Discovery of a pathway for terminal-alkyne amino acid biosynthesis.

Living systems can generate an enormous range of cellular functions, from mechanical infrastructure and signalling networks to enzymatic catalysis and information storage, using a notably limited set of chemical functional groups. This observation is especially notable when compared to the breadth of functional groups used as the basis for similar functions in synthetically derived small molecules and materials. The relatively small cross-section between biological and synthetic reactivity space forms the foundation for the development of bioorthogonal chemistry, in which the absence of a pair of reactive functional groups within the cell allows for a selective in situ reaction1-4. However, biologically 'rare' functional groups, such as the fluoro5, chloro6,7, bromo7,8, phosphonate9, enediyne10,11, cyano12, diazo13, alkene14 and alkyne15-17 groups, continue to be discovered in natural products made by plants, fungi and microorganisms, which offers a potential route to genetically encode the endogenous biosynthesis of bioorthogonal reagents within living organisms. In particular, the terminal alkyne has found broad utility via the Cu(I)-catalysed azide-alkyne cycloaddition 'click' reaction18. Here we report the discovery and characterization of a unique pathway to produce a terminal alkyne-containing amino acid in the bacterium Streptomyces cattleya. We found that L-lysine undergoes an unexpected reaction sequence that includes halogenation, oxidative C-C bond cleavage and triple bond formation through a putative allene intermediate. This pathway offers the potential for de novo cellular production of halo-, alkene- and alkyne-labelled proteins and natural products from glucose for a variety of downstream applications.

Raman scattering and growth disorders in single as-grown TiO2 nanowires.

An oxidation procedure has been developed to grow single-crystalline TiO(2) nanowires of the pure rutile phase, allowing subsequent characterizations of SEM, XRD, Raman, and TEM without any post-growth preparations. TEM observations support that the 1D anisotropic growth is dominated by oriented attachment processes, leading to typical growth-induced defects in the nanowires. Spatial variations of the rutile E(g) and A(1g) Raman modes were unambiguously revealed on single nanowires while scanned along the growth direction parallel to the rutile [110]. Symmetry-sensitive deviations were identified by comparing the Raman data with the spatial correlation model calculations based on realistic dispersion relations of the rutile, reflecting morphology-correlated defect distributions along single nanowires. This work provides an efficient, non-destructive in situ characterization approach for guiding growth design in future nanotechnology.

Size-effect induced short-range magnetic ordering in germanium nanostructures.

Formation of ordered magnetic states in germanium nanostructures embedded in SiO2 has been investigated. Samples with the nanostructures were prepared by sputtering deposition on Si(100) substrates, followed by thermal annealing in vacuum. Transmission electron microscopy, energy dispersive X-ray spectrometry, and Raman spectroscopy have been used to characterize the samples. Magnetic measurements were performed using a superconducting quantum interference device. Size-effect induced magnetic orderings in the germanium nanostructures were found to be present at room temperatures and below. Superparamagnetic behavior was observed at temperatures above 230 K, whereas thermal excitation of spin reorientation and magnetic coupling has been revealed at temperatures below 60 K. Inverted hysteresis loops with negative remanences and multiple plateaus revealed the ferri- or antiferromagnetic nature of the coupling. Inter-domain coupling and effect of magnetic anisotropy will be discussed based on the experimental results and simulations with a spin reorientation model.

Effect of K201, a novel antiarrhythmic drug on calcium handling and arrhythmogenic activity of pulmonary vein cardiomyocytes.

BACKGROUND AND PURPOSE: Pulmonary veins are the most important focus for the generation of atrial fibrillation. Abnormal calcium homeostasis with ryanodine receptor dysfunction may underlie the arrhythmogenic activity in pulmonary veins. The preferential ryanodine receptor stabilizer (K201) possesses antiarrhythmic effects through calcium regulation. The purpose of this study was to investigate the effects of K201 on the arrhythmogenic activity and calcium regulation of pulmonary vein cardiomyocytes. EXPERIMENTAL APPROACH: The ionic currents and intracellular calcium were studied in isolated single cardiomyocytes from rabbit pulmonary vein before and after the administration of K201, by the whole-cell patch clamp and indo-1 fluorimetric ratio techniques. KEY RESULTS: K201 (0.1, 0.3, 1 microM) reduced the firing rates in pulmonary vein cardiomyocytes, decreased the amplitudes of the delayed afterdepolarizations and prolonged the action potential duration. K201 decreased the L-type calcium currents, Na(+)/Ca(2+) exchanger currents, transient inward currents and calcium transients. K201 (1 microM, but not 0.1 microM or 0.3 microM) also reduced the sarcoplasmic reticulum calcium content. Moreover, both the pretreatment and administration of K201 (0.3 microM) decreased the isoprenaline (10 nM)-induced arrhythmogenesis in pulmonary veins. CONCLUSIONS AND IMPLICATIONS: K201 reduced the arrhythmogenic activity of pulmonary vein cardiomyocytes and attenuated the arrhythmogenicity induced by isoprenaline. These findings may reveal the anti-arrhythmic potential of K201.

Swelling activated chloride currents in the electrical activity of pulmonary vein cardiomyocytes.

BACKGROUND: Pulmonary veins (PVs) contain cardiomyocytes with a high arrhythmogenicity for inducing atrial fibrillation. The swelling-activated outwardly rectifying Cl(-) currents (I(Cl,swell)) are important in the electrical activity of cardiomyocytes. This study was to investigate whether I(Cl,swell) play a role in the PV electrophysiological characteristics. MATERIALS AND METHODS: A whole-cell patch clamp was used to investigate the action potentials and I(Cl,swell) in isolated rabbit single PV and atrial cardiomyocytes during immersion in isotonic (290-300 mosm L(-1)) and hypotonic (220-230 mosm L(-1)) solutions. The cell length and cell width were measured using confocal microscopy. RESULTS: Hypotonic solution induced larger I(Cl,swell) in the PV cardiomyocytes with pacemaker activity than those in the PV cardiomyocytes without pacemaker activity or atrial cardiomyocytes. Hypotonic solution shortened the action potential duration and increased the cell width to a greater extent in the PV cardiomyocytes than in the atrial cardiomyocytes. Moreover, hypotonic solution decreased the PV firing with a decrease in the transient inward currents and delayed after depolarizations. CONCLUSIONS: These findings suggest that the I(Cl,swell) plays an important role in the electrical activity of the PV cardiomyocytes.

Effects of rapid atrial pacing on the arrhythmogenic activity of single cardiomyocytes from pulmonary veins: implication in initiation of atrial fibrillation.

BACKGROUND: Pulmonary veins (PVs) are important sources of paroxysmal atrial fibrillation. Long-term rapid atrial pacing (RAP) changes atrial electrophysiology and facilitates the maintenance of atrial fibrillation. It is not clear whether RAP alters the arrhythmogenic activity of PVs. The purpose of this study was to isolate single PV cardiomyocytes from control and RAP dogs and evaluate their electrophysiological characteristics. METHODS AND RESULTS: The action potential and ionic currents were investigated in PV cardiomyocytes from control and long-term (6 to 8 weeks) RAP (780 bpm) dogs by use of the whole-cell clamp technique. Dissociation of PVs yielded rod-shaped single cardiomyocytes without (n=91, 60%) or with (n=60, 40%) pacemaker activity. Compared with the control group, the RAP dog PV cardiomyocytes had faster beating rates (0.86+/-0.28 versus 0.45+/-0.07 Hz, P<0.05) and shorter action potential duration. The RAP dog PV cardiomyocytes with pacemaker activity have a higher incidence of delayed (59% versus 7%, P<0.001) or early (24% versus 0%, P<0.005) after depolarization. The RAP dog PV cardiomyocytes with pacemaker activity had smaller slow inward and transient outward but larger transient inward (0.017+/-0.004 versus 0.009+/-0.002 pA/pF, P<0.05) and pacemaker (0.111+/-0.019 versus 0.028+/-0.008 pA/pF, P<0.001) currents. The RAP dog PV cardiomyocytes without pacemaker activity had only smaller slow inward and transient outward and larger pacemaker currents. CONCLUSIONS: PVs contain multiple cardiomyocytes with distinct electrophysiological characteristics. RAP changes the electrophysiological characteristics and arrhythmogenic activity of PVs.

Extragenic suppressors of mar2(sir3) mutations in Saccharomyces cerevisiae.

The silent mating-type genes (HML and HMR) of Saccharomyces cerevisiae are kept under negative transcriptional control by four trans-acting MAR (or SIR) loci. We have isolated extragenic suppressors of the mar2-1 mutation which, based on genetic complementation tests, define two additional loci involved in regulating the expression of HML and HMR. A strain with the genotype HMLa MAT alpha HMRa mar2-1 is sterile due to the simultaneous expression of a and alpha information. Two mutants exhibiting an alpha phenotype (which may result from the restoration of MAR/SIR repression) were isolated and genetically characterized. The mutations in these strains: (1) are recessive, (2) are capable of suppressing a mar2-deletion mutation, (3) are unlinked to MAT, (4) complement one another as well as the previously identified sum1-1 mutation, and (5) are not new alleles of the known MAR/SIR loci. We designate these new regulatory loci SUM2 and SUM3 (suppressor of mar). Unlike the sum1-1 mutation, suppression by sum2-1 and sum3-1 is mar2-locus specific. Both sum2-1 and sum3-1 affect the expression of a information at the HM loci. Transcript analysis shows a significant reduction in HMLa and HMRa gene transcription in mar2-1 sum2-1 and mar2-1 sum3-1 cells. Furthermore, we have found genetic evidence to suggest that mar2-1 sum2-1 cells exhibit only partial expression of silent alpha information. We conclude that the SUM2 and SUM3 gene products are required for expression of the HM loci and act downstream of the MAR2 (SIR3) gene function. Possible mechanisms for the action of the SUM gene products are discussed.

Depressant effect of adenosine in isolated human and canine atrial fibres.

UNLABELLED: STUDY OBJECTIVE0--he aim was to explore the cellular mechanisms responsible for the depressant effects of adenosine in human atrial tissues. DESIGN: Conventional microelectrode technique was used to record transmembrane action potential of human atrial tissues obtained at cardiac surgery. Effects of adenosine (0.1-100 microM) on action potential characteristics and contractile force of human atrial fibres in the absence and presence of an adenosine receptor antagonist (DPSPX) were evaluated. Results were then compared with those obtained from the canine atrial tissues. EXPERIMENTAL MATERIAL: Atrial tissues obtained from hearts of 25 patients undergoing corrective cardiac surgery were used. Seven mongrel dogs were anaesthetised and strands of atrial muscle were removed and used for comparison. MEASUREMENTS AND MAIN RESULTS: In human atrial fibres showing fast response action potential (mean dV/dtmax around 100 V.s-1) in normal [K]o (4 mM) Tyrode solution, adenosine (1 nM-10 microM) did not induce consistent effects on the action potential characteristics. When the fibres were depolarised in high [K]o (27 mM) or in atrial fibres showing slow response action potential (dV/dtmax less than 50 V.s-1), however, 10 microM adenosine reduced the upstroke velocity and the amplitude of action potential significantly and markedly depressed the contractile force. In atrial fibres spontaneously active in normal Tyrode solution (maximum diastolic potential around -50 mV), adenosine inhibited rate of spontaneous discharge in a concentration dependent manner. Delayed afterdepolarisation and aftercontraction induced by adrenaline or/and high [Ca]o were also suppressed. The depressant effects of adenosine were blocked after pretreatment with 50 microM DPSPX, a specific antagonist for adenosine receptor. CONCLUSIONS: These findings show that adenosine may abolish abnormal automatic rhythms and triggered activity in human atria.

Pacemaker current, membrane resistance, and K+ in sheep cardiac Purkinje fibres.

OBJECTIVE: The pacemaker current in cardiac Purkinje fibres has been attributed to either a decrease in potassium conductance or an increase in a non-specific (Na-K) conductance. The former mechanism would be associated with an increase in membrane resistance (Rm) and the latter with a decrease in Rm. The aim of this study was to obtain evidence in support of one or other mechanism by measuring Rm during the pacemaker current (Idd) under conditions where there is a small or no extracellular potassium depletion. METHODS: Hearts were obtained from anaesthetised sheep and thin strands of ventricular Purkinje fibres were shortened to less than or equal to 1.6 mm. Purkinje fibres were voltage clamped to potentials positive and negative to the potassium equilibrium potential (EK) using a two microelectrode technique. Small current pulses were superimposed on Idd to measure Rm changes. Procedures were used that decrease either the background potassium current IKl or Idd in order to dissect changes in Rm due to K depletion from those due to Idd. RESULTS: Rm increased during Idd, whether the pacemaker current increased or decreased as a function of time. Increasing [K]o from 2.7 to 5.4 mmol.litre-1 decreased Rm and during hyperpolarising steps increased the instantaneous current but did not change Idd amplitude. In 2.7 mmol.litre-1 K, caesium (Cs, 2 mmol.litre-1) increased the holding current (Ih), had little effect on the instantaneous current, and eliminated Idd and associated Rm changes. In 5.4 and 10.8 mmol.litre-1 K, Cs increased Ih and decreased Idd amplitude and in 10.8 mmol.litre-1 K Cs decreased the instantaneous current on hyperpolarisation. If the current was reversed, Cs decreased but did not abolish it. In normal [K]o, barium (Ba, 0.05-0.5 mmol.litre-1) increased Ih and Rm, reduced the instantaneous current but did not increase Idd amplitude. In high [K]o, Ba instead increased the amplitude and rate of development of Idd. When Cs was applied in the presence of Ba, Idd was reduced or eliminated depending on [K]o. CONCLUSIONS: The changes in membrane resistance during the pacemaker current cannot be accounted for by K depletion and suggest that in the range of diastolic depolarisation the pacemaker current results predominantly from a time dependent decrease in K conductance.

Electromechanical effects of caffeine in isolated human atrial fibres.

Effects of caffeine on the action potential and contractile force of human atrial fibres obtained at cardiac surgery were studied with standard microelectrode technique. In 4 mmol . litre-1 [K]o, the only significant action produced by 0.3 to 3 mmol . litre-1 caffeine on the electro-mechanical activity of relatively normal atrial fibres was a slight shortening of the action potential duration at 50% repolarisation. When the fibres were depolarised in 27 mmol . litre-1 [K]o or in atrial fibres showing slow responses in 4 mmol . litre-1 [K]o, however, caffeine could increase the upstroke of slow response and the force. In 18% of atrial fibres showing slow responses in 4 mmol . litre-1 [K]o, caffeine induced spontaneous discharges and potentiated afterdepolarisations. The positive inotropic and the arrhythmogenic effects of caffeine could be diminished by pretreating the fibres with propranolol or Ca antagonists (diltiazem and verapamil). In fibres beating spontaneously in normal [K]o, caffeine accelerated spontaneous rhythms initially and then depressed them. Propranolol potentiated the later depression but did not block the initial acceleration. The results suggest that caffeine increases the transmembrane Ca influx and enhances the release of Ca from the intracellular stores in human atrial fibres. As a consequence, caffeine could induce arrhythmias in atria from certain individuals.

Arrhythmogenic activity of cardiac muscle in pulmonary veins of the dog: implication for the genesis of atrial fibrillation.

OBJECTIVE: Pulmonary veins are important foci of ectopic beats to initiate paroxysmal atrial fibrillation. The purpose of this study were to investigate the electrophysiological characteristics of excitable cells in canine pulmonary veins obtained from healthy and chronic rapid atrial pacing dogs and their responses to cardioactive agents. METHODS: Transmembrane action potentials (APs) were recorded from multiple sites of pulmonary veins isolated from 17 healthy dogs and 14 dogs with chronic (6-8 weeks) rapid atrial pacing (780 bpm). RESULTS: In normal superfusate, several types of electrical activities were identified, including silent electrical activity, fast response APs driven by electrical stimulation, and spontaneous fast or slow response APs (with or without early afterdepolarizations). The incidences of AP with an early afterdepolarization (93% versus 41%) was greater in chronic pacing dogs. The spontaneous activities were depressed by beta-adrenoceptor blocker, calcium channel blocker, adenosine and acetylcholine. High frequency (>8 Hz) irregular rhythms occurred spontaneously or were induced by cardioactive agents or electrical stimuli. The incidence of spontaneously occurring tachyarrhythmias was much higher in preparations from chronic pacing dogs (93%) than from control (12%). The tachyarrhythmias were suppressed by sodium channel blocker, potassium channel blocker or magnesium. CONCLUSIONS: Pulmonary veins have arrhythmogenic ability through spontaneous activities or high-frequency irregular rhythms. The higher incidence of spontaneously occurring high-frequency irregular rhythms in chronic rapid atrial pacing dogs may account for the increased risk of atrial fibrillation in these dogs.

Suppressive effects of somatostatin in dog Purkinje fibres.

1. The effects of somatostatin (SS, 1 nM-3 microM) on the electrical and mechanical activities of isolated Purkinje fibres of the dog were studied. 2. In most Purkinje fibres driven electrically in normal [K]o Tyrode solution, SS decreased the force of contraction slightly and had very little effect on the fast response action potential. However, in sensitive fibres SS induced a moderate reduction of action potential duration and contractile force in normal [K]o and depressed the slow response action potentials in high [K]o. 3. In spontaneously beating Purkinje fibres, SS decreased the regular rhythms slightly but abolished bursts of fast rhythms at a concentration as low as 1 nM. 4. When the fibres were depolarized in the presence of 0.2 mM barium or in Na-free solution, SS suppressed the Ca-dependent slow response action potentials. 5. These findings suggest that SS may suppress abnormal automatic activity of dog Purkinje fibres through a reduction of transmembrane Ca influx or a modulation of intracellular calcium.

Ionic mechanisms responsible for the antiarrhythmic action of dehydroevodiamine in guinea-pig isolated cardiomyocytes.

1. Dehydroevodiamine alkaloid (DeHE), an active ingredient of a Chinese herbal medicine Wu-Chu-Yu (Evodiae frutus), has been shown to decrease aterial blood pressure in experimental animals and prolong action potential duration in cardiac cells. The aim of the present study was to explore the ionic basis of its possible antiarrhythmic effects. 2. Guinea-pig atrial and ventricular myocytes were isolated enzymatically and the ionic currents were recorded under whole-cell patch-clamp with single suction pipettes. 3. DeHE at a concentration of 0.1 microM inhibited reversibly the time-dependent outward K current (delayed rectifier, Ik) and the Na-dependent inward current (INa). 4. In low-K (1 mM) and high-Ca (9 mM) solution, DeHE also depressed the delayed afterdepolarizations (DAD) and the transient inward current (Iti) induced by 2 microM strophanthidin. On the other hand, DeHE occasionally induced early afterdepolarizations and slow response action potentials at a depolarized level. 5. At higher concentrations (1 microM and above), the L-type Ca current (ICa,L) was moderately inhibited. 6. The present findings indicate that DeHE may depress triggered arrhythmias in Ca-overloaded guinea-pig cardiac myocytes through its inhibitory actions on INa, Iti and, to a smaller extent, ICa. DeHE may also exert class III antiarrhythmic effect through a reduction of outward K currents (Ik) across the sarcolemma.

Abnormal activation of K(+) channels in aortic smooth muscle of rats with endotoxic shock: electrophysiological and functional evidence.

1. This study examined the role of K(+) channels in vascular hyporeactivity of rats with endotoxic shock ex vivo. 2. At the end of the in vivo experiments, thoracic aortas were removed from endotoxaemic and control rats. After removal of the endothelium, aortic segments were mounted in myographs for recording of isometric tension and smooth muscle membrane potential. 3. Membrane potentials recorded from endotoxaemic rats were hyperpolarized compared to those of the controls. This hyperpolarization was partially reversed by tetraethylammonium, charybdotoxin or glibenclamide, but not significantly affected by apamin. The hyperpolarization was also partially attenuated by N(omega)-nitro-L-arginine methyl ester (L-NAME) or 1H:-[1,2,4]oxadiazolo[4,3-a]quinoxalin-l-one (ODQ). 4. In phenylephrine-contracted aortic rings, both agonists of K(+) channels, NS1619 and pinacidil, induced greater relaxations and re-polarizations in the preparations obtained from endotoxaemic rats. The NS1619-induced relaxation and re-polarization in arteries from endotoxaemic rats were partially inhibited by tetraethylammonium and completely inhibited by charybdotoxin, L-NAME or ODQ, but not significantly affected by apamin. Similarly, the greater relaxation and re-polarization induced by pinacidil in arteries from endotoxaemic rats were also inhibited by glibenclamide, L-NAME or ODQ. However, these inhibitors had no significant effect on relaxations and re-polarizations induced by NS1619 and pinacidil in arteries from controls. 5. This study provides the electrophysiological and functional evidence showing an abnormal activation of K(+) channels in vascular smooth muscle in animals with endotoxic shock. Our observations suggest that overproduction of nitric oxide causes an activation of large conductance Ca(2+)-activated K(+) channels and ATP-sensitive K(+) channels which contributes to endotoxin-mediated vascular hyporeactivity.

Depressant Effects of Prostacyclin in Human Atrial Fibers and Cardiomyocytes.

The aim of this study was to characterize the electropharmacological effects of prostacyclin (PGI(2)) in human atrial fibers and cardiomyocytes. Atrial tissues obtained from the hearts of 28 patients undergoing corrective cardiac surgery were used. Transmembrane action potentials were recorded using a conventional microelectrode technique, and twitch force by a transducer. Effects of PGI(2) (1 nM-10 &mgr;M) on action potential characteristics and contraction of atrial fibers were evaluated in normal [K](o) (4 mM) and high [K](o) (27 mM) in the absence and presence of cardiotonic agents. In addition, atrial and ventricular myocytes were isolated enzymatically from atrial tissues and hearts of 4 patients undergoing cardiac transplant. The effects of PGI(2) on Na- and Ca-dependent inward currents (I(Na) and I(Ca)) of cardiomyocytes were tested. In 9 human atrial fibers showing fast-response action potentials (mean dV/dt(max) = 101 +/- 15 Vs(-1)) in 4 mM [K](o), PGI(2) did not influence dV/dt(max) of phase 0 depolarization even at 1 &mgr;M. However, at a concentration as low as 10 nM, PGI(2) depressed spontaneous rhythms or slow-response action potentials in high-K-depolarized fibers. PGI(2) also depressed delayed afterdepolarizations and aftercontractions induced by cardiotonic agents. In isolated cardiomyocytes, PGI(2) reduced I(Ca) but not I(Na). The present findings show that, in human atrial fibers and cardiomyocytes, PGI(2) induces greater depressant effects on the slow-response action potential, I(Ca) and triggered activity than on the fast-response action potential. It is suggested that PGI(2) may act through a selective reduction of transmembrane Ca influx. Copyright 1994 S. Karger AG, Basel

The antiarrhythmic effect of potassium and rubidium in strophanthidin toxicity.

The effect of potassium and rubidium on the electrical and mechanical activity of canine Purkinje fibers were studied in vitro in the presence and absence of strophanthidin. High (5.4mM) K or 2.7 Rb decreased the force of contraction. In the presence of these ions, strophanthidin increased the force of contraction as usual but the onset of arrhythmias was delayed. During the toxic stage of strophanthidin, high K or Rb increased the force of contraction, abolished the arrhythias and improved markedly the action potential. In the presence of calcium overload induced by exposure to a K-poor or Na-free solution, K and Rb induced an increase in force of contraction. And in ventricular muscle these ions relaxed the contracture induced by strophanthidin. It is concluded that K and Rb (in addition to other mechanisms) exert an antiarrhythmic action by increasing potassium conductance and by reducing the calcium overload induced by strophanthidin.

Differential inotropic effects of halothane and isoflurane in dog ventricular tissues.

The differential effects of halothane (0.25-0.75%) and isoflurane (0.5-1.25%) on the electromechanical activity of canine ventricular tissues were compared in vitro. In Purkinje fibres, halothane but not isoflurane could induce an initial increase of contractile force which was not blocked by diltiazem or propranolol. In ventricular muscles, halothane decreased the resting state contraction more markedly than isoflurane. The results suggest that halothane induces a greater negative inotropy than isoflurane through a differential alteration of intracellular Ca2+ stores.

Inhibitory effect of endothelin-1 on the isoproterenol-induced chloride current in human cardiac myocytes.

It is still controversial whether the cAMP-activated Cl(-) current (I(Cl,cAMP)) is expressed in human cardiomyocytes. The whole-cell configuration of the voltage-clamp technique was used to examine in detail the I(Cl,cAMP) in single human atrial and ventricular myocytes. Human cardiomyocytes were enzymatically isolated from atrial or ventricular specimens obtained from open-heart surgery or cardiac transplantation, respectively. Isoproterenol (1 microM) or forskolin (10 microM) was used to activate the cAMP second-messenger system. The isoproterenol- or forskolin-induced Cl(-) current was elicited in 12 of 54 atrial myocytes but was completely absent from ventricular myocytes. The isoproterenol-induced Cl(-) current in atrial myocytes was time-independent and had a reversal potential close to zero. Endothelin-1 (30 nM) inhibited the isoproterenol-induced Cl(-) current by 75+/-6% (n=4). This inhibitory effect of endothelin-1 was attenuated by pretreating atrial myocytes with the endothelin ET(A) receptor antagonist, BQ485, but not with the ET(B) receptor antagonist, BQ-788. The results provide evidence that the I(Cl,cAMP) exists in human atria, but not ventricle, and is inhibited by endothelin-1.

Hyperpolarization contributes to vascular hyporeactivity in rats with lipopolysaccharide-induced endotoxic shock.

We have examined the role of membrane hyperpolarization in mediating vascular hyporeactivity induced by bacterial lipopolysaccharide (LPS) in endothelial-denuded strips of rat thoracic aorta ex vivo. The injection of rats with LPS caused a significant fall of blood pressure and a severe vascular hyporeactivity to norepinephrine. The membrane potential recording showed that endotoxemia caused a hyperpolarization when compared to the control. This hyperpolarization was fully restored by methylene blue (MB; 10 microM) and partially reversed by Nomega-nitro-L-arginine methyl ester (L-NAME; 0.3 mM), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 1 microM), tetraethylammonium (TEA; 10 mM), charybdotoxin (CTX; 0.1 microM), or glibenclamide (GB; 10 microM), however, this hyperpolarization was not significantly affected by apamin (0.1 microM), 4-aminopyridine (4-AP; 1 mM), or Ba2+ (50 microM). In addition, the basal tension of the tissues obtained from endotoxemic rats was enhanced by the following order: MB > or = ODQ > TEA > or = L-NAME > or = CTX > GB; whereas apamin, 4-AP or Ba2+ had no significant effects on these tissues. In contrast, none of these inhibitors had significant effects on the membrane potential or the basal tension in control tissues. Our electrophysiological results further confirmed previous studies showing that in addition to nitric oxide, the large conductance Ca2+-activated K+-channels and ATP-sensitive K+-channels are, most likely, responsible for endotoxin-mediated hyporeactivity to vasoconstrictor agents in vascular smooth muscle.