarizing factors (ADHFs) that may partly act by opening myocyte K⁺ channels. The present study in rat and mouse mesenteric arteries aimed to identify the myocyte K⁺ channel activated by PVAT and to determine whether adiponectin contributed to the hyperpolarizing effects of PVAT.

Experimental Approach

Myocyte membrane potential was recorded from de-endothelialized, non-contracted rat and mouse mesenteric arteries in the presence and absence of PVAT.

Key Results

The β₃-adrenoceptor agonist, CL-316,243 (10 μM), generated PVAT-dependent, iberiotoxin-sensitive myocyte hyperpolarizations resulting from BK_Ca channel opening and which were partially blocked by L-NMMA (100 μM). Adiponectin (5 μg·mL⁻¹) also produced iberiotoxin-sensitive hyperpolarizations in PVAT-denuded arterioles. Activation of myocyte AMP-activated protein kinase (AMPK) using 5 μM A-769662 also induced BK_Ca-mediated hyperpolarizations. Dorsomorphin abolished hyperpolarizations to CL-316,243, adiponectin and A-769662. In vessels from Adipo^−/− mice, hyperpolarizations to CL-316,243 were absent whereas those to A-769662 and adiponectin were normal. In rat vessels, adipocyte-dependent hyperpolarizations were blocked by glibenclamide and clotrimazole but those to NS1619 (33 μM) were unaltered.

Conclusions and Implications

Under basal, non-contracted conditions, β₃-adrenoceptor stimulation of PVAT releases an ADHF, which is probably adiponectin. This activates AMPK to open myocyte BK_Ca channels indirectly and additionally liberates NO, which also contributes to the observed PVAT-dependent myocyte hyperpolarizations. Clotrimazole and glibenclamide each reversed hyperpolarizations to adiponectin and A-769662, suggesting the involvement of myocyte TRPM4 channels in the ADHF-induced myocyte electrical changes mediated via the opening of BK_Ca channels.     

Electrophysiological effects of anandamide on rat myocardium

Background and purpose:

The endocannabinoid, anandamide, has anti-arrhythmic effects. The aim of the present study was to explore the electrophysiological effects of anandamide on rat myocardium.

Experimental approach:

Evoked action potentials (APs) were recorded using intracellular recording technique in rat cardiac papillary muscles. In addition, L-type Ca²⁺ current was measured and analysed using whole-cell patch-clamp recording technique in isolated rat cardiac ventricular myocytes.

Key results:

In cardiac papillary muscles, anandamide (1, 10, 100 nM) decreased AP duration in a concentration-dependent manner. Furthermore, 100 nM anandamide decreased AP amplitude, overshoot and V_max in partially depolarized papillary muscles. These effects were abolished by AM251 (100 nM), a selective antagonist for CB₁ receptors, but not AM630 (100 nM), a CB₂ receptor antagonist. Furthermore, an agonist of L-type Ca²⁺ channels, Bay K 8644 (0.5 µM), a K⁺ channel blocker tetraethylammonium chloride (20 mM) and the nitric oxide synthase inhibitor l-NAME (1 mM) had no effect on anandamide-induced decrease in AP duration. In isolated ventricular myocytes, anandamide (1, 10, 100 nM) decreased L-type Ca²⁺ current concentration-dependently, and shifted the current–voltage relationship curve of the Ca²⁺ current. Anandamide (100 nM) shifted the steady-state inactivation curve to the left and the recovery curve to the right. Blockade of CB₁ receptors with AM251 (100 nM), but not CB₂ receptors with AM630 (100 nM), eliminated the effect of anandamide on L-type Ca²⁺ currents.

Conclusions and implications:

These data suggest that anandamide suppressed AP and L-type Ca²⁺ current in cardiac myocytes through CB₁ receptors.     

Effect of chronic ethanol treatment in vivo on excitability in mouse cortical neurones in vitro

1. The effects of cessation of chronic ethanol ingestion on seizure activity in vivo and on the characteristics of the evoked synaptic potentials in cortical neurones in vitro have been investigated in mice. Withdrawal from chronic ethanol treatment increased handling seizure ratings in mice between 4 and 16 h post-withdrawal. This ethanol-induced increase in seizure rating was unaffected by carbamazepine (30 mg kg⁻¹) but significantly reduced at a higher concentration (130 mg kg⁻¹).
2. Intracellular recordings were made from cortical layer II neurones in vitro from control mice and from mice following chronic ethanol ingestion. Evoked synaptic potentials were generated in these neurones through intralaminar stimulation.
3. Neurones from control mice displayed an evoked potential consisting of a fast excitatory postsynaptic potential (e.p.s.p.) mediated by AMPA-type glutamate receptors and an inhibitory postsynaptic potential (i.p.s.p.) mediated via GABA_A receptors. Application of pentylenetetrazole (PTZ) or bicuculline onto these neurones inhibited the i.p.s.p., caused a large increase in both the amplitude and duration of the e.p.s.p. and initiated spontaneous excitatory activity. The resulting large evoked e.p.s.p. was mediated via both NMDA- and AMPA-type glutamate receptors.
4. Most neurones (77%) from ethanol treated mice displayed an evoked potential which comprised a large e.p.s.p. and no i.p.s.p. The e.p.s.p. consisted of several distinct components and in addition these neurones displayed spontaneous paroxysmal depolarizing shifts. This multi-component e.p.s.p. was mediated through both NMDA- and AMPA-type glutamate receptors. A population (23%) of neurones from ethanol treated mice exhibited evoked potentials which possessed both inhibitory and excitatory components and these neurones were effectively identical to those obtained from control mice.
5. Carbamazepine reduced the duration of the e.p.s.p. in neurones from ethanol treated mice and in PTZ-treated control neurones.
6. Prolonged ethanol ingestion is known to create a neurochemical imbalance in cortical neurones resulting in abnormal neurotransmission. The present study highlights the functional consequences that arise as a result of these neurochemical changes leading to over-excitation of neurones and pronounced epileptiform activity.

     

K(ATP) channel expression and pharmacological in vivo and in vitro studies of the K(ATP) channel blocker PNU-37883A in rat middle meningeal arteries

BACKGROUND AND PURPOSE: Dilatation of cerebral and dural arteries causes a throbbing, migraine-like pain, indicating that these structures are involved in migraine.Clinical trials suggest that adenosine 5'-triphosphate-sensitive K(+) (K(ATP)) channel opening may cause migraine by dilatating intracranial arteries, including the middle meningeal artery (MMA). We studied the K(ATP) channel expression profile in rat MMA and examined the potential inhibitory effects of the K(ATP) channel blocker PNU-37883A on K(ATP) channel opener-induced relaxation of the rat MMA, using the three K(ATP) channel openers levcromakalim, pinacidil and P-1075. EXPERIMENTAL APPROACH: mRNA and protein expression of K(ATP) channel subunits in the rat MMA were studied by quantitative real-time PCR and western blotting, respectively. The in vivo and in vitro effects of the K(ATP) channel drugs on rat MMA were studied in the genuine closed cranial window model and in myograph baths, respectively. KEY RESULTS: Expression studies indicate that inwardly rectifying K(+) (Kir)6.1/sulphonylurea receptor (SUR)2B is the major K(ATP) channel complex in rat MMA. PNU-37883A (0.5 mg kg(-1)) significantly inhibited the in vivo dilatory effect of levcromakalim (0.025 mg kg(-1)), pinacidil (0.38 mg kg(-1)) and P-1075 (0.016 mg kg(-1)) in rat MMA. In vitro PNU-37883A significantly inhibited the dilatory responses of the three K(ATP) channel openers in rat MMA at 10(-7) and 3 x 10(-7) M. CONCLUSIONS AND IMPLICATIONS: We suggest that Kir6.1/SUR2B is the major functional K(ATP) channel complex in the rat MMA. Furthermore, we demonstrate the potent in vivo and in vitro blocking potentials of PNU-37883A on K(ATP) channel opener-induced relaxation of the rat MMA.     

Block of potassium currents in rat isolated sympathetic neurones by tricyclic antidepressants and structurally related compounds.

1. The block of K+ currents by the tricyclic antidepressants (TCAs), imipramine and amitriptyline and three structurally related compounds, chlorpromazine, tacrine and carbamazepine was investigated in rat isolated sympathetic neurones by whole-cell voltage-clamp recording. 2. At a concentration of 10 microM, imipramine, amitriptyline and chlorpromazine all blocked the delayed rectifier K+ current (IKv) by about the same extent, 54%, 47% and 53%. Tacrine was less effective (10%) while carbamazepine was ineffective at all concentrations tested. 3. The degree of block by the four effective compounds was relatively independent of the size of the voltage-step. Neither the activation nor the inactivation rates of IKv were altered by the blocking drugs. 4. Concentration-response relationships for imipramine and tacrine showed that imipramine was about 7 fold more potent than tacrine but that the maximum inhibition and the Hill slope were the same for both compounds. 5. Amitriptyline, chlorpromazine and imipramine (at 10 microM) were 2-3 fold more potent at inhibiting the sustained K+ current (mostly IKv) than the transient K+ current (mostly IA). Tacrine, however, was equally effective in blocking both components.     

A comparative study of the effects of Cl(-) channel blockers on mesenteric vascular conductance in anaesthetized rat

There is evidence to suggest that niflumic acid is capable of selectively inhibiting Ca(2+)-dependent Cl(-) channels. Furthermore, it has been demonstrated that niflumic acid is capable of antagonizing contractile responses due to activation of alpha(1)-adrenoceptor in mesenteric vasculature. Here, we have examined the effects of three Cl(-) channel blockers, niflumic acid, indanyloxyacetic acid 94 (IAA-94) and diphenylamine-2-carboxylic acid (DPC) on cirazoline-mediated vasoconstriction in mesenteric blood vessel in vivo. Infusion of cirazoline produced a dose-dependent increase in blood pressure, decrease in superior mesenteric blood flow, mesenteric vascular conductance and heart rate. While niflumic acid and IAA-94 did not have any impact on cirazoline-induced changes in blood pressure, DPC accentuated the pressor effect of cirazoline. Neither agent affected cirazoline-mediated reflex reduction in the heart rate. Niflumic acid, IAA-94 and DPC attenuated alpha(1)-adrenoceptor mediated decrease in mesenteric blood flow and vascular conductance. Based on the profile of the actions of these compounds, it may be suggested that IAA-94 did not appear to act as selective inhibitor of Ca(2+)-activated Cl(-) channels when compared to niflumic acid in the mesenteric blood vessels. In addition, while DPC seems to be as effective as niflumic acid in its effects on mesenteric blood vessels, its actions may be attributed to other pharmacological effects.     

Large-conductance K+ channel openers NS1619 and NS004 as inhibitors of mitochondrial function in glioma cells

Recently, it has been reported that large-conductance Ca(2+)-activated potassium channels, also known as BK(Ca)-type potassium channels, are present in the inner mitochondrial membrane of the human glioma LN229 cell line. Hence, in the present study, we have investigated whether BK(Ca)-channel openers (BK(Ca)COs), such as the benzimidazolone derivatives NS004 (5-trifluoromethyl-1-(5-chloro-2-hydroxyphenyl)-1,3-dihydro-2H-benzimidazole-2-one) and NS1619 (1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimidazol-2-one), affect the functioning of LN229 glioma cell mitochondria in situ. We examined the effect of BK(Ca)COs on mitochondrial membrane potential, mitochondrial respiration and plasma membrane potassium current in human glioma cell line LN229. We found that BK(Ca)COs decrease the mitochondrial membrane potential with an EC(50) value of 3.6+/-0.4 microM for NS1619 and 5.4+/-0.8 microM for NS004. This mitochondrial depolarization was accompanied by an inhibition of the mitochondrial respiratory chain. Both BK(Ca)COs induced whole-cell potassium current blocked by charybdotoxin, as measured by the patch-clamp technique. The BK(Ca)COs had no effect on membrane bilayer conductance. Moreover, the inhibition of mitochondrial function by NS004 and NS1619 was without effect on cell survival, as measured by lactate dehydrogenase release from the cells.     

Inhibitory effects of sevoflurane on pacemaking activity of sinoatrial node cells in guinea-pig heart

BACKGROUND AND PURPOSE

The volatile anaesthetic sevoflurane affects heart rate in clinical settings. The present study investigated the effect of sevoflurane on sinoatrial (SA) node automaticity and its underlying ionic mechanisms.

EXPERIMENTAL APPROACH

Spontaneous action potentials and four ionic currents fundamental for pacemaking, namely, the hyperpolarization-activated cation current (I_f), T-type and L-type Ca²⁺ currents (I_Ca,T and I_Ca,L, respectively), and slowly activating delayed rectifier K⁺ current (I_Ks), were recorded in isolated guinea-pig SA node cells using perforated and conventional whole-cell patch-clamp techniques. Heart rate in guinea-pigs was recorded ex vivo in Langendorff mode and in vivo during sevoflurane inhalation.

KEY RESULTS

In isolated SA node cells, sevoflurane (0.12–0.71 mM) reduced the firing rate of spontaneous action potentials and its electrical basis, diastolic depolarization rate, in a qualitatively similar concentration-dependent manner. Sevoflurane (0.44 mM) reduced spontaneous firing rate by approximately 25% and decreased I_f, I_Ca,T, I_Ca,L and I_Ks by 14.4, 31.3, 30.3 and 37.1%, respectively, without significantly affecting voltage dependence of current activation. The negative chronotropic effect of sevoflurane was partly reproduced by a computer simulation of SA node cell electrophysiology. Sevoflurane reduced heart rate in Langendorff-perfused hearts, but not in vivo during sevoflurane inhalation in guinea-pigs.

CONCLUSIONS AND IMPLICATIONS

Sevoflurane at clinically relevant concentrations slowed diastolic depolarization and thereby reduced pacemaking activity in SA node cells, at least partly due to its inhibitory effect on I_f, I_Ca,T and I_Ca,L. These findings provide an important electrophysiological basis of alterations in heart rate during sevoflurane anaesthesia in clinical settings.