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PubMed1992

Effects of BRL 38227 on neurally-mediated responses in the guinea-pig isolated bronchus.

1. In guinea-pig isolated bronchus treated with indomethacin (2.8 microM), electrical field stimulation (EFS; 10 Hz, 0.5 ms, 60-70 V, for 10 s) evoked a tetrodotoxin (3 microM)-sensitive, biphasic contraction comprising a rapid, atropine (1 microM)-sensitive cholinergic response succeeded by a slowly developing, capsaicin (10 microM)-sensitive, non-adrenergic, non-cholinergic excitatory (NANCe) response. 2. BRL 38227 (0.3-3 microM), salmeterol (0.003-3 microM) and ketotifen (1.0-300 microM) each produced concentration-dependent inhibition of both NANCe and cholinergic responses to EFS in guinea-pig isolated bronchus. 3. Substance P (SP; 1 microM) and neurokinin A (NKA; 0.07 microM) produced contractions equivalent in magnitude to the NANCe response to EFS, which were inhibited by salmeterol (1 microM), but not by BRL 38227 (3 microM) or ketotifen (100 microM). 4. Acetylcholine (ACh; 6 microM) was equi-effective with the electrical activation of cholinergic neurones. BRL 38227 (3 microM) slightly inhibited responses to ACh (6 microM). Salmeterol (1 microM) and ketotifen (100 microM) markedly inhibited responses to ACh (6 microM). 5. In bronchial rings pre-contracted with ACh (100 microM), BRL 38227 (0.1-30 microM), salmeterol (0.001-3 microM) and ketotifen (0.1-100 microM) each produced concentration-dependent relaxation. Unlike ketotifen, BRL 38227 and salmeterol only partially (18.8 +/- 2.1% and 51.8 +/- 3.9% respectively) reversed the ACh-induced contraction. 6. The (+)-analogue of BRL 38227, BRL 38226 (0.3-100 microM), was without effect on responses to EFS and had no effect on the inhibition caused by BRL 38227. The K+-channel activators pinacidil (3.0-30 microM) and RP 52891 (3.0-30 microM) exerted similar inhibitory actions on responses to EFS as BRL 38227, but were less potent. Glibenclamide (0.1-1.O microM) and phentolamine (3 microM) antagonized the inhibitory effects of BRL 38227 on responses to EFS.7. It is concluded that BRL 38227 and ketotifen can inhibit NANCe neuroeffector transmission at concentrations exerting little or no inhibitory effects on responses to exogenously applied tachykinins.By contrast, in addition to suppressing NANCe responses to EFS, salmeterol also markedly inhibits responses to SP and NKA. At concentrations markedly suppressing cholinergic neuroeffector transmission, BRL 38227 has only minor effects on responses to exogenously-applied ACh. Salmeterol and ketotifen both depress responses to ACh within the concentration-range over which they inhibit cholinergic responses to EFS. The inhibitory effects of BRL 38227 on responses to EFS exhibit stereo-specificity and may involve the opening of a neuronal K+-channel. This K+-channel is glibenclamide-and phentolamine-sensitive and appears similar to the smooth muscle K+-channel which is modulated by BRL 38227.

Albuterol↗
PubMed1993

Effects of atrial natriuretic peptide and nitroprusside on isolated pulmonary resistance and conduit arteries from rats with pulmonary hypertension.

1. The relaxant effects of atrial natriuretic peptide (ANP) and nitroprusside were studied on prostaglandin F2 alpha (PGF2 alpha)-contracted preparations of pulmonary resistance vessels (internal diameter 200-500 microns) and main pulmonary arteries taken from rats with pulmonary hypertension induced by monocrotaline (105 mg kg-1, s.c., 4 weeks previously). Control rats received saline. 2. In preparations from monocrotaline-treated rats, the potencies (negative log EC50) of ANP on resistance vessels (8.45) and main pulmonary arteries (8.25) were similar to those obtained in vessels from control rats (8.78 and 8.53 respectively), whereas those of nitroprusside were significantly less than in controls in both resistance vessels (7.13 compared with 7.63 in controls; three fold decrease in potency) and main pulmonary arteries (6.92 compared with 8.17 in controls; 18 fold decrease in potency). 3. On pulmonary resistance vessels from monocrotaline-treated rats, the maximum relaxant responses to ANP and nitroprusside, and also to pinacidil, were increased compared with controls, and reversal of the PGF2 alpha-induced contraction by these drugs was greater than 100%. In contrast, on main pulmonary arteries from monocrotaline-treated rats, the maximum relaxations to ANP and nitroprusside were not increased relative to controls, and reversal of PGF2 alpha was not greater than 100%. 4. Since the high potency of ANP on pulmonary resistance and conduit arteries is retained in vessels from rats with pulmonary hypertension, whether induced by monocrotaline (this study) or by chronic hypoxia (previous findings), it is postulated that elevation of plasma levels of ANP by use of drugs that inhibit the breakdown of this endogenous peptide, may be one approach to the pharmacological treatment of pulmonary hypertension.

Animals↗
PubMed1994

Resting and action potentials of nonischemic and chronically ischemic human ventricular muscle.

INTRODUCTION: The effect of chronic ischemia on the electrical properties of human cardiac tissue is not well understood. METHODS AND RESULTS: Membrane potentials were studied using microelectrode techniques in isolated human ventricular tissues obtained from nonischemic (n = 17) or chronically ischemic (n = 7) myocardium. In normal Tyrode's solution, resting potential (Vr) was lower in ischemic (-70.1 +/- 2.12 mV) than in nonischemic muscles (-77.6 +/- 0.93 mV; mean +/- SEM; P < 0.05). In high [K]o (> 10 mM) media, Vr was of similar magnitude in both types of tissue (in 21.6 mM [K]o, Vr was -53.1 +/- 2.24 mV in nonischemic and -49.6 +/- 2.03 mV in ischemic preparations; n = 7 each; P > 0.05). Lowering [K]o caused persistent hyperpolarization in nonischemic muscles, but caused depolarization in chronically ischemic preparations (in 2.7 mM [K]o, Vr was -84.9 +/- 2.74 mV and -61.7 +/- 7.72 mV, respectively; n = 7; P < 0.05). Pinacidil (100 microM) normalized the response of chronically ischemic preparations to [K]o. Action potentials (APs) from nonischemic tissues varied in shape and could show aberrations. Epinephrine (1.5 microM) and 4-aminopyridine (3 mM) increased the AP duration, while butanedione monoxime (20 mM) and tetrodotoxin (1 microM) shortened it. In chronically ischemic muscles, the AP was characterized by the absence of a plateau and the presence of a slow phase of final repolarization. CONCLUSION: The differential effect of low [K]o on the resting membrane potential of nonischemic and chronically ischemic tissues suggests a change in the properties or the regulation of background K+ channels during chronic ischemia.

4-Aminopyridine↗
PubMed2006

Effect of cyclic guanosine-monophosphate on porcine retinal vasomotion.

PURPOSE: Vasomotion refers to periodic oscillations in vascular tone that ensure the intermittent supply of blood to adjacent microvascular units. Previous evidence from vessels outside the eye suggests that cyclic guanosine-monophosphate (cGMP) is involved in the regulation of vasomotion, but it is unknown whether this compound has an effect on vasomotion in retinal vessels. METHODS: Retinal arterioles from porcine eyes were studied in a wire myograph. After initiation of vasomotion, the vessels were stimulated with increasing concentrations of the cGMP agonist 8-Br-cGMP (n = 6), the phosphodiesterase inhibitor zaprinast (n = 6) and the cGMP synthesis inhibitor L-NAME (n = 6). High concentrations of L-NAME blocked vasomotion, and control experiments (n = 20) using 8-Br-cGMP, S-nitroso-N-acetylpenicillamine (SNAP), adenosine and pinacidil were carried out to elucidate whether this effect was related to changes in the general tone of the vessel. Additionally, the relationship between oscillations in vascular tone and intracellular calcium concentration was studied. RESULTS: Induction of cGMP agonistic activity with either 8-Br-cGMP or zaprinast lowered the vasomotion frequency significantly, whereas L-NAME-induced inhibition of cGMP increased this frequency. Neither of the agents affected the amplitude of the oscillations. The control experiments indicated that the effect of cGMP on vasomotion frequency was independent of the accompanying increase in tone. The oscillations in tone during vasomotion were accompanied by similar oscillations in intracellular calcium concentration. CONCLUSION: Cyclic GMP lowers the frequency without affecting the amplitude of vasomotion in isolated porcine retinal arterioles.

Animals↗
PubMed1995

Inhibition of ATP-sensitive potassium channels in cardiac myocytes by the novel class III antiarrhythmic agent MS-551.

The novel class III antiarrhythmic agent, MS-551, has recently been shown to attenuate the decrease in ventricular effective refractory period and to prevent the subsequent ventricular fibrillation induced by pinacidil and hypoxia in isolated perfused rabbit hearts (Friedrichs et al. 1994). We studied the effects of MS-551 on single ATP-sensitive potassium channels in isolated rabbit ventricular myocytes using standard patch-clamp methods. MS-551 in the range from 1 microM to 100 microM produced a concentration-dependent reduction of the open probability of the ATP-sensitive potassium channel, with an apparent ED50 of 30 microM. This reduced channel activity was due to a smaller number of channel openings per unit time, and the average duration of each opening of the channel was unaffected. This property of MS-551 is likely to be of most significance in ischaemic tissue, where the ATP-sensitive channels are thought to carry the predominant current that shortens the duration of the action potential.

Action Potentials↗
PubMed1994

Rhythmic contractions in isolated small arteries of rat: role of K+ channels and the Na+,K(+)-pump.

Small mesenteric arteries from Wistar rats display rhythmic tension oscillations, associated with oscillations in membrane potential, when stimulated with noradrenaline. The purpose of this study was to investigate the role of potassium conductance and Na+, K(+)-pump activity in the generation of these oscillations. The effect on the rhythmic contractions of several agents, interacting with K+ channels, was studied. Application of apamin, pinacidil or glibenclamide did not affect the rhythmic activity. Tetraethylammonium (TEA) increased the frequency of the rhythmic contractions, while application of 4-aminopyridine (4-AP) increased the amplitude by approximately 50%, with no changes in frequency. Ba2+, on the other hand, impaired the rhythmic contractions or converted them to irregular oscillations in the presence of functional endothelium, but did not affect oscillations in endothelium-denuded vessels. Ouabain or exposure to K(+)-free solution, procedures known to inhibit the Na+,K(+)-pump, abolished the rhythmic contractions. This effect was immediate, suggesting that it was due to elimination of the electrogenic action of the Na+,K(+)-ATPase, rather than to a change in intracellular ion concentrations. Exposure to an extracellular potassium concentration of more than 20 mM also inhibited the oscillation activity. The results suggest that the oscillations are not caused by, but may be modulated by, variations in potassium conductance. The Na+,K(+)-pump seems to play an important role in the generation of rhythmic contractions in these vessels.

4-Aminopyridine↗
PubMed1994

Membrane stretch evoked by cell swelling increases contractile activity in vascular smooth muscle through dihydropyridine-sensitive pathways.

Effects of the dihydropyridine calcium antagonist felodipine and of calcium reduction were studied on osmotically induced contractile responses in the vascular smooth muscle of the rat isolated portal vein. Previous studies have shown that changes in osmolarity that cause cell swelling are accompanied by increased contractile activity in this smooth muscle (Johansson & Jonsson 1968). A transient enhancement of the contractile activity developed in the portal vein on return to standard Krebs solution after exposure to 60 mM urea. This osmotic response was dependent on extracellular Ca2+ (abolished in Ca2+ free solution +0.1 mM EGTA) and was reduced in proportion to the decrease in spontaneous phasic contractile activity when Ca2+ was lowered from the standard 2.5 mM concentration. Felodipine, 3 nM, reduced the spontaneous activity to approximately 50% but showed an even more pronounced inhibitory effect on the osmotic responses which were reduced to less than 20% of control. Other calcium antagonists such as verapamil, 60 nM and diltiazem, 300 nM, were also more effective in inhibiting the osmotic responses than the spontaneous activity. In contrast, the K+ channel opener, pinacidil, 100-200 nM, reduced the spontaneous activity to 50% but had only minor inhibitory effect on the osmotic responses, about 75% still persisting. It is suggested that stretch of the cell membrane in response to variations in osmolarity induces contractile activity in vascular smooth muscle by mechano-electrical coupling involving dihydropyridine-sensitive pathways.

Animals↗
PubMed1997

Properties of cloned ATP-sensitive K+ currents expressed in Xenopus oocytes.

1. We have studied the electrophysiological properties of cloned ATP-sensitive K+ channels (KATP channels) heterologously expressed in Xenopus oocytes. This channel comprises a sulphonylurea receptor subunit (SUR) and an inwardly rectifying K+ channel subunit (Kir). 2. Oocytes injected with SUR1 and either Kir6.2 or Kir6.1 exhibited large inwardly rectifying K+ currents when cytosolic ATP levels were lowered by the metabolic inhibitors azide or FCCP. No currents were observed in response to azide in oocytes injected with Kir6.2, Kir6.1 or SUR1 alone, indicating that both the sulphonylurea receptor (SUR1) and an inward rectifier (Kir6.1 or Kir6.2) are needed for functional channel activity. 3. The pharmacological properties of Kir6.2-SUR1 currents resembled those of native beta-cell ATP-sensitive K+ channel currents (KATP currents): the currents were > 90% blocked by tolbutamide (500 microM), meglitinide (10 microM) or glibenclamide (100 nM), and activated 1.8-fold by diazoxide (340 microM), 1.4-fold by pinacidil (1 mM) and unaffected by cromakalim (0.5 mM). 4. Macroscopic Kir6.2-SUR1 currents in inside-out patches were inhibited by ATP with a Ki of 28 microM. Kir6.1-SUR1 currents ran down within seconds of patch excision preventing analysis of ATP sensitivity. 5. No sensitivity to tolbutamide or metabolic inhibition was observed when SUR1 was coexpressed with either Kir1.1a or Kir2.1, suggesting that these proteins do not couple in Xenopus ocytes. 6. Our data demonstrate that the Xenopus oocyte constitutes a good expression system for cloned KATP channels and that expression may be assayed by azide-induced metabolic inhibition.

Adenosine Triphosphate↗
PubMed2002

Muscle contraction under capillaries in hamster muscle induces arteriolar dilatation via K(ATP) channels and nitric oxide.

We tested the hypothesis that adenosine and nitric oxide can be sensed by capillaries and are implicated in the remote arteriolar dilatation initiated by muscle contraction. We also explored a role for K(ATP) channel activity in this response. Small bundles of muscle fibres underlying a group of capillaries in cremaster muscles of anaesthetized hamsters were electrically stimulated to contract for 2 min at each of 2, 4 and 8 Hz. Diameter changes were measured in the inflow arteriole to the group of capillaries after muscle contraction in the presence or absence of 10(-6) M xanthine amine congener (XAC) to block A(1) and A(2) adenosine receptors, 10(-4) or 10(-3) M N(omega)-nitro-L-arginine (LNNA) to block nitric oxide production, or 10(-5) M glibenclamide to block K(ATP) channel activity. Dilatations were unchanged with XAC (3.0 +/- 0.5, 3.9 +/- 0.7 and 6.1 +/- 1.0 microm), but were significantly reduced with LNNA (to 1.8 +/- 0.6, 3.5 +/- 0.7 and 4.9 +/- 0.7 microm) or glibenclamide (to 0.4 +/- 0.3, 0.8 +/- 0.7 and 1.9 +/- 0.6 microm). Neither K(ATP) channel activity nor nitric oxide was required for transmission or manifestation of the dilator response. Thus, muscle contraction can be sensed by capillaries and the signalling mechanism for the ensuing remote dilatation depends on K(ATP) channel activity and on NO, but not adenosine. Local application of 10(-4) M adenosine, 10(-4) M sodium nitroprusside or 10(-5) M pinacidil directly to capillaries initiated remote arteriolar dilatations. Thus, capillaries can respond directly to known mediators of metabolic vasodilatation, but these signalling pathways are not invariably implicated in the response to muscle contraction.

ATP-Binding Cassette Transporters↗
PubMed2004

SUR2A C-terminal fragments reduce KATP currents and ischaemic tolerance of rat cardiac myocytes.

C-terminal fragments of the sulphonylurea receptor SUR2A can alter the functional expression of cloned ATP-sensitive K(+) channels (K(ATP)). To investigate the protective role of K(ATP) channels during metabolic stress we transfected SUR2A fragments into adult rat cardiac myocytes. A fragment comprising residues 1294-1358, the A-fragment, reduced sarcolemmal K(ATP) currents by over 85% after 2 days (pinacidil-activated current densities were: vector alone 7.04 +/- 1.22; and A-fragment 0.94 +/- 0.07 pA pF(-1), n= 6,6, P < 0.001). An inactive fragment (1358-1545, current density 6.30 +/- 0.85 pA pF(-1), n= 6) was used as a control. During metabolic inhibition (CN and iodoacetate) of isolated myocytes stimulated at 1 Hz, the A-fragment delayed action potential shortening and contractile failure, but accelerated rigor contraction and increased Ca(2+) loading. On reperfusion, A-fragment-transfected cells also showed increased intracellular Ca(2+) and the proportion of cells recovering contractile function was reduced from 40.0 to 9.5% (P < 0.01). The protective effect of pretreatment with 2,4-dinitrophenol, measured from increased functional recovery and reduced Ca(2+) loading, was abolished by the A-fragment. Our data are consistent with a role for K(ATP) channels in causing action potential failure and reduced Ca(2+) loading during metabolic stress, and with a major role in protection by preconditioning. The effects of the A-fragment may arise entirely from reduced expression of the sarcolemmal K(ATP) channel, but we also discuss the possibility of mitochondrial effects.

2,4-Dinitrophenol↗
PubMed2005

Lack of manifestations of diazoxide/5-hydroxydecanoate-sensitive KATP channel in rat brain nonsynaptosomal mitochondria.

Pharmacological modulation of the mitochondrial ATP-sensitive K+ channel (mitoKATP) sensitive to diazoxide and 5-hydroxydecanoate (5-HD) represents an attractive strategy to protect cells against ischaemia/reperfusion- and stroke-related injury. To re-evaluate a functional role for the mitoKATP in brain, we used Percoll-gradient-purified brain nonsynaptosomal mitochondria in a light absorbance assay, in radioisotope measurements of matrix volume, and in measurements of respiration, membrane potential (DeltaPsi) and depolarization-induced K+ efflux. The changes in mitochondrial morphology were evaluated by transmission electron microscopy (TEM). Polyclonal antibodies raised against certain fragments of known sulphonylurea receptor subunits, SUR1 and SUR2, and against different epitopes of K+ inward rectifier subunits Kir 6.1 and Kir 6.2 of the ATP-sensitive K+ channel of the plasma membrane (cellKATP), were employed to detect similar subunits in brain mitochondria. A variety of plausible blockers (ATP, 5-hydroxydecanoate, glibenclamide, tetraphenylphosphonium cation) and openers (diazoxide, pinacidil, chromakalim, minoxidil, testosterone) of the putative mitoKATP were applied to show the role of the channel in regulating matrix volume, respiration, and DeltaPsi and K+ fluxes across the inner mitochondrial membrane. None of the pharmacological agents applied to brain mitochondria in the various assays pinpointed processes that could be unequivocally associated with mitoKATP activity. In addition, immunoblotting analysis did not provide explicit evidence for the presence of the mitoKATP, similar to the cellKATP, in brain mitochondria. On the other hand, the depolarization-evoked release of K+ suppressed by ATP could be re-activated by carboxyatractyloside, an inhibitor of the adenine nucleotide translocase (ANT). Moreover, bongkrekic acid, another inhibitor of the ANT, inhibited K+ efflux similarly to ATP. These observations implicate the ANT in ATP-sensitive K+ transport in brain mitochondria.

ATP-Binding Cassette Transporters↗
PubMed2006

Glucose reduces endothelin inhibition of voltage-gated potassium channels in rat arterial smooth muscle cells.

Prolonged hyperglycaemia impairs vascular reactivity and inhibits voltage-activated K(+) (Kv) channels. We examined acute effects of altering glucose concentration on the activity and inhibition by endothelin-1 (ET-1) of Kv currents of freshly isolated rat arterial myocytes. Peak Kv currents recorded in glucose-free solution were reversibly reduced within 200 s by increasing extracellular glucose to 4 mm. This inhibitory effect of glucose was abolished by protein kinase C inhibitor peptide (PKC-IP), and Kv currents were further reduced in 10 mm glucose. In current-clamped cells, membrane potentials were more negative in 4 than in 10 mm glucose. In 4 mm d-glucose, 10 nm ET-1 decreased peak Kv current amplitude at +60 mV from 23.5 +/- 3.3 to 12.1 +/- 3.1 pA pF(-1) (n = 6, P < 0.001) and increased the rate of inactivation, decreasing the time constant around fourfold. Inhibition by ET-1 was prevented by PKC-IP. When d-glucose was increased to 10 mm, ET-1 no longer inhibited Kv current (n = 6). Glucose metabolism was required for prevention of ET-1 inhibition of Kv currents, since fructose mimicked the effects of d-glucose, while l-glucose, sucrose or mannitol were without effect. Endothelin receptors were still functional in 10 mm d-glucose, since pinacidil-activated ATP-dependent K(+) (K(ATP)) currents were reduced by 10 nm ET-1. This inhibition was nearly abolished by PKC-IP, indicating that endothelin receptors could still activate PKC in 10 mm d-glucose. These results indicate that changes in extracellular glucose concentration within the physiological range can reduce Kv current amplitude and can have major effects on Kv channel modulation by vasoconstrictors.

Animals↗
PubMed2003

Semicarbazide-sensitive amine oxidase substrates potentiate hydralazine hypotension: possible role of hydrogen peroxide.

The relation between inhibition of semicarbazide-sensitive amine oxidase (SSAO) and vasodilation by hydralazine (HYD) was evaluated in chloralose/urethane-anesthetized rats pretreated with various substrates of the enzyme and subsequently administered a threshold hypotensive dose of the vasodilator. The SSAO substrates benzylamine, phenethylamine, and methylamine potentiate the hypotensive response to HYD. Methylamine, which was studied in greater detail because of its status as a possible endogenous SSAO substrate, does not influence the response to the reference vasodilator pinacidil; it does enhance HYD relaxation in aortic rings obtained from pretreated rats. Experiments designed to identify the product of SSAO activity responsible for potentiation by methylamine suggest involvement of hydrogen peroxide (H2O2), as evidenced by the findings that such potentiation is abolished by additional pretreatment with the H2O2-metabolizing enzyme catalase, and that the plasma concentration of H2O2 is increased by methylamine and decreased by HYD. These results are interpreted as a substantiation of the relation between the known SSAO inhibitory effect of HYD and its vasodilator activity. Pretreatment with the SSAO substrates would increase production of H2O2 in vascular smooth muscle and thus magnify the influence of this vasoconstrictor agent on vascular tone. In these conditions, the decrease in H2O2 production and hence in vascular tone caused by SSAO inhibition by HYD would also be magnified. It is speculated that inhibition of vascular SSAO could represent a novel mechanism of vasodilation.

Amine Oxidase (Copper-Containing)↗
PubMed2004

A new ATP-sensitive potassium channel opener reduces blood pressure and reverses cardiovascular remodeling in experimental hypertension.

Some potassium channel openers (KCOs) are potent vasodilators that mainly target the ATP-sensitive potassium channels in vascular smooth muscle cells. Their lack of tissue selectivity limits their clinical use in hypertension therapy. Iptakalim [2,3-dimethyl-n-(1-methylethyl)-2-butylamine], which belongs to a novel chemical type of KCO, possesses unique pharmacological characteristics. In vitro experiments have shown that iptakalim could limit its vasorelaxing actions to resistance vessels. In this study, we investigate the antihypertensive effects of iptakalim on two different experimental hypertensive models: stroke-prone, spontaneously hypertensive rats (SHRsps) and two-kidney with one-clip renal hypertensive dogs (2K1C RHD). In acute hypotensive tests, iptakalim showed stable, long-lasting antihypertensive effects in SHRsps and 2K1C RHDs. Mean-while, it had little effect on heart rate when compared with pinacidil, nifedipine, captopril, or bisoprolol. In experimental therapeutic tests, repeated doses in SHRsps for 30 days or in 2K1C RHDs for 14 days produced consistent antihypertensive effects without causing tolerance. In separate experiments, chronic administration of iptakalim resulted in reversing hypertensive vascular remodeling in spontaneously hypertensive rats and hypertensive cardiac remodeling in SHRsps. These results suggest that iptakalim is a promising antihypertensive drug.

Adenosine Triphosphate↗
PubMed2005

Bepridil, an antiarrhythmic drug, opens mitochondrial KATP channels, blocks sarcolemmal KATP channels, and confers cardioprotection.

Bepridil, which is clinically useful in the treatment of arrhythmias, has been reported to inhibit sarcolemmal ATP-sensitive K(+) (sarcK(ATP)) channels. However, the effect of bepridil on mitochondrial ATP-sensitive K(+) (mitoK(ATP)) channels remains unclear. The objective of the present study was to determine whether bepridil activates mitoK(ATP) channels and confers cardioprotection. SarcK(ATP) channels composed of Kir6.2+SUR2A in human embryonic kidney (HEK) 293 cells were examined using the patch-clamp technique. Flavoprotein fluorescence in guinea pig ventricular cells and matrix volume in isolated rat heart mitochondria were measured to assay mitoK(ATP) channel activity. Mitochondrial Ca(2+) concentration ([Ca(2+)](m)) was measured by loading cells with rhod-2 fluorescence. Coronary-perfused guinea pig ventricular muscles were subjected to 35-min no-flow ischemia followed by 60-min reperfusion. Bepridil (10 microM) completely inhibited the pinacidil-induced Kir6.2+SUR2A channel current expressed in HEK 293 cells. Bepridil reversibly oxidized the flavoprotein and increased mitochondrial matrix volume in a concentration-dependent manner. Furthermore, bepridil significantly attenuated the ouabain-induced increase of [Ca(2+)](m). Pretreatment with bepridil for 5 min before ischemia improved the recovery of developed tension measured after 60 min of reperfusion. These effects of bepridil were abolished by the mitoK(ATP) channel blocker 5-hydroxydecanoate (500 microM) and by the nonselective K(ATP) channel blocker glisoxepide (10 microM). Our results indicate that bepridil is an opener of mitoK(ATP) channels but an inhibitor of sarcK(ATP) channels and exerts a direct cardioprotective effect on native cardiac myocytes. This is the first report of a unique modulator of K(ATP) channels; bepridil would be expected to mitigate ischemic injury while blunting arrhythmias.

Algorithms↗
PubMed2004

The size of a single residue of the sulfonylurea receptor dictates the effectiveness of K ATP channel openers.

K(ATP) channel openers are a diverse group of molecules able to activate ATP-sensitive K(+) channels in a tissue-dependent manner by binding to the channel regulatory subunit, the sulfonylurea receptor (SUR), an ATP-binding cassette protein. Residues crucial to this action were previously identified in the last transmembrane helix of SUR, transmembrane helix 17. This study examined the residue at the most important position, 1253 in the muscle isoform SUR2A and the matching 1290 in the pancreatic/neuronal isoform SUR1 (rat numbering). At this position in either isoform, a threonine enables action of openers, whereas a methionine prohibits it. Using single-point mutagenesis, we have examined the physicochemical basis of this phenomenon and discovered that it relied uniquely on side chain volume and not on shape, polarity, or hydrogen-bonding capacity of the residue. Moreover, the aromatic nature of neighboring residues conserved in SUR1 and SUR2A was found necessary for SUR2A to sustain the wild-type levels of channel activation by the openers tested, the cromakalim analog SR47063 [4-(2-cyanimino-1,2-dihydro-1-pyridyl)-2,2-dimethyl-6-nitrochromene] and the pinacidil analog P1075 [N-cyano-N'-(1,1-dimethylpropyl)-N'-3-pyridylguanidine]. These observations suggest that these residues can interact with openers via nonspecific stacking interactions provided that the adjacent 1253/1290 residue does not obstruct access. The smaller Thr1253 of SUR2A would permit activation, whereas the bulky Met1290 of SUR1 would not. This hypothesis is discussed in the context of a simple molecular model of transmembrane helix 17.

ATP-Binding Cassette Transporters↗
PubMed1997

Nucleotide regulation and characteristics of potassium channel opener binding to skeletal muscle membranes.

[3H]P1075 binding to membrane preparations of rabbit skeletal muscle were observed in the presence of nucleotide triphosphates or diphosphates but not AMP, cAMP, adenosine, tripolyphosphate, or pyrophosphate. Nonhydrolyzable or poorly hydrolyzable ATP analogs inhibited MgATP-supported binding. The EC50 value for MgATP-supported binding (0.4 mM) was decreased approximately 10-fold in the presence of an ATP-regenerating system, and significant metabolism by membrane nucleotidases was confirmed by high performance liquid chromatographic analysis. [3H]P1075 bound to skeletal muscle with a Kd value of 37 +/- 3 nM and a Bmax value of 280 +/- 14 fmol/mg of protein. [3H]P1075 binding to subcellular fractions was highest in membranes enriched in T tubules. Specific binding was reversible, trypsin-sensitive, maximal at pH 8, and stereoselective for the (3S,4R)-enantiomer of cromakalim. Potassium channel openers exhibited a rank order of potency of P1075 > pinacidil > levcromakalim = BMS-180448 > nicorandil > diazoxide = BRL 38226. Fluorescein analogs (ethyleosin, phloxine B, and rose bengal) were relatively potent inhibitors of binding (Ki = 200-300 nM). The potassium channel openers cromakalim and BMS-180448 were competitive inhibitors of [3H]P1075 binding. In contrast, rose bengal and the ATP-regulated potassium channel antagonist glyburide increased the rate of [3H]P1075 dissociation in a manner consistent with noncompetitive interaction.

Adenosine Triphosphate↗
PubMed2002

Interaction of K(ATP) channel modulators with sulfonylurea receptor SUR2B: implication for tetramer formation and allosteric coupling of subunits.

Sulfonylurea receptors (SURs) are subunits of ATP-sensitive K(+) channels (K(ATP) channels); they mediate the channel-closing effect of sulfonylureas such as glibenclamide and the channel-activating effect of K(ATP) channel openers such as the pinacidil analog P1075. We investigated the inhibition by MgATP and P1075 of glibenclamide binding to SUR2B, the SUR subtype in smooth muscle. To increase specific binding, experiments were also performed using SUR2B(Y1206S), a mutant with higher affinity for glibenclamide than for the wild-type (K(D )= 4 versus 22 nM, respectively) but otherwise exhibiting similar pharmacological properties. In the absence of MgATP, [(3)H]glibenclamide binding to both SURs was homogenous. MgATP inhibited [(3)H]glibenclamide binding to both SURs to 25% by reducing the apparent number of glibenclamide binding sites, leaving the affinity unchanged. In the absence of MgATP, P1075 inhibited [(3)H]glibenclamide binding in a monophasic manner with K(i) approximately 1 microM. In the presence of MgATP (1 mM), inhibition was biphasic with one K(i) value resembling the true affinity of P1075 for SUR2B (2-6 nM) and the other resembling K(i) in the absence of MgATP (approximately 1 microM). The data show that (1) MgATP induces heterogeneity in the glibenclamide sites; (2) the high-affinity glibenclamide sites remaining with MgATP are linked to two classes of P1075 sites; and (3) P1075 interacts specifically with SUR2B also in the absence of MgATP. The data are discussed with the assumption that SUR2B, expressed alone, forms tetramers; that MgATP induces allosteric interactions between the subunits; and that mixed SUR2B-glibenclamide-P1075 complexes can exist at equilibrium.

ATP-Binding Cassette Transporters↗