The effect of oxygen free radicals on calcium current and dihydropyridine binding sites in guinea-pig ventricular myocytes

1. We used electrophysiological and binding techniques to determine the effects of oxygen free radicals (OFRs) generated by dihydroxyfumaric acid (DHF, 5 mM) on calcium current and dihydropyridine binding sites in guinea-pig isolated ventricular myocytes. 2. Binding of [3H]-PN200-110 to isolated ventricular myocytes revealed one population of binding sites with a KD of 0.11 +/- 0.01 nM and Bmax of 139.1 +/- 6.9 fmol mg-1 protein (n = 24). After 15 min of exposure to DHF, the density, but not the affinity of [3H]-PN200-110 binding sites was significantly (P < 0.01) reduced to 35% of the control value (Bmax = 49.4 +/- 3.7 fmol mg-1 protein, KD = 0.11 +/- 0.01 nM, n = 15). In the presence of superoxide dismutase (SOD) and catalase (CAT) the reduction in [3H]-PN200-110 binding sites was almost completely prevented (Bmax = 120.5 +/- 7.4 in control, n = 4 and 98.8 +/- 7.4 fmol mg-1 protein in DHF plus SOD and CAT, n = 4). KD values were not modified (0.08 +/- 0.01 in control and 0.09 +/- 0.01 nM in DHF plus SOD and CAT). 3. The time-course of the reduction of [3H]-PN200-110 binding sites by OFRs was paralleled by the decrease in L-type calcium current (Ica,L) measured in patch-clamped guinea-pig ventricular myocytes either in the absence or in the presence of EGTA in the patch pipette. In the former conditions OFRs induced the appearance of calcium-dependent alterations, i.e. the transient inward current, within 10 min. After 30 min of incubation with DHF, [3H]-PN200-110 binding sites were reduced to 25% of the control value. 4. In myocytes incubated with the antilipoperoxidant agent, butylated hydroxytoluene (BHT, 50 microM), the decrease in [3H]-PN200-110 binding sites caused by DHF was partially prevented (Bmax values after 30 min exposure to DHF were 55.5 +/- 1.9 and 23.7 +/- 5.9 fmol mg-1 protein in the presence and in the absence of BHT respectively, P < 0.05). BHT did not affect the decrease in [3H]-PN200-110 binding sites during the first 15 min of exposure to DHF, but was able to prevent completely the further decrease occurring during the following 15 min of incubation with OFRs. 5. Our results demonstrate that the OFR-induced decrease in calcium current is associated with a reduction in DHP binding sites. The decrease in calcium current and in calcium channels may be implicated in the mechanical dysfunction associated with oxidative stress.     

Effects of volatile anesthetic isoflurane on ATP-sensitive K+ channels in rabbit ventricular myocytes

It has been suggested that volatile anesthetic, isoflurane mediates cardioprotective effects via activation of the ATP-sensitive K+ (KATP) channels. However, no direct evidence has been provided to define whether isoflurane activates cardiac KATP channels using patch-clamp technique. We examined the effects of isoflurane on the KATP channels in rabbit ventricular myocytes by use of patch-clamp technique. Contrary to the results of the in vivo experiments, isoflurane inhibited the channel activity without a change in the single-channel conductance. Isoflurane decreased the channel activity by a decrease in burst duration and an increase in the inter-burst duration. On the other hand, isoflurane diminished the ATP sensitivity of KATP channels, indicating an increased probability of KATP channel opening for a given concentration of ATP after isoflurane anesthesia. The result supports, at least in part, the hypothesis that isoflurane mediates cardioprotective effects via KATP channel activation.     

Electropharmacological actions of propofol on calcium current in guinea-pig ventricular myocytes

Propofol, a widely-used intravenous anesthetic, causes bradycardia, depression in contractility and hypotension. The cellular mechanisms responsible for these cardiac toxicity remain unclear. In this study, we examined the cellular electropharmacological actions of propofol on calcium current in guinea-pig heart. Single ventricular myocytes were freshly isolated from guinea-pig using modified enzymatic method. Whole-cell voltage-clamp technique was applied with one suction pipette. Transmembrane L-type calcium current (ICa(L)) was separated from other ionic currents by voltage-control, ionic channel blockers and ion substitution methods. Our results show that propofol decreased ICa(L) in a concentration-dependent manner (KD = 54.2 microM). Slope conductance of current-voltage relation was decreased by 56 microM propofol. Propofol did not affect the steady-state activation curve, but shifted the inactivation curve to hyperpolarizing direction. Recovery from inactivation was slowed down by propofol. Marked resting block and use-dependent block were noted. In conclusion, our results indicate that propofol inhibits cardiac L-type calcium current mainly by shifting inactivation curve and retarding the recovery from inactivation.     

Effects of hydroxyl radicals on purified angiotensin I converting enzyme

Somatic angiotensin-converting enzyme (ACE) is a protein which contains two similar domains (N and C), each possessing a functional active site. The relationship between ACE, its natural substrates and oxygen free radicals is starting to be explored. On one hand, superoxide anions production is induced by angiotensin II and on the other hand, activated polynuclear neutrophils, through free radicals generation, alter endothelial ACE activity. In this study, we examined the impact of hydroxyl radicals (.OH) on purified ACE. .OH were produced using a generator: 2,2'-azo-bis 2-amidinopropane (GRH) provided by Lara-Spiral (Fr). GRH (3 mM), in a time-dependent fashion, inhibited ACE activity. When ACE was co-incubated for 4 h with GRH, its activity decreased by 70%. Addition of dimethylthiourea (DMTU: 0.03 to 1 mM) or mannitol + methionine (20/10 mM), two sets of .OH scavengers, produced a dose-dependent protection on ACE activity. To examine whether oxidation of thiol groups in the ACE molecule could be involved in the action of GRH, the effects of thiol reducing agents: mercaptoethanol and dithiotreitol (DTT) were investigated. These compounds produced a dose-dependent and significant protection; with 100% protection at 0.2 and 0.3 mM for mercaptoethanol and at 0.1 mM for DTT. The hydrolysis of two natural and domain-specific substrates were also explored. The hydrolysis of angiotensin I preferentially cleaved by the C domain was significantly (p < 0.01) inhibited by 57, 58 and 69% in contact with 0.3, 1 and 3 mM GRH [in nmol angio II formed/min/nmol of ACE, n = 4; 35.9 +/- 0.6 (control), 15.5 +/- 2.8 (GRH : 0.3 mM), 15.1 +/- 0.5 (1), 10.9 +/- 0.6 (3)]. The hydrolysis of the hemoregulatory peptide (hp), preferential substrate for the N domain was not affected by GRH at 0.3 mM and inhibited by 28% (not significant) by 1 mM GRH [in nmol ph hydrolized/min/nmol ACE, n = 4; 12.6 +/- 1.9 (control), 14.9 (GRH : 0.3 mM), 8.3 +/- 4.0 (1). These results demonstrated that .OH affect ACE activity and could suggest a privileged impact of GRH on the C domain. The precise sites of action of .OH remain unknown. The Cys residues near the active centers, by forming disulphide bridges during the oxidation could be of critical importance. Further studies will be needed to determine whether oxidative stress again ACE can be involved in the genesis of inflammatory vascular pathologies.     

Gadolinium blocks the delayed rectifier potassium current in isolated guinea-pig ventricular myocytes

The effect of Gd3+ on the delayed rectifier potassium current (IK) in single guinea-pig ventricular myocytes was tested using whole-cell patch-clamp techniques. It was found that Gd3+ blocked 70% of the IK tail current at a concentration of 100 microM. The EC50 was 24 microM. Action potential durations were, however, reduced, consistent with a predominant effect on depolarizing L-type Ca2+ current (Ica.L). In the presence of 5 microM nifedipine Gd3+ prolonged the action potential. Using carbon fibres to stretch cells we observed that 10 microM Gd3+ was not effective in reducing a large stretch-activated increase in resting calcium. Modelling studies using the OXSOFT HEART program suggest that this lack of response is influenced by blockade of repolarizing current but is best reproduced by additional blockade of Ca2+ extrusion via the Na(+)-Ca2+ exchanger. When Gd3+ is used as a blocker of stretch-activated channels its actions upon both Ica.L and IK must therefore be accounted for.     

The voltage dependence of contraction at different stimulation rates in guinea-pig ventricular myocytes

Ventricular myocytes, isolated from the guinea-pig, were stimulated to contract by 100 ms long voltage clamp pulses from -80 to 0 mV at 0.5 and 3 Hz. An increase in frequency from 0.5 to 3 Hz led to a positive inotropic effect. Contraction-voltage relationships (CVR) were determined at each frequency. The CVR at 0.5 Hz was bell shaped and peaked between 0 and +20 mV, displaying a voltage dependence similar to the L-type Ca2+ current (ICa). At 3 Hz, contractions continued to increase at positive voltages, giving a more sigmoidal CVR. At 0.5 Hz, TTX reduced the size of steady-state contractions to 91 +/- 2% of control values, but had no effect on the shape of the CVR. At 3 Hz, TTX significantly reduced (P < 0.05) the magnitude of contractions at positive voltages (> or = +20 mV) but had no significant effect on contractions at voltages negative to 0 mV. These data illustrate that intracellular sodium activity (aNa(i)) and, in particular, Na+ entry due to the sodium current (INa) are important in determining the voltage dependence of contraction at positive voltages. Thapsigargin (2.5 microM), a blocker of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase, reduced the size of steady-state contractions at 0 mV to 65 +/- 7% at 0.5 Hz. Increasing frequency to 3 Hz abolished the positive inotropy seen under control conditions. With thapsigargin present, contractions at 0.5 Hz were reduced at all potentials and the CVR was bell shaped. At 3 Hz the CVR was sigmoidal in shape. Contractions were significantly inhibited by thapsigargin at all potentials, but most significantly at more positive potentials (> or = +20 mV). These data show that, at normal body temperature, the shape of the CVR of guinea-pig ventricular myocytes changes with stimulation rate. Due to the voltage dependence of ICa, contractions evoked at positive voltages at 3 Hz must be supported by other mechanisms. The sensitivity of such contractions to TTX and thapsigargin suggests the involvement of both a Na(+)-dependent process and the SR. One possibility is that when aiNa and the Ca2+ content of the SR are raised at higher stimulation rates, enhanced Ca2+ entry via reverse Na(+)-Ca2+ exchange leads to a direct activation of the myofilaments and, to a lesser extent, the release of Ca2+ from the SR.     

Activation of KATP channels by Na/K pump in isolated cardiac myocytes and giant membrane patches

Strophanthidin inhibits KATP channels in 2,4-dinitrophenol-poisoned heart cells (). The current study shows that the Na/K pump interacts with KATP current (IK-ATP) via submembrane ATP depletion in isolated giant membrane patches and in nonpoisoned guinea pig cardiac cells in whole-cell configuration. IK-ATP was inhibited by ATP, glibenclamide, or intracellular Cs+. Na/K pump inactivation by substitution of cytoplasmic Na+ for Li+ or N-methylglucamine decreased both IK-ATP by 1/3 (1 mM ATP, zero calcium), and IC50 of ATP for IK-ATP (0.3 +/- 0.1 mM) by 2/5. The Na+/Li+ replacement had no effect on IK-ATP at low pump activity ([ATP] 相似文献   

Divalent cation channels activated by phenothiazines in membrane of rat ventricular myocytes

Phenothiazines (PTZ) such as chlorpromazine (CPZ) or trifluoperazine (TPZ) induced a sustained divalent cation-permeable channel activity when applied on either side of inside-out patches or on external side of cell-attached patches of adult rat ventricular myocytes. The percentage of active patches was approximately 20%. In the case of CPZ, the Kd of the dose-response curve was 160 microM. CPZ-activated channels were potential-independent in the physiological range of membrane potential and were permeable to several divalent ions (Ba2+, Ca2+, Mg2+, Mn2+). At least three levels of currents were usually detected with conductances of 23, 50 and 80 pS in symmetrical 96 mM Ba2+ solution and 17, 36 and 61 pS in symmetrical 96 mM Ca2+ solution. Saturation curves corresponding to the three main conductances determined in Ba2+ symmetrical solutions (tonicity compensated with choline-Cl) gave maximum conductances of 36, 81 and 116 pS (with corresponding half-saturating concentration constants of 31.5, 38 and 34.5 mM). The corresponding conductance values were estimated to 1.7, 3.3 and 5.2 pS in symmetrical 1.8 mM Ba2+ and to 1.1, 2.4 and 3.7 pS in symmetrical 1.8 mM Ca2+ (the value in normal Tyrode solution). Channels were poorly permeable to monovalent cations, such as Na, with a PBa/PNa ratio of 10. A PTZ-induced channel activity similar to that described in cardiac cells was also observed in cultured rat aortic smooth muscle cells but not in cultured neuroblastoma cells. PTZ-activated channels described in cardiac cells appear very similar to the sporadically active divalent ion permeable channels described in a previous paper (Coulombe et al., 1989). Surprisingly, when 100 microM CPZ were applied to myocytes studied in the whole-cell configuration, and maintained at a holding potential of -80 mV in the presence of 24 mM external Ca2+ or Ba2+, no detectable macroscopic inward current could be observed, whereas the L-type Ca2+ current triggered by depolarizing pulses was markedly and reversibly reduced. The possible reasons are discussed.     

Regional differences in action potential characteristics and membrane currents of guinea-pig left ventricular myocytes

Regional differences in action potential characteristics and membrane currents were investigated in subendocardial, midmyocardial and subepicardial myocytes isolated from the left ventricular free wall of guinea-pig hearts. Action potential duration (APD) was dependent on the region of origin of the myocytes (P < 0.01, ANOVA). Mean action potential duration at 90 % repolarization (APD90) was 237 +/- 8 ms in subendocardial (n = 30 myocytes), 251 +/- 7 ms in midmyocardial (n = 30) and 204 +/- 7 ms in subepicardial myocytes (n = 36). L-type calcium current (ICa) density and background potassium current (IK1) density were similar in the three regions studied. Delayed rectifier current (IK) was measured as deactivating tail current, elicited on repolarization back to -45 mV after 2 s step depolarizations to test potentials ranging from -10 to +80 mV. Mean IK density (after a step to +80 mV) was larger in subepicardial myocytes (1.59 +/- 0.16 pA pF-1, n = 16) than in either subendocardial (1.16 +/- 0.12 pA pF-1, n = 17) or midmyocardial (1. 13 +/- 0.11 pA pF-1, n = 21) myocytes (P < 0.05, ANOVA). The La3+-insensitive current (IKs) elicited on repolarization back to -45 mV after a 250 ms step depolarization to +60 mV was similar in the three regions studied. The La3+-sensitive tail current, (IKr) was greater in subepicardial (0.50 +/- 0.04 pA pF-1, n = 11) than in subendocardial (0.25 +/- 0.05 pA pF-1, n = 9) or in midmyocardial myocytes (0.38 +/- 0.05 pA pF-1, n = 11, P < 0.05, ANOVA). The contribution of a Na+ background current to regional differences in APD was assessed by application of 0.1 microM tetrodotoxin (TTX). TTX-induced shortening of APD90 was greater in subendocardial myocytes (35.7 +/- 7.1 %, n = 11) than in midmyocardial (15.7 +/- 3. 8 %, n = 10) and subepicardial (20.2 +/- 4.3 %, n = 11) myocytes (P < 0.05, ANOVA). Regional differences in action potential characteristics between subendocardial, midmyocardial, and subepicardial myocytes isolated from guinea-pig left ventricle are attributable, at least in part, to differences in IK and Na+-dependent currents.     

Effects of cytisine on hydroxyl radicals in vitro and MPTP-induced dopamine depletion in vivo

Energy content of feedstuffs has to be determined by animal experiments. For practical purposes energy content of feeds was predicted by data on in vivo digestibility of nutrients, in vitro digestibility and different chemical parameters. Especially chemical and in vitro parameters were used in different combinations as predictors. The accuracy of such equations has to be discussed as well as cost and feasibility.     

The effect of alterations to action potential duration on beta-adrenoceptor-mediated aftercontractions in human and guinea-pig ventricular myocytes

Aftercontractions induced by beta-adrenoceptor stimulation in human and guinea-pig cardiomyocytes may be related to changes in action potential duration (APD). We investigated the effects of altering APD during the occurrence of isoproterenol-induced aftercontractions, using the KATP channel openers cromakalim and lemakalim or the action potential voltage clamp technique, in guinea-pig and human ventricular cardiomyocytes. Contractile responses were measured at 32 degrees C using a video-based edge-detection system. In guinea-pig myocytes, action potentials, Indo-1 fluorescence and contraction were measured at 22 degrees C. Isoproterenol (< or = 12 nM) had variable effects on APD but induced aftercontractions, the majority (14/19 cells) of which occurred during the action potential. Short action potentials were produced using K+ channel openers. These compounds reduced or completely abolished the isoproterenol-induced aftercontractions. Increasing isoproterenol in the presence of K+ channel opener restored the main contraction to a level similar to or above those with isoproterenol alone, but without the reappearance of aftercontractions. When cells were stimulated to contract under action potential voltage clamp, isoproterenol-induced aftercontractions were abolished by voltage clamping with action potentials of short duration. It was possible to induce aftercontractions in some cells without application of isoproterenol if voltage clamp-imposed action potentials of very long duration were used. These aftercontractions were also abolished by shortening action potential duration. We conclude that K+ channel openers or the imposition of action potentials of short duration can dissociate positively inotropic beta-adrenoceptor stimulation from aftercontraction formation and that action potentials of long duration can be pro-arrhythmic.     

The effects of propofol and enflurane on single calcium channel currents of guinea-pig isolated ventricular myocytes

1. The effects of the anaesthetics, propofol (100 microM) and enflurane (3%, 1.46 mM), on single L type calcium channel currents were investigated in single myocytes isolated from guinea-pig ventricles. Channel activity was recorded from membrane patches by use of the 'cell-attached' patch-clamp technique (pipette solution containing 110 mM BaCl2, 5 microM Bay K 8644, 5 microM HEPES, pH 7.4; temperature 36 degrees C). 2. Channel conductance was calculated from the slope of the relationship between single channel current and membrane potential during step depolarizations to activate the channel over a range of approximately -20 to +20 mV. Neither propofol (6 cells) nor enflurane (7 cells) caused any significant reduction in channel conductance. 3. Both propofol (7 cells) and enflurane (9 cells) decreased the probability of the channel being open during depolarizations to +10 mV (measured from histograms of the fraction of time spent by the channel at different current levels, taking areas under the Gaussian curves fitted to the open and closed components of the distributions to represent the proportion of time spent in the two states). 4. A fraction of the current traces showed no detectable channel openings in response to step depolarizations to +10 mV. Both propofol and enflurane significantly increased the fraction of silent traces. 5. Transitions across a threshold halfway between the open and closed levels were used to define periods spent in the open and closed states. Both propofol (7 cells) and enflurane (9 cells) reduced the mean open times and increased the mean closed times of the calcium channel. 6. Histograms were plotted showing the distributions of times spent by the channels in the open and closed states. Two exponentials were fitted to the open and closed time distributions. Both propofol (7 cells) and enflurane (9 cells) shortened both time constants fitted to the open times and lengthened both time constants fitted to the closed times.7. It is concluded that both propofol and enflurane appear to alter the kinetics of opening and closing of calcium channels to favour shut channels without altering channel conductance. This effect would be expected to result in a reduction of the macroscopic calcium current and thus contribute to the negative inotropic action of these anaesthetics.     

The role of Gi-proteins and beta-adrenoceptors in the age-related decline of contraction in guinea-pig ventricular myocytes

A decline in contractility in myocytes from ageing guinea-pig hearts was demonstrated, which is more pronounced for maximum beta-adrenoceptor-stimulated activity than contraction in high Ca2+. In this study the role of the inhibitory G-proteins (Gi) in this process was investigated. Comparisons were made between young (Y, < 400 g, < 4 weeks), adult (A. > 600 g, > 8 weeks) and senescent guinea pigs (S, 58-65 weeks, 1136 +/- 30 g). Gi alpha activity, detected by pertussis toxin-catalysed ADP ribosylation, was significantly increased in senescent compared to young animals, but immunodetectable levels of Gi alpha were unchanged, beta-adrenoceptor number was decreased by 27% in senescent compared with young animals (P < 0.002). Pertussis toxin treatment increased the maximum response to isoproterenol in contacting myocytes so that there was no longer any significant decline with age. Maximum contraction amplitudes (sarcomere length change, micron) with isoproterenol before pertussis toxin were 0.144 +/- 0.011 (Y, n = 22 animals), 0.104 +/- 0.009 (A. 18) and 0.098 +/- 0.009 (S. 14), P < 0.01 by analysis of variance (ANOVA). Following toxin treatment amplitudes were 0.140 +/- 0.012 (Y. 12), 0.117 +/- 0.010 (A. 10) and 0.117 +/- 0.018 (S. 8), P = N.S. Pertussis toxin treatment also reversed the effects of ageing on contraction and relaxation velocity in isoproterenol. In contrast, the effect of age on contraction amplitude or velocity in maximum Ca2+ was more pronounced after toxin treatment. The EC50 value for isoproterenol increased with age: pertussis treatment decreased the EC50 in each group, but the effect was especially pronounced for senescent animals. There was no significant difference in the concentration-response curves for the negative inotropic effect of adenosine (in the presence of isoprotenerol) between the three age groups before toxin treatment. All effects of adenosine were abolished after pertussis exposure. We conclude that increased Gi alpha activity is likely to contribute to the decreased response to isoproterenol, but not to high Ca2+, in myocytes from ageing guinea-pigs.     

B-type Ca2+ channels activated by chlorpromazine and free radicals in membrane of human atrial myocytes

The present study demonstrates that background or B-type calcium channel activity can be recorded in excised inside-out and cell-attached membrane patches from human atrial myocytes. In control conditions, with Ba2+ or Ca2+ as charge carrier, single-channel activity spontaneously appeared in irregular bursts separated by quiescent periods of 2-17 min, in nearly 25% of tested patches. Channel activity was recorded at steady-state applied membrane potentials including the entire range of physiological values, and displayed no "rundown" in excised patches. During activity, a variety of kinetic behaviors could be observed with more or less complex gating patterns. This type of channel activity was triggered or markedly increased when chlorpromazine (CPZ 20 or 50 microM) was applied to internal face of inside-out patches, with a proportion of active patches of approximately 25%. CPZ-activated channels were potential-independent in the physiological range of membrane potential. In 96 mM Ba2+ solution, three conductance levels: 23, 42 and 85 pS were routinely observed in the same excised membrane patch, sometimes combining to give a larger level. As previously observed by Wang et al. (1995) in membrane of rat ventricular myocytes, increasing free-radicals level and metabolic poisoning readily enhanced B-type channel activity in human atrial myocytes. Application of H2O2 (from 0.1-10 mM) in cell-attached mode induced an activation of Ba2+ permeable channel activity in a dose-dependent manner, with an estimated EC50 of 9.7 mM. In the same type of experiments, 10 mM deoxyglucose also induced similar Ba2+ permeable channel activity. When 500 microM CPZ were applied to myocytes studied in the whole-cell configuration and maintained at a holding potential of -80 mV in the presence of 5 mM external Ca2+, a noticeable inward current could be observed. The mean CPZ-activated current density determined from seven myocytes was 0.63 pA/pF.     

Effects of insulin on potassium currents of rat ventricular myocytes in streptozotocin diabetes

Membrane currents of ventricular cardiomyocytes isolated from control, diabetic and insulin-treated diabetic Wistar rats have been measured using the whole cell configuration of the patch-clamp technique. Insulin restored the density of the 4-aminopyridine-sensitive early transient component of the calcium-independent outward potassium currents which decreased in diabetes. The inactivation rate of the transients increased in diabetes and was normalised by insulin. The late 4-aminopyridine-insensitive component of the outward currents showed the same diabetes- and insulin-related changes. This current could reflect the activation of the delayed rectifier channels although pharmacological identification of this component could not be achieved.     

Effects of myosin light chain kinase inhibitors on carbachol-activated nonselective cationic current in guinea-pig gastric myocytes

The effects of myosin light chain kinase inhibitors on muscarinic stimulation-activated nonselective cationic current (ICCh) in guinea-pig gastric antral myocytes were studied using the whole-cell patch-clamp technique. ICCh was induced by carbachol (CCh, 50 microM) at a holding potential of -30 mV or -60 mV. ML-7, a chemical inhibitor of myosin light chain kinase (MLCK), inhibited ICCh concentration dependently in a reversible manner (53 +/- 8.6% at 1 microM, mean +/- SE, n = 11). In addition, amplitudes of ICCh were only 37 +/- 2.7% of the daily control values following the addition of a peptide inhibitor of MLCK to the pipette solution. On the other hand, ML-7 had an inhibitory effect on voltage-operated Ca2+ channel current. The peak value of Ba2+ current at 0 mV was reduced to 35 +/- 7.4% (n = 9) by 3 microM of ML-7. As ICCh is known to have an intracellular Ca2+ dependence, we tried to exclude the possibility that ML-7 inhibited ICCh indirectly via suppression of Ca2+ current and the similar inhibitory effects of ML-7 on ICCh were confirmed under the following conditions: (1) clamp of membrane potential at -60 mV; (2) clamp of intracellular [Ca2+] to 1 microM by 10 mM BAPTA; (3) pre-inhibition of Ca2+ channel by verapamil. Different from the effects on ICCh, ML-7 barely inhibited the same cationic current induced by guanosine 5'-O-(3-thiotriphosphate) (GTP[gammaS], 0.2 mM) in the pipette solution. These results suggest that a Ca2+/calmodulin-MLCK-dependent pathway can modulate the activation of ICCh in guinea-pig gastric antral myocytes.     

Membrane phospholipid control of nucleotide sensitivity of KATP channels

Adenosine triphosphate (ATP)-sensitive potassium (KATP) channels couple cell metabolism to electrical activity. Phosphatidylinositol phosphates (PIPs) profoundly antagonized ATP inhibition of KATP channels when applied to inside-out membrane patches. It is proposed that membrane-incorporated PIPs can bind to positive charges in the cytoplasmic region of the channel's Kir6.2 subunit, stabilizing the open state of the channel and antagonizing the inhibitory effect of ATP. The tremendous effect of PIPs on ATP sensitivity suggests that in vivo alterations of membrane PIP levels will have substantial effects on KATP channel activity and hence on the gain of metabolism-excitation coupling.     

Toward understanding the assembly and structure of KATP channels

Adenosine 5'-triphosphate-sensitive potassium (KATP) channels couple metabolic events to membrane electrical activity in a variety of cell types. The cloning and reconstitution of the subunits of these channels demonstrate they are heteromultimers of inwardly rectifying potassium channel subunits (KIR6.x) and sulfonylurea receptors (SUR), members of the ATP-binding cassette (ABC) superfamily. Recent studies indicate that SUR and KIR6.x associate with 1:1 stoichiometry to assemble a large tetrameric channel, (SUR/KIR6.x)4. The KIR6.x subunits form the channel pore, whereas SUR is required for activation and regulation. Two KIR6.x genes and two SUR genes have been identified, and combinations of subunits give rise to KATP channel subtypes found in pancreatic beta-cells, neurons, and cardiac, skeletal, and smooth muscle. Mutations in both the SUR1 and KIR6.2 genes have been shown to cause familial hyperinsulinism, indicating the importance of the pancreatic beta-cell channel in the regulation of insulin secretion. The availability of cloned KATP channel genes opens the way for characterization of this family of ion channels and identification of additional genetic defects.     

Production of hydroxyl radicals in contracting skeletal muscle of cats

Reactive oxygen species increase during exhaustive contraction of skeletal muscle, but characterization of the specific species involved and their rates of production during nonexhaustive muscle contraction have not been investigated. We hypothesized that the production rate of hydroxyl radical (.OH) increases in contracting muscle and that this rate is attenuated by pretreatment with deferoxamine (Def) or dimethylthiourea (DMTU). We measured the rate of production of .OH before, during, and after 5 min of intermittent static contraction of the triceps surae muscles in cats (n = 6) using the formation of p-, m-, and o-tyrosines by hydroxylation of phenylalanine. L-Phenylalanine (30 mg/kg i.v.) was administered to each animal 3 min before contraction. Blood samples were collected from the popliteal vein 1 min before contraction; 1, 3, and 4.5 min during contraction; and 1 min after contraction. During and after contraction, the cumulative production rates of p-, m-, and o-tyrosines were elevated by 42.84 +/- 5.41, 0.25 +/- 0.04, and 0.21 +/- 0.03 nmol.min-1.g-1, respectively, compared with noncontracting triceps surae muscles. Pretreatment with Def (10 mg/kg i.v.; n = 5) or DMTU (10 mg/kg i.v.; n = 4) decreased the cumulative rates of production of p-, m-, and o-tyrosines during and after contraction. Additionally, the rate of tyrosine production increased in proportion to the percentage of maximal tension developed by the triceps surae muscles. These results directly demonstrate that .OH is produced in vivo in the skeletal muscle of cats during intermittent static contraction and that production can occur before the onset of fatigue.