Effects of monocarboxylic acid-derived Cl- channel blockers on depolarization-activated potassium currents in rat ventricular myocytes

The effects of monocarboxylic acid-derived Cl(-) channel blockers on cardiac depolarization-activated K(+) currents were investigated. Membrane currents in rat ventricular myocytes were recorded using the whole-cell configuration of the patch-clamp technique. 5-Nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) and niflumic acid (NFA) induced an outward current at 0 mV. Both NPPB and NFA failed to induce any current when used intracellularly or after K(+) in the bath and pipette solutions was replaced by equimolar Cs(+). Voltage pulse protocols revealed that NPPB and NFA enhanced the steady-state K(+) current but inhibited the transient outward K(+) current. Genistein, a tyrosine kinase (PTK) inhibitor, inhibited NPPB- and NFA-induced outward current. Another PTK inhibitor, lavendustin A, produced a comparable effect. In contrast, the inactive analogue of genistein, daidzein, was ineffective. Orthovanadate, a tyrosine phosphatase inhibitor, markedly slowed the deactivation of the outward current induced by NPPB and NFA. The protein kinase A (PKA) inhibitor H-89 inhibited NPPB-induced outward current at 0 mV. In contrast, the protein kinase C (PKC) inhibitor H-7 was without significant effect on the action of NPPB. Pretreatment of the myocytes with genistein or H-89 prevented the enhancing effect of NPPB. Increasing intracellular Cl(-) from 22 to 132 mm slightly reduced NPPB-induced outward current at 0 mV. These results demonstrate that the monocarboxylic acid-derived Cl(-) channel blockers NPPB and NFA enhance cardiac steady-state K(+) current, and suggest that the enhancing effect of the Cl(-) channel blockers is mediated by stimulation of PKA and PTK signalling pathways.     

Inhibitory effects of potassium channel blockers on carbachol-induced contraction in rat detrusor muscle

We present accidental findings that potassium channel blockers, such as tetraethyl-ammonium (TEA) or 4-aminopyridine (4-AP), inhibit the sustained tonic contraction induced by carbachol in rat detrusor muscle strips. The relatively lower concentrations (<2 mM) of TEA and 4-AP inhibited phasic and tonic contractions induced by 5 micro M carbachol, whilst the relatively higher concentrations of TEA and 4-AP (>5 mM) potentiated phasic contractions. The potentiation of phasic contraction was not observed in nicardipine pretreated condition. In nicardipine pretreated condition, the concentration-response curves for the negative inotropic effect of potassium channel blockers were shifted to the right by the increasing concentration of carbachol from 0.5 microM to 5 microM. IC50 was changed significantly from 0.19 to 0.64 mM (TEA) and from 0.21 to 0.96 (4-AP). Such inhibitory effects were also observed in Ca2+ depleted condition, where 0.1 mM EGTA and 1 microM thapsigargin were added into Ca2+ free solution. In conclusion, inhibitory effects of potassium channel blockers on carbachol-induced contraction may be ascribed to the direct inhibition of receptor-agonist binding.     

Effects of chloride and potassium channel blockers on apoptotic cell shrinkage and apoptosis in cortical neurons

K⁺ and Cl^– homeostasis have been implicated in cell volume regulation and apoptosis. We addressed the hypothesis that K⁺ and Cl^– efflux may contribute to apoptotic cell shrinkage and apoptotic death in cultured cortical neurons. CLC-2 and CLC-3 chloride channels were detected in cultured cortical neurons. The Cl^– channel blockers 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS), 4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid (SITS) and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) inhibited the outwardly rectifying Cl^– current, prevented apoptotic cell shrinkage, and mildly attenuated cell death induced by staurosporine, C₂-ceramide, or serum deprivation. Cl^– channel blockers, however, at concentrations that prevented cell shrinkage had no significant effects on caspase activation and/or DNA fragmentation. Cell death in the presence of a Cl^– channel blocker was still sensitive to blockade by the caspase inhibitor z-Val-Ala-Asp(OMe)-fluoromethyl ketone (z-VAD-fmk). Electron microscopy revealed that, although DIDS prevented apoptotic cell shrinkage, certain apoptotic ultrastructural alterations still took place in injured neurons. On the other hand, the K⁺ channel blocker tetraethylammonium (TEA), clofilium, or the caspase inhibitor z-VAD-fmk prevented cell shrinkage as well as caspase activation and/or DNA damage, and showed stronger neuroprotection against apoptotic alterations and cell death. The results indicate that neurons may undergo apoptotic process without cell shrinkage and imply distinct roles for Cl^– and K⁺ homeostasis in regulating different apoptotic events.     

钙通道阻滞剂对大鼠肾上腺嗜铬细胞烟碱受体通道电流的影响

目的:研究钙通道阻滞剂(CCB)对大鼠肾上腺嗜铬细胞(RAMC) 烟碱受体通道电流(I_NIC)的作用。方法: 利用全细胞膜片钳技术,用烟碱(NIC)诱发RAMC的NIC受体通道电流,分别观察硝苯吡啶(NIF)、ω-conotoxin GVIA和ω-agatoxin IVA急性灌洗细胞前后I_NIC的变化。结果: 不同类型和浓度的CCB分别灌洗RAMC ,均能明显抑制NIC所诱发的细胞I_NIC,灌洗5 min时 10 μmol/L NIF、400 nmol/L ω-conotoxin GVIA和100 nmol/L ω-agatoxin IVA对I_NIC的峰值抑制率分别为(61.7±5.1)%、(29.3±7.4)%和(17.6±7.5)%。结论: 不同类型的CCB急性作用于RAMC能明显阻滞NIC所诱发的I_NIC,提示CCB可能通过直接阻滞NIC受体通道来抑制RAMC儿茶酚胺的分泌过程。     

Effects of noradrenaline on some potassium currents in CA1 neurones in rat hippocampal slices

Pyramidal (CA1) cells in rat hippocampal slices were voltage clamped using a single electrode voltage clamp. In the presence of tetrodotoxin (TTX), depolarizing pulses from holding potentials of −60 to −70 mV elicited a slow inward calcium (Ca²⁺) current and two outward potassium (K⁺) currents: an A current and a slower, Ca²⁺-dependent K⁺ current. Noradrenaline (NA) (20 μM) depressed the amplitude of the K⁺ currents without affecting the Ca²⁺ current. The effect of NA could be blocked with propranolol and was mimicked by isoprenaline, suggesting that NA depresses the K⁺ currents by binding to β-receptors.     

Effects of T-type, L-type, N-type, P-type, and Q-type calcium channel blockers on stimulus-induced pre-and postsynaptic calcium fluxes in rat hippocampal slices

The contribution of T-, L-, N-, P-, and Q-type Ca²⁺ channels to pre-and postsynaptic Ca²⁺ entry during stimulus-induced high neuronal activity in area CA1 of rat hippocampal slices was investigated by measuring the effect of specific blockers on stimulus-induced decreases in extracellular Ca²⁺ concentration ([Ca²⁺]₀). [Ca²⁺]₀ was measured with ion-selective electrodes in stratum radiatum (SR) and stratum pyramidale (SP), while Ca²⁺ entry into neurons was induced with stimulus trains (20 Hz for 10 s) alternately delivered to SR and the alveus, respectively. The [Ca²⁺]₀ decreases recorded in SR in response to SR stimulation represented mainly presynaptic Ca²⁺ entry (Ca_pre), while [Ca²⁺]⁰ decreases recorded in SP in response to alvear stimulation were predominantly based on postsynaptic Ca²⁺ entry (Ca_post). Ethosuximide and trimethadione were ineffective m concentrations up to 1 mM. At 10 mM, they reduced Ca_post and, much less, also Ca_pre Nimodipine (25 M) reduced Ca_post and, to a minor extent, Ca_pre. -Agatoxin IVA (0.4–1 M) and -conotoxin MVIIC (1 M) also reduced both Ca_pre and Ca_post, but with a stronger action on Ca_pre. -Conotoxin GVIA (3–8 M) reduced Ca_post without effect on Ca_pre. We conclude that during stimulus-induced, high-frequency neuronal activity Ca_post is carried by P/Q-, N-, and L-type channels and probably a further channel type different from these channels. Ca_pre includes at least P/Q-and possibly L-type channels. N-type channels did not contribute to Ca_pre in our experiments. Since ethosuximide and trimethadione were only effective in high concentrations, their action may be unspecific. Thus, T-type channels do not seem to play a major part in Ca²⁺ entry in this situation.     

Nonlinear effects of potassium channel blockers on endothelium‐dependent hyperpolarization

In a number of published studies on endothelium‐dependent hyperpolarization and relaxation, the results of the effects of K⁺ blockers have been difficult to interpret. When the effects of two blockers have been studied, often either blocker by itself had little effect, whereas the two blockers combined tended to abolish the responses. Explanations suggested in the literature include an unusual pharmacology of the K⁺ channels, and possible blocker binding interactions. In contrast, when we applied the same blockers to segments of small blood vessels under voltage clamp, the blockers reduced the endothelium‐dependent K⁺ current in a linearly additive manner. Resolution of these contrasting results is important as endothelium‐derived hyperpolarization (EDH) makes its greatest contribution to vasorelaxation in arterioles and small resistance arteries, where it can exert a significant role in tissue perfusion and blood pressure regulation. Furthermore, EDH is impaired in various diseases. Here, we consider why the voltage‐clamp results differ from earlier free‐running membrane potential and contractility studies. We fitted voltage‐clamp‐derived current–voltage relationships with mathematical functions and considered theoretically the effects of partial and total block of endothelium‐derived K⁺‐currents on the membrane potential of small blood vessels. When the K⁺‐conductance was partially reduced, equivalent to applying a single blocker, the effect on EDH was small. Total block of the endothelium‐dependent K⁺ conductance abolished the hyperpolarization, in agreement with various published studies. We conclude that nonlinear summation of the hyperpolarizing response evoked by endothelial stimulation can explain the variable effectiveness of individual K⁺ channel blockers on endothelium‐dependent hyperpolarization and resulting relaxation.     

Effect of Cl- channel blockers on aconitine-induced arrhythmias in rat heart

The effects of Cl- channel blockers 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and niflumic acid (NFA) on aconitine-induced arrhythmias were investigated. Left ventricular pressure and electrocardiogram were monitored in Langendorff-perfused rat hearts. Whole-cell patch-clamp and current-clamp techniques were used to measure sodium current (I(Na)) and action potential (AP), respectively, in single rat cardiac ventricular myocytes. Addition of the Na+ channel agonist aconitine (0.1 microM) to the perfusion solution produced polymorphic ventricular arrhythmias with a latent period of 25.5 +/- 6.3 s. NPPB could reverse aconitine-induced arrhythmias. A similar effect was observed by using NFA. NPPB and NFA reversibly depressed the upstroke of the AP in a dose-dependent manner with IC50 values of approximately 12.3 and approximately 73.1 microM, respectively, without significantly affecting the resting potential of rat ventricular myocytes. Both Cl- channel blockers inhibited I(Na) and induced a leftward shift of the steady-state inactivation of I(Na). In conclusion, the results of this study demonstrate that NPPB as well as NFA can suppress aconitine-induced arrhythmias in rat hearts mainly by inhibiting cardiac I(Na).     

N-methylaspartate-activated calcium channels in rat brain cortex slices. Effect of calcium channel blockers and of inhibitory and depressant substances

N-Methyl-DL-aspartate, L-glutamate, kainate and DL-homocysteate were found to increase the initial rate and the maximal uptake of 45Ca into the non-inulin space of rat brain cortex slices incubated in vitro. The N-methylaspartate-stimulated calcium uptake was blocked by cadmium and cobalt ions, but not by the organic calcium channel blocker nifedipine or by tetrodotoxin, both of which stimulated the N-methylaspartate-independent calcium influx. gamma-Aminobutyrate increased the spontaneous calcium influx, and also reduced that stimulated by N-methylaspartate to the same level, as found with gamma-aminobutyrate alone. Adenosine (1-100 microM), ethanol (0.1 M), pentobarbital (10-100 microM) and morphine (0.2 mM), were unable to inhibit the N-methylaspartate-activated calcium influx. Ethanol (0.1 M), had no effect on the glutamate- or kainate-activated calcium influx. These findings suggest that the excitatory amino acids, because of their neuronal depolarizing action in brain cortex, lead to the opening of voltage-sensitive calcium channels, which may be blocked by cadmium, but not by the organic calcium channel antagonist, nifedipine. The activation of calcium channels by the excitatory amino acid N-methylaspartate, was entirely unaffected by the depressants ethanol, pentobarbital or morphine, or by the endogenous inhibitory substance, adenosine, thus suggesting that their inhibitory or depressant effects occur through interference with a neuronal mechanism unrelated to the one studied here. gamma-Aminobutyrate, on the other hand, considerably inhibited N-methylaspartate-induced calcium uptake, an effect interpreted as due to a gamma-aminobutyrate-induced increase in chloride conductance, that "clamps" the membrane potential and does not allow further depolarization by N-methylaspartate.     

Suppression of epileptiform burst discharges in CA3 neurons of rat hippocampal slices by the organic calcium channel blocker,verapamil

Summary We studied the effects of the organic calcium channel blocker, verapamil, on spontaneous and bicuculline-induced epileptiform burst discharges in CA3 pyramidal cells of hippocampal slices. A transient increase of burst discharge rate was observed in most cells within 30 min after the addition of verapamil (100 M) to the perfusing medium. Prolonged verapamil perfusions gradually reduced the rate and duration of burst discharges, then abolished them in all tested slices (over periods of 50–150 min) without blocking synaptic transmission. Responses to intracellular injections of current pulses were also gradually affected by verapamil: Action potential amplitude was decreased, action potential duration increased, frequency adaptation increased, amplitude of the fast hyperpolarization following a single action potential decreased, and amplitude and duration of the slow afterhyperpolarization markedly reduced. The amplitude of calcium spikes elicited in slices perfused with tetrodotoxin-containing medium was not affected by verapamil, but the mean velocity of depolarization near the peak of the calcium spike was decreased. Membrane resting potential and input resistance were not affected by verapamil. These results confirm that verapamil is able to suppress epileptiform activity, but suggest that this effect is rather non-specific, due to inhibition of both postsynaptic sodium and calcium conductances.     

ROS介导线粒体ATP敏感性钾通道开放剂对缺氧脑的保护作用

目的：观察线粒体ATP敏感性钾通道(mitoKATP)及活性氧（ROS）在缺氧脑保护中的作用及其相互关系。 方法： 采用脑片灌流及电生理学技术，细胞外记录海马CA1区的群体锋电位(PS)和缺氧去极化电位(HD)。 结果： 用mitoKATP开放剂diazoxide (300 μmol/L) 预处理海马脑片，可延长HD的潜伏期及缺氧后PS消失的时间，提高复氧后PS的恢复率。该作用可被mitoKATP阻断剂5-hydroxydecanoic acid (200 μmol/L) 所阻断。以ROS清除剂N-2-mercaptopropionyl glycine (MPG) (500 μmol/L) 预处理海马脑片，可减弱diazoxide 的作用。单独使用MPG对PS及HD无明显影响。 结论： ROS介导了mitoKATP开放剂对缺氧脑的保护作用。     

吗啡对大鼠海马神经元钾、钙通道的作用

吗啡是常见的阿片类镇痛药,长时间应用可导致吗啡耐受和依赖,其作用机制十分复杂。海马中同时包含μ,κ,δ阿片受体,与吗啡耐受及依赖有一定的关系,并参与机体的痛觉调制过程,因而了解吗啡对海马神经元离子通道的作用对阐明吗啡的镇痛、耐受及依赖机制具有重要的理论及实际意义。本文报导吗啡对培养的海马神经元电压门控性钾、钙     

Inhibition of the generation of cytotoxic lymphocytes by potassium ion channel blockers.

Recent studies with patch-clamp technique have shown the presence of voltage-gated K+ channels in human T lymphocytes and natural killer cells. Blockers of voltage-gated K+ channel currents (4-Aminopyridine, 4-AP, and tetraethylammonium, TEA), were used here in a pharmacological approach to examine a role of K+ channels in the differentiation of precursors of cytotoxic cells into functionally active cytotoxic lymphocytes. The data presented here demonstrated that activation of peripheral blood lymphocytes with CCRF-HSB-2, 3163 and other allogeneic lymphoid cells for 5 days in mixed lymphocyte culture (MLC) renders them cytotoxic to the respective target cells. Both 4-AP and TEA (2-4 mM), when added to cultures, inhibited the development of cytotoxic effectors in a dose-dependent manner. Maximum inhibition of the generation of cytotoxic lymphocytes occurred when 4-AP was present at the start of cultures. Little or no inhibition was, however, observed when 4-AP was added 1 day of incubation. The results also demonstrate that the addition of recombinant IL-2 (rIL-2) overcame the 4-AP- or TEA-mediated inhibition of the generation of cytotoxic lymphocytes in a dose-dependent manner. The maximum reversal of 4-AP-induced inhibition occurred when exogenous IL-2 was added at Day 0 or 1. Taken together, these data suggest a role of K+ channels in the generation of cytotoxic lymphocytes.     

Changes in sensomotor cortical peptide levels in the rat brain on the appearance of poststimulus paroxysmal discharges

Research Institute of General Pathology and Pathological Physiology, Academy of Medical Sciences of the USSR, Moscow. Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 113, No. 2, pp. 118–120, February, 1992.     

Effects of calcium channel blockers on isolated carotid baroreceptors and baroreflex

Our study determined the effects of the calcium antagonists, nifedipine and verapamil, on the carotid sinus baroreceptors and baroreflex. The left carotid sinus region in dogs was vascularly isolated and filled with oxygenated physiological salt solution. Steady-state multiunit activity was recorded from the carotid sinus nerve for sinus pressures of 50-200 mmHg after bathing the carotid sinus region in a solution containing no drug, 10 micrograms/ml nifedipine (n = 6), or 5 micrograms/ml verapamil (n = 5). The slopes of the curves relating carotid sinus nerve activity (% of maximum control) to carotid sinus pressure were control, 0.81 +/- 0.06; nifedipine, 1.29 +/- 0.14; and verapamil, 0.48 +/- 0.06%/mmHg, indicating that nifedipine increased and verapamil decreased the sensitivity of the carotid sinus baroreceptors. Additional studies with bilateral carotid sinus isolation (carotid sinus nerves intact) indicated that nifedipine enhanced and verapamil attenuated carotid baroreflex control of renal sympathetic nerve activity. Pressure-volume curves generated in the isolated carotid sinus showed that effects on smooth muscle do not account for the opposing effects of the two Ca2+ antagonists. Omitting Ca2+ from the physiological solution resulted in increased carotid sinus nerve activity, an effect blocked by verapamil but not nifedipine. Verapamil, but not nifedipine, inhibited veratrine-induced (Na+-dependent) excitation of carotid baroreceptors. Thus the excitatory effects of nifedipine on the carotid sinus baroreceptors are dependent on Ca2+ mechanisms, whereas the inhibitory effects of verapamil may be due mainly to interference with the inward Na+ current.     

Potassium channel blockers inhibit anion secretion in cultured rat epididymal epithelium

The effect of putative K channel blockers on anion secretion has been studied in primary monolayer cultures of rat epididymal cells using the short circuit current technique. Under basal conditions, monolayers had a transepithelial potential difference of about 2-3 mV, apical side negative and a short circuit current (SCC) of about 2 microA.cm-2. The transepithelial resistance was about 500 omega.cm2. Addition of adrenaline (0.23 microM, basolaterally) caused the SCC to rise to a peak value of about 10.5 microA.cm-2 and then stabilized at about 4 microA.cm-2 after 15 min. This rise in the short circuit current has previously been shown to be due to an increase in net anion secretion from the basolateral to the apical medium. In tissues stimulated with adrenaline, addition of barium (Ba) to the apical side did not affect the adrenaline-induced SCC, but addition to the basolateral side caused a dose-dependent inhibition of the current with an IC50 value (concentration required to inhibit 50% of the current) of 0.92 mM. At Ba concentration of 5 mM, the adrenaline-induced SCC was completely abolished. There was no effect on transepithelial resistance. Addition of tetraethylamonium (TEA) (16 mM) to the apical or basolateral side had no significant effect on the adrenaline-stimulated SCC. Lidocaine and quinidine inhibited the adrenaline-stimulated SCC when added either to the apical or basolateral bathing solution. The IC50 values for lidocaine were 0.42 mM and 0.35 mM for basolateral and apical application, respectively. The IC50 values for quinidine were 0.062 mM and 0.050 mM for basolateral and apical application, respectively. In all cases there was no change in tissue resistance. It is proposed that in the basolateral membrane of the epididymal cells, there is a component which is sensitive to putative K channel blockers. It is likely that it is a K channel. As in other secretory cells, this channel plays an important role in secretion.     

Suramin-sensitive suppression of paired-pulse inhibition by adenine nucleotides in rat hippocampal slices

In order to assess the possible presence of presynaptic P2 receptors for nucleotides in the hippocampus, adenosine triphosphate and betagamma-methyleneATP have been examined on paired-pulse inhibition in rat hippocampal slices. Both compounds reproduced the effects of adenosine and reduced the amount of paired-pulse inhibition at an interpulse interval of 10 ms and increased the amount of facilitation at intervals of 20 and 50 ms. These effects were prevented by 8-phenyltheophylline and adenosine deaminase, indicating their mediation by adenosine. The effects were also reduced by suramin at 50 microM, suggesting the possible activation of P2 receptors. It is suggested that a population of P2 receptors may exist which promote the release of endogenous adenosine in the hippocampus.