Activation of recombinant h 5-HT1B and h 5-HT1D receptors stably expressed in C6 glioma cells produces increases in Ca2+-dependent K+ current

The putative coupling between stably expressed recombinant h 5-HT_1B or h 5-HT_1D receptors and K⁺ channels which regulate excitability was investigated in C6 glioma cells. Outward K⁺ currents (I _K) were examined in non-transfected C6 glioma cells and in cells expressing cloned h 5-HT_1B or h 5-HT_1D receptors using the patch-clamp technique in the whole-cell configuration. I _K was elicited by a depolarizing step from a holding potential of –60 mV. In C6 glioma cells expressing either recombinant h 5-HT_1B or h 5-HT_1D receptors, sumatriptan similarly increased I _K in a concentration-dependent manner (maximum increase 19.4±7.2%, n=8, P<0.05 and 25.1±3.9%, n=6, P<0.001, respectively) with EC₅₀ values (geometric mean with 95% confidence intervals in parentheses) of 56.3 nM (7.9–140 nM) and 68.7 nM (16–120 nM), respectively. Sumatriptan failed to elicit increases in I _K in non-transfected cells, confirming a specific involvement of the respective membrane h 5-HT_1B and h 5-HT_1D receptors in transfected C6 cells. In the presence of the mixed 5-HT_1B/D receptor antagonist GR 127935 (0.1 μM), sumatriptan (1 μM) failed to significantly increase I _K in C6 cells expressing h 5-HT_1B receptors (–7.5±3.5%, P=NS, n=6), although a higher concentration of GR 127935 (1 μM) was required to significantly inhibit sumatriptan-evoked increases in I _K in C6 cells expressing h 5-HT_1D receptors (–1.8±3.5%, P=NS, n=6), confirming that sumatriptan-evoked responses were indeed mediated by h 5-HT_1B and h 5-HT_1D receptors, respectively. In C6 cells expressing either cloned h 5-HT_1B or h 5-HT_1D receptors, sumatriptan-induced increases in I _K were prevented by the calcium chelator EGTA (5 mM) when included in the patch pipette (maximum increase 0.57±0.6%, n=3, P=NS and –2.8±1.6%, n=5, P=NS, respectively). In C6 cells expressing cloned h 5-HT_1B receptors, sumatriptan (1 μM) similarly failed to significantly increase I _K in the presence of dibutyryl cAMP (10 μM) or when a nominally Ca²⁺-free medium was included in the patch pipette (–19.4±5.1%, n=5 and –5.2±4.3%, n=5, respectively, P=NS in each case). In addition, the Ca²⁺-dependent K⁺ channel blockers iberiotoxin (0.1 μM) and tetraethylammonium (TEA, 1 mM) abolished sumatriptan-induced increases in I _K (–0.5±1.0%, n=4 and –3.9±3.1%, n=4, respectively, P=NS in each case) in C6 cells expressing h 5-HT_1B receptors, confirming the involvement of Ca²⁺-dependent K⁺ channels. In C6 cells expressing cloned h 5-HT_1B receptors, sumatriptan (1 μM) similarly failed to significantly increase I _k after 30-min incubation with thapsigargin (1 μM) or when heparin (2 mg/ml) was included in the patch pipette (1.1±0.4%, n=5 and 1.2±2.4%, n=5, respectively, P=NS). In conclusion, evidence is provided that both recombinant h 5-HT_1B and h 5-HT_1D receptors stably transfected in C6 glioma cells are positively coupled to Ca²⁺-dependent K⁺ channels, and the outward hyperpolarizing current mediated by these channels is dependent upon IP₃ receptor-mediated intracellular Ca²⁺ release. Received: 15 April 1998 / Accepted: 9 September 1998     

Inhibition of IK,ACh current may contribute to clinical efficacy of class I and class III antiarrhythmic drugs in patients with atrial fibrillation

Inward rectifier potassium currents I_K1 and acetylcholine activated I_K,ACh are implicated in atrial fibrillation (AF) pathophysiology. In chronic AF (cAF), I_K,ACh develops a receptor-independent, constitutively active component that together with increased I_K1 is considered to support maintenance of AF. Here, we tested whether class I (propafenone, flecainide) and class III (dofetilide, AVE0118) antiarrhythmic drugs inhibit atrial I_K1 and I_K,ACh in patients with and without cAF. I_K1 and I_K,ACh were measured with voltage clamp technique in atrial myocytes from 58 sinus rhythm (SR) and 35 cAF patients. The M-receptor agonist carbachol (CCh; 2 μM) was employed to activate I_K,ACh. In SR, basal current was not affected by either drug indicating no effect of these compounds on I_K1. In contrast, all tested drugs inhibited CCh-activated I_K,ACh in a concentration-dependent manner. In cAF, basal current was confirmed to be larger than in SR (at −80 mV, −15.2 ± 1.2 pA/pF, n = 88/35 vs. −6.5 ± 0.4 pA/pF, n = 194/58 [myocytes/patients]; P < 0.05), whereas CCh-activated I_K,ACh was smaller (−4.1 ± 0.5 pA/pF vs. −9.5 ± 0.6 pA/pF; P < 0.05). In cAF, receptor-independent constitutive I_K,ACh contributes to increased basal current, which was reduced by flecainide and AVE0118 only. This may be due to inhibition of constitutively active I_K,ACh channels. In cAF, all tested drugs reduced CCh-activated I_K,ACh. We conclude that in cAF, flecainide and AVE0118 reduce receptor-independent, constitutively active I_K,ACh, suggesting that they may block I_K,ACh channels, whereas propafenone and dofetilide likely inhibit M-receptors. The efficacy of flecainide to terminate AF may in part result from blockade of I_K,ACh.     

Kinetics and state-dependent effects of verapamil on cardiac L-type calcium channels

The voltage dependence and the kinetics of block by verapamil of L-type calcium current (I_Ca) were investigated in ventricular myocytes from rat hearts using the whole-cell patch-clamp technique. I_Ca was elicited repetitively in response to depolarizing voltage pulses from –80 mV to 0 mV at different pulse intervals and durations. Verapamil reduced the magnitude of I_Ca in a frequency-dependent manner without tonic component. The time course of I_Ca remained unchanged suggesting that not open but inactivated channels were affected by the drug. The interaction of verapamil with inactivated channels was investigated by the application of twin pulses. In the presence of verapamil, the duration of the first pulse significantly determined the magnitude of I_Ca during the second pulse. Variation of the duration of the first pulse between 12 and 3000 ms, followed by a pulse interval of 100 ms, resulted into a gradual decrease of I_Ca during the second pulse (180 ms), described by concentration-dependent monoexponential decay curves (t = 1060 ± 138 ms at 0.3 μM (n = 3); t = 310 ± 24 ms at 1 μM (n = 6), and t = 125 ± 7 ms at 10 μM (n = 5); means ± SEM). Under control conditions, the changes in I_Ca were comparably negligible. The recovery of I_Ca from block was analyzed by the application of a twin pulse protocol in which two depolarising voltage pulses at fixed length (1. pulse at 3 s and 2. pulse at 180 ms) were interrupted by variable pulse intervals (6 ms–60 s). Under control conditions, recovery from inactivation was fast (t = 11 ± 0.7 ms; means ± SEM; n = 3). In the presence of verapamil, recovery from block was about 500times slower than under control conditions, independent of the drug concentration (t = 5.05 ± 0.44 s at 0.3 μM (n = 3), t = 6.7 ± 0.69 s at 1 μM (n = 4), and t = 6.02 ± 0.9 s at 10 μM (n = 5); means ± SEM). Since development of block was dependent on the concentration of verapamil, whereas recovery from block was independent from the drug concentration, it is assumed that the described time constants for block and unblock reflect voltage-dependent net binding (τ_on) and unbinding (τ_off), respectively, of verapamil at its receptor sites. A computer simulation, including the time constants of block development at 0 mV and of recovery from block at –80 mV, predicted reasonably well the observed frequency-dependent block of I_Ca by verapamil. The development of either measured or calculated block of I_Ca, using 180 ms depolarising voltage pulses from –80 mV to 0 mV, was fitted by identical monoexponential association curves (t = 7 s each at 0.2 Hz and t = 1.7 s each at 1 Hz). When Ba²⁺ was used as the charge carrier, which removes the calcium-dependent inactivation of the current, verapamil (3 μM) was less efficient: I_Ca was decreased by 57 ± 6 % (means ± SEM; n = 6), whereas I_Ba was decreased by 24 ± 4 % (means ± SEM; n = 5). It is proposed that verapamil binds to calcium channels in their inactivated state at more positive potentials and dissociates from the channels in the resting state at more negative potentials. In the proposed scheme of periodical drug binding and unbinding, dependent on the state of the channels, the development of frequency-dependent block of I_Ca by verapamil is adequately predicted by the construction of cumulative association/dissociation curves which include the experimentally determined time constants of development and recovery from block at 0 mV and –80 mV, respectively. Received: 17 May 1996 / Accepted: 23 September 1996     

Effect of disopyramide on potassium currents in rabbit ventricular myocytes

The effects of disopyramide (1–30 μM) on the 4-aminopyridine sensitive transient outward current (I _to), on the rapid component of the delayed rectifier potassium current (I _Kr) and on the inward rectifier potassium current (I _k1) were studied in single rabbit ventricular myocytes at 35° C by applying the whole-cell configuration of the patch clamp technique. Disopyramide signifiantly decreased the amplitude of I _to (from 1510 ± 122 pA at control to 1015 ± 21 pA after 30 μM disopyramide at +50 mV; n = 5). This effect was not voltage- or use-dependent. Disopyramide (10 μM) influenced neither the recovery from inactivation of I _to nor the steady-state inactivation curve. The drug dose dependently decreased the time constant of the fast component of the decay of I _to (τ_f = 6.41 ± 0.25 ms, n = 24 for control; and 2.20 ± 0.38 ms, n = 5 after 30 μM disopyramide at +50 mV). The fractional block caused by 30 μM disopyramide as a function of time was well fitted by a single exponential function with time constant of 1.48 ± 0.18 ms (n = 5), most likely reflecting the binding kinetics of the drug to the open channel. The offset kinetics of the drug was estimated by using a double-pulse protocol and its time constant was 3.9 ± 0.5 ms. Disopyramide (30 μM) did not influence significantly the onset of inactivation measured at –20 mV. The estimated EC₅₀ value for the I _to block by disopyramide was 14.1 μM. Our results are consistent with an open-channel block of I _to by disopyramide, however, a weak, drug-induced increase of the rate of inactivation and a moderate tonic block cannot be excluded. The amplitude of the outward tail current attributed to I _Kr was depressed dose dependently by disopyramide (after clamping the cells back to the holding potential from +30 mV, 139.5 ± 10.9 pA for control, and 30.7 ± 3.2 pA in the presence of 10 μM disopyramide; n = 11). The estimated EC₅₀ was 1.8 μM. I _to is thus less sensitive to disopyramide than I _Kr. I _k1 was not influenced significantly by disopyramide, even when applied in the highest tested concentration (30 μM). It is concluded that in rabbit ventricular myocytes disopyramide blocks not only I _Kr, but also I _to, both of which may play an important role in the well established repolarization lengthening and antiarrhythmic effects of the drug. Received: 1 September 1997 / Accepted: 10 November 1997     

Adenosine inhibits the L-type calcium current in human atrial myocytes

The effects of adenosine on the L-type Ca ²⁺ current (I _Ca) were studied in human atrial myocytes using the whole-cell voltage clamp technique. I _Ca was recorded under physiological calcium concentrations (1.8 mmol/l) at 37?°C. Under these conditions the current density of basal I _Ca averaged 4.0 pA/pF. Isoprenaline (1 µmol/l) increased basal I _Ca to 249.7%. Adenosine (100 µmol/l) in the presence of isoprenaline (1 µmol/l) decreased I _Ca from the level obtained with isoprenaline to 87.5% of basal I _Ca. Adenosine (0.1 to 100 µmol/l) also reduced basal I _Ca, maximally to 64.5% of control. Activation and inactivation parameters of basal I _Ca were not significantly different between adenosine (100 µmol/l) and control recordings. Our results show that adenosine affects both basal and isoprenaline stimulated I _Ca in human atrial myocytes. Although a considerable decrease of basal I _Ca was seen, we conclude that the action of adenosine on L-type Ca ²⁺ current in human atrial myocytes is mainly antiadrenergic. Both effects may contribute to the antiarrhythmic properties of adenosine.     

Pharmacological evidence for altered src kinase regulation of <Emphasis Type="Italic">I</Emphasis><Subscript>Ca,L</Subscript> in patients with chronic atrial fibrillation

A reduction in l-type Ca²⁺ current (I _Ca,L) contributes to electrical remodeling in chronic atrial fibrillation (AF). Whether the decrease in I _Ca,L is solely due to a reduction in channel proteins remains controversial. Protein tyrosine kinases (PTK) have been described as potent modulators of I _Ca,L in cardiomyocytes. We studied α_1C l-type Ca²⁺ channel subunit expression and the regulation of I _Ca,L by PTK in chronic AF using PTK inhibitors: genistein, a nonselective inhibitor of PTK, and 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo-3,4-d-pyrimidine (PP1), a selective inhibitor of src kinases. Furthermore, type-1 and type-2A protein phosphatase activity was measured with phosphorylase as substrate in whole-cell lysates derived from atrial tissue of AF patients. Right atrial appendages were obtained from patients undergoing open-heart surgery. Protein levels of α_1C l-type Ca²⁺ channel subunit were determined using Western blot analysis and normalized to the protein amounts of calsequestrin as internal control. The protein concentrations of α_1C did not differ between AF and sinus rhythm (SR; α_1C/calsequestrin: 1.0 ± 0.1 and 1.2 ± 0.2, respectively, n = 8 patients). In cardiomyocytes from patients in SR (n = 20 patients), genistein and PP1 both evoked similar increases in I _Ca,L from 3.0 ± 0.3 to 6.1 ± 0.8 pA/pF and from 2.8 ± 0.4 to 6.1 ± 0.6 pA/pF, respectively. In cells from AF patients (n = 10 patients), basal I _Ca,L was significantly lower. In this case, genistein lead to the same relative increase in I _Ca,L as in SR cells (from 1.46 ± 0.30 to 3.2 ± 1.0 pA/pF), whereas no increase was elicited by PP1 suggesting impaired regulation of I _Ca,L by src kinases in AF. Total and type 1 and type 2A-related phosphatase activities were higher in tissue from patients with chronic AF compared to SR (4.8 ± 0.4, 2.1 ± 0.2, and 2.7 ± 0.4 nmol/mg/min and 3.6 ± 0.4, 1.3 ± 0.2, and 2.4 ± 0.3 nmol/mg/min, respectively, n = 7 patients per group). Downregulation of I _Ca,L in AF is not due to a reduction in l-type Ca²⁺ channel protein expression. Indirect evidence for an impaired src kinase regulation of I _Ca,L together with an increased phosphatase activity suggests that a complex alteration in the kinase/phosphatase balance leads to I _Ca,L dysregulation in chronic AF.     

Electrophysiological effects of EGIS-7229, a new antiarrhythmic agent, in isolated mammalian and human cardiac tissues

The cellular electrophysiological effects of EGIS-7229 (5-chlor-4-[N-(3,4-dimethoxy-phenyl-ethyl)-amino-propylamino]-3(2H)-pyridazinone fumarate), a novel antiarrhythmic agent, was studied using conventional microelectrode techniques in canine cardiac Purkinje fibers and papillary muscle preparations obtained from man, rabbits and guinea pigs. Low concentration of EGIS-7229 (3 μmol/l) selectively lengthened action potential duration (both APD₅₀ and APD₉₀) in all preparations. The effect of higher concentrations (30–100 μmol/l) of EGIS-7229 on action potential duration was variable depending on the preparation studied: in rabbit and human papillary muscles both APD₅₀ and APD₉₀ were lengthened, in canine Purkinje fibers APD₉₀ was lengthened but APD₅₀ was shortened, while in guinea pig papillary muscles both APD₅₀ and APD₉₀ were shortened by high concentrations of the drug. At these higher concentrations EGIS-7229 also decreased the maximum velocity of action potential upstroke (V _max) and depressed the plateau of action potentials without affecting the resting membrane potential or action potential amplitude. Both reduction of V _max and lengthening of APD were frequency dependent. The former effect was more prominent at higher pacing frequencies, while the latter was more pronounced at lower driving rates. In guinea pig papillary muscle, the time constant of recovery from V _max-block was 719 ± 33 ms (n = 18) and the rate of onset of the block was 1.81 ± 0.06 AP^–1 (n = 16) in the presence of 100 μmol/l EGIS-7229. EGIS-7229 had a complex action on refractoriness in guinea pig papillary muscles: ERP was lengthened at low concentrations (3 to 10 μmol/l), unchanged at 30 μmol/l and shortened at 100 μmol/l. The ratio of ERP/APD₉₀, however, was significantly increased at concentrations higher than 3 μmol/l. In canine Purkinje fiber, when the delayed rectifier K current (I_K) was blocked by d-sotalol (60 μmol/l) and APD was shortened back to its control value by additional application of nicorandil (15 μmol/l), APD was not affected by 3 μmol/l but was shortened by 30 μmol/l of EGIS-7229. 100 μmol/l EGIS-7229 shortened APD in guinea pig papillary muscle. This effect of EGIS-7229 was effectively prevented by nifedipine pretreatment (10 μmol/l). In this preparation, EGIS-7229 also decreased the V _max of the slow action potential, evoked in the presence of 20 mmol/l external K⁺ plus 0.5 mmol/l Ba²⁺. It is likely that EGIS-7229 at low concentrations blocks I_K in human, canine, rabbit and guinea pig cardiac preparations, but at higher concentrations also inhibits Ca and Na currents. Therefore, EGIS-7229 appears to carry mixed class III, IV and IB antiarrhythmic properties. Received: 8 July 1996 / Accepted: 23 October 1996     

Activity of glibenclamide-sensitive K+ channels under unstimulated conditions in smooth muscle cells of pig proximal urethra

The membrane potential in pig proximal urethra was examined by use of the microelectrode technique. In cells 1–2cm from the bladder neck the membrane potential was quiescent, with a value of –37.2±2.5 mV (n=16). In some cells small spontaneous de- and hyperpolarizations were seen. Glibenclamide (1μM) caused a small but significant depolarization in tissue strips (12±3mV, n=3) and also in dispersed cells using whole-cell patch electrodes (13±3mV, n=5). In the conventional whole-cell voltage-clamp configuration, glibenclamide reduced the noise level of the basal membrane current at –50mV and inhibited the membrane current in symmetrical 140mM K⁺ conditions. In cell-attached patches, brief openings of a glibenclamide-sensitive 43pS K⁺ channel (K_GS-43pS) were seen even under unstimulated conditions and greater activation occurred in the same membrane patch on subsequent application of 100μM levcromakalim. These results provide direct evidence that glibenclamide-sensitive K⁺ channels may play a role in maintaining the resting membrane potential of pig proximal urethra under unstimulated conditions. Received: 7 January 1997 / Accepted: 19 May 1997     

Different inhibition patterns of tedisamil for fast and slowly inactivating transient outward current in rat ventricular myocytes

Tedisamil has been described as a selective inhibitor of a fast inactivating transient outward current (i_to,f) in rat ventricular myocytes. Because recent reports demonstrated the existence of a second slowly inactivating transient component (i_to,s) we investigated i_to,s and differentiated the effects of tedisamil on both transient outward current components and their influence on action potential duration. Standard electrophysiological techniques were used for whole cell recordings at 24–26° C from enzymatically isolated myocytes. Inhibition of i_to,f by tedisamil was the result of an acceleration of inactivation at positive test potentials with a concentration for halfmaximal inhibition (EC₅₀) of 4–7 μmol/l, which is confirmatory to reports from other investigators. Our new results show that i_to,s is more sensitive to tedisamil with an EC₅₀ of 0.5 μmol/l. Furthermore the pattern of i_to,s inhibition is different compared with i_to,f, because inactivation of i_to,s is not accelerated by tedisamil. Instead the amplitude of the steady state inactivation curve of i_to,s is attenuated which indicates a reduction of maximally available current. I_to,s was evaluated by three different methods as time-dependently inactivating current (7.5 s test pulse duration), voltage-dependently inactivated current and tedisamil-sensitive current. All approaches yield similar inactivation curves. The potential for halfmaximal inactivation of i_to,s lies about 35 mV more negative than that for i_to,f and the slope factor (K = –23 mV) is different to that of i_to,f (K = –3 mV). Effectiveness of tedisamil-induced modulation of i_to,f and i_to,s on action potential repolarization was tested. Action potentials stimulated at 0.5 Hz were not prolonged by 1 μmol/l tedisamil (dominant i_to,s block) at a repolarization level of 0 mV but prolonged to about 120% of control at –70 mV. This indicates that i_to,f was sufficient to guarantee a regular early repolarization whereas decrease of i_to,s delayed the final repolarization. In conclusion, the observation that tedisamil inhibits i_to,f and i_to,s differently supports the hypothesis that the two i_to-components are related to two different channel populations expressed in rat ventricular myocytes. Received: 28 July 1997 / Accepted: 3 December 1997     

Effects of 17β-estradiol on action potentials and ionic currents in male rat ventricular myocytes

This study describes electrophysiological effects of estrogens in isolated male rat ventricular myocytes. According to the literature these cells do not express the nuclear estrogen receptor. Action potentials or membrane currents were recorded in the whole-cell configuration with standard techniques. Action potential durations (APD) measured at a level of 0 mV (APD 0) and –70 mV (APD –70) were prolonged by 17β-estradiol (0.5 Hz stimulation frequency, 24–26° C). Threshold concentration was 1 μmol/l. At the highest concentration used (30 μmol/l) no saturation of the response was reached and APD 0 was 162% and APD –70 was 230% of the respective control. The resting potential remained unaffected in most cells. The prolongation induced by 17β-estradiol developed rapidly and reached a steady state 10 min after start of hormone superfusion. Effects of estrogen were completely reversible during 10–15 min wash-out with hormone-free solution. The extent of prolongation (10 μmol/l 17β-estradiol) was frequency dependent. Expressed as percentage of the respective control APD 0 (or APD –70) was 115% (188%) at 0.05 Hz, 118% (163%) at 0.5 Hz and 99% (129%) at 5 Hz stimulation frequency. The response was stereoselective, because 30 μmol/l 17α-estradiol did not prolong action potentials (APD 0: 101%, APD –70: 104% of the respective control, 0.5 Hz stimulation frequency). The endogenous estrogens estrone and estriol were less effective than 17β-estradiol. With 30 μmol/l estrone (0.5 Hz stimulation frequency) APD 0 was 103% and ADP-70 148% of control and with 30 μmol/l estriol APD 0 was 135% and APD –70 137% of control. The prolongation of action potentials can be explained by inhibition of transient outward current which, in rat ventricle, is composed of fast (i _to,f) and slowly (i _to,s) inactivating components. At 30 μmol/l 17β-estradiol i _to,f was reduced to 50% and i _to,s to 43% of their maximal amplitudes. The voltage sensor of i _to,f or i _to,s was hardly affected. Additionally, 17β-estradiol decreased the calcium current (i _Ca,L) to 76% (10 μmol/l) and 38% at 30 μmol/l. The inwardly rectifying potassium current (i _K1) was reduced partly with 30 μmol/l 17β-estradiol and its amplitude was 72% of control at –90 mV (inward current flow) and 65% at –40 mV (outward current flow). These results show that 17β-estradiol is active in cardiac cells which do not express the nuclear estrogen receptor. The hormone exerts class III activity and reduces calcium inward current. These effects, however, occur in vitro with concentrations above the physiological level and therefore may be without significance in vivo. Received: 6 May 1997 / Accepted: 18 October 1997     

Dual effects of amiodarone on pacemaker currents in hypertrophied ventricular myocytes isolated from spontaneously hypertensive rats

The pacemaker current I_f conducted by hyperpolarization‐activated cyclic nucleotide‐gated (HCN) channels plays a critical role in the regulation of cardiac automaticity, with I_f density increased in hypertrophied ventricular myocytes. Amiodarone, a highly effective anti‐arrhythmic agent, blocks human HCN currents and native I_f under normal conditions. To determine the effects of amiodarone under pathological conditions, we monitored I_f under after both acute (0.01, 0.1, 1, 10 and 100 μmol/L) and chronic (10 μmol/L) amiodarone treatment in ventricular myocytes from spontaneously hypertensive rats (SHR) with left ventricular hypertrophy using the whole‐cell patch‐clamp technique. The I_f current density was significantly greater in SHR ventricular myocytes than in cells from healthy normotensive control Wistar‐Kyoto (WKY) rats. Acute application of amiodarone significantly decreased I_f density in myocytes from both SHR and WKY rats. The inhibition was concentration dependent with an IC₅₀ of 4.9 ± 1.2 and 6.9 ± 1.3 μmol/L in myocytes from SHR and WKY rats, respectively. Amiodarone increased the activation and deactivation times of I_f in myocytes from SHR, although it did not alter the relationship of voltage‐dependent activation and the reversal potential of I_f in myocytes from SHR. Chronic exposure of myocytes from SHR to amiodarone potently inhibited I_f and downregulated HCN2 and HCN4, the major channel subtypes underlying native I_f, at both the mRNA and protein level. These findings indicate that amiodarone inhibits I_f under hypertrophied conditions through dual mechanisms: (i) direct channel blockade of I_f currents; and (ii) indirect suppression via negative regulation of HCN channel gene expression. These unique properties of amiodarone may contribute to its anti‐arrhythmic properties under pathological conditions.     

High affinity regulation of cardiac Cl– and Ca2+ conductances by (13R)-spiroforskolin

The effects of a new forskolin derivative, (13R)-spiroforskolin, on the ventricular cAMP-activated chloride current (I_Cl(cAMP)) and the atrial L-type calcium current (I_Ca,L) were measured by means of whole-cell recording from isolated guinea-pig cardiac myocytes at 30°C and 20–22°C, respectively. In contrast to forskolin, the derivative contains a tetrahydrofuran rather than a tetrahydropyran moiety. (13R)-spiroforskolin activated I_Cl(cAMP) in 58% of the ventricular myocytes studied. The concentration required for the half maximal effect (EC₅₀ value) amounted to 9.6×10^–11 M and was lower than the EC₅₀ value for forskolin (2.4×10^–8 M). (13R)-spiroforskolin evoked a smaller maximal I_Cl(cAMP) amplitude than forskolin. The rundown of the (13R)-spiroforskolin-activated I_Cl(cAMP) was faster than that of the forskolin-induced current. Neither forskolin nor (13R)-spiroforskolin in maximally effective concentrations increased I_Cl(cAMP) in cells containing high concentrations of cAMP. Furthermore, as an activator of atrial I_Ca,L (13R)-spiroforskolin displayed a smaller activation and a lower EC₅₀ value (5.8×10^–10 M) than forskolin (EC₅₀ value: 3.7×10^–7 M). The effect of (13R)-spiroforskolin was observed in only 30% of the atrial cells studied. None of the drugs exerted a stimulatory effect in atrial cells containing a high [cAMP]. The washout of the drug effect was significantly faster in (13R)-spiroforskolin- than in forskolin-treated atrial myocytes. We conclude that (13R)-spiroforskolin as a forskolin derivative displays unique characteristics. It is a more potent but less efficacious activator of cardiac ionic conductances than the parent compound. The results suggest that (13R)-spiroforskolin, like forskolin, most probably exerts its effects via stimulation of the adenylyl cyclase. Received: 25 May 1998 / Accepted: 30 July 1998     

Positive inotropic response to 5-HT in human atrial but not in ventricular heart muscle

Summary The effects of 5-hydroxytryptamine (5-HT) on force of contraction (F_C), action potential (AP) and calcium current (I_Ca) were studied in human right atrial and left ventricular heart muscle. 5-HT exerted a concentration-dependent increase in F_C in multicellular atrial preparations; the EC₅₀ was approximately 3 × 10^–7 mol/l. Maximal increases in F_C (252±58% of control values; means ± SEM, n=6) were obtained at 5-HT 10^–5 mol/l. At this concentration, I_Ca was increased four- to sevenfold in enzymatically isolated atrial myocytes. In contrast, ventricular preparations did not respond to 5-HT; F_C, AP and I_Ca remained unaffected. In the same preparations, F_C was increased by isoprenaline three- to fourfold. These results confirm the observation that 5-HT induces a positive inotropic effect in the human atrium, possibly mediated by activation of the adenylyl cyclase — cyclic AMP system. Our study demonstrates, however, the complete lack of functional 5-HT receptors, with respect to changes in F_C, in the human ventricle. Since the positive inotropic effect of 5-HT in the human heart is obviously restricted to the atrium, our findings question the concept of developing 5-HT receptor agonists for the treatment of heart failure. Correspondence to: H. Nawrath at the above address     

Modulation of the delayed K+ current by histamine in guinea pig ventricular myocytes

Summary The effects of histamine on delayed K⁺ current (I_K) were investigated in patch-clamped single guinea pig ventricular myocytes. Histamine increased I_K with a maximal fractional response of 2.7 and a k_d of 9.4 × 10^–7 mol/l. At a concentration of 10^–8 mol/l, histamine did not increase I_K significantly, but increased I_Ca by 52% ± 12%. The voltage-dependence of I_K activation, the reversal potential and the time course of the I_K tail decay were not changed by histamine. Under pretreatment with 10^–4 mol/l of ranitidine, neither histamine (10^–6 mol/l) nor 2-pyridylethylamine (10^–4 mol/l) caused any sizable increase in I_K. When the cell was pretreated with a saturating dose of isoproterenol (10^–6 mol/l), histamine did not additively enhance I_K. The I_K enhancement by 3 × 10^–7 mol/l histamine was partially antagonized by concurrent exposure to 5 × 10^–6 mol/l carbachol. Whereas, use of a higher concentration of histamine (10^–6 mol/l) obscured the inhibitory effect of carbachol. It is concluded that histaminergic action of I_K is attributed exclusively to H₂ receptor-mediated reactions involving G_s protein and adenylate cyclase. Send offprint requests to Y. Habuchi at the above address     

Effects of the atrial antiarrhythmic drug AVE0118 on cardiac ion channels

Previous studies in pigs and goats have demonstrated that AVE0118 prolongs atrial refractoriness without any effect on the QT-interval. The purpose of the present study was to investigate the effect of the compound on various cardiac ion channels. AVE0118 blocked the pig Kv1.5 and the human Kv1.5 expressed in Xenopus oocytes with IC₅₀ values of 5.4±0.7 M and 6.2±0.4 M respectively. In Chinese hamster ovary (CHO) cells, AVE0118 decreased the steady-state hKv1.5 current with an IC₅₀ of 1.1±0.2 M. The hKv4.3/KChIP2.2 current in CHO cells was blocked by AVE0118 by accelerating the apparent time-constant of inactivation (_inact), and the integral current was inhibited with an IC₅₀ of 3.4±0.5 M. At 10 M AVE0118 _inact decreased from 9.3±0.6 ms (n=8, control) to 3.0±0.3 ms (n=8). The K_ACh current was investigated in isolated pig atrial myocytes by application of 10 M carbachol. At a clamp potential of –100 mV the I_KACh was half-maximally blocked by 4.5±1.6 M AVE0118. In the absence of carbachol, AVE0118 had no effect on the inward current recorded at –100 mV. Effects on the I_Kr current were investigated on HERG channels expressed in CHO cells. AVE0118 blocked this current half-maximally at approximately 10 M. Comparable results were obtained in isolated guinea pig ventricular myocytes, where half-maximal inhibition of the I_Kr tail current occurred at a similar concentration of AVE0118. Other ionic currents, like the I_Ks, I_KATP (recorded in guinea pig ventricular myocytes), and L-type Ca²⁺ (recorded in pig atrial myocytes) were blocked by 10 M AVE0118 by 10±3% (n=6), 28±7% (n=4), and 22±13% (n=5) respectively. In summary, AVE0118 preferentially inhibits the atrial K⁺ channels I_Kur, I_to and I_KACH. This profile may explain the selective prolongation of atrial refractoriness described previously in pigs and goats.     

Pharmacology and mode of action of bradykinin on mouse-isolated trachea

In the present study, the effect of bradykinin on basal and precontracted mouse-isolated trachea was investigated. In basal conditions mouse-isolated tracheal rings do not respond to bradykinin. However, when the tracheal rings were precontracted with carbachol (10^–7M) a relaxation with bradykinin (3·10^–9–3·10^–7) was found. The maximal response amounted 69.7±4.1% (n=15) with a pD₂ value of 7.2±0.21. The selective bradykinin B₂ receptor antagonist HOE 140 (10^–10–10^–8M) antagonized the bradykinin-induced relaxation, while the bradykinin B₁ receptor antagonist des-Arg⁹-Leu⁸-bradykinin (10^–6M) had no influence. The selective bradykinin B₁ receptor agonist des-Arg⁹-bradykinin (10^–6M) caused a small relaxation (8.4±2.5%, n=6), which could be antagonized completely by the selective bradykinin B₁ receptor antagonist des-Arg⁹-Leu⁸-bradykinin (10^–6M) while addition of the selective bradykinin B₂ receptor antagonist HOE 140 (10^–8M) was without effect. In the presence of indomethacin (10^–6M) the relaxation of bradykinin was completely abolished. Pretreatment of the tracheal rings with capsaicin, or the presence of the selective NK₁ receptor antagonist RP 67851 (10^–6M) or the presence of the nitric oxide synthase inhibitor L-NAME (3·10^–4M) had no effect on the bradykinin-induced relaxation. In conclusion, these results demonstrate that the mouse-isolated tracheal is a preparation in which bradykinin exerts a relaxant response via stimulation of bradykinin B₂ receptors. This response is probably mediated by prostaglandines. Received: 14 November 1996 / Accepted: 5 March 1997     

Diminution of contractile response by κ-opioid receptor agonists in isolated rat ventricular cardiomyocytes is mediated via a pertussis toxin-sensitive G protein

Opioids directly decrease the contractile response of isolated ventricular cardiomyocytes to electrical stimulation. To investigate whether these effects are mediated via GTP-binding G_i/o proteins we examined the influence of pertussis toxin on the effects of the κ-opioid receptor agonist trans-(±)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]-benzeneacetamide (U-50,488) methanesulphonate and on the as yet undescribed effects of the opioid peptide dynorphin A (1–8) on contraction. In isolated, electrically driven, rat ventricular cardiomyocytes both agents concentration dependently reduced cell shortening within 15 min, decreasing the contractile response by 79±4% (n=5) and 62±2% (n=6) of control values at maximal effective concentrations of 10 μM (U-50,488) and 1 μM [dynorphin A (1–8)], respectively. Pertussis toxin pre-treatment (200 ng/ml; 4.5–5 h) completely abolished the effects of U-50,488 and dynorphin A (1–8) on the contractile response, indicating that these effects are mediated via G_i/o proteins. In addition, the non-selective opioid receptor antagonist (–)-naloxone and the κ-opioid receptor antagonist nor-binaltorphimine antagonized the effects of U-50,488 and dynorphin A (1–8) on the contractile response. Furthermore, the μ- and δ-opioid receptor agonist (D-Ala², D-Leu⁵)-enkephalin (DADLE) had no effects on contraction. These results indicate that the decrease in cell shortening is due to stimulation of κ-opioid receptors. The direct effect of κ-opioid receptor agonists on the contractile response thus represents an additional mechanism for decreasing cardiac contractility, besides the M-cholinoceptor- or adenosine receptor-mediated pathway. It is conceivable that increased release of endogenous dynorphins from the heart during hypoxia may protect the heart in a similar manner to adenosine. Received: 16 September 1997 / Accepted: 15 June 1998     

抗疟药青蒿素抗心律失常的作用机制

目的：探讨青蒿素抗心律失常的离子电流基础。方法：用全细胞膜片钳技术和双电极电压钳技术。结果：当细胞超极化到-100 mV时,青蒿素以浓度依赖方式明显抑制家兔心室肌细胞I_k1,50μmol.L^-1青蒿素可使家兔心室肌细胞I_k1从对照组的-2.36±0.39　nA减少到-1.43±0.31nA。给予非洲蛙卵母细胞注射K_{ir 2.1}　cRNA后,用不同浓度青蒿素灌注,可减低K_{ir 2.1}钾通道电流,此作用呈电压和浓度依赖性。青蒿素对K_{ir 2.1}钾通道的阻断作用呈可逆性。结论：青蒿素能有效抑制离体心肌细胞I_k1,其抗心律失常作用机理与其抑制心肌细胞I_k1及阻断K_{ir 2.1}通道电流有关。