Methods: Male Sprague-Dawley rats were anesthetized and the chest was opened. All animals were subjected to 30 min of occlusion of the left coronary artery and 2 h of reperfusion. Before the 30-min occlusion, rats received either preconditioning by ischemia (ischemic preconditioning, 5-min occlusion, 5-min reperfusion x 3) or pretreatment with remifentanil, performed with the same regime (3 x 5-min infusions) using 0.2, 0.6, 2, 6, or 20 [mu]g[middle dot]kg-1[middle dot]min-1 intravenously. The experiment was repeated with naltrindole (a selective [DELTA]-opioid receptor antagonist, 5 mg/kg), nor-binaltorphimine (a selective [kappa]-OR antagonist, 5 mg/kg), or CTOP (a selective [mu]-opioid receptor antagonist, 1 mg/kg) administered before remifentanil-induced preconditioning or ischemic preconditioning, respectively. Infarct size, as a percentage of the area at risk, was determined by 2,3,5-triphenyltetrazolium staining.
Results: There was a dose-related reduction in infarct size/area at risk after treatment with remifentanil that was similar to that seen with ischemic preconditioning. This effect was prevented or significantly attenuated by coadministration of a [mu], [kappa], or [DELTA]-opioid antagonist. The infarct-sparing effect of ischemic preconditioning was abolished by blockade of [kappa]-opioid receptors or [DELTA]-opioid receptors but not by [mu]-opioid receptors. 相似文献
Methods: Remote preconditioning was induced in anesthetized male Sprague-Dawley rats by three cycles of 5 min of right femoral artery occlusion followed by 5 min of reperfusion. Myocardial ischemia-reperfusion was achieved by ligation of the left anterior descending coronary artery for 30 min and then reperfusion for 120 min. Infarct size was determined by 2,3,5-triphenyltetrazolium chloride staining. Levels of lactate dehydrogenase, dynorphin, and met-enkephalin in plasma were measured. The opening of the mitochondrial permeability transition pore was monitored with fluorescent calcein in isolated ventricular myocytes.
Results: Both remote preconditioning and U-50,488H (10 mg/kg intravenous), a [kappa]-opioid receptor agonist, significantly decreased the infarct size and plasma lactate dehydrogenase level induced by ischemia-reperfusion, and these effects were attenuated by nor-binaltorphimine (10 mg/kg intravenous), a [kappa]-opioid receptor antagonist, and atractyloside (5 mg/kg intravenous), a mitochondrial permeability transition pore activator. However, administration of naltrindole (5 mg/kg), a [delta]-opioid receptor antagonist, had no effect on the cardioprotection by remote preconditioning. The dynorphin plasma level was increased after remote preconditioning treatment, but the met-enkephalin level did not change. In isolated ventricular myocytes loaded with calcein, U-50,488H (300 [mu]m) decreased the mitochondrial permeability transition pore opening induced by calcium (200 [mu]m), and this effect was attenuated by cotreatment with nor-binaltorphimine (5 [mu]m) or atractyloside (20 [mu]m). 相似文献
Methods: Ventricular myocytes were enzymatically isolated from rabbit hearts. Action potential and membrane currents were recorded in current and voltage clamp modes.
Results: Morphine at concentrations from 0.01 to 1 [mu]m significantly prolonged cardiac action potential, and at 0.1 and 1 [mu]m slightly but significantly hyperpolarized the resting membrane potential. In addition, morphine at 0.1 [mu]m significantly augmented ICa.L (at +10 mV) from 5.9 +/- 1.9 to 7.3 +/- 1.7 pA/pF (by 23%; P < 0.05 vs. control) and increased IK1 (at -60 mV) from 2.8 +/- 1.0 to 3.5 +/- 0.9 pA/pF (by 27%; P < 0.05 vs. control). Five [mu]m naltrindole (a selective [delta]-opioid receptor antagonist) or 5 [mu]m norbinaltorphimine (a selective [kappa]-opioid receptor antagonist) prevented the increase in ICa.L induced by morphine, but 5 [mu]m CTOP (a selective [mu]-opioid receptor antagonist) did not. The three types of opioid antagonists did not affect the augmentation of IK1 by morphine. Morphine had no effect on IK. 相似文献
Methods: Cerebellar brain slices from adult Sprague-Dawley rats were incubated with morphine at 0.1-10 [mu]m in the presence or absence of various antagonists for 30 min. They were then kept in morphine- and antagonist-free buffer for 30 min before they were subjected to simulated ischemia (oxygen-glucose deprivation) for 20 min. After being recovered in oxygenated artificial cerebrospinal fluid for 5 h, they were fixed for morphologic examination to determine the percentage of undamaged Purkinje cells.
Results: The survival rate of Purkinje cells was significantly higher in slices preconditioned with morphine (>= 0.3 [mu]m) before the oxygen-glucose deprivation (57 +/- 4% at 0.3 [mu]m morphine) than that of the oxygen-glucose deprivation alone (39 +/- 3%, P < 0.05). This morphine preconditioning-induced neuroprotection was abolished by naloxone, a non-type-selective opioid receptor antagonist, by naltrindole, a selective [delta]-opioid receptor antagonist, or by 7-benzylidenenaltrexone, a selective [delta]1-opioid receptor antagonist. However, the effects were not blocked by the [mu]-, [kappa]-, or [delta]2-opioid receptor antagonists, [beta]-funaltrexamine, nor-binaltorphimine, or naltriben, respectively. Morphine preconditioning-induced neuroprotection was partially blocked by the selective mitochondrial adenosine triphosphate-sensitive potassium channel antagonist, 5-hydroxydecanoate, or the mitochondrial electron transport inhibitor, myxothiazol. None of the inhibitors used in this study alone affected the simulated ischemia-induced neuronal death. 相似文献
Methods: The authors recorded isometric contraction of human right atrial trabeculae suspended in oxygenated Tyrode's solution (34[degrees]C; stimulation frequency, 1 Hz). Before a 30-min anoxic period, 3, 6, and 9% desflurane was administered during 15 min. Desflurane, 6%, was also administered in the presence of 10 [mu]m glibenclamide, a KATP channels antagonist; 10 [mu]m HMR 1098, a sarcolemmal KATP channel antagonist; 800 [mu]m 5-hydroxy-decanoate (5-HD), a mitochondrial KATP channel antagonist; 1 [mu]m phentolamine, an [alpha]-adrenoceptor antagonist; 1 [mu]m propranolol, a [beta]-adrenoceptor antagonist; and 100 nm 8-cyclopentyl-1,3-dipropylxanthine (DPX), the adenosine A1 receptor antagonist. Developed force at the end of a 60-min reoxygenation period was compared (mean +/- SD).
Results: Desflurane at 3% (95 +/- 13% of baseline), 6% (86 +/- 6% of baseline), and 9% (82 +/- 6% of baseline) enhanced the recovery of force after 60 min of reoxygenation as compared with the control group (50 +/- 11% of baseline). Glibenclamide (60 +/- 12% of baseline), 5-HD (57 +/- 21% of baseline), DPX (63 +/- 19% of baseline), phentolamine (56 +/- 20% of baseline), and propranolol (63 +/- 13% of baseline) abolished desflurane-induced preconditioning. In contrast, HMR 1098 (85 +/- 12% of baseline) did not modify desflurane-induced preconditioning. 相似文献
Methods: Four days after administration of Freund adjuvant into the hind paw of male Wistar rats, antinociceptive effects of intraplantar and subcutaneous injection of FE 200665 and FE 200666 were measured by paw pressure algesiometry and compared with the [kappa]-agonist U-69,593. Peripheral and [kappa]-receptor selectivity was assessed by the antagonists naloxone methiodide (NLXM) and nor-binaltorphimine, respectively. Antiinflammatory effects were evaluated by paw volume plethysmometry and histologic score.
Results: Similar to intraplantar U-69,593, intraplantar FE 200665 (3-100 [mu]g) and FE 200666 (1-30 [mu]g) resulted in significant and dose-related increases of paw pressure thresholds. Higher doses of FE 200665 (0.2-20 mg) and FE 200666 (0.06-6 mg) were required by subcutaneous route to produce similar antinociceptive responses, supporting a peripheral site of action. nor-Binaltorphimine dose-dependently antagonized this effect, implying [kappa]-opioid selectivity. Analgesic effects of subcutaneous FE 200665 and FE 200666 were abolished by intraplantar nor-binaltorphimine, and both subcutaneous and intraplantar effects were dose-dependently antagonized by subcutaneous NLXM, further demonstrating a peripheral site of action. One to 6 days after Freund adjuvant inoculation, single and repeated intraplantar injections of FE 200665, FE 200666, and U-69,593 significantly reduced paw volume and histologic scores. Both changes were reversed by intraplantar nor-binaltorphimine and subcutaneous NLXM. 相似文献
Methods: Isolated rat hearts (n = 80) underwent 30 min of no-flow ischemia and 60 min of reperfusion. Two groups with eight hearts each underwent the protocol without intervention (control-1 and control-2), and, in eight hearts, preconditioning was elicited by two 5-min periods of ischemia before the 30 min ischemia. In the six treatment groups (each n = 8), ketamine, R (-)- or S (+)-ketamine were administered at concentrations of 2 or 20 [mu]g/ml before preconditioning. Eight hearts received 20 [mu]g/ml R (-)-ketamine before ischemia. Left ventricular (LV) developed pressure and creatine kinase (CK) release during reperfusion were determined as variables of ventricular function and cellular injury.
Results: Baseline LV developed pressure was similar in all groups: 104 +/- 28 mmHg (mean +/- SD). Controls showed a poor recovery of LV developed pressure (17 +/- 8% of baseline) and a high CK release (70 +/- 17 IU/g). Ischemic preconditioning improved recovery of LV developed pressure (46 +/- 14%) and reduced CK release (47 +/- 17 IU/g, both P < 0.05 vs. control-1). Ketamine (2 [mu]g/ml) and 2 or 20 [mu]g/ml S (+)-ketamine had no influence on recovery of LV developed pressure compared with preconditioning (47 +/- 18, 43 +/- 8, 49 +/- 36%) and CK release (39 +/- 8, 30 +/- 14, 41 +/- 25 IU/g). After administration of 20 [mu]g/ml ketamine and 2 or 20 [mu]g/ml R (-)-ketamine, the protective effects of preconditioning were abolished (LV developed pressure-recovery, 16 +/- 14, 22 +/- 21, 18 +/- 11%; CK release, 67 +/- 11, 80 +/- 21, 82 +/- 41 IU/g; each P < 0.05 vs. preconditioning). Preischemic treatment with R (-)-ketamine had no effect on CK release (74 +/- 8 vs. 69 +/- 9 IU/g in control-2, P = 0.6) and functional recovery (LV developed pressure 12 +/- 4 vs. 9 +/- 2 mmHg in control-2, P = 0.5). 相似文献
Methods: Adult rat cardiomyocytes were exposed to oxidative stress with 200 [mu]m H2O2 and 100 [mu]m FeSO4. Myocyte survival was determined based on morphologic characteristics and trypan blue exclusion. To induce preconditioning, the myocytes were pretreated with isoflurane. The involvement of sarcolemmal and mitochondrial KATP channels was investigated using specific inhibitors HMR-1098 and 5-hydroxydecanoic acid. Data are expressed as mean +/- SD.
Results: Oxidative stress induced cell death in 47 +/- 14% of myocytes. Pretreatment with isoflurane attenuated this effect to 26 +/- 8%. Blockade of the sarcolemmal KATP channels abolished the protection by isoflurane pretreatment when HMR-1098 was applied throughout the experiment (50 +/- 21%) or only during oxidative stress (50 +/- 12%), but not when applied during isoflurane pretreatment (29 +/- 13%). Inhibition of the mitochondrial KATP channels abolished cardioprotection irrespective of the timing of 5-hydroxydecanoic acid application. Cell death was 42 +/- 23, 45 +/- 23, and 46 +/- 22% when 5-hydroxydecanoic acid was applied throughout the experiment, only during isoflurane pretreatment, or only during oxidative stress, respectively. 相似文献
Methods: Isolated canine pulmonary venous rings without endothelium were suspended in modified Krebs-Ringer's buffer for measurement of isometric tension. The effects of nonspecific PKC inhibition (bisindolylmaleimide I; 3 x 10-6 m) and conventional PKC isoform inhibition (Go7936 10-6 m) on the acetylcholine dose-response relation were assessed. The expression of conventional PKC isoforms ([alpha], [beta], [gamma]), novel PKC isoforms ([delta], [varepsilon], [theta]), and atypical PKC isoforms ([zeta], [iota], [mu]) was measured in PVSM cells by Western blot analysis. The immunofluorescence technique and confocal microscopy were used to localize the cellular distribution of PKC isoforms before and after the addition of acetylcholine.
Results: Acetylcholine caused dose-dependent contraction in E-pulmonary veins. Pretreatment with bisindolylmaleimide I or Go7936 attenuated acetylcholine contraction. PKC-[alpha], -[iota], -[mu], and -[zeta] were expressed, whereas PKC-[beta], -[gamma], -[delta], -[varepsilon], and -[theta] were not expressed in PVSM cells. Immunofluorescence staining for PKC isoforms showed that in unstimulated cells, PKC-[alpha] and PKC-[mu] were detected only in the cytoplasm. PKC-[iota] and PKC-[zeta] also exhibited a cytoplasmic immunofluorescence pattern, which was especially abundant in the perinuclear zone. Activation with acetylcholine induced translocation of PKC-[alpha] from cytoplasm to membrane, whereas acetylcholine had no effect on the other PKC isoforms. Translocation of PKC-[alpha] in response to acetylcholine was blocked by the muscarinic receptor antagonist, atropine. 相似文献
Methods: The sarcKATP current was measured in ventricular myocytes isolated from guinea pig hearts using the whole cell configuration of the patch clamp technique. Peptides that induced the translocation of specific PKC isozymes were used to activate PKC-[epsilon] and PKC-[delta].
Results: Under whole cell conditions, isoflurane alone was unable to elicit the opening of the sarcKATP channel. Pretreatment with the specific PKC-[epsilon] activator, PP106, primed the sarcKATP channel to open in the presence of isoflurane. The resulting sarcKATP current densities in the presence of 0.88 mm isoflurane were 6.5 +/- 6.0 pA/pF (n = 7) and 40.4 +/- 18.2 pA/pF (n = 7) after pretreatment with 100 and 200 nm PP106, respectively. The PKC-[epsilon] antagonist PP93 abolished this effect. A scrambled peptide of the PKC-[epsilon] activator PP105 did not prime the sarcKATP channel. The PKC-[delta] activator PP114 was significantly less effective in priming the sarcKATP channel. 5-Hydroxydecanoate significantly attenuated the effect of the PKC-[epsilon] activator on the sarcKATP channel. In addition, immunohistochemical analysis showed that the PKC-[epsilon] isoform translocated to both the mitochondria and sarcolemma after anesthetic-induced preconditioning, whereas the PKC-[delta] isoform translocated to the mitochondria. 相似文献
Methods: Using the whole cell patch clamp technique, the authors investigated the presynaptic inhibitory actions of [mu]-, [delta]-, and [kappa]-opioid receptor agonists on primary afferent-evoked excitatory postsynaptic currents (EPSCs) in substantia gelatinosa neurons of adult rat spinal cord slices.
Results: The [mu] agonist DAMGO (0.1, 1 [mu]m) reduced the amplitude of glutamatergic monosynaptic A[delta]- or C fiber-evoked EPSCs. C fiber-evoked EPSCs were inhibited to a greater extent than A[delta] fiber-evoked EPSCs. The [delta] agonist DPDPE (1, 10 [mu]m) produced modest inhibition of A[delta]- or C fiber-evoked EPSCs. In contrast, the [kappa] agonist U69593 (1 [mu]m) did not affect the amplitude of either A[delta] or C fiber-evoked EPSCs. 相似文献
Methods: The authors used the hot plate and tail-pinch tests to evaluate in mice (C57BL/6 females; weight, 25-30 g) the effects of caffeine, a nonspecific adenosine receptor antagonist, on the antinociceptive effect induced by the intracerebroventricular administration of oxymorphone as a [mu] agonist, SNC80 as a [delta] agonist, or U69593 as a [kappa] agonist. They also investigated the effect of these opioid receptor agonists on the uptake of adenosine by whole brain synaptosomes.
Results: Caffeine decreased the analgesic effects induced by oxymorphone or SNC80 but not those induced by U69593. Oxymorphone and SNC80 inhibited adenosine uptake by brain cells, but U69593 did not. 相似文献
Methods: Whole cell and single channel patch clamp techniques were used to investigate the effects of isoflurane and the PTK inhibitor genistein on the cardiac sarcolemmal KATP channel in acutely dissociated guinea pig ventricular myocytes.
Results: At 0.5 mm internal ATP, genistein (50 [mu]m) elicited whole cell KATP current (22.5 +/- 7.9 pA/pF). Genistein effects were concentration-dependent, with an EC50 of 32.3 +/- 1.4 [mu]m. Another PTK inhibitor, tyrphostin B42, had a similar effect. The inactive analog of genistein, daidzein (50 [mu]m), did not elicit KATP current. Isoflurane (0.5 mm) increased genistein (35 [mu]m)- activated whole cell KATP current from 14.5 +/- 3.1 to 32.5 +/- 6.6 pA/pF. Stimulation of receptor PTKs with epidermal growth factor, nerve growth factor, or insulin attenuated genistein and isoflurane effects, and the protein tyrosine phosphatase inhibitor orthovanadate (1 mm) prevented their actions on KATP current. In excised inside-out membrane patches, and at fixed 0.2 mm internal ATP, genistein (50 [mu]m) increased channel open probability from 0.053 +/- 0.016 to 0.183 +/- 0.039, but isoflurane failed to further increase open probability (0.162 +/- 0.051) of genistein-activated channels. However, applied in the presence of genistein and protein tyrosine phosphatase 1B (1 [mu]g/ml), isoflurane significantly increased open probability to 0.473 +/- 0.114. 相似文献
Methods: Chick cardiomyocytes were studied in a flow-through chamber while pH, flow rate, oxygen, and carbon dioxide tension were controlled. Cell viability was quantified by nuclear stain propidium iodide, and oxygen radicals were quantified using molecular probe 2',7'-dichlorofluorescin diacetate.
Results: Morphine (1 [mu]m) or the selective [delta]-opioid receptor agonist BW373U86 (10 pm) given for 10 min before 1 h of ischemia and 3 h of reoxygenation reduced cell death (31 +/- 5%, n = 6, and 28 +/- 5%, n = 6 [P < 0.05], respectively, 53 +/- 6%, n = 6, in controls) and generated oxygen radicals before ischemia (724 +/- 53, n = 8, and 742 +/- 75, n = 8 [P < 0.05], respectively, vs. 384 +/- 42, n = 6, in controls, arbitrary units). The protection of morphine was abolished by naloxone, or the selective [delta]1-opioid receptor antagonist 7-benzylidenenaltrexone. Reduction in cell death and increase in oxygen radicals with BW373U86 were blocked by the selective mitochondrial KATP channel antagonist 5-hydroxydecanoate or diethyldithiocarbamic acid (1000 [mu]m), which inhibited conversion of O2- to H2O2. The increase in oxygen radicals was abolished by the mitochondrial electron transport inhibitor myxothiazol. Reduction in cell death was associated with attenuated oxidant stress at reperfusion. 相似文献
Methods: Ventricular myocytes were isolated from guinea pig hearts for the whole cell patch clamp recordings of the sarcolemmal KATP channel current (IKATP). Free radical scavengers N-acetyl-l-cysteine, carnosine, superoxide dismutase, and catalase were used to investigate whether reactive oxygen species mediate isoflurane facilitation of the channel opening by pinacidil. A possible role of the mitochondrial KATP channels was tested using a blocker of these channels, 5-hydroxydecanoate.
Results: The mean density (+/- SEM) of IKATP elicited by pinacidil (20 [mu]m) was 18.9 +/- 1.8 pA/pF (n = 11). In the presence of isoflurane (0.55 mm), the density of pinacidil-activated IKATP increased to 38.5 +/- 2.4 pA/pF (n = 9). Concurrent application of isoflurane and N-acetyl-l-cysteine decreased the sensitization effect by isoflurane in a concentration-dependent manner, whereby the densities of IKATP were 32.6 +/- 1.4 (n = 6), 26.2 +/- 2.3 (n = 6), and 19.4 +/- 2.1 pA/pF (n = 8) at 100, 250, and 500 [mu]m N-acetyl-l-cysteine, respectively. Concurrent application of isoflurane and carnosine (100 [mu]m), superoxide dismutase (100 U/ml), or catalase (100 U/ml) attenuated the densities of IKATP to 27.9 +/- 2.6, 27.2 +/- 2.9, and 25.9 +/- 2.2 pA/pF, respectively. None of the scavengers affected activation of IKATP by pinacidil alone. 5-Hydroxydecanoate (100 [mu]m) did not alter the sensitization effect by isoflurane, and the density of IKATP in this group was 37.1 +/- 3.8 pA/pF (n = 6). 相似文献
Methods: Barbiturate-anesthetized dogs (n = 88) were acutely instrumented for measurement of aortic and left ventricular pressures. All dogs were subjected to a 60-min left anterior descending coronary artery occlusion followed by 3-h reperfusion. In four separate groups, dogs received vehicle (0.9% saline) or the nonselective KATP channel antagonist glyburide (0.1 mg/kg intravenously) in the presence or absence of 1 minimum alveolar concentration desflurane. In four additional groups, dogs received 45-min intracoronary infusions of the selective sarcolemmal (HMR 1098; 1 [mu]g [middle dot] kg-1 [middle dot] min-1) or mitochondrial (5-hydroxydecanoate [5-HD]; 150 [mu]g [middle dot] kg-1 [middle dot] min-1) KATP channel antagonists in the presence or absence of desflurane. Myocardial perfusion and infarct size were measured with radioactive microspheres and triphenyltetrazolium staining, respectively.
Results: Desflurane significantly (P < 0.05) decreased infarct size to 10 +/- 2% (mean +/- SEM) of the area at risk as compared with control experiments (25 +/- 3% of area at risk). This beneficial effect of desflurane was abolished by glyburide (25 +/- 2% of area at risk). Glyburide (24 +/- 2%), HMR 1098 (21 +/- 4%), and 5-HD (24 +/- 2% of area at risk) alone had no effects on myocardial infarct size. HMR 1098 and 5-HD abolished the protective effects of desflurane (19 +/- 3% and 22 +/- 2% of area at risk, respectively). 相似文献
Methods: The actions of norepinephrine (50 [mu]m) on glutamatergic transmission were examined by using the whole cell patch clamp technique in substantia gelatinosa neurons of an adult rat spinal cord slice with an attached dorsal root.
Results: Norepinephrine inhibited the amplitude of monosynaptically evoked A[delta]-fiber and C-fiber excitatory postsynaptic currents in a reversible manner. When compared in magnitude between the A[delta]-fiber and C-fiber excitatory postsynaptic currents, the former inhibition (50 +/- 4%, n = 20) was significantly larger than the latter one (28 +/- 4%, n = 8). Both actions of norepinephrine were mimicked by an [alpha]2 adrenoceptor agonist, clonidine (10 [mu]m), and an [alpha]2A agonist, oxymetazoline (10 [mu]m), but not by an [alpha]1 agonist, phenylephrine (10 [mu]m), and a [beta] agonist, isoproterenol (40 [mu]m). The inhibitory actions were antagonized by an [alpha]2 antagonist, yohimbine (1 [mu]m), all of the results of which indicate an involvement of [alpha]2 adrenoceptors. Norepinephrine did not affect the amplitude of miniature excitatory postsynaptic current and of a response of substantia gelatinosa neurons to AMPA, indicating that its action on evoked excitatory postsynaptic currents is presynaptic in origin. 相似文献