The rate zonal separation of organelles from dilute suspensions: the problem of a large sample volume

We previously showed that preoperative nicorandil, a hybrid potassium channel opener and nitrate compound, conferred cardioprotective effects in a hypoxia/reoxygenation model of isolated human atrial muscle by using functional recovery as an end point, and that ischaemic preconditioning surprisingly abolished the protection afforded by nicorandil. In view of this paradoxic result, this study was undertaken to assess whether ischaemic preconditioning influences any protective effect of nicorandil by using infarct size as an end point. In addition, we investigated the underlying mechanisms of the protective action of nicorandil. Rabbits underwent a midline sternotomy under anaesthesia. A left coronary branch was occluded for 30 min followed by 120 min of reperfusion. Nicorandil (100 microg/kg bolus + 10 microg/kg/min) was given intravenously 30 min before coronary occlusion and continued to the time of reperfusion (early treatment) or 5 min before reperfusion and continued throughout reperfusion (late treatment). Ischaemic preconditioning was achieved by a single episode of 5-min coronary occlusion followed by 10-min reperfusion before the 30-minute occlusion in the presence or absence of nicorandil. Risk volume and infarct volume were determined by fluorescent microspheres and tetrazolium staining, respectively. Early treatment with nicorandil conferred a significant decrease in percentage of infarct size within the risk zone (24.9 +/- 2.9%) when compared with control (39.2 +/- 4.3%; p < 0.01). Late treatment with nicorandil had no effect on infarct size (43.5 +/- 3.4%). Ischaemic preconditioning also resulted in significant reduction in infarct size (13.4 +/- 4.3%; p < 0.01 vs. control). The combination of ischaemic preconditioning with nicorandil (early treatment) showed an intermediate protective efficacy between early treatment with nicorandil alone and ischaemic preconditioning alone (18.1 +/- 4.2%; p < 0.01 vs. control). Nitroglycerin (10 microg/kg bolus + 1 microg/kg/kg/min, i.v.) given before and during ischaemia tended to reduce infarct size, but the effect was not statistically significant (28.9 +/- 2.9%; p > 0.05 vs. control). Although an adenosine triphosphate (ATP)-sensitive potassium channel blocker, 5-hydroxydecanoate (5 mg/kg, i.v.) by itself had no effect on infarct size (38.8 +/- 3.6%), the protective effect of nicorandil was abolished by 5-hydroxydecanoate (37.7 +/- 5.8%; p < 0.05 vs. early treatment of nicorandil). There were no differences in area at risk or haemodynamics between groups. Our results show that nicorandil has a protective effect against myocardial infarction in our rabbit model when infused before and during ischaemia, but not during reperfusion, and the protective effect is abolished by an ATP-sensitive potassium channel blocker. Furthermore, the addition of ischaemic preconditioning does not detrimentally influence the effect of nicorandil. This suggests that nicorandil can confer an infarct-limiting effect by opening of ATP-sensitive potassium channels with or without intermittent ischaemia, as may happen in patients with unstable angina.     

Coordinate interaction between ATP-sensitive K+ channel and Na+, K+-ATPase modulates ischemic preconditioning

BACKGROUND: We reported that digoxin abolishes the infarct size (IS)-limiting effect of ischemic preconditioning (IPC). Because ATP-sensitive K+ (KATP) channels are involved in IPC, we studied whether Na+,K+-ATPase and KATP channels functionally interact, thereby modulating IPC. METHODS AND RESULTS: Rabbits received 30 minutes of coronary artery occlusion followed by 3 hours of reperfusion. IPC was elicited by 5 minutes of occlusion followed by 10 minutes of reperfusion. The IS, expressed as a percentage of the area at risk, was 40.2+/-2.8% in control and 39.8+/-5.0% in digoxin pretreatment rabbits. Both IPC and pretreatment with cromakalim, a KATP channel opener, reduced IS to 11.8+/-1.8% and 13.4+/-2.6% (P<0. 05 versus control). Digoxin abolished the reduction in IS induced by IPC (33.5+/-3.3%), whereas it did not change that induced by cromakalim (18.8+/-3.0%). In patch-clamp experiments, digoxin was found to inhibit the opening of KATP channels in single ventricular myocytes in which ATP depletion had been induced by metabolic stress. In contrast, digoxin had little effect on the channel opening induced by cromakalim. Moreover, the inhibitory action of digoxin on channel activities was dependent on subsarcolemmal ATP concentration. CONCLUSIONS: The IS-limiting effect of IPC is modulated by an interaction between KATP channels and Na+,K+-ATPase through subsarcolemmal ATP.     

Inhibitory effects of glibenclamide and pertussis toxin on the attenuation of ischemia-induced myocardial acidosis following ischemic preconditioning in dogs

Ischemic preconditioning is known to be mediated by several humoral factors, such as adenosine, norepinephrine, and bradykinin. We examined intracellular signal transduction of ischemic preconditioning following receptor stimulation. Alterations in the pH of the ischemic bed were monitored to assess the response of control and ischemic-preconditioned myocardium to glibenclamide and pertussis toxin. Pentobarbital-anesthetized open-chest dogs were subjected to 40 min of ligation of the left anterior descending coronary artery. Ischemic preconditioning was elicited by 25-min periods of coronary ligation followed by 5 min of reperfusion before a 40-min period of ligation. Glibenclamide (0.3 mg/kg)was given i.v. 20 min before the onset of ischemic preconditioning. Pertussis toxin (6-10 micrograms/kg) was given i.v. 3 days before the experiment. Tissue myocardial pH was measured by a glass micro-pH electrode. Ischemia for 5 min decreased myocardial pH and reperfusion returned it to the preischemic levels. Ischemia for 40 min decreased the myocardial pH from 7.43 +/- 0.06 to 6.43 +/- 0.08. Ischemic preconditioning significantly attenuated the decrease in myocardial pH (6.57 +/- 0.06) induced by 40 min of ischemia. Pretreatment with either glibenclamide or pertussis toxin completely abolished the effect of ischemic preconditioning on ischemic myocardial acidosis. Ischemic preconditioning can attenuate ischemia-induced myocardial acidosis in dogs, and this effect is mediated by activation of adenosine triphosphate-sensitive potassium channels and pertussis toxin-sensitive guanosine triphosphate-binding protein.     

Effect of age on the vasorelaxation elicited by cromakalim. Role of K+ channels and cyclic GMP

The objective of this study was to investigate whether age induces changes on vasodilator response induced by cromakalim, an ATP-sensitive K+ (K(ATP)) channel opener, as well as the underlying mechanism involved in this possible alteration. For this purpose, aortic segments from young (3-5 months) and old (3 years) rabbits were used, which were precontracted with noradrenaline (NA, 1 microM). The vasodilator response induced by cromakalim (0.01-100 microM) was reduced in intact segments from old rabbits, and endothelium removal reduced and did not modify this effect in young and old animals, respectively. In both groups of animals, glibenclamide (10 microM), a blocker of K(ATP) channels, significantly reduced the response elicited by cromakalim, which was not modified by the large conductance Ca2+-activated K+ channel blocker charybdotoxin (ChTX, 0.4 microM). Acetylcholine (ACh, 10 microM) and sodium nitroprusside (SNP, 100 microM) induced a lesser vasodilator effect in aortic segments from old compared with young rabbits. In segments precontracted with NA, 10 microM ACh or 100 microM SNP similarly increased cGMP levels in both groups of animals. However, basal cGMP level was reduced in segments from old rabbits. Incubation with 8-bromo-cGMP (100 microM) increased the response induced by cromakalim in both groups of animals, reaching similar maximum values in young and old rabbits. The response induced by cromakalim plus 8-bromo-cGMP was markedly decreased by glibenclamide and unmodified by ChTx in both types of animals. These results suggest that aging decreases the vasodilator response to cromakalim, mechanism in which appears to be involved the maintained low cGMP levels observed in old rabbits, and that this messenger modulates the degree of K(ATP) channel activation.     

Concerning the effect of the K+ channel blocking agent glibenclamide on ischaemic and reperfusion arrhythmias

Reports on effects of ATP-dependent K+ channel modulating drugs on ischaemia-induced cardiac arrhythmias have been scarce and contradictory. The channel blocking agent glibenclamide (glyburide) has been considered as an antiarrhythmic candidate, because it antagonizes the ischaemic K+ efflux and the shortening of the refractory period. In the present investigation its effects were tested, therefore, in rat hearts with coronary occlusion and reperfusion. In untreated hearts, tachyarrhythmias occurred during the reperfusion, and less pronounced during the coronary occlusion itself. Large amounts of adenosine and its degradation products were released during the coronary reperfusion, particularly from hearts which developed ventricular fibrillation. Glibenclamide (0.1 and 1.0 micromol/l perfusion fluid) neither antagonized the ischaemic nor the reperfusion arrhythmias. Ischaemic arrhythmias were even intensified. Also in control hearts without coronary occlusion, pro-arrhythmic effects of glibenclamide were observed. Furthermore, the coronary flow was considerably decreased by the drug, and the release of adenosine and its metabolites was significantly increased. Sodium nitroprusside antagonized the glibenclamide-induced decrease in the coronary flow, but did not prevent the arrhythmias. The Ca2+ channel blocking agent gallopamil increased the coronary flow, decreased the adenosine release, and antagonized the arrhythmias in hearts with and without glibenclamide. In conclusion, the present findings do not favour the idea of an antiarrhythmic effect of glibenclamide. Rather, some propensity to the occurrence of arrhythmias can be produced by the drug.     

Effects of MK-886, a leukotriene biosynthesis inhibitor, in a rabbit model of endotoxic shock

The aim of this study was to characterise the release of calcitonin gene-related peptide (CGRP) by capsaicin, low pH and prostacyclin in terms of Ca2+ channel dependence, interactions with K(ATP) channels and the role of action potential propagation, in the isolated, perfused guinea-pig heart. The Ca2+ channel blocker omega-conotoxin reduced CGRP release evoked by 10(-7) M capsaicin, as well as CGRP release evoked by pH 7. CGRP release caused by capsaicin at low (10(-7) M) but not high (10(-6) M) concentrations was also attenuated by tetrodotoxin, indicating partial dependence on action potential propagation. CGRP release caused by prostacyclin was not altered by any of the tested drugs. The K(ATP) channel activator cromakalim and the K(ATP) channel blocker glibenclamide had no effect on CGRP release. Previous findings that low pH and capsaicin stimulate capsaicin-sensitive afferents in the isolated heart at least partly through common mechanisms are thus supported. Attenuation of capsaicin-evoked release of CGRP by tetrodotoxin suggests recruitment of additional nerve terminals by a local axon reflex.     

Sulphonylurea treatment of NIDDM patients with cardiovascular disease: a mixed blessing?

Non-insulin-dependent diabetic (NIDDM) patients show a high incidence of cardiovascular disease, with greater risk of recurrent myocardial infarction and a less favourable clinical outcome than non-diabetic patients. The majority of NIDDM patients are treated with sulphonylurea (SU) derivatives. In the 1970's the University Group Diabetes Program concluded that tolbutamide treatment caused increased cardiovascular mortality; the study, which led to curtailment of oral antidiabetic treatment in the USA, was received with scepticism in Europe. Later criticism of its methodology reduced the impact of the study; however, the question of the safety of SU in NIDDM patients with cardiovascular disease has been re-opened in the face of new experimental data. The heart and vascular tissues do have prerequisites for SU action, i.e. SU receptors and ATP-dependent K+ (K+ATP) channels. These channels play an important role in the protection of the myocardium against ischaemia-reperfusion damage, and their closure by SU could lead to amplified ischaemic damage. Here we review evidence from animal and human studies for deleterious SU effects on ischaemia-induced myocardial damage, either by direct action or through diminished cardioprotective preconditioning. Closure of K+ATP channels by SU can lead to reduction of post-infarct arrhythmias; the drug has also been claimed to improve various atherosclerosis risk factors. The evidence for these beneficial effects of SU is also reviewed. We look at the major difficulties that hamper transfer of information from experimental studies to clinical decision-making: a) The affinity of SU for heart K+ATP channels is orders of magnitude lower than for beta-cell channels; is it reasonable to expect in vivo cardiac effects with therapeutic 'pancreatic' SU doses? b) Most studies utilized high doses of acutely administered SU; are effects similar in the chronic steady-state of the SU-treated diabetic patient? c) Convincing SU effects have been demonstrated in acutely induced ischaemia by acutely administering the drug; do such effects persist in the clinical situation of gradually progressive ischaemia? d) Ischaemia and modification of K+ATP channel activity induce complex events, some with opposing effects; what is the net result of SU action, and do different SU derivatives lead to different outcomes? e) In the chronic (and hence clinically relevant) situation, how can direct (deleterious or beneficial) SU effects be separated from beneficial effects mediated by the metabolic action of the drug? Only large prospective clinical studies, making use of advanced technology for assessment of cardiovascular function, can answer these questions. Millions of NIDDM patients are treated with SU derivatives; many are in the age group where cardiovascular risks are extremely high. The question of whether SU derivatives are beneficial or deleterious for these patients must finally be settle unequivocally.     

Ischaemic preconditioning and cardiac surgery

OBJECTIVE: This review discusses the phenomenon of ischaemic preconditioning and its potential application to cardiac surgery. The biology of ischaemic preconditioning is explained and the more limited evidence suggesting that the human heart can be preconditioned is discussed. METHODS AND RESULTS: It is now accepted that the heart is capable of short-term rapid adaptation in response to brief ischaemia so that during a subsequent, more severe ischaemic insult myocardial necrosis is delayed-ischaemic preconditioning. The infarct-delaying properties of ischaemic preconditioning have been observed in all species studied. Five minutes of ischaemia is enough to initiate preconditioning and the protective period lasts for 1-2 h. Laboratory experiments have demonstrated that the stimulation of adenosine receptors initiates preconditioning and the intracellular signal transduction mechanisms involve protein kinase C and ATP-dependent potassium channels, although there may be some differences between species. An analysis of studies on myocardial infarction in humans has revealed that some patients reporting angina in the days before infarction have a better outcome and this may be due to the ischaemia causing preconditioning. More direct evidence has come from an investigation of patients undergoing percutaneous transluminal angioplasty in whom the ST-segment changes induced by balloon inflation were more marked during the first inflation than the second. In patients undergoing coronary artery bypass grafting the decline in ATP content during the first 10 min of ischaemia was reduced in patients subjected to a brief preconditioning protocol. CONCLUSIONS: Preconditioning is a powerful and reproducible method of protecting the myocardium from irreversible ischaemic injury. There is now evidence indicating that the human heart can be preconditioned. However, more trials are necessary in patients undergoing cardiac surgery before the role of preconditioning as a means of myocardial protection can be assessed.     

Preconditioning improves myocardial function and reflow, but not vasodilator reactivity, after ischaemia and reperfusion in anaesthetized dogs

1. The present study examines whether three cycles of brief coronary artery occlusion and reperfusion (i.e. ischaemic preconditioning; PC) can prevent vasodilator dysfunction and the impairment of myocardial reflow caused by prolonged ischaemia. Coronary blood flow, left ventricular dP/dt, systemic arterial blood pressure and heart rate were measured in open-chest anaesthetized dogs. 2. Sixty minute occlusion of the left circumflex coronary artery (LCx) and 60 min LCx reperfusion (ISC/REP; group 1) significantly reduced resting coronary blood flow (CBF, initial 29 +/- 3 mL/min; ISC/REP 20 +/- 3 mL/min, P < 0.05 vs initial) and increased coronary vascular resistance (CVR, initial 4.1 +/- 0.6 mmHg/min per mL; ISC/REP 5.8 +/- 1.0 mmHg/min per mL, P < 0.05 vs initial). By contrast CBF and CVR were not affected in dogs subjected to preconditioning before ischaemia (group 2: CBF, initial 24 +/- 4 mL/min; PC+ISC/REP 23 +/- 4 mL/min; CVR, initial 4.7 +/- 0.6 mmHg/min per mL; PC+ ISC/REP 5.3 +/- 1.0 mmHg/min per mL). These data suggest that ischaemic preconditioning prevents the ischaemia-induced impairment of myocardial reflow. 3. Ischaemia and reperfusion impaired coronary dilator responses to the endothelium-dependent dilator acetylcholine (delta CBF, after ISC/REP: 50 +/- 6% of initial) and the endothelium-independent dilator glyceryl trinitrate (delta CBF, ISC/REP: 46 +/- 6% of initial). Despite the improvement in reperfusion in the preconditioned group, there was no significant improvement in responses to acetylcholine (PC+ISC/REP 52 +/- 6% of initial) or glyceryl trinitrate (PC+ISC/REP 59 +/- 6% of initial) after ischaemia and reperfusion. 4. The reduction in left ventricular dP/dt after ischaemia and reperfusion was significantly smaller in the preconditioned group indicating a lower level of impairment of cardiac contractility. In addition, we confirmed that preconditioning caused a significant reduction in infarct size and a reduction in the release of lactate dehydrogenase indicating less cardiac injury. 5. These results suggest that although ischaemic preconditioning was able to improve both myocardial reperfusion and contractility, it was not able to preserve vasodilator function. Such a reduction in vasodilator reserve could prevent adequate myocardial perfusion under conditions of elevated oxygen demand.     

Pituitary adenylate cyclase activating peptides relax human pulmonary arteries by opening of KATP and KCa channels

BACKGROUND: Pituitary adenylate cyclase activating peptides (PACAPs) are potent endothelium independent dilators of human coronary arteries; however, their effects on human pulmonary arteries are unknown. METHODS: The vasorelaxant effects of PACAP27 on human pulmonary segmental arteries were studied and the specific potassium (K+) channel regulatory mechanisms in the vasorelaxant effects were tested by means of isometric contraction experiments. RESULTS: PACAP27 produced dose dependent relaxations of 10 microM rings preconstricted with prostaglandin F2 alpha (PGF2 alpha) with half maximal relaxation (IC50) at 17 nM. Pretreatment of the vessels with the ATP sensitive K+ (KATP) channel blocker glibenclamide (1 microM) or with the Ca2+ activated K+ (KCa) channel blocker iberiotoxin (100 nM) inhibited the PACAP27 induced relaxation. CONCLUSIONS: These results provide evidence that PACAPs are potent vasodilators of human pulmonary arteries and that this relaxation might be mediated by opening of KATP and KCa channels.     

Flexor reflex afferents reset the step cycle during fictive locomotion in the cat

It has been reported that antiarrhythmic drugs possessing the property of potassium channel blockade were most effective in preventing halothane-epinephrine induced arrhythmias. Recent attention has focused on ATP-sensitive potassium (K(ATP)) channels because of their contribution to the cardiovascular actions of volatile anesthetics. The present study was designed to evaluate whether K(ATP) channels or transient outward potassium channels (Ito) were involved in the mechanism of halothane-epinephrine arrhythmias in rat. Rats were anesthetized with halothane (1.5%), and the lungs were mechanically ventilated. The arrhythmogenic thresholds of epinephrine during halothane anesthesia were determined in 74 rats receiving saline or one of tested agents. The arrhythmogenic dose of epinephrine (ADE) was significantly increased by a K(ATP) channel opener, JTV506 (P < 0.01), and had a tendency to be increased by other K(ATP) channel openers, cromakalim, nicorandil, KRN2391 and Y 26763, but were not affected by a K(ATP) channel blocker, glibenclamide. The Ito blocker, 4-aminopyridine, also significantly increased the ADE. Epinephrine produced second-degree or complete atrioventricular block in 4 out of 7 rats receiving glibenclamide. These results suggest that Ito but not K(ATP) channels might be involved in the mechanism in producing halothane-epinephrine arrhythmias.     

Pharmacological analysis in rat of the role of the ATP-sensitive potassium channel as a potential target for antifibrillatory intervention in acute myocardial ischaemia

We tested the hypothesis that blockade of the ATP-sensitive K+ channel (IK(ATP)) is an antiarrhythmic mechanism in acute myocardial ischaemia, using an opener of the channel (10 microM RP 49356, RP) and a blocker of the channel (10 microM glibenclamide, GL) and a combination of the two drugs (GL+RP, 10 microM each) in a randomised blinded study. Isolated rat hearts (n = 8 per group) were subjected to 30-min left regional ischaemia. GL and GL+RP widened QT interval after 10-min ischaemia (197 +/- 39 and 203 +/- 20 ms, respectively vs. 154 +/- 12 ms in controls), whereas RP significantly shortened QT interval (123 +/- 6 ms). GL and GL+RP decreased coronary flow (p < 0.05). RP caused slight increase in flow during ischaemia. These effects are all consistent with modulation of vascular and cardiac IK(ATP). RP alone had no effect on ischaemia-induced arrhythmias. Neither did GL have any effect on the incidence of ventricular fibrillation (VF: 88 vs. 100% in controls). However, GL reduced the incidence of sustained VF (VF lasting continuously for > 2 min) to 14% vs. 88% in controls (p < 0.05). Therefore, GL had defibrillatory activity. Surprisingly, in view of these findings, the GL+RP combination significantly reduced the incidence of VF to 25% (from 100% in control hearts, p < 0.05) i.e., had an antifibrillatory effect. So, two agents that produce pharmacological effects attributable to block and opening of IK(ATP) when administered singly had no effects on the incidence of ischaemia-induced VF.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tissue specificity of sulfonylureas: studies on cloned cardiac and beta-cell K(ATP) channels

Sulfonylureas stimulate insulin secretion from pancreatic beta-cells by closing ATP-sensitive K+ (K(ATP)). The beta-cell and cardiac muscle K(ATP) channels have recently been cloned and shown to possess a common pore-forming subunit (Kir6.2) but different sulfonylurea receptor subunits (SUR1 and SUR2A, respectively). We examined the mechanism underlying the tissue specificity of the sulfonylureas tolbutamide and glibenclamide, and the benzamido-derivative meglitinide, using cloned beta-cell (Kir6.2/SUR1) and cardiac (Kir6.2/SUR2A) K(ATP) channels expressed in Xenopus oocytes. Tolbutamide inhibited Kir6.2/SUR1 (Ki approximately 5 micromol/l), but not Kir6.2/SUR2A, currents with high affinity. Meglitinide produced high-affinity inhibition of both Kir6.2/SUR1 and Kir6.2/SUR2A currents (Kis approximately 0.3 micromol/l and approximately 0.5 micromol/l, respectively). Glibenclamide also blocked Kir6.2/SUR1 and Kir6.2/SUR2A currents with high affinity (Kis approximately 4 nmol/l and approximately 27 nmol/l, respectively); however, only for cardiac-type K(ATP) channels was this block reversible. Physiological concentrations of MgADP (100 micromol/l) enhanced glibenclamide inhibition of Kir6.2/SUR1 currents but reduced that of Kir6.2/SUR2A currents. The results suggest that SUR1 may possess separate high-affinity binding sites for sulfonylurea and benzamido groups. SUR2A, however, either does not possess a binding site for the sulfonylurea group or is unable to translate the binding at this site into channel inhibition. Although MgADP reduces the inhibitory effect of glibenclamide on cardiac-type K(ATP) channels, drugs that bind to the common benzamido site have the potential to cause side effects on the heart.     

Modulation by endogenous ATP of the light-evoked release of ACh from retinal cholinergic neurones

The retina is an area of the central nervous system that possesses intrinsic cholinergic neurones which release acetylcholine (ACh) in response to stimulation with flickering light. Using an eye-cup preparation in anaesthetized rabbits we found that when the retina was exposed to the P2-purinoceptor antagonist, PPADS, the light-evoked release of ACh was strikingly increased (by over 40%). In contrast, ATP reduced the light-evoked release of ACh by 20%. The inhibitory effect of ATP was not due to its catabolism to adenosine because it was not affected by the A1-adenosine receptor antagonist, DPCPX, in combination with adenosine deaminase. The actions of both ATP and PPADS were completely blocked by strychnine. We conclude that during physiological stimulation of the retina with light, ATP is co-released with ACh and partially inhibits ACh release by activating (with ACh) an inhibitory glycinergic feedback loop.     

An association study between a transcriptional polymorphism in the serotonin transporter gene and panic disorder in a Japanese population

1. To determine the effects of an acute oral dose of glibenclamide on blood pressure (BP), basal forearm vascular resistance (FVR) and FVR responses to the K+ATP channel activating vasodilator diazoxide, a placebo-controlled, double-blind cross-over study was performed in eight male volunteers with non-insulin-dependent diabetes mellitus. 2. Changes in vascular responses to progressively increasing concentrations of diazoxide (3.75-30 mg/kg per min) and noradrenaline (25-100 ng/kg per min) were measured by venous occlusion plethysmography. 3. Glibenclamide significantly lowered plasma glucose levels compared with placebo (P < 0.02) and attenuated the decrease in FVR (P < 0.05) and the decrease in systolic BP (P < 0.05) that followed a meal. However, vasodilator responses to diazoxide were potentiated by the administration of oral glibenclamide (P < 0.01). 4. Acute administration of oral glibenclamide attenuates the normal decrease in FVR and systolic BP that follows a meal and potentiates rather than inhibits forearm vasodilator responses to intra-arterial diazoxide, probably via indirect humoral effects. These results suggest that glibenclamide has direct or indirect vasoconstrictor effects that antagonize the normal increase in forearm blood flow that follows a meal and that the inhibition of vascular K+ATP channels following acute oral glibenclamide administration is clinically insignificant compared with other indirect vascular effects of the drug.     

The high prevalence of bipolar II and associated cyclothymic and hyperthymic temperaments in HIV-patients

The role of K+ channels in mediating vasorelaxation induced by two prostacyclin analogues was investigated in guinea-pig aorta. Iloprost caused substantial relaxation of tissues contracted with phenylephrine or 25 mM K+ but not 60 mM K+. In endothelial-denuded tissues, maximal relaxations to iloprost, cicaprost or isoprenaline were inhibited by approximately 40-50% with tetraethylammonium or iberiotoxin, both blockers of large conductance Ca2+-activated K+ (BKCa) channels. In contrast, the response to forskolin, an activator of adenylate cyclase was marginally inhibited by tetraethylammonium. The K(ATP) channel blocker, glibenclamide significantly augmented the response to iloprost but not cicaprost. These effects were largely inhibited by the EP1 receptor antagonist, 8-chlorodibenz[b,f][1,4]oxazepine-10(11H)-carboxylic acid 2-[1-oxo-3(4-pyridinyl)propyl]hydrazide, monohydrochloride (SC-51089) and partially by indomethacin, suggesting that iloprost relaxation is counterbalanced by activation of EP1 receptors, in part through a constrictor prostaglandin. We conclude that BKCa channels play an important role in mediating the effects of iloprost and cicaprost and raises the possibility that cyclic AMP-independent pathways might be involved.     

Rejuvenation of the aging forehead and brow

Long-lasting myocardial ischaemia reduces the density of sarcolemmal L-type calcium channels (LCC). Ischaemic preconditioning protects the myocardium against development of infarction. The aim of this study was to investigate if ischaemia-induced loss in LCC is affected by ischaemic preconditioning. Specific (+) - [3H]isradipine binding to LCC was compared in membranes and homogenates from control and ischaemic regions of non-preconditioned and ischaemically preconditioned hearts [two 10 min left anterior descending coronary artery (LAD) occlusions, each followed by 30 min reperfusion]. Biopsies were sampled after 60 min mid LAD occlusion from ischaemic and control (supplied by circumflex artery) regions. Sixty min ischaemia reduced binding density of specific (+) - [3H]isradipine in membranes by 23 +/- 11% (n = 7, P < 0.05) in the non-preconditioned group and by 20 +/- 8% (n = 6, P < 0.05) in the preconditioned group. Binding density in homogenates was reduced by 36 +/- 5% (n = 5. P < 0.05) in the non-preconditioned group and by 21 +/- 5% (n = 5. P < 0.05) in the preconditioned group. The reductions in the two groups and reductions in membranes and homogenates were not statistically different. The dissociation constant of binding was similar in the groups. In conclusion, 60 min of ischaemia reduced the binding density of (+)-[3H]isradipine in membranes and homogenates by 20-36%. The reduction in density of binding sites was not caused by redistribution of sarcolemmal LCC to an intracellular compartment. Ischaemic preconditioning did not affect the decline in binding density as hypothesized.     

Roles of calcium-activated and voltage-gated delayed rectifier potassium channels in endothelium-dependent vasorelaxation of the rabbit middle cerebral artery

1. The cellular mechanism(s) of action of endothelium-derived vasodilator substances in the rabbit middle cerebral artery (RMCA) were investigated. Specifically, the subtypes of potassium channels involved in the effects of endothelium-derived relaxing factors (EDRFs) in acetylcholine (ACh)-induced endothelium-dependent vasorelaxation in this vessel were systematically compared. 2. In the endothelium-intact RMCA precontracted with histamine (3 microM), ACh induced a concentration-dependent vasorelaxation, which was sensitive to indomethacin (10 microM) or N(G)-nitro-L-arginine (L-NOARG; 100 microM); pD2 values 8.36 vs 7.40 and 6.38, P < 0.01 for both, n = 6 and abolished by a combination of both agents. ACh caused relaxation in the presence of high K+ PSS (40 mM KCl), which was not affected by indomethacin, but abolished by L-NOARG and a combination of indomethacin and L-NOARG. 3. In the presence of indomethacin, relaxation to ACh in the endothelium-intact RMCA precontracted with histamine was unaffected by either glibenclamide (10 microM), an ATP-sensitive K+ channel (K[ATP]) blocker, 4-aminopyridine (4-AP, 1 mM) or dendrotoxin (DTX, 0.1 microM), delayed rectifier K channel (Kv) blockers. However, relaxation responses to ACh were significantly inhibited by either LY83583 (10 microM) and 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ, 10 microM), guanylyl cyclase inhibitors, or charybdotoxin (CTX; 0.1 microM), iberiotoxin (ITX, 0.1 microM) and apamin (APA, 0.1 microM), large conductance Ca2+-activated K+ channels (BK[Ca]) blocker and small conductance Ca2+-activated K+ channel (SK[Ca]) blocker, respectively. 4. In the presence of L-NOARG, relaxation to ACh was unaffected by glibenclamide or the cytochrome P450 mono-oxygenase inhibitor, clotrimazole (1 microM), but was significantly inhibited by either 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ 22,536, 10 microM) and 2',3'-dideoxyadenosine (2',3'-DDA, 30 microM), adenylyl cyclase inhibitors, or 4-AP, DTX, CTX, ITX and APA. 5. In the endothelium-denuded RMCA precontracted with histamine, authentic NO-induced relaxation was unaffected by glibenclamide, 4-AP and DTX, but significantly reduced by ODQ, ITX and APA. Authentic prostaglandin I2 (PGI2)-induced relaxation was unaffected by glibenclamide, but significantly reduced by 2',3'-DDA, 4-AP, DTX, ITX and APA. Forskolin-induced relaxation was significantly inhibited by high K+, CTX and 4-AP. 6. These results indicate that: (1) in the RMCA the EDRFs released by ACh are NO and a prostanoid (presumably PGI2), and there is no evidence for the release of a non-NO/PGI2 endothelium-derived hyperpolarizing factor (EDHF), (2) K(Ca) channels are involved in NO-mediated relaxation of the RMCA but both K(Ca) and Kv channels are involved in PGI2-mediated relaxation.     

Terikalant, an inward-rectifier potassium channel blocker, does not abolish the cardioprotection induced by ischemic preconditioning in the rat

Recent results have shown that the sulfonylurea receptor couples to several types of inward-rectifier potassium (KIR) channels, which suggests that sensitivity to blockade of a pathophysiological phenomenon such as ischemic preconditioning (PC) by glibenclamide may not be the result of this compound selectively blocking the ATP-sensitive potassium (KATP) channel. Therefore, to address this possibility, a role for myocardial KIR v KATP channels in ischemic PC was evaluated in the rat. To test this hypothesis, anesthetized, open-chest, male Wistar rats were assigned to one of seven experimental protocols. Animals assigned to group I (control) received 30 min of occlusion and 2 h of reperfusion. Ischemic PC was produced by 3x5-min occlusion and 2-h reperfusion periods (group II). Terikalant (TK), an inward-rectifier potassium channel blocker, was used to test the role of other K+ channels, most notably the KIR, in the cardioprotective effect of ischemic PC in the rat. TK was given at a dose of 3 mg/kg, i.v., 15 min before the prolonged occlusion and reperfusion periods (group III). In groups IV, V, and VI terikalant (1, 3 and 6 mg/kg, i.v.) was given 15 min before ischemic PC (lowTK+PC, medTK+PC and hiTK+PC, respectively). Group VII consisted of glibenclamide (0.3 mg/kg, i.v.) given 30 min prior to ischemic PC (GLY+PC). Infarct size (IS) as a percent of the area at risk (AAR) was measured using the histochemical stain, 2,3, 5-triphenyltetrazolium chloride. The average IS/AAR for the control was 49.9+/-2.1%. Ischemic PC markedly reduced infarct size (8.6+/-1. 8%; * P<0.05 v control). Terikalant (TK; 1, 3 and 6 mg/kg, i.v.) did not abolish the cardioprotective effect of ischemic PC at any dose (15.5+/-6.4, 16.4+/-5.2 and 8.8+/-1.6%, respectively; * P<0.05 v control). TK itself had no effect on infarct size. GLY completely abolished the cardioprotective effect of ischemic PC (48.2+/-6.4%). In addition, the high dose of TK significantly (P<0.05) increased the action potential duration at 50% repolarization from 48+/-3 to 64+/-4 ms and 30 microM of TK, a concentration which produced a 39% decrease in the inward-rectifier potassium channel current in isolated guinea-pig ventricular myocytes in the whole-cell patch-clamp mode did not block the increase in K ATP current produced by the KATP opener bimakalim (3 microM). These results demonstrate that although the myocardial KATP channel belongs to the K IR superfamily, the endogenous myocardial KIR channel does not mediate ischemic PC in the rat heart; however, the K ATP channel does mediate its cardioprotective effect.     

Effects of hydroxyl radicals on KATP channels in guinea-pig ventricular myocytes

We studied the effects of oxygen free radicals on the ATP-sensitive potassium channel (KATP channel) of guinea-pig ventricular myocytes. Single KATP channel currents were recorded from inside-out patches in the presence of symmetrical K+ concentrations (140 mM in both bath and pipette solutions). Reaction of xanthine oxidase (0.1 U/ml) on hypoxanthine (0.5 mM) produced superoxide anions (.O2-) and hydrogen peroxide (H2O2). Exposure of the patch membrane to.O2- and H2O2 increased the opening of KATP channels, but this activation was prevented by adding 1 microM glibenclamide to the bath solution. In the presence of ferric iron (Fe3+: 0.1 mM), the same procedure produced hydroxyl radicals (.OH) via the iron-catalysed Haber-Weiss reaction.OH also activated KATP channels; however, this activation could not be prevented by, even very high concentrations of glibenclamide (10 microM). These different effects of glibenclamide suggest that the mode of action of these oxygen free radicals on KATP channels is different and that.OH is more potent than.O2-/H2O2 in activating KATP channels in the heart.