"Early" class III drugs for the treatment of atrial fibrillation: efficacy and atrial selectivity of AVE0118 in remodeled atria of the goat.

BACKGROUND: Currently available antiarrhythmic drugs are only moderately effective against atrial fibrillation (AF) and may cause ventricular proarrhythmia. AVE0118 is a blocker of atrium-specific early K+ currents (I(Kur)/I(to)). METHODS AND RESULTS: Effects of intravenous AVE0118 and dofetilide on atrial effective refractory period (AERP) and inducibility of AF were measured before and after 48-hours of AF-induced electrical remodeling in the goat. During persistent AF (53+/-19 days), the cardioversion efficacy and effects on atrial wavelength of AVE0118, dofetilide, and ibutilide were evaluated. QT durations were measured during atrial pacing and persistent AF. After 48 hours of AF, the effect of dofetilide on AERP was reduced, and induction of AF was not prevented. In contrast, the class III action of AVE0118 was enhanced, and AF inducibility decreased from 100% to 32% (P<0.001). At 1, 3, and 10 mg x kg(-1) x h(-1), AVE0118 terminated persistent AF in 1 of 8, 3 of 8, and 5 of 8 goats, respectively. Dofetilide and ibutilide terminated AF in 1 of 5 and 2 of 7 goats. AVE0118 0.5, 1.5, and 5 mg/kg prolonged the AERP during AF and increased the fibrillation wavelength from 6.7+/-0.6 to 8.5+/-0.5, 9.7+/-0.5, and 11.2+/-0.9 cm (P<0.01). Whereas dofetilide and ibutilide prolonged QT duration, AVE0118 had no appreciable effect. CONCLUSIONS: AVE0118 markedly prolongs the AERP during AF without affecting QT duration. Cardioversion of AF was due to an approximately 2-fold increase in fibrillation wavelength. Atrium-selective class III drugs like AVE0118 may be a promising new option for safe and effective cardioversion of AF.

Inhibitors of ATP-sensitive potassium channels in guinea pig isolated ischemic hearts.

During heart ischemia, ATP-sensitive potassium channels in the sarcolemmal membrane (sarcK(ATP)) open and cause shortening of the action potential duration. This creates heterogeneity of repolarization, being responsible for the development of re-entry arrhythmias and sudden cardiac death. Therefore, the aim is to develop selective blockers of the cardiac sarcK(ATP) channel. In the present study we established an in vitro model and classified 5 K(ATP) channel inhibitors with respect to their potency and selectivity between cardiomyocytes and the coronary vasculature and compared the results with inhibition of Kir6.2/SUR2A channels expressed in HEK293 cells, recorded with the Rb(+)-efflux methods. We used Langendorff-perfused guinea pig hearts, where low-flow ischemia plus hypoxia was performed by reducing the coronary flow (CF) to 1.2 ml/min and by gassing the perfusion solution with N(2) instead of O(2). Throughout the experiment, the monophasic action potential duration at 90% repolarization (MAPD(90)) was recorded. In separate experiments, high-flow hypoxia was produced by oxygen reduction in the perfusate from 95% to 20%, which caused an increase in the coronary flow. Under normoxic conditions, the substances glibenclamide, repaglinide, meglitinide, HMR 1402 and HMR 1098 (1 microM each) reduced the CF by 34%, 38%, 19%, 12% and 5%, respectively. The hypoxia-induced increase in CF was inhibited by the compounds half-maximally at 25 nM, approximately 200 nM, 600 nM, approximately 9 microM and >100 microM, respectively. In control experiments after 5 min low-flow ischemia plus hypoxia, the MAPD(90) shortened from 121+/-2 to 99+/-2 ms ( n=29). This shortening was half-maximally inhibited by the substances at concentrations of 95 nM, 74 nM, 400 nM, 110 nM and 550 nM, respectively. In HEK293 cells the Rb(+)-efflux through KIR6.2/SUR2A channels was inhibited by the compounds with IC(50) values of 21 nM, 67 nM, 205 nM, 60 nM and 181 nM, respectively. In summary, the present data demonstrate that the sulfonylurea glibenclamide, and the carbamoylbenzoic acid derivatives repaglinide and meglitinide are unselective blockers of K(ATP) channels in cardiac cells and in the cardiac vascular system, whereas the sulfonylthioureas HMR 1402, and especially HMR 1098 selectively blocked the cardiac sarcK(ATP) channel. Blockade of Kir6.2/SUR2A channels in HEK293 cells occurred with comparable efficacy as in the cardiac tissue, indicating that the expression system is suited for screening for novel inhibitors.

Characterization of a novel Kv1.5 channel blocker in Xenopus oocytes, CHO cells, human and rat cardiomyocytes.

The inhibitory effects of the novel Kv1.5 channel blocker, S9947 (2'-(benzyloxycarbonylaminomethyl)biphenyl-2-carboxylic acid 2-(2-pyridyl)ethylamide), on cloned human Kv1.5 (hKv1.5), expressed in both Xenopus oocytes and Chinese hamster ovary (CHO) cells, and on native cardiac ultrarapid delayed rectifier potassium currents (IKur) in rat (ventricle myocytes) and human (atrial myocytes) were investigated. The influence of S9947 on the action potential was examined in rat ventricular myocytes. Using the two-electrode voltage-clamp technique in Xenopus oocytes and the patch-clamp technique (whole cell configuration) in CHO cells, hKv1.5 was inhibited by S9947 with IC50 values of 0.65 microM and 0.42 microM, respectively. In addition, inhibition of human Kv4.3 (hKv4.3) and HERG by 10 microM S9947 was low (approximately 20%) and absent, respectively. Using the patch-clamp technique in the whole cell configuration, IKur currents in rat ventricular (rIKur) cardiomyocytes and human atrial (hIKur) cardiomyocytes were inhibited by S9947 with IC50 values of 0.96 microM and 0.07 microM, respectively. In contrast, rat cardiac inward rectifier current (rIK1) and rat (rIto) and human (hIto) cardiac transient outward currents were only inhibited by approximately 20% with 10 microM S9947. In rat cardiomyocytes, using the patch-clamp technique, action potential duration was increased by S9947 in a concentration-dependent (0.3-10 microM) and rate-independent manner. The data show that S9947 suppresses both cloned (Kv1.5) and native (IKur) cardiac potassium currents. Furthermore, S9947 prolongs rat action potential in a rate-independent manner.

Effects of the cardioselective KATP channel blocker HMR 1098 on cardiac function in isolated perfused working rat hearts and in anesthetized rats during ischemia and reperfusion.

It has been argued that activation of KATP channels in the sarcolemmal membrane of heart muscle cells during ischemia provides an endogenous cardioprotective mechanism. In order to test whether the novel cardioselective KATP channel blocker HMR 1098 affects cardiac function during ischemia, experiments were performed in rat hearts during ischemia and reperfusion. Isolated perfused working rat hearts were subjected to 30 min of low-flow ischemia in which the coronary flow was reduced to 10% of its control value, followed by 30-min reperfusion. In the first set of experiments the hearts were electrically paced at 5 Hz throughout the entire protocol. At the end of the 30-min ischemic period the aortic flow had fallen to 44 +/- 2% (n=8) of its nonischemic value in vehicle-treated hearts, whereas in the presence of 0.3 micromol/l and 3 micromol/l HMR 1098 it had fallen to 29 +/- 7% (n=5, not significant) and 8 +/- 2% (n=12, P<0.05), respectively. Glibenclamide (3 micromol/l) reduced the aortic flow to 9.5 +/- 7% (n=4, P<0.05). In control hearts the QT interval in the electrocardiogram shortened from 63 +/- 6 ms to 36 +/- 4 ms (n=10, P<0.05) within 4-6 min of low-flow ischemia. This shortening was completely prevented by 3 micromol/l HMR 1098 (60 +/- 5 ms before ischemia, 67 +/- 6 ms during ischemia, n=9, not significant). When rat hearts were not paced, the heart rate fell spontaneously during ischemia, and HMR 1,098 (3 micromol/l) caused only a slight, statistically non-significant reduction in aortic flow during the ischemic period. In order to investigate whether HMR 1098 shows cardiodepressant effects in a more pathophysiological model, the left descending coronary artery was occluded for 30 min followed by reperfusion for 60 min in anesthetized rats. Treatment with HMR 1098 (10 mg/kg i.v.) had no statistically significant effects on mean arterial blood pressure and heart rate during the control, ischemia and reperfusion periods. At the end of the reperfusion period, aortic blood flow was slightly reduced by HMR 1098, without reaching statistical significance (two-way analysis of ANOVA, P=0.15). Myocardial infarct size as a percentage of area at risk was not affected by HMR 1098 (vehicle: 75 +/- 3%, HMR 1098: 72 +/- 2%, n=7 in each group). In conclusion, cardiodepressant effects of HMR 1098 were observed only in isolated perfused working rat hearts which were continuously paced during global low-flow ischemia. In the model of anesthetized rats subjected to regional ischemia, HMR 1098 had no significant effect on cardiac function or infarct size.

The K(ATP) channel blocker HMR 1883 does not abolish the benefit of ischemic preconditioning on myocardial infarct mass in anesthetized rabbits.

Previous experimental studies showed that the benefit of ischemic preconditioning (IPC) is abolished by K(ATP) channel blockade with glibenclamide. However, the newly discovered K(ATP) channel blocker HMR 1883 (1-[[5-[2-(5-chloro-o-anisamido)ethyl]-methoxyphenyl]sulfonyl]-3-m ethylthiourea) shows marked antifibrillatory activity in the dose range of 3 mg/kg to 10 mg/kg i.v. in various experimental models without affecting blood glucose levels. In order to investigate in a head to head comparison glibenclamide and HMR 1883 with respect to their influence on IPC, experiments were performed in rabbits with ischemia-reperfusion using myocardial infarct mass as final read out. Male New Zealand White rabbits (2.6-3.0 kg) were subjected to 30-min occlusion of a branch of the left descending coronary artery (LAD) followed by 2-h reperfusion. For IPC experiments the LAD was additionally occluded for two periods of 5 min, each followed by 10-min reperfusion, before the long-term ischemia. Infarct mass was evaluated by TTC staining and expressed as a percentage of area at risk. Rabbits (n=7/group) were randomly selected to receive (i.v.) saline vehicle 5 min prior to the 30-min occlusion period in infarct studies without IPC or to receive glibenclamide (0.3 mg/kg) or HMR 1883 (3 mg/kg) in IPC experiments, these substances being given 5 min prior to the first preconditioning or 5 min prior to the long-term ischemia of 30 min. Myocardial risk mass as a percentage of left ventricular mass did not differ between groups. The same was true for the ratio of left ventricular mass to 100 g body weight. Myocardial infarct mass as a percentage of the area at risk in the saline vehicle group without IPC was 41+/-3%. Whereas glibenclamide significantly increased infarct mass (from 41+/-3% to 55+/-4%), HMR 1883 did not affect it. IPC reduced infarct mass from 41+/-3% to 21+/-4% (P<0.05 vs. control without IPC). Glibenclamide given prior to IPC or prior to the long-term ischemia totally abolished the IPC effect (42+/-2% and 55+/-4%, respectively; P<0.05 vs. control). In contrast, HMR 1883 under the same conditions did not affect infarct size when given prior to IPC or prior to the long-term ischemia (21+/-3% and 26+/-2%, respectively). The monophasic action potential duration (MAP50) was reduced from 103+/-3 ms under normoxic conditions to 82+/-2 ms, 5 min after ischemia in the absence of drugs. This ischemia-induced shortening of the MAP was prevented by both HMR 1883 (MAP50 103+/-3 ms) and glibenclamide (MAP50 106+/-3 ms). In conclusion, although both K(ATP) channel blockers prevented ischemia-induced shortening of MAP, HMR 1883 did not abolish the beneficial effects of IPC on myocardial infarct mass in rabbits, whereas glibenclamide totally reversed this cardioprotective effect of IPC. This suggests that the sarcolemmal ATP-sensitive potassium channels are not involved in the mechanism of IPC.

K(ATP) channel blocker HMR 1883 reduces monophasic action potential shortening during coronary ischemia in anesthetised pigs.

ATP-sensitive potassium channels (KATP) open during myocardial ischemia. The ensuing repolarising potassium efflux shortens the action potential. Accumulation of extracellular potassium is able to partially depolarise the membrane, reducing the upstroke velocity of the action potential and thereby impairing impulse conduction. Both mechanisms are believed to be involved in the development of reentrant arrhythmias during cardiac ischemia. The sulfonylthiourea HMR 1883 (1-[[5-[2-(5-chloro-O-anisamido)ethyl]-methoxyphenyl]sulfonyl]-3-m ethylthiourea) was designed as a cardioselective KATP channel blocker for the prevention of arrhythmic sudden death in patients with ischemic heart disease. The aim of this study was to show that this compound, which has already shown antifibrillatory efficacy in dogs and rats, is able to inhibit ischemic changes of the action potential induced by coronary artery occlusion in anesthetised pigs. Action potentials were taken in situ with the technique of monophasic action potential (MAP) recording. In a control group (n=7), three consecutive occlusions of a small branch of the left circumflex coronary artery resulted in reproducible reductions in MAP duration and a decrease in upstroke velocity. In a separate group (n=7), HMR 1883 (3 mg/kg i.v.) significantly (P<0.05) reduced the ischemia-induced shortening of the MAP: during the first and second control occlusion of the coronary artery in the HMR 1883-group, MAP50 duration shortened from 218.5 +/- 3.0 ms to 166.7 +/- 3.3 ms and from 219.7 +/- 4.5 ms to 164.9 +/- 1.8 ms, respectively. After HMR 1883, during the third occlusion, MAP duration decreased from 226.9 +/- 3.6 ms to 205.3 +/- 4.3 ms only corresponding to 59% inhibition. HMR 1883 also improved the upstroke velocity of the MAP, which was depressed by ischemia: in the two preceding control occlusions ischemia prolonged the time to peak of the MAP, an index for upstroke velocity, from 10.83 +/- 0.43 ms to 39.42 +/- 1.60 ms and from 12.97 +/- 0.40 ms to 37.17 +/- 2.98 ms, respectively. With HMR 1883, time to peak during ischemia rose from 12.42 +/- 0.51 ms to 25.53+/-2.51 ms only, corresponding to an average inhibitory effect of 53.4%. The irregular repolarisation contour of the ischemic MAP was also improved. In conclusion, the present results indicate that HMR 1883 effectively blocks myocardial KATP channels during coronary ischemia in anesthetised pigs, preventing an excessive shortening of the action potential and improving excitation propagation.

Inhibition of IKs channels by HMR 1556.

Chromanol HMR 1556 [(3R,4S)-(+)-N-[3-hydroxy-2,2-dimethyl-6-(4,4,4-trifluorobutoxy)chroman-4-yl]-N-methylmethanesulfonamide], a novel inhibitor of the slow component of the delayed outward current in heart muscle cells (IKs), has been characterized in several in-vitro systems. mRNA encoding for the human protein minK was injected into Xenopus oocytes, leading to the expression of IKs channels. HMR 1556 inhibited this current half-maximally at a concentration of 120 nmol/l (IC50). Expression of the K+ channels Herg, Kv 1.5, Kv 1.3 and Kir2.1, and also the cationic current HCN2, were blocked little or not at all by 10 micromol/l HMR 1556. In isolated ventricular myocytes from the guinea pig the whole-cell patch-clamp method revealed inhibition of the IKs current with an IC50, of 34 nmol/l. Other current components, like IKr and IK1. were only slightly blocked at an HMR 1556 concentration of 10 micromol/l, whereas 10 micromol/l HMR 1556 inhibited the transient outward current I(to) and the sustained outward current I(sus) in rat ventricular myocytes by 25% and 36%, respectively. The L-type Ca2+ channel in guinea pig cardiomyocytes was blocked by 10 micromol/l HMR 1556 by 31%. Guinea pig right papillary muscles were investigated by the micropuncture technique at various pacing rates. In the frequency range of 0.5-7 Hz HMR 1556 (1 micromol/l) caused a prolongation of the action potential duration at 90% repolarization (APD90) by 19%-27%. In the presence of isoproterenol (10 micromol/l) the prolongation of the APD90 was more pronounced at low pacing rates (47% at 0.5 Hz and 35% at 1 Hz, compared with 25% at 7 Hz). The monophasic action potential was recorded in Langendorff-perfused guinea pig hearts. In spontaneously beating preparations, HMR 1556, at 0.1 micromol/l and 1 micromol/l, prolonged the MAPD90 by 3% and 10%, respectively, with no further prolongation at 10 micromol/l. The prolongation was much greater at low pacing rates [25% at 100 beats per min (bpm) and 13% at 150 bpm] than at fast pacing rates (9% at 350 bpm). The left ventricular pressure LVPmax was not affected at 1 micromol/l HMR 1556, but it decreased by 15% at 10 micromol/l. Other parameters, like the heart rate and coronary flow, were only slightly decreased at 1 micromol/l HMR 1556. In conclusion, HMR 1556 is a potent and selective inhibitor of the IKs current in guinea pig ventricular myocytes. The prolongation of the action potential duration is maintained at fast pacing rates.

Molecular basis, pharmacology and physiological role of cardiac K(ATP) channels.

ATP-dependent potassium (K(ATP)) channels exist in high density in the sarcolemmal membrane of heart muscle cells. Under normoxic conditions these channels are closed, but they become active when the intracellular ATP level falls. This leads to a shortening of the action potential duration, rendering the heart susceptible for life-threatening arrhythmias. Molecular biology has revealed that K(ATP) channels consist of heteromultimers of the inwardly rectifying channel Kir6.2 and the sulfonylurea receptor SUR. To date, three types of SURs were identified, representing the pancreatic (SUR1), the cardiac (SUR2A) and the smooth muscle (SUR2B) K(ATP) channel. In order to develop a novel therapeutic principle against ischemia-induced life-threatening arrhythmias leading to sudden cardiac death, the cardioselective K(ATP) channel blocker HMR 1883 was developed. This substance inhibits the sarcolemmal cardiac K(ATP) channel activated by the channel opener rilmakalim half-maximally at concentrations of 0.6-2.2 micromol/l, and substantially affects pancreatic K(ATP) channels at 9-50 times higher concentrations. K(ATP) channels of the coronary vascular system are only slightly blocked by HMR 1883 when activated by hypoxia. The substance was potently effective in preventing ventricular fibrillation in a conscious dog model, and thus can be considered to be a potential novel drug candidate against sudden cardiac death.

HMR 1883, a novel cardioselective inhibitor of the ATP-sensitive potassium channel. Part I: effects on cardiomyocytes, coronary flow and pancreatic beta-cells.

The novel sulfonylthiourea HMR 1883 was investigated in in vitro systems. The rilmakalim-induced shortening of the APD90 in guinea pig right papillary muscle at pHo = 6.0 was antagonized half-maximally by glibenclamide and HMR 1883 with 0.14 microM and 0. 6 microM, respectively. Hypoxia-induced shortening of the APD90 was significantly attenuated by the sulfonylureas when applied 60 min after induction of hypoxia. In isolated guinea pig ventricular myocytes the APD90 as well as the whole-cell current was measured with the patch-clamp technique. The rilmakalim-induced shortening of the APD90 was half-maximally antagonized by glibenclamide and HMR 1883 with 10 nM and 0.4 microM, respectively (pHo = 6.5). The rilmakalim-induced whole-cell current (at 0 mV clamp-potential) was inhibited by glibenclamide and HMR 1883 half-maximally with 20 nM and 0.8 microM, respectively (pHo = 7.4). In isolated perfused guinea pig hearts, the coronary flow (CF) was increased by perfusion with hypoxic solution (20% O2). Whereas 1 microM glibenclamide completely inhibited the hypoxia-induced increase in CF, 10 microM HMR 1883 reduced it by only 18%. Pancreatic effects were investigated in rat insulinoma cells (RINm5F), which were hyperpolarized with 100 microM diazoxide. Addition of glibenclamide or HMR 1883 depolarized the cell potential half-maximally with concentrations of 9 nM and approximately 20 microM, respectively. In conclusion, the sulfonylthiourea HMR 1883 blocks KATPs in cardiac muscle cells with 10-50 fold higher potency than in pancreatic beta-cells and has little effect on the coronary vascular system. Therefore, HMR 1883 has pharmacological selectivity for cardiac myocytes and thereby may be a promising substance for the prevention of ischemia-induced ventricular fibrillation.

KVLQT channels are inhibited by the K+ channel blocker 293B.

Previous data have indicated that the chromanol 293B blocks a cAMP activated K+ conductance in the colonic crypt, a K+ conductance in pig cardiac myocytes and the K+ conductance induced by IsK protein expression in Xenopus oocytes. We have also shown that cAMP-activated cystic fibrosis transmembrane conductance regulator (CFTR) up-regulates, apart from the typical Cl- current, a 293B- inhibitable K+ current. Very recently it has been shown that the IsK protein interacts with KVLQT subunits to produce a K+ channel. These data have prompted us to ask the following questions: Is the 293B-inhibitable current in oocytes expressing CFTR and activated by cAMP caused by an endogenous Xenopus KVLQT (XKVLQT), and is mouse KVLQT (mKVLQT) expressed in oocytes inhibited by 293B? Antisense and sense probes for XKVLQT were coinjected with CFTR cRNA into oocytes. After 3-4 days the oocytes were examined by two electrode voltage clamp. It was found that in control oocytes expressing CFTR and stimulated by isobutylmethylxanthine (IBMX, 1 mmol/l) 293B (10 micromol/l) reduced the conductance (Gm). In oocytes coinjected with the sense probe for XKVLQT and pretreated with IBMX 293B still reduced Gm, whilst the 293B-inhibitable Gm was almost completely absent in oocytes coinjected with XKVLQT antisense. In another series a full length clone for mKVLQT was generated by PCR techniques and the cRNA was injected into oocytes. After several days these oocytes, unlike water injected ones, were found to be strongly hyperpolarized and their Gm was increased significantly. The oocytes were depolarized significantly and their Gm was reduced reversibly by 10 micromol/l 293B. These data indicate that CFTR activation by IBMX indeed co-activates an endogenous oocyte XKVLQT channel and that this channel is inhibited by a new class of channel blockers, of which 293B is the prototype.

Effects of the Na+/H+-exchange inhibitor Hoe 642 on intracellular pH, calcium and sodium in isolated rat ventricular myocytes.

The inhibitors of the Na+/H+-exchange (NHE1) system Hoe 694 and Hoe 642 possess cardioprotective effects in ischaemia/reperfusion. It is assumed that these effects are due to the prevention of intracellular sodium (Nai) and calcium (Cai) overload. The purpose of the present study was to investigate the effects of Hoe 642 on intracellular pH, Na+ and Ca2+ (pHi, Nai and Cai) in isolated rat ventricular myocytes under anoxic conditions or in cells in which oxidative phosphorylation had been inhibited by 1.5 mmol/l cyanide. In cells which were dually loaded with the fluorescent dyes 2, 7-biscarboxyethyl-5,6-carboxyfluorescein (BCECF) and Fura-2, anoxia caused acidification of the cells (from pHi 7.2 to pHi 6.8) and an increase in Cai from about 50 nmol/l to about 1 micromol/l. The decrease in pHi began before the cells underwent hypoxic (rigor) contracture, whereas Cai only began to rise after rigor shortening had taken place. After reoxygenation, pHi returned to its control value and Cai oscillated and then declined to resting levels. It was during this phase that the cells rounded up (hypercontracture). When 10 micromol/l Hoe 642 was present from the beginning of the experiment, pHi and Cai were not significantly different from control experiments. At reoxygenation, pHi did not recover, but Cai oscillated and returned to its resting level. To monitor Nai, the cells were loaded with the dye SBFI. After adding 1.5 mmol/l cyanide or 100 micromol/l ouabain, Nai increased from the initial 8 mmol/l to approximately 16 mmol/l. Hoe 642 or Hoe 694 (10 micromol/l) did not prevent the increase in Nai. In contrast, the blocker of the persistent Na+ current R56865 (10 micromol/l) attenuated the CN--induced rise in Nai. The substance ethylisopropylamiloride was not used because it augmented considerably the intensity of the 380 nm wavelength of the cell's autofluorescence. In conclusion, the specific NHE1 inhibitor Hoe 642 did not attenuate anoxia-induced Cai overload, nor CN--induced Nai and Cai overload. Hoe 642 prevented the recovery of pHi from anoxic acidification. This low pHi maintained after reoxygenation may be cardioprotective. Other possible mechanisms of NHE1 inhibitors, such as prevention of Ca2+ overload in mitochondria, cannot be ruled out. The increase in Nai during anoxia is possibly due to an influx of Na+ via persistent Na+ channels.

Simultaneous recording of ATP-sensitive K+ current and intracellular Ca2+ in anoxic rat ventricular myocytes. Effects of glibenclamide.

We investigated the temporal relationship between the adenosine triphosphate-sensitive K current (KATP current), hypoxic shortening and Ca accumulation in cardiomyocytes exposed to anoxia or metabolic inhibition. Whole-cell, patch-clamp experiments were performed with nonstimulated isolated rat heart ventricular muscle cells loaded with the Ca-sensitive fluorescent dye 1-[2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy]-2-(2'- amino-5'-methylphenoxy) ethane-N,N,N',N'-tetraacetic acid (fura-2) via the patch pipette. After approximately 8 min anoxia, the KATP current started to rise and reached a maximum of 21.3 +/- 3.7 nA (n = 5, recorded at 0 mV clamp potential) within 1-3 min. At that time hypoxic contracture also occurred. Resting cytoplasmic free calcium (Cai) did not change significantly before hypoxic shortening. After hypoxic contracture, the KATP current decreased and Cai started to rise, reaching about 1 micromol/l. The presence of glibenclamide (10 micromol/l) in the bath reduced the anoxia-induced KATP current by more than 50%, but did not significantly influence the time dependence of current, hypoxic shortening and Cai, or the magnitude of Cai. Metabolic inhibition with 1.5 mmol/l CN resulted in KATP current increase and hypoxic shortening, occurring somewhat earlier than under anoxia, but all other parameters were comparable. In non-patch-clamped cells loaded with fura-2 AM ester and field-stimulated with 1 Hz, 1 micronol/l glibenclamide had no significant effect on the magnitude of the Cai increase caused by exposure of the cells to 1.5 mmol/l CN-. After CN- wash-out in non-patch-clamped cells, Cai declined, oscillated and finally returned to control values. It can be concluded that glibenclamide inhibits anoxia-induced KATP currents only partially and has no significant effect on anoxia-induced rise in resting Cai.

Adenosine triphosphate-dependent K currents activated by metabolic inhibition in rat ventricular myocytes differ from those elicited by the channel opener rilmakalim.

Adenosine triphosphate (ATP) dependent potassium channels (KATP channels) in heart ventricular muscle cells can be activated by depletion of intracellular ATP stores as well as by channel openers. In the present study we examined whether properties of KATP channels are dependent on the mode of activation. Whole-cell and single-channel currents were investigated by use of the patch-clamp technique in isolated ventricular rat myocytes. The channel opener rilmakalim dose dependently activated whole-cell currents [concentration for half-maximal activation (EC50) = 1.1 microM, Hill coefficient = 3.1, saturation concentration 10 microM]. Metabolic inhibition with 2-deoxy-D-glucose (10 mmol/l) also activated KATP currents after a time lag of several minutes. These currents were about two-fold higher than the rilmakalim-activated currents (rilmakalim-activated current 3.9 +/- 0.2 nA, 2-deoxy-D-glucose-activated current 8.1 +/- 0.9 nA; both recorded at 0 mV clamp potential). While the rilmakalim-activated current could be blocked completely and with high affinity by the sulphonylurea glibenclamide [concentration for half-maximal inhibition (IC50) = 8 nM, Hill coefficient = 0.7] the 2-deoxy-D-glucose-activated current could only be blocked partially (by maximally 46%) and higher glibenclamide concentrations were needed (IC50 = 480 nM, Hill coefficient = 0.8). The partial loss of blocking efficiency after metabolic inhibition was not restricted to glibenclamide but was also observed with the sulfonylureas glimepiride and HB 985, as well as with the non-sulfonylureas HOE 511 and 5-hydroxy-decanoate. Single-channel studies were in accordance with these whole-cell experiments. Both rilmakalim and metabolic inhibition with the uncoupler carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) activated single channels in the attached mode, where the number of current levels was significantly higher in the case of FCCP. Rilmakalim-activated channels were completely blocked by 10 microM glibenclamide, whereas several single-channel levels appeared in the presence of 100 microM glibenclamide after metabolic inhibition. In conclusion, after metabolic inhibition the amplitude of the activated KATP current is about twice as high as under saturating concentrations of the opener rilmakalim. Moreover, channels activated by metabolic inhibition lost part of their sensitivity to known channel blockers.

Protective effects of HOE642, a selective sodium-hydrogen exchange subtype 1 inhibitor, on cardiac ischaemia and reperfusion.

OBJECTIVE: The aim was to characterise the new compound HOE642 as a selective and cardioprotective Na+/H+ exchange inhibitor in various models. METHODS: The effect of HOE642 was tested in the osmotically activated Na+/H+ exchange of rabbit erythrocytes and in propionate induced swelling of human thrombocytes. Recovery of pH after an NH4Cl prepulse and effects on other ion transport systems by patch clamp technique were investigated in rat cardiomyocytes. NHE subtype specifity of the compound was determined by 22Na+ uptake inhibition in a fibroblast cell line separately expressing subtype isoforms 1-3. Protective effects of HOE642 in cardiac ischaemia and reperfusion by ligation of coronary artery were investigated in isolated working rat hearts and in anaesthetised rats. RESULTS: HOE642 concentration dependently inhibited the amiloride sensitive sodium influx in rabbit erythrocytes, reduced the swelling of human platelets induced by intracellular acidification, and delayed pH recovery in rat cardiomyocytes. In the isolated working rat heart subjected to ischaemia and reperfusion HOE642 dose dependently reduced the incidence and the duration of reperfusion arrhythmias. It also reduced the the release of lactate dehydrogenase and creatine kinase, and preserved the tissue content of glycogen, ATP, and creatine phosphate. In anaesthetised rats undergoing coronary artery ligation intravenous and oral pretreatment with HOE642 caused a dose dependent reduction or a complete prevention of ventricular premature beats, ventricular tachycardia, and ventricular fibrillation. The compound was well tolerated and neutral to circulatory variables. Other cardiovascular agents tested in this model were not, or were only partly, effective at doses showing marked cardiodepressive effects. CONCLUSIONS: HOE642 is a very selective NHE subtype 1 inhibitor showing cardioprotective and antiarrhythmic effects in ischaemic and reperfused hearts. Further development of well tolerated compounds like HOE642 could lead to a new therapeutic approach in clinical indications related to cardiac ischaemia and reperfusion.

Inhibition of K+ channels by chlorpromazine in rat ventricular myocytes.

Isolated rat ventricular myocytes were investigated with the whole-cell patch-clamp technique. Chlorpromazine inhibited inward-rectifying K+ currents (IC50 = 6.1 microM), time-independent outward currents (IC50 = 16 microM) and transient outward K+ currents. In the latter case, 100 microM of chlorpromazine reduced the amplitude of the peak current recorded at a clamp potential of 50 mV from 2.14 +/- 0.59 nA to 1.38 +/- 0.20 nA (n = 4) and decreased the time course of fast inactivation from 8.29 +/- 1.17 msec to 4.01 +/- 0.90 msec (n = 4). In addition, chlorpromazine blocked the ATP-dependent K+ current, which was activated either by the channel opener rilmakalim (10 microM) or by metabolic inhibition with carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP, 500 nM; IC50 for rilmakalim = 2.5 microM; IC50 for FCCP = 11.5 microM). The drug caused marked depolarization of the resting potential at higher concentrations (50 microM) from -79 +/- 3 mV to -27 +/- 11 mV (n = 4). The reversibility from channel block was slow and only partial for time-independent currents, especially inward-rectifying K+ currents, but it was relatively fast and complete for time-independent currents. Thus chlorpromazine blocks a variety of K+ channels in heart muscle cells. Inasmuch as the potency of inhibition is less than the previously reported use-dependent block of Na+ channels, the cardiovascular adverse effects of chlorpromazine are probably caused mainly by the latter effect.

Ca2+ influx through stretch-activated cation channels activates maxi K+ channels in porcine endocardial endothelium.

The endocardial endothelium is an important modulator of myocardial function. The present study demonstrates the existence of a stretch-activated Ca(2+)-permeable cation channel and of a Ca(2+)-activated K+ channel in the endocardial endothelium of the porcine right atrium. The stretch-activated channel is permeable for K+, Na+, Ca2+, and Ba2+, with mean conductances of approximately 32 pS for the monovalent cations and approximately 13 pS for divalent cations. The Ca(2+)-activated K+ channel has a mean conductance of 192 pS in symmetrical KCl. solution. Channel activity is strongly dependent on membrane potential and the cytosolic Ca2+ concentration. Half-maximal activation occurs at a cytosolic Ca2+ concentration of approximately 5 microM. The influx of Ca2+ through the stretch-activated channel is sufficient to activate the Ca(2+)-activated K+ channel in cell-attached patches. Upon activation of the stretch-activated channel, the cytosolic Ca2+ concentration increases, at least locally, to values of approximately 0.5 microM, as deduced from the open probability of the Ca(2+)-dependent K+ channel that was activated simultaneously. The stretch-activated channels are capable of inducing an intracellular Ca2+ signal and may have a role as mechanosensors in the atrial endothelium, possibly activated by atrial overload.

Enhancement of cytosolic calcium, prostacyclin and nitric oxide by bradykinin and the ACE inhibitor ramiprilat in porcine brain capillary endothelial cells.

We studied whether primary cultured porcine brain capillary endothelial cells (PBCEC) respond to bradykinin with an enhanced intracellular cytosolic calcium concentration [Ca2+]i with subsequent formation of nitric oxide (NO) and prostacyclin (PGI2). In addition we examined whether these cells synthetize and release kinins that may accumulate during angiotensin-converting enzyme (ACE) inhibition. [Ca2+]i was assessed by the fluorescent dye Fura-2, NO formation by determination of intracellular cyclic GMP and PGI2 by a specific radioimmunoassay for 6-ketoprostaglandin F1 alpha. Bradykinin and the ACE inhibitor ramiprilat concentration-dependently increased the formation of cyclic GMP which was completely prevented by the stereospecific inhibitor of NO synthase, NG-nitro-L-arginine. Also the specific B2-kinin receptor antagonist icatibant (Hoe 140) abolished the increase in cyclic GMP as well as the ramiprilat-induced increase in PGI2 formation. The data demonstrate the existence of B2-kinin receptors and ACE activity in PBCEC. Moreover PBCEC are capable of producing and releasing kinins in amounts that lead via stimulation of B2-kinin receptors to an enhanced [Ca2+]i as well as NO and PGI2 synthesis and release, provided that degradation of kinins is prevented by inhibition of endothelial ACE activity.

Effects of forskolin on crypt cells of rat distal colon. Activation of nonselective cation channels in the crypt base and of a chloride conductance pathway in other parts of the crypt.

We recently showed that prostaglandin E2 (PGE2) causes depolarization in cells at the base of isolated crypts from rat distal colon by activating nonselective cation channels. In order to investigate whether PGE2 acts via intracellular cyclic adenosine monophosphate (cAMP), the effect of forskolin on cell potential and on whole-cell current was investigated using the slow whole-cell patch-clamp method with nystatin. In addition, effects of forskolin in cells at other sites along the crypt were investigated. At the crypt base, the unstimulated cells had a resting potential of -70.6 +/- 1.3 mV (n = 25). When forskolin was added to the bath, the cells depolarized to -21.1 +/- 1.5 mV (n = 25). This depolarization was inhibited by substitution of all Na+ in the bath solution by N-methyl-D-glucamine (NMDG+) or by addition of flufenamic acid (50 mumol/l), a blocker of nonselective cation channels, to the bath. In contrast, the Cl- channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, 50 mumol/l) did not affect the depolarization. Moving along the crypt, the resting potential was -66.8 +/- 1.8 mV (n = 11) in the mid-crypt and -48.1 +/- 2.9 mV (n = 9) in cells of the upper part of the crypt. Forskolin caused a strong depolarization to about -20 mV in all parts of the crypt. In contrast to cells at the base, this depolarization was only partly diminished by substitution of Na+ by NMDG+, whereas substitution of bath Cl- by gluconate caused an initial further depolarization, followed by a repolarization to the cell's resting potential.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanosensitive nonselective cation channels in the antiluminal membrane of cerebral capillaries (blood-brain barrier).

Single stretch-activated (SA) cation channels have been investigated in the antiluminal membrane of freshly isolated brain capillaries. SA-channels did not distinguish between K+ and Na+ ions and were also permeable to Ca2+ and Ba2+ ions. With monovalent cations in the patch pipette the single-channel conductance was 37 pS and with the divalent cations Ba2+ and Ca2+ slope conductance was 16 and 19 pS, respectively. The open probability of the SA-channel increased with increasing negative pressure as well as with depolarization. Cell swelling induced by hypotonic shock activated the SA-channels in cell-attached experiments. The contribution of SA-channels to the regulation of cerebrospinal fluid in brain edema is discussed.