Effects of intermediate-conductance Ca2+-activated K+ channel modulators on human prostate cancer cell proliferation

The effects of 1-ethyl-2-benzimidazolinone (1-EBIO) and riluzole on human prostate cancer cells, LNCaP and PC-3, were evaluated using rubidium (86Rb(+)) efflux and proliferation assays. 1-EBIO and riluzole evoked concentration-dependent increases in 86Rb(+) efflux from LNCaP and PC-3 cells that were sensitive to inhibition by intermediate-conductance Ca(2+)-activated K(+) channel (IK(Ca)) blockers clotrimazole and charybdotoxin. Blockers of large-conductance Ca(2+)-activated K(+) (BK(Ca)) channel, iberiotoxin, or small-conductance Ca(2+)-activated K(+) (SK(Ca)) channel, apamin or scyllatoxin, had no effect. Concurrently, both 1-EBIO and riluzole evoked concentration-dependent increases in proliferation from human prostate cancer cell lines (LNCaP and PC-3 cells). Clotrimazole and charybdotoxin, but not iberiotoxin, apamin or scyllatoxin, inhibited 1-EBIO- and riluzole-evoked increases in proliferation from LNCaP and PC-3 cells. N-(3-(trifluoromethyl)phenyl)-N'-(2-hydroxy-5-chlorophenyl)urea (NS-1608) and 2-amino-5-(2-fluorophenyl)-4-methyl-1H-pyrrole-3-carbonitrile (NS-8), BK(Ca) channel openers had no effect on LNCaP and PC-3 proliferation. These results demonstrate that IK(Ca) channels play an important role in the regulation of human prostate cancer cell proliferation.     

Diethyl pyrocarbonate, a histidine-modifying agent, directly stimulates activity of ATP-sensitive potassium channels in pituitary GH(3) cells

The ATP-sensitive K(+) (K(ATP)) channels are composed of sulfonylurea receptor and inwardly rectifying K(+) channel (Kir6.2) subunit. These channels are regulated by intracellular ADP/ATP ratio and play a role in cellular metabolism. Diethyl pyrocarbonate (DEPC), a histidine-specific alkylating reagent, is known to modify the histidine residues of the structure of proteins. The objective of this study was to determine whether DEPC modifies K(ATP)-channel activity in pituitary GH(3) cells. Steady-state fluctuation analyses of macroscopic K(+) current at -120 mV produced power spectra that could be fitted with a single Lorentzian curve in these cells. The time constants in the presence of DEPC were increased. Consistent with fluctuation analyses, the mean open time of K(ATP)-channels was significantly increased during exposure to DEPC. However, DEPC produced no change in single-channel conductance, despite the ability of this compound to enhance K(ATP)-channel activity in a concentration-dependent manner with an EC(50) value of 16 microM. DEPC-stimulated K(ATP)-channel activity was attenuated by pretreatment with glibenclamide. In current-clamp configuration, DEPC decreased the firing of action potentials in GH(3) cells. A further application of glibenclamide reversed DEPC-induced inhibition of spontaneous action potentials. Intracellullar Ca(2+) measurements revealed the ability of DEPC to decrease Ca(2+) oscillations in GH(3) cells. Simulation studies also demonstrated that the increased conductance of K(ATP)-channels used to mimic DEPC actions reduced the frequency of spontaneous action potentials and fluctuation of intracellular Ca(2+). The results indicate that chemical modification with DEPC enhances K(ATP)-channel activity and influences functional activities of pituitary GH(3) cells.     

A-272651, a nonpeptidic blocker of large-conductance Ca2+-activated K+ channels, modulates bladder smooth muscle contractility and neuronal action potentials

BACKGROUND AND PURPOSE: The large-conductance Ca(2+)-activated K(+) channel (BK(Ca), K(Ca)1.1) links membrane excitability with intracellular Ca(2+) signaling and plays important roles in smooth muscle contraction, neuronal firing, and neuroendocrine secretion. This study reports the characterization of a novel BK(Ca) channel blocker, 2,4-dimethoxy-N-naphthalen-2-yl-benzamide (A-272651). EXPERIMENTAL APPROACH: (86)Rb(+) efflux in HEK-293 cells expressing BK(Ca) was measured. Effects of A-272651 on BK(Ca) alpha- and BK(Ca) alphabeta1-mediated currents were evaluated by patch-clamp. Effects on contractility were assessed using low-frequency electrical field stimulated pig detrusor and spontaneously contracting guinea pig detrusor. Effects of A-272651 on neuronal activity were determined in rat small diameter dorsal root ganglia (DRG). KEY RESULTS: A-272651 (10 microM) inhibited (86)Rb(+) efflux evoked by NS-1608 in HEK-293 cells expressing BK(Ca) currents. A-272651 concentration-dependently inhibited BK(Ca) currents with IC(50) values of 4.59 microM (Hill coefficient 1.04, measured at +40 mV), and 2.82 microM (Hill coefficient 0.89), respectively, for BK(Ca) alpha and BK(Ca) alphabeta1-mediated currents. Like iberiotoxin, A-272651 enhanced field stimulated twitch responses in pig detrusor and spontaneous contractions in guinea pig detrusor with EC(50) values of 4.05+/-0.05 and 37.95+/-0.12 microM, respectively. In capsaicin-sensitive DRG neurons, application of A-272651 increased action potential firing and prolonged action potential duration. CONCLUSIONS AND IMPLICATIONS: These data demonstrate that A-272651 modulates smooth muscle contractility and neuronal firing properties. Unlike previously reported peptide BK(Ca) blockers, A-272651 represents one of the first small molecule BK(Ca) channel blockers that could serve as a useful tool for further characterization of BK(Ca) channels in physiological and pathological states.     

Large conductance Ca2+-activated K+ (BKCa) channels are involved in the vascular relaxations elicited by piceatannol isolated from Rheum undulatum rhizome

We previously reported that piceatannol isolated from the rhizome extract of RHEUM UNDULATUM has a potent vasorelaxant activity. In the present study, the mechanisms underlying the direct vascular relaxant effect of piceatannol were investigated in isolated rat aorta. Piceatannol induced a concentration-dependent relaxation in aortic preparations precontracted with phenylephrine (EC (50) : 2.4 +/- 0.4 microM), which was completely inhibited by endothelial removal, N(omega)-nitro- L-arginine (nitric oxide synthase inhibitor), methylene blue and 1 H- oxadiazolo [4,3- A]quinoxalin-1-one (guanylyl cyclase inhibitor). The piceatannol-induced relaxation was also blocked by raising the extracellular K (+) (45 mM), 4-aminopyridine (voltage-sensitive K (+) channel blocker) and tetraethylammonium [the non-selective Ca (2+)-activated K (+) (K (Ca)) channel blocker] but not by indomethacin (cyclooxygenase inhibitor), atropine (muscarinic receptor antagonist), propranolol (beta-adrenoceptor antagonist), verapamil and nifedipine (L-type voltage-gated Ca (2+) channel blocker), barium chloride (inward rectifier K (+) channel inhibitor) and glibenclamide (ATP-sensitive K (+) channel blocker). In further studies investigating the role of Ca (2+)-activated K (+) (K (Ca)) channels, piceatannol-induced relaxant responses were decreased by charybdotoxin [large (BK (Ca))- and intermediate (IK (Ca))-conductance K (Ca) channel blocker], iberiotoxin (selective BK (Ca) channels blocker), but not by apamin [small-conductance K (Ca) (SK (Ca)) channel blocker], TRAM-34 [intermediate-conductance K (Ca) (IK (Ca)) channel blocker]. The present results demonstrate that piceatannol-induced vascular relaxation in rat aorta may be mediated by an endothelium-dependent nitric oxide signaling pathway, at least partially, through the activation of BK (Ca).     

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.     

Inhibition of ATP-sensitive K+ channels by taurine through a benzamido-binding site on sulfonylurea receptor 1

ATP-sensitive potassium (K(ATP)) channels in pancreatic beta-cells comprise sulfonylurea receptor (SUR) 1 and inwardly-rectifying potassium channel (Kir) 6.2 subunits. We have evaluated the effect of intracellular taurine on K(ATP) channel activity in rat pancreatic beta-cells using the patch-clamp technique. The mechanism of taurine action was also examined using recombinant K(ATP) channels. The islets and single beta-cells from male Sprague-Dawley rats were collected by collagenase digestion technique. Single K(ATP) channel currents were recorded by the inside-out mode at a membrane potential of -60mV. Cytosolic free-Ca(2+) concentration ([Ca(2+)](c)) and insulin secretory capacity were measured by the dual-excitation fluorimetry and radioimmunoassay, respectively. The native beta-cell K(ATP) channel was directly inhibited by taurine in a dose-dependent manner. Taurine did not influence ATP-mediated inhibition or MgADP-induced activation of the channel activity. The sensitivity of the K(ATP) channel to glybenclamide, but not gliclazide, was enhanced by taurine. Glybenclamide elicited a greater increase in [Ca(2+)](c) and increased insulin secretion in the beta-cells when pretreated with taurine. Taurine did not inhibit Kir6.2DeltaC36 currents, a truncated form of Kir6.2, expressed in Xenopus oocytes without SUR. These results demonstrate that taurine inhibits the K(ATP) channel activity in the beta-cells, interacting with a benzamido-binding site on SUR1, but not Kir6.2.     

Large-conductance Ca2+- activated K+ channels:physiological role and pharmacology

Large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels differ from most of other K(+) channels in that their activation is under dual control, i.e., activated by either increase in intracellular Ca(2+) or membrane depolarization. These channels, which are widely distributed in a variety of cells, can control Ca(2+) influx as well as a number of Ca(2+)-dependent physiological processes. In neurons or neuroendocrine cells, BK(Ca) channels are believed to play an important role in controlling hormonal secretion by altering the duration and frequency of action potentials. The activity of BK(Ca) channels functionally expressed in vascular endothelial cells can control K(+) efflux and affect intracellular Ca(2+) concentration. Experimental observations have revealed that a variety of compounds can directly modulate BK(Ca) channel activity. Epoxyeicosatrienoic acids, a metabolite of arachidonic acid, and the increase in intracellular cyclic GMP with vinpocetine or YC-1 can stimulate BK(Ca) channel activity. The increased activity of BK(Ca) channels thus serves as a negative feedback mechanism to limit Ca(2+) influx in excitable cells. Clotrimazole, an imidazole P-450 inhibitor used for the management of sickle cell anemia, can directly suppress BK(Ca) channel activity. Riluzole, a drug used for the treatment of amyotrophic lateral sclerosis, can directly enhance channel activity in neuroendocrine cells. This effect may explain its inhibitory action on excitatory neurotransmission. 2-Methoxyestradil, an endogenous metabolite of 17beta-estradiol, suppresses BK(Ca) channel activity, whereas resveratrol, a natural phytoalexin present in grapes and wine, directly stimulates BK(Ca) channel activity in vascular endothelial cells. These effects may be responsible for their actions on functional activities of endothelial cells. The fenamates, a family of nonsteroidal anti-inflammatory drugs, are also openers of BK(Ca) channels. Therefore, the modulation of BK(Ca) channel activity in excitable and non-excitable cells can be important for therapeutic interventions.     

(+/-)-Naringenin as large conductance Ca(2+)-activated K+ (BKCa) channel opener in vascular smooth muscle cells

BACKGROUND AND PURPOSE. The aim of this study was to investigate, in vascular smooth muscle cells, the mechanical and electrophysiological effects of (+/-)-naringenin. EXPERIMENTAL APPROACH: Aorta ring preparations and single tail artery myocytes were employed for functional and patch-clamp experiments, respectively. KEY RESULTS: (+/-)-Naringenin induced concentration-dependent relaxation in endothelium-denuded rat aortic rings pre-contracted with either 20 mM KCl or noradrenaline (pIC(50) values of 4.74 and 4.68, respectively). Tetraethylammonium, iberiotoxin, 4-aminopyridine and 60 mM KCl antagonised (+/-)-naringenin-induced vasorelaxation, while glibenclamide did not produce any significant antagonism. Naringin [(+/-)-naringenin 7-beta-neohesperidoside] caused a concentration-dependent relaxation of rings pre-contracted with 20 mM KCl, although its potency and efficacy were significantly lower than those of (+/-)-naringenin. In rat tail artery myocytes, (+/-)-naringenin increased large conductance Ca(2+)-activated K(+) (BK(Ca)) currents in a concentration-dependent manner; this stimulation was iberiotoxin-sensitive and fully reversible upon drug wash-out. (+/-)-Naringenin accelerated the activation kinetics of BK(Ca) current, shifted, by 22 mV, the voltage dependence of the activation curve to more negative potentials, and decreased the slope of activation. (+/-)-Naringenin-induced stimulation of BK(Ca) current was insensitive either to changes in the intracellular Ca(2+) concentration or to the presence, in the pipette solution, of the fast Ca(2+) chelator BAPTA. However, such stimulation was diminished when the K(+) gradient across the membrane was reduced. CONCLUSIONS AND IMPLICATIONS: The vasorelaxant effect of the naturally-occurring flavonoid (+/-)-naringenin on endothelium-denuded vessels was due to the activation of BK(Ca) channels in myocytes.     

Nitric oxide stimulates a large-conductance Ca-activated K+ channel in human skin fibroblasts through protein kinase G pathway

In order to investigate the large-conductance Ca(2+)-activated K(+) (BK(Ca)) channel and determine the effects of nitric oxide (NO) on the channel in human skin fibroblasts, we performed electrophysiological patch clamp recordings on 5th-passage cells of human genital skin cultures. The whole-cell outward K(+) current was increased with depolarization, and proved to be sensitive to NS1619 (a selective BK(Ca) channel activator) and iberiotoxin (a specific BK(Ca )channel inhibitor). The single-channel currents showed 226 pS of mean conductance in symmetrical K(+). Sodium nitroprusside (SNP; an NO donor) significantly increased the K(+) current amplitude in the whole-cell mode, and open probability of the channel (NPo) in the cell-attached mode, but not in the inside-out mode. S-nitroso-N-acetylpenicillamine (an NO donor) and 8-Br-cGMP (a membrane-permeant cGMP analogue) also increased the BK(Ca )channel activity. The stimulatory effect of SNP on BK(Ca) channels was inhibited by pretreatment with 1H-[1,2,4]-oxadiazolo[4,3-a]quinoxalin-1-one (a soluble guanylyl cyclase inhibitor), or KT5823 [a specific protein kinase G (PKG) inhibitor]. Cytoplasmic PKG also increased the channel activity in inside-out patches. In conclusion, the present data indicate that BK(Ca) channels constitute a significant fraction of K(+) current in human skin fibroblasts, and that NO increases NPo of BK(Ca) channels, which are mediated via the cGMP/PKG pathway, without direct effects on the channel.     

Pulses of external ATP aid to the synchronization of pancreatic beta-cells by generating premature Ca(2+) oscillations

Pancreatic beta-cells respond to glucose stimulation with increase of the cytoplasmic Ca(2+) concentration ([Ca(2+)](i)), manifested as membrane-derived slow oscillations sometimes superimposed with transients of intracellular origin. The effect of external ATP on the oscillatory Ca(2+) signal for pulsatile insulin release was studied by digital imaging of fura-2 loaded beta-cells and small aggregates isolated from islets of ob/ob-mice. Addition of ATP (0.01-100 microM) to media containing 20mM glucose temporarily synchronized the [Ca(2+)](i) rhythmicity in the absence of cell contact by eliciting premature oscillations. External ATP triggered premature [Ca(2+)](i) oscillations also when the sarcoendoplasmic reticulum Ca(2+)-ATPase was inhibited with 50 microM cyclopiazonic acid and phospholipase C inhibited with 10 microM U-73122. The effect of ATP was mimicked by other activators of cytoplasmic phospholipase A(2) (10nM acetylcholine, 0.1-1 micro M of the C-terminal octapeptide of cholecystokinin and 2 microg/ml melittin) and suppressed by an inhibitor of the enzyme (50 microM p-amylcinnamoylanthranilic acid). Premature oscillations generated by pulses of ATP sometimes triggered subsequent oscillations. However, prolonged exposure to high concentrations of the nucleotide (10-100 microM) had a suppressive action on the beta-cell rhythmicity. The early effects of ATP included generation of transients induced by inositol (1,4,5) trisphosphate and superimposed on the premature oscillation or on an ordinary oscillation induced by glucose. The results support the idea that purinergic activation of phospholipase A(2) has a co-ordinating effect on the beta-cell rhythmicity by triggering premature [Ca(2+)](i) oscillations mediated by closure of ATP-sensitive K(+) channels.     

Functional analysis of large conductance Ca2(+)-activated K(+) channels: ion flux studies by atomic absorption spectrometry

Although techniques such as (86)Rb(+) flux provide a sensitive measure of K(+) channel activity, the relatively short half-life and high-energy emission, together with the quantities of radioactive material generated, hinder the usefulness of flux-based formats in high throughput screening efforts. This study elaborates on the utilization of flame atomic absorption spectrometry (AAS) techniques for a nonradioactive rubidium efflux assay for large conductance Ca(2+)-activated K(+) channels (BK(Ca)) channels. Utilizing human embryonic kidney (HEK293) cells expressing the BK(Ca) alpha subunit, a 96-well cell-based nonradioactive rubidium efflux screen for channel openers and inhibitors was established. Known BK(Ca) channel openers, including NS1608, NS1619, and NS-8, activated rubidium efflux with EC(50) values ranging from 1 to 4 microM in both radioactive and nonradioactive efflux formats. Compounds such as iberiotoxin, paxilline, and charybdotoxin inhibited rubidium efflux responses evoked by the BK(Ca) channel opener NS1608 in both radioactive and nonradioactive efflux formats. The IC(50) values of the inhibitors in AAS format were comparable to those derived from (86)Rb(+) efflux assays. The present studies show that the pharmacological profiles of BK(Ca) channels assessed by AAS compare well with those obtained using the (86)Rb(+) efflux assay, and support the utility of nonradioactive efflux format for higher throughput screening campaigns for novel K(+) channel modulators.     

NPPB block of the intermediate-conductance Ca2+-activated K+ channel

We have shown that the Cl(-) channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) also blocks the intermediate-conductance Ca(2+)-activated K(+) (IK(Ca)) current in human leukemic HL-60 and glioblastoma GL-15 cell lines. The macroscopic IK(Ca) current was activated by ionomycin plus 1-EBIO, and identified as intermediate conductance by being fully blocked by charybdotoxin, clotrimazole, nitrendipine (L-type Ca(2+) channel blocker), and NS1619 (BK(Ca) channel opener), but not by D-tubocurarine or TEA. The IK(Ca) current was blocked by NPPB in a reversible dose-dependent manner, with an IC(50) of 39 microM in HL-60 and 125 microM in GL-15 cells. The block of the IK(Ca) current was also recorded at the single channel level in excised inside-out patches. As expected, NPPB also blocked the volume-activated Cl(-) current expressed by GL-15 cells, with an IC(50) of 44 microM. The functional implications of IK(Ca) current block by NPPB are discussed.     

Quercetin-induced Growth Inhibition in Human Bladder Cancer Cells Is Associated with an Increase in Ca-activated K Channels

Quercetin (3,3',4',5,7-pentahydroxyflavone) is an attractive therapeutic flavonoid for cancer treatment because of its beneficial properties including apoptotic, antioxidant, and antiproliferative effects on cancer cells. However, the exact mechanism of action of quercetin on ion channel modulation is poorly understood in bladder cancer 253J cells. In this study, we demonstrated that large conductance Ca(2+)-activated K(+) (BK(Ca)) or MaxiK channels were functionally expressed in 253J cells, and quercetin increased BK(Ca) current in a concentration dependent and reversible manner using a whole cell patch configuration. The half maximal activation concentration (IC(50)) of quercetin was 45.5±7.2 μM. The quercetin-evoked BK(Ca) current was inhibited by tetraethylammonium (TEA; 5 mM) a non-specific BK(Ca) blocker and iberiotoxin (IBX; 100 nM) a BK(Ca)-specific blocker. Quercetin-induced membrane hyperpolarization was measured by fluorescence-activated cell sorting (FACS) with voltage sensitive dye, bis (1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC(4)(3); 100 nM). Quercetin-evoked hyperpolarization was prevented by TEA. Quercetin produced an antiproliferative effect (30.3±13.5%) which was recovered to 53.3±10.5% and 72.9±3.7% by TEA and IBX, respectively. Taken together our results indicate that activation of BK(Ca) channels may be considered an important target related to the action of quercetin on human bladder cancer cells.     

Beta-cell toxicity of ATP-sensitive K+ channel-blocking insulin secretagogues

A prolonged exposure of isolated pancreatic islets to insulin secretagogues, the imidazolines phentolamine, alinidine and idazoxan (100microM each), the sulfonylurea tolbutamide (500microM), or the alkaloid quinine (100microM) resulted in morphological damage of 4-18% of beta-cells compared to less than 2% in controls. Thus, the question arose whether K(ATP) channel-blocking insulin secretagogues are beta-cell toxic as has already been suggested for sulfonylureas. The concentration- and time-dependency of the secretagogue-associated toxicity was documented by viability assays in insulin-secreting HIT T15 cells. Treatment for 24h with idazoxan reduced MTT conversion by 50% at 100microM and by 98% at 1000microM. Phentolamine and quinine reduced viability comparably at 1000microM, but were less toxic at 100microM. On the other hand, the imidazoline alinidine and the sulfonylurea tolbutamide were only moderately toxic (less than 40% viability loss at 1000microM). The imidazoline efaroxan appeared even to be non-toxic. Apoptotic DNA fragmentation and DEVD-caspase activation was observed at 100microM of idazoxan and phentolamine, whereas at 1000microM signs of necrosis predominated. Alinidine, tolbutamide and quinine treatment did not increase markers of apoptotic cell death. Blocking Ca(2+) influx by D600 did not diminish secretagogue-associated toxicity. Electron microscopy confirmed the validity of these observations for beta-cells in intact mouse islets. In summary, beta-cell toxicity of the tested insulin secretagogues varied widely and did not depend on a prolonged Ca(2+) influx via L-type Ca(2+) channels. Thus, secretagogue-mediated closure of K(ATP) channels is apparently not per se beta-cell toxic.     

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.     

Antagonism of the insulinotropic action of first generation imidazolines by openers of K(ATP) channels

The antagonism between K(ATP) channel-blocking insulinotropic imidazolines - phentolamine, alinidine, idazoxan and efaroxan - and K(ATP) channel openers, diazoxide and nucleoside diphosphates, was studied in mouse pancreatic islets and B-cells. In inside-out patches from B-cells, 500muM MgGDP abolished the inhibitory effect of the imidazolines. 300muM diazoxide further increased channel activity. The depolarizing effect of all imidazolines (100muM) on the B-cell membrane potential was practically completely antagonized by 300muM diazoxide. In contrast, diazoxide was unable to decrease the cytosolic Ca(2+) concentration ([Ca(2+)](i)) which was elevated by phentolamine, whereas the [Ca(2+)](i) increases induced by the other imidazolines were promptly antagonized. The effects on [Ca(2+)](i) were reflected by the secretory activity in that the stimulatory effects of alinidine, idazoxan and efaroxan, but not that of phentolamine were antagonized by diazoxide. Metabolic inhibition of intact B-cells by 250muM NaCN, most likely by a decrease of the ATP/ADP ratio, significantly diminished the K(ATP) channel-blocking effect of a low concentration of alinidine (10muM), whereas efaroxan proved to be susceptible even at a highly effective concentration (100muM). This may explain the oscillatory pattern of the [Ca(2+)](i) increase typically produced by efaroxan in pancreatic B-cells. In conclusion, the inhibitory effect of imidazolines on K(ATP) channels, which is exerted at the pore-forming subunit, Kir6.2, is susceptible to the action of endogenous and exogenous K(ATP) channel openers acting at the regulatory subunit SUR, which confers tissue specificity. With intact cells this antagonism can be obscured, possibly by intracellular accumulation of some imidazolines.     

Gabapentin-mediated inhibition of voltage-activated Ca2+ channel currents in cultured sensory neurones is dependent on culture conditions and channel subunit expression

We have used the whole cell patch clamp method and fura-2 fluorescence imaging to study the actions of gabapentin (1-(aminoethyl) cyclohexane acetic acid) on voltage-activated Ca(2+) entry into neonatal cultured dorsal root ganglion (DRG) neurones and differentiated F-11 (embryonic rat DRG x neuroblastoma hybrid) cells. Gabapentin (2.5 microM) in contrast to GABA (10 microM) did not influence resting membrane potential or input resistance. In current clamp mode gabapentin failed to influence the properties of evoked single action potentials but did reduce the duration of action potentials prolonged by Ba(2+). Gabapentin attenuated high voltage-activated Ca(2+) channel currents in a dose- and voltage- dependent manner in DRG neurones and reduced Ca(2+) influx evoked by K(+) depolarisation in differentiated F-11 cells loaded with fura-2. The sensitivity of DRG neurones to gabapentin was not changed by the GABA(B) receptor antagonist saclofen but pertussis toxin pre-treatment reduced the inhibitory effects of gabapentin. Experiments following pre-treatment of DRG neurones with a PKA-activator and a PKA-inhibitor implicated change in phosphorylation state as a mechanism, which influenced gabapentin action. Sp- and Rp-analogues of cAMP significantly increased or decreased gabapentin-mediated inhibition of voltage-activated Ca(2+) channel currents. Culture conditions used to maintain DRG neurones and passage number of differentiated F-11 cells also influenced the sensitivity of Ca(2+) channels to gabapentin. We analysed the Ca(2+) channel subunits expressed in populations of DRG neurones and F-11 cells that responded to gabapentin had low sensitivity to gabapentin or were insensitive to gabapentin, by Quantitative TaqMan PCR. The data obtained from this analysis suggested that the relative abundance of the Ca(2+) channel beta(2) and alpha(2)delta subunit expressed was a key determinant of gabapentin sensitivity of both cultured DRG neurones and differentiated F-11 cells. In conclusion, gabapentin inhibited part of the high voltage-activated Ca(2+) current in neonatal rat cultured DRG neurones via a mechanism that was independent of GABA receptor activation, but was sensitive to pertussis toxin. Gabapentin responses identified in this study implicated Ca(2+) channel beta(2) subunit type as critically important to drug sensitivity and interactions with alpha(1) and alpha(2)delta subunits may be implicated in antihyperalgesic therapeutic action for this compound.     

Cyclic AMP-mediated inhibition of gallbladder contractility: role of K+ channel activation and Ca2+ signaling

We have examined the mechanisms of cAMP-induced gallbladder relaxation by recording isometric tension and membrane potential in the intact tissue, and global intracellular calcium concentrations ([Ca(2+)](i)) and F-actin content in isolated myocytes. Both the phosphodiesterase (PDE) inhibitor, IBMX (100 microM) and the adenylate cyclase activator, forskolin (2 microM) caused decreases in basal tone that exhibited similar kinetics. IBMX and forskolin both caused concentration dependent, right-downward shifts in the concentration-response curves of KCl and cholecystokinin (CCK). IBMX and forskolin elicited a membrane hyperpolarization that was almost completely inhibited by the ATP-sensitive K(+) channel (K(ATP)) channel blocker, glibenclamide (10 microM). IBMX also induced an increase in large-conductance Ca(2+)-dependent K(+) (BK) channel currents, although the simultaneous blockade of BK and K(ATP) channels did not block IBMX- and forskolin-induced relaxations. Ca(2+) influx activated by L-type Ca(2+) channel activation or store depletion was also impaired by IBMX and forskolin, indicating a general impairment in Ca(2+) entry mechanisms. IBMX also decreases [Ca(2+)](i) transients activated by CCK and 3,6-Di-O-Bt-IP(4)-PM, a membrane permeable analog of inositol triphosphate, indicating an impairment in Ca(2+) release through IP(3) receptors. Ionomycin-induced [Ca(2+)](i) transients were not altered by IBMX, but the contractile effects of the Ca(2+) ionophore were reduced in the presence of IBMX, suggesting that cAMP can decrease Ca(2+) sensitivity of the contractile apparatus. A depolymerization of the thin filament could be reason for this change, as forskolin induced a decrease in F-actin content. In conclusion, these findings suggest that multiple, redundant intracellular processes are affected by cAMP to induce gallbladder relaxation.     

Large conductance Ca2+ -activated K+ channel activation with NS1619 decreases myogenic and neurogenic contractions of rat detrusor smooth muscle

Large conductance voltage- and Ca(2+)-activated K(+) (BK) channels are important in regulating detrusor smooth muscle (DSM) function. Here, we examined systematically how the BK channel pharmacological activation modulates DSM contractility. NS1619, a potent BK channel activator, was utilized as a pharmacological tool to investigate the effect of BK channel activation on rat DSM contractility. Isometric tension recordings of DSM strips isolated from rat urinary bladder were performed systematically under various experimental conditions. NS1619 (30 μM) substantially diminished DSM spontaneous contraction amplitude, muscle force integral, frequency, duration and muscle tone. This effect was blocked by iberiotoxin, a BK channel selective inhibitor. NS1619 inhibited the phasic and tonic contractions in DSM strips pre-contracted with either the cholinergic agonist, carbachol (0.1 μM), or the depolarizing agent, KCl (20mM). In the presence of elevated KCl (60 mM KCl), the inhibitory effect of NS1619 was significantly reduced, indicating that BK channel activation is the underlying mechanism of NS1619 action. BK channel activation with NS1619 dramatically decreased the amplitude of electrical field stimulation (EFS)-induced contractions under a range of stimulation frequencies (0.5-50 Hz). In the presence of specific neurotransmitter inhibitors, BK channel activation with NS1619 significantly decreased both cholinergic and purinergic components of EFS-induced contractions. We conclude that BK channel activation with NS1619 significantly inhibited spontaneous, pharmacologically induced and nerve-evoked DSM contractions. Targeting the BK channel with selective openers may offer a unique opportunity to control DSM contractile activity, including pathophysiological conditions such as overactive bladder and detrusor overactivity, regardless of the underlying cause.     

Insulin activates native and recombinant large conductance Ca(2+)-activated potassium channels via a mitogen-activated protein kinase-dependent process

Evidence is accumulating that, in addition to regulating peripheral energy metabolism, insulin is an important modulator of neuronal function. Indeed, high levels of insulin and insulin receptors are expressed in several brain regions including the hippocampus. We have shown previously that insulin inhibits aberrant synaptic activity in hippocampal neurons via activation of large conductance Ca(2+)-activated K+ (BK) channels. In this study, we have examined further the effects of insulin on native hippocampal and recombinant (hSlo) BK channels expressed in human embryonic kidney (HEK) 293 cells. Pipette or bath application of insulin evoked a rapid increase in hippocampal BK channel activity, an action caused by activation of insulin receptors because insulin-like growth factor 1 (IGF-1) failed to mimic insulin action. In parallel studies, insulin, applied via the pipette or bath, also activated hSlo channels expressed in HEK293 cells. Although phosphoinositide 3-kinase is a key component of insulin and IGF-1 receptor signaling pathways, activation of this lipid kinase does not underlie the effects of insulin because neither 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002) nor wortmannin inhibited or reversed insulin action. However, specific inhibitors of mitogen-activated protein kinase (MAPK) activation, 2'-amino-3'-methoxyflavone (PD98059) or 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)-butadiene (U0126), attenuated insulin action, indicating that a MAPK-dependent mechanism underlies this process. Furthermore, insulin activation of this pathway enhances BK channel activity by shifting the Ca(2+)-sensitivity such that BK channels are active at more hyperpolarized membrane potentials. Because postsynaptic BK channels are important regulators of neuronal hyperexcitability, insulin-induced activation of BK channels, via stimulation of a MAPK-dependent pathway, may be an important process for regulating hippocampal function under normal and pathological conditions.