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We developed an assay to detect wild-type CFTR in respiratory epithelial cells with the objective to evaluate the efficacy of DNA delivery during in vivo gene transfer. The method is based on the previous observation that the common delta F508-CFTR mutant does not reach the apical membrane as does the transgene product. We thus used a monoclonal antibody, MATG 1031, raised against the first extracellular loop sequence of the CFTR protein and an immunodetection protocol lacking premature fixation or permeabilization. Specificity of MATG 1031 for its epitope was controlled by immunoblotting. In HT29-19A, 184, CAPAN-1 human cell lines, and in respiratory primary cultures, staining with MATG 1031, examined by confocal scanning laser microscopy, appeared as small dots restricted to the apical surface. No such staining was observed in NIH-3T3 fibroblasts, in the cystic fibrosis cell line CFPAC-1 or in primary cultures from cystic fibrosis patients. Apical immunostaining with MATG 1031 was restored in CFPAC-1 cells cultured at a low temperature (30 degrees C) and in CFPAC-1 cells transfected with wild-type CFTR Recombinant CFTR was also recognized in CF respiratory cells lipotransfected with wild-type CFTR plasmid DNA MATG 1031 immunostaining was further investigated under blinded conditions in primary cultures derived from nasal curettage. In all the cell cultures examined, our protocol allowed discrimination between non-CF and CF cells. We propose that this method is convenient to detect apical CFTR and may be used to monitor in vivo gene transfer.

By making use of immunoblotting and immunocytochemical analysis, we explored whether stimulation of adenosine A1 receptors would promote the activation of delta-protein kinase C (delta-PKC) immunolabeled with a polyclonal antibody. Immunoblot analysis of Triton X-100-soluble cell membrane and cytosolic fractions revealed the presence of a specific 75-kD band reactive to the delta-PKC polyclonal antibody. In freshly isolated rat cardiac myocytes, 28% of the total immunoreactive delta-PKC was associated with the membrane fraction, whereas 72% was associated with the soluble fraction. Under stimulation with the tumor-promoting phorbol 12-myristate 13-acetate (PMA, 50 nmol/L) used as a positive control, delta-PKC translocated to the cell membrane, with the membrane fraction representing 88% and the cytosolic fraction representing 12% of the total immunoreactive delta-PKC. Transverse optical sections performed with confocal laser microscopy showed that immunostaining with anti-delta-PKC antibody was distributed in the cytosol membrane under PMA stimulation. In the membrane fraction of cells pretreated with adenosine (100 mumol/L) or with the adenosine A1 agonist (--)-N6-(2-phenylisopropyl)-adenosine (R-PIA, 1 mumol/L), the 75-kD band corresponding to delta-PKC increased by 57% and 66%, respectively, when compared with nonstimulated cells processed under the same experimental conditions. In cells exposed to either of the purine agonists, specific fluorescence staining decorated the cell membrane, a pattern that was not observed in control cells. Activation of membrane delta-PKC produced either by adenosine itself or by its analogue R-PIA was fully antagonized by the specific A1 antagonist 8-cyclopentyl-1,3-dipropylxanthine (1 mumol/L). From these data, we conclude that adenosine A1 stimulation activates delta-PKC in freshly isolated rat ventricular myocytes.

Preconditioning is a temporary tolerance to ischaemia acquired by the myocardium after a short period of ischaemia. It results in the limitation of the infarct size induced by prolonged coronary occlusion. The mechanism of this cytoprotection remains poorly understood. The A1 adenosine receptors, the ATP-sensitive potassium channels and protein-kinase C seem to play prominent roles. The effects of preconditioning on the complications of ischaemia/reperfusion such as myocardial stunning, ventricular arrhythmias or decreased coronary reserve are not well known. Several studies suggest that the cytoprotection resulting from preconditioning could be applied to human myocardium and constitute a preventive anti-ischaemic therapy during coronary angioplasty, cardiac surgery or the conservation of transplant grafts.

The elementary K+ conductance activated by the cAMP or the Ca2+ second messenger pathways was investigated in the model salt-secreting epithelium, the human T84 cell line. Under Cl(-)-free conditions, an inwardly rectifying whole-cell K+ current was evoked by either forskolin 10 (mumol/l) or acetylcholine 1 (mumol/l) and blocked by extracellular charybdotoxin 10 (nmol/l). In the cell-attached mode, both secretory agonists induced the opening of a channel showing inward rectification with a unitary chord conductance of 36.8 +/- 2.5 pS (n = 26) for inward currents. In inside-out patches, a 35-pS inwardly rectifying K+ channel that corresponded to the channel recorded in the cell-attached configuration was recorded in the presence of 0.3 mumol/l free Ca2+ at the inner side of the membrane. This channel was blocked by Ba2+ (5 mumol/l) and by charybdotoxin (50 nmol/l). Its open probability was enhanced by intracellular Ca2+ with and EC50 of 0.25 mumol/l and strongly reduced by intracellular MgATP with an IC50 of 600 mumol/l. In the continuous presence of ATP, the channel activity was consistently increased by 125 kU/l catalytic subunit of cAMP-dependent protein kinase. In the cystic fibrosis pancreatic duct cell line CFPAC-1, a K+ channel was also recorded, with similar characteristics and regulation as the 35-pS channel in T84 cells. We conclude that an ATP-sensitive K+ channel regulated by intracellular Ca2+ and phosphorylation supports the main K+ current activated by secretory agonists in normal cystic fibrosis cell lines.(ABSTRACT TRUNCATED AT 250 WORDS)

Phenotypical expression of cystic fibrosis (CF) includes decreased epithelial chloride secretion and increased sodium absorption. These anomalies produce increased water absorption and a dehydrated mucus responsible for decreased mucociliary clearance. Identification of the gene responsible for the genetic disease (CFTR for cystic fibrosis transmembrane conductance regulator) together with a more accurate comprehension of complexes interactions that exist between the CFTR gene product and other epithelial ionic channels has created novel opportunities for discovering specific pharmacological drugs to treat the disease. Amiloride, which limits sodium hyperabsorption, has demonstrated both efficacy and safety in vivo in a restricted number of adult patients. Nucleotides such as ATP or UTP, prescribed in association with amiloride, increase chloride secretion. Potassium channel openers, by stimulating transepithelial chloride transport, may represent an additional innovative approach. Specific pharmacology to CF is not competitive but rather complementary to gene therapy.

The cystic fibrosis gene product, CFTR, is a Cl- channel that possesses specific binding sites for cytosolic ATP and is activated by cAMP-dependent protein kinase. Most recently, it was reported that CFTR localizes at the surface apical compartment of normal airway epithelial cells, but accumulates in the cytosol of airway cells from CF patients with the delta F508 mutation. In order to explore whether the same difference exists in normal and CF established cell lines that are commonly used in physiological and pharmacological investigations of the CF defect, we employed monoclonal antibodies raised against synthetic peptides corresponding to two different regions of the CFTR protein. One antibody (MATG 1061) was generated against amino acids 503-515 delta 508 in the nucleotide binding domain 1, whereas the other (MATG 1031) was generated against amino acids 107-117 situated in a putative external loop. We used confocal laser scanning microscopy to localize the CFTR protein in T84 (a colonic derived carcinoma), CAPAN-1 (a pancreatic carcinoma), and in CFPAC-1 (a pancreatic carcinoma homozygous for the delta F508 deletion) cell lines. In permeabilized T84 and CAPAN-1 cells, immunolabeling with MATG 1061 predominated at the apical domain. By contrast, CFTR staining with MATG 1061 was homogeneously distributed in the cytoplasm of CFPAC-1 cells. In non-permeabilized non-CF cell lines, MATG 1031 specifically labeled an apical membrane surface epitope. No such labeling was present in CFPAC-1 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

The cystic fibrosis gene product (CFTR) is a Cl- channel that possesses specific binding sites for cytosolic adenosine triphosphate (ATP) and is activated by cyclic adenosine monophosphate (cAMP)-dependent protein kinases. We explored the possibility that CFTR shares a common pharmacology with another ATP-regulated channel protein, the ATP-sensitive K+ channel that is blocked by sulphonylureas and activated by diazoxide. cAMP-stimulated Cl- effluxes were measured with 36Cl- in the epithelial cell line T84 which stably expresses CFTR. Neither glibenclamide (30 microM), tolbutamide (1 mM) nor diazoxide (100 microM) significantly affected forskolin-activated 36Cl- effluxes in T84 cells. In patch-clamp experiments, glibenclamide exerted only weak inhibitory effects on the whole-cell currents through CFTR with an IC50 of around 0.1 mM. Tolbutamide at 1 mM, but not at 0.1 mM, blocked a current of small amplitude which reversed near the equilibrium potential for K+ ions. We conclude that sulphonylureas and diazoxide are not effective antagonists of endogenous CFTR Cl- channels.

Antiarrhythmic drugs, especially the Class I family, exert a negative inotropic effect on the myocardium which is particularly undesirable in patients with depressed left ventricular function. Therefore, research has been directed to the development of new, more specific molecules of the Class III family. The authors studies the mechanical effects of RP 62719 on guinea pig left ventricular papillary muscle. This new molecule is a pure Class III antiarrhythmic, known to lengthen the duration of the cardiac action potential by selectively blocking the potassium current iK1 (inward rectifier K+ current). The mechanical parameters were determined during the phases of contraction and relaxation under isotonic and isometric conditions. At 0.2 and 2 microM concentrations, RP 62719 improved cardiac contraction under both isotonic and isometric conditions with an increase of about 30% of Vmax (p < 0.001), the maximum unloaded shortening velocity delta 1 (p < 0.001), the peak isometric active force normalized per cross-sectional area [AF/S (p < 0.001)]. At these two concentrations, a positive lusitropic effect (improved relaxation) was demonstrated by an increase in negative peak of derivative per mm2-dF/s and maximum lengthening velocity VR max (p < 0.01). At higher concentrations (20 microM), the inotropic and lusitropic effects were less marked with a bell-shaped form of the dose-effect curve. This study indicates that RP 62719 has moderate but significant positive inotropic and lusitropic effects. These actions could provide significant therapeutic advantages especially in patients cardiac failure.

The effects of 1-500 microM riluzole, a novel psychotropic agent, were studied on the nodal K current of isolated nerve fibres of the frog. When added to the external solution, the substance rapidly and reversibly inhibited slow, fast 1 and fast 2 K components of the tail K current. The concentrations of riluzole inducing half maximum reduction of slow, fast 1 and fast 2 K conductances were 413 microM, 24 microM and 21 microM respectively. It is concluded that the substance is about 20 times more effective in blocking fast than slow K channels.

Normally-polarized tissue from the human atrial myocardium usually exhibits a diastolic depolarization phase which can be suppressed reversibly by Cs+ or enhanced by inhibiting the inward rectifier K+ current, iK1, with Ba2+. (Escande et al., 1986). Because the suppression of the diastolic slope by Cs+ leads to a hyperpolarization of the cell membrane at the end of the diastolic phase, it was suggested that Cs+ might inhibit an inward current responsible for diastolic depolarization. Among the ionic mechanisms underlying the diastolic depolarization phase of cardiac tissues, the hyperpolarization-activated inward current, if, fits well to explain the small diastolic slope of human atrial fibres. In other preparations, this inward current carried both by Na+ and K+ ions is rapidly deactivated during the action potential and entirely blocked by millimolar concentrations of Cs+ (DiFrancesco 1981; DiFrancesco, et al., 1986; Kokubun et al., 1982; Callewaert et al., 1984; Denyer and Brown, 1990). Such a current in human myocardial cells has not been characterized so far although its existence in human atrial trabeculae was previously reported in an abstract (Carmeliet, 1984). In the present study, we describe an inward current which activates upon hyperpolarization in patch-clamped single human atrial cells and shares similar characteristics with the if pacemaker current described in unicellular and intact preparations of mammalian cardiac tissues.

The present article presents an overview of the pharmacologic profile of the benzopyran derivative RP 58866, a racemic mixture, and of RP 62719 (terikalant), its active enantiomer. In normal cardiac tissues studied in vitro, both drugs dose-dependently prolonged the atrial and ventricular action potential but affected neither the upstroke of the action potential nor the diastolic potential. Patch-clamp experiments demonstrated that the prolongation of the action potential induced by the drugs is due to a specific blockade of the inward rectifier K+ current. In vivo, intravenous administration to anesthetized dogs of low doses of RP 62719 consistently induced bradycardia and prolonged the atrial, nodal, and ventricular refractory periods, but did not affect the conduction velocity. Because of these properties, RP 58866 and RP 62719 exert potent antiarrhythmic and antifibrillatory actions both at the atrial and ventricular levels in various experimental models of arrhythmia. Our results demonstrate that RP 58866 and RP 62719 are K(+)-channel blockers acting as pure class III antiarrhythmic drugs.

The effects of 0.15-250 microM riluzole, a novel psychotropic agent with anticonvulsant properties, were studied on voltage-clamped nodes of Ranvier of isolated nerve fibres of the frog. When added to the external solution, the drug rapidly and reversibly inhibited both K and Na currents with an apparent dissociation constant of 0.09 mM. The riluzole-induced decrease of these currents was not "use-dependent". At concentrations up to 100 microM, the drug had no noticeable effect on the time course of Na current inactivation nor on the shape and the position along voltage axis of the Na conductance/voltage relationship. On the other hand, it induced substantial shifts towards negative voltages of the steady-state Na inactivation/voltage curve. From these results, according to the modulated-receptor model, an apparent dissociation constant of 0.29 microM could be calculated for riluzole-induced blockage of inactivated Na channels. The recovery from Na current inactivation was also affected by the drug. It is concluded that riluzole is a highly specific blocker of inactivated Na channels, which is more than 300 times more effective on these channels than on K or resting Na channels.

We investigated the contribution of opening of the ATP-sensitive K+ channel to extracellular accumulation of K+ during ischemia with the use of glibenclamide, a specific blocker of this K+ channel. To characterize the electrophysiological effects of glibenclamide during metabolic inhibition (by either application of dinitrophenol or hypoxia) we performed patch-clamp studies in isolated membrane patches of guinea pig myocytes and in intact guinea pig myocytes and studied action potential parameters in isolated superfused guinea pig papillary muscle. We studied the effect of glibenclamide on extracellular accumulation of K+ and H+ in isolated retrogradely perfused globally ischemic hearts of rat, guinea pig, and rabbit. Experimental evidence is presented that supports the conclusions that glibenclamide 1) effectively blocks open K+ATP channels, 2) reverses the dinitrophenol-induced increase of the outward current and prevents the hypoxia-induced shortening of the action potential, 3) decreases the rate of K+ accumulation during the first minutes of ischemia in stimulated hearts, an effect which was entirely absent in quiescent hearts, and 4) does not influence the rate and extent of ischemia-induced extracellular acidification.

In a previous article (Escande et al. 1988a), we have shown that cromakalim (BRL 34915), a potassium channel opener (PCO), is a potent activator of ATP-sensitive K+ channels in cardiac cells. In the present article, the influence on K+ channels of two other potassium channel openers chemically unrelated to cromakalim, RP 49356 and pinacidil, has been investigated in patch-clamped isolated cardiac myocytes. In the whole-cell configuration, K+ currents were recorded in the presence of 50 microM TTX and 3 microM nitrendipine or 3 mM cobalt. Like cromakalim, RP 49356 or pinacidil activated a time-independent outward current at 33-35 degrees C but not at 19-21 degrees C, which showed little voltage-dependency in the potential range -60 to +60 mV. Its amplitude was a function of the agonist concentration, e.g. it was 2.1 +/- 0.4 nA at +60 mV with 30 microM RP 49356 and 4.3 +/- 0.8 nA with 300 microM. In control conditions, glibenclamide, a blocker of K+-ATP channels in pancreatic and heart cells, affected neither the inward rectifier, iK1, nor the delayed K+ current, iK. At 3 microM, glibenclamide fully prevented the effects of 300 microM RP 49356 or pinacidil. At lower concentrations, glibenclamide partially counteracted the activation by PCOs of a K+ current. In the cell-attached configuration, externally applied RP 49356 or pinacidil caused opening of large channels which reversed around O mV in a high K+ external medium. In inside-out patches, both RP 49356 or pinacidil activated K+-ATP channels by increasing the time period for which the channels remained in the open state. It is concluded that, like cromakalim, RP 49356 and pinacidil are potent activators of K+-ATP channels in cardiac myocytes.

Foci of abnormal automaticity are the result of either 1) an abnormal pacemaker discharging spontaneously in the absence of an initiating stimulation usually arising in the midst of depolarised myocardial fibres but also from zones of physiological subsidiary automaticity normally inhibited during sinus rhythm, or 2) automatic activity triggered by an initiating stimulus related to oscillating after-depolarisation or early after-depolarisation. Oscillating after-depolarisation, the clinical expression of which was thought initially to be limited to digitalis toxicity, can arise in any experimental situation associated with calcium overload of the cell. Early after-depolarisation which often occurs during the terminal phase of the action potential of the Purkinje fibres, arises under different experimental conditions to those giving rise to oscillating after-depolarisation. Cellular electrophysiological techniques demonstrate the mechanism responsible for abnormal automaticity and the sites of action of antiarrhythmic drugs. Close collaboration between cellular electrophysiologists and clinicians is necessary to establish the responsibility of mechanisms demonstrated in vitro in the genesis of clinical arrhythmias.

In cardiac myocytes, cromakalim (BRL 34915), a potassium channel opener, activates a time-independent K+ current exhibiting poor voltage-sensitivity. This effect of cromakalim is antagonized by low concentrations of glibenclamide, a specific blocker of ATP-dependent K+ channels in cardiac cells. Direct recording of the activity of K+ channels in inside-out membrane patches, confirmed that cromakalim is a potent activator of ATP-dependent K+ channels in cardiac myocytes.

The intracellular electrophysiological properties of a new antiarrhythmic agent, penticainide (5 x 10(-6) to 5 x 10(-5) M) were studied in isolated driven human right atrial appendage and papillary muscle superfused with oxygenated Tyrode's solution. In atrial fibers, penticainide decreased the amplitude, maximum rate of rise (dV/dtmax), plateau amplitude, and duration (APD) of action potentials (AP). In ventricular fibers, the main AP modification induced by penticainide was a dV/dtmax diminution. All those effects were frequency and concentration dependent. Penticainide decreased resting potential at 5 x 10(-5) M only. Ventricular APD variations were relatively weak: in most of the cases, 5 x 10(-6) M decreased APD and 5 x 10(-5) M shortened long APD (greater than 300 ms) and lengthened short APD (less than 300 ms). The class I antiarrhythmic property (dV/dtmax decrease) of penticainide was rate dependent in both human fibers and was obtained at lower drug concentrations than those used in other species. The relatively rapid rate of onset and the rather slow recovery kinetics of dV/dtmax block suggest a common mechanism of action of penticainide on sodium channels in human heart and others mammals.

The capability of generating spontaneous diastolic depolarization and automaticity was investigated in vitro by means of standard microelectrode techniques in 50 human atrial preparations. Samples were classified within two groups: group 1 was composed of 12 well-polarized preparations exhibiting action potentials that were fast responses (mean maximum diastolic potential: -75.5 mV and Vmax greater than 100 V/s); group 2 was composed of 38 partially-depolarized samples (mean maximum diastolic potential: -50.3 mV and Vmax less than 10 V/s) and was further divided into two subgroups. Subgroup 2A consisted of 20 spontaneously beating preparations and subgroup 2B consisted of 18 non-automatic partially-depolarized specimens. Highly-polarized fibers from group 1, although exhibiting a slight diastolic depolarization which was almost entirely suppressed by 2 mM caesium, never presented spontaneous activity under our experimental conditions. 90% of automatic fibers from subgroup 2A were sampled from dilated atria. In automatic preparations, diastolic depolarization was usually separated into two phases: an initial phase, also present in non-automatic fibers, and a late phase. Changes in the initial phase were not accompanied by concomitant changes in the spontaneous rate. Abnormal automaticity was clearly related to the late diastolic phase (absent in non-automatic fibers), the generation of which appeared to be a specific property of automatic fibers. The use of K conductance inhibitors (caesium, 4-aminopyridine, barium, low K solutions) provided indirect evidence that neither delayed outward ix current nor if type inward current are principally responsible for abnormal automaticity.

Among other characteristics, the steady-state current-voltage relationship of patch-clamped single atrial myocytes from guinea-pig hearts is defined by an outward current hump in the potential region -15 to +40 mV. This hump was reversibly suppressed by Co2+ (3 mM) or nitrendipine (5 microM) and enhanced by Bay K 8644 (5 microM). The maintained outward current component suppressed by Co2+ extended between -15.2 +/- 1.9 mV and +39.5 +/- 1.7 mV (mean +/- SEM of 14 cells) and has an amplitude of 95.7 +/- 9.4 pA at +10 mV. In isochronal I-V curves, the hump was already visible at 400 ms with essentially the same amplitude as at 1500 ms. The Co2+-sensitive outward current underlying the hump was poorly time-dependent during 1.5 s voltage pulses but slowly relaxed upon repolarization. Tail currents reversed near the K+ equilibrium potential under our experimental conditions. The current hump of the steady-state I-V curve was also abolished by caffeine (10 mM) or ryanodine (3 microM), both drugs that interfere with sarcoplasmic reticulum function. Apamin (1 microM) or quinine (100 microM) but not TEA (5-50 mM) markedly reduced its amplitude. However, at similar concentrations as required to inhibit the hump, both apamin and quinine appeared to be poorly specific for Ca2+-activated K+ currents in heart cells since they also inhibited the L-Type Ca2+ current. It is concluded that a long lasting Ca2+-activated outward current, probably mainly carried by K+ ions but not sensitive to TEA, exists in atrial myocytes which is responsible for the current hump of the background I-V curve.

Little information is available about rate dependent changes in electrical activity of human myocardial cells. We therefore studied, in vitro, the electrical activity of adult human atrial fibres driven at frequencies near that of atrial flutter by means of the standard microelectrode technique. Thirty two atrial samples exhibiting "normal" responses with fast upstroke were selected. At very high frequencies, the action potential (AP) upstroke arose from the repolarisation phase of the preceding AP in spite of marked frequency induced shortening of the plateau. As the stimulation rate was progressively increased, the take off potential (TOP) was less and less negative and the maximal rate of depolarisation (Vmax) decreased. Moreover, in most preparations, a clear alternation between two types of action potentials occurred. Calcium channel inhibitors cobalt (5 mM) or diltiazem (5 x 10(-6) M) shortened AP duration, increased Vmax and markedly reduced alternation. Sodium channel inhibitors, tetrodotoxin (7.5 10(-6) M) or lignocaine (10(-5) M) shortened AP duration and induced a transient increase in Vmax. Ouabain (10(-6) M) prolonged AP duration, decreased Vmax, enhanced alternation and finally suppressed the 1:1 capture of the atrial tissue. Our results show that, at high driving rates corresponding to the frequencies of atrial flutter, slight variations in action potential duration induced by drugs are associated with marked concomitant variations in Vmax and probably with consequent modifications of the conduction velocity.

35 human atrial samples, presenting electrophysiological characteristics within "normal" limits, were studied in vitro. 19 were sampled from adult patients and 16 were sampled from very young patients. Maximum diastolic potential did not significantly differ between adult and young, either in mean value or in variability. Action potential duration increased with age, particularly during the earliest years of life. The variability of action potential duration, which is considered as an arrhythmogenic factor, is greater in the adult.