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Openers of KATP channels are known to inhibit KCl-, carbachol- and also electrically induced contractions in detrusor muscle strips from various species. Contractions of isolated strips of urinary bladder are usually of higher amplitude when the urothelium has been removed. This has been explained by the release of an urothelium-derived relaxing factor. In this study we examined whether intact urothelium may modulate the effect of the selective KATP channel opener rilmakalim. Contractile responses to 85 mM KCl and 10 μM carbachol were measured in detrusor strips from mouse, pig and man. In the presence of an intact urothelium, contractions were significantly reduced in strips from all three species investigated. In preparations with urothelium rilmakalim reduced KCl contractions with similar potency and efficacy [−logIC50 (M) 4.6 to 5.1; Effmax reduction to 14-30% of control]. However, in urothelium-denuded strips rilmakalim was more potent in pig (−logIC50 5.5) than in mouse and man (−logIC50 4.7 and 4.4, respectively). The order of potency for rilmakalim to suppress carbachol-induced contractions was pig (—logIC50 6.7) > man (5.8) > mouse (4.7); contractions were significantly more reduced in pig (Effmax reduction to 11±2%, n=10) and in mouse (21±2%, n=8) than in human detrusor (55±5%, n=5). The presence of urothelium did not affect the concentration–response curves for rilmakalim, with the exception of KCl-induced contractions in pig. Only the rilmakalim-induced relaxation of carbachol-mediated contractions in pig were prevented by the KATP channel blocker glibenclamide. We conclude that with this one exception, the responses to rilmakalim in detrusor contractions were not mediated by KATP channel opening. [ABSTRACT FROM AUTHOR]

1 Openers of ATP-sensitive K+ channels (KATP channels) are thought to act by enhancing the ATPase activity of sulphonylurea receptors (SURs). the regulatory channel subunits. At higher concentrations, some openers activate KATP channels also in the absence of MgATP. Here, we describe binding and effect of structurally diverse openers in the absence of Mg2+ and presence of EDTA. 2 Binding of openers to SUR2B was measured using a mutant with high affinity for [3H]glibenclamide (3H]GBC). In the absence of Mg2+, 'typical' openers (benzopyrans, cyanoguanidines and aprikalim) inhibited [3H]GBC binding with Ki values ∼ 200 × higher than in the presence of MgATP. Minoxidil sulphate and nicorandil were inactive, whereas binding of diazoxide was unaffected by MgATP. 3 In the absence/presence of MgATP, N-cyano-N′-(1,1-dimethylpropyl)-N″-3-pyridylguanidine (PI075) activated the Kir6.2 SUR2B channel in inside-out patches with EC50 = 2000/67nM and Emax = 32/134%. In the absence of Mg2+, responses were variable with only a small part of the variability being explained by a decrease in channel responsiveness with time after patch excision and to differences in the ATP sensitivity between patches. 4 The rank order of efficacy of the openers was P1075>rilmakalim ∼ nicorandil > diazoxide > minoxidil sulphate. 5 The data show that structurally diverse openers are able to bind to, and to activate the Kir6.2/ SUR2B channel by a pathway independent of ATP hydrolysis. These effects are observed at concentrations used to define the biochemical mechanism of the openers in the presence of MgATP and allow the openers to be classified into 'typical' and 'atypical' KCOs with diazoxide standing apart. [ABSTRACT FROM AUTHOR]

1 Openers of ATP-sensitive K+ channels (KATP channels) are thought to act by enhancing the ATPase activity of sulphonylurea receptors (SURs). the regulatory channel subunits. At higher concentrations, some openers activate KATP channels also in the absence of MgATP. Here, we describe binding and effect of structurally diverse openers in the absence of Mg2+ and presence of EDTA. 2 Binding of openers to SUR2B was measured using a mutant with high affinity for [3H]glibenclamide (3H]GBC). In the absence of Mg2+, 'typical' openers (benzopyrans, cyanoguanidines and aprikalim) inhibited [3H]GBC binding with Ki values ∼ 200 × higher than in the presence of MgATP. Minoxidil sulphate and nicorandil were inactive, whereas binding of diazoxide was unaffected by MgATP. 3 In the absence/presence of MgATP, N-cyano-N′-(1,1-dimethylpropyl)-N″-3-pyridylguanidine (PI075) activated the Kir6.2 SUR2B channel in inside-out patches with EC50 = 2000/67nM and Emax = 32/134%. In the absence of Mg2+, responses were variable with only a small part of the variability being explained by a decrease in channel responsiveness with time after patch excision and to differences in the ATP sensitivity between patches. 4 The rank order of efficacy of the openers was P1075>rilmakalim ∼ nicorandil > diazoxide > minoxidil sulphate. 5 The data show that structurally diverse openers are able to bind to, and to activate the Kir6.2/ SUR2B channel by a pathway independent of ATP hydrolysis. These effects are observed at concentrations used to define the biochemical mechanism of the openers in the presence of MgATP and allow the openers to be classified into 'typical' and 'atypical' KCOs with diazoxide standing apart. [ABSTRACT FROM AUTHOR]

Adenosine triphosphate (ATP) dependent potassium channels (K 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 K 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 (EC) = 1.1 μM, Hill coefficient = 3.1, saturation concentration 10 μM]. Metabolic inhibition with 2-deoxy- d-glucose (10 mmol/l) also activated K 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 (IC) = 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 (IC = 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 K 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. [ABSTRACT FROM AUTHOR]

Many openers of ATP-dependent potassium channels (K channel openers) cause bronchorelaxation, whereas only a few of them have been claimed to reverse airways hyperreactivity. We investigated whether the antihyperreactive effect is a general feature of K channel openers and whether this property is linked to their ability to relax airways smooth muscle. For this purpose, the potency of the four K channel openers, bimakalim, rilmakalim, levcromakalim and SDZ PCO 400 ((−)-(3S,4R)3,4-dihydro-3-hydroxy-2,2-dimethyl-4-(3-oxo-cyclopent-lenyloxy)-2H-1-benzopyran-6-carbonitrile), to inhibit bombesin-or histamine-induced bronchoconstriction and to reverse immune complex-induced airways hyperreactivity to histamine in guinea pigs, was compared to salbutamol, following intratracheal administration to minimize pharmacokinetic differences. Total lung resistance (R) was determined in anaesthetized, ventilated guinea pigs. Bronchoconstriction, measured as increase in R, was elicited in normoreactive animals by i.v. infusion of bombesin (100 ng/kg/min) or by i.v. injection of histamine (1.8-10 µg/kg). Airways hyperreactivity was induced by acute i.v. administration of preformed immune complexes. I.v. bolus injections of histamine were used to define the sensitivity of the airways prior to and after the exposure to immune complex. Levcromakalim (ED = 150 μg/kg), bimakalim (ED = 4 μg/kg), rilmakalim (ED = 40 μg/kg) and SDZ PCO 400 (ED = 280 μg/kg) reversed bombesin-induced bronchoconstriction with lower potency than salbutamol (ED = 1 μg/kg). The four K channel openers and salbutamol also reversed immune complex-induced airways hyperreactivity to histamine with ED values which were markedly lower than those for reversal of bombesin-induced bronchoconstriction; the rank order of potency was rilmakalim (ED = 0.2 μg/kg) > bimakalim (ED = 0.5 μg/kg) > SDZ PCO 400 (ED = 3.2 μg/kg) > levcromakalim (ED = 22 μg/kg). Salbutamol (ED = 0.008 μg/kg) was the most potent compound in this test. Bimakalim, levcromakalim and SDZ PCO 400 did not inhibit histamine-induced bronchoconstriction in normoreactive guinea pigs at doses which completely reversed immune complex-induced airways hyperreactivity to histamine. For rilmakalim and salbutamol, 60-130 times higher doses were needed for protection against histamine-induced bronchoconstriction in normoreactive guinea pigs than for reversal of airways hyperreactivity. There was a poor correlation between the ED values for inhibition of histamine- or bombesin-induced bronchoconstriction in normoreactive guinea pigs and the reversal of immune cimplex-induced airways hyperreactivity. It is thus concluded that the ability of K channel openers to reverse immune complex-induced airways hyperreactivity is independent of their ability to reverse or prevent bronchoconstriction and thus from their ability relax airway smooth muscle. [ABSTRACT FROM AUTHOR]