Bimodal effects of the Kv7 channel activator retigabine on vascular K+ currents.

BACKGROUND AND PURPOSE: This study investigated the functional and electrophysiological effects of the Kv7 channel activator, retigabine, on murine portal vein smooth muscle. EXPERIMENTAL APPROACH: KCNQ gene expression was determined by reverse transcriptase polymerase chain reaction (RT-PCR) and immunocytochemical experiments. Whole cell voltage clamp and current clamp were performed on isolated myocytes from murine portal vein. Isometric tension recordings were performed on whole portal veins. K+ currents generated by KCNQ4 and KCNQ5 expression were recorded by two-electrode voltage clamp in Xenopus oocytes. KEY RESULTS: KCNQ1, 4 and 5 were expressed in mRNA derived from murine portal vein, either as whole tissue or isolated myocytes. Kv7.1 and Kv7.4 proteins were identified in the cell membranes of myocytes by immunocytochemistry. Retigabine (2-20 microM) suppressed spontaneous contractions in whole portal veins, hyperpolarized the membrane potential and augmented potassium currents at -20 mV. At more depolarized potentials, retigabine and flupirtine, decreased potassium currents. Both effects of retigabine were prevented by prior application of the K(v)7 blocker XE991 (10 muM). Recombinant KCNQ 4 or 5 channels were only activated by retigabine or flupirtine. CONCLUSIONS AND IMPLICATIONS: The Kv7 channel activators retigabine and flupirtine have bimodal effects on vascular potassium currents, which are not seen with recombinant KCNQ channels. These results provide support for KCNQ4- or KCNQ5-encoded channels having an important functional impact in the vasculature.

Sub-plasmalemmal [Ca2+]i upstroke in myocytes of the guinea-pig small intestine evoked by muscarinic stimulation: IP3R-mediated Ca2+ release induced by voltage-gated Ca2+ entry.

Membrane depolarization triggers Ca(2+) release from the sarcoplasmic reticulum (SR) in skeletal muscles via direct interaction between the voltage-gated L-type Ca(2+) channels (the dihydropyridine receptors; VGCCs) and ryanodine receptors (RyRs), while in cardiac muscles Ca(2+) entry through VGCCs triggers RyR-mediated Ca(2+) release via a Ca(2+)-induced Ca(2+) release (CICR) mechanism. Here we demonstrate that in phasic smooth muscle of the guinea-pig small intestine, excitation evoked by muscarinic receptor activation triggers an abrupt Ca(2+) release from sub-plasmalemmal (sub-PM) SR elements enriched with inositol 1,4,5-trisphosphate receptors (IP(3)Rs) and poor in RyRs. This was followed by a lesser rise, or oscillations in [Ca(2+)](i). The initial abrupt sub-PM [Ca(2+)](i) upstroke was all but abolished by block of VGCCs (by 5 microM nicardipine), depletion of intracellular Ca(2+) stores (with 10 microM cyclopiazonic acid) or inhibition of IP(3)Rs (by 2 microM xestospongin C or 30 microM 2-APB), but was not affected by block of RyRs (by 50-100 microM tetracaine or 100 microM ryanodine). Inhibition of either IP(3)Rs or RyRs attenuated phasic muscarinic contraction by 73%. Thus, in contrast to cardiac muscles, excitation-contraction coupling in this phasic visceral smooth muscle occurs by Ca(2+) entry through VGCCs which evokes an initial IP(3)R-mediated Ca(2+) release activated via a CICR mechanism.

Pharmacological activators of AMP-activated protein kinase have different effects on Na+ transport processes across human lung epithelial cells.

BACKGROUND AND PURPOSE: AMP-activated protein kinase (AMPK) is activated by metformin, phenformin, and the AMP mimetic, 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR). We have completed an extensive study of the pharmacological effects of these drugs on AMPK activation, adenine nucleotide concentration, transepithelial amiloride-sensitive (I(amiloride)) and ouabain-sensitive basolateral (I(ouabain)) short circuit current in H441 lung epithelial cells. EXPERIMENTAL APPROACH: H441 cells were grown on permeable filters at air interface. I(amiloride), I(ouabain) and transepithelial resistance were measured in Ussing chambers. AMPK activity was measured as the amount of radiolabelled phosphate transferred to the SAMS peptide. Adenine nucleotide concentration was analysed by reverse phase HPLC and NAD(P)H autofluorescence was measured using confocal microscopy. KEY RESULTS: Phenformin, AICAR and metformin increased AMPK (alpha1) activity and decreased I(amiloride). The AMPK inhibitor Compound C prevented the action of metformin and AICAR but not phenformin. Phenformin and AICAR decreased I(ouabain) across H441 monolayers and decreased monolayer resistance. The decrease in I(amiloride) was closely related to I(ouabain) with phenformin, but not in AICAR treated monolayers. Metformin and phenformin increased the cellular AMP:ATP ratio but only phenformin and AICAR decreased cellular ATP. CONCLUSIONS AND IMPLICATIONS: Activation of alpha1-AMPK is associated with inhibition of apical amiloride-sensitive Na(+) channels (ENaC), which has important implications for the clinical use of metformin. Additional pharmacological effects evoked by AICAR and phenformin on I(ouabain), with potential secondary effects on apical Na+ conductance, ENaC activity and monolayer resistance, have important consequences for their use as pharmacological activators of AMPK in cell systems where Na+K+ATPase is an important component.

Molecular expression and pharmacological identification of a role for K(v)7 channels in murine vascular reactivity.

BACKGROUND AND PURPOSE: This study represents a novel characterisation of KCNQ-encoded potassium channels in the vasculature using a variety of pharmacological and molecular tools to determine their role in contractility. EXPERIMENTAL APPROACH: Reverse transcriptase polymerase chain reaction (RT-PCR) experiments were undertaken on RNA isolated from mouse aorta, carotid artery, femoral artery and mesenteric artery using primers specific for all known KCNQ genes. RNA isolated from mouse heart and brain were used as positive controls. Pharmacological experiments were undertaken on segments from the same blood vessels to determine channel functionality. Immunocytochemical experiments were performed on isolated myocytes from thoracic aorta. KEY RESULTS: All blood vessels expressed KCNQ1, 4 and 5 with hitherto 'neuronal' KCNQ4 being, surprisingly, the most abundant. The correlated proteins K(v)7.1, K(v)7.4 and K(v)7.5 were identified in the cell membranes of aortic myocytes by immunocytochemistry. Application of three compounds known to activate K(v)7 channels, retigabine (2 -20 microM), flupirtine (20 microM) and meclofenamic acid (20 microM), relaxed vessels precontracted by phenylephrine or 1 mM 4-aminopyridine but had no effect on contractions produced by 60 mM KCl or the K(v)7 channel blocker XE991 (10 microM). All vessels tested contracted upon application of the K(v)7 channel blockers XE991 and linopirdine (0.1-10 microM). CONCLUSIONS AND IMPLICATIONS: Murine blood vessels exhibit a distinctive KCNQ expression profile with 'neuronal' KCNQ4 dominating. The ion channels encoded by KCNQ genes have a crucial role in defining vascular reactivity as K(v)7 channel blockers produced marked contractions whereas K(v)7 channel activators were effective vasorelaxants.

Pharmacological and molecular evidence for the involvement of Kv4.3 in ultra-fast activating K+ currents in murine portal vein myocytes.

BACKGROUND AND PURPOSE: The aim of this study was to determine the molecular identity of a transient K+ current (termed IUF) in mouse portal vein myocytes using pharmacological and molecular tools. EXPERIMENTAL APPROACH: Whole cell currents were recorded using the ruptured patch con from either acutely dispersed single smooth muscle cells from the murine portal vein or human embryonic kidney cells. Reverse transcriptase polymerase reaction (RT-PCR) experiments were undertaken on RNA isolated from mouse portal vein using primers specific for various voltage-dependent K+ channels, auxillary subunits and calcium-binding proteins. Immunocytochemistry was undertaken using an antibody specific for Kv4.3. KEY RESULTS: IUF had a mean amplitude at +40 mV of 558 +/- 50 pA (n = 32) with a mean time to peak at +40 mV of approximately 4 ms. IUF activated and inactivated with a half maximal voltage of -12 +/- 2 mV and -85 +/- 2 mV, respectively. IUF was relatively resistant to 4-aminopyridine (5 mM produced 30 +/- 6 % block at +20 mV) but was inhibited effectively by flecainide (IC50 value was 100 nM) and phrixotoxin II. This pharmacological profile is consistent with IUF being comprised of Kv4.x proteins and this is supported by the results from the quantitative PCR and immunocytochemical experiments. CONCLUSIONS AND IMPLICATIONS: These data represent a rigorous investigation of the molecular basis of vascular transient K+ currents and implicates Kv4.3 as a major component of the channel complex.

TRPC3 properties of a native constitutively active Ca2+-permeable cation channel in rabbit ear artery myocytes.

Previously we have described a constitutively active, Ca2+-permeable, non-selective cation channel in freshly dispersed rabbit ear artery myocytes which has similar properties to some of the canonical transient receptor potential (TRPC) channel proteins. In the present work we have compared the properties of constitutive channel activity with known properties of TRPC proteins by investigating the effect of selective anti-TRPC antibodies and pharmacological agents on whole-cell and single cation channel activity. Bath application of anti-TRPC3 antibodies markedly reduced channel activity in inside-out patches and also produced a pronounced reduction of both current amplitude and variance of constitutively active whole-cell cation currents whereas anti-TRPC1/4/5/6/7 antibodies had no effect on channel activity. In the presence of antigenic peptide, anti-TRPC3 antibodies had no effect on whole-cell or single cation channel activity. Bath application of flufenamic acid, Gd3+, La3+ and Ca2+ inhibited spontaneous channel activity in outside-out patches with IC50 values of 6.8 microm, 25 nm, 1.5 microm and 0.124 mm, respectively, which are similar values to those against TRPC3 proteins. Immunocytochemical studies combined with confocal microscopy showed expression of TRPC3 proteins in ear artery myocytes, and these were predominately distributed at, or close to, the plasma membrane. These data provide strong evidence that native constitutively active cation channels in rabbit ear artery myocytes have similar properties to TRPC3 channel proteins and indicate that these proteins may have an important role in mediating this conductance.

Interstitial cells in the vasculature.

Interstitial cells of Cajal are believed to play an important role in gastrointestinal tissues by generating and propagating electrical slow waves to gastrointestinal muscles and/or mediating signals from the enteric nervous system. Recently cells with similar morphological characteristics have been found in the wall of blood vessels such as rabbit portal vein and guinea pig mesenteric artery. These non-contractile cells are characterised by the presence of numerous processes and were easily detected in the wall of the rabbit portal vein by staining with methylene blue or by antibodies to the marker of Interstitial Cells of Cajal c-kit. These vascular cells have been termed "interstitial cells" by analogy with interstitial cells found in the gastrointestinal tract. Freshly dispersed interstitial cells from rabbit portal vein and guinea pig mesenteric artery displayed various Ca2+-release events from endo/sarcoplasmic reticulum including fast localised Ca2+ transients (Ca2+ sparks) and longer and slower Ca2+ events. Single interstitial cells from the rabbit portal vein, which is a spontaneously active vessel, also demonstrated rhythmical Ca2+ oscillations associated with membrane depolarisations, which suggests that in this vessel interstitial cells may act as pacemakers for smooth muscle cells. The function of interstitial cells from the mesenteric arteries is yet unknown. This article reviews some of the recent findings regarding interstitial cells from blood vessels obtained by our laboratory using electron microscopy, immunohistochemistry, tight-seal patch-clamp recording, and fluorescence confocal imaging techniques.

Smooth muscle cells and interstitial cells of blood vessels.

A rise in intracellular ionised calcium concentration ([Ca(2+)](i)) at sites adjacent to the contractile proteins is a primary signal for contraction in all types of muscles. Recent progress in the development of imaging techniques with special accent on the fluorescence confocal microscopy and new achievements in the synthesis of organelle- and ion-specific fluorochromes provide an experimental basis for study of the relationship between the structural organisation of the living smooth muscle myocyte and the features of calcium signalling at subcellular level. Applying fluorescent confocal microscopy and tight-seal recording of transmembrane ion currents to freshly isolated vascular myocytes we have demonstrated that: (1) Ca(2+) sparks originate from clustered opening of ryanodine receptors (RyRs) and build up a cell-wide increase in [Ca(2+)](i) upon myocyte excitation; (2) spontaneous Ca(2+) sparks occurred at the highest rate at certain preferred locations, frequent discharge sites (FDS), which are associated with a prominent portion of the sarcoplasmic reticulum (SR) located close to the cell membrane; (3) Ca(2+)-dependent K(+) and Cl(-) channels sense the local changes in [Ca(2+)](i) during a calcium spark and thereby couple changes in [Ca(2+)](i) within a microdomain to changes in the membrane potential, thus affecting excitability of the cell; (4) an intercommunication between RyRs and inositol trisphosphate receptors (IP(3)Rs) is one of the important determinants of intracellular calcium dynamics that, in turn, can modulate the cell membrane potential through differential targeting of calcium dependent membrane ion channels. Furthermore, using immunohystochemical approaches in combination with confocal imaging we identified non-contractile cells closely resembling interstitial cells (ICs) of Cajal (which are considered to be pacemaker cells in the gut) in the wall of portal vein and mesenteric artery. Using electron microscopy, tight-seal recording and fluorescence confocal imaging we obtained information on the morphology of ICs and their possible coupling to smooth muscle cells (SMCs), calcium signalling in ICs and their electrophysiological properties. The functions of these cells are not yet fully understood; in portal vein they may act as pacemakers driving the spontaneous activity of the muscle; in artery they may have other a yet unsuspected functions.

Calcium release events in excitation-contraction coupling in smooth muscle.

Although smooth muscle cells are not organized in sarcomeres, as are striated muscles, nevertheless Ca2+ for contraction is released from the sarcoplasmic reticulum (SR) at certain preferred sites. These sites commonly discharge packets of Ca2+ spontaneously and have been called frequent discharge sites (FDSs). Each spontaneous release of a Ca2+ packet usually leads to a burst of openings of Ca2+-activated K+ channels in the cell membrane which produces a spontaneous transient outward current (STOC) in smooth muscle cells under voltage clamp. When fluorescent Ca2+ indicators such as Fluo-3 became available, the spontaneous transient increases in [Ca2+]i produced by Ca2+ packets released from the SR were also detected in cardiac muscle as flashes of fluorescence or 'sparks'. Sparks in smooth muscle consist of smaller Ca2+ packets that can give rise to 'microsparks'. In some smooth muscles which have Ca2+-activated Cl- channels, STICs (spontaneous transient inward currents) are also found to be associated with sparks. FDSs have been found to be important initiating sites for a Ca2+ wave in response to an action potential or in response to receptor activation and possibly other stimuli, such as stretch. In both cases Ca2+-induced Ca2+ release seems to be crucially involved.

[Non-selective cationic current--the basis of depolarization in smooth muscle]. / Neselektívny katiónový prúd--základ depolarizácie hladkého svalu.

The activation of smooth muscle muscarinic receptors leads to its contraction. Electrophysiological and biochemical methods have gradually, over the time span of roughly 30-40 past years, helped to reveal this phenomenon's underlying processes. One element of this cascade of processes is the influx of cations into the smooth muscle cell--non-selective cationic current--causing depolarisation of the cell membrane and subsequent opening of voltage-operated calcium channels. This mini-review provides the reader with up-to-date knowledge on non-selective cationic current, supplemented with information on muscarinic receptors of smooth muscle, their coupling with effectors via G proteins, as well as on muscarinic modulation of calcium and potassium channels of smooth muscle. The conclusion is dedicated to pointing out the future trends of research in this area. (Tab. 2, Fig. 2, Ref. 82.)

Effects of superoxide generating systems on muscle tone, cholinergic and NANC responses in cat airway.

To study the possible role of reactive oxygen species in airway hyperreactivity, we examined the effects of the superoxide anion radical (O(2)(-)) generating systems, pyrogallol and xanthine with xanthine oxidase, on muscle tone, excitatory and inhibitory neurotransmission in the cat airway. Smooth muscle contraction or non-adrenergic non-cholinergic (NANC) relaxation evoked by electrical field stimulation (EFS) were measured before or after O(2)(-) generating systems with or without diethydithiocarbamic acid (DEDTCA), an inhibitor of endogenous superoxide dismutase (SOD). Resting membrane potential or excitatory junction potential (EJP) were also measured in vitro. Both pyrogallol and xanthine/xanthine oxidase produced biphasic changes in basal and elevated (by 5-HT) muscle tone. After SOD pretreatment, both systems consistently produced a prolonged contraction, thereby indicating that O(2)(-) was converted to H(2)O(2) by the action of SOD and as a result the actions of O(2)(-) were lost but those of H(2)O(2) introduced. The O(2)(-) showed no significant effect on smooth muscle contraction or EJP evoked by EFS, however after DEDTCA pretreatment, it evoked initial enhancement followed by suppression of the contraction and EJP. DEDTCA pretreatment ameliorated the inhibitory action of pyrogallol and xanthine/xanthine oxidase on the NANC relaxation, probably because O(2)(-) could combine with endogenous NO to form peroxynitrite. These results indicate that the O(2)(-) generating systems have multiple actions, presumably due to the presence and simultaneous action of at least two different reactive oxygen species (O(2)(-) and H(2)O(2)). While H(2)O(2) seems to be responsible for elevation of muscle tone and augmentation of smooth muscle contraction by EFS, O(2)(-) inhibits muscle tone, cholinergic and NANC neurotransmission.

Reactive oxygen species induced smooth muscle responses in the intestine, vessels and airways and the effect of antioxidants.

Numerous experimental data confirm the importance of reactive oxygen species (ROS) in physiological activities of smooth muscles and in the pathogenesis of various diseases with altered function of smooth muscles. The present study shows that smooth muscles of the intestine, airways and vessels, as well as their epithelium, endothelium and innervations, might be important targets of the ROS action. We demonstrated differences among the actions of various ROS (endogenous, exogenous, produced enzymatically, non-enzymatically) as well as among their actions in different smooth muscle tissues. Our results indicate that ROS are involved in changes in muscle tone, membrane conductance, calcium homeostasis, calcium-dependent processes, as well as in eicosanoid and nitric oxide metabolism. The effects of antioxidative enzymes (superoxide dismutase, catalase), of several drugs of natural origin (e.g. Kampo Medicines) and synthetic agents (e.g. stobadine, nitrosopine, ACE inhibitors) suggest that smooth muscle tissues are useful models to study ROS action and drug intervention in ROS induced injuries.

Muscarinic cation current and suppression of Ca2+ current in guinea pig ileal smooth muscle cells.

Cationic current (Icat) and inhibition of the voltage-dependent Ca2+ current (ICa) evoked by muscarinic receptor activation with carbachol were studied using whole-cell patch clamp technique in smooth muscle cells isolated from longitudinal muscle of guinea pig small intestine. With low buffering of [Ca2+]i (0.1 mM BAPTA [1,2-bis-(2-aminophenoxy)-ethane-N,N, N', N'-tetraacetic acid] in pipette solution) Icat and ICa inhibitory responses had a rapid onset to an initial peak followed by a sustained phase. The sustained phase of ICa suppression was bigger than in the case when [Ca2+]i was clamped to 100 nM, but decreased with repeated stimulation. Upon repeated stimulation with 50 microM carbachol in cells where [Ca2+]i was clamped to 100 nM and when GTP was absent, Icat amplitude decreased strongly and more substantially compared to ICa inhibition, but both responses declined only slightly when 1 mM GTP was present in the pipette solution. GDP-betaS (1 or 5 mM) in pipette solution or pre-treatment of cells with pertussis toxin (6 microg/ml, for 4 h or longer) blocked Icat more than ICa suppression by carbachol, whereas L-NAME (N-omega-nitro-L-arginine methyl ester hydrochloride) (100 microM in pipette solution) affected neither of them significantly. We conclude that the cationic current and the suppression of the voltage-dependent Ca2+ current evoked by muscarinic receptor activation are mediated by pertussis toxin-sensitive G-protein(s) but the latter response was less sensitive to blockade by GDP-betaS and to GTP deficiency in the cell.

A comparative study of guinea pig and rat tracheal reactivity.

Species differences in behavior and responses of guinea pig (GPT) and rat (RT) tracheal smooth muscle to electrical field stimulation (EFS) and chemicals were investigated under semi-isometric conditions. Indomethacin augmented and papaverine reduced the contractions elicited by EFS in both tissues. They relaxed the GPT and did not affect the tone decline in the RT. Experiments with different load, cartilage content, thread and rubber strips suggest the role of elastic elements in passive elongation of the RT. This was independent of muscarinic, adrenergic, histaminergic and serotoninergic receptors, nitric oxide, eicosanoids and metabolic processes. The second response on repeated administration of acetylcholine was augmented and that of serotonin attenuated in both GPT and RT. Upon repeated elevation of [K+]o the tissues responded in an opposite manner. Further data are provided on similarities (modulation of cholinergic transmission by prostaglandins, sensitization to acetylcholine and desensitization to serotonin) and differences (innervation, responses to repeated elevation of [K+]o, presence/absence of prostaglandin-regulated basal tone and spontaneous activity) in reactivity of GPT and RT.

[Smooth muscle preparations as models for studies of drug effects on organs]. / Hladkosvalové preparáty ako modelové orgány stúdia úcinku farmák.

The action of drugs on processes in smooth muscles, in their innervation or mucosa results in changes in contractility of the gut, airways, vessels and urogenital system. Noteworthy insight has been gained into the basic common characteristics ot smooth muscles as well as into special properties of individual smooth muscle types whose fundamental properties have become adapted to a particular situation. This insight along with knowledge on the subcellular and cellular organization of smooth muscle cells and of their innervation, on the role of the mucosa, and introduction of sophisticated electrophysiological, biochemical, isotopic and morphological methods makes smooth muscle suitable for investigation of elemental physiological and pathophysiological processes and of targets of drug action. The complexity of the smooth muscle tissue allows to study the mechanisms of drug action on the peripheral cholinergic, adrenergic, nonadrenergic-noncholinergic nerves and their neuromediators, on the epithelial and endothelial cells and the biologically active substances which they release, on the membrane and subcellular receptors, receptor coupled processes, ion channels, enzymes, Ca2+ availability, etc. Since most of these mechanisms operate also in other tissues, the obtained results may characterize drug action in other systems as well.

Involvement of different Ca2+ sources in changes of responsiveness of guinea-pig trachea to repeated administration of histamine and acetylcholine.

The role of Ca(i) and Ca(o) in changes of responsiveness of guinea pig tracheal smooth muscle strips to repeated applications of histamine and acetylcholine was investigated. Homologous desensitization to histamine developed when the airways were exposed to concentrations higher than 10(-5) mol/l, while sensitization to acetylcholine was recorded even when its highest concentration did not exceed 10(-5) mol/l. The maximum of the concentration response curves (CRC) was reduced upon repeated histamine, and enhanced upon repeated acetylcholine administration. There was, however, no significant difference in EC50 values for repeated CRCs of the stimulants. In Ca2+ free, EGTA (10(-4) mol/l) containing solution the second contraction elicited by single (10(-3) mol/l) or cumulative (10(-9)-10(-3) mol/l) histamine application was significantly smaller, while that elicited by acetylcholine did not differ significantly from the first one. In Ca(2+)-free, caffeine (10(-2) mol/l) and EGTA containing solution the contractile responses to repeated additions of Ca2+ (2.7 mmol/l) in histamine and acetylcholine (10(-3) mol/l) treated tracheae was decreased and unchanged, respectively. Addition of nifedipine (10(-6) mol/l) to this solution fully prevented Ca2+ in inducing contraction in histamine treated tracheae, while Ca2+ still induced contraction in acetylcholine treated tracheae. TMB-8 (10(-5) mol/l) was ineffective in blocking the remaining acetylcholine induced contractions. The present data suggest that contractions of the guinea pig trachea elicited by histamine and acetylcholine are due to release of intracellular Ca2+ from a caffeine sensitive store and to influx of Ca2+ from the extracellular space via voltage operated channels (VOC). Moreover, acetylcholine activated Ca2+ entry into guinea pig tracheal smooth muscle cells via the nifedipine insensitive mechanism, probably receptor operated channels (ROC). It is concluded that desensitization to histamine in the guinea pig trachea is most probably due to alterations in intracellular Ca2+ mobilization and Ca2+ influx via VOC. In contrast, sensitization to acetylcholine involved primarily enhanced Ca2+ influx via VOC and Ca2+ induced Ca2+ release.