The Ba2+ block of the ATP-sensitive K+ current of mouse pancreaticβ-cells

We studied the block of whole-cell ATP-sensitive K⁺ (K_ATP) currents in mouse pancreatic-cells produced by external Ba²⁺. Ba²⁺ produced a time- and voltage-dependent block of K_ATP currents, both the rate and extent of the block increasing with hyperpolarization. With 5.6 mM [K⁺]_o, the relationship between the steady-state KATP current and [Ba²⁺]_o, was fit by the Hill equation with aK _d of 12.5 ± 2.8 M at –123 mV and of 0.18 ± 0.02 mM at –62 mV The Hill coefficient (n) was close to 1 at all potentials indicating that binding of a single Ba²⁺ ion is sufficient to block the channel. When [K⁺]_o was raised to 28 mM the K_d was little changed (12.4 ± 4.1 gM at –123 mV 0.27 ± 0.05 mM at –62 mV) and n was unaffected, suggesting that K⁺ does not interact with the Ba²⁺ binding site. The kinetics of Ba²⁺ block were slow, 10 M Ba²⁺ blocking the K_ATP current with a time constant of 20 ms at –123 mV in 28 mM [K⁺]_o. The blocking rate constant was calculated as 1.7 mM^–1 ms^–1 and the unblocking rate as 0.02 ms^–1, at –123 mV The data are discussed in terms of a model in which Ba²⁺ binds to a site at the external mouth of the channel to inhibit the K_ATP channel.     

The control of chloride conductance in rat parotid isolated acinar cells investigated by photorelease of caged compounds

The control of Cl^– conductance in rat parotid isolated acinar cells was studied by combined use of whole-cell recording and flash photolysis techniques. Cells were voltage-clamped either at a membrane potential of –40 mV or stepped between –85 mV and 0 mV. Bath-applied carbachol and noradrenaline evoked Cl^– current at –85 mV and K⁺ current at 0 mV. Similar current activations resulted from the photolytic release of either inositol trisphosphate (InsP ₃) or Ca²⁺ by a brief near-UV flash. The peak amplitudes of the Cl^– conductance (at –85 mV), measured relative to the K⁺ conductance (at 0 mV), evoked by application of carbachol, noradrenaline or direct manipulation of cytosolic free calcium ([Ca²⁺]_i), were very similar, being 0.56±0.09 (mean±SEM,n=9), 0.52 ± 0.01 (n=7) and 0.46±0.06 (n=7). In contrast, the relative amplitude of the Cl^– conductance evoked by InsP₃ was much larger: 1.49±0.24 (n=9). Neither bath application of isoprenaline nor photolysis of caged cAMP induced any detectable membrane current. The most probable interpretation of these results is that the observed activation of Cl^– conductance by agonists can be explained by the elevation of [Ca²⁺]_i alone. In addition, the present results provide further support for the previously reported suggestion that the Cl^– channels and the Ca²⁺-release sites are co-localised [10].     

KATP channel current increases in postinfarction remodeled cardiomyocytes

Adenosintriphosphate-sensitive potassium channels (K_ATP channels) are an important linkage between the metabolic state of a cell and electrophysiological membrane properties. In this study, K_ATP channels were studied in myocytes of normal and remodeled myocardium of the rat. Myocardial infarction was induced by ligature of the left anterior descending artery. Remodeled myocytes were obtained from the hypertrophied posterior left ventricular wall and interventricular septum 3 months after infarction. The current through K_ATP channels was measured in whole-cell and inside-out patches by using the patch-clamp technique. After myocardial infarction, the heart weight/body weight ratio was doubled and the myocytes were hypertrophied yielding a cell capacitance of 266±16 pF compared to 122±12 pF in control cells. The amount of Kir6.2 protein was indistinguishable in corresponding regions of control and remodeled hearts. The ATP sensitivity of K_ATP channels in remodeled cells was significantly lower than in control cells (half maximum block at 115 μmol/l ATP in remodeled and at 71 μmol/l ATP in control cells). The maximum I _KATP density induced by metabolic inhibition was higher in small remodeled (176±15 pA/pF) than in control cells (127±11 pA/pF), but was unchanged in large remodeled cells. Both, the higher I _KATP density and the lower sensitivity of the K_ATP channels to ATP suggest that remodeled cardiomyocytes develop an improved tolerance to ischemia by stabilizing the resting potential and decreasing excitability.     

Intracellular ADP activates ATP-sensitive K+ channels in vascular smooth muscle cells of the guinea pig portal vein

Vasodilatation following tissue ischemia is assumed to partially result from activation of ATP-dependent K⁺ channels (K_ATP). To assess the effect of cytosolic adenosine nucleotides, the balance of which depends on tissue pO₂, on K_ATP, we have measured steady state outward currents (SSC) by the whole-cell clamp technique in smooth muscle cells of the guinea pig portal vein at different concentrations of ATP and ADP in the pipette solution. Glibenclamide, a selective inhibitor of K_ATP, was used as a pharmacological tool. — With no nucleotides in the pipette solution (Ca²⁺-free), the SSC determined at +20 mV was unaffected by glibenclamide, while with 0.1 mM ATP or with 0.1 mM ADP, the SSC exhibited a glibenclamide-sensitive component indicating activation of K_ATP. At 5 mM ATP and no ADP, hardly any effect of glibenclamide on the SSC was detected, suggesting inhibition of K_ATP by this high concentration of ATP. With 0.1 mM ADP at 5 mM ATP however, activation of K_ATP was achieved. — At 10^–7 M Ca²⁺ in the pipette solution, an increased SSC was measured, but the responses to the nucleotides and/or glibenclamide were not modified. — These findings suggest that in vivo, ADP may be involved in the regulation of vascular K_ATP, linking tissue pO₂ with vascular tone and tissue perfusion.     

Different modulation of Ca-activated K channels by the intracellular redox potential in pulmonary and ear arterial smooth muscle cells of the rabbit

Astract We investigated the electrical responses of Ca-activated K (KCa) currents induced by hypoxia and reduction or oxidation of the channel protein in pulmonary (PASMC) and ear (EASMC) arterial smooth muscle cells using the patch-clamp technique. In cell-attached patches, in the presence of a high K solution (containing 0.316 (M Ca²⁺), the activity of K_Ca channels from PASMC was decreased (by 49±7% compared to control, pipette potential = –70 mV) by changing to a hypoxic solution (1 mM Na₂S₂O₄, aeration with 100% N₂ gas). EASMC channels did not respond to hypoxia. In order to investigate the possible mechanisms involved, using inside-out patches bathed symmetrically in 150 mM KCl, we applied redox couples to the intracellular side. Reducing agents, such as dithiothreitol (DDT, 5 mM), reduced glutathione, (GSH, 5 mM), and nicotinamide adenine dinucleotide reduced (NADH, 2 mM) decreased PASMC, but not EASMC, K_Ca channel activity. However, oxidizing agents such as 5,5-dithio-bis(2-nitrobenzoic acid) (DTNB, 1 mM), oxidized glutathione (GSSG, 5 mM) and NAD (2 mM) increased K_Ca channel activity in both PASMC and EASMC. The increased activity due to oxidizing agents was restored by applying reducing agents. From these results, we could suggest that the basal redox state of the EASMC K_Ca channel is more reduced than that of the PASMC channel, since the response of K_Ca channels of the EASMC to intracellular reducing agents differs from that of the PASMC. This difference may be related to the different responses of PASMC and EASMC K_Ca channels to hypoxia.     

Demonstration of A-currents in pancreatic islet cells

Voltage-activated K⁺ currents resistant to TEA but blockable by 4-AP were recorded from mouse pancreatic islet cells. These currents first become observable during depolarizations to voltages more positive than –40 mV, reaching a peak amplitude of 120±34 pA at +6 mV (n=4), display rapid turn on (=3.3±1.1 ms at +6 mV) and inactivate completely within 250 ms (=65±5 at +6 mV). The current is subject to steady-state inactivation. The midpoint (V _h) of the inactivation curve (h) was observed at –72±2 mV. The properties of this current resemble those reported for the A-current in neurons.     

Sodium current in single myocardial mouse cells

Morphologically intact single myocardial cells of the adult mouse show a length of 132±20 m, a width of 21±5 , and a height of 10±4 m (all mean ± SD) and are brick-like in shape. A one suction pipette method is used for voltage clamp of those single cells. The determined time constant of capacitive current =35±14 s is very short. Series resistancer _s, membrane resistancer _m, and membrane capacityc _m are calculated to be 192±48 k, 6.1±1.1 M, and 186±92 pF (all mean ± SD), respectively. Assuming the specific unit membrane capacitance of 1 F/cm², a total membrane area of 1.86×10^–4 cm² is determined yielding a specific membrane resistanceR _m of 1,134 cm². Settling time of voltage clamp is 30 s. TTX-block of sodium current is described by 1:1 binding with aK _D value of 1.4×10^–6M. Using a reduced extracellular sodium concentration the maximum Na current is between 25 and 40 nA at voltages between –40 and –30 mV. Currents of between +20 and +30 mV reverse in an outward direction. Inward currents are approximated by a m³h model. The time constant of activation decreases from 0.7 ms at –60 mV to 0.12 ms at +20 mV. The time constant of inactivation falls from 9.1 ms at –60 mV to 0.6 ms at +20 mV.Steady state inactivationh is characterized by the half maximum valueV _H=–76.1±4.3 mV and the slope parameters=–6.3±1.1 mV (mean ± SD). A prepulse duration of 500 ms is essential for real steady state inactivation. Steady state activationm and inactivationh overlap each other defining a maximum window current at –65 mV.     

Comparative roles of ATP-sensitive K channels and Ca-activated BK channels in posthypoxic hyperexcitability and rapid hypoxic preconditioning in hippocampal CA1 pyramidal neurons in vitro

The aim of this study was to investigate the comparative effects of glibenclamide (GC), a selective blocker of K⁺_ATP channels, and iberiotoxin (IbTX), a selective blocker of BK⁺_Ca channels, on the repeated brief hypoxia-induced posthypoxic hyperexcitability and rapid hypoxic preconditioning in hippocampal CA1 pyramidal neurons in vitro. The method of field potentials measurement in CA1 region of the rat hippocampal slices was used. In contrast to GC (10 μM), IbTX (10 nM) significantly abolished both posthypoxic hyperexcitability and rapid hypoxic preconditioning induced by brief hypoxic episodes. These effects of IbTX did not depend on its ability to reduce the hypoxia-induced decrease of population spike (PS) amplitude during hypoxic episodes since GC (10 μM), comparatively with IbTX (10 nM), significantly reduced the depressive effect of hypoxia on the PS amplitude during hypoxic episodes but did not abolish both posthypoxic hyperexcitability and rapid hypoxic preconditioning in CA1 pyramidal neurons. Our results indicated that BK⁺_Ca channels, in comparison with K⁺_ATP channels, play a more important role in such repeated brief hypoxia-induced forms of neuroplasticity in hippocampal CA1 pyramidal neurons as posthypoxic hyperexcitability and rapid hypoxic preconditioning.     

Characterization of the ATP-inhibited K+ current in canine coronary smooth muscle cells

Intracellular adenosine triphosphate (ATP)-inhibited K⁺ currents (I _{K, ATP} ) in canine coronary artery smooth muscle cells were characterized in the wholecell configuration using the suction pipette method. Cells dialysed internally with solutions containing 5 mM ATP (ATP_i) showed little detectable whole-cell current at potentials more negative than –30 mV. However, cells dialysed with ATP_i-free solutions developed a time- and voltage-independent current which reached a maximum of 132±25 pA at –40 mV about 10 min following patch rupture. After run-up, the current showed little run-down. Concentration-dependent inhibition by ATP_i yielded an inhibition constant (K _i of 350 M and a Hill coefficient of 2.3. In ATP_i-free solutions, the large current at –40 mV was reduced by glibenclamide with aK _i of 20 nM and a Hill coefficient of 0.95. Conversely, in 1 mM ATP_i solutions, the small current at –40 mV was increased by P-1075 from 8±2 pA to 143±33 pA, with a dissociation constant (K _d) of 0.16 M and a Hill coefficient of 1.7. The effect of P-1075 was antagonized by glibenclamide. Maximal current density elicited by either ATP_i depletion or external application of the channel opener P-1075 was similar with slope conductances of 81±10 pS/pF and 76±13 pS/pF respectively in the potential range of –90 to –40 mV. External Ca²⁺ had no effect on this current. Finally, in 1 mM ATP_i, 20 and 50 M adenosine increased the current slope conductance by 36±15% and 73±10% respectively between –90 to –40 mV. TheI _{K, ATP} although very small in these cells, was extremely effective in causing membrane potential hyperpolarization.     

cAMP-dependent activation of small-conductance Cl− channels in HT29 colon carcinoma cells

The present study was performed to examine the conductance properties in the colon carcinoma cell line HT₂₉ and the activation of Cl^– channels by cAMP. A modified cell-attached nystatin patch-clamp technique was used, allowing for the simultaneous recording of the cell membrane potential (PD) and the conductance properties of the cell-attached membrane. In resting cells, PD was –56±0.4 mV (n=294). Changing the respective ion concentrations in the bath indicate that these cells possess a dominating K⁺ conductance and a smaller Cl^– conductance. A significant non-selective cation conductance, which could not be inhibited by amiloride, was only observed in cells examined early after plating. The K⁺ conductance was reversibly inhibited by 1–5 mmol/l Ba²⁺. Stimulation of the cells by the secretagogues isoproterenol and vasointestinal polypeptide (VIP) depolarized PD and induced a Cl^– conductance. Similar results were obtained with compounds increasing cytosolic cAMP: forskolin, 3-isobutyl-1-methylxanthine, cholera toxin and 8-bromoadenosine cyclic 3,5-monophosphate (8-Br-cAMP). VIP (1 nmol/l, n=10) and isoproterenol (1 umol/l, n=12) depolarized the cells dose-dependently and reversibly by 12±2 mV and 13±2 mV. The maximal depolarization was reached after some 20 s. The depolarization was due to increases in the fractional Cl^– conductance. Simultaneously the conductance of the cellattached membrane increased from 155±31 pS to 253±40 pS (VIP, n=4) and from 170±43 pS to 268±56 pS (isoproterenol, n=11), reflecting the gating of Cl^– channels in the cell-attached membrane. 5-Nitro-2-(3-phenylpropylamino)-benzoate (1 mol/l) was without significant effects in resting and in forskolin-stimulated HT₂₉ cells. The agonist-induced conductance increase of the cell-attached nystatin patches was not paralleled by the appearance of detectable single-channel events in these membranes. These data suggest activation of small, non-resolvable Cl^– channels by cAMP.Supported by DFG Gr 480/10 and BMFT 01 GA 88/6     

Adenosine triphosphate-dependent K currents activated by metabolic inhibition in rat ventricular myocytes differ from those elicited by the channel opener rilmakalim

Adenosine triphosphate (ATP) dependent potassium channels (K_ATP channels) in heart ventricular muscle cells can be activated by depletion of intracellular ATP stores as well as by channel openers. In the present study we examined whether properties of K_ATP channels are dependent on the mode of activation. Whole-cell and single-channel currents were investigated by use of the patch-clamp technique in isolated ventricular rat myocytes. The channel opener rilmakalim dose dependency activated whole-cell currents [concentration for half-maximal activation (EC₅₀) = 1.1 M, Hill coefficient = 3.1, saturation concentration 10 M]. Metabolic inhibition with 2-deoxy-d-glucose (10 mmol/l) also activated K_ATP currents after a time lag of several minutes. These currents were about two-fold higher than the rilmakalim-activated currents (rilmakalim-activated current 3.9 ±0.2nA, 2-deoxy-d-glucose-activated current 8.1±0.9 nA; both recorded at 0 mV clamp potential). While the rilmakalim-activated current could be blocked completely and with high affinity by the sulphonylurea glibenclamide [concentration for half-maximal inhibition (IC₅₀) = 8 nM, Hill coefficient = 0.7] the 2-deoxy-d-glucose-activated current could only be blocked partially (by maximally 46%) and higher glibenclamide concentrations were needed (IC₅₀ = 480 nM, Hill coefficient = 0.8). The partial loss of blocking efficiency after metabolic inhibition was not restricted to glibenclamide but was also observed with the sulfonylureas glimepiride and HB 985, as well as with the non-sulfonylureas HOE 511 and 5-hydroxydecanoate. Single-channel studies were in accordance with these whole-cell experiments. Both rilmakalim and metabolic inhibition with the uncoupler carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) activated single channels in the attached mode, where the number of current levels was significantly higher in the case of FCCP. Rilmakalim-activated channels were completely blocked by 10 M glibenclamide, whereas several single-channel levels appeared in the presence of 100 M glibenclamide after metabolic inhibition. In conclusion, after metabolic inhibition the amplitude of the activated K_ATP current is about twice as high as under saturating concentrations of the opener rilmakalim. Moreover, channels activated by metabolic inhibition lost part of their sensitivity to known channel blockers.     

Contribution of prostaglandins in hypoxia-induced vasodilatation in isolated rabbit hearts. Relation to adenosine and KATP channels

The mechanism of hypoxia-induced coronary vasodilatation was studied in isolated, saline-perfused rabbit hearts under constant flow conditions. Reduction in the perfusion solution PO₂ (from 520±6 to 103±9 mm Hg) under control conditions halved the coronary resistance and was accompanied by a significant release of the prostaglandin (PG) 6-keto-PGF₁ (from 1.8±0.3 to a maximum of 4.4±0.9 pmol min^–1 g^–1). The cyclooxygenase inhibitor, diclofenac (1 M), blocked the release of PGI₂ and reduced hypoxia-induced vasodilatation (from 47±8% to 25±5%, P<0.05). The relative contribution of adenosine, prostaglandins, and adenosine triphosphate (ATP)-sensitive K⁺ channel (K_ATP channel) activation in hypoxia-induced vasodilatation was assessed by comparing the differential change (control response minus response after treatment) in coronary perfusion pressure (CPP) during infusion of 8-phenyltheophylline (8-PT), diclofenac, and glibenclamide, respectively. The differential change in CPP with 8-PT and diclofenac given together (–48 ±7%) was found to be equivalent to the sum of their respective effects (–24±7 and –19±4%, respectively). Glibenclamide (0.3 M) reduced significantly hypoxia-induced vasodilatation (differential change in CPP of –27±6%) as well as the dilator response to 10 M adenosine and to the stable PGI₂-analogue, iloprost. Forskolin-induced coronary vasodilatation in arrested hearts was slightly, but significantly, reduced by glibenclamide. Our results suggest that both cyclooxygenase products and adenosine, acting independently and concomitantly, contribute to the dilator response of coronary resistance vessels to hypoxia, in part through the activation of K_ATP channels. K_ATP channel activation by prostacyclin and adenosine may involve both cyclic adenosine monophosphate-dependent and independent pathways.     

Regulation of smooth muscle delayed rectifier K+ channels by protein kinase A

We identified voltage-activated K⁺ channels in freshly dispersed smooth muscle cells from the circular layer of the canine colon in patch-clamp experiments using 200 nM charybdotoxin to suppress 270-pS Ca²⁺-activated K⁺ channels (BK channels). Three channel types were distinguished in symmetrical 140 mM KCl solutions: 19.5 ± 1.7 pS channels (K_DR1), 90.6 ± 5.4 pS channels (K_DR2) and 149 ± 4 pS intermediate-conductance Ca²⁺-activated K⁺ channels (IK channels). All three types showed an increase in open probability with membrane depolarization. Ensemble average current from K_DR1 channels inactivated with a time constant of 1.7 ± 0.1 s at +60 mV test potential, while K_DR2 and IK channels did not show inactivation. IK channels were activated by free cytoplasmic [Ca²⁺] (10⁻⁶M) but were insensitive to 4-aminopyridine (4-AP, 10 mM) and intracellular tetraethylammonium (TEA, 1 mM). K_DR1 channels were sensitive to 4-AP (10 mM) and intracellular TEA (1–10 mM) but not to Ca²⁺. K_DR2 channels did not have a consistent pharmacological profile, suggesting that this class may be comprised of several subtypes. At +40 mV membrane potential, the catalytic subunit of protein kinase A (PKA) increased the open probability of K_DR1 channels 3.4-fold and of K_DR2 channels 3.9-fold, but had no effect on IK channels. In the absence of Mg-ATP, PKA did not affect channel open probabilities. At physiological membrane potentials (−60 mV) only openings of K_DR1 channels could be induced by PKA, suggesting that these 4-AP-sensitive 20-pS K⁺ channels are primarily responsible for the cAMP-mediated hyperpolarization of colonic smooth muscle cells. Received: 20 June 1995/Received after revision: 25 January 1996/Accepted: 7 February 1996     

Simultaneous recording of ATP-sensitive K+ current and intracellular Ca2+ in anoxic rat ventricular myocytes. Effects of glibenclamide

We investigated the temporal relationship between the adenosine triphosphate-sensitive K current (K _ATP current), hypoxic shortening and Ca accumulation in cardiomyocytes exposed to anoxia or metabolic inhibition. Whole-cell, patch-clamp experiments were performed with nonstimulated isolated rat heart ventricular muscle cells loaded with the Ca-sensitive fluorescent dye 1-[2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy]-2-(2′-amino-5′-methylphenoxy) ethane-N,N,N′,N′-tetraacetic acid (fura-2) via the patch pipette. After approximately 8 min anoxia, the K _ATP current started to rise and reached a maximum of 21.3 ± 3.7 nA (n = 5, recorded at 0 mV clamp potential) within 1–3 min. At that time hypoxic contracture also occurred. Resting cytoplasmic free calcium (Ca_i) did not change significantly before hypoxic shortening. After hypoxic contracture, the K _ATP current decreased and Ca_i started to rise, reaching about 1 μmol/l. The presence of glibenclamide (10 μmol/l) in the bath reduced the anoxia-induced K _ATP current by more than 50%, but did not significantly influence the time dependence of current, hypoxic shortening and Ca_i, or the magnitude of Ca_i. Metabolic inhibition with 1.5 mmol/l CN resulted in K _ATP current increase and hypoxic shortening, occurring somewhat earlier than under anoxia, but all other parameters were comparable. In non-patch-clamped cells loaded with fura-2 AM ester and field-stimulated with 1 Hz, 1 μmol/l glibenclamide had no significant effect on the magnitude of the Ca_i increase caused by exposure of the cells to 1.5 mmol/l CN⁻. After CN⁻ wash-out in non-patch-clamped cells, Ca_i declined, oscillated and finally returned to control values. It can be concluded that glibenclamide inhibits anoxia-induced K _ATP currents only partially and has no significant effect on anoxia-induced rise in resting Ca_i. Received: 3 November 1995/Received after revision: 9 January 1996/Accepted: 16 January 1996     

Catecholamine secretion from rat foetal adrenal chromaffin cells and hypoxia sensitivity

The adrenal medulla chromaffin cells (AMCs) secrete catecholamines in response to various types of stress. We examined the hypoxia-sensitivity of catecholamine secretion by rat foetal chromaffin cells in which the innervation by the splanchnic nerve is not established. The experiments were performed in primary cultured cells from two different ages of foetuses (F15 and F19). Membrane potential of AMCs was monitored with the patch clamp technique, and the catecholamine secretion was detected by amperometry. We found that: (1) AMCs from F19 foetuses showed hypoxia-induced catecholamine release. (2) This hypoxia-induced secretion is produced by membrane depolarization generated by an inhibition of Ca²⁺-activated K⁺ current [I _K(Ca)] current. (3) Chromaffin precursor cells from F15 foetuses secrete catecholamine. The quantal release is calcium-dependent, but the size of the quantum is reduced. (4) In the precursor cells, a hypoxia-induced membrane hyperpolarization is originated by an ATP-sensitive K⁺ current [I _K(ATP)] activation. (5) During the prenatal period, at F15, the percentage of the total outward current for I _K(ATP) and I _K(Ca) was 50 and 29.5%, respectively, whereas at F19, I _K(ATP) is reduced to 14%, and I _K(Ca) became 64% of the total current. We conclude that before birth, the age-dependent hypoxia response of chromaffin cells is modulated by the functional activity of K_ATP and K_Ca channels.     

The protective roles of mitochondrial ATP-sensitive potassium channels during hypoxia–ischemia–reperfusion in brain

The role of ATP-sensitive potassium (K_ATP) channels in cerebral ischemia–reperfusion has been well documented. K_ATP channel openers protect neuron by mimicking ischemic preconditioning. However, the different protection between the mitochondrial and sarcolemma K_ATP openers has been seldom studied. In the experiment, we investigated the effects of K_ATP channel openers diazoxide and pinacidil on the hypoxia–ischemia–reperfusion in cultured hippocampal neurons and gerbil brain. The cultured hippocampal neurons and gerbil brain were pretreated with diazoxide or pinacidil before oxygen-glucose deprivation (OGD) and cerebral ischemia–reperfusion, respectively. Survival rate, apoptosis rate and lactate dehydrogenase (LDH) releasing after the reperfusion were subsequently detected. Then the subunits mRNA was detected by RT-PCR. The survival rate and LDH content in diazoxide group increased more than that in pinacidil group (86.21 ± 2.73% vs. 78.59 ± 1.94%, P < 0.05; 133.29 ± 15.00 U/L vs. 193.47 ± 3.39 U/L, P < 0.01). The apoptosis rate in diazoxide group decreased significantly more than that in pinacidil group (23.82 ± 0.14% vs. 37.05 ± 0.67%, P < 0.01). Diazoxide pretreatment increased the expression of Kir6.1 mRNA obviously. The results suggested that mitoK_ATP channels opener diazoxide played a major protective role on cerebral ischemia–reperfusion. Furthermore, diazoxide might become a new treatment for cerebral ischemia diseases through increasing the expression of Kir6.1 mRNA.     

Regulation of membrane excitability by intracellular pH (pHi) changers through Ca2+-activated K+ current (BK channel) in single smooth muscle cells from rabbit basilar artery

Employing microfluorometric system and patch clamp technique in rabbit basilar arterial myocytes, regulation mechanisms of vascular excitability were investigated by applying intracellular pH (pH_i) changers such as sodium acetate (SA) and NH₄Cl. Applications of caffeine produced transient phasic contractions in a reversible manner. These caffeine-induced contractions were significantly enhanced by SA and suppressed by NH₄Cl. Intracellular Ca²⁺ concentration ([Ca²⁺]_i) was monitored in a single isolated myocyte and based the ratio of fluorescence using Fura-2 AM (R _340/380). SA (20 mM) increased and NH₄Cl (20 mM) decreased R _340/380 by 0.2 ± 0.03 and 0.1 ± 0.02, respectively, in a reversible manner. Caffeine (10 mM) transiently increased R _340/380 by 0.9 ± 0.07, and the ratio increment was significantly enhanced by SA and suppressed by NH₄Cl, implying that SA and NH₄Cl may affect [Ca²⁺]_i (p < 0.05). Accordingly, we studied the effects of SA and NH₄Cl on Ca²⁺-activated K⁺ current (IK_Ca) under patch clamp technique. Caffeine produced transient outward current at holding potential (V _h) of 0 mV, caffeine induced transient outward K⁺ current, and the spontaneous transient outward currents were significantly enhanced by SA and suppressed by NH₄Cl. In addition, IK_Ca was significantly increased by acidotic condition when pH_i was lowered by altering the NH₄Cl gradient across the cell membrane. Finally, the effects of SA and NH₄Cl on the membrane excitability and basal tension were studied: Under current clamp mode, resting membrane potential (RMP) was −28 ± 2.3 mV in a single cell level and was depolarized by 13 ± 2.4 mV with 2 mM tetraethylammonium (TEA). SA hyperpolarized and NH₄Cl depolarized RMP by 10 ± 1.9 and 16 ± 4.7 mV, respectively. SA-induced hyperpolarization and relaxation of basal tension was significantly inhibited by TEA. These results suggest that SA and NH₄Cl might regulate vascular tone by altering membrane excitability through modulation of [Ca²⁺]_i and Ca²⁺-activated K channels in rabbit basilar artery.     

Identification of an ATP-sensitive K+ channel in rat cultured cortical neurons

To determine whether membranes of mammalian central neurons contain an ATP-sensitive K⁺ (K_ATP) channel similar to that present in pancreatic cells, the patch-clamp technique was applied to cultured neurons prepared from the neonatal rat cerebral cortex and hippocampus. In whole-cell experiments with hippocampal neurons, extracellular application of 0.5 mM diazoxide (a K_ATP channel activator) elicited a hyperpolarization concomitant with an increase in membrane conductance, whereas application of 0.5 mM tolbutamide (a K_ATP channel blocker) induced a depolarization with a decrease in conductance. Similar results were obtained with cortical neurons. In outside-out patch experiments with cortical neurons, a K⁺ channel sensitive to these drugs was found. The channel was completely blocked by 0.5 mM tolbutamide and activated by 0.5 mM diazoxide. The single-channel conductance was 65 pS under symmetrical 145 mM K⁺ conditions and 24 pS in a physiological K⁺ gradient. In inside-out patch experiments, this channel was demonstrated to be inhibited by an application of 0.2–1 mM ATP to the cytoplasmic surface of the patch membrane. These results indicate that the membranes of rat cortical neurons contain a K_ATP channel that is quite similar to that found in pancreatic cells. It is also suggested that the same or a similar K⁺ channel may exist in membranes of hippocampal neurons.     

ATP reduces voltage-activated K+ current in cultured rat hippocampal neurons

Effects of extracellular ATP were investigated in cultured rat hippocampal neurons using whole-cell voltage-clamp techniques. When a depolarizing step to +10 mV was applied from a holding potential of -60 mV, an outward K⁺ current was activated. ATP (3 to 300 μM) reduced the K⁺ current. Among adenosine derivatives, ADP (100 μM) slightly inhibited the K⁺ current, and AMP or adenosine (100 μM) was ineffective. UTP was as potent as ATP and α,β-methylene ATP was less effective than ATP. The inhibition by ATP of the K⁺ current was abolished by inclusion of 2 mM GDPβS in the intracellular solution. The results indicate that ATP inhibits K⁺ channels in rat hippocampal neurons through UTP-responsive P₂-purinoceptors coupled with GTP-binding proteins.     

Impaired hippocampal Ca homeostasis and concomitant K channel dysfunction in a mouse model of rett syndrome during anoxia

Methyl-CpG-binding protein 2 (MeCP2) deficiency causes Rett syndrome (RTT), a neurodevelopmental disorder characterized by severe cognitive impairment, synaptic dysfunction, and hyperexcitability. Previously we reported that the hippocampus of MeCP2-deficient mice (Mecp2^−/y), a mouse model for RTT, is more susceptible to hypoxia. To identify the underlying mechanisms we now focused on the anoxic responses of wildtype (WT) and Mecp2^−/y CA1 neurons in acute hippocampal slices. Intracellular recordings revealed that Mecp2^−/y neurons show only reduced or no hyperpolarizations early during cyanide-induced anoxia, suggesting potassium channel (K⁺ channel) dysfunction. Blocking adenosine-5′-triphosphate-sensitive K⁺ channels (K_ATP-) and big-conductance Ca²⁺-activated K⁺ channels (BK-channels) did not affect the early anoxic hyperpolarization in either genotype. However, blocking Ca²⁺ release from the endoplasmic reticulum almost abolished the anoxic hyperpolarizations in Mecp2^−/y neurons. Single-channel recordings confirmed that neither K_ATP- nor BK-channels are the sole mediators of the early anoxic hyperpolarization. Instead, anoxia Ca²⁺-dependently activated various small/intermediate-conductance K⁺ channels in WT neurons, which was less evident in Mecp2^−/y neurons. Yet, pharmacologically increasing the Ca²⁺ sensitivity of small/intermediate-conductance K_Ca channels fully restored the anoxic hyperpolarization in Mecp2^−/y neurons. Furthermore, Ca²⁺ imaging unveiled lower intracellular Ca²⁺ levels in resting Mecp2^−/y neurons and reduced anoxic Ca²⁺ transients with diminished Ca²⁺ release from intracellular stores. In conclusion, the enhanced hypoxia susceptibility of Mecp2^−/y hippocampus is primarily associated with disturbed Ca²⁺ homeostasis and diminished Ca²⁺ rises during anoxia. This secondarily attenuates the activation of K_Ca channels and thereby increases the hypoxia susceptibility of Mecp2^−/y neuronal networks. Since cytosolic Ca²⁺ levels also determine neuronal excitability and synaptic plasticity, Ca²⁺ homeostasis may constitute a promising target for pharmacotherapy in RTT.