Anomalous effect of anthracene-9-carboxylic acid on calcium-activated chloride currents in rabbit pulmonary artery smooth muscle cells

1 Ca(2+)-activated Cl(-) currents (I(Cl(Ca))) evoked by K(+)-free pipette solutions containing 500 nM Ca(2+) were recorded in rabbit pulmonary artery smooth muscle cells. A voltage step protocol in which the cells were stepped to +70 mV and then to -80 mV produced outward and inward Cl(-) currents respectively that exhibited distinctive voltage- and time-dependent kinetics that remained consistent for the recording period. 2 Application of the Cl(-) channel inhibitor anthracene-9-carboxylic acid (A-9-C, 500 micro M), produced a small inhibition of the maximum outward Cl(-) current at +70 mV (21+/-10%) but augmented the amplitude of the instantaneous inward relaxation at -80 mV by 321+/-34% (n=12). 3 The current recorded in the absence and presence of A-9-C reversed at the theoretical Cl(-) equilibrium potential and the reversal potential was shifted by about -40 mV upon replacement of external chloride ion by the more permeant anion thiocyanate. Currents in the absence and presence of A-9-C were similarly affected by 100 micro M niflumic acid. 4 Augmentation of the inward current at -80 mV by A-9-C required prior depolarization, i.e. A-9-C did not simply activate a Cl(-) current at negative membrane potentials. Moreover the degree of augmentation was independent of the internal Ca(2+) for concentrations between 100 nM and 1 micro M Ca(2+). 5 The data from the present study confirm previous observations that the inhibitory effect of Cl(-) channel blockers is modified when [Ca(2+)](i) is maintained at higher than normal resting concentrations.     

NMDA enhances a depolarization-activated inward current in subthalamic neurons

Previous studies have shown that N-methyl-D-aspartate (NMDA) receptor stimulation evokes Ca2+- and Na+-dependent burst firing in subthalamic nucleus (STN) neurons. Using whole-cell patch pipettes to record currents under voltage-clamp, we identified a time-dependent depolarization-activated inward current (DIC) that may underlie NMDA-induced burst firing in STN neurons in rat brain slices. Continuous superfusion with NMDA (20 microM) elicited a marked TTX-insensitive inward current when the membrane was depolarized to the level of -70 or -50 mV, from a holding potential of -100 mV. This current had a long duration, and its peak amplitude occurred at a test potential of -60 mV. DIC could not be evoked using the non-NMDA receptor agonist D,L-alpha-amino-3-hydroxy-5-methylisoxalone-4-propionic acid (AMPA). DIC was blocked by either intracellular BAPTA or by removal of extracellular Ca2+, but selective blockers of T-type (mibefradil), L-type (nifedipine) and N-type (omega-conotoxin GVIA) Ca2+ channels did not. Perfusing slices with a low extracellular concentration of sodium abolished the NMDA-induced DIC, implying that both Ca2+ and Na+ are necessary for the expression of DIC. Transient receptor potential (TRP) channel blockers flufenamic acid and SKF96365 severely reduced DIC amplitude, whereas NMDA-gated currents were either increased or were unchanged. These results suggest that the activation of NMDA receptors enhances a Ca2+-activated non-selective cation current that may be mediated by a member of the TRP channel family in STN neurons.     

Bicuculline free base blocks voltage-activated K+ currents in rat medial preoptic neurons

The effects of the well-known GABA(A)-receptor blocker bicuculline on voltage-gated K(+) currents were studied in neurons from the medial preoptic nucleus (MPN) of rat. Whole-cell currents were recorded using the perforated-patch technique. Voltage steps from -54 to +6 mV resulted in tetraethylammonium-sensitive K(+) currents of delayed rectifier type. The total K(+) current (at 300 ms), including Ca(2+)-dependent and Ca(2+)-independent components, was reversibly reduced (17 +/- 4%) by 100 microM bicuculline methiodide and (37 +/- 5%) by 100 microM bicuculline as free base. The Ca(2+)-independent fraction (77 +/- 2%) of K(+) current evoked by a voltage step was, however, reduced (54 +/- 6%) only by bicuculline free base, but was not affected by bicuculline methiodide. The half-saturating concentration of bicuculline free base for blocking this purely voltage-gated K(+) current was 113 microM, whereas for blocking a steady Ca(2+)-dependent K(+) current it was 36 microM. The bicuculline-sensitive voltage-gated K(+) current was composed of 4-AP-sensitive and 4-AP-resistant components with different kinetic properties. No component of the purely voltage-gated K(+) current was affected neither by 100 nM alpha-dendrotoxin nor by 100 nM I-dendrotoxin. The possible K(+)-channel subtypes mediating the bicuculline-sensitive current in MPN neurons are discussed.     

Ca(2+)-activated K+ current induced by external ATP in PC12 cells

1. The effect of external ATP on the membrane current was investigated in PC12 cells by whole-cell voltage-clamp techniques. 2. Lower concentrations of ATP (1 or 10 mumol/L) induced only an inward current at 1 mmol/L EGTA in the K+ pipette solution, while higher concentrations of ATP (100 mumol/L and 1 mmol/L) induced an outward current following the inward current. 3. Lowering the EGTA concentration in the pipette solution induced a larger outward current following ATP application. The membrane potential at which the outward current crossed with the control before ATP application was more negative at lower concentrations of EGTA in the pipette. 4. The development of the outward current was blocked by a Ca(2+)-free external solution, 5 mmol/L tetraethylammonium and a Cs+ pipette solution instead of K+, indicating that the outward current was a Ca(2+)-activated K+ current. 5. Charybdotoxin (0.1 mumol/L) and iberiotoxin (0.1 mumol/L), but not apamin (0.2 mumol/L) blocked the development of the outward current, indicating the ATP-induced outward current is a BK-type Ca(2+)-activated K+ channel current and not the SK type. 6. UTP had no effect on the membrane current, indicating that the ATP-induced current change was not mediated by P2u but by P2x purinoceptor. 7. In conclusion, stimulation of P2x purinoceptors by ATP induces a Ca(2+)-permeable inward current that results in increases in intracellular Ca2+ concentrations and activation of a BK-type Ca(2+)-activated K+ current in PC12 cells.     

Characterization of a charybdotoxin-sensitive intermediate conductance Ca2+-activated K+ channel in porcine coronary endothelium: relevance to EDHF

1. This study characterizes the K(+) channel(s) underlying charybdotoxin-sensitive hyperpolarization of porcine coronary artery endothelium. 2. Two forms of current-voltage (I/V) relationship were evident in whole-cell patch-clamp recordings of freshly-isolated endothelial cells. In both cell types, iberiotoxin (100 nM) inhibited a current active only at potentials over +50 mV. In the presence of iberiotoxin, charybdotoxin (100 nM) produced a large inhibition in 38% of cells and altered the form of the I/V relationship. In the remaining cells, charybdotoxin also inhibited a current but did not alter the form. 3. Single-channel, outside-out patch recordings revealed a 17.1+/-0.4 pS conductance. Pipette solutions containing 100, 250 and 500 nM free Ca(2+) demonstrated that the open probability was increased by Ca(2+). This channel was blocked by charybdotoxin but not by iberiotoxin or apamin. 4. Hyperpolarizations of intact endothelium elicited by substance P (100 nM; 26.1+/-0.7 mV) were reduced by apamin (100 nM; 17.0+/-1.8 mV) whereas those to 1-ethyl-2-benzimidazolinone (1-EBIO, 600 microM, 21.0+/-0.3 mV) were unaffected (21.7+/-0.8 mV). Substance P, bradykinin (100 nM) and 1-EBIO evoked charybdotoxin-sensitive, iberiotoxin-insensitive whole-cell perforated-patch currents. 5 A porcine homologue of the intermediate-conductance Ca(2+)-activated K(+) channel (IK1) was identified in endothelial cells. 6. In conclusion, porcine coronary artery endothelial cells express an intermediate-conductance Ca(2+)-activated K(+) channel and the IK1 gene product. This channel is opened by activation of the EDHF pathway and likely mediates the charybdotoxin-sensitive component of the EDHF response.     

Potentiation of potassium currents by beta-adrenoceptor agonists in human urinary bladder smooth muscle cells: a possible electrical mechanism of relaxation

We examined the effects of beta-adrenoceptor agonists on the membrane currents of smooth muscle cells from the human urinary bladder using a whole-cell patch clamp to investigate the involvement of Ca(2+)-activated K(+) (K(Ca)) channels in relaxation by beta-adrenergic agonists. With 0.05 mmol/l EGTA in the patch pipette, depolarizing pulses evoked outward rectifying currents. Isoproterenol (1 micromol/l) significantly increased the membrane currents by 75% at +80 mV with 0.05 mmol/l EGTA pipette solution. BRL 37344 (1 micromol/l) significantly increased the membrane currents by 44% at +80 mV. Iberiotoxin (100 nmol/l) significantly decreased the membrane currents by 60% at +80 mV. In the presence of iberiotoxin, the potentiation of the outward currents by isoproterenol was greatly suppressed and, in the presence of iberiotoxin and apamin (1 micromol/l), the potentiation by isoproterenol was totally abolished. On the other hand, with 5 mmol/l EGTA pipette solution, depolarizing pulses evoked smaller outward currents. Isoproterenol (1 micromol/l) did not change the membrane currents with 5 mmol/l EGTA pipette solution. The real-time PCR analysis revealed the expression of beta(2)-adrenoceptors in the cells. These results suggest that Ca(2+)-activated and iberiotoxin- and apamin-sensitive currents via both large-conductance and small-conductance K(Ca) channels could be increased by stimulation of beta(2)-adrenoceptors.     

A delayed ATP-elicited K+ current in freshly isolated smooth muscle cells from mouse aorta

Adenosine 5'-triphosphate (ATP) activated two sequential responses in freshly isolated mouse aortic smooth muscle cells. In the first phase, ATP activated Ca(2+)-dependent K(+) or Cl(-) currents and the second phase was the activation of a delayed outward current with a reversal potential of -75.9 +/- 1.4 mV. A high concentration of extracellular K(+) (130 mM) shifted the reversal potential of the delayed ATP-elicited current to -3.5 +/- 1.3 mV. The known K(+)-channel blockers, iberiotoxin, charybdotoxin, glibenclamide, apamin, 4-aminopyridine, Ba(2+) and tetraethylammonium chloride all failed to inhibit the delayed ATP-elicited K(+) current. Removal of ATP did not decrease the amplitude of the ATP-elicited current back to the control values. The simultaneous recording of cytosolic free Ca(2+) and membrane currents revealed that the first phase of the ATP-elicited response is associated with an increase in intracellular Ca(2+), while the second delayed phase develops after the return of cytosolic free Ca(2+) to control levels.ATP did not activate Ca(2+)-dependent K(+) currents, but did elicit Ca(2+)-independent K(+) currents, in cells dialyzed with ethylene glycol-bis (2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA). The delay of activation of Ca(2+)-independent currents decreased from 10.5 + 3.4 to 1.27 +/- 0.33 min in the cells dialyzed with 2 mM EGTA. Adenosine alone failed to elicit a Ca(2+)-independent K(+) current but simultaneous application of ATP and adenosine activated the delayed K(+) current. Intracellular dialysis of cells with guanosine 5'-O-(2-thiodiphosphate) transformed the Ca(2+)-independent ATP-elicited response from a sustained to a transient one. A phospholipase C inhibitor, U73122 (1 microM), was shown to abolish the delayed ATP-elicited response. These results indicate that the second phase of the ATP-elicited response was a delayed Ca(2+)-independent K(+) current activated by exogenous ATP. This phase might represent a new vasoregulatory pathway in vascular smooth muscle cells.     

Copper-induced non-selective permeability changes in intracellularly perfused snail neurons.

The effect of extracellularly applied Cu2+ was studied on isolated intracellularly perfused Helix pomatia neurons. It was found that the Cu(2+)-activated current (ICu) is biphasic and composed of overlapping outward and inward components. The outward component of ICu is the result of a blockade by Cu2+ of the steady-state outward Cl- current. The inward component is assumed to flow through Ca(2+)-activated non-selective cationic channels. The washing-out procedure resulted in a large inward current (Iw), which was composed of transient and steady-state components. It is most likely that the activation of metabolic pumps is responsible for the transient component and the steady-state component is the result of increased neuronal membrane permeability for Cl-. Moreover, both ICu and Iw were highly Ca(2+)- and temperature-dependent processes. It is concluded that Cu2+ application resulted in complex permeability changes in the Helix pomatia neurons.     

Chelerythrine and bisindolylmaleimide I prolong cardiac action potentials by protein kinase C-independent mechanism

Effects of chelerythrine and bisindolylmaleimide I on action potential duration and on voltage-activated K(+) and Ca(2+) currents in rat ventricular myocytes were studied using perforated patch-clamp technique. The action potentials were markedly prolonged after application of 20 microM chelerythrine or 100 nM bisindolylmaleimide I. Chelerythrine and bisindolylmaleimide I reduced the amplitude of sustained current (I(K,sus)) significantly. Transient K(+) current (I(to)) was inhibited only by chelerythrine. Ca(2+) current was reduced only with highest chelerythrine concentration (50 microM). Application of chelerythrine and bisindolylmaleimide I inhibited outward K(+) currents significantly also in ruptured patch-clamp configuration. Bisindolylmaleimide V, an inactive analogue of bisindolylmaleimide I, decreased I(K,sus) substantially. However, I(to) and I(K,sus) were not affected by calphostin C. Direct protein kinase C activators resulted in decrease of outward K(+) currents. Chelerythrine blocked I(to) in a use-dependent manner and the block did not recover during a 4-min washout. I(K,sus) was not blocked by this mechanism by either inhibitor. We conclude that chelerythrine and bisindolylmaleimide I inhibit outward K(+) currents independently of protein kinase C inhibition.     

Oscillatory transient inward currents in ventricular myocytes of healthy versus myopathic Syrian hamster

The present experiments were performed in order to study abnormal action potential configuration and ion channel activity in ventricular myocytes obtained from 23 male myopathic Syrian hamsters (Biobreeders strain 14.6, 32-52 weeks old) compared with 10 age-matched healthy control hamsters (Biobreeders F1B) by means of whole-cell patch-clamp techniques. The results show that the myopathic myocytes had a longer action potential duration, a reduced transient outward K(+) current on depolarization and a smaller transient inward current on repolarization after prolonged depolarizing pulses (> 500 msec). However, the L-type Ca(2+) current and the inwardly rectifing K(+) current were not significantly different from those of healthy myocytes. The oscillatory transient inward currents could be diminished by treatment with ryanodine (0.01-1 micromol/L), a sarcoplasmic reticulum (SR) Ca(2+) release channel blocker, or with Na(+)-free superfusate. We conclude that the hereditary myopathic hamsters are less likely to develop delayed after depolarization-related transient inward currents and triggered arrhythmias owing to a smaller SR Ca(2+) content.     

Postsynaptic actions of substance P on rat periaqueductal grey neurons in vitro

The postsynaptic actions of substance P on rat midbrain periaqueductal grey (PAG) neurons were examined using whole-cell patch-clamp recordings in brain slices. Substance P produced an inward current in a subpopulation (60%) of PAG neurons. The substance P induced current was concentration dependent (EC50=27 nM) and was reduced by the NK1, NK2 and NK3 antagonists L-732,138 (20 microM), GR 159897 (3 microM) and SB 218795 (3 microM). The selective NK1, NK2 and NK3 agonists [Sar9,Met(O2)11]-Substance P (100 nM), GR 64349 (300-500 nM) and senktide (300 nM) also produced inward currents in subpopulations of neurons. A greater proportion of substance P-sensitive neurons (70%) than substance P-insensitive neurons (31%) responded to the mu/delta opioid agonist met-enkephalin (10 microM). Substance P reduced the outward current produced by met-enkephalin. The reversal potential of the substance P induced current varied from -5 mV to below -140 mV in the absence of met-enkephalin, and was -105 mV in the presence of met-enkephalin. These results indicate that substance P acts via NK1, NK2 and NK3 receptors to excite subpopulations of opioid-sensitive and insensitive PAG neurons by increasing a non-selective cation conductance and by reducing a K+ current. In addition, substance P has anti-opioid actions that are largely mediated by a reduction in the opioid induced K+ current.     

Calcium-activated currents in cultured neurones from rat dorsal root ganglia.

1. Voltage-activated Ca2+ currents and caffeine (1 to 10 mM) were used to increase intracellular Ca2+ in rat cultured dorsal root ganglia (DRG) neurones. Elevation of intracellular Ca2+ resulted in activation of inward currents which were attenuated by increasing the Ca2+ buffering capacity of cells by raising the concentration of EGTA in the patch solution to 10 mM. Low and high voltage-activated Ca2+ currents gave rise to Cl- tail currents in cells loaded with CsCl patch solution. Outward Ca2+ channel currents activated at very depolarized potentials (Vc + 60 mV to + 100 mV) also activated Cl- tail currents, which were enhanced when extracellular Ca2+ was elevated from 2 mM to 4 mM. 2. The Ca(2+)-activated Cl- tail currents were identified by estimation of tail current reversal potential by use of a double pulse protocol and by sensitivity to the Cl- channel blocker 5-nitro 2-(3-phenyl-propylamino) benzoic acid (NPPB) applied at a concentration of 10 microM. 3. Cells loaded with Cs acetate patch solution and bathed in medium containing 4 mM Ca2+ also had prolonged Ca(2+)-dependent tail currents, however these smaller tail currents were insensitive to NPPB. Release of Ca2+ from intracellular stores by caffeine gave rise to sustained inward currents in cells loaded with Cs acetate. Both Ca(2+)-activated tail currents and caffeine-induced inward currents recorded from cells loaded with Cs acetate were attenuated by Tris based recording media, and had reversal potentials positive to 0 mV suggesting activity of Ca(2+)-activated cation channels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Charybdotoxin-sensitive small conductance K(Ca) channel activated by bradykinin and substance P in endothelial cells

1 In cultured porcine coronary artery endothelial cells, we have recently shown that substance P and bradykinin stimulated different types of Ca(2+)-dependent K(+) (K(Ca)) current. A large part of this current was insensitive to iberiotoxin and apamin. The aim of the present study was to characterize the K(Ca) channel responsible for this current. 2 In cell-attached configuration and asymmetrical K(+) concentration, 100 nM bradykinin or substance P activated a 10 pS K(+) channel. In inside-out configuration, the channel was half-maximally activated by 795 nM free Ca(2+). 3 Apamin (1 micro M) added to the pipette solution failed to inhibit the channel activity while charybdotoxin (50 nM), completely blocked it. Perfusion at the intracellular face of the cell, of an opener of intermediate conductance K(Ca) channel, 500 micro M 1-ethyl-benzimidazolinone (1-EBIO) increased the channel activity by about 4.5 fold. 4 In whole-cell mode, bradykinin and substance P stimulated an outward K(+) current of similar amplitude. Charybdotoxin inhibited by 75% the bradykinin-induced current and by 80% the substance P-induced current. Charybdotoxin plus iberiotoxin (50 nM each) inhibited by 97% the bradykinin-response. Charybdotoxin plus apamin did not increase the inhibition of the substance P-response obtained in the presence of charybdotoxin alone. 5 1-EBIO activated a transient outward K(+) current and hyperpolarized the membrane potential by about 13 mV. Charybdotoxin reduced the hyperpolarization to about 3 mV. 6 Taken together these results show that bradykinin and substance P activate a 10 pS K(Ca) channel, which largely contributes to the total K(+) current activated by these agonists. Despite its small conductance, this channel shares pharmacological characteristics with intermediate conductance K(Ca) channels.     

Ginseng saponins induce store-operated calcium entry in Xenopus oocytes

1 We investigated the effect of the active ingredients of Panax ginseng, ginsenosides, on store-operated Ca2+ entry (SOCE) using a two-electrode voltage clamp technique in Xenopus oocytes in which SOCE is monitored through Ca(2+)-activated Cl- currents. 2 Under hyperpolarizing voltage clamp conditions, treatment with ginsenosides produced a biphasic Ca(2+)-activated Cl- current consisting of a rapid transient inward current and a slowly developing secondary sustained inward current. The transient inward current was inactivated rapidly, whereas the sustained inward current persisted for nearly 10 min. The effect of ginsenosides on the biphasic current was dose-dependent and reversible. The EC50 was 42.8+/-11.6 and 46.6+/-7.1 microg ml(-1) for the transient and sustained inward current, respectively. 3 In the absence of extracellular Ca2+ ginsenosides induced only a transient inward current but in the presence of extracellular Ca2+ ginsenosides induced the biphasic current. Magnitudes of the sustained currents were dependent on extracellular Ca2+ concentration. Sustained inward current induced by ginsenosides, but not transient inward current, and ginsenoside-induced store-operated Ca2+ (SOC) currents (ISOC) were blocked by La3+, a Ca2+ channel blocker, suggesting that the sustained inward current and ISOC was derived from an influx of extracellular Ca2+. 4 Treatment with 2-APB and heparin, which are IP3 receptor antagonists, inhibited the ginsenoside-induced biphasic current. Treatment with the PLC inhibitor, U73122, also inhibited the ginsenoside-induced biphasic current. Intraoocyte injection of ATP-gammaS, but not adenylyl AMP-PCP, induced a persistent activation of ginsenoside-induced sustained current but did not affect the transient current. 5 In rat hippocampal neurons, ginsenosides inhibited both carbachol-stimulated intracellular Ca2+ release and intracellular Ca2+ depletion-activated SOCE. 6 These results indicate that ginsenoside might act as a differential regulator of intracellular Ca2+ levels in neurons and Xenopus oocytes.     

Electrophysiological effects of endothelin-1 and their relationship to contraction in rat renal arterial smooth muscle

The electophysiological effects of endothelin-1 (ET-1) and their relationship to contraction remain unclear in the renal circulation. Using endotheliumdenuded arteries from the main branch of the renal artery proximal to the kidney of the rat, we have examined its effects on tension and conducted parallel patch-clamp measurements using freshly isolated smooth muscle cells from this tissue. Pharmacological experiments revealed that ET-1 produced constriction of renal arteries dependent on the influx of extracellular Ca(2+), mediated solely through ET(A) receptor stimulation. Current-clamp experiments revealed that renal arterial myocytes had a resting membrane potential of approximately 32 mV, with the majority of cells exhibiting spontaneous transient hyperpolarizations (STHPs). Application of ET-1 produced depolarization and in those cells exhibiting STHPs, either caused their inhibition or made them occur regularly. Under voltage-clamp conditions cells were observed to exhibit spontaneous transient outward currents (STOCs) inhibited by iberiotoxin. Application of voltage-ramps revealed an outward current activated at approximately -30 mV, sensitive to both 4-AP and TEA. Taken together these results suggest that renal arterial myocytes possess both delayed rectifying K(+) (K(V)) and Ca(2+)-activated K(+) (BK(Ca)) channels. Under voltage-clamp, ET-1 attenuated the outward current and reduced the magnitude and incidence of STOCs: effects mediated solely as a consequence of ET(A) receptor stimulation. Thus, in conclusion, activation of ET(A) receptors by ET-1 causes inhibition of K(V) and BK(Ca) channel activity, which could promote and/or maintain membrane depolarization. This effect is likely to favour L-type Ca(2+) channel activity providing an influx pathway for extracellular Ca(2+) essential for contraction.     

Characterization of an apamin-sensitive small-conductance Ca(2+)-activated K(+) channel in porcine coronary artery endothelium: relevance to EDHF

1. The apamin-sensitive small-conductance Ca(2+)-activated K(+) channel (SK(Ca)) was characterized in porcine coronary arteries. 2. In intact arteries, 100 nM substance P and 600 microM 1-ethyl-2-benzimidazolinone (1-EBIO) produced endothelial cell hyperpolarizations (27.8 +/- 0.8 mV and 24.1 +/- 1.0 mV, respectively). Charybdotoxin (100 nM) abolished the 1-EBIO response but substance P continued to induce a hyperpolarization (25.8 +/- 0.3 mV). 3. In freshly-isolated endothelial cells, outside-out patch recordings revealed a unitary K(+) conductance of 6.8 +/- 0.04 pS. The open-probability was increased by Ca(2+) and reduced by apamin (100 nM). Substance P activated an outward current under whole-cell perforated-patch conditions and a component of this current (38%) was inhibited by apamin. A second conductance of 2.7 +/- 0.03 pS inhibited by d-tubocurarine was observed infrequently. 4. Messenger RNA encoding the SK2 and SK3, but not the SK1, subunits of SK(Ca) was detected by RT - PCR in samples of endothelium. Western blotting indicated that SK3 protein was abundant in samples of endothelium compared to whole arteries. SK2 protein was present in whole artery nuclear fractions. 5. Immunofluorescent labelling confirmed that SK3 was highly expressed at the plasmalemma of endothelial cells and was not expressed in smooth muscle. SK2 was restricted to the peri-nuclear regions of both endothelial and smooth muscle cells. 6. In conclusion, the porcine coronary artery endothelium expresses an apamin-sensitive SK(Ca) containing the SK3 subunit. These channels are likely to confer all or part of the apamin-sensitive component of the endothelium-derived hyperpolarizing factor (EDHF) response.     

Modulation of spontaneous transient Ca2+-activated K+ channel currents by cADP-ribose in vascular smooth muscle cells

Transient local releases of Ca(2+) from the sarcoplasmic reticulum activate nearby Ca(2+)-activated K(+) channels to produce spontaneous transient outward current (STOC) in smooth muscle cells. We examined if cADP-ribose, an endogenous mediator of Ca(2+) release channels of the sarcoplasmic reticulum, could modify STOC activity. In freshly isolated rat tail arterial cells, cADP-ribose (5 microM) increased STOC frequency significantly from 308+/-26.2 to 398.8+/-28.8 per minute. The average current at a test potential of -20 mV was increased significantly from 47.8+/-0.7 to 101.1+/-0.7 pA in the presence of cADP-ribose. The cell permeant antagonist 8-bromo-cADP-ribose (50 microM) reduced significantly the STOC frequency to 52.5+/-7.5 per minute and the average current to 24.7+/-0.1 pA. The STOCs were inhibited significantly by ryanodine (1 microM) and charybodotoxin (150 nM). These findings suggest the presence of basal cADP-ribose activity in resting vascular smooth muscle cells and that STOC activity is stimulated by cADP-ribose.     

Elevated extracellular K(+) concentrations inhibit N-methyl-D-aspartate-induced Ca(2+) influx and excitotoxicity.

Although extracellular [K(+)] ([K(+)](E)) is highly elevated during brain ischemia, in vitro studies aimed at explaining the mechanisms of excitotoxicity have been conducted at low [K(+)](E). Whether high [K(+)](E) affects excitotoxicity has not been formally addressed. Therefore this study, using digital fluorescence microscopy, tested how the elevation of [K(+)](E) from 5.6 to 60 mM affects N-methyl-D-aspartate (NMDA)-induced Ca(2+) and Na(+) influx, plasma membrane (PM) potential, mitochondrial Ca(2+) load, and viability of primary cultures of rat cerebellar granule cells. High [K(+)](E) curtailed the NMDA-induced Ca(2+) and Na(+) influx and mitochondrial Ca(2+) overload, and prevented neuronal death. Surprisingly, the inhibitory effect of high [K(+)](E) on the NMDA-induced Ca(2+) influx could not be linked to depolarization of the PM. Apparently, the PM of cerebellar granule cells exposed to NMDA was more depolarized at low than at high [K(+)](E), probably because the NMDA-induced Na(+) influx was greatly enhanced when the extracellular [Na(+)]/[K(+)] ratio was increased. When this ratio was small, i.e., at high [K(+)](E), the NMDA-induced increase in cytoplasmic [Na(+)] was suppressed, preventing Ca(2+) influx via the reverse operation of the Na(+)/Ca(2+) exchanger, which may explain the inhibitory effect of high [K(+)](E) on NMDA-induced Ca(2+) influx and excitotoxicity.     

Ionic currents and inhibitory effects of glibenclamide in seminal vesicle smooth muscle cells.

1. Whole-cell voltage-clamp recordings were made from smooth muscle cells isolated from guinea-pig seminal vesicle. 2. When the recording pipette solution contained 130 mM KCl and a low concentration of EGTA (0.2 mM), a dominant outward current was elicited by depolarization to positive of -30 mV from a holding potential of -50 mV. The current was non-inactivating, stimulated by intracellular Ca2+ and blocked by bath-applied 1 mM tetraethylammonium but not 1 mM 3,4 diaminopyridine. 3. If 10 mM EGTA was added to the KCl pipette solution and the holding potential was -50 mV, or more negative, the major current elicited by depolarization to positive of -30 mV was an A-type K(+)-current. This current inactivated rapidly (within 100 ms) and was blocked by bath-applied 1 mM 3,4-diaminopyridine but not 10 mM tetraethylammonium. 4. An inward voltage-gated Ca channel current was observed on depolarization to positive of -30 mV with 1.5 mM Ca2+ or 10 mM Ba2+ in the bath solution and when Ca+ replaced K+ in the pipette. The Ba(2+)-current was shown to be abolished by bath-applied 100 microM Cd2+ and inhibited by 90% by 1 microM nifedipine, and thus appeared to be carried by L-type Ca channels. 5. High concentrations of glibenclamide (10-500 microM) inhibited A-type K(+)-current, Ba(2+)-current and contraction of the whole tissue induced by noradrenaline or electrical field stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of functionally protective K(+) channels by methylmercury in rat alveolar macrophages

Methylmercury (MeHg) is generally known as a neurotoxic heavy metal while its effect on alveolar macrophages is still rarely studied. In this paper, we attempted to use whole cell and cell-attached patch-clamp recording technique and fura-2 fluorescence measurement to elucidate the effects of MeHg on rat alveolar macrophages. The results showed that extracellular application of MeHg induced a transient outward current I(O)(MeHg), 10-20 s in duration, 100-1000 pA in amplitude at -40 mV associated with a marked increase in conductance. The reversal potential depended distinctly on the external K(+) concentration. Removal of external Ca(2+) as well as bath applied verapamil caused a depression of I(O)(MeHg), and intracellular dialysis with 5 mM EGTA completely abolished I(O)(MeHg). Heparin (5 mg/ml) applied by intracellular dialysis greatly accelerated a run-down of I(O)(MeHg) induced by pressure ejection of MeHg. K(+) channel blockers such as quinine, and 4-aminopyridine especially low concentrations of dequalinium and apamin, but not tetraethylammonium inhibited I(O)(MeHg). Cell-attached single-channel recordings with the pipette solution containing 145 mM KCl revealed that the activation of single-channel currents with a conductance of 12 pS could be induced by application of MeHg outside the patch. Since MeHg increased [Ca(2+)](i), in a concentration-dependent manner which was partially blocked by either verapamil or Ca(2+)-free medium containing 1 mM EGTA, it is concluded that MeHg activates a Ca(2+)-dependent K(+) conductance by an increase of [Ca(2+)](i) through an influx from outside the cells as well as mobilization from intracellular store. A possibility that this membrane hyperpolarizing K(+) current may exhibit a functioning modulator in response to the harmful cytotoxic increase in [Ca(2+)](i) caused by MeHg was tested. Accordingly, this working hypothesis is verified by an increase of MeHg-induced cytotoxicity of cultured rat alveolar macrophages through a blockade of this Ca(2+)-activated K(+) channel by dequalinium.