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Diphenylphosphinic amides and diphenylphosphine oxides have been synthesized and tested as inhibitors of the Kv1.5 potassium ion channel as a possible treatment for atrial fibrillation. In vitro structure-activity relationships are discussed and several compounds with Kv1.5 IC(50) values of

Accumulating evidence suggest that drug-induced QT prolongation per se poorly predicts repolarisation-related proarrhythmia liability. We examined whether beat-by-beat variability of the QT interval may be a complementary proarrhythmia marker to QT prolongation.Anaesthetised rabbits sensitized towards developing torsades de pointes (TdP) were infused for 30 min maximum with explorative antiarrhythmic compounds characterised as mixed ion channel blockers. Based on the outcome in this model the compounds were classified as having a low (TdPlow; n=5), intermediate (TdPintermediate; n=7) or high (TdPhigh; n=10) proarrhythmic potential. Dofetilide (n=4) was included as a representative of a selective IKr-blocking antiarrhythmic with known high proarrhythmic potential. QT interval prolongation and beat-by-beat QT variability (quantified as the short-term variability, STV) were continuously assessed during the infusion or up to the point where ventricular proarrhythmias were induced.All compounds significantly prolonged the QT interval. For TdPlow and TdPhigh compounds the QT interval maximally increased from 169 ± 14 to 225 ± 28 ms (p0.05) and from 186 ± 21 to 268 ± 42 ms (p0.01), respectively. Likewise, in the dofetilide-infused rabbits the QT interval maximally increased from 177 ± 11 to 243 ± 25 ms (p0.01). In contrast, whereas the STV in rabbits administered the TdPhigh compounds or dofetilide significantly increased prior to proarrhythmia induction (from 1.6 ± 0.4 to 10.5 ± 5.6 ms and from 1.6 ± 0.5 to 5.9 ± 1.8 ms, p0.01) it remained unaltered in the TdPlow group (1.3 ± 0.6 to 2.2 ± 0.9 ms). In the TdPintermediate group, rabbits experiencing TdP had a similar maximal QT prolongation as the non-susceptible rabbits whereas the change in the STV was significantly different (from 0.9 ± 0.5 to 8.7 ± 7.3 ms vs 0.8 ± 0.3 to 2.5 ± 1.1 ms).It is concluded from the present series of experiments in a sensitive rabbit model of TdP that increased beat-by-beat QT interval variability precedes drug-induced TdP. In addition, assessment of this potential proarrhythmia marker may be useful in discriminating highly proarrhythmic compounds from compounds with a low proarrhythmic potential.

A wide range of ion channels have been considered as potential targets for pharmacological treatment of atrial fibrillation. The Kv1.5 channel, carrying the IKur current, has received special attention because it contributes to repolarization in the atria but is absent or weakly expressed in ventricular tissue. The dog serves as an important animal model for electrophysiological studies of the heart and mathematical models of the canine atrial action potential (CAAP) have been developed to study the interplay between ionic currents. To enable more-realistic studies on the effects of Kv1.5 blockers on the CAAP in silico, two continuous-time Markov models of the guarded receptor type were formulated for Kv1.5 and subsequently inserted into the Ramirez-Nattel-Courtemanche model of the CAAP. The main findings were: 1), time- and state-dependent Markov models of open-channel Kv1.5 block gave significantly different results compared to a time- and state-independent model with a downscaled conductance; 2), the outcome of Kv1.5 block on the macroscopic system variable APD90 was dependent on the precise mechanism of block; and 3), open-channel block produced a reverse use-dependent prolongation of APD90. This study suggests that more-complex ion-channel models are a prerequisite for quantitative modeling of drug effects.

Atrial fibrillation (AF) is an arrhythmia of growing clinical concern that is increasing in prevalence and is associated with significant morbidity and mortality. Pharmacological agents remain the first-line therapy for the AF patient, and the potential advantages of sinus rhythm maintenance motivate continued efforts to identify novel pharmacological means to restore and maintain sinus rhythm. Traditional antiarrhythmic agents only moderately suppress AF and present problematic concerns of proarrhythmia and extracardiac toxicity. Current investigational or recently approved strategies for improving efficacy and safety of anti-AF agents include (i) specific or predominant blockade of atrial ion channels; (ii) "upstream therapies" affecting non-ion channel targets that influence electrical and structural remodeling, inflammation and oxidative stress; (iii) amiodarone derivatives with an improved safety profile; (iv) intracellular calcium handling; and (v) therapies aiming at alleviating conduction disturbances (gap junction coupling enhancers). This review provides a succinct overview of some of these strategies.

AZD7009 is a novel antiarrhythmic compound in early clinical development for management of atrial fibrillation. Electrophysiological studies in animals have shown high antiarrhythmic efficacy, predominant action on atrial electrophysiology, and low proarrhythmic activity. AZD7009 has previously been shown to inhibit hERG and hNav1.5 currents. The main objective of the present study was to characterize the effects of AZD7009 on hKv1.5 and hKv4.3/hKChIP2.2 currents to get a deeper understanding of the ion channel-blocking properties of the compound. hKv1.5 and hKv4.3/hKChIP2.2 currents were expressed in CHO cells. Currents were measured using the whole-cell configuration of the voltage-clamp technique. AZD7009 inhibited hKv1.5 and hKv4.3/hKChIP2.2 currents with equal potency: the IC50 for hKv1.5 block was 27.0 +/- 1.6 muM (n = 6), and the IC50 for hKv4.3/hKChIP2.2 block was 23.7 +/- 4.4 muM (n = 5). Block of the hKv4.3/hKChIP2.2 current was frequency dependent with larger block at higher frequency, whereas block of the hKv1.5 current was slightly decreased at higher frequency. In conclusion, AZD7009 inhibits both the hKv1.5 and the hKv4.3/hKChIP2.2 currents. These effects likely contribute to the effects described in animals in vivo.

The mucosa of the esophagus consists of stratified squamous epithelium that has a considerable resistance to injury. Intercellular glycoconjugates appear to constitute a major permeability barrier in the superficial portion of the esophageal mucosa. In the present study, we used a panel of lectins to investigate the differences in glycoconjugate production among different mammalian species. A battery of 12 lectins was used to study binding in sections from the esophagus of 6 mammalian species, including man. In general, the strongest staining was obtained in the stratum superficiale and the weakest staining in the stratum germinativum. In rabbit esophagus, exposure to pepsin/HCl produced a superficial damage to the epithelium, a considerable decrease in electrical resistance and a decreased staining of the esophageal epithelium with selected lectins. Pretreatment of the esophageal mucosa with sucrose octasulfate, a compound with protective properties, prevented, to some extent, the decrease in resistance and lectin staining.

We present a capillary electrophoresis-patch clamp detection system optimized for screening of antagonists and inhibitors of ligand-gated ion channels. In this system, highly selective receptor agonists are delivered through the electrophoresis capillary to the cell surface where they continuously activate a receptor, resulting in increased steady-state transmembrane currents. Thus, receptor selection and biosensor functionality is simply achieved by selection of an appropriate agonist. The antagonists are fractionated in the same electrophoresis capillary and inhibit the agonist-evoked response, resulting in transiently decreased steady-state transmembrane currents. Specifically, a mixture containing 6-cyano-7-nitroquinoxaline-2,3-dione, that reversibly blocks alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate and kainate receptors, and 6,7-dichloro-3-hydroxy-2-quinoxaline-carboxylate, a broad-spectrum glutamate receptor antagonist, were separated and detected by kainate-activated patch-clamped interneurons freshly dissociated from rat brain olfactory bulb. In addition, Mg2+ that reversibly blocks the N-methyl-D-aspartate receptor in a voltage-dependent way was detected using the same cell detector system when activated by N-methyl-D-aspartate and the co-agonist glycine. The presented method offers new possibilities for drug screening and for identifying endogenous receptor antagonists and to determine their mode of action on any ionotropic receptor system of interest.

A series of lactam sulfonamides has been discovered and optimized as inhibitors of the Kv1.5 potassium ion channel for treatment of atrial fibrillation. In vitro structure–activity relationships from lead structure C to optimized structure 3y are described. Compound 3y was evaluated in a rabbit PD-model and was found to selectively prolong the atrial effective refractory period at submicromolar concentrations.

Objective: To examine the electrophysiological, hemodynamic, and antiarrhythmic effects of the novel antiarrhythmic agent AZ13395438. Methods: The ion channel-blocking potency of AZ13395438 was assessed in Chinese hamster ovary cells stably expressing various human cardiac ion channels and in human atrial myocytes. The in vivo electrophysiological, hemodynamic, and antiarrhythmic effects of intravenously administered AZ13395438 were examined in anesthetized rabbits, in anesthetized naive dogs, and in dogs subjected to rapid atrial pacing (RAP) for 8 weeks. Pharmacokinetic/pharmacodynamic (PKPD) modeling was applied to predict the potency of AZ13395438 in increasing atrial and ventricular refractoriness. Results: AZ13395438 potently and predominantly blocked the atrial repolarizing potassium currents IKur, IAch, and Ito in vitro. In vivo, AZ13395438 caused a concentration-dependent and selective increase in atrial refractoriness with no or small effects on ventricular refractoriness and repolarization and on hemodynamics in both rabbits and dogs. The PKPD modeling predicted unbound plasma concentrations of AZ13395438 of 0.20 ± 0.039, 0.38 ± 0.084, and 0.34 ± 0.057 µmol/L to increase the right atrial effective refractory period by 20 milliseconds in the rabbit and in the naive and the RAP dogs, respectively. In the RAP dog with atrial fibrillation (AF), AZ13395438 significantly increased AF cycle length and successfully converted AF to sinus rhythm in 12 of the 12 occasions at an unbound plasma concentration of 0.48 ± 0.076 µmol/L. During saline infusion, conversion was seen only in 4 of the 10 occasions ( P = .003 vs AZ13395438). Furthermore, AZ13395438 reduced AF inducibility by burst pacing from 100% to 25% ( P < .001). Conclusion: AZ13395438 can be characterized as a mixed potassium ion channel-blocking agent that selectively prolongs atrial versus ventricular refractoriness and shows promising antiarrhythmic efficacy in a clinically relevant animal model of AF.

Gamma-aminobutyrate acid, L-glutamate, and N-methyl-D-aspartate were separated by capillary electrophoresis and detected by the use of whole-cell and outside-out patch-clamp techniques on freshly dissociated rat olfactory interneurons. These neuroactive compounds could be identified from their electrophoretic migration times, unitary channel conductances, and power spectra that yielded corner frequencies and mean single-channel conductances characteristic for each of the different agonist-receptor interactions. This technique has the sensitivity to observe the opening of a single ion channel for agonists separated by capillary electrophoresis.

The possible role of glutamate, aspartate, sulfur-containing excitatory amino acids and γ-glutamyl peptides as major transmitters in the rat optic nerve was evaluated. Four days following optic nerve lesion the K + -evoked Ca + -dependent glutamate release was reduced to 31 ± 16% (±S.D., n = 9 ) comparing release from slices of the denervated (contralateral to the lesion) and non-denervated (ipsilateral) superior colliculus, indicative of a major transmitter function for glutamate. However, significant decreases in glutamate release could not be detected seven days following the lesion ( n = 5 ). Other studies have shown that optic nerve denervation induce formation of synapses of non-retinal origin and cause other cellular changes which may reduce the effect of deafferentation on glutamate release after 7 days. No significant change was observed in aspartate release following the lesion. The concentrations of cysteine sulfinate, cysteate, homocysteine sulfinate, homocysteate and O -sulfo-serine in the optic layers of the superior colliculus were below 1 nmol/g tissue ( n = 6 ). Theoretical considerations indicate that this level is too low for a function of any of these as a major optic nerve transmitter. All postsynaptic components in the rat superior colliculus response, evoked by electrical optic nerve stimulation, were reduced by kynurenate (1–10 mM), a broad spectrum glutamate-receptor antagonist. The study gives further support for the view that glutamate is a major transmitter in the rat optic nerve.

Optimization of argon ion laser-induced (UV multiline) fluorescence detection in liquid chromatography (1 mm i.d. columns) was performed aiming for ultratrace determination of o-phthalaldehyde/β-mercaptoethanol-labeled neuroactive amino acids and peptides. The derivatives are shown to undergo facile photochemical decomposition with photodestruction quantum yields, Φ D , in the range 0.009-0.14. In agreement with theoretical predictions, the optimal signal-to-noise (S/N) ratio was obtained at laser irradiances much lower than are required for prompt fluorescence saturation

Pharmacological treatment of atrial fibrillation (AF) exhibits limited efficacy. Further developments require a comprehensive characterization of ionic modulators of electrophysiology in human atria. Our aim is to systematically investigate the relative importance of ionic properties in modulating excitability, refractoriness, and rotor dynamics in human atria before and after AF-related electrical remodeling (AFER). Computer simulations of single cell and tissue atrial electrophysiology were conducted using two human atrial action potential (AP) models. Changes in AP, refractory period (RP), conduction velocity (CV), and rotor dynamics caused by alterations in key properties of all atrial ionic currents were characterized before and after AFER. Results show that the investigated human atrial electrophysiological properties are primarily modulated by maximal value of Na+/K+ pump current ( GNaK) as well as conductances of inward rectifier potassium current ( GK1) and fast inward sodium current ( GNa). GNaK plays a fundamental role through both electrogenic and homeostatic modulation of AP duration (APD), APD restitution, RP, and reentrant dominant frequency (DF). GK1 controls DF through modulation of AP, APD restitution, RP, and CV. GNa is key in determining DF through alteration of CV and RP, particularly in AFER. Changes in ionic currents have qualitatively similar effects in control and AFER, but effects are smaller in AFER. The systematic analysis conducted in this study unravels the important role of the Na+/K+ pump current in determining human atrial electrophysiology.

The concentration of γ-l-glutamyl-l-glutamate (γ-glu-glu), a potent displacer of excitatory amino acid receptor binding, and other structurally related dipeptides were determined in extracts of the rat olfactory bulb by a novel liquid chromatographic method. Whole-cell patch clamp recordings of currents from freshly isolated neurons showed that γ-glu-glu produced inward currents at negative holding potentials, provided μM concentrations of glycine were added and no Mg2+-ions were present, indicating activation of N-methyl-d-aspartate receptors. Consistently, geometry optimization of γ-glu-glu using molecular mechanics calculations, suggested a bent conformer with structural features supporting this view.

After 6–12 h of recovery from transient cerebral ischemia, the pyramidal cells of the hipppocampal CA1 region take up excessive amounts of calcium upon electrical stimulation, which has been suggested to be important for the development of delayed neuronal death. The aim of this study was to further characterize this enhanced calcium uptake with respect to time-course of development, relationship to neuronal damage, and amplitude of evoked field potentials as well as the dependency on N-methyl-d-aspartate (NMDA) and non-NMDA receptors. Adult Wistar rats were used and calcium-sensitive microelectrodes were placed in the stratum radiatum of the CA1 hippocampus for recording of the extracellular calcium concentration ([Ca2+]ec) during 20 min of ischemia and for 6 h of reflow. High-frequency stimulation of the perforant pathway elicited burst firing in CA1 and a transient decrease in [Ca2+]ec which reflects neuronal uptake. Shifts in [Ca2+]ec could not be evoked 0–1 h after ischemia. However, from 1–2 h burst firing could be evoked and the accompanying shift in [Ca2+]ec increased thereafter in amplitude with prolonged reflow, exceeded preischemic levels after 4 h, and reached 250 ± 116% (mean ± SD) of control after 6 h of reflow ( p < 0.05). The extracellular reference potential shift during electrical stimulation and the amplitude of evoked field potentials were still subnormal after 6 h [85 ± 25% and 83 ± 25%, respectively (mean ± SD)]. There was a significant correlation between the degree of stimulated calcium uptake at 6 h postischemia and the extent of CA1 damage evaluated 7 days after the ischemic insult ( r = 0.849; p < 0.001). The shifts in [Ca2+]ec were reduced by the NMDA antagonist MK-801 (0.5–2 mg/kg, i.v.) to approximately 50% of the initial level during both control and postischemic conditions ( p < 0.01). The non-NMDA antagonist 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo[ F]quinoxaline (NBQX) (42 ± 13 mg/kg, i.p.; mean ± SD) decreased the amplitude of the evoked field potentials (to 30 ± 28% of control, p < 0.05) and completely abolished the evoked shifts in [Ca2+]ec. In conclusion, the uptake of calcium into CA1 pyramidal cells during electrical stimulation was enhanced already 4 h after ischemia in spite of the fact that other measures of excitability were subnormal. This calcium uptake correlated to the extent of CA1 pyramidal cell damage and was dependent on both NMDA and non-NMDA receptor activation.

We have used the whole cell patch clamp technique to investigate the action of pentobarbitone on the nicotinic channels of bovine chromaffin cells. Application of agonists induced an inward current associated with a large increase in current noise. The noise could be fitted by Lorentzian functions with time constants of 17 +/- 2 ms for 10 microM acetylcholine and 10 +/- 1 ms for 10 microM carbachol. The single channel conductance estimated from the current variance was about 25 pS in each case. Pentobarbitone decreased the time constants in a concentration-dependent fashion, but the unit conductances were unaffected. Single channel events were recorded in chromaffin cells held under voltage clamp. Pentobarbitone did not reduce the amplitude of channel openings or the probability of channel opening but reduced the mean channel open time. This reduction was sufficient to account for the decrease in inward current produced by pentobarbitone.

This study addresses the possible involvement of an agonist-induced postischemic hyperactivity in the delayed neuronal death of the CA1 hippocampus in the rat. In two sets of experiments, dialytrodes were implanted into the CA1 either acutely or chronically (24 h of recovery). During 20 min of cerebral ischemia (four-vessel occlusion model) and 8 h of reflow, we followed extracellular amino acids and multiple-unit activity. Multiple-unit activity ceased within 20 sec of ischemia and remained zero during the ischemic insult and for the following 1 h of reflow. During ischemia, extracellular aspartate, glutamate, taurine, and gamma-aminobutyric acid increased in both acute and chronic experiments (seven- to 26-fold). Multiple-unit activity recovered to preischemic levels following 4-6 h of reflow. In the group with dialytrodes implanted acutely, the continuous increase in multiple-unit activity reached 110% of basal at 8 h of reflow. In the group with dialytrodes implanted chronically, multiple-unit activity recovered faster and reached 140% of control at 8 h, paralleled by an increase in extracellular aspartate (5.5-fold) and glutamate (twofold). In conclusion, the postischemic increase of excitatory amino acids and the recovery of the neuronal activity may stress the CA1 pyramidal cells, which could be detrimental in combination with, e.g., postsynaptic impairments.

Glutamate (glu) is a major excitatory transmitter and a toxin in the brain. In the present study, the immature rat hippocampal slice was used to determine the morphology, topography, ionic mediation and receptor specificity of glu toxicity. Slices were exposed to glu for 30 min, and the damage was evaluated after 3 h of recovery in regular medium. The effects on glu toxicity of changes of [Ca2+], [Cl-] and [Na+] were determined. The receptor preference of glu was assessed by using the N-methyl-D-aspartate (NMDA) antagonist MK-801 and the kainate (KA)/quisqualate (QA) antagonist DNQX, alone or in combination. Further, to see whether glu produces cytotoxicity via osmolysis, the effects of hyperosmolal sucrose on glu toxicity were studied. Glu toxicity was similar to the previously described NMDA toxicity with regard to cytopathology, but differed in some aspects from that caused by KA and QA. The severity of the lesion was determined by the proximity of neurons to the incubation fluid, probably as a consequence of cellular accumulation of the amino acid. Omission of Ca2+ abolished glu toxicity in all neurons except the granule cells of the outer blade. This population was completely protected when Ca2+ was omitted and [Cl-] was reduced. Elevation of [Ca2+] markedly aggravated the lesion caused by glu. Substitution of isethionate for Cl- worsened the glu-induced damage, whilst the amino acid produced qualitatively different neuropathology when choline substituted for Na+. Apparently glu did not damage hippocampal nerve cells through an osmolytic mechanism as medium supplemented with 100 mM sucrose increased the toxicity of glu. Since the lesion produced by glu was more widespread in the presence of high [Ca2+], the effects of receptor antagonists were studied under this condition. MK-801 inhibited glu toxicity whereas DNQX had no effect. Combination of MK-801 and DNQX did not offer better protection than did MK-801 alone. The results suggest that Ca2+ is the main (but not single) determinant of glu toxicity in the immature hippocampal slice. The ionic requirements of glu neurotoxicity are identical to those of NMDA, but differ from those of KA and QA. The notion that glu is a selective NMDA agonist in the present model was confirmed by the protection of MK-801, and by the lack of an effect of DNQX. This is the first report demonstrating that the toxicity of glu is mediated by NMDA receptors in brain tissue which has developed normally. The findings indicate that specific blockade of NMDA receptors may be the most rational strategy in the prevention of glu-related neuronal death occurring in certain neurological anomalies.

AZD7009 (tert-Butyl-2-(7-[(2S)-3-(4-cyanophenoxy)-2-hydroxypropyl]-9-oxa-3,7-diazabicyclo[3.3.1]non-3-yl)ethylcarbamate) is an antiarrhythmic agent that increases atrial refractoriness, shows high antiarrhythmic efficacy and has low proarrhythmic potential. This study was primarily undertaken to determine the effects of AZD7009 on the late sodium current and to examine the impact of late sodium current inhibition on action potential duration in various myocardial cells. AZD7009 inhibited the late sodium current in Chinese Hamster Ovary K1 (CHO K1) cells expressing hNa(v)1.5 with an IC(50) of 11+/-2 microM. The late sodium current in isolated rabbit atrial and ventricular myocytes was also concentration dependently inhibited by AZD7009. Action potentials were recorded during exposure to 5 microM E-4031 (1-[2-(6-methyl-2pyridyl)ethyl]-4-(4-methylsulfonyl aminobenzoyl)piperidine), a compound that selectively inhibits the rapid delayed rectifier potassium current (I(Kr)), and to E-4031 in combination with AZD7009 or lidocaine in rabbit atrial and ventricular tissue and Purkinje fibres. In Purkinje fibres, but not in ventricular tissue, AZD7009 and lidocaine attenuated the E-4031-induced action potential duration prolongation. In atrial cells, AZD7009, but not lidocaine, further prolonged the E-4031-induced action potential duration. E-4031 induced early afterdepolarisations (EADs) in Purkinje fibres, EADs that were totally suppressed by AZD7009 or lidocaine. In conclusion, excessive action potential duration prolongation induced by E-4031 was attenuated by AZD7009 and lidocaine in rabbit Purkinje fibre, but not in atrial or ventricular tissue, most likely by inhibiting the late sodium current. Furthermore, the opposite effect by AZD7009 on action potential duration in atrial tissue suggests that AZD7009, in addition to inhibiting I(Kr), also inhibits other repolarising currents in the atria.

Aims To compare the electrophysiological and antiarrhythmic effects of AZD7009, azimilide, and AVE0118 in the acutely dilated rabbit atria in vitro. Methods and results In the isolated Langendorf-perfused rabbit heart, the atrial effective refractory period (AERP) and the inducibility of atrial fibrillation (AF) were measured at increasing concentrations of AZD7009 (0.1–3 mM), azimilide (0.1–3 mM), and AVE0118 (0.3–10 mM). In separate groups of atria, termination of sustained AF was assessed. In non-dilated atria, the AERP was 82+ 1.3 ms (mean+ SEM) and AF could not be induced. Dilation significantly reduced the AERP to 49+ 1.0 ms (P , 0.001) and 92% of the atria became inducible. Perfusion with AZD7009, azimilide, and AVE0118 concentrationdependently increased the AERP and reduced the AF inducibility. At the highest concentrations of AZD7009, azimilide, and AVE0118, AERP and AF inducibility changed from 50+ 4.5 to 136+ 6.6 ms and 80 to 0% (both P , 0.001) from 51+ 3.0 to 105+ 9.9 ms (P , 0.001) and 80 to 0% (P , 0.01) and from 46+ 2.8 to 85+ 6.0 ms and 90 to 0% (both P , 0.001). Restoration of sinus rhythm was seen in 6/6, 5/6, and 5/6 hearts perfused with AZD7009, azimilide, and AVE0118, respectively. Conclusion In the dilated rabbit atria, AZD7009, azimilide, and AVE0118 concentration-dependently increased AERP, effectively prevented AF induction, and rapidly restored sinus rhythm.

Blocking Characteristics of hERG, hNav1.5, and hKvLQT1/hminK after Administration of the Novel Anti-Arrhythmic Compound AZD7009. Introduction: AZD7009 is a novel anti- arrhythmic compound under development for short- and long-term management of atrial fibrillation and flutter. Electrophysiological studies in animals have shown high anti-arrhythmic efficacy, predominant action on atrial electrophysiology, and low proarrhythmic activity. The main aim of this study was to characterize the blocking effects of AZD7009 on the human ether-a-go-go-related gene (hERG), the hNav1.5, and the hKvLQT1/hminK currents. Methods and Results: hERG, hKvLQT1/hminK, and hNav1.5 were expressed in CHO K1 cells. Currents were measured using the whole-cell configuration of the voltage-clamp technique. AZD7009 inhibited the hERG current with an IC50 of 0.6 ± 0.07 µ M( n= 6). AZD7009 1 µ Mh yperpolarized the potential for half-maximal activation from −8.2 ± 0.1 mV to −18.0 ± 0.6 mV (P < 0.001, n = 14) and induced pre-pulse potentiation at potentials near the activation threshold. The hNav1.5 current was blocked with an IC50 of 4.3 ± 1.20 µ Ma t 10 Hz (n= 6) and block developed use-dependently. Recovery from use-dependent block was slow, τ = 131 seconds. AZD7009 inhibited the hKvLQT1/hminK current only at high concentrations (IC50 = 193 ± 20 µM, n = 6). Conclusion: AZD7009 inhibits both the hERG and the hNav1.5 current, and it is most likely this combined current block that underlies the prolongation of the refractoriness and the low proarrhythmic activity demonstrated in animals in vivo. (J Cardiovasc Electrophysiol, Vol. 16, pp. 329-341, March 2005) Antiarrhythmic drug, hERG, hNav1.5, voltage-clamp

Introduction: The aim of this study was to analyze the block of HERG-carried membrane currents caused by H 345/52, a new antiarrhythmic compound with low proarrhythmic activity, in transfected mouse fibroblasts. Methods and Results: Using the whole-cell configuration of the voltage patch clamp technique, it was demonstrated that H 345/52 concentration-dependently blocked HERG-carried currents with an IC50 of 230 nM. H 345/52 preferentially bound to the open channel with unusually rapid kinetics and was trapped by channel closure. Voltage-independent behavior of H 345/52 was observed during both square-pulse and action potential clamp protocols. In contrast, the Class III agents dofetilide (10 nM) and almokalant (250 nM) demonstrated significant membrane potential-dependent effects during square-pulse clamp protocols. When using action potential clamp protocols, voltage dependence was seen with dofetilide but not with almokalant. Mathematical simulations of human ventricular action potentials predicted that the different voltage-dependent behaviors would not produce marked variations in action potential duration prolongation patterns. Conclusion: We propose that block of IKr is the principal mechanism by which H 345/52 delays repolarization in human myocardium. The voltage independence of HERG/IKr block is unlikely to underlie the low proarrhythmic potential, and ancillary effects on other membrane currents must be considered. (J Cardiovasc Electrophysiol, Vol. 14, pp. 651-658, June 2003)

In the present paper we studied early acid-induced changes in the passive electrical properties of the rabbit esophageal epithelium in vivo by measurements of the transluminal potential difference (PD) during acid perfusion and by estimating the transmucosal electrical resistance (Rm) using cable analysis. Perfusion with acid (pH 1) for 45 min produced a rapid (1 min) negative shift in the lumen-negative PD followed by a slow lumen-negative drift. The acid-induced change in PD was dependent on the accompanying anion, the largest anion (sulfate) producing the largest change. The acid-induced changes in PD were parallelled by reductions in Rm, these reductions also being dependent on the accompanying anion. Interpretation of resistance and net current (estimated by Ohm's law) time curves suggest that the initial acid-induced changes of the PD reflect properties of the naive mucosa whereas the later drift will reflect a diffusion driven increase in transmucosal proton permeability. Further, coapplication of the protective drug sucrose octasulfate attenuated the hydrochloric acid-induced changes of all measured and estimated electrophysiological parameters. The electrophysiological results were to some extent corroborated by light microscopic findings, although no large acid-induced change in mucosal appearance was observed.

Efficient techniques for identifying endogenous and synthetic ligands of ion channels are important in understanding neuronal communication and for screening drug libraries. This paper describes a technique based on capillary electrophoresis (CE) separation coupled to patch-clamp (PC) detection where a pulsed-flow superfusion scheme was implemented for improved detection. The nicotinic acetylcholine receptor (nAChr) agonists acetylcholine, carbachol, and (-)-nicotine were fractionated and detected by patch-clamped pheochromcytoma detector cells. The high-conductance state of the nAChr during CE-PC detection was maintained and repetitively resensitized using pulsed-flow superfusion with agonist-free buffer. In this way, each agonist evoked an ensemble of peak currents that reflected the spatiotemporal distribution for the ligand at the cell surface. The technique takes advantage of the intrinsic high selectivity and sensitivity of membrane-expressed receptors and allowed for resolution and identification of closely migrating ligands. The method was employed for determination of acetylcholine content in cell lysates.

By coupling CE to voltage-clamp biosensors, a highly selective and sensitive means for analyzing biologically active components in complex mixtures with minimum sample handling is obtained. In contrast to traditional techniques for chemical analyses, this system can be engineered for sensitivity to specific biological species, thereby improving the response selectivity and greatly simplifying the challenge of chemical analysis. CE-PC and CE-VC are complementary techniques. CE-PC detection is a highly sensitive and information-rich technique that enables the study of electrophoretically separated ligands activating a single ion channel. The technique suffers from short lifetimes of the patch pipettes and is therefore mainly qualitative. CE-VC detection, on the other hand, might become a quantitative technique because the detector cells are viable for several hours. Even if mean single-channel conductances and I-V relationships can be obtained with CE-VC, the trade-off with this format is that much less information can be extracted from the current traces compared to patch-clamp recorded data.

Many areas of research in the life sciences have been hampered by the inability to analyse microenvironments for important biologically active species. For example, the identities of many fast acting neurotransmitter substances in the mammalian brain are unknown, even after at least thirty years of active research, and endogenous ligands to numerous orphan receptors (e.g., plasma membrane opiate receptors) already cloned, remain to be discovered. During the past twenty years, development in instrumentation for microanalysis has resulted in techniques capable of sample handling in the low femtoliter range (10–15 L). Yet the development of detection devices for bioactive compounds that are compatible with these instrumentations has been extremely slow.

Systemic administration of N-methyl-D-aspartate (NMDA) receptor antagonists induces a well defined behaviour in rodents characterized by, for example increased locomotion and ataxia. It is not clear in what brain region(s) NMDA antagonists induce this behaviour. We have studied the possible involvement of olfactory pathways by making adult mice anosmic via intranasal injection of zinc sulphate, a procedure that is known to destroy the olfactory epithelium. The NMDA antagonist MK-801 was given intraperitoneally (0.1-1.0 mg/kg) and the animals were scored for locomotion and ataxia 60-90 min later. Before MK-801 administration, olfactory-lesioned mice did not differ from non-lesioned controls with regard to locomotion or ataxia. MK-801 caused locomotor activation (> or = 0.2 mg/kg) and ataxia (> or = 0.5 mg/kg) in both groups. In general, olfactory-lesioned animals showed more locomotion and less ataxia after MK-801 administration than non-lesioned animals. Lesioned animals displayed 2.0- (P < 0.05) and 3.7-fold (P < 0.05) more extensive locomotor activation than non-lesioned animals after 0.5 and 1.0 mg/kg of MK-801, respectively. No difference in the degree of ataxia was seen between the two groups at 0.5 mg/kg, whereas non-lesioned animals showed a 2.1-fold higher degree of ataxia after 1.0 mg/kg of MK-801, indicating that the enhanced MK-801-induced locomotor activation in olfactory-lesioned mice was not simply due to less ataxia. These results suggest that olfactory input is involved in NMDA antagonist-induced behaviour.

An excessive activation of the excitatory amino acid system has been proposed as one possible mediator of the ischemia-induced delayed death of CA1 pyramidal cells in the hippocampus. Using dialytrodes in the CA1 of the rat, we have investigated multiple-unit activity and extracellular changes in acidic sulfur-containing amino acids and gamma-glutamyl peptides during ischemia (20-min, four-vessel occlusion) and during 8 h of reflow. Multiple-unit activity was abolished during ischemia and for the following 1 h, but then recovered, gradually reaching preischemic levels after 8 h of reflow. Extracellular cysteate, cysteine sulfinate, and gamma-glutamyltaurine increased (1.5- to threefold) during ischemia, and extracellular glutathione and gamma-glutamylaspartate plus gamma-glutamylglutamine increased during early reflow (two- to threefold). The recovery of neuronal activity at 4-8 h was paralleled by an increase in extracellular cysteine sulfinate (2.5-fold at 8 h of reflow). Perfusion with 10 microM tetrodotoxin at 8 h of reflow abolished the multiple-unit activity and reduced extracellular cysteine sulfinate. Considering the glutamate-like properties of cysteine sulfinate, the observed postischemic increase may be involved in the development of the delayed neuronal death.

Publisher Summary This chapter explains the use of microdialysis techniques for studying brain amino acids in the extracellular fluids. The chapter describes two types of microdialysis probes that are used in animal experiments: (1) the trans-hippocampal probe and (2) the stereotactically implantable probe. The transverse microdialysis probe, which is introduced on one side of the skull and has its outlet on the opposite side, was developed for acute experiments by Ungerstedt and co-workers. Dialysis probes for stereotactic implantation are designed and constructed in several laboratories and are commercially available. There is an increasing interest in the use of microdialysis in clinical investigation on a number of body organs and tissues. Among the organs outside the CNS, subcutaneous tissue is easily available and particularly useful for the investigation of various methodological aspects. Technically, the trans-hippocampal fiber is inserted in the subcutis of the lower abdomen with a fine cannula and without anesthesia. This is non-traumatic and harmless for the patient. Dialysis perfusion has in a couple of years attained the position of a most promising approach to the biochemistry of cell-to-cell signaling in the brain.

Retinal proteins were labeled by intraocular injections of radioactive amino acids. Tissue slices of the superior colliculus (SC) were prepared 18-20 hr later, i.e., when the rapid phases of the axonal transport had reached the SC terminals. The effect of depolarizing pulses of high K and of Ca withdrawal on the scretion of radioactivity was studied in a perfusion system. The effluents were separated into a trichloroacetic acid (TCA) precipitable fraction and a TCA-soluble fraction. High K evoked a release of TCA-soluble radioactivity when [(3)H]glycine, [(3)H]leucine, or [(3)H]proline were used as protein precursors. Small changes occurred for TCA-precipitable fractions. The evoked release of radioactivity was Ca dependent and particularly prominent after labeling with [(3)H]glycine. Ca withdrawal increased the efflux of exogenous GABA, primary amines, and TCA-precipitable radioactivity but not of TCA-soluble radioactivity when normal media were used. The formation of TCA-soluble radioactivity was measured by incubating combined homogenates of SC and the lateral geniculate body (LGB), containing labeled proteins transported by the slow or rapid phase. The proteolytic activity was highly Ca dependent, for the rapidly transported proteins the half maximum was at approximately 0.1 mM Ca. The formation of TCA-soluble radioactivity was inhibited by p-chloromercuriphenylsulfonic acid (PCMS). Other divalent cations could not substitute for Ca. The rate of formation of TCA-soluble radioactivity and the influence of Ca ions was smaller when proteins of the slow phase were used as substrate.

Extracellular (EC) adenosine, hypoxanthine, xanthine, and inosine concentrations were monitored in vivo in the striatum during steady state, 15 min of complete brain ischemia, and 4 h of reflow and compared with purine and nucleotide levels in the tissue. Ischemia was induced by three-vessel occlusion combined with hypotension (50 mm Hg) in male Sprague-Dawley rats. EC purines were sampled by microdialysis, and tissue adenine nucleotides and purine catabolites were extracted from the in situ frozen brain at the end of the experiment. ATP, ADP, and AMP were analyzed with enzymatic fluorometric techniques, and adenosine, hypoxanthine, xanthine, and inosine with a modified HPLC system. Ischemia depleted tissue ATP, whereas AMP, adenosine, hypoxanthine, and inosine accumulated. In parallel, adenosine, hypoxanthine, and inosine levels increased in the EC compartment. Adenosine reached an EC concentration of 40 microM after 15 min of ischemia. Levels of tissue nucleotides and purines normalized on reflow. However, xanthine levels increased transiently (sevenfold). In the EC compartment, adenosine, inosine, and hypoxanthine contents normalized slowly on reflow, whereas the xanthine content increased. The high EC levels of adenosine during ischemia may turn off spontaneous neuronal firing, counteract excitotoxicity, and inhibit ischemic calcium uptake, thereby exerting neuroprotective effects.

The lipophilic cation [3H]triphenylrnethylphosphonium bromide ([3H]TPMP+) was investigated as a measure of the membrane potential of synaptosomes. Conditions under which [3H]TPMP+ achieved an equilibrium distribution were tested. The toxicity of TPMP has been studied relative to its inhibitory effects on [3H]y-aminobutyric acid ([3H]GABA) transport. In some experiments the distribution of 86RbZ+ and [3H]TPMP+ was changed upon incubation in the presence of elevated levels of K+, ouabain, or KCN, or at 0°C in a way that would be expected from the membrane potential. In normal incubation conditions a membrane potential of ∼−60 mv was calculated.

The effects of severe hypoglycemia on perforant path-evoked field potentials were examined in the rat hippocampus. Although a complete loss of this response was noted when blood glucose concentration fell below 1 mM, this occurred before cessation of electroencephalogram (EEG) activity. Both spontaneous and evoked responses recovered partially following glucose readministration. D-2-Amino-5-phosphonovalerate, an NMDA-sensitive acidic amino acid receptor antagonist, facilitated this recovery from the hypoglycemic challenge when administered via a dialysis probe.

Brain ischemia was induced for 10 or 30 min by clamping the common carotid arteries in rabbits whose vertebral arteries had previously been electrocauterized. EEG and tissue content of high energy phosphates were used to verify the ischemic state and to evaluate the degree of postischemic recovery. Extracellular levels and total contents of amino acids were followed in the hippocampus during ischemia and 4 h of recirculation. At the end of a 30-min ischemic period, GABA had increased 250 times, glutamate 160 times, and aspartate and taurine 30 times in the extracellular phase. The levels returned to normal within 30 min of reflow. A delayed increase of extracellular phosphoethanolamine and ethanolamine peaked after 1–2 h of reflow. Ten minutes of ischemia elicited considerably smaller but similar effects. With respect to total amino acids in the hippocampus, glutamate and aspartate decreased to 30–50% of control while GABA appeared unaffected after 4 h of reflow. Alanine, valine, phenylalanine, leucine, and isoleucine increased severalfold. The importance of toxic extracellular levels of excitatory amino acids, as well as of high extracellular levels of inhibitory amino acids, are considered in relation to the pathophysiology of neuronal cell loss during cerebral ischemia.

Publisher Summary The amino acids aspartate, GABA, glutamate, glycine, as well as taurine and p-alanine, have been suggested as neurotransmitters in the mammalian central nervous system. A criterion for a transmitter is that it should be released from its synaptic store upon depolarization. The physiological release of neurotransmitters from nerve terminals in vivo is mimicked in vitro by the application of elevated [K + ] or veratridine to slices of nervous tissue. In order to try to ascertain the synaptic origin of the released material, the Ca-dependence and tetrodotoxin (TTX) sensitivity of the release are used. Experiments are carried out by monitoring the efflux of exogenously applied amino acids or by measurement of the efflux of endogenous amino acids. During preloading of the tissue with radio-labeled amino acids, problems with metabolism and compartmentation are often encountered that may make the results difficult to interpret. This chapter reports on the release of endogenous amino acids from rabbit SC slices. As comparison, results from similar experiments, with slices from rat hippocampus, will be presented.

The effect of ouabain on extracellular amino acid levels was investigated in the rabbit olfactory bulb using brain dialysis. Extracellular field potentials, elicited by stimulation of the lateral olfactory tract (LOT), were recorded simultaneously. Ouabain (100 μM) induced a rapid increase in extracellular aspartate, glutamate and γ-aminobutyric acid. LOT-evoked potentials changed concomitantly, suggesting a neuronal depolarization.

Free amino acids were studied in the olfactory bulb of the rabbit during basal conditions and veratridine-induced depolarization, in vitro with a tissue slice preparation and in vivo with a perfusion-dialysis technique. In vivo, basal extracellular concentrations of GABA, beta-alanine and aspartate were low, while glutamine showed the highest level. The basal steady-state concentration ratio between the total tissue pool of free amino acids and amino acids in the extracellular fluid was high for GABA, aspartate and glutamate, while low for glutamine and other 'non-transmitter' amino acids. Veratridine induced a marked TTX-sensitive release of GABA (40-50 times the control) both in vivo and in vitro. In vivo, the GABA release showed a peak during the first minutes of veratridine perfusion. The TTX-sensitive release of aspartate and glutamate, on the other hand, was approximately 5 times higher in vitro than in vivo. Furthermore, a prolonged response to veratridine was seen for glutamate and aspartate in vivo consisting of an early peak, followed by a sustained release. Taurine showed a time-delayed veratridine response, both in vivo and in vitro, whereas glutamine displayed a slow, TTX-sensitive decrease. No effect of veratridine was seen on beta-alanine or carnosine-threonine levels.

The chemically evoked efflux of endogenous amino acids from perfused rabbit superior colliculus (SC) slices was studied. Amino acids in the perfusates were determined fluorimetrically with a precolumn derivatisation method and subsequent separation on an HPLC column. Potassium-induced depolarisation caused a calcium-dependent release of beta-alanine, GABA, glutamate, and aspartate. Veratridine-evoked efflux was essentially similar in selectivity and could be blocked by tetrodotoxin (TTX). The results are indicative of a neurotransmitter role of beta-alanine, GABA, glutamate, and aspartate in the SC of the rabbit.

The effect of kainic acid (KA) on the extracellular content of amino acids in the rabbit olfactory bulb was investigated, both in vivo with the brain dialysis technique and in vitro with a superfused tissue slice preparation. Olfactory bulb EEG activity and lateral olfactory tract (LOT)-evoked field potentials were monitored simultaneously during dialysis experiments. KA induced a rapid (within 5 min) increase of extracellular aspartate, glutamate, GABA, phosphoethanolamine and taurine in vivo. LOT-evoked potentials were altered concomitantly in a concentration- and time-dependent manner. The antidromic invasion of mitral/tufted cells was depressed and the synaptic activation of granule cells was abolished in the presence of KA. Olfactory bulb EEG activity was also affected. Oscillatory bursts in olfactory bulb EEG were abolished by 10 mM in most experiments, whereas sustained oscillations were induced by 1 mM KA. The effects of KA may partly be due to a blockade of amino acid reuptake since dihydrokainate (DKA) perfusion was found to increase extracellular aspartate and glutamate. However, DKA had no significant effect on EEG or evoked potentials. In vitro, aspartate and glutamate were selectively increased during KA perfusion.

Neuroexcitatory amino acids are considered to act as transmitters in several pathways of the mammalian brain. Very recently, disturbances in glutamatergic and/or aspartatergic systems have been associated with various pathological states. Thus, there is evidence to suggest that an uncontrolled release of excitatory amino acids and a subsequent post-synaptic overexcitation is involved in the induction of neuronal damage which occurs after ischemia (Benveniste et al., 1984; Simon et al., 1984; Hagberg et al., 1985; Wieloch et al., 1985) and hypoglycemia (Sandberg et al., 1985; Wieloch, 1985). Similar mechanisms are proposed to operate during status epilepticus-induced neuronal necrosis (Griffiths et al., 1982). No direct measurements of extracellular amino acids during status epilepticus have been reported and we have therefore addressed this issue using the brain dialysis method.

Although acidic amino acids are widely believed to act as chemical transmitters at a number of synapses in the brain, the precise nature of the transmitter substance remains unclear. Several researchers have attempted to evaluate the identity of the neurotransmitter utilised in these pathways by examining the in vitro release of transmitter candidates such as glutamate and as-partate (Fagg and Lane, 1979; Fagg and Foster, 1983; Fonnum et al., 1983). This approach has indeed provided some relevant information especially when used in combination with lesion experiments. However, any extrapolation of in vitro data to the in vivo situation is fraught with difficulties, and in vivo neurochemical studies will clearly be necessary before a physiological role for any putative transmitter can be confirmed.

Extracellular amino acids were followed in the hippocampus during sustained seizures induced by systemic administration of kainic acid (KA) or bicuculline (BC). KA epilepsy was associated with marked increases in phosphoethanolamine (PEA) and taurine. Alanine and ethanolamine were moderately raised while other amino acids were unaffected. BC seizures encompassed a slightly different pattern of alterations. In contrast to KA seizures, BC epilepsy had no effect on taurine. Significant increments were observed for PEA and alanine while elevations of ethanolamine were subtle. In both types of seizures, glutamate and GABA remained unaffected extracellularly, probably due to efficient recapture mechanisms.

For high sensitivity analysis of neuroactive amino acids, liquid chromatography employing precolumn derivatisation with o-phthalaldehyde (OPA) is suitable for several reasons. The OPA reagent is non-fluorescent per se, the reaction occurs rapidly in alkaline aqueous solutions and forms highly fluorescent derivatives with primary amines.