Sulfonylureas, ATP-sensitive K+ channels, and cellular K+ loss during hypoxia, ischemia, and metabolic inhibition in mammalian ventricle

Sulfonylurea derivatives glibenclamide and tolbutamide are selective blockers of ATP-sensitive K+ (KATP) channels. However, their ability to prevent cellular K+ loss and shortening of action potential duration during ischemia or hypoxia in the intact heart is modest compared with their efficacy at blocking KATP channels in excised membrane patches. In the isolated arterially perfused rabbit interventricular septum, the increase in unidirectional K+ efflux and shortening of action potential duration during substrate-free hypoxia were effectively blocked by glibenclamide, but only by very high concentrations (100 microM); during hypoxia with glucose present, glibenclamide was only partially effective at reducing K+ loss. During total global ischemia (10 minutes), up to 100 microM glibenclamide or 1 mM tolbutamide attenuated shortening of action potential duration but only reduced [K+]0 accumulation by a maximum of 32 +/- 6%. In isolated patch-clamped guinea pig ventricular myocytes in which the whole-cell ATP-sensitive K+ current was activated by exposure to the metabolic inhibitors, glibenclamide (up to 100 microM) and tolbutamide (10 mM) were only partially effective at blocking the whole-cell ATP-sensitive K+ current (maximum block, 51 +/- 10% and 50 +/- 9%, respectively), especially when ADP was included in the patch electrode solution. In inside-out membrane patches excised from these myocytes, glibenclamide blocked unitary currents through KATP channels with a Kd of 0.5 microM and a Hill coefficient of 0.5 in the absence of ADP at the cytosolic membrane surface, but block was incomplete when 100 microM ADP (+2 mM free Mg2+) was present. ADP had a similar effect on block of KATP channels by tolbutamide. These findings suggest that free cytosolic [ADP], which rises rapidly to the 100 microM range during early myocardial ischemia and hypoxia, may account for the limited efficacy of sulfonylureas at blocking ischemic and hypoxic cellular K+ loss under these conditions.     

ATP-sensitive potassium channel modulation of the guinea pig ventricular action potential and contraction

The role of ATP-sensitive potassium (KATP) channels in modulating the action potential and contraction of guinea pig ventricular myocytes was investigated. Under voltage clamp, the maximum whole-cell KATP channel conductance was estimated (195 +/- 10 nS, n = 6) by exposing the cells to complete metabolic blockade (2 mM cyanide in the presence of 10 mM 2-deoxy-glucose). In isolated inside-out membrane patches, the ATP dependence of KATP channel activity under relevant conditions was measured (half-maximal inhibition at 114 microM). Under current clamp (with intracellular ATP concentration = 5 mM), the effect of graded KATP channel activation on the action potential and the twitch was estimated by injection of a current (proportional to voltage) that simulated the KATP conductance. As this "conductance" was increased, the action potential was shortened, and contractile amplitude declined, as expected. From the results of these experiments, the quantitative dependence of the action potential duration on intracellular ATP concentration was estimated, without relying on a mathematical model of the cell membrane. The results imply that KATP-dependent action potential shortening is likely to occur if ATP concentration falls below normal levels (approximately 5 mM), as may happen regionally, or globally, during myocardial ischemia.     

Inhibition of Na(+)-K+ pump alleviates the shortening of action potential duration caused by metabolic inhibition via blockade of KATP channels in coronary perfused ventricular muscles of guinea-pigs

The Na(+)-K+ pump is a consumer of intracellular ATP. We therefore examined whether blockade of the Na(+)-K+ pump by cardiac glycosides could inhibit ATP-sensitive K+ (KATP) channels and prolong the action potential duration (APD) of the guinea-pig ventricular muscles perfused with Tyrode's solution via the coronary artery and stimulated at 3 Hz. The metabolic inhibition (MI) achieved by application of 0.1 microM carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (a mitochondrial uncoupler) shortened the APD in a time-dependent manner. When dihydroouabain (DHO, 5 microM) was introduced 5 min but not 10 min after introduction of MI, the APD shortening was significantly attenuated. Application of glibenclamide (1 microM), a blocker of KATP channels, introduced both 5 and 10 min after MI also alleviated the APD shortening: DHO did not alleviate the APD shortening effect produced by cromakalim (5 microM), a KATP-channel opener. In separate experiments using whole-cell patch-clamp techniques, we found that this concentration of DHO (5 microM) depressed the Na(+)-K+ pump current of the guinea-pig ventricular myocytes from 210 to 100 pA (at 0 mV) or by 49.5%. We conclude that, during early phase (approximately 5 min) of MI, the APD shortening mostly results from the activation of KATP channels, and that even a approximately 50% inhibition of the Na(+)-K+ pump by DHO leads to the blockade of KATP channels and eventual lengthening of the APD.     

A role for a glibenclamide-sensitive, relatively ATP-insensitive K+ current in regulating membrane potential and current in rat aorta

OBJECTIVE: ATP-sensitive K+ channels have been classified based on their inhibition by cytoplasmic ATP. Recent evidence in vascular smooth muscle has suggested that these channels show weak sensitivity to intracellular ATP. However, it is not known whether these channels regulate the resting K+ conductance in vascular smooth muscles. Therefore, the aim of the present investigation was to characterize this current in rat aorta myocytes and to examine whether it contributes to setting the membrane potential. METHODS: The conventional and nystatin-permeablised whole cell patch clamp techniques were used to characterize the effect of glibenclamide on membrane potential and K+ current in enzymatically dispersed rat aorta myocytes. RESULTS: The mean resting potential measured in current clamp mode using the permeabilized patch approach was -54 +/- 5 mV (n = 8). Glibenclamide (10 microM) caused a reversible 24-mV depolarization in these cells. In symmetrical K+ (135 mM) solution an inward glibenclamide-sensitive (10 microM) current (-4.1 +/- 0.7 pA/pF; n = 5), hereafter termed Iglib, was observed at a membrane potential of -80 mV when cells held at -60 mV were ramped from -80 to +80 mV. In the absence of any nucleotide in the pipette solution, Iglib measured by the conventional whole-cell method was -23.69 +/- 4.65 pA/pF (n = 9). With 1 and 3 mM ATP in the pipette, the average current density was -25 +/- 6.3 pA/pF (n = 8), and -9.4 +/- 2.7 pA/pF (n = 9), respectively. In the absence of ATP, 1 mM GDP significantly (P < 0.01) increased Iglib (-44.8 +/- 8.4 pA/pF; n = 13). Inclusion of 1 mM ATP in the GDP-containing pipette solution had no significant effect on the current amplitude (-56.4 +/- 10.7 pA/pF; n = 7). Iglib fell to -11.0 +/- 2.9 pA/pF (n = 10) if 1 mM GDP and 3 mM ATP were present. In symmetrical K+, the Iglib observed in the presence of 1 mM ATP in the pipette was increased by more than two-fold in the presence of 10 microM levcromakalim. In PSS containing 5 mM K+, a significant glibenclamide-sensitive current was observed at -45 mV membrane potential when cells dialyzed with 1 mM ATP were ramped between -80 to 30 mV. CONCLUSION: These results demonstrate that Iglib channels in rat aorta myocytes differ from classical KATP channels, being relatively insensitive to intracellular ATP. Iglib therefore appears to have an important role in contributing to the maintenance of the resting potential in rat aortic smooth muscle.     

Role of cardiac ATP-regulated potassium channels in differential responses of endocardial and epicardial cells to ischemia

Epicardial cells are more susceptible to the electrophysiological effects of ischemia than are endocardial cells. To explore the ionic basis for the differential electrophysiological responses to ischemia at the two sites, we used patch-clamp techniques to study the effects of ATP depletion on action potential duration and the ability of ATP-regulated K+ channels in single cells isolated from feline left ventricular endocardial and epicardial surfaces. During ATP depletion by treatment with 1 mM cyanide (CN-), shortening of action potential durations was significantly greater in epicardial cells than in endocardial cells. Thirty minutes after initiating exposure to 1 mM CN-, action potential duration at 90% repolarization was reduced to 0.70 +/- 0.12 of the control value for endocardial cells versus 0.39 +/- 0.18 for epicardial cells (p less than 0.01), and action potential duration at 20% repolarization was reduced to 0.72 +/- 0.13 for endocardial cells versus 0.12 +/- 0.09 for epicardial cells (p less than 0.01). In both endocardial and epicardial cells, the shortening of action potential by CN- treatment was partially reversed by 0.3 microM glibenclamide; the magnitude of reversal, however, was much greater in epicardial cells. After exposure to 1 mM CN-, the activity of ATP-regulated K+ channels in cell-attached membrane patches was significantly greater in epicardial cells than in endocardial cells. To study the dose-response relation between ATP concentration and open-state probability of the channels, intracellular surfaces of inside-out membrane patches containing ATP-regulated K+ channels were exposed to various concentrations of ATP (10-1,000 microM). The concentration of ATP that produced half-maximal inhibition of the channel was 23.6 +/- 21.9 microM in endocardial cells and 97.6 +/- 48.1 microM in epicardial cells (p less than 0.01). These data indicate that ATP-regulated K+ channels are activated by a smaller reduction in intracellular ATP in epicardial cells than in endocardial cells. The differential ATP sensitivity of ATP-regulated K+ channels in endocardial and epicardial cells may be responsible for the differential shortening in action potentials during ischemia at the two sites.     

Comparative studies of ATP sensitive potassium channels in heart and pancreatic beta cells using Vaughan-Williams class Ia antiarrhythmics.

OBJECTIVE: Actions of cibenzoline and disopyramide, agents with Vaughan-Williams class Ia antiarrhythmic action, on ATP sensitive K+ (KATP) channels were examined in heart and pancreatic beta cells. METHODS: Single ventricular myocytes and beta cells were prepared enzymatically from adult Wistar rat hearts and pancreatic islets. Using patch clamp techniques, KATP channel activities were recorded in whole cell and single channel modes. In whole cell experiments, myocytes were bathed with Tyrode's medium (34 degrees C); inside out patches were bathed with internal solutions (22-24 degrees C) containing 1 microM ATP and varying concentrations of cibenzoline or disopyramide. Myocytes were voltage clamped at -40 mV and glibenclamide blockade conductance was produced by cromakalim. RESULTS: Micromolar concentrations of both cibenzoline and disopyramide suppressed cromakalim induced conductance. When applied to the cytosolic surface of the cell membrane in inside out configuration, both drugs reversibly inhibited single KATP channel activities. Neither unitary conductance nor intraburst fast kinetics was affected by the compounds. At a holding potential of -40 mV under symmetrical approximately 150 mM K+ conditions, half maximum doses (IC50) were 0.9 microM [Hill coefficient (h) = 1.3] for cibenzoline induced block of cardiac KATP channels and 1.8 microM (h = 1.0) for disopyramide block. At +40 mV, IC50 for cibenzoline block was 1.4 microM (h = 0.9). Thus there was little voltage dependence in cibenzoline induced channel block. A similar IC50 value of 2.5 microM (h = 1.2 at -60 mV under symmetrical approximately 150 mM K+) was observed for cibenzoline induced block of KATP channels. CONCLUSIONS: Near therapeutic concentrations of cibenzoline and disopyramide inhibit KATP channel activities in both heart and pancreatic beta cells. This may be causally related to the fasting hypoglycaemia which is sometimes reported in patients receiving the drugs. These antiarrhythmic agents may also modulate myocardial electrical properties during hypoxia or ischaemia.     

Unique properties of the ATP-sensitive K⁺ channel in the mouse ventricular cardiac conduction system

Background- The specialized cardiac conduction system (CCS) expresses a unique complement of ion channels that confer a specific electrophysiological profile. ATP-sensitive potassium (K(ATP)) channels in these myocytes have not been systemically investigated. Methods and Results- We recorded K(ATP) channels in isolated CCS myocytes using Cntn2-EGFP reporter mice. The CCS K(ATP) channels were less sensitive to inhibitory cytosolic ATP compared with ventricular channels and more strongly activated by MgADP. They also had a smaller slope conductance. The 2 types of channels had similar intraburst open and closed times, but the CCS K(ATP) channel had a prolonged interburst closed time. CCS K(ATP) channels were strongly activated by diazoxide and less by levcromakalim, whereas the ventricular K(ATP) channel had a reverse pharmacological profile. CCS myocytes express elevated levels of Kir6.1 but reduced Kir6.2 and SUR2A mRNA compared with ventricular myocytes (SUR1 expression was negligible). SUR2B mRNA expression was higher in CCS myocytes relative to SUR2A. Canine Purkinje fibers expressed higher levels of Kir6.1 and SUR2B protein relative to the ventricle. Numeric simulation predicts a high sensitivity of the Purkinje action potential to changes in ATP:ADP ratio. Cardiac conduction time was prolonged by low-flow ischemia in isolated, perfused mouse hearts, which was prevented by glibenclamide. Conclusions- These data imply a differential electrophysiological response (and possible contribution to arrhythmias) of the ventricular CCS to K(ATP) channel opening during periods of ischemia.     

Diazoxide causes early activation of cardiac sarcolemmal KATP channels during metabolic inhibition by an indirect mechanism

OBJECTIVE: We have used isolated myocytes to investigate the effects of diazoxide on sarcolemmal KATP channel (sarcoKATP) activity and action potential failure during metabolic inhibition, and the role of these channels in protection of functional recovery on reperfusion. MATERIALS AND METHODS: Isolated adult rat ventricular myocytes were exposed to metabolic inhibition (NaCN and iodoacetate) and reperfusion. Functional recovery was assessed from the ability of cells to contract on electrical stimulation and to recover calcium homeostasis, measured with fura-2. Action potentials and KATP currents were measured using patch clamp. RESULTS: Pretreatment with diazoxide (100 microM, 5 min) increased the proportion of cells that recovered contractile function after MI and reperfusion from 16.8 +/- 2.4% to 65.0 +/- 2.2% (p<0.001) and the proportion of cells in which [Ca2+]i recovered to <250 nM. Pretreatment also accelerated action potential and contractile failure during MI. In cell-attached patches, MI activated sarcoKATP channels after 224 +/- 11 s, and diazoxide pretreatment decreased this to 145 +/- 24 s (p<0.01). However, diazoxide present in the patch pipette did not accelerate sarcoKATP channel activation. Intracellular Mg2+ rose earlier in diazoxide-pretreated cells. The sarcoKATP blocker HMR 1883 delayed action potential failure and reduced diazoxide protection. CONCLUSIONS: Diazoxide pretreatment increases recovery of function and [Ca2+]i following reperfusion. Protection is coupled with early action potential failure, due to early activation of sarcoKATP channels during metabolic inhibition (MI), which is likely to involve an indirect effect of diazoxide.     

Interrelation between pinacidil and intracellular ATP concentrations on activation of the ATP-sensitive K+ current in guinea pig ventricular myocytes

The patch-clamp technique was used to study the relation between pinacidil and intracellular ATP concentration [( ATP]i) on the activation of the outward K+ current in guinea pig ventricular myocytes. Pinacidil shortened the action potential duration, exhibiting stronger effect at 2 mM [ATP]i than at 5 mM [ATP]i. Pinacidil at 5 microM or higher concentrations activated the time-independent outward current at potentials positive to -80 mV, and the pinacidil-activated current was suppressed by increasing [ATP]i from 2 to 5 mM. The dose-response curve of pinacidil at different [ATP]i showed a shift to the right and a depression of the maximum response at increased [ATP]i. The pinacidil-induced shortening of the action potential duration and outward current were inhibited by application of 0.3-1.0 microM glibenclamide. In single-channel current recordings, pinacidil activated the intracellular ATP-sensitive K+ channel current without changing the unitary amplitude, and increased open probability of the channel, an effect dependent on [ATP]i. The pinacidil-activated single-channel current was blocked by glibenclamide. These results prove the notion that pinacidil activates the ATP-sensitive K+ channel current, which explains the action potential shortening in cardiac cells after application of pinacidil.     

Cardiac IK1 underlies early action potential shortening during hypoxia in the mouse heart

It is established that prolonged hypoxia leads to activation of K(ATP) channels and action potential (AP) shortening, but the mechanisms behind the early phase of metabolic stress remain controversial. Under normal conditions IK1 channels are constitutively active while K(ATP) channels are closed. Therefore, early changes in IK1 may underlie early AP shortening. This hypothesis was tested using transgenic mice with suppressed IK1 (AAA-TG). In isolated AAA-TG hearts AP shortening was delayed by approximately 24 s compared to WT hearts. In WT ventricular myocytes, blocking oxidative phosphorylation with 1 mM cyanide (CN; 28 degrees C) led to a 29% decrease in APD90 within approximately 3-5 min. The effect of CN was reversed by application of 100 microM Ba2+, a selective blocker of IK1, but not by 10 microM glybenclamide, a selective blocker of KATP channels. Accordingly, voltage-clamp experiments revealed that both CN and true hypoxia lead to early activation of IK1. In AAA-TG myocytes, neither CN nor glybenclamide or Ba2+ had any effect on AP. Further experiments showed that buffering of intracellular Ca2+ with 20 mM BAPTA prevented IK1 activation by CN, although CN still caused a 54% increase in IK1 in a Ca2+ -free bath solution. Importantly, both (i) 20 microM ruthenium red, a selective inhibitor of SR Ca2+ -release, and (ii) depleting SR by application of 10 microM ryanodine+1 mM caffeine, abolished the activation of IK1 by CN. The above data strongly argue that in the mouse heart IK1, not KATP, channels are responsible for the early AP shortening during hypoxia.     

Mechanisms for the positive inotropic effect of alpha 1-adrenoceptor stimulation in rat cardiac myocytes.

alpha 1-Adrenoceptor activation can enhance myocardial contractility, and two possible inotropic mechanisms are an increase in myofilament Ca2+ sensitivity and action potential prolongation, which can increase net Ca2+ entry into cells. In adult rat ventricular myocytes (bath Ca2+, 1 mM; stimulated at 0.2-0.5 Hz), the drug 4-aminopyridine and the whole-cell voltage clamp have been used to control Ca2+ entry and differentiate between the two mechanisms. At 22-23 degrees C the specific alpha 1-adrenoceptor agonist methoxamine (100 microM) prolonged action potential duration at 50% repolarization from 55 +/- 2 to 81 +/- 5 msec, delayed time to peak contraction, and increased shortening amplitude from 5.3 +/- 0.6 to 7.8 +/- 1 microns (n = 18). Reduction of the transient outward current and other K+ currents by methoxamine was the major cause of action potential prolongation in rat myocytes with little change in the L-type calcium current. Block of the transient outward current with 2 mM 4-aminopyridine prolonged action potential duration from 52 +/- 6 to 98 +/- 12 msec and increased unloaded cell shortening from 2.9 +/- 0.4 to 6.6 +/- 0.6 microns (n = 4). Subsequently, methoxamine no longer increased cell shortening, although significant potentiation of twitch amplitude was still seen after a brief rest interval. In voltage-clamp experiments, with 70-500-msec pulses, although membrane currents were reduced, methoxamine had no positive inotropic effect and reduced cell shortening from 5.3 +/- 0.7 to 4.97 +/- 0.8 microns at pulse potentials positive to -40 mV. Similar alpha 1-adrenoceptor responses were observed at 35 degrees C during action potential and voltage-clamp experiments, which could be blocked by 10 microM prazosin. In myocytes loaded with the Ca2+ indicator indo-1, alpha 1-adrenoceptor stimulation or 4-aminopyridine both increased cell contraction and intracellular Ca2+ transients by similar amounts. As in unloaded cells, prior exposure to 4-aminopyridine prevented any inotropic effect of methoxamine without changing the systolic intracellular Ca2+ transient. The results indicated that under our experimental conditions positive inotropy in rat cardiomyocytes on exposure to alpha 1-adrenoceptor agonists was strongly correlated with the action potential prolongation that accompanied K+ current reduction. In addition, modulation of K+ channels could occur independent of changes in contractility and/or [Ca2+]i.     

Verapamil diminishes action potential changes during metabolic inhibition by blocking ATP-regulated potassium currents.

Verapamil has beneficial effects on ischemic myocardium, including reduction in electrophysiological derangements, prevention of intracellular K+ loss, and preservation of high-energy phosphates, but the mechanisms underlying these actions are not clear. Recent studies have demonstrated a role of ATP-regulated K+ (KATP) current in action potential shortening and K+ loss during ischemia and metabolic inhibition. Therefore, we studied the effects of verapamil on KATP current in feline ventricular myocytes to test the hypothesis that the drug prevents ischemic electrophysiological disturbances by affecting the KATP channel. Membrane potentials and currents were recorded using standard patch-clamp techniques. During 15-minute superfusion with 1 mM CN-, action potential duration measured at 90% repolarization was reduced from 259 +/- 12 to 98 +/- 15 msec (62% reduction) in the absence of verapamil and from 266 +/- 11 to 183 +/- 16 msec (31% reduction) in the presence of 2 microM verapamil (p less than 0.01). In inside-out membrane patches, the KATP current, activated in the absence of ATP, was significantly suppressed by intracellular application of 2 microM verapamil, but the single-channel conductance was not changed. Verapamil did not change the mean open and closed times of the channel within bursts (e.g., the mean open time was 1.92 +/- 0.18 and 1.82 +/- 0.21 msec in the absence and presence of 2 microM verapamil, respectively), but it shortened the mean lifetime of bursts from 41.1 +/- 3.5 to 24.9 +/- 2.8 msec (p less than 0.01) and prolonged the closed time between bursts from 39.4 +/- 4.6 to 78.5 +/- 5.1 msec (p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

Hypoxia does not activate ATP-sensitive K+ channels in arteriolar muscle cells

OBJECTIVE: To test the hypothesis that hypoxia activates ATP-sensitive K+ (KATP) channels in cremasteric arteriolar muscle cells, resulting in membrane hyperpolarization and inhibition of norepinephrine-induced contraction. METHODS: Arteriolar muscle cells were isolated enzymatically from second- and third-order arterioles that were surgically removed from hamster cremaster muscles. The effects of hypoxia (PO2 = 12-15 mm Hg) were then examined on norepinephrine-induced contraction, membrane currents, and membrane potential in these cells at room temperature. Whole-cell currents and membrane potential were recorded using the perforated patch technique. RESULTS: Hypoxia (12-15 mm Hg PO2) reversibly inhibited norepinephrine-induced contraction to 52 +/- 6% of the response in normoxic solutions (156 mm Hg, n = 12 digests, p < 0.05). These effects of hypoxia could be prevented by superfusion of the cells with either solutions containing the KATP channel antagonist glibenclamide (1 microM) or solutions containing 35 mM K+ to reduce the electrochemical gradient for K+ diffusion. Cromakalim, an activator of KATP channels, also inhibited norepinephrine-induced contraction to a similar extent as hypoxia, and in a glibenclamide and 35 mM K(+)-sensitive manner. These results are consistent with the KATP channel hypothesis. In contrast, hypoxia had no effect on estimated whole-cell membrane conductance between -40 and -90 mV in voltage-clamp experiments; on holding current measured at -60 mV in cells superfused with 143 mM K+ under voltage-clamp conditions; or on membrane potential in current-clamp experiments, despite positive effects of cromakalim in all three protocols. These electrophysiological data lead to rejection of the hypothesis that hypoxia activates KATP channels. CONCLUSIONS: Hypoxia inhibits norepinephrine-induced contraction of cremasteric arteriolar muscle cells by a mechanism that does not involve KATP channels. It is speculated that the inhibitory effects of glibenclamide and 35 mM K+ on the effects of hypoxia on contraction resulted from depolarization induced by these treatments rather than specific inhibition of KATP channels.     

Regulation of ATP sensitive potassium channel of isolated guinea pig ventricular myocytes by sarcolemmal monocarboxylate transport.

OBJECTIVE: The aim was to describe the effects of extracellular application of monocarboxylates (pyruvate, lactate, or acetate) on current through KATP channels (iK,ATP) in isolated guinea pig ventricular myocytes. METHODS: The iK,ATP was elicited during whole cell voltage clamping by application of metabolic poisons, 2,4-dinitrophenol (150 microM) or glucose free cyanide (1 mM) and could be blocked by glibenclamide (3 microM). RESULTS: Extracellular application of monocarboxylates, pyruvate (0.1-10 mM), L-lactate (0.1-10 mM), and acetate (10 mM) led to a rapid inhibition of iK,ATP--an effect which was fully reversible upon washout. Substances without any effect on iK,ATP were (10 mM each) gluconate, citrate, glutamate, creatine, succinate, and glycine. The mechanism underlying the effects of monocarboxylates on iK,ATP was unlikely to be related to an increased ATP production, since D-lactate (10 mM) essentially had the same effect on iK,ATP as the L-isomer of lactate. Furthermore, with intracellular dialysis of alpha-cyano-4-hydroxycinnamate (0.1-0.5 mM), which inhibits pyruvate uptake into mitochondria, extracellular pyruvate exerted the same inhibitory effect on iK,ATP. High concentrations of extracellular alpha-cyano-4-hydroxycinnamate (4 mM), which blocks the sarcolemmal monocarboxylate carrier, prevented the effects on iK,ATP by pyruvate, L-lactate, D-lactate, and acetate. Furthermore, intracellular dialysis with D-lactate (10 mM) led to a more rapid onset of iK,ATP when activated by ATP free dialysis. Activity of isolated KATP channels, measured in isolated membrane patches in the inside out or outside out configuration, typically had a single channel conductance of around 80 pS and was blocked by glibenclamide (3-9 microM). No significant effect of pyruvate was observed in either patch configuration. CONCLUSIONS: In cardiac tissue there may be some modulatory role involving monocarboxylate transport on KATP channel activity, the nature of which is unclear at present but which may involve cytosolic pH changes. Physiological and pathophysiological implications of these findings are discussed.     

Modulation of norepinephrine release by ATP-dependent K(+)-channel activators and inhibitors in guinea-pig and human isolated right atrium.

OBJECTIVE: The aim of this study was to show, whether ATP sensitive K+ channels (KATP channels), are involved in the modulation of norepinephrine (NE) release from the sympathetic nerves innervating the guinea-pig and human right atrium. METHODS: The resting and stimulation-evoked release of [3H]norepinephrine ([3H]NE) was measured from the isolated guinea-pig and human right atrium and the effect of activators and inhibitors of ATP sensitive K+ channels was studied. RESULTS: Cromakalim (30-300 microM), a KATP channel-agonist decreased concentration-dependently the stimulation-evoked release of NE from the guinea-pig atrium, an effect, antagonized by glibenclamide, a KATP channel-antagonist (30 microM). Diazoxide (30-300 microM), another activator of the KATP channels reduced the resting release of NE, and also attenuated the evoked release at a single concentration (100 microM), and this latter action was also counteracted by glibenclamide (30 microM). Pinacidil, increased dose-dependently the resting and stimulation-evoked release of NE in a glibenclamide-sensitive manner and reversed the inhibitory effect of cromakalim (100 microM), suggesting that it acts as an antagonist. Glibenclamide (30-300 microM), by itself enhanced the stimulation-evoked release of [3H]NE, and also increased the resting release of NE. On the other hand, 5-hydroxydecanoate, an ischemia-selective inhibitor of cardiac KATP channels did not change NE release. Adenosine, (30-300 microM), an A1-receptor agonist, clonidine (3 microM), an alpha 2-adrenoceptor agonist and oxotremorine, a muscarinic receptor agonist (30 microM) all reduced the evoked release of [3H]NE, but these effects were not modified by glibenclamide (300 microM), indicating that neuronal adenosine (A1), adrenergic (alpha 2) and muscarinic (M3) receptors do not act on KATP channels. In the human right atrium, cromakalim, and diazoxide did not affect significantly the release of [3H]NE. However, glibenclamide (30-300 microM) and pinacidil (30-300 microM) enhanced dose-dependently the evoked-release of NE, and pinacidil also augmented the resting release. CONCLUSIONS: Our results indicate that sympathetic nerve endings of the human and guinea-pig atrium are endowed with ATP-sensitive K+ channels. These channels responded to agonists and antagonists under the experimental conditions applied and they could modulate the release of NE thereby affecting the autonomic control of cardiac function under various physiological and pathophysiological conditions.     

Effects of 2,4-dinitrophenol or low [ATP]i on cell excitability and action potential propagation in guinea pig ventricular myocytes.

Inhibition of aerobic metabolism leads to a major disruption of cardiac cell homeostasis. The purpose of the present study was twofold: 1) We determined the relative importance of junctional and nonjunctional membrane resistance (Rj and Rm, respectively) in the development of propagation failure during inhibition of aerobic metabolism in guinea pig ventricular cell pairs. 2) We used the patch-action potential clamp technique in single ventricular myocytes to study some of the properties of the membrane channels that are responsible for shortening of action potential duration and eventual failure of cell excitation after metabolic blockade. In most experiments, whole-cell patch pipettes were filled with a solution containing 1 mM EGTA, 5 mM HEPES, and 5 mM ATP. Our results in cell pairs showed that pharmacological inhibition of aerobic metabolism with the mitochondrial uncoupler 2,4-dinitrophenol (DNP) led to a drop in Rm followed by an increase in Rj. The increase in Rj was not sufficient to cause a measurable delay in cell-to-cell propagation, whereas the drop in Rm consistently led to failure of cell excitation. Similar results were obtained in additional experiments in which the EGTA concentration in the pipette was reduced to 50 microM. Similar results were also obtained by loading the recording patch pipettes with a solution containing only 0.1 mM ATP. Our patch-action potential clamp experiments, on the other hand, revealed that DNP induced the opening of time- and voltage-independent membrane channels, with a unitary conductance of 23 pS. The channels allowed for the passage of outward current in the voltage range of the action potential, and the increase in membrane patch conductance correlated with the observed shortening of action potential duration during DNP superfusion. Our experiments provide the first simultaneous recordings of action potentials and DNP-induced channel currents in guinea pig ventricular myocytes. Overall, the data provide new evidence for the understanding of the cellular and subcellular mechanisms involved in the development of slow conduction velocity and propagation block after metabolic blockade.     

Glimepiride (Hoe490) inhibits the rilmakalim induced decrease in intracellular free calcium and contraction of isolated heart muscle cells from guinea pigs to a lesser extent than glibenclamide

Glibenclamide is a potent inhibitor of the ATP-dependent potassium channel. Opening of the ATP-dependent potassium channel is regarded as a mechanism of ischemic preconditioning. This in vitro study examines the influence of glibenclamide and glimepiride, a new sulfonylurea, on the negative inotropic action of the potassium channel opener rilmakalim in isolated ventricular myocytes. Cardiac myocytes were isolated from adult guinea pig hearts by collagenase perfusion and incubated with rilmakalim (concentration range 0.1-12.0 microM), glibenclamide (concentration range 0.03-3.0 microM) plus rilmakalim (3.0 or 7.5 microM), and glimepiride (0.03-9.0 microM) plus rilmakalim (3.0 or 7.5 microM) and paced by electrical field stimulation. Contractility of the myocytes was evaluated by digital image analysis, intracellular free calcium was determined by means of fura-2 fluorescence measurements, and cell viability was assessed morphologically as well as by measurement of lactate dehydrogenase activity. Rilmakalim reduced the systolic intracellular free calcium and contractility of ventricular myocytes in a concentration dependent manner. This effect was antagonized by glibenclamide at lower concentrations (0.3 microM) than glimepiride (3.0 microM). The smaller antagonistic action of glimepiride on the negative inotropic effect of rilmakalim as compared with glibenclamide most likely reflects a less potent inhibition of ATP-dependent potassium channels by glimepiride.     

Decreased ATP-sensitive K(+) current density during chronic human atrial fibrillation

Chronic atrial fibrillation (AF) is associated with shortening of action potential duration (APD), which involves modified activity of atrial ion currents. However, little is known about the activity of ATP-sensitive K(+) channels (I(K,ATP)) during chronic AF. An AF-related increase in the activity of I(K,ATP) would reduce APD and could contribute to initiation and/or perpetuation of AF. Here, we studied the activity of I(K,ATP) in atrial myocytes from patients with sinus rhythm (SR) and chronic AF. Human atrial myocytes were isolated from atrial tissue obtained from patients undergoing open-heart surgery. Inward rectifier currents were measured with the whole-cell patch-clamp technique by applying a depolarizing ramp pulse (1245 ms) from -100 to +40 mV (0.5 Hz). I(K,ATP) was activated with the I(K,ATP) channel opener rilmakalim. The inward rectifier I(K1) and I(K,ATP) were identified by their sensitivity to 1 mM Ba(2+). Density of I(K1) did not differ between cells from patients with AF (at -100 mV: -14.8 +/- 1.3 pA/pF, n = 38/10 (cells/patients)) and SR (-13.8 +/- 1.5 pA/pF, n = 33/16). In both types of cells, rilmakalim stimulated I(K,ATP) (defined as rilmakalim-inducible current) in a concentration-dependent manner (0.3-10 microM). However, maximum activation of I(K,ATP) with 10 microM rilmakalim was smaller in AF than in SR cells (at -100 mV: -5.3 +/- 0.8 pA/pF, n = 22/7 vs. -11.2 +/- 2.9 pA/pF, n = 19/9; at +40 mV: +9.6 +/- 2.1 pA/pF, n = 22/7 vs. +23.7 +/- 3.4 pA/pF, n = 19/9 for AF and SR, respectively; P < 0.05). Only aortic valve disease and pulmonary hypertension were found to be independent contributors to I(K,ATP) current density. We provide evidence that chronic AF is associated with a downregulation of ATP-sensitive K(+) currents. These changes may provide an additional molecular mechanism for electrical remodeling in chronic AF.     

Nitric oxide activates the sarcolemmal K(ATP) channel in normoxic and chronically hypoxic hearts by a cyclic GMP-dependent mechanism

Chronic myocardial hypoxia results in elevated nitric oxide (NO) production and increased current through the sarcolemmal K(ATP) channel. We hypothesized these two processes are related and determined whether NO alters the electrophysiology of Purkinje fibers obtained from rabbits (n=12/group) raised in a normoxic (F(I)O2=0.21) or hypoxic (F(I)O2=0.12) environment from birth to 9 days of age. Action potential duration (APD)(90) was shorter (112+/-3 ms v 126+/-3 ms) and maximum diastolic potential (MDP) was more negative (-84+/-2 mV v-80+/-1 mV) in hypoxic hearts compared with normoxic controls. In normoxic hearts the NO donors, S-nitrosoglutathione (GSNO) 50 microM and spermine NONOate (50 microM) shortened APD(90) and increased MDP to levels present in chronically hypoxic hearts. This effect was completely abolished by the K(ATP) channel blocker glibenclamide (3 microM) and by a nitric oxide trap, Carboxy-PTIO (100 microM). The NO carrier glutathione (50 microM) and decomposed spermine NONOate had no effect on APD(90) or MDP. GSNO had no effect in hypoxic hearts; however, when GSNO was combined with glibenclamide APD(90) increased, and MDP decreased to normoxic values. 8-Bromo cGMP (100 microM) shortened APD(90) and increased MDP to levels present in chronically hypoxic hearts. This effect was abolished by glibenclamide. A soluble guanylyl cyclase inhibitor, ODQ (10 microM), had no effect on action potentials in normoxic hearts but in hypoxic hearts resulted in an increase in APD(90) to levels present in normoxic hearts and a decrease in MDP. The effect of ODQ could not be reversed by GSNO. We conclude that NO activates the sarcolemmal K(ATP) channel in normoxic and chronically hypoxic hearts by a cyclic GMP-dependent mechanism.     

Activation of the cardiac ATP-sensitive K+ channel by ER-001533, a newly synthesized vasorelaxant.

Effects of ER-001533 (ER), a newly synthesized vasorelaxant, on the membrane currents were examined in single ventricular cells of guinea pigs. The patch-clamp technique was used in the "whole-cell" and "inside-out" patch configurations. In the whole-cell clamp condition, ER induced a time-independent K(+)-dominant current, which was inhibited by glibenclamide (1-3 microM), suggesting that ER activated the cardiac ATP-sensitive K+ channel (KATP). To elucidate the mechanism of ER-mediated KATP channel activation, the drug was applied to the inside-out patches before and after channel "run-down." Since nucleotide diphosphates could induce the channel openings after complete run-down, effects of the drug on the nucleotide diphosphate-induced channel openings were also examined. Before run-down, ER activated the KATP channel only in the presence of ATP. ER shifted the relation between [ATP]i and the channel activity to the right in a concentration-dependent fashion without a significant alteration of the slope. After channel run-down, ER did not affect the channel openings either in the absence or in the presence of UDP. However, ER could relieve the ATP-gamma-S inhibition of the UDP-induced channel openings. Thus, we conclude that ER antagonizes the inhibitory effect of ATP on the KATP channel in a competitive manner, thereby enhancing the channel openings.