Selective blockade of endothelial Ca2+-activated small- and intermediate-conductance K+-channels suppresses EDHF-mediated vasodilation

1. Activation of Ca(2+)-activated K(+)-channels (K(Ca)) has been suggested to play a key role in endothelium-derived hyperpolarizing factor (EDHF)-mediated vasodilation. However, due to the low selectivity of commonly used K(Ca)-channel blockers it is still elusive which endothelial K(Ca)-subtypes mediate hyperpolarization and thus initiate EDHF-mediated vasodilation. 2. Using the non-cytochrome P450 blocking clotrimazole-derivatives, 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (TRAM-34) and 2-(2-chlorophenyl)-2,2-diphenylacetonitrile (TRAM-39) as highly selective IK1-inhibitors, we investigated the role of the intermediate-conductance K(Ca) (rIK1) in endothelial hyperpolarization and EDHF-mediated vasodilation. 3. Expression and function of rIK1 and small-conductance K(Ca) (rSK3) were demonstrated in situ in single endothelial cells of rat carotid arteries (CA). rIK1-currents were blocked by TRAM-34 or TRAM-39, while rSK3 was blocked by apamin. In current-clamp experiments, endothelial hyperpolarization in response to acetylcholine was abolished by the combination of apamin and TRAM-34. 4. In phenylephrine-preconstricted CA, acetylcholine-induced NO and prostacyclin-independent vasodilation was almost completely blocked by ChTX, CLT, TRAM-34, or TRAM-39 in combination with the SK3-blocker apamin. Apamin, TRAM-34, and CLT alone or sulphaphenzole, a blocker of the cytochrome P450 isoform 2C9, were ineffective in blocking the EDHF-response. 5. In experiments without blocking NO and prostacyclin synthesis, the combined blockade of SK3 and IK1 reduced endothelium-dependent vasodilation. 6. In conclusion, the use of selective IK1-inhibitors together with the SK3-blocker apamin revealed that activation of both K(Ca), rIK1 and rSK3 is crucial in mediating endothelial hyperpolarization and generation of the EDHF-signal while the cytochrome P450 pathway seems to play a minor or no role in rat CA.     

Role of nitric oxide and K+-channels in vascular hyporeactivity induced by endotoxin

This study was to investigate possible mechanisms associated with vascular hyporeactivity to vasoconstrictor agents in rats with endotoxaemia. Wistar-Kyoto rats were anaesthetised and injected with endotoxin [E. coli lipopolysaccharide (LPS); 10 mg/kg, i.v.] for 4 h. Pressor responses to noradrenaline (NA; 1 μg/kg, i.v.) were determined prior to and at every hour after LPS injection. After the in vivo experiment, rat thoracic aortas were excised and prepared as rings 3–4 mm in width. The endothelium was mechanically removed to evaluate K⁺-channel activity and the effects of nitric oxide (NO) on the vascular smooth muscle. Our results demonstrated that: (1) injection of LPS caused a significant fall in blood pressure and a severe vascular hyporeactivity to NA in the anaesthetised rat, (2) the relaxation induced by the K⁺-channel opener cromakalim was greater in rings obtained from endotoxaemic rats and this enhanced relaxation was partially inhibited by pretreatment of these rings with 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of the NO/cGMP pathway, (3) endotoxaemia for 4 h was also associated with a profound vascular hyporeactivity to NA ex vivo and this vascular hyporesponsiveness was partially inhibited by ODQ, tetraethylammonium (TEA, a non-selective inhibitor of K⁺-channels) and charybdotoxin [CTX, a selective inhibitor of large conductance calcium-activated K⁺ channels (BK_Ca)], but not by apamin, and (4) the combination of TEA or CTX with ODQ completely restored that vascular responsiveness to normal. These results suggest that activation of BK_Ca and overproduction of NO in the vascular smooth muscle simultaneously contribute to vascular hyporeactivity to vasoconstrictor agents in endotoxaemia. Received: 14 December 1998 / Accepted: 22 March 1999     

Spasmogen action in guinea-pig isolated trachealis: involvement of membrane K+-channels and the consequences of K+-channel blockade.

1. Acetylcholine (ACh), histamine, prostaglandin E2 and potassium chloride (KCl) each evoked concentration-dependent spasm of guinea-pig isolated trachealis treated with indomethacin (2.8 microM). 2. Neither tetraethylammonium (TEA; 0.1-10 mM) nor procaine (0.1-10 mM) potentiated these spasmogens. Indeed, procaine (10 mM) depressed the log concentration-effect curves of all the spasmogens while TEA (1-10 mM) caused some depression of the log concentration-effect curve of prostaglandin E2. 3. Intracellular electrophysiological recording was performed in trachealis bathed by normal Krebs solution or by Krebs solution containing 2.8 microM indomethacin. In either medium the majority of trachealis cells exhibited spontaneous electrical slow waves while some cells were electrically quiescent. In either medium the spasmogenic effects of ACh (1 mM) and histamine (0.2 mM) were accompanied by depolarization and abolition of slow wave discharge. In many cases the record of membrane potential subsequently exhibited noise which incorporated fast, hyperpolarizing transients. 4. In the absence and presence of indomethacin, TEA (10 mM) and procaine (5 mM) markedly reduced the membrane noise and hyperpolarizing transients evoked by ACh or histamine without augmenting the evoked tension. 5. It is concluded that slow wave discharge does not depend on prostaglandin synthesis. The membrane noise and hyperpolarizing transients evoked by ACh and histamine represent the opening of membrane K+-channels. While such K+-channel opening may offset spasmogen-induced depolarization it does not moderate the evoked tension.     

NA+- and K+-channels as molecular targets of the alkaloid ajmaline in skeletal muscle fibres

BACKGROUND AND PURPOSE: Ajmaline is a widely used antiarrhythmic drug. Its action on voltage-gated ion channels in skeletal muscle is not well documented and we have here elucidated its effects on Na(+) and K(+) channels. EXPERIMENTAL APPROACH: Sodium (I(Na)) and potassium (I(K)) currents in amphibian skeletal muscle fibres were recorded using 'loose-patch' and two-microelectrode voltage clamp techniques (2-MVC). Action potentials were generated using current clamp. KEY RESULTS: Under 'loose patch' clamp conditions, the IC(50) for I(Na) was 23.2 microM with Hill-coefficient h=1.21. For I(K), IC(50) was 9.2 microM, h=0.87. Clinically relevant ajmaline concentrations (1-3 microM) reduced peak I(Na) by approximately 5% but outward I(K) values were reduced by approximately 20%. Na(+) channel steady-state activation and fast inactivation were concentration-dependently shifted towards hyperpolarized potentials ( approximately 10 mV at 25 microM). Inactivation curves were markedly flattened by ajmaline. Peak-I(K) under maintained depolarisation was reduced to approximately 30% of control values by 100 microM ajmaline. I(K) activation time constants were increased at least two-fold. Lower concentrations (10 or 25 microM) reduced steady-state-I(K) slightly but peak-I(K) significantly. Action potential generation threshold was increased by 10 microM ajmaline and repolarisation prolonged. CONCLUSIONS AND IMPLICATIONS: Ajmaline acts differentially on Na(+) and K(+) channels in skeletal muscle. This suggests at least multiple sites of action including the S4 subunit. Our data may provide a first insight into specific mechanisms of ajmaline-ion channel interaction in tissues other than cardiac muscle and could suggest possible side-effects that need to be further evaluated.     

Impaired small-conductance Ca2+-activated K+ channel-dependent EDHF responses in Type II diabetic ZDF rats

We have examined the relative contributions of small- and intermediate-conductance Ca(2+)-activated K(+) channels (SK(Ca) and IK(Ca)) to the endothelium-derived hyperpolarizing factor (EDHF) pathway response in small mesenteric arteries of Zucker Diabetic Fatty (ZDF) rats, before and after the development of Type II diabetes, together with Lean controls. Smooth muscle membrane potential was recorded using sharp microelectrodes in the presence of 10 microM indomethacin plus 100 microM N(omega)-nitro-L-arginine. SK(Ca) was selectively inhibited with 100 nM apamin, whereas IK(Ca) was blocked with 10 microM TRAM-39 (2-(2-chlorophenyl)-2,2-diphenylacetonitrile). Resting membrane potentials were similar in arteries from 17- to 20-week-old control and diabetic rats (approximately -54 mV). Responses elicited by 1 and 10 microM acetylcholine (ACh) were significantly smaller in the diabetic group (e.g. hyperpolarizations to -69.5 +/- 0.8 mV (ZDF; n = 12) and -73.2 +/- 0.6 mV (Lean; n = 12; P < 0.05) evoked by 10 microM ACh). The IK(Ca)-mediated components of the ACh responses were comparable between groups (hyperpolarizations to approximately -65 mV on exposure to 10 microM ACh). However, SK(Ca)-mediated responses were significantly reduced in the diabetic group (hyperpolarizations to -63.1 +/- 1.0 mV (ZDF; n = 6) and -71.5 +/- 1.2 mV (Lean; n = 6; P < 0.05) on exposure to 10 microM ACh. Impaired ACh responses were not observed in arteries from 5- to 6-week-old (pre-diabetic) animals. SK(Ca) subunit mRNA expression was increased in the diabetic group. The EDHF pathway, especially the SK(Ca)-mediated response, is impaired in Type II diabetic ZDF rats without a reduction in channel gene expression. These results may be particularly relevant to the microvascular complications of diabetes. The functional separation of SK(Ca) and IK(Ca) pathways is discussed.     

Role of ATP-sensitive K+ -channels in hemodynamic effects of peroxynitrite in anesthetized rats

The aim of this study was to determine whether the hypotensive and vasodilator actions of peroxynitrite in pentobarbital-anesthetized rats involve the activation of ATP-sensitive K+-channels (K+ATP-channels). The effects of the K+ATP-channel agonist, cromakalim (9-36 microg/kg, iv), peroxynitrite (0.5-10 micromol/kg iv), and L-S-nitrosocysteine (12.5-200 nmol/kg, iv) on mean arterial blood pressure (MAP) and mesenteric (MR) and hindquarter (HQR) vascular resistances were determined before and after injection of the K+ATP-channel blocker, glibenclamide (40 micromol/kg, iv). Cromakalim, peroxynitrite, and L-S-nitrosocysteine produced dose-dependent reductions in MAP, MR, and HQR. Administration of glibenclamide did not affect resting hemodynamic parameters but markedly attenuated the hemodynamic actions of cromakalim. The maximal falls in MAP and HQR produced by peroxynitrite were attenuated by glibenclamide whereas the maximal falls in MR were not affected. In addition, the duration of the hypotensive and vasodilator effects of peroxynitrite in the mesenteric and hindquarter beds were markedly diminished by glibenclamide. In contrast, glibenclamide did not affect the maximal hypotensive or vasodilator effects of L-S-nitrosocysteine or the duration of these responses. These results suggest that the hypotensive and vasodilator actions of peroxynitrite in anesthetized rats involve the activation of K+ATP-channels whereas the hemodynamic actions of L-S-nitrosocysteine do not.     

Restricted usefulness of tetraethylammonium and 4-aminopyridine for the characterization of receptor-operated K+-channels.

1. Recently, we suggested that the D2-dopamine receptor involved in the inhibition of evoked [3H]-acetylcholine release from rat striatum is coupled to K+-channels. 2. In the present study, an attempt was made to elucidate further the role of these K+-channels, using the K+-channel blocking agents tetraethylammonium and 4-aminopyridine. With a superfusion method, the effects of both drugs on the D2-dopamine receptor-mediated inhibition of the electrically evoked release of [3H]-acetylcholine from rat striatal tissue slices was investigated. 3. Both tetraethylammonium (30 mM) and 4-aminopyridine (0.1 mM) significantly stimulated the electrically evoked release of [3H]-acetylcholine and completely abolished the effect of the selective D2-receptor agonist LY 171555 (1 microM) on evoked acetylcholine release. In addition, tetraethylammonium (0.03-30 mM) and 4-aminopyridine (0.003-1 mM) strongly increased the basal (non-evoked) release of radioactivity in a concentration-dependent manner. The results suggest that the effect of the drugs on the basal release of radioactivity and on the electrically evoked release of acetylcholine cannot exclusively be explained by their action on K+-channels. 4. Furthermore, with the use of a receptor binding assay, data were obtained on the affinity of tetraethylammonium and 4-aminopyridine for D2-receptors and various other neurotransmitter recognition sites. At concentrations in which both drugs are known to block K+-channels, they were found to inhibit the specific binding of selective radioligands to their respective recognition sites. 5. It is concluded that due to their 'side-effects', both tetraethylammonium and 4-aminopyridine are of only limited value in the investigation of the alleged interaction between neurotransmitter receptors and K+-channels.     

Action of di- and tri-valent cations on calcium-activated K+-efflux in rat erythrocytes

Isolated rat erythrocytes were labelled with [86Rb] as a tracer for intracellular K+. It was demonstrated that rat erythrocytes possess a Ca2+-mediated K+-efflux mechanism similar to that reported for human erythrocytes. This model was used to investigate the interactions of di- and tri-valent cations on potassium [86Rb] permeability in intact cells. Low concentrations of Ag2+ and Hg2+ haemolysed erythrocytes and Pb2+ produced a selective increase in [86Rb] efflux which became self-inhibitory at concentrations above 100 microM. The effects of Pb2+ were potentiated by A23187. Ni2+, Cu2+, Co2+, Zn2+, Fe2+, Mn2+, Y2+ and Ba2+ did not initiate [86Rb] efflux, even in the presence of 0.5 microM A23187 and at concentrations as high as 1 mM. All of these cations, except Ba2+, were potent inhibitors of [86Rb] efflux evoked by 50 microM Ca2+ + 0.5 microM A23187. The lanthanides Tb3+, Gd3+, Eu3+, Sm3+ and La3+ increased [86Rb] efflux at low concentrations in the presence of A23187, but were self inhibitory at higher concentrations. They also inhibited Ca2+-mediated [86Rb]-efflux. It is concluded that the effectiveness of a cation in activating [86Rb] efflux is, in part, related to its non-hydrated crystalline ionic radius, and that the site of activation may only accommodate ionic radii between 0.95 and 1.00 A.     

Signalling pathways in bradykinin- and nitric oxide-induced hypotension in the normotensive rat; role of K+-channels

1. Bradykinin and nitric oxide (NO) are potent hypotensive agents. In the present study, the role of K⁺-channels in the signalling pathways responsible for their hypotensive action was investigated in normotensive, anaesthetized rats. The rats were treated with ion-channel inhibitors before administration of bradykinin (2.8, 5.6, 28 and 56 nmol kg⁻¹, i.v.) followed in some of the protocols by nitroprusside (1.1, 3.5, 7, 14, and 28 nmol kg⁻¹, i.v.).
2. No attenuation of the hypotensive response to bradykinin was detected for inhibitors of the Na-K-Cl-cotransporter (30 μmol kg⁻¹ furosemide), the ATP-sensitive K⁺-channel (40 μmol kg⁻¹ glibenclamide), high conductance Ca²⁺-activated K⁺-channel (180 μmol kg⁻¹ tetraethylammonium, 54 μmol kg⁻¹ tetrabutylammonium, 35 nmol kg⁻¹ iberiotoxin, 35 nmol kg⁻¹ charybdotoxin) or the low conductance Ca²⁺-activated K⁺-channel (74 nmol kg⁻¹ apamin).
3. However, the voltage-sensitive K⁺-channel (I_A) inhibitor 4-aminopyridine (4.05–40.5 μmol kg⁻¹) induced a concentration-dependent (P<0.0001) attenuation of the hypotensive response (P<0.0001). Bradykinin had no effect on heart rate in anaesthetized rats and this observation was not altered by pretreatment with 4-aminopyridine.
4. 4-Aminopyridine (53 μmol kg⁻¹) also significantly attenuated the hypotensive response to nitroprusside (P<0.0003) without altering the heart rate concentration-response curve. Of the two Ca²⁺-activated K⁺-channel inhibitors tested on nitroprusside-induced hypotension, tetrabutylammonium induced a slight attenuation (P<0.0101), whereas iberiotoxin had no effect.
5. We therefore concluded that, although the acute hypotensive response to bradykinin in the normotensive rat is not mediated through nitric oxide synthesis, the hypotensive response to both agents was mediated through opening of voltage-sensitive K⁺-channels (I_A), resulting in a decrease in peripheral vascular resistance.

     

Inhibition of EDHF by two new combinations of K+-channel inhibitors in rat isolated mesenteric arteries

It is widely established that in rat mesenteric arteries, endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxation evoked by acetylcholine is abolished by a combination of charybdotoxin plus apamin. 4-Aminopyridine, an inhibitor of voltage-gated (Kv) K(+)-channels, in combination with apamin had moderate effects on the EDHF-mediated relaxation. Maurotoxin (MTX), an inhibitor of Kv and intermediate-conductance Ca(2+)-activated K(+)-channels (IK), had no effect on EDHF-mediated relaxation. However, MTX in combination with apamin completely abolished EDHF-mediated relaxation and endothelial cell hyperpolarization. The selective IK inhibitor 2-(2-chlorophenyl)-2,2-diphenyl acetonitrile (TRAM-39) had no significant effect on EDHF-mediated relaxation. EDHF-mediated vasorelaxation and hyperpolarization was abolished by a combination of TRAM-39 and apamin. These data demonstrate two new combinations of K(+)-channel inhibitors for the investigation of EDHF. Furthermore, by using TRAM-39, a potent selective inhibitor of IK channels, we provide the first direct evidence that abolition of EDHF requires the simultaneous presence of intermediate- and small-conductance Ca(2+)-activated K(+)-channel inhibitors.     

Effects of inhibitors of small- and intermediate-conductance calcium-activated potassium channels, inwardly-rectifying potassium channels and Na(+)/K(+) ATPase on EDHF relaxations in the rat hepatic artery

1. In the rat hepatic artery, the SK(Ca) inhibitors UCL 1684 (300 nM) completely blocked, and scyllatoxin (1 microM) and d-tubocurarine (100 microM) partially inhibited EDHF relaxations when each of them was combined with charybdotoxin (300 nM). 2. The IK(Ca) inhibitors clotrimazole (3 microM) and 2-chlorophenyl-bisphenyl-methanol (3 microM) strongly depressed EDHF relaxations when each of them was combined with apamin (300 nM). The cytochrome P450 mono-oxygenase inhibitor ketoconazole (10 microM) had no effect in the presence of apamin. 3. Ciclazindol (10 microM), which abolishes EDHF relaxations in the presence of apamin, almost completely prevented the calcium ionophore (A23187) stimulated (86)Rb(+) influx via the Gardos channel (IK(Ca)) in human erythrocytes. 4. The Na(+)/K(+) ATPase inhibitor ouabain (500 microM) and the K(IR) blocker Ba(2+) (30 microM) neither alone nor in combination inhibited EDHF relaxations. Ba(2+) was also without effect in the presence of either apamin or charybdotoxin. 5. In contrast to EDHF, an increase in extracellular [K(+)] from 4.6 mM to 9.6, 14.6 and 19.6 mM inconsistently relaxed arteries. In K(+)-free physiological salt solution, re-admission of K(+) always caused complete and sustained relaxations which were abolished by ouabain but unaffected by Ba(2+). 6. The present study provides pharmacological evidence for the involvement of SK(Ca) and IK(Ca) in the action of EDHF in the rat hepatic artery. Our results are not consistent with the idea that EDHF is K(+) activating Na(+)/K(+) ATPase and K(IR) in this blood vessel.     

Characterization of a charybdotoxin-sensitive intermediate conductance Ca2+-activated K+ channel in porcine coronary endothelium: relevance to EDHF

1. This study characterizes the K(+) channel(s) underlying charybdotoxin-sensitive hyperpolarization of porcine coronary artery endothelium. 2. Two forms of current-voltage (I/V) relationship were evident in whole-cell patch-clamp recordings of freshly-isolated endothelial cells. In both cell types, iberiotoxin (100 nM) inhibited a current active only at potentials over +50 mV. In the presence of iberiotoxin, charybdotoxin (100 nM) produced a large inhibition in 38% of cells and altered the form of the I/V relationship. In the remaining cells, charybdotoxin also inhibited a current but did not alter the form. 3. Single-channel, outside-out patch recordings revealed a 17.1+/-0.4 pS conductance. Pipette solutions containing 100, 250 and 500 nM free Ca(2+) demonstrated that the open probability was increased by Ca(2+). This channel was blocked by charybdotoxin but not by iberiotoxin or apamin. 4. Hyperpolarizations of intact endothelium elicited by substance P (100 nM; 26.1+/-0.7 mV) were reduced by apamin (100 nM; 17.0+/-1.8 mV) whereas those to 1-ethyl-2-benzimidazolinone (1-EBIO, 600 microM, 21.0+/-0.3 mV) were unaffected (21.7+/-0.8 mV). Substance P, bradykinin (100 nM) and 1-EBIO evoked charybdotoxin-sensitive, iberiotoxin-insensitive whole-cell perforated-patch currents. 5 A porcine homologue of the intermediate-conductance Ca(2+)-activated K(+) channel (IK1) was identified in endothelial cells. 6. In conclusion, porcine coronary artery endothelial cells express an intermediate-conductance Ca(2+)-activated K(+) channel and the IK1 gene product. This channel is opened by activation of the EDHF pathway and likely mediates the charybdotoxin-sensitive component of the EDHF response.     

Some degree of overlap exists between the K+-channels opened by cromakalim and those opened by minoxidil sulphate in rat isolated aorta

Summary The effects of the K⁺ channel opening drugs minoxidil sulphate and cromakalim, on ⁴²K⁺ and ⁸⁶Rb⁺ efflux and on vasorelaxation in rat isolated aorta, were compared. In rat aortic rings precontracted with noradrenaline (100 nmol/l), minoxidil sulphate and cromakalim concentration-dependently inhibited induced tension by up to 90%, with pD₂ values of 7.35±0.1 and 7.17±0.1, respectively. Glibenclamide (300 nmol/l), produced 2200- and 19-fold rightward shifts in the concentration-relaxation curves to minoxidil sulphate and cromakalim, respectively, without an effect on the maximum relaxation.Both minoxidil sulphate and cromakalim increased the efflux of ⁴²K⁺ and ⁸⁶Rb⁺ from aorta in a concentration-dependent manner, with midpoints in the µmol/l range; the maximum efflux induced by minoxidil sulphate being approximately one tenth of that induced by cromakalim. The ratio of stimulated ⁸⁶Rb⁺/⁴²K⁺ efflux increased from 0.22 to 0.48 with increasing cromakalim concentrations, but was approximately constant (0.39) when the minoxidil sulphate concentration was varied. In the presence of minoxidil sulphate, the effects of cromakalim on ⁴²K⁺ and ⁸⁶Rb⁺ efflux were inhibited in a concentration-dependent manner, by up to 60%. In the continuing presence of cromakalim (300 nmol/l), minoxidil sulphate (10 µmol/l)-induced increases in ⁴²K⁺ and ⁸⁶Rb⁺ efflux were inhibited by 45%, whereas conditioning with cromakalim (1 µmol/l) inhibited the ⁸⁶Rb⁺ efflux stimulated by additional superfusion of cromakalim (1 µmol/l) by 85%. Glibenclamide inhibited minoxidil sulphate (10 µmol/l)- and cromakalim (1 µmol/l)-induced increases in ⁴²K⁺ and ⁸⁶Rb⁺ efflux in a concentration-dependent manner with IC₅₀ values of approximately 80 nmol/l.In conclusion, the efflux data suggest that considerable overlap exists between the channels opened by minoxidil sulphate and those opened by cromakalim in rat aorta. Minoxidil sulphate has a weak efficacy as a K⁺ channel opener, and may act to open a homogeneous population of K⁺ channels. In contrast, the actions of cromakalim (1 µmol/l) are associated with large increases in tracer efflux, which are probably mediated via a heterogeneous population of K⁺ channels. However, only a small proprtion of this induced efflux appears to be required for relaxation. The differential inhibition by glibenclamide of the vasorelaxant effects of minoxidil sulphate and cromakalim may result from (a) the partial agonist properties of minoxidil sulphate in opening K⁺ channels and/or (b) additional mechanisms of vasorelaxation, which differ in their sensitivity to glibenclamide. Send offprint requests to U. Quasi at the above address     

Inhibition of ATP-sensitive K(+)-channels by a sulfonylurea analogue with a phosphate group

Hypoglycaemic sulfonylureas initiate insulin secretion by direct inhibition of ATP-sensitive K(+)-channels in the pancreatic beta-cells. These channels are composed of two proteins, a pore-forming subunit (K(IR)6.2 in the case of beta-cells) and a regulatory subunit, the sulfonylurea receptor (SUR). In the present study we characterised the interaction with SURs of the new sulfonylurea analogues 5-chloro-N-[2-(4-hydroxyphenyl)ethyl]-2-methoxybenzamide (compound I) and (4-[2-(5-chloro-2-methoxybenzamido)ethyl]phenyl)phosphate (compound II). Compounds I and II differ from the sulfonylurea analogue meglitinide only in so far as the carboxylic group of meglitinide is replaced by a hydroxyl group or a phosphate group, respectively. The binding affinities of compound II for the SUR subtypes SUR1 (identified in beta-cells) and SUR2A (identified in heart and skeletal muscle) were higher by 55 or 21-fold, respectively, than the corresponding affinities for compound I. In inside-out patch-clamp experiments compound II inhibited ATP-sensitive K(+)-channels of the SUR1/K(IR)6.2-type (characteristic of beta-cells) with an IC(50) value of 0.16 microM which is 6-fold lower than the corresponding value for meglitinide. These findings strongly support the conclusion that the interaction of sulfonylureas and acidic analogues with SURs is favoured by the anionic group of these drugs and that a phosphate group allows more efficient ligand interaction with SUR1 than a carboxylic group.     

Myocardial tolerance to arrhythmogenic exposure and pharmacological activation of K(ATP)-channels in rats

The cardioselective KATP channel activator BMS 180448 (3 mg/kg) administered intravenously 15 min before the coronary artery occlusion (10 min) decreased the incidence of ischemic and reperfusion arrhythmias in rats. A similar antiarrhythmic effect was observed when BMS 180448 was infused 2 min before reperfusion. Pretreatment with BMS 180448 also prevented the occurrence of CsCl induced arrhythmias, but but did not affect the incidence of epinephrine induced arrhythmias. On the contrary, BMS 180448 potentiated the arrhythmogenic action of CaCl2. The mechanism of the antiarrhythmic activity of BMS 180448 is discussed.     

The Properties of Voltage-operated Ca2+-channels in Bovine Isolated Trachealis Cells

Summary: Freshly-dispersed bovine trachealis cells were used for recording by the patch clamp technique of whole-cell and unitary currents through Ca²⁺-channels. Whole-cell Ca²⁺-current (I_Ca) activated at -40 mV and appeared to be carried by a single type of Ca²⁺-channel. Inactivation of I_Ca was increased by increasing the concentration of free Ca²⁺ within the recording pipette but reduced by using Ba²⁺ as the charge carrier. Steady-state inactivation studies showed that the Ca²⁺-channels were half-maximally available following a conditioning depolarization to -35 mV. A two-pulse protocol showed that I_Ca induced by the step to a test potential was inversely related to I_Ca induced by the step to the conditioning potential. Unitary Ba²⁺-currents were activated at a threshold of -30 mV and had a reversal potential of +41 mV. The channel carrying the unitary Ba²⁺-currents had a slope conductance of 23 pS. Steady-state inactivation studies showed that the unitary Ba²⁺-currents were half-maximally available at a holding potential of -28 mV. I_Ca and unitary Ba²⁺-currents were inhibited by nifedipine (10 nM-1 μM) but augmented by Bay K 8644 (10 μM). It is concluded that the plasmalemma of bovine trachealis muscle contains a single population of voltage-dependent Ca²⁺-channels of the L-type. These channels may be subject to inactivation primarily by an increase in the concentration of free Ca²⁺ on the cytosolic side of the plasmalemma and secondarily by a voltage-dependent mechanism. Overlap of the inactivation and activation curves of I_Ca may allow the passage of 'window current' through the Ca²⁺-channels during sustained depolarization.     

A novel family of insect-selective peptide neurotoxins targeting insect large-conductance calcium-activated K+ channels isolated from the venom of the theraphosid spider Eucratoscelus constrictus

Spider venoms are actively being investigated as sources of novel insecticidal agents for biopesticide engineering. After screening 37 theraphosid spider venoms, a family of three new "short-loop" inhibitory cystine knot insecticidal toxins (κ-TRTX-Ec2a, κ-TRTX-Ec2b, and κ-TRTX-Ec2c) were isolated and characterized from the venom of the African tarantula Eucratoscelus constrictus. Whole-cell patch-clamp recordings from cockroach dorsal unpaired median neurons revealed that, despite significant sequence homology with other theraphosid toxins, these 29-residue peptides lacked activity on insect voltage-activated sodium and calcium channels. It is noteworthy that κ-TRTX-Ec2 toxins were all found to be high-affinity blockers of insect large-conductance calcium-activated K(+) (BK(Ca)) channel currents with IC(50) values of 3 to 25 nM. In addition, κ-TRTX-Ec2a caused the inhibition of insect delayed-rectifier K(+) currents, but only at significantly higher concentrations. κ-TRTX-Ec2a and κ-TRTX-Ec2b demonstrated insect-selective effects, whereas the homologous κ-TRTX-Ec2c also resulted in neurotoxic signs in mice when injected intracerebroventricularly. Unlike other theraphosid toxins, κ-TRTX-Ec2 toxins induce a voltage-independent channel block, and therefore, we propose that these toxins interact with the turret and/or loop region of the external entrance to the channel and do not project deeply into the pore of the channel. Furthermore, κ-TRTX-Ec2a and κ-TRTX-Ec2b differ from other theraphotoxins at the C terminus and positions 5 to 6, suggesting that these regions of the peptide contribute to the phyla selectivity and are involved in targeting BK(Ca) channels. This study therefore establishes these toxins as tools for studying the role of BK(Ca) channels in insects and lead compounds for the development of novel insecticides.