Inhibitory action of ICI-182,780, an estrogen receptor antagonist, on BK(Ca) channel activity in cultured endothelial cells of human coronary artery

ICI-182,780 is known to be a selective inhibitor of the intracellular estrogen receptors. The effect of ICI-182,780 on ion currents was studied in cultured endothelial cells of human coronary artery. In whole-cell current recordings, ICI-182,780 reversibly decreased the amplitude of K(+) outward currents. The decrease in outward current caused by ICI-182,780 could be counteracted by further application of magnolol or nordihydroguaiaretic acid, yet not by 17beta-estradiol. Under current-clamp condition, ICI-182,780 (3microM) depolarized the membrane potentials of the cells, and magnolol (10 microM) or nordihydroguaiaretic acid (10 microM) reversed ICI-182,780-induced depolarization. In inside-out patches, ICI-182,780 added to the bath did not alter single-channel conductance of large-conductance Ca(2+)-activated K(+) channels (BK(Ca) channels), but decreased their open probability. ICI-182,780 reduced channel activity in a concentration-dependent manner with an IC(50) value of 3 microM. After BK(Ca) channel activity was suppressed by 2-methoxyestradiol (3 microM), subsequent application of ICI-182,780 (3 microM) did not further reduce the channel activity. The application of ICI-182,780 shifted the activation curve of BK(Ca) channels to positive potentials. Its decrease in the open probability primarily involved a reduction in channel open duration. ICI-182,780 also suppressed the proliferation of these endothelial cells with an IC(50) value of 2 microM. However, in coronary smooth muscle cells, a bell-shaped concentration-response curve for the ICI-182,780 effect on BK(Ca) channel activity was observed. This study provides evidence that ICI-182,780 can inhibit BK(Ca) channels in vascular endothelial cells in a mechanism unlikely to be linked to its anti-estrogen activity. The inhibitory effects on these channels may partly contribute to the underlying mechanisms by which ICI-182,780 affects endothelial function.     

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.     

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

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

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.     

Insulin inhibits rat hippocampal neurones via activation of ATP-sensitive K+ and large conductance Ca2+-activated K+ channels

In this study, we have used a combination of immunocytochemical and Ca(2+) imaging techniques to determine the functional localisation of insulin receptors as well as the potential role for insulin in modulating hippocampal synaptic activity. Comparison of insulin receptor and MAP2 labelling demonstrated extensive insulin receptor immunoreactivity on the soma and dendrites of cultured hippocampal neurones. Dual labelling with synapsin 1 also showed punctate insulin receptor labelling associated with synapses. In functional studies, insulin inhibited spontaneous Ca(2+) oscillations evoked in cultured hippocampal neurones following Mg(2+) removal. This action of insulin was mimicked by the ATP-sensitive K(+) (K(ATP)) channel opener diazoxide or the large conductance Ca(2+)-activated K(+) (BK) channel activator NS-1619. Furthermore, application of the K(ATP) channel blocker glybenclamide or the BK channel inhibitors iberiotoxin or charybdotoxin attenuated the actions of insulin, whereas prior incubation with a combination of glybenclamide and iberiotoxin completely blocked insulin action. The ability of insulin to modulate the Ca(2+) oscillations was reduced by the inhibitors of MAPK activation PD 98059 and U0126, but not by the PI 3-kinase inhibitors LY 294002 or wortmannin, indicating that a MAPK-driven process underlies insulin action. In conclusion, insulin inhibits spontaneous Ca(2+) oscillations via a process involving MAPK-driven activation of BK and K(ATP) channels. This process may be a useful therapeutic target for the treatment of epilepsy and certain neurodegenerative diseases.     

Molecular and functional characterization of the human platelet Na(+) /Ca(2+) exchangers

BACKGROUND AND PURPOSE The Na(+) /Ca(2+) exchanger is a bi-directional transporter that plays an important role in maintaining the concentration of cytosolic Ca(2+) ([Ca(2+) ](i) ) of quiescent platelets and increasing it during activation with some, but not all, agonists. There are two classes of Na(+) /Ca(2+) exchangers: K(+) -independent Na(+) /Ca(2+) exchanger (NCX) and K(+) -dependent Na(+) /Ca(2+) exchanger (NCKX). Platelets have previously been shown to express NCKX1. However, initial studies from our laboratory suggest that NCX may also play a role in platelet activation. The objective of this study was to determine if the human platelet expresses functional NCXs. EXPERIMENTAL APPROACH RT-PCR, DNA sequencing and Western blot analysis were utilized to characterize the human platelet Na(+) /Ca(2+) exchangers. Their function during quiescence and collagen-induced activation was determined by measuring [Ca(2+) ](i) with calcium-green/fura-red in response to: changes in the Na(+) and K(+) gradient, NCX pharmacological inhibitors (CBDMB, KB-R7943 and SEA0400) and antibodies specific to extracellular epitopes of the exchangers. KEY RESULTS Human platelets express NCX1.3, NCX3.2 and NCX3.4. The NCXs operate in the Ca(2+) efflux mode in resting platelets and also during their activation with thrombin but not collagen. Collagen-induced increase in [Ca(2+) ](i) was reduced with the pharmacological inhibitors of NCX (CBDMB, KB-R7943 or SEA0400), anti-NCX1 and anti-NCX3. In contrast, anti-NCKX1 enhanced the collagen-induced increase in [Ca(2+) ](i) . CONCLUSIONS AND IMPLICATIONS Human platelets express K(+) -independent Na(+) /Ca(2+) exchangers NCX1.3, NCX3.2 and NCX3.4. During collagen activation, NCX1 and NCX3 transiently reverse to promote Ca(2+) influx, whereas NCKX1 continues to operate in the Ca(2+) efflux mode to reduce [Ca(2+) ](i) .     

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.     

Characterization of beta-leptinotarsin-h and the effects of calcium flux antagonists on its activity.

beta-Leptinotarsin-h, purified from the hemolymph of the beetle Leptinotarsa haldemani, is a potent ( approximately 1 nM) neuroactive protein that rapidly (few seconds) stimulates Ca(2+) influx and neurotransmitter release. Our goals were to further characterize beta-leptinotarsin-h and to test the hypothesis that it stimulates Ca(2+) influx through presynaptic Ca(2+) channels. Analysis of partial amino acid sequences revealed that beta-leptinotarsin-h is a unique protein with significant similarity to only one other protein, the juvenile hormone esterase of Leptinotarsa decemlineata, commonly known as the Colorado potato beetle. We have examined the effect of beta-leptinotarsin-h on Ca(2+) current, Ca(2+) uptake, Ca(2+) levels, and neurotransmitter release in synaptosomes, cell lines, and neuronal systems. We found that its preferred site of action appears to be mammalian presynaptic nerve terminals. We tested antagonists of Ca(2+) flux for their effects on beta-leptinotarsin-h-stimulated Ca(2+) uptake in rat brain synaptosomes. The non-selective Ca(2+) channel blockers flunarizine, Ni(2+), ruthenium red, high-concentration thapsigargin, and SKF 96365 inhibited beta-leptinotarsin-h's activity, but none of the tested selective blockers of voltage-operated Ca(2+) channels (omega-agatoxin IVA, omega-conotoxin GVIA, omega-conotoxin MVIIC, nicardipine, nifedipine, SNX-482) was inhibitory. Selective inhibitors of ligand-operated, store-operated, and transduction-operated channels were also not inhibitory. beta-Leptinotarsin-h did not stimulate Na(+) uptake, ruling out Na(+) channels and many non-selective cation channels as targets. We conclude that beta-leptinotarsin-h stimulated Ca(2+) uptake through presynaptic Ca(2+) channels; which channel is yet to be determined. beta-Leptinotarsin-h may prove to be a useful tool with which to investigate calcium channels and calcium flux.     

Regulatory effect of sulphatides on BKCa channels

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

Halothane attenuates the cerebroprotective action of several Na+ and Ca2+ channel blockers via reversal of their ion channel blockade

We have previously shown the involvement of Na(+) channel as well as N-type and P/Q-type Ca(2+) channels in the oxygen and glucose deprivation-induced injury in rat cerebrocortical slices. In the present study, we investigated the influence of halothane on the cerebroprotective effects of a variety of Na(+) and Ca(2+) channel blockers in rat cerebrocortical slices. The hypoxic injury was attenuated by Na(+) channel blockers including tetrodotoxin, lidocaine and dibucaine, and Ca(2+) channel blockers, such as verapamil, omega-agatoxin IVA and omega-conotoxin GVIA. Halothane abolished the protective effects of lidocaine, dibucaine and verapamil, all of which block the respective cation channels in a voltage-dependent manner, without affecting the actions of tetrodotoxin, omega-agatoxin IVA and omega-conotoxin GVIA, which reveal voltage-independent blockade. On the other hand, the nitric oxide synthesis estimated from the extracellular cyclic GMP formation was elevated during exposure to hypoxia. All channel blockers tested here attenuated hypoxia-evoked nitric oxide synthesis. Halothane blocked almost completely these actions of lidocaine and verapamil. Moreover, the Na(+) and Ca(2+) channel blockade by these compounds, as determined by veratridine- and KCl-stimulated nitric oxide synthesis, respectively, was also reversed by halothane. These findings suggest that an anesthetic agent halothane reversed the Na(+) and Ca(2+) channel blockade of several voltage-dependent ion channel blockers, leading to the attenuation of their cerebroprotective actions. Therefore, the influence of halothane anesthesia should be taken into consideration for the evaluation of neuroprotective action of Na(+) and Ca(2+) channel blockers.     

Biphasic effects of oxethazaine,a topical anesthetic,on the intracellular Ca(2+) concentration of PC12 cells

There have been few reports on the mechanism(s) of action of oxethazaine (OXZ) despite its potent local anesthetic action. Generally, local anesthetics (LAs) not only inhibit Na(+) channels but also affect various membrane functions. In the present study, using PC12 cells as a nerve cell model, the effects of OXZ on intracellular Ca(2+) concentration ([Ca(2+)](i)) were examined in relation to cytotoxicity and dopamine release. [Ca(2+)](i) was determined by the quin2 method. In resting cells, (6-10)x10(-5)M OXZ produced lactate dehydrogenase leakage, which was Ca(2+)-dependent, inhibited by metal Ca(2+) channel blockers, and preceded by a marked increase in [Ca(2+)](i). Some other LAs showed no cytotoxicity at these concentrations. In K(+)-depolarized cells, however, lower concentrations of OXZ (10(-6)-10(-7)M), that had no effect on resting [Ca(2+)](i), inhibited both the dopamine release and the increase of [Ca(2+)](i) in parallel. The inhibitory potency against the [Ca(2+)](i) increase was in the order of nifedipine>OXZ approximately verapamil>diltiazem, and OXZ acted additively on the Ca(2+) channel blockers. OXZ showed the least effect on K(+)-depolarization as determined by bisoxonol uptake. OXZ also inhibited the increase in [Ca(2+)](i) induced by S(-)-BAY K 8644, a Ca(2+) channel agonist. These observations suggested that low concentrations of OXZ interact with L-type Ca(2+) channels. The biphasic effects of OXZ on Ca(2+) movement may be due to a unique chemical structure, and may participate in and complicate the understanding of the potent pharmacological and toxicological actions of OXZ.     

Role of sodium in intracellular calcium elevation and leukotriene B4 formation by receptor-mediated activation of human neutrophils

The role of Na(+) and Na(+) exchangers in intracellular Ca(2+) elevation and leukotriene B(4) (LTBs) formation was investigated in granulocyte macrophage colony-stimulating factor (GM-CSF)-primed, fMLP-stimulated human neutrophils. Isotonic substitution of extracellular Na(+) with N-methyl-D-glucamine(+) (NMDG(+)) resulted in over 85% inhibition of the LTBs generation observed (from 14.1+/-0.9pmol/10(6) neutrophils to 1.7+/-1.0pmol/10(6) neutrophils at 0.3 microM fMLP). Isotonic substitution of Na(+) with NMDG(+) also induced a significant inhibition of fMLP-induced rise in cytosolic Ca(2+) concentration ([Ca(2+)](i)) (from 2.17- to 0.78-fold increase over basal levels). Pretreatment with an inhibitor of the Na(+)/Ca(2+) exchanger (benzamil) did not inhibit either [Ca(2+)](i) rise or LTBs production, indicating that the observed effects of extracellular Na(+)-deprivation were unrelated to the Na(+)/Ca(2+) exchanger in receptor-mediated Ca(2+) influx, as previously hypothesized. LTBs production by thapsigargin-activated neutrophils was not affected by Na(+) depletion, but was totally abolished in the presence of EGTA, suggesting that store depletion-driven extracellular Ca(2+) influx is required for leukotriene synthesis and that this process is independent of Na(+)-deprivation. Exposure to Na(+)-free medium for the time of GM-CSF priming led to a significant decrease of intracellular pH values, suggesting a role of the Na(+)/H(+) exchanger in intracellular Na(+) depletion. Reducing the time of Na(+)-deprivation totally reversed the observed effect on LTBs production, resulting in enhanced, rather than inhibited, formation of LTBs. These results indicate that LTBs generation and [Ca(2+)](i) rise in human neutrophils primed by GM-CSF and stimulated with fMLP is dependent on intracellular Na(+) concentration, and, at variance with previously published results, unrelated to the Ca(2+) influx through the Na(+)/Ca(2+) exchanger.     

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.     

Vasopressin V(1) receptor-mediated activation of central sympatho-adrenomedullary outflow in rats

High-threshold Ca(2+) channels and tetrodotoxin-resistant Na(+) channels are highly expressed in small dorsal root ganglion neurons. In acutely isolated rat dorsal root ganglion neurons, the effects of neomycin, one of the aminoglycoside antibiotics, on high-threshold Ca(2+) currents and tetrodotoxin-resistant Na(+) currents were examined using whole-cell patch recording. We showed for the first time that neomycin dose-dependently inhibited peak high-threshold Ca(2+) currents and peak tetrodotoxin-resistant Na(+) currents with half-maximal inhibitory concentrations at 3.69 microM (n=20) and 1213.44 microM (n=25), respectively. Inactivation properties of high-threshold Ca(2+) currents and activation properties of tetrodotoxin-resistant Na(+) currents were also affected by neomycin with reduction of excitability of small dorsal root ganglion neurons. Half-maximal inactivation voltage of high-threshold Ca(2+) currents was -45.56 mV before and -50.46 mV after application of neomycin (n=10). Half-maximal activation voltage of tetrodotoxin-resistant Na(+) currents was -19.93 mV before and -11.19 mV after administration of neomycin (n=15). These results suggest that neomycin can inhibit high-threshold Ca(2+) currents and tetrodotoxin-resistant Na(+) currents in small dorsal root ganglion neurons, which may contribute to neomycin-induced peripheral and central analgesia.     

The gasotransmitter hydrogen sulphide decreases Na⁺ transport across pulmonary epithelial cells

BACKGROUND AND PURPOSE The transepithelial absorption of Na(+) in the lungs is crucial for the maintenance of the volume and composition of epithelial lining fluid. The regulation of Na(+) transport is essential, because hypo- or hyperabsorption of Na(+) is associated with lung diseases such as pulmonary oedema or cystic fibrosis. This study investigated the effects of the gaseous signalling molecule hydrogen sulphide (H(2) S) on Na(+) absorption across pulmonary epithelial cells. EXPERIMENTAL APPROACH Ion transport processes were electrophysiologically assessed in Ussing chambers on H441 cells grown on permeable supports at air/liquid interface and on native tracheal preparations of pigs and mice. The effects of H(2)S were further investigated on Na(+) channels expressed in Xenopus oocytes and Na(+) /K(+)-ATPase activity in vitro. Membrane abundance of Na(+) /K(+)-ATPase was determined by surface biotinylation and Western blot. Cellular ATP concentrations were measured colorimetrically, and cytosolic Ca(2+) concentrations were measured with Fura-2. KEY RESULTS H(2)S rapidly and reversibly inhibited Na(+) transport in all the models employed. H(2)S had no effect on Na(+) channels, whereas it decreased Na(+) /K(+)-ATPase currents. H(2)S did not affect the membrane abundance of Na(+) /K(+)-ATPase, its metabolic or calcium-dependent regulation, or its direct activity. However, H(2)S inhibited basolateral calcium-dependent K(+) channels, which consequently decreased Na(+) absorption by H441 monolayers. CONCLUSIONS AND IMPLICATIONS H(2) S impairs pulmonary transepithelial Na(+) absorption, mainly by inhibiting basolateral Ca(2+)-dependent K(+) channels. These data suggest that the H(2)S signalling system might represent a novel pharmacological target for modifying pulmonary transepithelial Na(+) transport.     

Effect of arvanil (N-arachidonoyl-vanillyl-amine), a nonpungent anandamide-capsaicin hybrid,on ion currents in NG108-15 neuronal cells

The effects of arvanil (N-arachidonoyl-vanillyl-amine), a structural hybrid between capsaicin and anandamide, on ion currents in a mouse neuroblastoma and rat glioma hybrid cell line, NG108-15, were examined with the aid of the whole-cell voltage-clamp technique. Arvanil (0.2-50 microM) caused an inhibition of voltage-dependent L-type Ca(2+) current (I(Ca,L)) in a concentration-dependent manner. Arvanil produced no change in the overall shape of the current-voltage relationship of I(Ca,L). The IC(50) value of arvanil-induced inhibition of I(Ca,L) was 2 microM. Arvanil (5 microM) could shift the steady-state inactivation curve of I(Ca,L) to a more negative potential by approximately -15mV. No effect of arvanil (20 microM) on delayed rectifier K(+) current (I(K(DR))) was observed; however, capsaicin (20 microM), glyceryl nonivamide (20 microM) and capsinolol (20 microM) suppressed it significantly. Arvanil (20 microM) caused a slight reduction in the amplitude of erg (ether-à-go-go-related)-mediated K(+) current (I(K(erg))) without modifying the activation curve of this current, while capsaicin and glyceryl nonivamide were more effective in suppressing I(K(erg)). Under current-clamp configuration, arvanil decreased the firing frequency of action potentials. Arvanil-mediated inhibition of I(Ca,L) appeared to be independent of its binding to either vanilloid or cannabinoid receptors. The channel-blocking properties of arvanil may, at least in part, contribute to the underlying mechanisms by which it affects neuronal or neuroendocrine function.     

(+/-)-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.     

Regulation of mastoparan-induced increase of paracellular permeability in T84 cells by RhoA and basolateral potassium channels

Mastoparan, a polypeptide known to activate heterotrimeric GTP-binding proteins, enhances the transport of Ca2+ and K+ across membranes. In the present study we investigated the influence of mastoparan on transepithelial resistance (TER) and on short circuit current (SCC) of the intestinal cell line T84. Mastoparan decreased the TER by 80% of baseline and induced a SCC of 8.34+/-1.38 microAcm(-2). The changes in paracellular conductance were estimated using the nystatin technique and showed that mastoparan increased the paracellular conductance 4-fold. Basolateral Cl(-)-free medium, or blockade of the basolateral Cl(-) uptake via the Na+/K+/2Cl(-) co-transporter with bumetanide, reduced SCC of T84 cells, but did not abolish the effect of mastoparan on the TER. Luminal addition of the Cl(-)-channel blocker DIDS or NPPB had no effect on the increase in SCC. In contrast, blocking the basolateral K(+)-channels by 2mM Ba2+ inhibited both the resistance decrease and elevation of the SCC, and further inhibited the mastoparan-induced increase in intracellular free Ca2. This indicates that mastoparan acts primarily via activating K+ channels with a secondary Cl(-) secretion and Ca2+ influx. Reduction of intracellular free Ca2+ did not alter the effect of mastoparan on TER. Stimulation with mastoparan led to a biphasic rearrangement of actin filaments and increased globular actin content in T84 cells. Depolymerization of actin filaments also correlated with inactivation of Rho-proteins, which are known regulators of the cytoskeleton. Mastoparan induced a 2-fold increase in GDI-complexed Rho.We conclude that mastoparan-induced changes in paracellular permeability are mediated via enhanced basolateral K+ conductance and Rho-protein inactivation. A secondary increase in intracellular Ca2+ or direct interaction of small GTPases with the cytoskeleton are likely mediators of the remodeling of the cytoskeleton with subsequent changes in paracellular permeability.     

Cromakalim activates the KATP and enhances spontaneous transient outward potassium currents in rat saphenous arterial myocytes

Potassium channel openers, e.g. cromakalim are held to relax smooth muscle by hyperpolarizing the cell membrane via activation of ATP-sensitive K(+) (K(ATP)) channels. A recent report indicates that members of this group dilate cerebral arteries also by enhancing the K(Ca)-based spontaneous transient outward currents (STOCs) through the activation of mitochondrial K(ATP) channels. We extended the study to rat saphenous arterial myocytes, a model for peripheral resistance vessels, to investigate the effects of cromakalim on K(ATP) and STOCs, and the underlaying mechanisms. Smooth muscle myocytes were enzymatically dissociated from the saphenous branch of the femoral artery. Macroscopic currents were recorded from acutely isolated cells using the perforated-patch and whole-cell variants of the patch-clamp technique. Predictably metabolic inhibitors and cromakalim activated a background K(+) current blocked by glibenclamide, identified as the K(ATP) channel. However, in addition, cromakalim markedly increased the amplitude and frequency of STOCs. The latter action was not sensitive to the specific K(ATP) channel blocker glibenclamide, excluding the participation of mitocondrial K(ATP) channels in this action. In conclusion, this study suggests that, in addition to the opening of K(ATP) channels, the increased STOC activity may have an important role in the vasorelaxing action of cromakalim, but through a mechanism different from that reported on cerebral artery.