[Neurosurgery of the spasticity syndrome in children cerebral palsy].

The review is devoted to main neurosurgical approaches to the treatment of the spasticity syndrome in children cerebral palsy. Neurosurgical procedures are divided into destructive and neuromodulating. The former included posterior selective rhizotomy, selective neurotomy and destructive operations on subcortical brain structures. The latter group included electrostimulation of brain and spinal cord structures and implantation of pumps for the chronic intrathecal baclofen (lioresal) infusion. Each method is considered in a historical aspect. Details of clinical application, positive and negative sides of the methods are described.

Anti-arrhythmic effects of I (Na), I (Kr), and combined I (Kr)-I (CaL) blockade in an experimental model of acute stretch-related atrial fibrillation.

INTRODUCTION: Atrial dilatation is commonly associated with atrial fibrillation (AF), but the electrophysiological mechanisms and the implications for anti-arrhythmic therapy are poorly understood. In a model of acute stretch-related AF in isolated rabbit hearts, we evaluated the electrophysiological effects of three different anti-arrhythmic drugs: dofetilide, flecainide and BRL-32872 (associating I (Kr) and I (CaL) blocking properties). METHODS: After 30 min of sustained stretch-related AF, we perfused BRL 10-7 M, BRL 3.10-7 M, BRL 10-6 M, flecainide 2.4 10-6 M and dofetilide 10-7 M and iteratively measured atrial effective refractory periods (ERPs), AF inducibility and AF cycle length (AFCL) 15, 30 and 60 min after drug perfusion, respectively. RESULTS: After a significant shortening of the ERPs by acute atrial stretch in the five groups individually (p < 0.001, stretch vs baseline for each group individually), drug perfusion led to a strong lengthening of AFCL, a very significant prolongation of ERPs (p < 0.001 vs stretch) and a reduction of AF inducibility (p < 0.01 vs control group) for each of the five experimental groups. The relative ERP increase was comparable in all groups, whereas a significantly lower AF inducibility was observed in the BRL 10-6 M group (p < 0.05 vs other BRL concentrations). CONCLUSION: In a model of acute stretch-related AF, dofetilide, flecainide and BRL-32872 terminated AF and prevented its immediate reinduction after having comparatively prolonged AFCL and ERPs. These comparative results suggest that those drugs are equally efficacious, albeit with different mechanisms, in the setting of acute atrial stretch.

[Ion transporters and cardiovascular diseases: pH control or modulation of intracellular calcium concentration]. / Transporteurs ioniques dans les pathologies cardiovasculaires: contrôle du pH ou régulation de la concentration calcique intracellulaire.

The regulation of the intracellular pH is under tight control by several ion transport systems including the sodium-proton exchanger, the sodium-bicarbonate cotransporter and the chlore-bicarbonate anion exchanger. While the activation of the anion exchange induces a cellular acidification, both the sodium-proton exchanger and the sodium-bicarbonate cotransporter are responsible for a protection against acidosis by extruding protons or importing bicarbonate. These transporters are transmembrane proteins whose activity is regulated by several mechanisms including phosphorylation, calcium binding and which are involved in several pathophysiologic processes such as ischemia, hypertrophy and arrhythmias. Recent studies suggest that the activation of these transporters during various diseases induces an increase in intracellular calcium concentration. Therefore, inhibiting these transporters could represent novel therapeutic strategies for the treatment of cardiovascular diseases.

Inhibition of the cardiac electrogenic sodium bicarbonate cotransporter reduces ischemic injury.

OBJECTIVE: Although it is believed that sodium-driven acid-base transport plays a central role in the development of the reperfusion injury that follows cardiac ischemia, research to date has demonstrated only a role for Na(+)/H(+) exchange (NHE). However, Na(+)-driven HCO(-)(3) transport, which is quantitatively as important as NHE in cardiac cells, has not been examined. METHODS AND RESULTS: Here the results show that a neutralizing antibody raised against the human heart electrogenic Na(+)/HCO(3)(-) cotransporter (hhNBC) blocked the recovery of pH after acidic pulse both in HEK-293 cells expressing hhNBC and in rat cardiac myocytes demonstrating the presence of an electrogenic NBC in rat cardiac myocytes similar to hhNBC. Administration of anti-NBC antibody to ischemic-reperfused rat hearts markedly protects systolic and diastolic functions of the heart during reperfusion. Furthermore, using a quantitative real-time RT-PCR (TaqMan) and Western blot analysis we demonstrated that in human cardiomyopathic hearts, mRNA and protein levels of hhNBC increase, whereas mRNA levels of the electroneutral Na(+)/HCO(3)(-) cotransporter (NBCn1) remain unchanged. CONCLUSION: Our data provide evidence that inhibition of hhNBC, whose role in cardiac pathologies could be amplified by overexpression, represents a novel therapeutic approach for ischemic heart disease.

In vivo myocardial protection from ischemia/reperfusion injury by the peroxisome proliferator-activated receptor-gamma agonist rosiglitazone.

BACKGROUND: Diabetes is associated with increased risk of mortality as a consequence of acute myocardial infarction. This study determined whether rosiglitazone (ROSI) could reduce myocardial infarction after ischemia/reperfusion injury. METHODS AND RESULTS: Male Lewis rats were anesthetized, and the left anterior descending coronary artery was ligated for 30 minutes. After reperfusion for 24 hours, the ischemic and infarct sizes were determined. ROSI at 1 and 3 mg/kg IV reduced infarct size by 30% and 37%, respectively (P<0.01 versus vehicle). Pretreatment with ROSI (3 mg. kg(-1). d(-1) PO) for 7 days also reduced infarct size by 24% (P<0.01). ROSI also improved ischemia/reperfusion-induced myocardial contractile dysfunction. Left ventricular systolic pressure and positive and negative maximal values of the first derivative of left ventricular pressure (dP/dt) were significantly improved in ROSI-treated rats. ROSI reduced the accumulation of neutrophils and macrophages in the ischemic heart by 40% and 43%, respectively (P<0.01). Ischemia/reperfusion induced upregulation of CD11b/CD18 and downregulation of L-selectin on neutrophils and monocytes; these effects were significantly attenuated in ROSI-treated animals. Likewise, intercellular adhesion molecule-1 expression in ischemic hearts was markedly diminished by ROSI, as was the ischemia/reperfusion-stimulated upregulation of monocyte chemoattractant protein-1. CONCLUSIONS: ROSI reduced myocardial infarction and improved contractile dysfunction caused by ischemia/reperfusion injury. The cardioprotective effect of ROSI was most likely due to inhibition of the inflammatory response.

EDG1 receptor stimulation leads to cardiac hypertrophy in rat neonatal myocytes.

Sphingosine 1 phosphate (S1P), an aminophospholipid, acts extracellularly as a ligand via the specific G protein-coupled receptors of the endothelial differentiation gene (EDG) 1, 3, 5, 6 and 8 receptors family and intracellularly as a second messenger in various cellular types. The aim of this work was to investigate biological activity of S1P in cardiomyocytes with respect to related sphingolipids. S1P was applied for 48 h on rat neonatal cardiomyocytes at 10 nM, 100 nM and 1 microM. S1P induced a concentration-dependent cellular hypertrophy evidenced by an increase in cell size, [3H]-phenylalanine incorporation, protein content and Brain Natriuretic Peptide (BNP) secretion. Among the lipids tested S1P exhibits the lower EC50 (67 nM) followed by dihydro-S1P (107 nM) and sphingosylphosphorylcholine (1.6 microM). The effect of S1P could be related to a stimulation of the EDG1 receptor since we showed that the EDG1 receptor is predominantly expressed at the mRNA and protein levels in rat cardiomyocytes and that specific anti-EDG1 antibodies inhibited the hypertrophic effect induced by S1P. Furthermore the expression level of most other EDG receptors for S1P appeared very low in cardiac myocytes. S1P (100 nM) increased the phosphorylation of p42/44MAPK, p38MAPK, JNK, Akt and p70(S6K), this effect being reversed by inhibitors of their respective phosphorylation which also rescue the hypertrophic phenotype. Finally, S1P stimulated actin stress fibre formation reverted by the Rho inhibitor, the C3 exoenzyme. Altogether, our results show that S1P induces cardiomyocyte hypertrophy mainly via the EDG1 receptor and subsequently via Gi through ERKs, p38 MAPK, JNK, PI3K and via Rho pathway.

Influence of atenolol on the kinetics of RT interval rate adaptation in conscious dogs.

The objective was to test an effect of atenolol independent of heart rate on electrocardiographic RT rate adaptation by investigating RT adaptation during spontaneous rate and after an abrupt change of atrial rate (study of RT delay). Digital electrocardiograms were recorded from eight conscious dogs. Analysis of RT interval (measured from QRS apex to end of T) was performed on a beat-to-beat basis. The protocol was repeated in the control state and after atenolol administration (2 mg/kg). Regarding spontaneous heart rate, an increased or decreased RR duration did not modify the beat-to-beat relative adaptation of RT to a change of RR (2.15 +/- 1% during control). Atenolol increased mean RR (p < 0.001) and decreased relative adaptation of RT (0.22 +/- 0.18%, p < 0.001). The inverse correlation between mean RR and the relative RT adaptation (r = -0.76, p < 0.05) disappeared after atenolol administration. Regarding RT delay, complete adaptation of RT required 3 min; 48 +/- 16% of this adaptation was observed after the first beat and 60 +/- 11% was observed after the 20th. Atenolol attenuated this adaptation during the first six beats following the abrupt cycle length change (p < 0.05). We concluded that the attenuation of RT rate adaptation after atenolol is related to heart rate modulation and to the time delay in RT rate adaptation.

The gamma-subunit of (Na+,K+)-ATPase: a representative example of human single transmembrane protein with a key regulatory role.

The (Na+,K+)-ATPase is a plasma membrane protein complex composed of at least three subunits (alpha,beta,gamma) that couples the exchange of three cytoplasmic Na+ ions with two extracellular K+ ions, to the hydrolysis of one molecule ofATP in most animal cells. The gamma-subunit is a 66 residue membrane protein associated with the active alpha/beta binary complex. It can be considered as an archetype of single transmembrane proteins (type I) which may play a modulatory role upon association with functional membrane partners. This paper highlights similar associations observed with other ATPases such as the sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA1/SERCA 2a), but also with Cl- and/or K+ currents, ionic channels (HERG, KCNQ1) and G-protein coupled receptors (adrenomedullin, CGRP and calcitonin) which are of particular interest in the cardiovascular field. Here is reviewed the assessed or suggested regulatory role of a family of small plasma/SR associated membrane proteins including gamma-subunit, phospholemman, Mat 8, KCNE (type 1, 2 and 3), RAMP (type 1, 2 and 3), sarcolipin and phospholamban, mainly found in muscular and vascular tissues. These proteins are critical in controlling important biological processes which derive from specific associations with a binding partner and particular subcellular localizations.

hKv4.3 channel characterization and regulation by calcium channel antagonists.

Relative expression pattern of short and long isoforms of hKv4.3 channels was evaluated by RT-PCR and RPA. Electrophysiological studies were performed in HEK293 cells transfected with short or long hKv4.3 cDNA. The long variant L-hKv4.3 was the only form present in lung, pancreas, and small intestine. The short variant S-hKv4.3 was predominant in brain whereas expression levels of the two isoforms were similar in cardiac and skeletal muscles. Properties of the ionic channels encoded by L-hKv4.3 and S-hKv4.3 cDNAs were essentially similar. Cadmium chloride and verapamil inhibited hKv4.3 current (with EC50s of 0.110 +/- 0.004 mM and 492.9 +/- 15.1 microM, respectively). Verapamil also accelerated current inactivation. Another calcium channel antagonist nicardipine was found inactive. In conclusion, this study confirms that both isoforms underlie the transient outward potassium current. Moreover, calcium channel inhibitors markedly affect hKv4.3 current, an effect which must be considered when evaluating transient outward potassium channel properties in native tissues.

Role of endothelium in ischaemia-induced myocardial dysfunction of isolated working hearts: cardioprotection by activation of adenosine A(2A) receptors.

1 This study aimed to determine the role of the vascular endothelium on recovery of contractile function following global low-flow ischaemia of guinea-pig isolated working hearts and the effects of adenosine analogues on this recovery. 2 Guinea-pig isolated spontaneously beating or paced working hearts were set up and coronary flow (CF), aortic output (AO) (as an index of cardiac function), heart rate (HR), left ventricular pressure (LVP) and dP/dt max recorded. The endothelium was either intact or removed by a blast of oxygen. 3 In spontaneously beating hearts, low-flow ischaemia for 30 min reduced CF and cardiac contractility (LVP, dP/dt max) but not AO. On reperfusion, CF, LVP and dP/dt max recovered, while AO fell precipitously followed by a gradual recovery, indicative of myocardial stunning. The effects of ischaemia did not differ between endothelium-intact and -denuded hearts, indicating no role of the endothelium in the changes observed. 4 The adenosine analogues, N6-cyclopentyladenosine (CPA, A1 selective), 5'-N-ethylcarboxamidoadenosine (NECA, two-fold A2 selective over A1) and 2-p-((carboxyethyl)-phenethylamino)-5'carboxamidoadenosine (CGS21680, A2A selective) were infused (3 x 10-7 M) from 10 min into the 30-min low-flow ischaemia of denuded hearts and during reperfusion. 5 CGS21680 increased CF and improved the postischaemic functional recovery, as measured by the AO. NECA and CPA were not cardioprotective. The A2A selective antagonist, ZM241385, attenuated the coronary vasodilatation by CGS21680 and abolished the improved recovery of AO on reperfusion. 6 Reperfusion of paced working hearts caused a dramatic fall in AO which failed to recover. Infusion of CGS21680 from 15 min into the ischaemic period produced vasodilatation but failed to restore AO, presumably because the ischaemic damage was irreversible. 7 Thus, the endothelium plays no role in myocardial dysfunction following low-flow global ischaemia and reperfusion of guinea-pig working hearts. The A2A adenosine receptor-selective agonist but not the non-selective A2 receptor agonist, NECA, attenuated ischaemia- and reperfusion-induced stunning. This was attributed to increased CF and was independent of the endothelium.

Functional specificity conferred by the unique plasticity of fully alpha-helical Ras and Rho GAPs.

Structural comparisons of the two GTPase activating proteins (GAPs) p120 and p50 in complex with Ras and Rho, respectively, allowed us to decipher the functional role of specific structural features, such as helix alpha8c of p120 and helix A1 of p50, necessary for small GTPase recognition. We identified important residues that may be critical for stabilization of the GAP/GTPase binary complexes. Detection of topohydrophobic positions (positions which are most often occupied by hydrophobic amino acids within a family of protein domains) conserved between the two GAP families led to the characterization of a common flexible four-helix bundle. Altogether, these data are consistent with a rearrangement of several helices around a common core, which strongly supports the assumption that p50 and p120 GAPs derive from a unique fold. Considered as a whole, the remarkable plasticity of GAPs appears to be a means used by nature to accurately confer functional specificity.

Cardiac loading conditions modify the ventricular repolarization in conscious dogs with heart failure.

Changes in myocardial loading conditions influence the ventricular action potential via mechanoelectric feedback, a mechanism impaired in pathology. In vivo the QT interval of the electrocardiogram which reflects the action potential duration allows appropriate determination of its modifications. The effects of changes in cardiac loading conditions (load reduction with trinitrin; volume loading with Plasmion) on regional function and ventricular local electrogram were investigated in conscious dogs before (control) and after 4 weeks of rapid pacing (dilated cardiomyopathy, DCM). In controls both interventions increased heart rate. Trinitrin increased end-diastolic wall thickness (EDWth, P<0.001) and reduced absolute QT interval duration (P<0.05). Plasmion decreased EDWth (P<0.001) and increased left ventricular end diastolic pressure (LVEDP, P<0.001) without QT interval alteration. However, the corrected QT interval was unchanged in both interventions. In DCM, trinitrin did not change the QT interval. Plasmion increased LVEDP (P<0.01) and prolonged QT and corrected QT intervals (P<0.01) despite the tachycardia. In controls the changes in cardiac loading conditions did not modify the QT interval, suggesting intact endogenous regulation of repolarization. The impaired adaptation observed in DCM contributed to an increase in QT interval following volume loading. In this model, a prolonged repolarization after abrupt volume loading may result from enhanced or disclosed mechanoelectric feedback.

Influence of sympathetic heart rate modulation on RT interval rate adaptation in conscious dogs.

The objective was to test if changes in autonomic tone still influenced the RT-RR relationship when full RT adaptation is completed, when heart rate is controlled, and when beat-to-beat variability is abolished by atrial pacing. Eight dogs (8-11 kg) were chronically instrumented with atrial pacing electrodes. Digital ECG (1,000 Hz, 12 bits) were recorded from healthy conscious dogs during spontaneous sinus rhythm and during atrial pacing. The protocol was repeated before and after atenolol (2 mg/kg), prazosin (0.5 mg/kg), or atenolol + prazosin. A vocal incitation was used as sympathetic stimulation. Beat-to-beat quantitative analysis of the RT interval (from QRS apex to end of T wave) was correlated with the preceding RR by linear regression. In spontaneous rhythm, atenolol increased RR (P < 0.001), RT (P < 0.001), and short-term heart rate variability (P < 0.01) and decreased RT-RR slopes (P < 0.001). Prazosin did not significantly modify any parameter. Sympathetic stimulation decreased RR (P < 0.001), RT (P < 0.05), and short-term heart rate variability (P < 0.01) and increased RT-RR slopes (P < 0.001). In atrial pacing, the RT-RR slopes were steeper during pacing than during spontaneous rhythm but were not modified by pharmacological manipulation of the autonomic nervous system. During sinus rhythm the RT-RR relationship is increased by sympathetic stimulation and decreased by beta-blockade. When heart rate modulation and the effects of the time delay in RT rate adaptation are abolished by atrial pacing, the influence of autonomic tone on RT rate adaptation disappears.

[The use of chronic epidural electrostimulation of the spinal cord in children with spastic diplegia--a type of infantile cerebral palsy]. / Primenenie khronicheskoi épidural'noi élektrostimuliatsii spinnogo mozga u detei s detskim tserebral'nym paralichom v forme spasticheskoi displegii.

The authors' first experience with spinal cord stimulation (SCS) at the lumbar level in the treatment of cerebral palsies (CP) in children aged 3-11 years who had lower spastic paraparesis of varying severity is presented. Complex clinical and physiological studies of all 9 children were made before and after surgery (follow-ups lasting 6-12 months). Preliminary results have indicated that the most significant changes occur primarily in pathological postural tonic reflexes. SCS has virtually no effect on fixed limb deformities, which requires further orthopedic surgical correction. A medical care schedule for CP children affected predominantly with lower spastic paraparesis should include, among its first priorities, electrode implantation for SCS. Due to the specific rehabilitative dynamics of motor functions, SCS should be made to treat these children before the deformities in the lower extremity joints get fixed.

Electrophysiological characterization of BRL-32872 in canine Purkinje fiber and ventricular muscle. Effect on early after-depolarizations and repolarization dispersion.

Amongst the different pharmacological approaches to the treatment of cardiac arrhythmias, compounds with multiple electrophysiological activities appear to exhibit a reduced adverse effect profile. BRL-32872 (N-(3,4-dimethoxyphenyl)-N-[3[[2-(3,4-dimethoxyphenyl) ethyl] propyl]-4-nitrobenzamide hydrochloride) is a typical example of an antiarrhythmic agent with combined K(+) and Ca(2+) blocking actions. In this study, we investigated the effects of BRL-32872 on early after-depolarizations and on dispersion of repolarization. Action potentials were recorded either in canine cardiac Purkinje fibers alone or in preparations containing both ventricular muscle and the attached Purkinje fibers. In Purkinje fibers, BRL-32872 (0. 3-10 microM) induced a bell-shaped concentration-dependent increase in action potential duration. At 90% of repolarization, the action potential was prolonged at concentrations up to 1 microM and was shortened when the concentration of BRL-32872 was further increased. In all 17 experiments, BRL-32872 did not cause early after-depolarizations in Purkinje fibers. On the contrary, BRL-32872 (3 microM) systematically suppressed early after-depolarizations induced by clofilium (4-chloro-N, N-diethyl-N-heptylbenzenebutanaminium tosylate, 1 microM), a selective inhibitor of the delayed rectifier K(+) current. A similar effect was observed once with 1 microM BRL-32872, a concentration able to prolong Purkinje fiber action potentials. Simultaneous recording of action potentials in ventricular and Purkinje preparations showed that increasing concentrations of BRL-32872 (0. 3-10 microM) induced a limited increase in the difference of repolarization time between the two tissues. The selective K(+) channel inhibitor E-4031 (N-(4-(1-[2-(6-methyl-2-pyridyl) ethyl]-4-piperidyl)-carbonyl] phenyl) methanesulfonamide dihydrochloride dihydrate) exhibited a significant concentration-dependent increase in dispersion of repolarization. We conclude from the present results that the Ca(2+) blocking activity of BRL-32872 (i) prevents the occurrence of early after-depolarizations associated with action potential prolongation and (ii) limits an excessive increase in action potential duration heterogeneity. These electrophysiological features might represent the basis for antiarrhythmic compounds with reduced proarrhythmic profile.

Identification and characterization of cvHsp. A novel human small stress protein selectively expressed in cardiovascular and insulin-sensitive tissues.

Starting with computational tools that search for tissue-selective expression of assembled expressed sequenced tags, we have identified by focusing on heart libraries a novel small stress protein of 170 amino acids that we named cvHsp. cvHsp was found as being computationally selectively and highly (0.3% of the total RNA) expressed in human heart. The cvHsp gene mapped to 1p36.23-p34.3 between markers D1S434 and D1S507. The expression of cvHsp was analyzed with RNA dot, Northern blots, or reverse transcription-polymerase chain reaction: expression was high in heart, medium in skeletal muscle, and low in aorta or adipose tissues. In the heart of rat models of cardiac pathologies, cvHsp mRNA expression was either unchanged (spontaneous hypertension), up-regulated (right ventricular hypertrophy induced by monocrotaline treatment), or down-regulated (left ventricular hypertrophy following aortic banding). In obese Zucker rats, cvHsp mRNA was increased in skeletal muscle, brown, and white adipose tissues but remained unchanged in the heart. Western blot analysis using antipeptide polyclonal antibodies revealed two specific bands at 23 and 25 kDa for cvHsp in human heart. cvHsp interacted in both yeast two-hybrid and immunoprecipitation experiments with alpha-filamin or actin-binding protein 280. Within cvHsp, amino acid residues 56-119 were shown to be important for its specific interaction with the C-terminal tail of alpha-filamin.

COOH-terminal truncated cardiac myosin-binding protein C mutants resulting from familial hypertrophic cardiomyopathy mutations exhibit altered expression and/or incorporation in fetal rat cardiomyocytes.

Mutations in human cardiac myosin-binding protein C (cMyBP-C) gene are associated with familial hypertrophic cardiomyopathy (FHC), and most of them are predicted to produce COOH-truncated proteins. To understand the molecular mechanism(s) by which such mutations cause FHC, we analyzed (i) the accumulation of human cMyBP-C mutants in fetal rat cardiomyocytes, and (ii) the protein sequence of the human wild-type (wt) cMyBP-C by hydrophobic cluster analysis with the aim of identifying new putative myosin-binding site(s). Accumulation and sarcomeric localization of the wt protein and of four FHC-mutant cMyBP-Cs (E542Q and three COOH-truncated proteins) were studied in cardiomyocytes by immunostaining and confocal microscopy after transfection with myc-tagged constructs. We found that: (i) 10 % of the cells expressing COOH-truncated mutants exhibit an incorporation into the A-band of the sarcomere without any alteration of the myofibrillar architecture versus 76 % of those expressing the wt or E542Q mutant cMyBP-Cs (p<0.001); (ii) 90 % of the cells expressing the truncated mutants show a diffuse localization of these proteins in the cardiomyocytes, out of which 45 % exhibit a significant alteration of the sarcomeric structure (p<0.0001 versus wt); and (iii) the two shortest mutant cMyBP-Cs accumulate at very low levels in fetal rat cardiomyocytes as compared to the wt (p<0.008). Protein sequence analysis indicated that a 45-residue sequence in the NH2-terminal C0 domain of cMyBP-C exhibits a consistent homology (sequence similarity score of 42 %) with a segment of the NH2-terminal domain of myomesin, another myosin-binding protein. This result suggests that the C0 domain of human cMyBP-C contains a novel putative myosin-binding site that could account for the A-band incorporation of the truncated mutants. In addition, the faint accumulation and the diffuse localization of truncated mutants could probably be explained by a low affinity of the C0 domain for myosin. We conclude that COOH-truncated cMyBP-Cs may act as poison polypeptides that disrupt the myofibrillar architecture and result in the defects observed in FHC.

Characterisation of Kv4.3 in HEK293 cells: comparison with the rat ventricular transient outward potassium current.

OBJECTIVE: The Shal (or Kv4) gene family has been proposed to be responsible for primary subunits of the transient outward potassium current (Ito). More precisely, Kv4.2 and Kv4.3 have been suggested to be the most likely molecular correlates for Ito in rat cells. The purpose of the present study was to compare the properties of the rat Kv4.3 gene product when expressed in a human cell line (HEK293 cells) with that of Ito recorded from rat ventricular cells. METHODS: The cDNA encoding the rat Kv4.3 potassium channel was cloned into the pHook2 mammalian expression vector and expressed into HEK293. Patch clamp experiments using the whole cell configuration were used to characterise the electrophysiological parameters of the current induced by Kv4.3 in comparison with the rat ventricular myocyte Ito current. RESULTS: The transfection of HEK293 cells with rat Kv4.3 resulted in the expression of a time- and voltage-dependent outward potassium current. The current activated for potentials positive to -40 mV and the steady-state inactivation curve had a midpoint of -47.4 +/- 0.3 mV and a slope of 5.9 +/- 0.2 mV. Rat ventricular Ito current was activated at potentials positive to -20 mV and inactivated with a half-inactivation potential and a Boltzmann factor of -29.1 +/- 0.7 mV and 4.5 +/- 0.5 mV, respectively. The time course of recovery from inactivation of rat Kv4.3 expressed in HEK293 cells and of Ito recorded from native rat ventricular cells were exponentials with time constants of 213.2 +/- 4.1 msec and 23. +/- 1.5 msec, respectively. Pharmacologically, Ito of rat myocytes showed a greater sensitivity to 4-aminopyridine than Kv4.3 since half-maximal effects were obtained with 1.54 +/- 0.13 mM and 0.14 +/- 0.02 mM on Kv4.3 and Ito, respectively. In both Kv4.3 and Ito, 4-aminopyridine appears to bind to the closed state of the channel. Finally, although a higher level of expression was observed in the atria compared to the ventricle, the distribution of the Kv4.3 gene across the ventricles appeared to be homogeneous. CONCLUSION: The results of the present study show that Kv4.3 channel may play a major role in the molecular structure of the rat cardiac Ito current. Furthermore, because the distribution of Kv4.3 across the ventricle is homogeneous, the blockade of this channel by specific drugs may not alter the normal heterogeneity of Ito current.

Cloning and characterization of a human electrogenic Na+-HCO-3 cotransporter isoform (hhNBC).

Our group recently cloned the electrogenic Na+-HCO-3 cotransporter (NBC) from salamander kidney and later from mammalian kidney. Here we report cloning an NBC isoform (hhNBC) from a human heart cDNA library. hhNBC is identical to human renal NBC (hkNBC), except for the amino terminus, where the first 85 amino acids in hhNBC replace the first 41 amino acids of hkNBC. About 50% of the amino acid residues in this unique amino terminus are charged, compared with approximately 22% for the corresponding 41 residues in hkNBC. Northern blot analysis, with the use of the unique 5' fragment of hhNBC as a probe, shows strong expression in pancreas and expression in heart and brain, although at much lower levels. In Xenopus oocytes expressing hhNBC, adding 1.5% CO2/10 mM HCO-3 hyperpolarizes the membrane and causes a rapid fall in intracellular pH (pHi), followed by a pHi recovery. Subsequent removal of Na+ causes a depolarization and a reduced rate of pHi recovery. Removal of Cl- from the bath does not affect the pHi recovery. The stilbene derivative DIDS (200 microM) greatly reduces the hyperpolarization caused by adding CO2/HCO-3. In oocytes expressing hkNBC, the effects of adding CO2/HCO-3 and then removing Na+ were similar to those observed in oocytes expressing hhNBC. We conclude that hhNBC is an electrogenic Na+-HCO-3 cotransporter and that hkNBC is also electrogenic.