Heinrich Christian Englert

Cl(-)-channel blockers in the thick ascending limb of the loop of Henle. Structure activity relationship.

On the basis of our findings with diphenylamine-2-carboxylate [5] we have searched for compounds which possess an even higher affinity for the Cl−-channels in the basolateral membrane of the thick ascending limb of the loop of Henle. To quantitiy the inhibitory potency, we performed measurements of the equivalent short circuit current, corresponding to the secondary active transport of Cl− [8] and measurements of the voltage across the basolateral membrane. A survey of 219 compounds reveals that relatively simple modifications in the structure of diphenylamine-2-carboxylate led to very potent blockers such as 5-nitro-2-(3-phenylpropylamino)-benzoate which inhibits the short circuit current half maximally (IC50) at 8·10−8 mol/l. A comparison of the structural formula and the respective IC50 values leads to several empirical conclusions: 1. The potent compounds are lipophilic due to the apolar residue (e.g. phenyl- or cycloalkyl group). Replacing this part of the molecule by an aliphatic chain (up to 4 C-atoms) leads to inactive compounds. 2. Most of the inhibitors are secondary amines. Linking other than with-NH- between the phenyl ring and the benzoic acid results in inactive compounds. Tertiary amines, such as in case of 2-(N,N-diphenylamine) benzoic acid or N-methylphenylaminebenzoic acid are poorly active. 3. The carboxylate group of the benzoate moiety must be in ortho position to the amino group. 4. Introduction of substituents into the benzoate moiety e.g.-NO2 (in meta position to the carboxylate group), or by-Cl (in para position to the carboxylate group) results in an increase of inhibitory potency. 5. A-CH2-,-C2H4-,-C3H6-) spacer between the amino bridge and the phenyl ring increases the affinity for the Cl−-channel by several orders of magnitude. The above described structure activity relationship renders it likely that these chloride channel blockers possess several sites of interaction: The negatively charged carboxylate group, the secondary amine group which probably carries a positive partial charge, and for the very potent agents (nos. 130, 143, 144, and 145) an additional negative partial charge at the respective-Cl or-NO2 substituent. Finally, also an apolar interaction with an cycloalkyl or cycloaryl residue seems to be required, and this site of interaction has a defined spacing from the secondary amino nitrogen.

Diphenylamine-2-carboxylate, a blocker of the Cl−-conductive pathway in Cl−-transporting epithelia

The present study examines the effects of diphenylamine-2-carboxylate (DPC) in Cl−-transporting epithelia. This substance blocks reversibly the Cl−-conductance present under normal circumstances in the basolateral membrane of the thick ascending limb of the loop of Henle (TAL) and in the apical membrane of shark rectal gland tubules (RGT). This leads to a reduction in active NaCl reabsorption (TAL) and NaCl secretion (RGT) respectively, as measured by the equivalent short circuit current. The cells hyperpolarize as the membrane voltage drifts from the control value (some compromise between the chemical potential of Cl− and K+) towards the chemical potential of K+. The resistance of the basolateral (TAL) or apical membrane (RGT) increases and this leads to a moderate increase in transepithelial resistance. In addition, the Cl−-concentration step induced membrane voltage changes, which can be produced under control conditions, disappear in the presence of the blocker. Finally, experiments in excised membrane patches indicate that this substance inhibits the single current events of individual Cl−-channels.

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 dependency activated whole-cell currents [concentration for half-maximal activation (EC50) = 1.1 μM, Hill coefficient = 3.1, saturation concentration 10 μM]. 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 ±0.2nA, 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-hydroxydecanoate. 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 μM glibenclamide, whereas several single-channel levels appeared in the presence of 100 μM 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.

Specificity of Classical and Putative Cl- Transport Inhibitors on Membrane Transport Pathways in Human Erythrocytes

The majority of anion transport inhibitors tend to be non-specific. This is problematic from a research point of view as caution is required when defining pathways purely based on pharmacology. Here we have tested a range of classical and putative Cl- transport inhibitors on three Cl- carrier systems (the KCl cotransporter (KCC), the NaK2Cl cotransporter (NKCC), and the Band 3 anion exchanger (AE)) found in human erythrocytes, using radiolabel tracer experiments. The study confirms the cross-reactivity of many anion transport inhibitors. However, two compounds, H25 and H156, were found to be both potent (IC50 values < 0.1 mM) and specific (at least 1000-fold more effective against one carrier compared to the other two) inhibitors of NKCC and AE, respectively.

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.

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

ATP-dependent potassium (KATP) 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 KATP 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) KATP channel. In order to develop a novel therapeutic principle against ischemia-induced life-threatening arrhythmias leading to sudden cardiac death, the cardioselective KATP channel blocker HMR 1883 was developed. This substance inhibits the sarcolemmal cardiac KATP channel activated by the channel opener rilmakalim halfmaximally at concentrations of 0.6–2.2 μmol/l, and substantially affects pancreatic KATP channels at 9–50 times higher concentrations. KATP 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.

Airway pharmacology of the potassium channel opener, HOE 234, in guinea pigs: in vitro and in vivo studies

The smooth muscle relaxant effects of the novel potassium channel opener, HOE 234, were investigated in guinea pig airways and compared with those of lemakalim (BRL 38227). Both agents evoked concentration-related reduction in spontaneous tracheal tone or in the tone induced by histamine, prostaglandin E2 or carbachol. HOE 234 was more potent, particularly against carbachol, and was considerably longer acting than lemakalim in a wash-out experiment. On testing for preventive efficacy against histamine-induced bronchoconstriction in anaesthetized animals a dose-related decrease of pulmonary resistance (RL) was observed. HOE 234 given either intravenously (i.v.) or by inhalation was longer acting and 3 and 6 times more potent than lemakalim. Administration of 30 micrograms/kg i.v. HOE 234 during continuous bronchoconstriction maintained by infusion of histamine decreased RL for more than 20 min whereas the effect of 100 micrograms/kg i.v. lemakalin disappeared within 4 min. These results show that HOE 234 is effective against contractile response induced by asthma mediators in guinea pig airways and compares favourably with lemakalim. Moreover it acts on acute existing bronchospasm and therefore has the potential to act against asthma attacks.

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 l-[2-(5-carboxyoxazol-2-yl)-6-amino-benzofuran-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 μmol/l. The presence of glibenclamide (10μmol/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/1 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 μmol/1 glibenclamide had no significant effect on the magnitude of the Cai increase caused by exposure of the cells to 1.5 mmol/1 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 anoxiainduced rise in resting Cai.

Characterization of human sweat duct chloride conductance by chloride channel blockers

To characterize the chloride conductance of human sweat duct the effect of various analogues of diphenylamine-2-carboxylate was investigated on the transepithelial potential difference (PDT) and resistance (RT) of isolated microperfused sweat ducts. Although the most powerful analogues which block Cl− channels in various secretory and absorptive epithelia were ineffective, a number of analogues (in particular Cl substituted ones) were found which at high concentrations significantly and reversibly increased PDT andRT. The data suggest that the main chloride conductance pathway of sweat duct epithelium resides in the cell membranes rather than in the tight junctions. In addition the different blocking spectra of the chloride conductances of sweat duct and tracheal epithelium (Welsh MJ, Science 232:1648, 1986) suggest that the combined impairment of both conductances in cystic fibrosis does not result from a molecular defect in the Cl− channels.

Blockers of the ATP-Sensitive Potassium Channel SUR2A / Kir6.2: A New Approach to Prevent Sudden Cardiac Death

The cardiac ATP sensitive potassium channel (K(ATP) channel) SUR2A/Kir6.2 is an emerging target for antiarrhythmic intervention. This channel accounts for known electrophysiological derangements soon after the onset of myocardial ischemia. Consequently, blockers of this channel have the potential to prevent ischemic malignant arrhythmias and sudden cardiac death in humans. Since cardiac K(ATP) channels are closed at physiological intracellular ATP concentrations (ATP(i)) and open only when ATP(i) falls below a critical value, these agents do not affect the normal cardiac action potential and should be devoid of proarrhythmic side effects. Due to the existence of isoforms of this channel, mainly in vascular smooth muscle cells, pancreatic beta-cells and cardiac mitochondria, only specific blockers of SUR2A/Kir6.2 will offer a reasonable option for the treatment of cardiovascular patients at risk of sudden cardiac death. Presently known K(ATP) blockers are derived from diverse classes of compounds with antidiabetic sulfonylureas being their most prominent members. Retrospective evaluations of clinical studies with the sulfonylurea glibenclamide in diabetics revealed antifibrillatory activity to be an important additional effect of this class of compounds. However, for the safe treatment of arrhythmias nearly all presently known blockers lack sufficient selectivity, either within the target family or with respect to other ion channels modulating the cardiac action potential. The present article illustrates the new principle in terms of molecular biology and electrophysiology and summarizes all presently known K(ATP) blockers. As a highlight, first strategies to come to selective SUR2A/Kir6.2 blockers, such as HMR 1883, are reviewed.