Induction of a novel cation current in cardiac ventricular myocytes by flufenamic acid and related drugs.

BACKGROUND AND PURPOSE: Interest in non-selective cation channels has increased recently following the discovery of transient receptor potential (TRP) proteins, which constitute many of these channels. EXPERIMENTAL APPROACH: We used the whole-cell patch-clamp technique on isolated ventricular myocytes to investigate the effect of flufenamic acid (FFA) and related drugs on membrane ion currents. KEY RESULTS: With voltage-dependent and other ion channels inhibited, cells that were exposed to FFA, N-(p-amylcinnamoyl)anthranilic acid (ACA), ONO-RS-082 or niflumic acid (NFA) responded with an increase in currents. The induced current reversed at +38 mV, was unaffected by lowering extracellular Cl(-) concentration or by the removal of extracellular Ca(2+) and Mg(2+), and its inward but not outward component was suppressed in Na(+)-free extracellular conditions. The current was suppressed by Gd(3+) but was resistant to 2-aminoethoxydiphenyl borate (2-APB) and to amiloride. It could not be induced by the structurally related non-fenamate anti-inflammatory drug diclofenac, nor by the phospholipase-A(2) inhibitors bromoenol lactone and bromophenacyl bromide. Muscarinic or alpha-adrenoceptor activation or application of diacylglycerol failed to induce or modulate the current. CONCLUSIONS AND IMPLICATIONS: Flufenamic acid and related drugs activate a novel channel conductance, where Na(+) is likely to be the major charge carrier. The identity of the channel remains unclear, but it is unlikely to be due to Ca(2+)-activated (e.g. TRPM4/5), Mg(2+)-sensitive (e.g. TRPM7) or divalent cation-selective TRPs.

Changes of beta2-adrenergic stimulation induced by hyperosmosis in human atrium.

PURPOSE: The purpose of the present study was to determine whether extracellular osmotic pressure modulates beta2-adrenergic stimulation of the contraction force and L-type Ca2+ current in human atrial myocytes. MATERIAL AND METHODS: Experiments were performed on human atrial trabeculae and myocytes isolated from the right atrium. The concentration dependent effect of salbutamol (SAL), a beta2-adrenoreceptor agonist, on peak tension (P) and L-type calcium current (ICaL) under isoosmolar (345 mOsm) and hyperosmolar (405 or 525 mOsm was achieved by adding of mannitol) conditions was studied. RESULTS: Salbutamol (10 nmol/L-10 micromol/L) added to the control solution increased P by 180.6 +/- 45.8% over control with a half-stimulation constant EC50 = 27 +/- 6 nmol/L. Under isoosmolar conditions SAL (0.1/10(3)nmol/L) increased ICaL by 182.3 +/- 19.8% over control with an EC50 2.9 +/- 0.9 nmol/L. In hyperosmolar solutions the same concentrations of SAL increased P and ICaL by 57.2 +/- 12.6% and 217.2 +/- 70.5% over control with EC50 = 640 +/- 260 nmol/L and 12 +/- 5 nmol/L respectively. CONCLUSIONS: These results indicated that hyperosmolarity reduced the effect of beta2-adrenergic stimulation, i.e. the dose-response curve of salbutamol on L-type calcium current was shifted to the higher concentration range and maximal increase in contraction force was diminished in human atrial cells.

Inhibition of G protein-coupled and ATP-sensitive potassium currents by 2-methyl-3-(3,5-diiodo-4-carboxymethoxybenzyl)benzofuran (KB130015), an amiodarone derivative.

2-Methyl-3- (3,5-diiodo-4-carboxymethoxybenzyl) benzofuran (KB130015; KB), a novel compound derived from amiodarone, has been proposed to have antiarrhythmic properties. Its effect on the G protein-coupled inward rectifying K+ current [IK(ACh) or IK(Ado)], ATP-sensitive K+ current [IK(ATP)], and background inward rectifying current (I(K1)) were studied in guinea pig atrial and ventricular myocytes by the whole-cell voltage-clamp technique. Receptor-activated IK(ACh/Ado), induced in atrial myocytes by the stimulation of either muscarinic or Ado receptors was concentration dependently (IC50 value of approximately 0.6-0.8 microM) inhibited by KB. Receptor-independent guanosine 5'-O-(3-thio)triphosphate-induced and background IK(ACh), which contributes to the resting conductance of atrial myocytes, were equally sensitive to KB (IC50 value of approximately 0.9 microM). IK(ATP) induced in atrial myocytes during metabolic inhibition with 2,4-dinitrophenol (DNP) was also suppressed by KB, whereas IK1 measured in ventricular myocytes was insensitive to the drug (KB < or =50 microM). Although being effective when applied from the outside, intracellular application of KB via the patch pipette affected neither IK(ACh) nor IK(ATP). 3,3',5-triodo-L-thyronin, which shares structural groups with KB, did not have an effect on the K+ currents. Consistent with the effects on single myocytes, KB did not depolarize the resting potential but antagonized the shortening of action potential duration by carbamylcholine-chloride or by DNP in multicellular preparations and antagonized the shortening of action potential duration by acetylcholine in single myocytes. It is concluded that KB inhibits IK(ACh) and IK(ATP) by direct drug-channel interaction at a site more easily accessible from extracellular side of the membrane.

Action potential changes associated with a slowed inactivation of cardiac voltage-gated sodium channels by KB130015.

1. We have studied the acute cardiac electrophysiological effects of KB130015 (KB), a drug structurally related to amiodarone. Membrane currents and action potentials were measured at room temperature or at 37 degrees C during whole-cell patch-clamp recording in ventricular myocytes. Action potentials were also measured at 37 degrees C in multicellular ventricular preparations. 2. The effects of KB were compared with those of anemone toxin II (ATX-II). Both KB and ATX-II slowed the inactivation of the voltage-gated Na(+) current (I(Na)). While KB shifted the steady-state voltage-dependent inactivation to more negative potentials, ATX-II shifted it to more positive potentials. In addition, while inactivation proceeded to completion with KB, a noninactivating current was induced by ATX-II. 3. KB had no effect on I(K1) but decreased I(Ca-L) The drug also did not change I(to) in mouse myocytes. 4. The action potential duration (APD) in pig myocytes or multicellular preparations was not prolonged but often shortened by KB, while marked APD prolongation was obtained with ATX-II. Short APDs in mouse were markedly prolonged by KB, which frequently induced early afterdepolarizations. 5. A computer simulation confirmed that long action potentials with high plateau are relatively less sensitive to a mere slowing of I(Na) inactivation, not associated with a persisting, noninactivating current. In contrast, simulated short action potentials with marked phase-1 repolarization were markedly modified by slowing I(Na) inactivation. 6 It is suggested that a prolongation of short action potentials by drugs or mutations that only slow I(Na) inactivation does not necessarily imply identical changes in other species or in different myocardial regions.

Slowing of the inactivation of cardiac voltage-dependent sodium channels by the amiodarone derivative 2-methyl-3-(3,5-diiodo-4-carboxymethoxybenzyl)benzofuran (KB130015).

-Methyl-3-(3,5-diiodo-4-carboxymethoxybenzyl)benzofuran (KB130015 or KB) is a new drug, structurally related to amiodarone and to thyroid hormones. Its effects on cardiac voltage-dependent Na+ current (I Na) were studied in pig single ventricular myocytes at 22 degrees C using the whole-cell (with [Na+]i = [Na+]o = 10 mM) and cell-attached patch-clamp techniques. KB markedly slowed I Na inactivation, due to the development of a slow-inactivating component (tau slow approximately equal 50 ms) at the expense of the normal, fast-inactivating component (tau fast approximately equal 2-3 ms). The effect was concentration-dependent, with a half-maximally effective concentration (K0.5) of 2.1 micro M. KB also slowed the recovery from inactivation and shifted the voltage-dependent inactivation (DeltaV(0.5) = -15 mV; K0.5 > or = 6.9 micro M) and activation to more negative potentials. Intracellular cell dialysis with 10 micro M KB had marginal or no effect on inactivation and did not prevent the effect of extracellularly applied drug. In cell-attached patches, extracellular KB prolonged Na+ channel opening. Amiodarone (10 micro M) and 10 micro M 3,5,-diiodo-L-thyropropionic acid had no effect on inactivation and did not prevent KB effects. 3,3',5-Triodo-L-thyronine (T3) also had no effect on inactivation, but at 10 micro M it increased I Na amplitude and partially prevented the slowing of inactivation by KB. These data suggest the existence of a binding site for KB and T3 on Na+ channels.

Suppression of transient outward potassium currents in mouse ventricular myocytes by imidazole antimycotics and by glybenclamide.

The whole-cell patch-clamp technique was used in adult mouse ventricular myocytes at 22 degrees C to study the transient outward current (I(to)) and its sensitivity to the antimycotics miconazole and clotrimazole, as well as to glybenclamide. I(to) elicited by depolarizing steps from a holding potential of -80 mV consisted of a fast inactivating component and a slowly inactivating component. In the presence of miconazole (IC50 of approximately 8 microM) or clotrimazole, I(to) peak amplitude was reduced and its inactivation accelerated, due to a selective suppression of the slow component, without an effect on the fast component or on the noninactivating current. The effect did not reverse upon washout, was not induced by intracellular drug application, and occurred without a change of the steady-state inactivation. In the presence of glybenclamide I(to) peak amplitude was reduced and its inactivation accelerated. In contrast to the antimycotics, glybenclamide suppressed both the fast and the slow components (IC50 of approximately 50 microM), its effect was reversible, and was associated with a negative shift of the steady-state inactivation. These data demonstrate a pharmacological separation of I(to) components using antimycotic drugs but not glybenclamide.

Modulation of the extracellular divalent cation-inhibited non-selective conductance in cardiac cells by metabolic inhibition and by oxidants.

The effect of metabolic inhibition and oxidative stress on the monovalent cation-permeable, extracellular divalent cation-inhibited non-selective conductance was investigated in ventricular myocytes at 22 degrees C. Under whole-cell voltage-clamp, with L-type Ca2+ channels blocked by nifedipine, and K+ currents blocked by Cs+ substitution for K+, removal of Ca2+(o)and Mg2+(o) induced a non-selective current (I(NS-(Ca)o)) in mouse, rabbit and rat cells. Removal of glucose increased I(NS-(Ca)o)in the absence of Ca2+(o) and Mg2+(o), but failed to induce this current in the presence of the divalent cations. Further inhibition of glycolysis by 2-deoxyglucose (DOG; 10 mM, in zero glucose) or of mitochondrial function by rotenone (10 microM) or NaCN (5 mM) also failed to induce I(NS-(Ca)o)in the presence of Ca2+(o) and Mg2+(o). Even when given together, DOG and rotenone did not induce I(NS-(Ca)o) in the presence of divalent cations. Preactivated I(NS-(Ca)o) was increased by the oxidants thimerosal (50 microM), diamide (500 microM) and pCMPS (50 microM). However, none of these drugs nor NEM (1 mM) did elicit I(NS-(Ca)o)in the presence of Ca2+(o) and Mg2+(o). Exposure of rat myocytes to Ag+ induced a current resembling I(NS-(Ca)o) (reversing at -5 mV; blocked by 100 microM Gd3+) even in the presence of divalent cations. The data indicate that metabolic inhibition only regulates activated I(NS-(Ca)o)but does not induce the opening of closed channels, and that small oxidants like Ag+ may induce I(NS-(Ca)o) activation by accessing at sites unavailable for larger molecules.

Influence of action potential duration and resting potential on effects of quinidine, lidocaine and ethmozin on Vmax in guinea-pig papillary muscles.

The effects of class I antiarrhythmic drugs (quinidine, lidocaine, ethmozin) on the maximum upstroke velocity (Vmax) of the action potential (AP) of guinea-pig papillary muscles were investigated in the presence and absence of nifedipine and a potassium-free solution. Nifedipine (10 microM) decreased AP duration from 208 +/- 13 ms to 148 +/- 17 ms (P less than 0.05, n = 4), but did not influence resting potential and Vmax. Removal of K+ from the perfusate increase the membrane potential from -86 +/- 4 mV to -103 +/- 7 mV (P less than 0.05, n = 4). Quinidine (20 microM) lidocaine (40 microM) and ethmozin (2 microM) decreased Vmax. Nifedipine and K(+)-free solution elevated Vmax when depressed by lidocaine and ethmozin. At 2 Hz rate of stimulation, Vmax increased from 69.5 +/- 12.0% to 76.0 +/- 10.6% and 98.5 +/- 3.2% (P less than 0.05, n = 7) for lidocaine and from 33.9 +/- 13.9% to 41.3 +/- 14.4% and 75.3 +/- 10.3% (P less than 0.05, n = 6) for ethmozin, compared to the control, when nifedipine or K(+)-solution was used, respectively. Nifedipine induced a slight decrease and potassium-free solution, a slight increase of Vmax in the case with quinidine from 49.9 +/- 11.8% to 48.8 +/- 7.2% and 52.7 +/- 6.7 (P greater than 0.05, n = 7), respectively. The time constant of recovery (tau r) from use-dependent block of Vmax decreased in K(+)-free solution containing lidocaine and ethmozin, but not quinidine. The guarded receptor hypothesis was used to stimulate the effects of these drugs. Our estimates of the drug affinities for activated, inactivated, and rested channels were: for quinidine 1.28 x 10(5), 1.15 x 10(4), 1.0 x 10(3); for lidocaine 1.7 x 10(4), 1.88 x 10(5) 2.5 x 10(1); and for ethmozin 1.5 x 10(6), 3.0 x 10(6), 1.5 x 10(4), respectively. The results suggest that the role of AP duration on lidocaine and ethmozin effectiveness is reduced when resting potential decreases and that removal of K+ from a perfusate containing. quinidine had a small effect on Vmax, despite a marked increase in AP duration and resting potential.

Dependence of Ca outflow and depression of frog myocardium contraction on ryodipine concentration.

The effect of ryodipine on calcium outflow from tissues, on contraction force, the duration of action potentials and the relaxation phase time-constant in the contraction cycles of myocardial strips was studied using frog heart preparations. It was found that calcium outflow (delta Ca) as a function on ryodipine concentration can be represented as: (formula; see text) A linear correlation exists between Ca2+, contraction blocking and the shortening of the action potential in the presence of various ryodipine concentrations. Ryodipine (10(-5) mol/l) decreased the relaxation time-constant by about 20% as compared to controls. It was concluded that calcium outflow from myocardial tissues in response to ryodipine is due to blockade of calcium entry into the cells and their output through the Na+--Ca2+ exchange system. Frog heart myocardial contractions are essentially under the control of calcium entry through sarcolemmal calcium channels.

Effect of slow calcium channel blockers on the electromechanical activity of frog myocardium in the presence of epinephrine.

The calcium channels blockers fenihidine (3.5 X 10(-5) mol/l), ryosidine (10(-5) mol/l), D-600 (10(-5) mol/l) and Mn ions (2 X 10(-3) mol/l or 5 X 10(-3) mol/l) block contraction force and shorten the duration of action potentials of the frog myocardial ventricle strand under normal conditions. When contraction force and the duration of action potentials were restored by epinephrine (10(-5) mol/l), these agents were unable to suppress these parameters. The increase in both contraction force and the duration of action potentials induced by epinephrine were blocked by acetylcholine. Recording by voltage clamp of inward calcium current (Ica) of the frog atrial trabeculae it was found that fenihidine decreases Ica activated by epinephrine to a smaller extent than observed at normal conditions. Let as assume that epinephrine increases Ica by means of increasing number of calcium channels so these data support the proposed existence of as many as two calcium channel fractions in frog myocardium, which differ in the sensitivity to calcium channels blockers.

Effect of ryodipine on electromechanical parameters of heart and vessels, cAMP phosphodiesterase activity and swelling-contraction cycle of mitochondria.

2,6-Dimethyl-3,5-dimethoxycarbonyl-4-(o-difluoromethoxyphenyl)-1,4 -dihydropyridine (ryodipine, PP-1466), an effective Ca2+ channel blocker, diminishes contraction force and decreases duration of action potential in the frog heart ventricle strips. Dissociation constants K0.5 are 2 x 10(-7), 5 x 10(-7), and 10(-6) mol/l for PP-1466, nifedipine and nicardipine, respectively (at 0.25-0.3 Hz stimulation). One molecule of PP-1466 or nifedipine apparently interacts with two receptors on the channel (n = 0.5), nicardipine with one receptor (n = 1). The binding energy of PP-1466 and nifedipine increases at closed and diminishes at open channels which is in contrast to nicardipine, whose effect is irreversible. Thus, the site of nicardipine action differs from that of PP-1466 and nifedipine. PP-1466 (10(-8) mol/l--10(-6) mol/l) suppresses contraction force and diminishes frequency of spontaneous contractions of the rabbit atria, and also displays antagonism to the effect of Ca2+ upon rabbit auricle contractions. In the isolated rabbit aorta and portal vein PP-1466 is more antagonistic to contractions caused by Ca2+ than by epinephrine. Both competitive and non-competitive types of antagonism can be distinguished.(ABSTRACT TRUNCATED AT 250 WORDS)