Determination of the kinetic properties of platycodin D for the inhibition of pancreatic lipase using a 1, 2-diglyceride-based colorimetric assay

A 1,2-diglyceride-based multi-step colorimetric assay to measure the pancreatic lipase activity was applied for the determination of the kinetic profiles of the lipase inhibition with a slight modification and the validity verification. With this assay method, our study revealed that platycodin D, one of major constituents of Platycodi Radix, inhibits the pancreatic lipase activity in a competitive type, with the value of KI being 0.18+/-0.02 mM. In addition, PD has affected the values of K(m,app) and Kcat/Km in a dose-dependent manner. The results shed a meaningful light on how PD mediates lipid metabolism in the intestinal tracts. On the other hand, since the revised assay is sensitive, rapid, and does not affect the accuracy to the kinetic properties, it is applicable not only to evaluation of the kinetic properties of the pancreatic lipase, but also to high-throughput screening of pancreatic lipase activity.     

ATP-sensitive K+ channels in the kidney

ATP-sensitive K⁺ channels (K_ATP channels) form a link between the metabolic state of the cell and the permeability of the cell membrane for K⁺ which, in turn, is a major determinant of cell membrane potential. K_ATP channels are found in many different cell types. Their regulation by ATP and other nucleotides and their modulation by other cellular factors such as pH and kinase activity varies widely and is fine-tuned for the function that these channels have to fulfill. In most excitable tissues they are closed and open when cell metabolism is impaired; thereby the cell is clamped in the resting state which saves ATP and helps to preserve the structural integrity of the cell. There are, however, notable exceptions from this rule; in pancreatic -cells, certain neurons and some vascular beds, these channels are open during the normal functioning of the cell.In the renal tubular system, K_ATP channels are found in the proximal tubule, the thick ascending limb of Henle's loop and the cortical collecting duct. Under physiological conditions, these channels have a high open probability and play an important role in the reabsorption of electrolytes and solutes as well as in K⁺ homeostasis. The physiological role of their nucleotide sensitivity is not entirely clear; one consequence is the coupling of channel activity to the activity of the Na-K-ATPase (pump-leak coupling), resulting in coordinated vectorial transport. In ischemia, however, the reduced ATP/ADP ratio would increase the open probability of the K_ATP channels independently from pump activity; this is particularly dangerous in the proximal tubule, where 60 to 70% of the glomerular ultrafiltrate is reabsorbed.The pharmacology of K_ATP channels is well developed including the sulphonylureas as standard blockers and the structurally heterogeneous family of channel openers. Blockers and openers, exemplified by glibenclamide and levcromakalim, show a wide spectrum of affinities towards the different types of K_ATP channels. Recent cloning efforts have solved the mystery about the structure of the channel: the K_ATP channels in the pancreatic -cell and in the principal cell of the renal cortical collecting duct are heteromultimers, composed of an inwardly rectifying K⁺ channel and sulphonylurea binding subunit(s) with unknown stoichiometry. The proteins making up the K_ATP channel in these two cell types are different (though homologous), explaining the physiological and pharmacological differences between these channel subtypes.     

Physicochemical and Biological Data for the Development of Predictive Organophosphorus Pesticide QSARs and PBPK/PD Models for Human Risk Assessment

ABSTRACT

A search of the scientific literature was carried out for physiochemical and biological data [i.e., IC₅₀, LD₅₀, K_p (cm/h) for percutaneous absorption, skin/water and tissue/blood partition coefficients, inhibition k_i values, and metabolic parameters such as V_max and K_m] on 31 organophosphorus pesticides (OPs) to support the development of predictive quantitative structure–activity relationship (QSAR) and physiologically based pharmacokinetic and pharmacodynamic (PBPK/PD) models for human risk assessment. Except for work on parathion, chlorpyrifos, and isofenphos, very few modeling data were found on the 31 OPs of interest. The available percutaneous absorption, partition coefficients and metabolic parameters were insufficient in number to develop predictive QSAR models. Metabolic kinetic parameters (V_max, K_m) varied according to enzyme source and the manner in which the enzymes were characterized. The metabolic activity of microsomes should be based on the kinetic activity of purified or cDNA-expressed cytochrome P450s (CYPs) and the specific content of each active CYP in tissue microsomes. Similar requirements are needed to assess the activity of tissue A- and B-esterases metabolizing OPs. A limited amount of acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and carboxylesterase (CaE) inhibition and recovery data were found in the literature on the 31 OPs. A program is needed to require the development of physicochemical and biological data to support risk assessment methodologies involving QSAR and PBPK/PD models.     

Incomplete dissociation of glibenclamide from wild-type and mutant pancreatic KATP channels limits their recovery from inhibition

Background and purpose:

The antidiabetic sulphonylurea, glibenclamide, acts by inhibiting the pancreatic ATP-sensitive K⁺ (K_ATP) channel, a tetradimeric complex of K_IR6.2 and sulphonylurea receptor 1 (K_IR6.2/SUR1)₄. At room temperature, recovery of channel activity following washout of glibenclamide is very slow and cannot be measured. This study investigates the relation between the recovery of channel activity from glibenclamide inhibition and the dissociation rate of [³H]-glibenclamide from the channel at 37°C.

Experimental approach:

K_IR6.2, K_IR6.2ΔN5 or K_IR6.2ΔN10 (the latter lacking amino-terminal residues 2–5 or 2–10 respectively) were coexpressed with SUR1 in HEK cells. Dissociation of [³H]-glibenclamide from the channel and recovery of channel activity from glibenclamide inhibition were determined at 37°C.

Key results:

The dissociation kinetics of [³H]-glibenclamide from the wild-type channel followed an exponential decay with a dissociation half-time, t_1/2(D) = 14 min; however, only limited and slow recovery of channel activity was observed. t_1/2(D) for K_IR6.2ΔN5/SUR1 channels was 5.3 min and recovery of channel activity exhibited a sluggish sigmoidal time course with a half-time, t_1/2(R) = 12 min. t_1/2(D) for the ΔN10 channel was 2.3 min; recovery kinetics were again sigmoidal with t_1/2(R) ∼4 min.

Conclusions and implications:

The dissociation of glibenclamide from the truncated channels is the rate-limiting step of channel recovery. The sigmoidal recovery kinetics are in quantitative agreement with a model where glibenclamide must dissociate from all four (or at least three) sites before the channel reopens. It is argued that these conclusions hold also for the wild-type (pancreatic) K_ATP channel.     

儿童静滴维生素K1注射液引起的17例药物不良反应分析

目的 通过探讨维生素K1注射液药物不良反应发生的影响因素及规律,为儿童安全、合理使用维生素K₁注射液提供依据。方法 通过对我院2012~2017年发生的17例维生素K₁注射液引起的不良反应病例进行统计分析。结果 维生素K1注射液发生药品不良反应与药物本身的质量、用药人群的生理特点及临床用药合理性等因素密切相关。结论 应加强维生素K₁注射液合理应用及管理,保证儿童用药安全。     

肌注维生素K1致湿疹型皮肤反应及文献回顾

目的：总结维生素K₁注射液肌肉注射后引起皮肤反应的临床特征及治疗方法。方法：对1例产妇肌注维生素K₁致湿疹型皮肤反应进行报道,并对相关文献报道的情况进行回顾性汇总分析。结果：维生素K₁注射液导致的皮肤反应为非Ig E介导的Ⅳ型过敏反应,主要由增溶剂聚山梨酯80所引起,抗过敏治疗效果不显著。结论：应严格掌握用药的适应证,加强用药监测,提高用药警惕性,保障患者用药安全。     

Novel ion channel targets in atrial fibrillation

Introduction: Atrial fibrillation (AF) is the most common arrhythmia in humans. It is progressive and the development of electrical and structural remodeling makes early intervention desirable. Existing antiarrhythmic pharmacological approaches are not always effective and can produce unwanted side effects. Additional atrial-selective antiarrhythmic strategies are therefore desirable.

Areas covered: Evidence for three novel ion channel atrial-selective therapeutic targets is evaluated: atrial-selective fast sodium channel current (I_Na) inhibition; small conductance calcium-activated potassium (SK) channels; and two-pore (K_2P) potassium channels.

Expert Opinion: Data from animal models support atrial-ventricular differences in I_Na kinetics and also suggest atrial-ventricular differences in sodium channel β subunit expression. Further work is required to determine whether intrinsic atrial-ventricular differences in human I_Na exist or whether functional differences occur due to distinct atrial and ventricular action and resting potentials. SK and K_2P channels (particularly K_2P 3.1) offer potentially attractive atrial-selective targets. Work is needed to identify the underlying basis of SK current that contributes to (patho)physiological atrial repolarization and settings in which SK inhibition is anti- versus pro-arrhythmic. Although K_2P3.1 appears to be a promising target with comparatively selective drugs for experimental use, a lack of selective pharmacology hinders evaluation of other K_2P channels as potential atrial-selective targets.     

Rosiglitazone selectively inhibits K(ATP) channels by acting on the K(IR) 6 subunit

BACKGROUND AND PURPOSE

Rosiglitazone is an anti-diabetic drug acting as an insulin sensitizer. We recently found that rosiglitazone also inhibits the vascular isoform of ATP-sensitive K⁺ channels and compromises vasodilatory effects of β-adrenoceptor activation and pinacidil. As its potency for the channel inhibition is in the micromolar range, rosiglitazone may be used as an effective K_ATP channel inhibitor for research and therapeutic purposes. Therefore, we performed experiments to determine whether other isoforms of K_ATP channels are also sensitive to rosiglitazone and what their sensitivities are.

EXPERIMENTAL APPROACH

K_IR6.1/SUR2B, K_IR6.2/SUR1, K_IR6.2/SUR2A, K_IR6.2/SUR2B and K_IR6.2ΔC36 channels were expressed in HEK293 cells and were studied using patch-clamp techniques.

KEY RESULTS

Rosiglitazone inhibited all isoforms of K_ATP channels in excised patches and in the whole-cell configuration. Its IC₅₀ was 10 µmol·L⁻¹ for the K_IR6.1/SUR2B channel and ∼45 µmol·L⁻¹ for K_IR6.2/SURx channels. Rosiglitazone also inhibited K_IR6.2ΔC36 channels in the absence of the sulphonylurea receptor (SUR) subunit, with potency (IC₅₀= 45 µmol·L⁻¹) almost identical to that for K_IR6.2/SURx channels. Single-channel kinetic analysis showed that the channel inhibition was mediated by augmentation of the long-lasting closures without affecting the channel open state and unitary conductance. In contrast, rosiglitazone had no effect on K_IR1.1, K_IR2.1 and K_IR4.1 channels, suggesting that the channel inhibitory effect is selective for K_IR6.x channels.

CONCLUSIONS AND IMPLICATIONS

These results suggest a novel K_ATP channel inhibitor that acts on the pore-forming K_IR6.x subunit, affecting the channel gating.

LINKED ARTICLE

This article is commented on by Dart, pp. 23–25 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2012.01990.x     

The K+ channels KCa3.1 and Kv1.3 as novel targets for asthma therapy

Asthma affects 10% of the UK population and is an important cause of morbidity and mortality at all ages. Current treatments are either ineffective or carry unacceptable side effects for a number of patients; in consequence, development of new approaches to therapy are important. Ion channels are emerging as attractive therapeutic targets in a variety of non-excitable cells. Ion channels conducting K⁺ modulate the activity of several structural and inflammatory cells which play important roles in the pathophysiology of asthma. Two channels of particular interest are the voltage-gated K⁺ channel K_v1.3 and the intermediate conductance Ca²⁺-activated K⁺ channel K_Ca3.1 (also known as IK_Ca1 or SK4). K_v1.3 is expressed in IFNγ-producing T cells while K_Ca3.1 is expressed in T cells, mast cells, macrophages, airway smooth muscle cells, fibroblasts and epithelial cells. Both channels play important roles in cell activation, migration, and proliferation through the regulation of membrane potential and calcium signalling. We hypothesize that K_Ca3.1- and/or K_v1.3-dependent cell processes are one of the common denominators in asthma pathophysiology. If true, these channels might serve as novel targets for the treatment of asthma. Emerging evidence lends support to this hypothesis. Further validation through the study of the role that these channels play in normal and asthmatic airway cell (patho)physiology and in vivo models will provide further justification for the assessment of small molecule blockers of K_v1.3 and K_Ca3.1 in the treatment of asthma.     

Concomitant facilitation of GABAA receptors and KV7 channels by the non-opioid analgesic flupirtine

BACKGROUND AND PURPOSE

Flupirtine is a non-opioid analgesic that has been in clinical use for more than 20 years. It is characterized as a selective neuronal potassium channel opener (SNEPCO). Nevertheless, its mechanisms of action remain controversial and are the purpose of this study.

EXPERIMENTAL APPROACH

Effects of flupirtine on native and recombinant voltage- and ligand-gated ion channels were explored in patch-clamp experiments using the following experimental systems: recombinant K_IR3 and K_V7 channels and α3β4 nicotinic acetylcholine receptors expressed in tsA 201 cells; native voltage-gated Na⁺, Ca²⁺, inward rectifier K⁺, K_V7 K⁺, and TRPV1 channels, as well as GABA_A, glycine, and ionotropic glutamate receptors expressed in rat dorsal root ganglion, dorsal horn and hippocampal neurons.

KEY RESULTS

Therapeutic flupirtine concentrations (≤10 µM) did not affect voltage-gated Na⁺ or Ca²⁺ channels, inward rectifier K⁺ channels, nicotinic acetylcholine receptors, glycine or ionotropic glutamate receptors. Flupirtine shifted the gating of K_V7 K⁺ channels to more negative potentials and the gating of GABA_A receptors to lower GABA concentrations. These latter effects were more pronounced in dorsal root ganglion and dorsal horn neurons than in hippocampal neurons. In dorsal root ganglion and dorsal horn neurons, the facilitatory effect of therapeutic flupirtine concentrations on K_V7 channels and GABA_A receptors was comparable, whereas in hippocampal neurons the effects on K_V7 channels were more pronounced.

CONCLUSIONS AND IMPLICATIONS

These results indicate that flupirtine exerts its analgesic action by acting on both GABA_A receptors and K_V7 channels.     

Endothelium-dependent hyperpolarization-related relaxations diminish with age in murine saphenous arteries of both sexes

Background and Purpose

We investigated the effects of aging on the contributions of NO and endothelium-dependent hyperpolarization (EDH) to endothelium-dependent relaxation in saphenous arteries of male and female C57BL/6J mice aged 12, 34 and 64 weeks.

Experimental Approach

Vasomotor responses of saphenous arteries were analysed by wire myography in the absence and presence of stimuli of the endothelium, inhibitors of NOS, and inhibitors and stimulants of small (K_Ca2.3) and intermediate (K_Ca3.1) conductance calcium-activated potassium channels.

Key Results

Arterial relaxing responses to sodium nitroprusside and to ACh in the absence of pharmacological inhibitors (indomethacin and L-NAME), were similar in all age groups and sexes, but those mediated by endothelium-derived NO were slightly but significantly increased in 64-week-old male mice. In the presence of inhibitors, 12-week-old animals showed pronounced ACh-induced relaxation, which was significantly reduced in 34- and 64-week-old mice of both sexes. The EDH-related component of ACh-induced relaxations was abolished by TRAM-34 (K_Ca3.1 blocker) or UCL 1684 (K_Ca2.3 blocker). Although the maximal relaxation induced by NS309 (K_Ca activator) was not affected by aging, the sensitivity for NS309 significantly decreased with aging. The presence of SKA-31 (K_Ca modulator) potentiated relaxations induced by ACh in arteries of 12-week-old but not older mice.

Conclusion and Implications

In a small muscular artery of mice of either sex, total endothelium-dependent relaxation is not affected by age. However, possibly due to changes in K_Ca channel function, the contribution of EDH to endothelium-dependent relaxations decreased with age. The contribution of endothelium-derived NO increases in old male mice.     

Kinetic evidence for a common binding site for substrates and inhibitors of the neuronal noradrenaline carrier

Summary The neuronal noradrenaline uptake mechanism (uptake₁) has been further characterized. For a number of substrates of uptake, the half-saturating concentration (K _m) and the maximal initial transport rate (V _max) were determined. Furthermore, the dissociation constants (K _D) for binding of these substrates to the desipramine binding site of the neuronal noradrenaline carrier were measured. The uptake experiments were done on rat phaeochromocytoma cells (PC12 cells), the binding experiments on purified plasma membranes of PC12 cells. The substrates differed markedly in respect of V _max, K _m, and K _D. Neither K _m and V _max nor K _D and V _max were found to be correlated. However, the discrepancy between K _m and K _D expressed as the ratio, K_m/K_D, was negatively correlated with V _max (r = – 0.9315, n = 7, p < 0.01).For the interpretation of these results a model on the basis of the steady-state assumption has been proposed for uptake₁. From the mathematics of that model the following conclusions can be drawn. (1) The half-saturating substrate concentration (K _m) is not identical with the dissociation constant for the binding of a substrate to the substrate recognition site (K _D). (2) The discrepancy between K _m and K _D is expected to be negatively correlated with the maximal initial transport rate of the substrate (V _max).The experimental results are in good agreement with the proposed model for uptake,. Especially the negative correlation between K _m/K _D and V_max supports the hypothesis that desipramine inhibits uptake, via binding to the substrate recognition site of the neuronal noradrenaline carrier.This study was supported by the Deutsche Forschungsgemeinschaft (SFB 176) Send offprint requests to E. Schömig at the above address     

Formulation development of Carvedilol compression coated tablet

Purpose: The aim of present research was to produce carvedilol compression coated tablet to provide biphasic drug release.

Method: A compressed coated tablet made of a sustained release core tablet and an immediate release coat tablet. Both the core and the coat contained carvedilol. The sustained release effect was achieved with polymers (HPMC K₄M and PEO WSR 205) to modulate the release of the drug. The powder blends for core and coat tablets were evaluated for angle of repose, bulk density, compressibility index, and drug content. Compressed coated tablets were evaluated for thickness, diameter, weight variation test, drug content, hardness, friability, disintegration and in vitro release studies.

Result: The powder blends showed satisfactory flow properties, compressibility, drug content and all the tablet formulations showed acceptable pharmaco-technical properties. Carvedilol contained in the fast releasing component was released within 3?min, whereas the drug in the core tablet was released at different times up to 24?h, depending on the composition of the matrix tablet. The mechanism of drug release was fickian diffusion or anomalous behavior.

Discussion: Batch F7, containing 10?mg PEO WSR 205 and 5?mg HPMC K₄M, showed maximum similarity with theoretical profile and zero order drug release kinetic.     

Interaction of N-benzoyl-D-phenylalanine and related compounds with the sulphonylurea receptor of β-cells

1. The structure activity relationships for the insulin secretagogues N-benzoyl-D-phenylalanine (NBDP) and related compounds were examined at the sulphonylurea receptor level by use of cultured HIT-T15 and mouse pancreatic β-cells. The affinities of these compounds for the sulphonylurea receptor were compared with their potencies for K_ATP-channel inhibition. In addition, the effects of cytosolic nucleotides on K_ATP-channel inhibition by NBDP were investigated.
2. NBDP displayed a dissociation constant for binding to the sulphonylurea receptor (K_D value) of 11 μM and half-maximally effective concentrations of K_ATP-channel inhibition (EC₅₀ values) between 2 and 4 μM (in the absence of cytosolic nucleotides or presence of 0.1 mM GDP or 1 mM ADP).
3. In the absence of cytosolic nucleotides or presence of GDP (0.1 mM) maximally effective concentrations of NBDP (0.1–1 mM) reduced K_ATP-channel activity to 47% and 44% of control, respectively. In the presence of ADP (1 mM), K_ATP-channel activity was completely suppressed by 0.1 mM NBDP.
4. The L-isomer of N-benzoyl-phenylalanine displayed a 20 fold lower affinity and an 80 fold lower potency than the D-isomer.
5. Introduction of a p-nitro substituent in the D-phenylalanine moiety of NBDP did not decrease lipophilicity but lowered affinity and potency by more than 30 fold.
6. Introduction of a p-amino substituent in the D-phenylalanine moiety of NBDP (N-benzoyl-p-amino-D-phenylalanine, NBADP) reduced lipophilicity and lowered affinity and potency by about 10 fold. This loss of affinity and potency was compensated for by formation of the phenylpropionic acid derivative of NBADP. A similar difference in affinity was observed for the sulphonylurea carbutamide and its phenylpropionic acid derivative.
7. Replacing the benzene ring in the D-phenylalanine moiety of NBDP by a cyclohexyl ring increased lipophilicity, and the K_D and EC₅₀ values were slightly lower than for NBDP. Exchange of both benzene rings in NBDP by cyclohexyl rings further increased lipophilicity without altering affinity and potency.
8. This study shows that N-acylphenylalanines interact with the sulphonylurea receptor of pancreatic β-cells in a stereospecific manner. Their potency depends on lipophilic but not aromatic properties of their benzene rings. As observed for sulphonylureas, interaction of N-acylphenylalanines with the sulphonylurea receptor does not induce complete inhibition of K_ATP-channel activity in the absence of inhibitory cytosolic nucleotides.

     

The insulinotropic effect of fluoroquinolones

Antimicrobial fluoroquinolones induce, with strongly varying frequency, life-threatening hypoglycemias, which is explained by their ability to block K_ATP channels in pancreatic B-cells and thus to initiate insulin secretion. In apparent contradiction to this, we observed that none of the fluoroquinolones in this study (gatifloxacin, moxifloxacin, ciprofloxacin, and a number of fluorophenyl-substituted compounds) initiated insulin secretion of perifused mouse islets when the glucose concentration was basal (5 mM). Only when the glucose concentration was stimulatory by itself (10 mM), the fluoroquinolones enhanced secretion. The fluoroquinolones were ineffective on SUR1 Ko islets, which do not have functional K_ATP channels. All of these fluoroquinolones depolarized the membrane potential of mouse B-cells (patch-clamping in the whole-cell mode). Using metabolically intact B-cells (perforated-patch mode) however, 100 μM of gatifloxacin, ciprofloxacin or moxifloxacin were unable to depolarize when the glucose concentration was 5 mM, whereas other K_ATP channel blockers (tolbutamide and efaroxan) remained effective. Only at a very high concentration (500 μM) gatifloxacin and moxifloxacin, but not ciprofloxacin induced repetitive depolarizations which could be antagonized by diazoxide. In the presence of 10 mM glucose all fluoroquinolones which enhanced secretion markedly elevated cytosolic calcium concentration ([Ca²⁺]_i). In the presence of 5 mM glucose gatifloxacin and moxifloxacin at 500 μM but not at 100 μM elevated [Ca²⁺]_i. It is concluded that fluoroquinolones in the clinically relevant concentration range are not initiators, but rather enhancers of glucose-induced insulin secretion. The block of K_ATP channels appears necessary but not sufficient to explain the hypoglycemic effect of fluoroquinolones.     

Cocaine inhibits cromakalim-activated K<Superscript>+</Superscript> currents in follicle-enclosed<Emphasis Type="Italic"> Xenopus</Emphasis> oocytes

The effect of cocaine on K⁺ currents activated by the K_ATP channel opener cromakalim was investigated in follicular cells of Xenopus oocytes. The results indicate that cocaine in the concentration range of 3–500 M reversibly inhibits cromakalim-induced K⁺ currents. The IC₅₀ value for cocaine was 96 M. Inhibition of the cromakalim-activated K⁺ current by cocaine was noncompetitive and voltage independent. Pretreatment with the Ca²⁺ chelator BAPTA did not modify the cocaine-induced inhibition of cromakalim-induced K⁺ currents, suggesting that Ca²⁺-activated second messenger pathways are not involved in the actions of cocaine. Outward K⁺ currents activated by the application of 8-Br-cAMP or forskolin were also inhibited by cocaine. The EC₅₀ and slope values for the activation of K⁺ currents by cromakalim were 184±19 M and 1.14 in the absence of cocaine as compared to 191±23 M and 1.03 in the presence of cocaine (300 M). Cocaine also blocked K⁺ currents mediated through C-terminally deleted form of Kir6.2 (KirC26) in the absence of sulfonylurea receptor with an IC₅₀ value of 87 M, suggesting that cocaine interacts directly with the channel forming Kir6.2 subunit. Radioligand binding studies indicated that cocaine (100 M) did not affect the binding characteristics of the K_ATP ligand, [³H]glibenclamide. These results demonstrate that cromakalim-activated K⁺ currents in follicular cells of Xenopus oocytes are modulated by cocaine.     

Evidence that BKCa channel activation contributes to K+ channel opener induced relaxation of the porcine coronary artery

The rank order of potency of a series of benzopyran and cyanoguanidine K⁺ channel openers (KCOs) for causing relaxation of the PGF₂-precontracted porcine coronary artery was determined. Glyburide, an inhibitor of K_ATP channels, showed an apparent competitive inhibition of the vasorelaxant activity of the KCOs. The pA₂ values of glyburide when cromakalim and CGP 14877 (P1060) were used as vasorelaxants were 7.66 and 7.83, respectively. Charybdotoxin (40 nM), an inhibitor of BK_Ca channels, also caused a significant inhibition of the cromakalim mediated relaxation of the porcine coronary artery. In order to clarify the site of action of these KCOs, we identified a K⁺ channel current in single porcine coronary arterial cells and measured channel activity in the presence of these compounds. The prominent K⁺ ion current in these cells had characteristics typical of the conventional large Ca²⁺-activated K⁺ channel BK_Ca present in other smooth muscle cells. Using symmetrical K⁺ concentrations, the channel had a conductance of 214 pS and was found to be sensitive to [Ca²⁺]_i and membrane potential. The KCOs were found to reversibly increase the open probability (P_o) of the channel without changing channel conductance. The potency of the KCOs to increase K⁺ channel opening was similar to the potency of these compounds to cause coronary artery relaxation. These results indicate that the porcine coronary artery contains the BK_Ca channel and that this channel, along with other types of K⁺ channels (K_ATP), mediate the vasorelaxant effects of K⁺ channel openers.     

Binding of [3H]-P1075, an opener of ATP-sensitive K+ channels, to rat glomerular preparations

ATP-sensitive K⁺ channels (K_ATP channels) in the kidney have been found in the tubular system and in the afferent arteriole. In this study we have examined the binding of [³H]-P1075 ([³H]-N-cyano-N-(1,1-dimethylpropyl)-N-3-pyridylguanidine), a selective opener of K_ATP channels, in rat glomerular preparations.Equilibrium (saturation, competition) and kinetic experiments indicated that [³H]-P1075 binds to a single class of sites with a dissociation constant of about 3 nM and a maximum binding capacity of 10 fmol mg^–1 glomerular protein. The association rate constant of the complex was 6,5×10⁷ M^–1 min^–1; dissociation occurred with a half-time of 6.2 min. Specific [³H]-P1075 binding was strongly reduced when the metabolic state of the glomerular preparation was impaired during the preparation procedure or the binding assay or when the preparation was subjected to mild collagenase treatment. In different metabolically competent preparations, the amount of specific [³H]-P1075 binding correlated well with the number of vascular endings adherent to the glomeruli; no specific binding was found in mesangial cells in culture. Specific [³H]-P1075 binding was inhibited by representatives of the different classes of K_ATP channel openers and by sulphonylureatype blockers with inhibition constants similar to those obtained in rat aortic rings.It is concluded that rat glomerular preparations possess specific binding sites for K_ATP channel openers with vascular characteristics. The sensitivity of binding to mild collagenase treatment suggests that these sites are located on a membrane protein; in addition, the data suggest that these sites are localized on smooth muscle and/or renin secreting cells of the afferent vascular endings attached to some of the glomeruli. Their estimated density (1,500 m^–2) is much higher than that of K_ATP channels in smooth muscle.     

Effect of the Ito activator NS5806 on cloned Kv4 channels depends on the accessory protein KChIP2

BACKGROUND AND PURPOSE

The compound NS5806 increases the transient outward current (I_to) in canine ventricular cardiomyocytes and slows current decay. In human and canine ventricle, I_to is thought to be mediated by K_V4.3 and various ancillary proteins, yet, the exact subunit composition of I_to channels is still debated. Here we characterize the effect of NS5806 on heterologously expressed putative I_to channel subunits and other potassium channels.

EXPERIMENTAL APPROACH

Cloned K_V4 channels were co-expressed with KChIP2, DPP6, DPP10, KCNE2, KCNE3 and K_V1.4 in Xenopus laevis oocytes or CHO-K1 cells.

KEY RESULTS

NS5806 increased K_V4.3/KChIP2 peak current amplitudes with an EC₅₀ of 5.3 ± 1.5µM and significantly slowed current decay. KCNE2, KCNE3, DPP6 and DPP10 modulated K_V4.3 currents and the response to NS5806, but current decay was slowed only in complexes containing KChIP2. The effect of NS5806 on K_V4.2 was similar to that on K_V4.3, and current decay was only slowed in presence of KChIP2. However, for K_V4.1, the slowing of current decay by NS5806 was independent of KChIP2. K_V1.4 was strongly inhibited by 10 µM NS5806 and K_V1.5 was inhibited to a smaller extent. Effects of NS5806 on kinetics of currents generated by K_V4.3/KChIP2/DPP6 with K_V1.4 in oocytes could reproduce those on cardiac I_to in canine ventricular myocytes. K_V7.1, K_V11.1 and K_ir2 currents were unaffected by NS5806.

CONCLUSION AND IMPLICATIONS

NS5806 modulated K_V4 channel gating depending on the presence of KChIP2, suggesting that NS5806 can potentially be used to address the molecular composition as well as the physiological role of cardiac I_to.