Hans Jochen Lang

Effects of the chromanol 293B, a selective blocker of the slow, component of the delayed rectifier K+ current, on repolarization in human and guinea pig ventricular myocytes

OBJECTIVES The slow component of the delayed rectifier K+ current (IKs) is believed to be important in cardiac repolarization, and may be a potential target for antiarrhythmic drugs, but its study has been limited by a lack of specific blockers. The chromanol derivate 293B blocks currents expressed by minK and not HERG in Xenopus oocytes, but little is known about its effects on native currents and action potentials. We aimed to establish the effects of 293B on K+, Na+ and Ca2+ currents and action potentials in human and guinea pig cardiomyocytes. METHODS Whole-cell patch clamp techniques were applied to assess the effects of 293B on isolated myocytes at 36 degrees C. RESULTS Delayed rectifier current (IK) elicited by pulses to +60 mV from a holding potential of -50 mV in guinea pig myocytes was strongly inhibited by 293B (maximum inhibition 96.9 +/- 0.8%; 50% inhibitory concentration, EC50, 1.02 microM), but IK during pulses to -10 mV was unaffected (3.9 +/- 8.4% inhibition at 50 microM). Half-activation voltages, current-voltage relations, and current densities of drug-resistant and drug-sensitive IK correspond to those of IKr and IKs respectively. Inward rectifier K+ current, Na+ current and L-type Ca2+ current were unaffected by 293B. Transient outward current in human ventricular myocytes was inhibited by 293B at an EC50 of 24 microM, less than one twentieth the potency for IKs inhibition in guinea pig myocytes. While dofetilide prolonged action potential duration (APD) with strong reverse use dependence, 293B prolonged guinea pig and human ventricular APD to a similar fractional extent at all frequencies. CONCLUSIONS 293B is a selective IKs blocker, and the frequency dependence of APD prolongation caused by this IKs blocker is different from that caused by IKr blockade: 293B may be an interesting tool to study the physiologic role of IKs and the antiarrhythmic potential of IKs blockade.

Hoe 694, a new Na+/H+ exchange inhibitor and its effects in cardiac ischaemia

1 The benzoylguanidine derivative Hoe 694 ((3‐methylsulphonyl‐4‐piperidino‐benzoyl) guanidine methanesulphonate) was characterized as an inhibitor of Na+/H+ exchange in rabbit erythrocytes, rat platelets and bovine endothelial cells. The potency of the compound was slightly lower or comparable to ethylisopropyl amiloride (EIPA). 2 To investigate a possible cardioprotective role of the Na+/H+ exchange inhibitor Hoe 694, rat isolated working hearts were subjected to ischaemia and reperfusion. In these experiments all untreated hearts suffered ventricular fibrillation on reperfusion. Addition of 10−7 m Hoe 694 to the perfusate almost abolished reperfusion arrhythmias in the rat isolated working hearts. 3 Hoe 694 reduced the release of lactate dehydrogenase (LDH) and creatine kinase (CK), which are indicators of cellular damage during ischaemia, into the venous effluent of the hearts by 60% and 54%, respectively. 4 The tissue content of glycogen at the end of the experiments was increased by 60% and the high energy phosphates ATP and creatine phosphate were increased by 240% and 270% respectively in the treated hearts as compared to control hearts. 5 Antiischaemic effects of the Na+/H+ exchange inhibitor, Hoe 694, were investigated in a second experiment in anaesthetized rats undergoing coronary artery ligation. In these animals, pretreatment with Hoe 694 caused a dose‐dependent reduction of ventricular premature beats and ventricular tachycardia as well as a complete suppression of ventricular fibrillation down to doses of 0.1 mg kg−1, i.v. Blood pressure and heart rate remained unchanged. 6 We conclude that the new Na+/H+ exchange inhibitor, Hoe 694, shows cardioprotective and antiarrhythmic effects in ischaemia and reperfusion in rat isolated hearts and in anaesthetized rats. In view of the role which Na+/H+ exchange seems to play in the pathophysiology of cardiac ischaemia these effects could probably be attributed to Na+/H+ exchange inhibition.

A new class of inhibitors of cAMP-mediated Cl− secretion in rabbit colon, acting by the reduction of cAMP-activated K+ conductance

Previously we have shown that arylamino-benzoates like 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), which are very potent inhibitors of NaCl absorption in the thick ascending limb of the loop of Henle, are only poor inhibitors of the cAMP-mediated secretion of NaCl in rat colon. This has prompted our search for more potent inhibitors of NaCl secretion in the latter system. The chromanole compound 293 B inhibited the equivalent short-circuit current (Isc) induced by prostaglandin E2 (n=7), vasoactive intestinal polypeptide (VIP,n=5), adenosine (n=3), cholera toxin (n=4) and cAMP (n=6), but not by ionomycin (n=5) in distal rabbit colon half maximally (IC50) at 2 μmol/l from the mucosal and at 0.7 μmol/l from the serosal side. The inhibition was reversible and paralleled by a significant increase in transepithelial membrane resistance [e.g. in the VIP series from 116±16 Ω·cm2 to 136±21 Ω·cm2 (n=5)]. A total of 25 derivatives of 293 B were examined and structure activity relations were obtained. It was shown that the racemate 293 B was the most potent compound with-in this group and that its effect was due to the enantiomer 434 B which acted half maximally at 0.25 μmol/l. Further studies in isolated in vitro perfused colonic crypts revealed that 10 μmol/l 293 B had no effect on the membrane voltage across the basolateral membrane (Vbl) in non-stimulated crypt cells: −69±3 mV versus −67±3 mV (n=10), whilst in the same cells 1 mmol/l Ba2+ depolarised (Vbl) significantly. However, 293 B depolarised (Vbl) significantly in the presence of 1 μmol/l forskolin: −45±4mV versus −39±5 mV (n=7). Similar results were obtained with 0.1 mmol/l adenosine. 293 B depolarised (Vbl) from −40±5 mV to −30±4 mV (n=19). This was paralleled by an increase in the fractional resistance of the basolateral membrane. VIP had a comparable effect. The hyperpolarisation induced by 0.1 mmol ATP was not influenced by 10 μmol/l 293 B: −75±6 mV versus −75±6 mV (n=6). Also 293 B had no effect on basal K+ conductance (n=4). Hence, we conclude that 293 B inhibits the K+ conductance induced by cAMP. This conductance is apparently relevant for Cl− secretion and the basal K+ conductance is insufficient to support secretion.

DCPIB is a novel selective blocker of ICl,swell and prevents swelling‐induced shortening of guinea‐pig atrial action potential duration

We identified the ethacrynic‐acid derivative DCPIB as a potent inhibitor of ICl,swell, which blocks native ICl,swell of calf bovine pulmonary artery endothelial (CPAE) cells with an IC50 of 4.1 μM. Similarly, 10 μM DCPIB almost completely inhibited the swelling‐induced chloride conductance in Xenopus oocytes and in guinea‐pig atrial cardiomyocytes. Block of ICl,swell by DCPIB was fully reversible and voltage independent. DCPIB (10 μM) showed selectivity for ICl,swell and had no significant inhibitory effects on ICl,Ca in CPAE cells, on chloride currents elicited by several members of the CLC‐chloride channel family or on the human cystic fibrosis transmembrane conductance regulator (hCFTR) after heterologous expression in Xenopus oocytes. DCPIB (10 μM) also showed no significant inhibition of several native anion and cation currents of guinea pig heart like ICl,PKA, IKr, IKs, IK1, INa and ICa. In all atrial cardiomyocytes (n=7), osmotic swelling produced an increase in chloride current and a strong shortening of the action potential duration (APD). Both swelling‐induced chloride conductance and AP shortening were inhibited by treatment of swollen cells with DCPIB (10 μM). In agreement with the selectivity for ICl,swell, DCPIB did not affect atrial APD under isoosmotic conditions. Preincubation of atrial cardiomyocytes with DCPIB (10 μM) completely prevented both the swelling‐induced chloride currents and the AP shortening but not the hypotonic cell swelling. We conclude that swelling‐induced AP shortening in isolated atrial cells is mainly caused by activation of ICl,swell. DCPIB therefore is a valuable pharmacological tool to study the role of ICl,swell in cardiac excitability under pathophysiological conditions leading to cell swelling.

Heteromeric KCNE2/KCNQ1 potassium channels in the luminal membrane of gastric parietal cells

Recently, we and others have shown that luminal K+ recycling via KCNQ1 K+ channels is required for gastric H+ secretion. Inhibition of KCNQ1 by the chromanol 293B strongly diminished H+ secretion. The present study aims at clarifying KCNQ1 subunit composition, subcellular localization, regulation and pharmacology in parietal cells. Using in situ hybridization and immunofluorescence techniques, we identified KCNE2 as the β subunit of KCNQ1 in the luminal membrane compartment of parietal cells. Expressed in COS cells, hKCNE2/hKCNQ1 channels were activated by acidic pH, PIP2, cAMP and purinergic receptor stimulation. Qualitatively similar results were obtained in mouse parietal cells. Confocal microscopy revealed stimulation‐induced translocation of H+,K+‐ATPase from tubulovesicles towards the luminal pole of parietal cells, whereas distribution of KCNQ1 K+ channels did not change to the same extent. In COS cells the 293B‐related substance IKs124 blocked hKCNE2/hKCNQ1 with an IC50 of 8 nm. Inhibition of hKCNE1‐ and hKCNE3‐containing channels was weaker with IC50 values of 370 and 440 nm, respectively. In conclusion, KCNQ1 coassembles with KCNE2 to form acid‐activated luminal K+ channels of parietal cells. KCNQ1/KCNE2 is activated during acid secretion via several pathways but probably not by targeting of the channel to the membrane. IKs124 could serve as a leading compound in the development of subunit‐specific KCNE2/KCNQ1 blockers to treat peptic ulcers.

Different types of blockers of the intermediate-conductance outwardly rectifying chloride channel in epithelia.

Epithelial chloride channels can be blocked by various inhibitors, which show considerable differences in their molecular structure. In the present patch-clamp study, we compared different blockers of one type of epithelial Cl− channel with respect to their inhibitory potency. We applied the blockers to excised inside-out-or outside-out-oriented membrane patches of cultured HT29 colon carcinoma and respiratory epithelial cells (REC) containing the outwardly rectifying intermediate-conductance (ICOR) chloride channel. Four types of inhibitory compounds were tested: stilbene disulphonate derivatives, indanyloxyacetic acid, amidine, and arylaminobenzoates. The concentrations for half-maximal inhibition (IC50) for the different channel blockers were (μmol/l): 4-acetamido-4′-isothiocyanato-stilbene-2,2′-disulphonic acid 100; 4,4′-diisothiocyanato-stilbene-2,2′-disulphonic acid 80; indanyloxyacetic acid 9; 4,4′-dinitrostilbene-2, 2′-disulphonic acid 8; amidine 8 and 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) 0.9. All compounds, when applied to the cytosolic side of the channel, induced a flicker-type block of the ICOR Cl− channel at lower concentrations and a complete channel inhibition at higher concentrations. The inhibitory potency of NPPB was much higher when it was added to the external surface of the channel in outside-out-oriented membrane patches. At 1 μmol/l the inhibition was complete. All blocker effects were fully reversible. The probe with the highest affinity (NPPB) and a closely related compound 5-nitro-2-(3-phenylethylamino)-benzoate (NPFB) were used to construct macromolecular probes by linking these blockers to aminopolyethyleneglycol (PEG) or aminoethyl-O-dextran (5 kDa). These macromolecular NPPB and NPEB derivatives inhibited the ICOR Cl− channels only from the outside but had no effect on the cytosolic side. In the case of PEG-NPPB an IC50 of 30 nmol/l was determined in outside-out patches. The data indicate that the interaction site for arylaminobenzoates is accessible from the outer aspects of the Cl− channel facing the extracellular medium. Furthermore, these data show that the macromolecular probes of arylaminobenzoates have affinities to the Cl− channel very similar to those of the respective parent compounds.

Effects of a New Na+/h+ Antiporter Inhibitor on Postischemic Reperfusion in Pig Heart

Summary We investigated the effects of a new compound (3-methylsulfonyl-4-piperidinobenzoyl) guanidine hydrochloride (HOE 694) known to inhibit the Na+/H + exchanger in a porcine model of ischemia/reperfusion. Ischemia was induced by coronary occlusion (twice for 10 min, with a 30-min reperfusion interval) followed by a 4-h reperfusion period. Treated animals (n = 8) received HOE 694 as a bolus (7 mg/kg) 20 min before ischemia and subsequently as a continuous infusion (0.07 mg/kg) throughout the experiment. Control pigs (n = 11) received vehicle. Regional wall function (percentage of segment shortening, % SS) of the treated animals was significantly improved as compared with that of controls after the 4-h reperfusion period (74.1 ± 2.5 vs. 50.9 ± 5.4, p < 0.005). Ventricular fibrillation (VF) could be prevented completely in treated pigs but occurred in 9 of 11 control animals (p < 0.001). Ultrastructural changes after ischemia and reperfusion were moderate and slightly abnormal in controls but much milder and completely recovered in the treated group, respectively. The tissue content of high-energy phosphates did not show a significant difference between groups. Inhibition of the sarcolemmal Na+/ H+ antiporter with HOE 694 is antiarrhythmic and diminishes myocardial ischemic cell injury by preventing Na+ overload.

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

Abstract 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 μmol/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 μmol/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 μmol/l ouabain, Nai increased from the initial 8 mmol/l to approximately 16 mmol/l. Hoe 642 or Hoe 694 (10 μmol/l) did not prevent the increase in Nai. In contrast, the blocker of the persistent Na+ current R56865 (10 μmol/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.

Effects of Na+/H+ exchange inhibitors in cardiac ischemia

To investigate a possible protective role of Na+/H+ exchange inhibition under ischemic conditions isolated rat hearts were subjected to regional ischemia and reperfusion. In these experiments all 6 untreated hearts suffered ventricular fibrillation on reperfusion. Addition of 1 x 10(-5) mol/l amiloride or 3 x 10(-7) mol/l 5-(N-ethyl-N-isopropyl)amiloride (EIPA) markedly decreased the incidence and duration of ventricular fibrillation or even suppressed fibrillation completely as in the case of 1 x 10(-6) mol/l EIPA. Both compounds diminished the activities of lactate dehydrogenase and creatine kinase in the venous effluent of the hearts during ischemia. At the end of the experiments tissue contents of glycogen, ATP and creatine phosphate were increased in the treated hearts as compared to control hearts. In an additional experiment the beneficial effects of Na+/H+ exchange inhibition during ischemia was confirmed in vivo with anaesthetized rats undergoing coronary artery ligation. In these animals amiloride or EIPA pretreatment caused a marked reduction of ventricular premature beats and ventricular tachycardia as well as a complete suppression of ventricular fibrillation. The concentration dependent inhibition of Na+ influx via Na+/H+ exchange by amiloride and EIPA was investigated in erythrocytes from hypercholesterolemic rabbits with Na+/H+ exchange activated by exposure to hyperosmotic medium. Furthermore the inhibition of Na+ influx by EIPA after intracellular acidification was studied in cardiac myocytes of neonatal rats. Both agents were effective in the same order of potency in the ischemic isolated working rat heart as in the erythrocyte model in which they inhibited Na+/H+ exchange.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of arylaminobenzoate-type chloride channel blockers on equivalent short-circuit current in rabbit colon

Arylaminobenzoates were examined in rabbit colon mounted in an Ussing chamber. The open-circuit transepithelial voltage (Vte) and resistance (Rte) were measured and the equivalent short-circuit current (ISC=Vte/ Rte) was calculated. After serosal (s) and mucosal (m) addition of indomethacin (1 μmol/l) ISC was −71±11 (n = 118) μA/cm2. Amiloride (0.1 mmol/l, m) inhibited this current and reversed the polarity to + 32±4 (n=118) μA/cm2. In the presence of amiloride and indomethacin, prostaglandin E2 (1 μmol/l, s), known to induce Cl− secretion, generated an ISC of -143 ± 8 (n = 92) μA/cm2. The arylaminobenzoate and Cl− channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) reduced ISC reversibly with a half-maximal inhibition (IC50) at approximately 0.35 mmol/l and 0.2 mmol/l for mucosal and serosal application respectively. To test whether the poor effect was caused by mucus covering the luminal surface, dose/response curves of the mucosal effect were repeated after several pretreatments. Acidic pH on the mucosal side reduced IC50 to approximately 0.1 mmol/l. A similar effect was observed after N-acetyl-l-cysteine (m) preincubation. Pretreatment with N-acetyl-l-cysteine (m) and carbachol (s), in order to exhaust mucus secretion, and l-homocysteine (m) were more effective and reduced IC50 to approximately 50 μmol/l. To test whether this effect of NPPB was caused by non-specific effects, the two enantiomers of 5-nitro-2-(+/−1-phenylethylamino)-benzoate were tested of which only the (+) form inhibited the Cl− conductance in the thick ascending limb of the loop of Henle (TAL). In the present study the (+) enantiomer inhibited significantly more strongly than the (−) form. This suggests that the inhibitory effect of NPPB, even though it requires rather high concentrations, is probably due to Cl− channel inhibition. For other arylaminobenzoates the sequence of potencies was different from that determined for the TAL. The present data indicate that substances that have been designed to block the Cl− conductance of the TAL segment also inhibit reversibly but with much lower affinity the PGE2-induced Cl− secretion in rabbit colon.