Blocker Of Na Research Articles

The Slack Sodium-Activated Potassium Channel Provides a Major Outward Current in Olfactory Neurons of Kv1.3−/− Super-Smeller Mice

The Kv1.3 voltage-dependent potassium channel is expressed at high levels in mitral cells of the olfactory bulb (OB). Deletion of the Kv1.3 potassium channel gene (Kv1.3-/-) in mice lowers the threshold for detection of odors, increases the ability to discriminate between odors, and alters the firing pattern of mitral cells. We have now found that loss of Kv1.3 produces a compensatory increase in Na(+)-activated K(+) currents (K(Na)) in mitral cells. Levels of the K(Na) channel subunit Slack-B determined by Western blotting are substantially increased in the OB from Kv1.3-/- animals compared with those of wildtype animals. In voltage-clamp recordings of OB slices, elevation of intracellular sodium from 0 to 60 mM increased mean outward currents by 15% in mitral cells from wildtype animals and by 40% in cells from Kv1.3-/- animals. In Kv1.3-/- cells, K(Na) current could even be detected with 0 mM Na(+) internal solutions, provided extracellular Na(+) was present, and this current could be abolished by TTX and ZD7288, blockers of Na(+) influx through voltage-dependent Na(+) channels and H-channels, respectively. The role of enhanced expression of Slack subunits in the increase of K(Na) current in Kv1.3-/- cells was also confirmed using an RNA interference (RNA(i)) approach to suppress Slack expression in primary cultures of olfactory neurons. In Kv1.3-/- neurons, treatment with Slack-specific RNA(i) inhibited approximately 75% of the net outward current, whereas in wildtype cells, the same treatment suppressed only about 25% of the total current. Scrambled and mismatched RNA(i) oligonucleotides failed to suppress currents. Our findings raise the possibility that the olfactory phenotype of Kv1.3-/- animals results in part from an enhancement of K(Na) currents.

Diphenidol inhibited sodium currents and produced spinal anesthesia

The aim of this study was to evaluate the effect of diphenidol on blocking Na(+) currents and spinal anesthesia. We used the patch-clamp method to examine if diphenidol blocked Na(+) currents. Lidocaine, a common used local anesthesia, was used as control. We also evaluated the potencies and durations of diphenidol and lidocaine on spinal blockades of motor function, proprioception, and nociception in rats. Lidocaine exhibited a concentration- and state-dependent effect on tonic blockade of voltage-gated Na(+) currents in mouse neuroblastoma N2A cells (IC(50) of 8.1 and 138.9 microM at holding potentials of -70 and -100 mV, respectively). Diphenidol was more potent (IC(50) of 0.77 and 62.6 microM at holding potentials of -70 and -100 mV, respectively). However, unlike lidocaine, block of Na(+) currents by diphenidol lacked use-dependence. We also found that diphenidol acted like lidocaine and produced dose-related spinal blockades of motor function, proprioception and nociception. Although diphenidol had similar potencies of spinal anesthesia compared with lidocaine it produced a much longer duration of spinal blockades than lidocaine. Our results demonstrated that intrathecal diphenidol produced a long duration and similar potency on spinal anesthesia compared with lidocaine in rats. The anesthetic effect of diphenidol could be in part due to its blockade of Na(+) currents.

Endolymphatic Sodium Homeostasis by Extramacular Epithelium of the Saccule

The saccule is a vestibular sensory organ that depends upon regulation of its luminal fluid, endolymph, for normal transduction of linear acceleration into afferent neural transmission. Previous studies suggested that endolymph in the saccule was merely derived from cochlear endolymph. We developed and used a preparation of isolated mouse saccule to measure transepithelial currents from the extramacular epithelium with a current density probe. The direction and pharmacology of transepithelial current was consistent with Na(+) absorption by the epithelial Na(+) channel (ENaC) and was blocked by the ENaC-specific inhibitors benzamil and amiloride. Involvement of Na(+),K(+)-ATPase and K(+) channels was demonstrated by reduction of the current by ouabain and the K(+) channel blockers Ba(2+), XE991, and 4-AP. Glucocorticoids upregulated the current via glucocorticoid receptors. Dexamethasone stimulated the current after 24 h and the stimulation was blocked by mifepristone but not spironolactone. No acute response was observed to elevated cAMP in the presence of amiloride nor to bumetanide, a blocker of Na(+),K(+),2Cl(-) cotransporter. The results are consistent with a canonical model of corticosteroid-regulated Na(+) absorption that includes entry of luminal Na(+) through apical membrane Na(+) channels and active basolateral exit of Na(+) via a Na(+) pump, with recycling of K(+) at the basolateral membrane via K(+)-permeable channels. These observations provide our first understanding of the active role played by saccular epithelium in the local regulation of the [Na(+)] of endolymph for maintenance of our sense of balance.

Potassium induced dilation in bovine coronary artery involves both inward rectifier potassium channels and Na+/K+ATPase

Increases in extracellular potassium (K+) concentration (up to 20 mM) cause dilation in some blood vessels. This may be particularly important in myocardial ischemia because in this condition K+ is released from ischemic cells. In this study, we investigated mechanisms of effect of increased K+ concentration on the tone of isolated bovine coronary artery. Bovine coronary arteries were isolated and mounted in organ baths for isometric tension recording. After an equilibration period, arteries were contracted with serotonin (1 microM). When serotonin contraction reached a steady-state, K+ concentration of organ baths was increased from physiological levels to 10 mM, 14 mM, 18 mM or 22 mM in four groups of the arteries. After a washout period, this procedure was repeated in presence of ouabain, a blocker of Na+ /K+ ATPase or a K+ channel blocker (tetraethylammonium, 4-aminopyridine, glibenclamide or barium). Increasing K+ concentration of the organ baths to 10 mM, 14 mM and 18 mM caused dilation in the arteries. Ouabain abolished the dilation and barium (a blocker of inward rectifier K + channels) inhibited the dilation significantly.According to our results there is K+ -induced dilation in bovine coronary artery and it involves activation of both Na+ /K+ ATPase and inward rectifier K+ channels.

The effect of different kinds of electrolyte and non‐electrolyte solutions on the survival rate and morphology of zebrafish Danio rerio embryos

The effect of electrolyte and non-electrolyte solutions on the survival and on the morphology of zebrafish Danio rerio embryos was investigated. Embryos in different ontogenetic stages were incubated in electrolyte (NaCl, KCl, MgCl2 and CaCl2) and non-electrolyte solutions [sucrose and polyvinylalcohol (PVA)] of different concentrations for 5-15 min. The embryos were hatched to the long-pec stage and the effective concentrations which caused a 50% decrease in embryo development (EC50) were determined. The morphometric changes, which were caused by the test solutions, were measured. Ion channel blockers were used to see if active ion transport played a role for embryo survival. Finally, dechorionated embryos were exposed to the test solutions to get indications about the importance of chorion and perivitelline space. For 12 hours post fertilization (hpf) embryos and a 15 min exposure period, EC50 was highest for MgCl2 (1.60 mol l(-1)), followed by sucrose (0.73 mol l(-1)), NaCl (0.49 mol l(-1)), KCl (0.44 mol l(-1)), CaCl2 (0.43 mol l(-1)) and PVA [0.0005 mol l(-1) (2.2%)]. EC50 were lower for early embryonic stages than for advanced stages for all solutions with exception of MgCl2 and sucrose. At the EC50, MgCl2 and CaCl2 solutions did not induce morphometric changes. NaCl and sucrose solutions induced reversible morphometric changes, which were compensated within 10 min. Only the EC50 of KCl and PVA solutions induced permanent morphometric changes, which could not be compensated. Incubation of embryos in electrolyte and non-electrolyte solutions together with ouabain (blocker of Na+- K+ ATPase), HgCl3 (dose-dependent inhibition of aquaporine channels), verapamil (inhibition of calcium and magnesium uptake) and amiloride (inhibition of sodium uptake) significantly decreased the per cent of embryos developing to the long-pec stage in comparison to the same solutions without blockers. Ouabain and HgCl(3) also induced morphometric changes. For dechorionated embryos the survival rates in water and in the different test solutions were similar to untreated embryos.

Different pathways with distinct properties conduct dilations in the microcirculation in vivo

Conduction of vasomotor signals along the vessel coordinates the behaviour of vascular cells and is attributed to the spread of hyperpolarizations through gap junctions. Intriguingly, conducted dilations encompass larger distances than can be expected by passive electrotonic spread. Because distances are quite distinct for different dilators, we hypothesized that separate pathways with distinct properties are involved. We characterized local and conducted responses elicited by acetylcholine (ACh) and adenosine (Ado) in the murine microcirculation in vivo. Local (and remote) ACh dilations were nearly abrogated by blockade of K(Ca) channels (charybdotoxin), but dilations to Ado were abolished by the K(ATP) blocker glibenclamide. Bupivacaine, a blocker of Na+ and K+ channels, and similarly the blockade of inwardly rectifying K+ channels (barium) revealed different conduction mechanisms, as the remote dilation to Ado, but not ACh, was abrogated. Surprisingly, expression of connexin37 (Cx37) was not detected in Cx40-deficient arterioles, although abundantly expressed in endothelium of wild-type arterioles. In contrast to the wild-type mice, the amplitude of conducted ACh and Ado dilations decreased similarly with distance in Cx40-deficient mice. Recordings of membrane potential in vivo showed endothelial hyperpolarization by approximately 10 mV in response to ACh, whereas Ado did not alter endothelial membrane potential. Distinct pathways conduct responses along the vessel wall which involve dissimilar K+ channels and connexins in initiation and spreading. Most likely, the endothelium is the preferential conduction pathway activated by ACh, whereas in the case of Ado the smooth muscle serves as the signalling pathway. However, in arterioles nearly devoid of Cx40 and Cx37, ACh responses can likewise be conducted along the smooth muscle.

Counter current transfer of beta-endorphin in the perihypophyseal cavernous sinus--carotid rete vascular complex of sheep.

This study was performed to answer the question of whether counter current retrograde transfer of beta-endorphin in the perihypophyseal cavernous sinus-carotid rete vascular complex depends on the reproductive activity of sheep and if this transfer depends on membrane Na+ K+ ATP-ase blocking by ouabain. Sheep were anaesthetised and the jugular vein and the carotid artery were cannulated on both sides. Multielectrolitical liquids (Solfin, Polfa "Kutno", Poland): 500 ml of Solfin with heparin (25,000 IU), or Solfin with heparin and ouabain (Sigma, St. Louis, USA) in concentrations of 10(-5) or 10(-4) mol 1(-1) were infused into the brain through the carotid artery. Heparinized blood was collected through the carotid artery. After exsanguination, the head with the neck was removed. The isolated head was supplied with oxygenated, heated, autologous blood diluted with Solfin (4:1) without or with ouabain in concentration of 10(-5) or 10(-4) mol 1(-1). Blood pressure and temperature were measured throughout the duration of the experiment. During the experiment 125I-beta-endorphin (7.9 x 107 dpm) dissolved in 10 ml of Solfin was infused for 5 min (5 ml) into each cavernous sinus through the angularis oculi veins. Blood samples for radioactivity measurements were collected each min from the carotid rete (through the opposite carotid artery to the artery supplying the brain with arterial blood) and from both jugular veins. In all the experiments significant 125I-beta-endorphin radioactivity was found in arterial blood supplying the brain and hypophysis in the early luteal phase in sheep. No radioactivity was found (with the exception of one animal) in sheep during seasonal anoestrus. A blockage of Na+ K+ ATP-ase by ouabain administered during exsanguination and during head perfusion with dose of 10(-4) mol 1(-1) reduced beta-endorphin counter current transfer to arterial blood, but this effect was not evident with the dose of 10(-5) mol 1(-1). Increased blood pressure was observed in all the experiments with either dose of ouabain.

Expression of the epithelial Na + channel and other components of an aldosterone response pathway in human adrenocortical cells

We have unexpectedly found expression of the epithelial Na + channel (ENaC) in human adrenocortical cells and tested the hypothesis that these cells contain the components of an aldosterone response pathway. Tissue was obtained from patients undergoing adrenalectomy and mRNA and protein expression of recognised components of an aldosterone-response pathway were determined by RT-PCR and Western blotting. The effects of mineralocorticoid receptor agonists and antagonists, amiloride analogues, and extracellular Na + on basal and stimulated aldosterone release from immortalised (H295R) cells were determined by radioimmunoassay. Expression of mRNA for α-, β- and γ-subunits of ENaC, the mineralocorticoid receptor, Nedd4L, Sgk1 and 11β hydroxysteroid dehydrogenase type II was confirmed in human adrenal cortex. Using Western blotting α-, β- and γ-ENaC expression was demonstrated in adrenocortical cells. Measurements of 24 h aldosterone release from H295R cells showed stimulation by K + and angiotensin II, suppression by both Na + and high-concentration 5-( N-ethyl- N-isopropyl) amiloride (EIPA, blocker of Na +–H + exchange) and no change with benzamil (ENaC blocker). 22Na-uptake into H295R cells was inhibited by EIPA, but not by benzamil. Our experiments suggest that the components of an aldosterone response pathway are present in human adrenal cortex. Studies in H295R cells, however, suggest that ENaC is not an important mediator of 22Na-uptake or aldosterone production. Further studies are required to determine the importance of an adrenal aldosterone response pathway.

Longitudinal analysis of the depressive effects of intravenous amiodarone on depolarization and repolarization: A case report

Intravenous amiodarone (AMD) induces multiple antiarrhythmic effects via blocking of Na(+), Ca(2+), and IKr channels, and beta receptors. A patient on chronic dialysis was administered AMD for nonsustained ventricular tachycardia after successful cardiopulmonary resuscitation. QT prolongation occurred 5 h after AMD administration. AMD was withdrawn at 24 h because of prolonged QTc interval (716 ms), which persisted for a further 48 h (661 ms). Ventricular premature contraction (VPC) was significantly decreased at 7h; however, VPC increased again after discontinuing AMD. Depolarization changes induced by the Na(+)-channel blocking action of AMD were analyzed. There was increasing filtered QRS-duration and duration of low-amplitude signals at voltage <40 µV, and decreasing root-mean-square voltage of signals in the last 40 ms of ventricular late potentials (LPs) within 7 h. However after stopping AMD, LPs were reversed. The blood concentration of AMD reached the effective level within 10 min but decreased immediately to an ineffective level. Onset and disappearance of the VPC-inhibiting effect corresponded to the depressive effect on depolarization but not with the increase in the prolonged repolarization effect and blood concentration. Even if the QT interval is sufficiently prolonged, the Na(+)-channel blocking action is required for AMD to induce the antiarrhythmic effect.

Chloride Is Essential for Capacitation and for the Capacitation-associated Increase in Tyrosine Phosphorylation

After epididymal maturation, sperm capacitation, which encompasses a complex series of molecular events, endows the sperm with the ability to fertilize an egg. This process can be mimicked in vitro in defined media, the composition of which is based on the electrolyte concentration of the oviductal fluid. It is well established that capacitation requires Na(+), HCO(3)(-), Ca(2+), and a cholesterol acceptor; however, little is known about the function of Cl(-) during this important process. To determine whether Cl(-), in addition to maintaining osmolarity, actively participates in signaling pathways that regulate capacitation, Cl(-) was replaced by either methanesulfonate or gluconate two nonpermeable anions. The absence of Cl(-) did not affect sperm viability, but capacitation-associated processes such as the increase in tyrosine phosphorylation, the increase in cAMP levels, hyperactivation, the zona pellucidae-induced acrosome reaction, and most importantly, fertilization were abolished or significantly reduced. Interestingly, the addition of cyclic AMP agonists to sperm incubated in Cl(-)-free medium rescued the increase in tyrosine phosphorylation and hyperactivation suggesting that Cl(-) acts upstream of the cAMP/protein kinase A signaling pathway. To investigate Cl(-) transport, sperm incubated in complete capacitation medium were exposed to a battery of anion transport inhibitors. Among them, bumetanide and furosemide, two blockers of Na(+)/K(+)/Cl(-) cotransporters (NKCC), inhibited all capacitation-associated events, suggesting that these transporters may mediate Cl(-) movements in sperm. Consistent with these results, Western blots using anti-NKCC1 antibodies showed the presence of this cotransporter in mature sperm.

TEA-induced long-term potentiation at hippocampal mossy fiber-CA3 synapses: Characteristics of its induction and expression

Potassium ion channel blockade by tetraethylammonium (TEA) reportedly induces long-term potentiation (LTP) at hippocampal mossy fiber (MF)-CA3 synapses, but the characteristics of induction, expression, and modulation of the LTP remain unclear. In the present study, these features of TEA-induced LTP at MF-CA3 synapses were electrophysiologically examined using rat hippocampal slices. Synaptic responses recorded from MF-CA3 synapses were enhanced long-term by TEA application even under the blockade of NMDA receptors with d-AP5, whereas selective pharmacological blockade of T-type voltage-dependent calcium channels (VDCCs) strongly inhibited TEA-induced LTP. Decrease of the paired-pulse facilitation ratio after LTP induction by TEA suggests the involvement of increased neurotransmitter release probability from MF terminals as LTP expression. The facilitative modulation of MF-CA3 LTP by GABAA receptor activation reported previously was reversed when bumetanide, a blocker of Na+–K+–Cl− co-transporters (NKCCs), was applied, suggesting that the region-specific modulation of TEA-induced LTP by GABAergic inputs at MF-CA3 synapses is due to the dominance of NKCC action at MF terminals.

077 The Bioelectric Field Imager: A New Tool for Measuring the Electric Field Around Skin Wounds

We are studying in the role of endogenous electric fields in mammalian skin wound healing. The epidermis generates a transepithelial potential (TEP) of 20–50 mV across itself, inside positive. Any wound or break in the epidermis creates a low resistance pathway and the TEP at the wound site is 0 mV. However, the TEP of the intact epidermis around the wound is still present, resulting in a lateral voltage gradient or electric field along the skin surrounding the wound. We have evidence that this lateral electric field stimulates keratinocytes in the area to migrate towards the wound with the optimal response occurring at a field strength of 100 mV/mm. In order to determine the actual field strength present in mammalian wounds, we have developed a new instrument called the Bioelectric Field Imager (BFI). The BFI vibrates a small sensor perpendicular to the skin about 200 μm above the surface and uses the capacitance signal to determine the surface potential of the epidermis just beneath the stratum corneum. By measuring this surface potential in many positions around the wound, we generate a spatial map of the surrounding electric field. We have measured the wound field near a 1 mm long, full thickness skin wound in mice. A field of 100–200 mV/mm is present immediately following wounding and persists until wound healing is complete. The topical application of a blocker of Na+ channels (amiloride) reduces this wound field by about 50%, supporting the hypothesis that it is generated by the TEP. Preliminary measurements on human skin wounds detect lateral electric fields that are similar to those measured in mice. Acknowledgments: Supported by NIH R43 GM069194 to RPN and NIH NCRR P41 RR001395 to P.J.S.S

Characterization of Voltage-Dependent Ca2+Currents in IdentifiedDrosophilaMotoneurons In Situ

Voltage-dependent Ca2+ channels contribute to neurotransmitter release, integration of synaptic information, and gene regulation within neurons. Thus understanding where diverse Ca2+ channels are expressed is an important step toward understanding neuronal function within a network. Drosophila provides a useful model for exploring the function of voltage-dependent Ca2+ channels in an intact system, but Ca2+ currents within the central processes of Drosophila neurons in situ have not been well described. The aim of this study was to characterize voltage-dependent Ca2+ currents in situ from identified larval motoneurons. Whole cell recordings from the somata of identified motoneurons revealed a significant influence of extracellular Ca2+ on spike shape and firing rate. Using whole cell voltage clamp, along with blockers of Na+ and K+ channels, a Ca2+-dependent inward current was isolated. The Drosophila genome contains three genes with homology to vertebrate voltage-dependent Ca2+ channels: Dmca1A, Dmca1D, and Dmalpha1G. We used mutants of Dmca1A and Dmca1D as well as targeted expression of an RNAi transgene to Dmca1D to determine the genes responsible for the voltage-dependent Ca2+ current recorded from two identified motoneurons. Our results implicate Dmca1D as the major contributor to the voltage-dependent Ca2+ current recorded from the somatodendritic processes of motoneurons, whereas Dmca1A has previously been localized to the presynaptic terminal where it is essential for neurotransmitter release. Altered firing properties in cells from both Dmca1D and Dmca1A mutants indicate a role for both genes in shaping firing properties.

Estradiol modulation of phenylephrine-induced excitatory responses in ventromedial hypothalamic neurons of female rats

Estrogens act within the ventromedial nucleus of the hypothalamus (VMN) to facilitate lordosis behavior. Estradiol treatment in vivo induces alpha(1b)-adrenoreceptor mRNA and increases the density of alpha(1B)-adrenoreceptor binding in the hypothalamus. Activation of hypothalamic alpha(1)-adrenoceptors also facilitates estrogen-dependent lordosis. To investigate the cellular mechanisms of adrenergic effects on VMN neurons, whole-cell patch-clamp recordings were carried out on hypothalamic slices from control and estradiol-treated female rats. In control slices, bath application of the alpha(1)-agonist phenylephrine (PHE; 10 microM) depolarized 10 of 25 neurons (40%), hyperpolarized three neurons (12%), and had no effect on 12 neurons (48%). The depolarization was associated with decreased membrane conductance, and this current had a reversal potential close to the K(+) equilibrium potential. The alpha(1b)-receptor antagonist chloroethylclonidine (10 microM) blocked the depolarization produced by PHE in all cells. From estradiol-treated rats, significantly more neurons in slices depolarized (71%) and fewer neurons showed no response (17%) to PHE. PHE-induced depolarizations were significantly attenuated with 4-aminopyridine (5 mM) but unaffected by tetraethylammonium chloride (20 mM) or blockers of Na(+) and Ca(2+) channels. These data indicate that alpha(1)-adrenoceptors depolarize VMN neurons by reducing membrane conductance for K(+). Estradiol amplifies alpha(1b)-adrenergic signaling by increasing the proportion of VMN neurons that respond to stimulation of alpha(1b)-adrenergic receptors, which is expected in turn to promote lordosis.

Both cell volume and calmodulin contribute to the regulation of macula densa nitric oxide

Elevations in luminal sodium chloride concentration ([NaCl]L) alter macula densa (MD) intracellular calcium concentration and result in the release of nitric oxide (NO). Also, concomitant elevations in [NaCl]L and luminal osmolality (osmL) lead to shrinkage of macula densa cells, while increases in [NaCl]L at constant osmL produce cell swelling. Currently, there is little information concerning the intracellular signaling event that leads to the activation of MD NO synthase. Current studies were performed to determine whether changes in MD cell volume and/or calmodulin‐dependent signaling activate MD NO synthase. Thick ascending limb‐glomerulus preparations were isolated and perfused from rabbits, and MD NO release was detected with fluorescence microscopy using DAF‐FM. Elevations in [NaCl]L (from 0 to 150 mmol/L) at constant osmL produced an increase in DAF‐FM fluorescence of 5±0.5%, while concomitant elevations in [NaCl]L and osmL led to an increase of 12±0.7%. Luminal application of furosemide, a blocker of Na+:2Cl‐:K+ cotransport, reversed [NaCl]L‐dependent NO release (‐0.04±0.2%). Also, application of the calmodulin‐inhibitor calmidazolium blocked [NaCl]L‐dependent NO release (1.8±1.8%). Thus, [NaCl]L‐dependent NO production by MD cells may be modulated by changes in cell volume and requires activation of calmodulin signaling.

E-Ring Isoprostanes Stimulate a Cl− Conductance in Airway Epithelium via Prostaglandin E2-Selective Prostanoid Receptors

Isoprostanes comprise a class of membrane lipid metabolites produced during oxidative stress, including asthma, chronic obstructive pulmonary disease, and cystic fibrosis. They are widely recognized to evoke a variety of biological responses in airway and pulmonary vascular smooth muscle, lymphatics, and innervation. However, their effects on airway epithelium are largely unstudied. We examined the electrophysiological responses evoked by several different isoprostane species in bovine airway epithelium using the Ussing chamber technique. The E-ring isoprostanes 15-E(1t)-IsoP and 15-E(2t)-IsoP evoked a substantial increase in short-circuit current (I(SC)), whereas four different F-ring isomers were ineffective. 15-E(2t)-IsoP-evoked I(SC) was mimicked by the prostaglandin E(2)-selective prostanoid receptor (EP)-agonist prostaglandin E(2) but not by agonists of EP(1)/EP(3)-, FP-, or TP receptors (sulprostone, fluprostenol, and U46619, respectively). This response was significantly reduced by the EP(4)-receptor blocker GW627386 but not by blockers of other prostanoid receptors (ICI 192,605 [TP-selective], SC19220 [EP(1)-selective], AH6809 [DP/EP(1)/EP(2)-selective], and AL8810 [FP-selective]). 15-E(2t)-IsoP-evoked I(SC) was reduced by blockers of Cl(-) channels (niflumic acid and 5-nitro-2-(3-phenylpropylamino)-benzoic acid), of Na(+)/K(+)/2Cl(-) co-transport (furosemide and bumetanide), of adenylate cyclase (MDL 12,330A), or of guanylate cyclase (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) but not by blockers of Na(+) conductances (amiloride). We conclude that 15-E(2t)-IsoP activates a transepithelial Cl(-) conductance in bovine airway epithelium through an EP(4) receptor coupled to adenylate cyclase and soluble guanylate cyclase.

Choice of cardiac tissue in vitro plays an important role in assessing the risk of drug-induced cardiac arrhythmias in human: Beyond QT prolongation

Introduction Recently we have demonstrated that to the choice of tissue type is important in identifying I Kr and I Ks-induced prolongation of the action potential. However, the differential sensitivity of cardiac tissues to other ionic current blockers or modulators is relatively unknown. The aim of the present study was therefore to evaluate tissue-specific effects of different ion channel blockers or activators on the action potential (AP), which can affect other parameters in addition to drug-induced APD/QT prolongation or shortening. Methods and Results Electrophysiological effects were measured in isolated rabbit Purkinje fibers, papillary muscles and ventricular trabeculae using a microelectrode technique under the following conditions: block of I to with 4-AP (1 × 10 − 3 M), block of Ca 2+ channels with diltiazem (1 × 10 − 5 M), block of Na + channels with flecainide (1 × 10 − 5 M), activation of Ca 2+ current with Bay-K-8644 (1 × 10 − 5 M), activation of K ATP channels with levcromakalim (1 × 10 − 5 M) or block of I K1 current with BaCl 2 ( n = 8 to 12 for each group). 4-AP prolonged APD significantly more in the Purkinje fiber than in the papillary muscle or the ventricular trabecula. 4-AP elicited 63% incidence of early afterdepolarizations but 0% in the papillary or trabeculae. Diltiazem and flecainide shortened APD 40 and APD 50 and increased triangulation more in the Purkinje fiber, whilst having little effect on these parameters in the papillary muscle or the ventricular trabecula. Bay-K-8644 significantly prolonged APD in the ventricular trabecula, but not in the Purkinje fiber or the papillary muscle. BaCl 2 prolonged APD 90 in all tissues, but significantly shortened APD 40 only in the Purkinje fiber. Levcromakalim shortened APD in all tissues, but significantly less in the Purkinje fibers. Conclusion The present study demonstrates that certain cardiac tissues respond differently to the same ion channel blockers/activators, which are not involved in APD/QT prolongation. As such the appropriate selection of tissue needs to be taken into careful consideration in cardiac safety assessments when exploring different mechanisms of drug-induced changes in the action potential.

NIP-141, a multiple ion channel blocker, terminates aconitine-induced atrial fibrillation and prevents the rapid pacing-induced atrial effective refractory period shortening in dogs

NIP-141 is a novel multiple ion channel blocker with atrial selective effects. In this study, we examined the effects of NIP-141 on aconitine-induced atrial fibrillation (AF) and rapid atrial pacing-induced atrial effective refractory period (ERP) shortening in dogs. Aconitine AF was induced by the application of aconitine on the right appendage. NIP-141 (10 mg/kg) converted AF to sinus rhythm in 5 of 6 dogs. The Na(+) channel blockers disopyramide (1 mg/kg) and phenytoin (10 mg/kg) also terminated AF, but the I(Kr) blocker (d-sotalol; 4 mg/kg) and a Ca(2+) channel blocker (verapamil; 0.3 mg/kg) did not terminate AF in this model. To clarify the mechanism of AF termination, we examined the effects on ERP and conduction time, but NIP-141 (10 mg/kg) had no significant effects. In a short-term rapid atrial pacing model, NIP-141 (2.5 mg/kg/10 min, followed by 0.033 mg/kg/min) prevented atrial ERP shortening. We also found NIP-141 bound to Na(+) channel site 2 receptor and L-type Ca(2+) channel, but not to Na(+) channel site 1 receptor using radioligands binding assay. NIP-141 terminated AF in aconitine-induced AF and prevented the atrial remodelling by short-term rapid pacing in dogs, possibly via the blocking of Na(+) and Ca(2+) channels.

Sources of axonal calcium loading during in vitro ischemia of rat dorsal roots

A detailed understanding of injury mechanisms in peripheral nerve fibers will help guide successful design of therapies for peripheral neuropathies. This study was therefore undertaken to examine the ionic mechanisms of Ca2+ overload in peripheral myelinated fibers subjected to chemical inhibition of energy metabolism. Myelinated axons from rat dorsal roots were co-loaded with Ca2+-sensitive (Oregon Green BAPTA-1) and Ca2+-insensitive (Alexa Fluor 594) dextran-conjugated fluorophores and imaged using confocal laser scanning microscopy. Axoplasmic regions were clearly outlined by the Ca2+-insensitive dye, from which axonal Ca2+-dependent fluorescence changes (FCa.ax) were measured. Block of Na+-K+ ATPase (ouabain), opening of Na+ channels (veratridine), and inhibiting energy metabolism (iodoacetate + NaN3) caused a rapid rise in FCa.ax to 96% above control after 30 min. Chemical ischemia (iodoacetate + NaN3) caused a more gradual increase in FCa.ax (54%), which was almost completely dependent on bath Ca2+, indicating that most of the Ca2+ accumulation occurred via influx across the axolemma. Na+ channel block (tetrodotoxin) reduced ischemic FCa.ax rise (14%); however, inhibition of L-type Ca2+ channels (nimodipine) had no effect (60%). In contrast, Na+-Ca2+ exchange inhibition (KB-R7943) significantly reduced ischemic FCa.ax rise (18%). Together our results indicate that the bulk of Ca2+ overload in injured peripheral myelinated axons occurs via reverse Na+-Ca2+ exchange, driven by axonal Na+ accumulation through voltage-gated tetrodotoxin-sensitive Na+ channels. This mechanism may represent a viable therapeutic target for peripheral neuropathies.

Normotonic Cell Shrinkage Induced by Na+ Deprivation Results in Apoptotic Cell Death in Human Epithelial HeLa Cells

Apoptosis is a major form of cell death that occurs in response to a variety of signals in both physiological and pathological situations. A hallmark of apoptosis is normotonic cell shrinkage, called apoptotic volume decrease (AVD), the process of which involves fluxes of K(+), Cl(-), and Na(+). Na(+) influx was suggested to be required in Fas-induced apoptosis in human Jurkat T cells, whereas Na(+) efflux was found to be associated with AVD and apoptosis in human HL-60 cells. Here we examined the effects of extracellular Na(+) deprivation on cell volume and viability in human epithelial HeLa cells. The incubation of HeLa cells in normotonic Na(+)-free Ringer solution resulted in persistent cell shrinkage after > or = 30 min and reduction in cell viability after > or = 1 h. After exposure to Na(+)-free solution for 5 h, a marked reduction in cell viability was found to be associated with an activation of caspase-3 without showing significant LDH release, indicating that the cells underwent apoptosis but not necrosis. Na(+) deprivation-induced cell shrinkage and apoptotic cell death were significantly inhibited by a blocker of Na(+)-K(+)-2Cl(-) cotransporter (NKCC) or of the reverse-mode operation of Na(+)/Ca(2+) exchanger (NCX), but not by a blocker of Na(+)/H(+) exchanger (NHE). Therefore it is concluded that Na(+) deprivation causes persistent cell shrinkage resulting from Na(+) efflux mainly via NKCC and NCX and thereafter leads to apoptotic death of HeLa cells. It is also suggested that normotonic cell shrinkage per se, if persistent, provides a sufficient condition for apoptosis induction.