A specific binding site for K+ channel openers in rat aorta.

The K+ channel openers, including cromakalim, pinacidil, minoxidil sulfate, diazoxide, and nicorandil, form a chemically heterogeneous group of compounds, which relax smooth muscle by opening plasmalemmal K+ channels. At present it is not known whether these drugs elicit their effects by binding to the same target, presumably the K+ channel. In order to address this question, a binding assay for K+ channel openers has been developed in vascular smooth muscle. The novel tritiated K+ channel opener, [3H]P1075, an analogue of pinacidil, binds with high affinity (KD = 6 +/- 1 nM) to endothelium-denuded rings of rat aorta. Inhibition studies indicate that the different families of K+ channel openers bind to a common target. Evidence is presented to suggest that the binding site for the sulfonylurea, glibenclamide, the major blocker of the K+ channel openers, is coupled in a negative allosteric manner to the binding site(s) for the openers. The binding assay described here may open the way to the biochemical characterization of the drug receptor for the K+ channel openers.     

Evidence for presence of ATP-sensitive K+ channels in rat colonic smooth muscle cells.

Coexpression of sulfonylurea receptor (SUR) and inward-rectifying K+ channel (Kir6.1 or 6.2) subunit yields ATP-sensitive K+ (K(ATP)) channels. Three subtypes of SUR have been cloned: pancreatic (SUR1), cardiac (SUR2A), and vascular smooth muscle (SUR2B). The distinct responses to K+ channel openers (KCOs) produced in different tissues may depend on the SUR isoform of K(ATP) channel. Therefore, we investigated the effects of pinacidil and diazoxide, two KCOs, on K(ATP) currents in intestinal smooth muscle cells of the rat colon (circular layer) using whole-cell voltage clamp. Pinacidil stimulated a time-independent K+ current evoked by various test potentials from a holding potential of -70 mV. The reversal potential of the stimulated current was about -75 mV, which is close to the equilibrium potential for K+ (E(K)). Both pinacidil and diazoxide dose-dependently stimulated K+ currents (evoked by ramp pulses), with EC50 values of 1.3 and 34.2 microM, respectively. The stimulated current was completely reversed by glybenclamide (3 microM). Since the EC50 values are close to those reported for vascular smooth muscle (VSM) cells, the SUR subtype may be similar to that in VSM cells, and could form the functional K(ATP) channel in rat colonic smooth muscle cells.     

钾通道活化剂对豚鼠气道平滑肌电压依赖性钾通道特性的影响

钾通道活化剂可激活钾离子通道并松驰支气管平滑肌,在急性分离的豚鼠支气管平滑肌细胞上,用膜片钳技术的细胞贴附式和内面向外式研究了其对电压依赖性钾通道的直接作用。结果证实：在全细胞记录条件下,卡吗克林和拉吗克林不影响静息膜电位。但在去极化时可使通道电导从７５．２±５．１ｐＳ分别增大到８５．９±１１．８ｐＳ和８２．１±５．５ｐＳ。通道动力学特性也发生了改变,通道平均开放时间的τｏ２值延长和开放概率显著增加,其中拉吗克林的作用更为强。两者均可诱发通道出现多级开放。表明这两类活化剂可使去极化时钾离子外流增加。     

2-alkyl-3-Alkylamino-2H-Benzo- and pyridothiadiazine 1,1-dioxides: from K+ATP channel openers to Ca++ channel blockers?

A series of 2-alkyl-3-alkylamino-2H-benzo- and 2-alkyl-3-alkylamino-2H-pyrido[4,3-e]-1,2,4-thiadiazine 1,1-dioxides, structurally related to BPDZ 44 and BPDZ 73, two potent pancreatic B-cells K+ATP channel openers, were synthesized and tested on rat pancreatic islets (endocrine tissue) as well as on rat aorta rings (vascular smooth muscle tissue). Alkylation of the 2-position led to double bond tautomerization and formation of compounds with a 2H-conformation. In contrast to the previously described pyridothiadiazine dioxides, such as BPDZ 44, and 7-chlorobenzothiadiazine dioxides, such as BPDZ 73, the 2-alkyl-substituted analogs were found to be poorly active on the insulin releasing process although most drugs exhibited a vasorelaxant activity. As a result, the new 2-alkyl-substituted pyridinic compounds expressed a selectivity profile (vascular smooth muscle tissue vs pancreatic tissue) opposite to that of their non-alkyl-substituted counterparts, i.e. BPDZ 44. Additional investigations revealed that, in contrast to their non 2-alkyl-substituted analogs, the most interesting 2-methyl-substituted derivatives did not express the pharmacological profile of classical K+ATP channel openers. The pharmacological results rather suggest that alkylation of the 2-position of the thiadiazine ring led to drugs that could act as Ca2+ channel blockers rather than as potassium channel openers.     

Design, synthesis, and BK channel-opening activity of hexahydrodibenzazepinone derivatives

In order to explore new scaffolds for large-conductance Ca2+ -activated K+ channel (BK channel) openers, we carried out molecular design and synthesis on the basis of the following two concepts: (1) introduction of a heteroatom into the dehydroabietic acid (BK channel opener) skeleton would allow easier introduction of substituents. (2) Because of the fourfold symmetrical structure of BK channels, dimeric compounds in which two pharmacophores are linked through a tether are expected to have a greater binding probability to the channels, resulting in increased channel-opening activity. Herein, we explore the usefulness of the hexahydrodibenzazepinone structure as a new scaffold for BK channel openers. The synthesized monomer compounds of hexahydrodibenzazepinone derivatives, which can be derived from dehydroabietic acid, were subjected to electrophysiological patch-clamp studies, followed by Magnus contraction-relaxation assay using rabbit urinary bladder smooth muscle strips to assess overall activities. Dimeric compounds were designed by linking the monomeric hexahydrodibenzazepinone derivatives through a diacetylenebenzene tether, and their channel-opening activities were evaluated by electrophysiological methods. Finally, we concluded that the critical structure for BK channel-opening activity is the hexahydrodibenzazepinone monomer substituted with a phenyl-bearing alkynyl substituent on the lactam amide.     

Synthesis and bladder smooth muscle relaxing properties of substituted 3-amino-4-aryl-(and aralkyl-)cyclobut-3-ene-1,2-diones

We have reported on the design, synthesis, and biological characterization of (R)-4-[3,4-dioxo-2-(1,2,2-trimethyl-propylamino)-cyclobut-1-enylamino]-3-ethyl-benzonitrile (1), a novel, potent, and selective adenosine 5'-triphosphate-sensitive potassium (K(ATP)) channel opener with potential utility for the treatment of urge urinary incontinence (UUI). Excising the aniline-derived nitrogen atom of 1 or replacing it with an aralkyl group, led to bladder smooth muscle relaxant chemotypes 3 and 4, respectively. Prototype compounds in these series were found to produce significant increases in an iberiotoxin (IbTx)-sensitive hyperpolarizing current, thus suggesting that these relatively modest structural modifications resulted in a switch in the mechanism of action of these smooth muscle relaxants from K(ATP) channel openers to activators of the large-conductance Ca2+-activated potassium channel (BK(Ca)). We report herein the syntheses and biological evaluation of a series of substituted 3-amino-4-aryl-(and aralkyl-)cyclobut-3-ene-1,2-diones.     

Potential role of potassium channel openers in the treatment of asthma and chronic obstructive pulmonary disease

Airway smooth muscle (ASM) cells express various types of potassium (K+) channels which play a key role in determining the resting membrane potential, a relative electrical stability and the responsiveness to both contractile and relaxant agents. In addition, K+ channels are also involved in modulation of neurotransmitter release from airway nerves. The most important K+ channels identified in airways include large and small Ca2+-activated, delayed-rectifier, and ATP-sensitive channels. These K+ channels are structurally and functionally different, thus playing distinct roles in airway electrophysiology and pharmacology. Many in vitro and in vivo studies, performed in both animals and humans, have shown that K+ channel openers are able to induce hyperpolarization of ASM cells, bronchodilation, suppression of airway hyperresponsiveness (AHR), and inhibition of neural reflexes. Therefore, airway K+ channels represent a suitable pharmacological target for the development of new effective therapeutic options in the treatment of asthma and chronic obstructive pulmonary disease (COPD).     

Effects of valinomycin on calcium mobilization in vascular smooth muscle cells induced by angiotensin II

The effect of the specific potassium (K+) ionophore valinomycin on increase in intracellular calcium concentration [( Ca2+]i) was studied in vascular smooth muscle cells (VSMC). Valinomycin at more than 10(-9) M dose-dependently suppressed phasic increase in [Ca2+]i in VSMC induced by angiotensin II (AII) in both control and Ca2+-free solution, indicating that it suppressed the release of Ca2+ from intracellular Ca2+ stores. Nicorandil and cromakalim, which are both K+ channel openers, also suppressed the increases in [Ca2+]i induced by AII in the Ca2+ free solution. However, valinomycin did not suppress AII-induced production of inositol 1,4,5-trisphosphate (IP3), which is known to mediate the release of Ca2+. These results indicate that decrease of intracellular K+ induced by valinomycin suppressed the release of Ca2+ from intracellular Ca2+ stores induced by IP3.     

Two different types of channels are targets for potassium channel openers in Xenopus oocytes

K+ channel openers elicit K+ currents in follicle-enclosed Xenopus oocytes. The most potent activators are the pinacidil derivatives P1075 and P1060. The rank order of potency to activate K+ currents in follicle-enclosed oocytes was: P1075 (K0.5:5 microM) greater than P1060 (K0.5:12 microM) greater than BRL38227 (lemakalim) (K0.5:77 microM) greater than RP61410 (K0.5:100 microM) greater than (-)pinacidil (K0.5:300 microM). Minoxidil sulfate, nicorandil, RP49356 and diazoxide were ineffective. Activation by the K+ channel openers could be abolished by the antidiabetic sulfonylurea glibenclamide. It was not affected by the blocker of the Ca(2+)-activated K+ channels charybdotoxin. The various K+ channel openers failed to activate glibenclamide-sensitive K+ channels in defolliculated oocytes, but BRL derivatives (K0.5 for BRL38226 is 150 microM) and RP61419 inhibited a background current. The channel responsible for this background current is K+ permeable but not fully selective for K+. It is resistant to glibenclamide. It is inhibited by Ba2+, 4-aminopyridine, Co2+, Ni2+ and La3+.     

Cardiovascular and biological effects of K+ channel openers, a class of drugs with vasorelaxant and cardioprotective properties

Several new chemical entities (RP 52891, cromakalim and its derivatives) are potent and specific openers of vascular K+ channels. This mechanism is also shared, at least partially, by drugs such as minoxidil, diazoxide, pinacidil and nicorandil. The opening of plasmalemma K+ channels produces loss of cytosolic K+. This effect results in cellular hyperpolarization and functional vasorelaxation. In normotensive or hypertensive rats, K+ channel activators decrease aortic blood pressure (by producing a directly mediated fall in systemic vascular resistance) and reflexly increase heart rate. The former effect is not modified by specific blockers of classical vascular receptors but it is completely antagonized by the hypoglycemic sulphonylurea, glibenclamide, an established blocker of ATP-regulated K+ channels. K+ channel openers produce selective coronary vasodilatation and afford functional and biochemical protection to the ischemic myocardium. This salutary effect is mediated via cardiac K+ channel modulation and may result from an improved myocardial oxygen balance in the ischemic region. K+ channel openers increase plasma renin activity in animals as well as in man. However, only diazoxide, but not cromakalim or RP 52891, lowers plasma insulin concentration. The dose of glibenclamide entirely blocking the latter effect is over 50-fold smaller than that antagonizing the hypotensive and hyper-reninemic responses to diazoxide. In conclusion, K+ channel activators are potent vasorelaxant and cardioprotective agents possessing an original mechanism of action which is the opening of plasmalemma ATP-regulated K+ channels. Their clinical use as antihypertensive agents may be accompanied by undesirable effects (characteristic of peripheral vasodilators) which are likely to be attenuated or avoided by controlled release formulations. However, inasmuch as low doses of K+ channel openers may be sufficient to produce selective coronary artery dilatation and cardioprotection, these compounds could be of particular value in treating patients with coronary artery disease efficaciously and possibly without adverse cardiovascular effects.     

ATPase activity of the sulfonylurea receptor: a catalytic function for the KATP channel complex.

ATP-sensitive K+ (KATP) channels are unique metabolic sensors formed by association of Kir6.2, an inwardly rectifying K+ channel, and the sulfonylurea receptor SUR, an ATP binding cassette protein. We identified an ATPase activity in immunoprecipitates of cardiac KATP channels and in purified fusion proteins containing nucleotide binding domains NBD1 and NBD2 of the cardiac SUR2A isoform. NBD2 hydrolyzed ATP with a twofold higher rate compared to NBD1. The ATPase required Mg2+ and was insensitive to ouabain, oligomycin, thapsigargin, or levamisole. K1348A and D1469N mutations in NBD2 reduced ATPase activity and produced channels with increased sensitivity to ATP. KATP channel openers, which bind to SUR, promoted ATPase activity in purified sarcolemma. At higher concentrations, openers reduced ATPase activity, possibly through stabilization of MgADP at the channel site. K1348A and D1469N mutations attenuated the effect of openers on KATP channel activity. Opener-induced channel activation was also inhibited by the creatine kinase/creatine phosphate system that removes ADP from the channel complex. Thus, the KATP channel complex functions not only as a K+ conductance, but also as an enzyme regulating nucleotide-dependent channel gating through an intrinsic ATPase activity of the SUR subunit. Modulation of the channel ATPase activity and/or scavenging the product of the ATPase reaction provide novel means to regulate cellular functions associated with KATP channel opening.     

Activation by cromakalim of pre- and post-synaptic ATP-sensitive K+ channels in substantia nigra

Membrane potentials and synaptic potentials were recorded using the patch clamp technique from neurons isolated from the substantia nigra. Intracellular perfusion of dopaminergic neurons with an ATP-free solution caused hyperpolarization and inhibition of firing. Intracellular perfusion with a solution containing 2 mM ATP prevented this hyperpolarization, but application of the K+ channel openers cromakalim and pinacidil caused a similar hyperpolarization as well as the disappearance of bicuculline-sensitive synaptic potentials. All these effects were reversed by sulfonylureas, indicating that they are mediated by ATP-sensitive K+ channels. It is concluded that K+ channel openers activate ATP-sensitive K+ channels both presynaptically on GABAergic terminals and postsynaptically on substantia nigra dopaminergic neurons.     

4-Aryl-3-(mercapto)quinolin-2-ones: novel maxi-K channel opening relaxants of corporal smooth muscle

A novel series of 4-aryl-3-(mercapto)quinolin-2-one derivatives was prepared and evaluated as openers of the cloned maxi-K channel hSlo expressed in Xenopus laevis oocytes by utilizing electrophysiological methods. The effect of these maxi-K openers on corporal smooth muscle was studied in vitro using isolated rabbit corpus cavernosum. In vivo efficacy has been demonstrated with a selective maxi-K opening relaxant in a rat model of erectile function.     

Partial structures of ketoconazole as modulators of the large conductance calcium-activated potassium channel (BK(Ca))

A series of partial structures of ketoconazole has been synthesized and tested for activity on the large conductance calcium-activated potassium channel (BK) in bovine smooth muscle cells. This has provided openers and blockers of the channel. The results suggest that the phenyl and phenoxy moieties are important for interaction with BK, whereas the imidazole group is unimportant. The properties of the phenoxy moiety seem to determine whether the compounds act to open or block the channel.     

Ca2+ channel antagonist actions in bladder smooth muscle: comparative pharmacologic and [3H]nitrendipine binding studies

The actions of a series of 15 Ca2+ channel antagonists including D-600, nifedipine, and diltiazem were examined against K+ depolarization and muscarinic receptor induced responses in guinea pig bladder smooth muscle. Responses of bladder are very dependent upon extracellular Ca2+ and sensitive to the Ca2+ channel antagonists, the tonic component more than the phasic component of response. Regardless of stimulant, K+ or methylfurmethide (MF), or component of response, the same rank order of antagonist activities is expressed, suggestive of a single structure-activity relationship and the existence of a single category of binding site which may, however, exist in several affinity states. High affinity binding of [3H]nitrendipine (KD = 1.1 X 10(-10) M) occurs in bladder membranes, and similar high affinity binding was found in microsomal preparations from other smooth muscles including guinea pig and rat lung, rat vas deferens, uterus, and stomach. [3H]nitrendipine binding in the bladder was sensitive to displacement by other 1,4-dihydropyridines, paralleling their pharmacologic activities and showing excellent agreement with binding data previously obtained for guinea pig ileal smooth muscle. Comparison of pharmacologic data for inhibition of K+- and MF-induced responses by a common series of Ca2+ channel antagonists in bladder and ileum revealed excellent correlations. Neither pharmacologic nor binding studies suggest significant differences in Ca2+ channel antagonist properties in smooth muscle from bladder and intestine.     

Opening of potassium channels modulates mitochondrial function in rat skeletal muscle

We have investigated the presence of diazoxide- and nicorandil-activated K+ channels in rat skeletal muscle. Activation of potassium transport in the rat skeletal muscle myoblast cell line L6 caused a stimulation of cellular oxygen consumption, implying a mitochondrial effect. Working with isolated rat skeletal muscle mitochondria, both potassium channel openers (KCOs) stimulate respiration, depolarize the mitochondrial inner membrane and lead to oxidation of the mitochondrial NAD-system in a strict potassium-dependent manner. This is a strong indication for KCO-mediated stimulation of potassium transport at the mitochondrial inner membrane. Moreover, the potassium-specific effects of both diazoxide and nicorandil on oxidative phosphorylation in skeletal muscle mitochondria were completely abolished by the antidiabetic sulfonylurea derivative glibenclamide, a well-known inhibitor of ATP-regulated potassium channels (K(ATP) channels). Since both diazoxide and nicorandil facilitated swelling of de-energised mitochondria in KSCN buffer at the same concentrations, our results implicate the presence of a mitochondrial ATP-regulated potassium channel (mitoK(ATP) channel) in rat skeletal muscle which can modulate mitochondrial oxidative phosphorylation.     

Effect of ATP-sensitive K+ channel regulators on cystic fibrosis transmembrane conductance regulator chloride currents.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl- channel that is regulated by cAMP-dependent phosphorylation and by intracellular ATP. Intracellular ATP also regulates a class of K+ channels that have a distinct pharmacology: they are inhibited by sulfonylureas and activated by a novel class of drugs called K+ channel openers. In search of modulators of CFTR Cl- channels, we examined the effect of sulfonylureas and K+ channel openers on CFTR Cl- currents in cells expressing recombinant CFTR. The sulfonylureas, tolbutamide and glibenclamide, inhibited whole-cell CFTR Cl- currents at half-maximal concentrations of approximately 150 and 20 microM, respectively. Inhibition by both agents showed little voltage dependence and developed slowly; > 90% inhibition occurred 3 min after adding 1 mM tolbutamide or 100 microM glibenclamide. The effect of tolbutamide was reversible, while that of glibenclamide was not. In contrast to their activating effect on K+ channels, the K+ channel openers, diazoxide, BRL 38227, and minoxidil sulfate inhibited CFTR Cl- currents. Half-maximal inhibition was observed at approximately 250 microM diazoxide, 50 microM BRL 38227, and 40 microM minoxidil sulfate. The rank order of potency for inhibition of CFTR Cl- currents was: glibenclamide < BRL 38227 approximately equal to minoxidil sulfate > tolbutamide > diazoxide. Site-directed mutations of CFTR in the first membrane-spanning domain and second nucleotide-binding domain did not affect glibenclamide inhibition of CFTR Cl- currents. However, when part of the R domain was deleted, glibenclamide inhibition showed significant voltage dependence. These agents, especially glibenclamide, which was the most potent, may be of value in identifying CFTR Cl- channels. They or related analogues might also prove to be of value in treating diseases such as diarrhea, which may involve increased activity of the CFTR Cl- channel.     

Decrease in activity of smooth muscle L-type Ca2+ channels and its reversal by NF-kappaB inhibitors in Crohn's colitis model

We investigated the mechanisms of dysmotility of the colonic circular muscle of the Crohn's disease rat model. Contractions induced by KCl, carbachol, and Bay K 8644 were decreased in circular smooth muscles isolated from 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis rat colon. However, the absolute force and Ca2+ sensitivity of contractile proteins were not affected as assessed in alpha-toxin permeabilized smooth muscle. The current density of the L-type Ca2+ channel in circular smooth muscle cells was significantly decreased in the TNBS-treated colonic cells. However, expressions of the L-type Ca2+ channel mRNA and protein did not differ between control and TNBS-treated preparations. Pretreatment with the NF-kappaB inhibitors pyrrolidinedithiocarbamate and sulfasalazine partially recovered the decreased contractility and current density of the L-type Ca2+ channel by TNBS treatment. These results suggest that the decrease in the contraction of circular smooth muscle isolated from TNBS-induced colitis rat colon, which may be related to gut dysmotility in Crohn's disease, is attributable to the decreased activity of the L-type Ca2+ channel. The dysfunction of the L-type Ca2+ channel may be mediated by NF-kappaB-dependent pathways.