Regional differences in action potential characteristics and membrane currents of guinea-pig left ventricular myocytes

Regional differences in action potential characteristics and membrane currents were investigated in subendocardial, midmyocardial and subepicardial myocytes isolated from the left ventricular free wall of guinea-pig hearts. Action potential duration (APD) was dependent on the region of origin of the myocytes (P < 0.01, ANOVA). Mean action potential duration at 90 % repolarization (APD90) was 237 +/- 8 ms in subendocardial (n = 30 myocytes), 251 +/- 7 ms in midmyocardial (n = 30) and 204 +/- 7 ms in subepicardial myocytes (n = 36). L-type calcium current (ICa) density and background potassium current (IK1) density were similar in the three regions studied. Delayed rectifier current (IK) was measured as deactivating tail current, elicited on repolarization back to -45 mV after 2 s step depolarizations to test potentials ranging from -10 to +80 mV. Mean IK density (after a step to +80 mV) was larger in subepicardial myocytes (1.59 +/- 0.16 pA pF-1, n = 16) than in either subendocardial (1.16 +/- 0.12 pA pF-1, n = 17) or midmyocardial (1. 13 +/- 0.11 pA pF-1, n = 21) myocytes (P < 0.05, ANOVA). The La3+-insensitive current (IKs) elicited on repolarization back to -45 mV after a 250 ms step depolarization to +60 mV was similar in the three regions studied. The La3+-sensitive tail current, (IKr) was greater in subepicardial (0.50 +/- 0.04 pA pF-1, n = 11) than in subendocardial (0.25 +/- 0.05 pA pF-1, n = 9) or in midmyocardial myocytes (0.38 +/- 0.05 pA pF-1, n = 11, P < 0.05, ANOVA). The contribution of a Na+ background current to regional differences in APD was assessed by application of 0.1 microM tetrodotoxin (TTX). TTX-induced shortening of APD90 was greater in subendocardial myocytes (35.7 +/- 7.1 %, n = 11) than in midmyocardial (15.7 +/- 3. 8 %, n = 10) and subepicardial (20.2 +/- 4.3 %, n = 11) myocytes (P < 0.05, ANOVA). Regional differences in action potential characteristics between subendocardial, midmyocardial, and subepicardial myocytes isolated from guinea-pig left ventricle are attributable, at least in part, to differences in IK and Na+-dependent currents.     

Human brain activity related to speed discrimination tasks

This study examined the ionic mechanism of ibutilide, a class III antiarrhythmic in clinical use, on freshly isolated human atrial cells. Cells had resting potentials of -71.4 +/- 2.4 mV, action potentials with overshoot of 36.8 +/- 1.8 mV, duration of 265 +/- 89 msec at 90% repolarization and slow repolarization (n = 16). Ibutilide, at 10(-7) M, markedly increased action potential duration. Four types of outward currents were detected: Ito, Iso, a delayed rectifier and IK1. Ibutilide had no inhibitory effect on these outward currents at 10(-7) M (n = 28). In K(+)-free solutions and -40 mV holding potential, mean peak inward current at 20 mV was -1478 +/- 103 pA (n = 12). Ibutilide increased this current to -2347 +/- 75 pA at 10(-7) M, with half maximal effect (Kd) of 0.1 to 0.9 nM between -10 and +40 mV (n = 21). At similar concentrations, the drug increased APD, with Kd of 0.7 and 0.23 nM at 70 and 90% repolarization, respectively (n = 8). Ibutilide shifted the mid-point of the steady-state inactivation curve from -21 to -12.2 mV (n = 6), and reduced current decline during repetitive depolarization (n = 5). The drug induced inward current was carried by Na+o through a nifedipine inhibited inward channel because Na+o removal eliminated the effect, and nifedipine abolished the inward current and the drug induced APD prolongation. We propose that a Na+ current through the L-type Ca++ channel mediates ibutilide's potent clinical class III antiarrhythmic action.     

Characterisation of xenobiotic-metabolising enzyme expression in human bronchial mucosa and peripheral lung tissues

We examined the effects of amiodarone (AMI) and desethylamiodarone (DAM) on whole-cell inward rectifying potassium current (IK1) in freshly isolated adult rabbit ventricular myocytes by using the whole-cell voltage-clamp technique, as an index of their effects on resting membrane resistance (Rm). Under control conditions, the current showed a strong inward rectification with a maximal inward current measured at -130 mV of -26.4 +/- 1.3 pA/pF and a maximal outward current measured at -50 mV of 3.5 +/- 0.3 pA/pF The current also exhibit a time-dependent activation, with a time constant of activation (tau(a)) that increased with depolarization. The maximal slope conductance normalized to cell capacitance was 0.509 +/- 0.019 nS/pE After exposure to both DAM (50 microM; n = 8) and AMI (50 microM; n = 7), rapid decrease in inward IK1 was observed. Block was restricted almost exclusively to the inward component. DAM caused a significant reduction of the maximal inward current (-20.0 +/- 2.0 pA/pF; p < 0.05), whereas AMI induced an even greater reduction of the same component (-14.1 +/- 1.2 pA/pF; p < 0.05 with respect to control and to DAM). The outward component of IK1 was not changed by either AMI or DAM (4.0 +/- 0.3 pA/pF and 3.4 +/- 0.4 pA/pF, respectively). AMI and DAM also decreased the maximal slope conductance significantly (0.297 +/- 0.019 nS/pF and 0.421 +/- 0.038 nS/pF, respectively). In addition, AMI but not DAM significantly increased the tau(a). However, the voltage dependence of the acceleration of tau(a) remained unchanged after both AMI and DAM exposure. These results allow us to conclude that AMI may induce a greater increase in the resting Rm than its main metabolite. This effect may counterbalance, at least in part, the conduction slowing due to its sodium channel-blocking properties.     

alpha 1-Adrenoceptor stimulation partially inhibits ATP-sensitive K+ current in guinea pig ventricular cells: attenuation of the action potential shortening induced by hypoxia and K+ channel openers

Effects of alpha 1-adrenoceptor stimulation on the action potential shortening produced by K+ channel openers (KCOs) or hypoxia and on the ATP-sensitive K+ current (IK.ATP) activated by KCOs were examined in guinea-pig ventricular cells by using conventional microelectrode and patch-clamp techniques. In papillary muscles, nicorandil (1 mM) or cromakalim (30 microM) markedly shortened the action potential duration (APD) (to 51 +/- 2% and 40 +/- 5% of each control value). Addition of 100 microM methoxamine, an alpha 1-adrenoceptor agonist, partially but significantly reversed the KCOs-induced APD shortening (to 69 +/- 3% and 50 +/- 4% of each control value). The APD-prolonging effect of methoxamine was antagonized by 1 microM prazosin (alpha 1-antagonist) and 100 nM WB4101 (alpha 1A-antagonist) but not by 10 microM chloroethylclonidine (alpha 1B-antagonist). In papillary muscles exposed to a hypoxic, glucose-free solution, APD declined gradually. In the presence of 100 microM methoxamine or 10 microM glibenclamide, the hypoxia-induced action potential shortening was significantly inhibited. In single ventricular myocytes, the KCOs increased a steady-state outward current that was abolished by glibenclamide (1 microM), thereby suggesting that these KCOs activate IK.ATP. Methoxamine (100 microM) significantly inhibited the nicorandil-induced IK.ATP by 18 +/- 5% and the cromakalim-induced IK.ATP by 16 +/- 2%. 4 beta-Phorbol 12-myristate 13-acetate (100 nM), a protein kinase C activator, failed to mimic the alpha 1-adrenoceptor-mediated inhibition of the nicorandil-induced outward current. Staurosporine (30 nM), a protein kinase C inhibitor, also failed to affect the partial inhibition of IK.ATP by methoxamine. Neither intracellular loading of heparin (100 micrograms/ml), an inositol 1,4,5-trisphosphate (IP3)-dependent Ca2+ release inhibitor, nor IP3 (20 microM) plus inositol 1,3,4,5-tetrakisphosphate (IP4 5 microM) could affect the inhibitory action of methoxamine. In conclusion, alpha 1A-adrenergic stimulation partially inhibits IK.ATP in cardiac cells. Neither protein kinase C activation nor IP3 formation appears to be involved in the partial inhibition of IK.ATP. The alpha 1A-adrenoceptor-mediated inhibition of IK.ATP may be deleterious for ischemic myocardium and partly offset the cardioprotective effect of KCOs because attenuation of action potential shortening may potentially increase Ca2+ influx in ischemic cells.     

The ABC maltose transporter

OBJECTIVE: To define the electrophysiologic mechanism(s) by which MCI-154, a putative Ca2+ sensitizer, produces a positive inotropic response without a positive chronotropic response, we examined effects of MCI-154 on the action potential of atrial preparations and the membrane currents of atrial myocytes. METHODS: The action potentias were recorded from left atrial and sinoatrial node preparations of guinea pigs by the use of standard microelectrode techniques. The whole-cell membrane currents were recorded from enzymatically-dissociated guinea pig atrial myocytes using conventional patch clamp techniques. RESULTS: In isolated left atria, MCI-154 increased the developed tension in a concentration-dependent manner. MCI-154 at concentrations of 10 and 100 microM increased the action potential duration (APD) in left atria stimulated at 0.5 Hz. In sinoatrial node preparations MCI-154 at a concentration of 100 microM produced a negative chronotropic response and prolonged APD. In single right atrial myocytes, MCI-154 at concentrations of 10 and 100 microM failed to increase the inward L-type Ca2+ current, but decreased the delayed rectifier K+ current (IK) in a concentration-dependent manner. MCI-154 decreased IK elicited by short depolarizing pulses more markedly than that induced by long depolarizing pulses. In addition, MCI-154 produced only a little inhibition of IK in the presence of E-4031, a specific blocker of rapidly activating component of IK (IKr). CONCLUSIONS: MCI-154 preferentially blocks IKr and the inhibitory action on IKr may be partly involved in the negative chronotropic and positive inotropic responses in atrial preparations.     

Effects of hypertrophy on regional action potential characteristics in the rat left ventricle: a cellular basis for T-wave inversion?

BACKGROUND: In cardiac hypertrophy, ECG T-wave changes imply an abnormal sequence of ventricular repolarization. We investigated the hypothesis that this is due to changes in the normal regional differences in action potential duration. We assessed the contribution of potassium- and calcium-dependent currents to these differences. Both the altered sequence of ventricular repolarization and the underlying cellular mechanisms may contribute to the increased incidence of ventricular arrhythmias in hypertrophy. METHODS AND RESULTS: Rats received daily isoproterenol injections for 7 days. Myocytes were isolated from basal subendocardial (endo), basal midmyocardial (mid), and apical subepicardial (epi) regions of the left ventricular free wall. Action potentials were stimulated with patch pipettes at 37 degrees C. The ratio of heart weight to body weight and mean cell capacitance are increased by 22% and 18%, respectively, in hypertrophy compared with controls (P<.001). Normal regional differences in action potential duration at 25% repolarization (APD25) are reduced in hypertrophy (control: endo, 11.4+/-0.9 ms; mid, 8.2+/-0.9 ms; epi, 5.1+/-0.4 ms; hypertrophy: endo, 11.6+/-0.9 ms; mid, 10.4+/-0.8 ms; epi, 7.8+/-0.6 ms). The regional differences in APD25 are still present in 3 mmol/L 4-aminopyridine. Hypertrophy affects APD75 differently, depending on the region of origin of myocytes (ANOVA P<.05). APD75 is shortened in subendocardial myocytes but is prolonged in subepicardial myocytes (control: endo, 126+/-7 ms; epi, 96+/-10 ms; hypertrophy: endo, 91+/-6 ms; epi, 108+/-7 ms). These changes in APD75 are altered by intracellular calcium buffering. CONCLUSIONS: Normal regional differences in APD and the changes observed in hypertrophy are only partially explained by differences in I(tol). In hypertrophy, the normal endocardial/epicardial gradient in APD75 appears to be reversed. This may explain the T-wave inversion observed and will have implications for arrhythmogenesis.     

Estimation of outward currents in isolated human atrial myocytes using inactivation time course analysis

The aim was to investigate outward currents in single, isolated, human, atrial myocytes and to determine the relative contribution of individual current components to the total outward current. Currents were recorded using the whole-cell patch-clamp technique at 36-37 degreesC. Individual outward current components were estimated from recordings of total outward current using a mathematical procedure based on the inactivation time course of the respective currents. This method allows estimation of outward currents without the use of drugs or conditioning voltage-clamp protocols to suppress individual current components. A rapidly activating and partially inactivating total outward current was recorded when myocytes were voltage clamped at potentials positive to -20 mV (peak current density 24. 0+/-0.97 pA/pF at +40 mV; n=107 cells, 33 patients). This total outward current comprised three overlapping currents: a rapidly inactivating, transient, outward current (Ito1) a slowly and partially inactivating current (ultrarapid delayed rectifier, IKur) and a third current component which most probably reflects a non selective cation current (not characterized). The average current densities at +40 mV were 8.92+/-0.44 pA/pF for Ito1 and 15.1+/-0.72 pA/pF for IKur (n=107 cells). Recovery from inactivation was bi-exponential for both currents and was faster for Ito1. A slowly activating delayed rectifier current (IK) was not found. The current densities of peak Ito1 and IKur varied strongly between individual myocytes, even in those from the same patient. The ratio IKur/Ito1 was 0.5-6.9 with a mean of 1.98+/-0.11 (n=107 cells), suggesting that IKur is the main repolarizing current. The amplitudes of the total outward current, Ito1 and IKur, and the ratio of the latter two were independent of patient age (16-87 years).     

Comparison of the rate-dependent properties of the class III antiarrhythmic agents azimilide (NE-10064) and E-4031: considerations on the mechanism of reverse rate-dependent action potential prolongation

INTRODUCTION: Reverse rate-dependence, a lessening in Class III antiarrhythmic agent action potential duration (APD) prolongation as heart rate is increased, has been proposed to be related to an incomplete deactivation of the slow component (IKs) of the delayed rectifier K+ current (IK). The rate-dependent properties of block of IK by azimilide were compared to E-4031, which selectively blocks the rapid component (IKr) of IK, in guinea pig ventricular muscle. METHODS AND RESULTS: Azimilide prolonged APD in isolated papillary muscles in a concentration-dependent manner and to a greater degree than E-4031. Both agents prolonged APD less at fast than slow rates, consistent with a similar reverse rate-dependent effect. Isolation of azimilide block of IKs by subtraction of APD during E-4031 plus azimilide from E-4031 alone revealed rate-independent prolongation of APD. In voltage clamp experiments on single ventricular myocytes, activation of IKs was similar following 30 seconds of conditioning pulses of physiological duration (125 to 200 msec) with either a fast (cycle length 250 msec) or slow (cycle length 2000 msec) rate. The block of IKs by azimilide 3 microM was greater after a fast conditioning pulse train. CONCLUSIONS: Selective block of IKs prolongs APD in a rate-independent manner. In voltage clamped myocytes, no evidence of a rate-dependent accumulation of IKs was observed. These findings support a mechanism of reverse rate-dependent APD prolongation by Class III antiarrhythmic agents that block IKr independent of IKs.     

Differential class III and glibenclamide effects on action potential duration in guinea-pig papillary muscle during normoxia and hypoxia/ischaemia

1. Microelectrode recording techniques were used to study the effects of several potassium channel blockers which are considered to be Class III antiarrhythmic compounds. The effects of (+)-sotalol, UK-66,914, UK-68,798 and E-4031 on action potential duration (APD) were determined in guinea-pig isolated papillary muscles. The compounds were evaluated under normoxic or hypoxic/ischaemic conditions at 36.5 degrees C and compared to glibenclamide, which is considered to be a blocker of ATP-dependent potassium channels. Prolongation of action potential duration at 90% repolarization (APD90) was taken as an indirect measure of potassium channel blockade. 2. Under normoxic conditions, the Class III compounds prolonged APD in a concentration-dependent manner. According to EC15 values, the order of potency of the Class III compounds was found to be UK-68,798 > E-4031 > UK-66,914 > (+)-sotalol. Glibenclamide did not significantly prolong APD90 under normoxic conditions. 3. Perfusion with an experimental hypoxic or ischaemic bathing solution produced qualitatively similar effects on action potentials. Over a period of 20-25 min in either of the experimental solutions, there was a small decrease in action potential amplitude (APA) and a prominent shortening of APD. The ischaemic solution also depolarized the resting membrane potential by about 15 mV. 4. (+)-Sotalol and UK-66,914 did not reverse the shortening of APD induced by perfusion with hypoxic Krebs solution. High concentrations of glibenclamide (10 microM) and UK-68,798 (30 and 60 microM) partially reversed the hypoxia-shortened APD. Glibenclamide was more potent and exhibited a greater time-dependent action than UK-68,798. 5. During experimental ischaemia, the Class III compound E-4031 (10 microM, n = 7) produced small, but significant, increases in the APD90 (11 +/-3 ms after 20 min) which were not clearly time-dependent(14 +/- 4 ms after 30 min). UK-68,798 (10 microM) also produced a small, but insignificant, increase in APD90(12 =/-6 ms at 20 min, n = 4). Higher concentrations of UK-68,798 (30 and 60 microM, n = 4) did not produce a consistently significant increase in APD90 during ischaemia: significance was only attained after 20 min in the presence of 60 microM UK-68,798 (24 +/- 12 ms). However, in marked contrast to the effects of the Class III compounds, glibenclamide (10 microM) produced large time-dependent increases in ischaemic APD90 (34 +/- 11 ms at 7 min, n = 9) which were significant 15 min or more after drug addition(52 +/- 12 ms at 20 min, n = 7; 74 +/- 5 ms at 30 min, n = 6).6. The present microelectrode data suggest that blockers of ATP-dependent potassium channels, such as glibenclamide, might prove to be more effective than Class III compounds against ischaemia-induced shortening of cardiac action potentials.     

Ion channel and exchange currents in single myocytes isolated from the rabbit atrioventricular node

OBJECTIVE: To review findings from the authors' laboratory in studies of electrophysiological properties of single rod- and spindle-shaped myocytes from rabbit atrioventricular node (AVN). DESIGN: Single cells were isolated from the AVN of the rabbit heart with the use of enzymatic and mechanical dispersion. For recording, cells were superfused with a Tyrode's solution at 33 to 37 degrees C, and recordings were made with microelectrodes or patch pipettes under 'current' or voltage' clamp conditions. Results are expressed as mean +/- SEM. RESULTS: AVN cells had a mean membrane capacitance of 40 +/- 3.9 pF and membrane resistance of 565 +/- 167 M omega (n = 9). Spontaneously active cells exhibited pacemaker activity showing a clear diastolic depolarization and overshooting action potential (AP) with a relatively slow upstroke velocity (7.4 +/- 0.9 V/s, n = 6) and a maximum diastolic potential of -70.5 +/- 2.9 mV. Under voltage clamp conditions, depolarizing pulses from 40 mV elicited L-type calcium currents sensitive to inhibition by nifedipine and managanese or cadmium ions, which could also block spontaneous APs. Depolarizing pulses also activated delayed rectifier potassium current (IK). IK showed rapid activation, and IK 'tails' in AVN cells were blocked by 5 microM E4031, consistent with the rapidly activating subtype of IK (IKr). IK was similar in AVN and ventricular myocytes, except for the time-course of deactivation, which was faster in AVN cells. In 80% to 90% of cells, hyperpolarizing voltage steps activated a small time-independent current. Ten per cent to 20% of cells showed the hyperpolarization-activated current (I(f)), but I(f) amplitude was only significant at potentials more negative than the pacemaker potential. AVN cells showed an apparent absence of inwardly rectifying potassium current. CONCLUSIONS: The high membrane resistance of AVN cells suggests that only small changes in ionic currents could significantly affect membrane potential. L-type calcium current is important in generating the AP upstroke, and IKr may play a role in both AP repolarization and diastolic depolarization. The ionic basis underlying spontaneous activity is not yet clear, but in some cells I(f) is not required because cells without I(f) can generate spontaneous APs.     

Inhibition by taurine of the inwardly rectifying K+ current in guinea pig ventricular cardiomyocytes

Effects of taurine on the inwardly rectifying K+ current (IK1) in isolated guinea pig ventricular cardiomyocytes were examined using patch voltage-clamp methods. All experiments were performed at 36 degrees C. Taurine (10-20 mM) increased the action potential duration, but failed to affect the resting potential. Holding potential was maintained at -30 mV. The current was activated with an inwardly going rectification, and was completely blocked by Ba2+ (2 mM). Taurine inhibited IK1 at - 120 mV by 28.3+/-1.1% (n=6, P < 0.05) at 10 mM and by 36.0+/-2.1% (n=6, P < 0.01) at 20 mM. The reversal potential was shifted in the hyperpolarizing direction by 3.7+/-0.6 mV (n=6) at 20 mM. In inside-out patch-clamp experiments, the amplitude of unitary channels was -2.7+/-0.3 pA (n=21) at -90 mV. Symmetrical high-K+ (150 mM) solutions in both bath and pipette were used. The channel conductance was 32+/-2 pS (n=9). Taurine did not affect channel conductance, but markedly decreased the open probability at - 120 mV of channel by 21.5+/-2.4% (n=8, P < 0.01) at 10 mM, and by 56.7+/-3.8% (n=8, P < 0.001) at 20 mM. These responses were almost reversible. These results suggest that taurine directly modulates the open probability of the inwardly rectifying K+ current, resulting in regulation of the functions of heart cells.     

Monensin-induced reversal of positive force-frequency relationship in cardiac muscle: role of intracellular sodium in rest-dependent potentiation of contraction

We have investigated the role of a rest-dependent inotropic factor in determining species-related differences in cardiac force-frequency relationships (FFR). Isolated rat, rabbit or guinea-pig papillary muscles, as well as guinea-pig ventricular myocytes were superfused with 1.8 mM Ca2+ Tyrode. In rat muscles, isometric force amplitude decreased, while in rabbit or guinea-pig muscles force increased with frequency (0.02-1 Hz). Paired-pulse pacing potentiated contraction markedly at all frequencies in rabbit muscles, but not at low frequencies in rat muscles. We tested the hypothesis that high intracellular Na+ levels (Nai) are responsible for negative FFR. The ionophore monensin increased Nai, reversed the FFR of rabbit and guinea-pig muscles from positive to negative, by increasing force mostly at low frequencies, and decreased the paired-pulse potentiation of contraction at low frequencies. Monensin added during rest also reversed rest-induced decay. In isolated myocytes, monensin had qualitatively similar effects on cell shortening as well as on Cai transients. Monensin also decreased the action potential duration (APD) but did not change the pattern of its variation with frequency. Cells intracellularly dialyzed with 20 mM Na+ via a patch pipette also showed rest potentiation of the Cai transients, in contrast to cells dialyzed with 10 mM Na+, which showed rest decay of the transients. APD was also shorter in myocytes dialyzed with 20 mM Na+ than in those dialyzed with lower Na+. The results indicate that in the presence of high Nai, sarcoplasmic reticular Ca2+ load is increased during diastole, possibly via reverse-mode Na+/Ca2+ exchange, and therefore that Nai is an important factor determining the FFR. In addition, the data suggest that short APDs in preparations showing negative FFR may be partly a consequence of increased Nai.     

A micromechanical model of lung tissue rheology

We evaluated the acute effects of ibuprofen and salicylic acid on cAMP-mediated Cl- secretion (Isc) in both colonic and airway epithelia. In T84 cells, ibuprofen inhibited the forskolin-dependent Isc in a concentration-dependent manner, having an apparent Ki of 142 microM. Salicylic acid inhibited Isc with an apparent Ki of 646 microM. We determined whether ibuprofen would also inhibit the forskolin-stimulated Isc in primary cultures of mouse trachea epithelia (MTE) and human bronchial epithelia (HBE). Similar to our results in T84 cells, ibuprofen (500 microM) inhibited the forskolin-induced Isc in MTEs and HBEs by 59+/-4% (n = 11) and 39+/-6% (n = 8), respectively. Nystatin was employed to selectively permeabilize the basolateral or apical membrane to determine the effect of ibuprofen on apical Cl- (ICl) and basolateral K+ (IK) currents after stimulation by forskolin. After forskolin stimulation, ibuprofen (500 microM) reduced both the ICl and IK; reducing ICl and IK by 60 and 15%, respectively. To determine whether this inhibition of ICl was due to the inhibition of CFTR, the effects of ibuprofen and salicylic acid on CFTR Cl- channels in excised, inside-out patches from L-cells were evaluated. Ibuprofen (300 microM) reduced CFTR Cl- current by 60+/-16% and this was explained by a short-lived block (approximately 1.2 ms) which causes an apparent reduction in single channel amplitude from 1.07+/-0.04 pA to 0.59+/-0.04 pA (n = 3). Similarly, salicylic acid (3 mM) reduced CFTR Cl- current by 50+/-8% with an apparent reduction in single channel amplitude from 1.08+/-0.03 pA to 0.48+/-0.06 pA (n = 4). Based on these results, we conclude that the NSAIDs ibuprofen and salicylic acid inhibit cAMP-mediated Cl- secretion in human colonic and airway epithelia via a direct inhibition of CFTR Cl- channels as well as basolateral membrane K+ channels. This may reduce their efficacy in conjunction with other therapeutic strategies designed to increase CFTR expression and/or function in secretory epithelia.     

Influence of postnatal changes in action potential duration on Na-Ca exchange in rabbit ventricular myocytes

Cardiac Na-Ca exchanger (NCX) expression and current density are significantly greater in newborn rabbit hearts compared with adults. However, the relatively short action potential (AP) at birth may limit the impact of increased NCX expression by diminishing Ca2+ entry via Na-Ca exchange current (INaCa). To address the interdependence of AP duration and NCX activity, we voltage-clamped newborn (NB, 1-5 day), juvenile (JV, 10-14 day) and adult (AD) rabbit myocytes with a series of APs of progressively increasing duration (APD90: 108-378 ms) under nominally chloride-free conditions. In each age group we quantified an increase in outward (QExout) and inward (QExin) Ni2+-sensitive charge movement in response to AP prolongation. QExout and QExin measured during age-appropriate APs declined postnatally [QEXout: NB (2 day) 0.19 +/- 0.02, JV (10 day) 0.10 +/- 0.01, AD 0.04 +/- 0.002; QEXin: NB -0. 2 +/- 0.01, JV -0.11 +/- 0.02; AD -0.04 +/- 0.003 pC/pF] despite the significantly shorter APD90 of newborn myocytes (NB 122 +/- 10; AD 268 +/- 22 ms). When Ca2+ fluxes by other transport pathways were blocked with nifedipine, ryanodine and thapsigargin, age-appropriate APs elicited contractions in NB and JV but not AD myocytes (NB 4.8 +/- 0.5, JV 1.2 +/- 0.3% resting length). These data demonstrate that a shorter AP does not negate the impact of increased NCX expression at birth.     

Consensus Conference on Platelet Transfusion, Royal College of Physicians of Edinburgh, 27-28 November 1997. Synopsis of background papers

The effects of 1-(2-amino-4-methanesulfonamidophenoxy)-2-[N-(3,4-dimethoxypheneth yl)-N-methylamino] ethane hydrochloride (KCB-328), in comparison with those of dofetilide, were studied on the action potentials (APs) of isolated guinea pig papillary muscles. KCB-328 (0.003-3 microM) concentration-dependently prolonged the AP duration at 90% repolarization (APD90) at 1- and 3-Hz pacing, and the concentration-response relations at 1 and 3 Hz resemble each other. Dofetilide (0.001-1 microM) also produced the concentration-dependent prolongation of APD90 but more pronouncedly at 1 than at 3 Hz, demonstrating the reverse frequency-dependent effect. KCB-328 at 0.03, 0.1, 0.3, and 1 microM increased APD90 by 11 +/- 1, 19 +/- 1, 25 +/- 1, and 29 +/- 1% at 3 Hz and by 9 +/- 1, 19 +/- 2, 27 +/- 2, and 33 +/- 2% at 1 Hz, respectively. Prolongation of the effective refractory period (ERP) by each drug is parallel to those of APD90 at each pacing frequency. KCB-328 modified neither the maximal velocity of depolarization, amplitude of AP, and resting membrane potential in the fast APs, nor any parameters of the slow APs. In a separate experiment, the effects of KCB-328 on the ERP of contractile response (ERPc) of excised guinea-pig papillary muscles also were studied at 1 and 3 Hz. KCB-328 (0.01-10 microM) lengthened the ERPc in a concentration-dependent and frequency-independent manner as in the electrophysiologic results. This frequency-independent ERPc prolongation by KCB-328 was not influenced by increased extracellular K+ concentration from 4 to 10 mM. These results suggest that KCB-328 might be a selective class III agent with effects that are relatively frequency independent.     

Effects of antiarrhythmic agents and Mg2+ on aconitine-induced arrhythmias

The effects of antiarrhythmic agents, including Classes I and IV and 3-10 mM Mg2+ on aconitine-induced arrhythmias were examined using a conventional microelectrode and patch clamp method in Langendorff-perfused rabbit hearts and isolated guinea-pig ventricular myocytes. Intracoronary application of 0.1 microM aconitine induced polymorphic ventricular tachycardia (PVT) which continued for more than 60 minutes. Application of aconitine to ventricular myocytes caused a prolonged action potential duration (APD) and the appearance of early afterdepolarization (EAD) together with the occurrence of an inward hump of the I-V curve around -60 to -40 mV and increased outward current at positive voltages. Application of 10 microM TTX and 5 mM or higher Mg2+ restored aconitine-induced PVT to sinus rhythm in Langendorff-perfused preparations and also shortened the prolonged APD, demonstrating the abolishment of EAD by aconitine in ventricular myocytes. However, antiarrhythmic agents did not exert such effects. In conclusion, the antiarrhythmic actions of Mg2+ and TTX in aconitine-induced arrhythmia are to abolish EAD and shorten the prolonged APD by suppression of the inward Na+ current around -60 to -40 mV.     

Modulation of atrial repolarization by site of pacing in the isolated rabbit heart

BACKGROUND: Single-site or multisite atrial pacing may reduce the incidence of atrial fibrillation in humans. The therapeutic mechanisms may include synchronization of atrial repolarization (repolarization "memory") and/or decreased dispersion of atrial repolarization. These responses have not been well documented in intact atria. METHODS AND RESULTS: Monophasic action potential recordings were made from six atrial epicardial sites in 39 isolated perfused rabbit heart preparations during 3 hours of continuous right atrial, left atrial, or biatrial pacing. Action potential recordings obtained at times 0, 45, 90, 135, and 180 minutes were computer analyzed for activation time (AT) and 90% action potential duration (APD) at each site. No consistent relationship could be demonstrated between APD and AT at any time during atrial pacing (all P > .05). On average, left atrial APDs were longer than right atrial APDs by up to 6.3 ms at all times, regardless of the site of pacing (P < or = .05). At all times, dispersion of atrial repolarization was minimized by left atrial pacing compared with right atrial pacing (21.6 +/- 9.1 versus 32.4 +/- 15.1 ms, respectively, at time 0; P < .05). Biatrial pacing provided no further reduction in dispersion of repolarization compared with left atrial pacing (all P > .05). CONCLUSIONS: No relationship can be demonstrated between atrial AT and APD in the isolated rabbit heart preparation. This differs from ventricular repolarization "memory," which is demonstrable under the same conditions. Left atrial APD is, on average, longer than right atrial APD, suggesting spatial heterogeneity in repolarization. Dispersion of atrial repolarization is minimized by left atrial pacing in this preparation with no further advantage to biatrial pacing.     

Accentuated antagonism in canine subendocardium is not altered by chronic exercise

Acetylcholine often affects cardiac action potential repolarization only during augmented adrenergic tone, i.e., the phenomenon of accentuated antagonism. Since chronic exercise involves repeated changes in autonomic outflow, we determined whether it also influenced adrenergic/cholinergic interactions in isolated canine cardiac tissue. Using standard micro-electrode techniques in thin ventricular subendocardial slices isolated from exercised (EX: 8-10 wk daily exercise) and sedentary (SED): 8-10 wk cage rest) dogs, we examined transmembrane potential responses to isoproterenol (ISO: 10(-8), 10(-7), 10(-6) M) and to ISO in the presence of ACH (10(-5) M). Control transmembrane characteristics at BCL = 500 ms were similar for EX (N = 8 dogs) and SED (N = 9 dogs). ISO (10(-6) M) decreased action potential duration at 50% repolarization (APD50): EX = -29 +/- 15 ms; SED = 11 ms and at 90% repolarization (APD90): EX = -37 +/- 17 ms; and SED = -24 +/- 14 ms (P > 0.05, EX vs SED). ACH alone did not alter APD. With ACH (10(-5) M), delta APD50 with ISO (10(-6) M) was -5 +/- ms and 0 +/- 5 ms for EX and SED, respectively; delta APD90 was -8 +/- 4 ms and -8 +/- 7 ms for EX and SED, respectively (P > 0.05, EX vs SED). Thus, ACH antagonized ISO-mediated acceleration of repolarization equally in both groups. Chronic daily exercise does not influence adrenergic/cholinergic interactions at the cellular level.     

Differential effects of cytochalasin D and 2,3 butanedione monoxime on isometric twitch force and transmembrane action potential in isolated ventricular muscle: implications for optical measurements of cardiac repolarization

INTRODUCTION: 2,3-Butanedione monoxime (BDM) has been widely used to inhibit contraction during optical recordings of cardiac membrane voltage changes, even though it markedly abbreviates cardiac action potentials. METHODS AND RESULTS: We compared the effects of BDM and of the F-actin disrupter cytochalasin D (cyto D) on isometric twitch force and transmembrane action potentials in isolated canine right ventricular trabeculae superfused with Tyrode's solution (2 mmol/L CaCl2, 37 degrees C) and stimulated at 0.5 Hz. BDM at 10 mmol/L and cyto D at 80 micromol/L were equally effective in reducing peak isometric force to 10%+/-3% (n = 6; mean+/-SEM) and 8%+/-1% (n = 8), respectively. Neither agent significantly altered resting tension. While 10 mmol/L BDM markedly shortened the action potential duration at 90% repolarization (APD90) from 198+/-7 msec to 146+/-9 msec (P < 0.001), 80 micromol/L cyto D had no significant effects on APD90 or on any other action potential parameter. The effects of BDM on peak isometric force and APD were completely reversible after 15 minutes of washout, whereas in the cyto D group contractile force continued to be reduced (13%+/-3%) and action potential characteristics did not show significant changes from control values after a 60-minute period of superfusion with cyto D-free Tyrode's solution. CONCLUSION: We conclude that cyto D should be considered an alternative excitation-contraction uncoupler for optical mapping studies of cardiac repolarization.     

Antagonist effects of seglitide (MK 678) at somatostatin receptors in guinea-pig isolated right atria

Somatostatin (SS) exerts a negative inotropic effect in isolated atria. Here we report that in guinea-pig isolated right atria, seglitide, a potent cyclic hexapeptide somatostatin agonist, behaves as a competitive somatostatin receptor antagonist with pA2 values against SS14, SS25 and SS28, of 6.50 +/- 0.40, 6.24 +/- 0.08 and 6.09 +/- 0.06, respectively. Seglitide had little or no effect on the negative inotropic action of carbachol or N6-cyclohexyladenosine. Our findings indicate that the receptor-response coupling characteristics of guinea-pig atria are such that in this preparation seglitide has low intrinsic activity and behaves specifically as a somatostatin receptor antagonist.