Methods: Whole-cell patch clamp recording techniques were used to investigate the effects of superfused isoflurane (0-1.5 minimum alveolar concentration) or sevoflurane (0-1.5 minimum alveolar concentration) on K+ and ClCa channel currents in dispersed smooth muscle cells.
Results: Isoflurane and sevoflurane inhibited whole-cell K+ currents to a greater degree in tracheal versus bronchial smooth muscle cells. More than 60% of the total K+ currents in tracheal smooth muscle appeared to be mediated through delayed rectifier K+ channels compared with less than 40% in bronchial smooth muscle. The inhibitory effects of the anesthetics were greater on the delayed rectifier K+ channels than on the remaining K+ channels. Cl- currents through ClCa channels were significantly inhibited by the anesthetics. The inhibitory potencies of the anesthetics on the ClCa channels were not different in tracheal and bronchial smooth muscle cells. 相似文献
Methods: Rings of rat thoracic aortas without endothelia were suspended for isometric force recording. Concentration-response curves were obtained in a cumulative fashion. During submaximal contractions to phenylephrine (3 x 10 (-7) M), relaxations to cromakalim (10-7 to 3 x 10-5 M), pinacidil (10-7 to 3 x 10-5 M), or diltiazem (10-7 to 3 x 10 (-4) M) were obtained. Lidocaine (10-5 to 3 x 10-4 M), mexiletine (10-5 to 10-4 M) or glibenclamide (5 x 10-6 M) was applied 15 min before addition of phenylephrine.
Results: During contractions to phenylephrine, cromakalim and pinacidil induced concentration-dependent relaxations. A selective ATP-sensitive K+ channel antagonist, glibenclamide (5 x 10-6 M), abolished these relaxations, whereas it did not alter relaxations to a voltage-dependent Ca (2+) channel inhibitor, diltiazem (10-7 to 3 x 10-4 M). Lidocaine (more than 10-5 M) significantly reduced relaxations to cromakalim or pinacidil in a concentration-dependent fashion, whereas lidocaine (3 x 10-4 M) did not affect relaxations to diltiazem. In contrast, mexiletine (more than 10-5 M) significantly augmented relaxations to cromakalim or pinacidil. Glibenclamide (5 x 10-6 M) abolished relaxations to cromakalim or pinacidil in arteries treated with mexiletine (10-4 M). 相似文献
Methods: Vessels were cannulated and held at a constant transmural pressure (40 mmHg). Image analysis and microfluorimetry were used to simultaneously measure vessel diameter and smooth muscle intracellular [Ca2+] concentration ([Ca2+]i). Myosin light chain (MLC) phosphorylation was measured using the Western blotting technique.
Results: Step increases in extracellular [Ca2+] concentration (0-10 mm) in high K+ (40 mm)-depolarized smooth muscle produced incremental increases in [Ca2+]i, MLC phosphorylation, and contraction. Halothane (0.5-4.5%) inhibited contraction in a concentration-dependent manner, but the decrease in [Ca2+]i was small, and there was a marked shift in the [Ca2+]i-contraction relationship to the right, indicating an important Ca2+ desensitizing effect. Halothane (0.5-4.5%) did not affect MLC phosphorylation or the [Ca2+]-MLC phosphorylation relationship, but the MLC phosphorylation-contraction relationship was also shifted rightward, indicating an "MLC phosphorylation" desensitizing effect. In contrast, control relaxations produced by the Ca2+ channel blocker nifedipine were accompanied by decreases in both [Ca2+]i and MLC phosphorylation, and nifedipine had no affect on the [Ca2+]i-contraction, [Ca2+]i-MLC phosphorylation, and MLC phosphorylation-contraction relationships. 相似文献
Methods: [Ca2+]i was monitored by measuring the 500-nm light emission ratio (F340/F380) of a Ca2+ indicator fura-2 with isometric tension of canine tracheal smooth muscle strip. During Ca2+-free conditions, carbachol (10-5 M) was introduced with pretreatment of halothane (0-3%). During Ca2+-free conditions, 20 mM caffeine, a Ca (2+-induced) Ca2+ release channel opener, was introduced with or without halothane. We measured [IP3]i during exposure to carbachol and halothane by radioimmunoassay technique.
Results: Pretreatment with halothane significantly diminished carbachol-induced increases in [Ca2+]i by 77% and muscle tension by 83% in a dose-dependent manner. Simultaneous administration of halothane significantly enhanced caffeine-induced transient increases in [Ca2+] (i) and muscle tension in a dose-dependent manner, by 97% and 69%, respectively. Pretreatment with halothane abolished these responses. Rapid increase in [IP3]i produced by carbachol was significantly inhibited by 32% by halothane in a dose-dependent manner. 相似文献
Methods: Intracellular concentration of free Ca2+ was monitored by the 500-nm light emission ratio of Ca2+ indicator fura-2. Isometric tension was measured simultaneously. Whole-cell patch clamp recording techniques were used to investigate the effects of volatile anesthetics on ICa in dispersed smooth muscle cells. Isoflurane (0-1.5 minimum alveolar concentration) or sevoflurane (0-1.5 minimum alveolar concentration) was introduced into a bath solution.
Results: The volatile anesthetics tested had greater inhibitory effects on carbachol-induced bronchial smooth muscle contraction than on tracheal smooth muscle contraction. These inhibitory effects by the anesthetics on muscle tension were parallel to the inhibitory effects on [Ca2+]i. Although tracheal smooth muscle cells had only L-type VDCs, some bronchial smooth muscle cells (~30%) included T-type VDC. Each of the two anesthetics significantly inhibited the activities of both types of VDCs in a dose-dependent manner; however, the anesthetics had greater inhibitory effects on T-type VDC activity in bronchial smooth muscle. 相似文献
Methods: Rings of rat aortas without endothelia were suspended for isometric force recording. Concentration-response curves of sodium nitroprusside (10-10 to 10-5 M) and 1-hydroxy-2-oxo-3-(N-methyl-3-aminopropyl)-3-methyl-1-triazene (NOC-7; 10-9 to 10-5 M) were obtained in the absence and in the presence of mexiletine, in combination with a soluble guanylate cyclase inhibitor, 1H-[1,2,4]oxadiazolo [4,3,-a]quinoxaline-1-one (ODQ), or inhibitors for ATP-sensitive K+ channels (glibenclamide), inward rectifier K+ channels (BaCl2), delayed rectifier K+ channels (4-aminopyridine), large conductance Ca2+-dependent K+ channels (iberiotoxin), or small conductance Ca2+-dependent K+ channels (apamin).
Results: Mexiletine (10-5 or 3 x 10-5 M) augmented relaxations to sodium nitroprusside and NOC-7. In arteries treated with glibenclamide (10-5 M), mexiletine (3 x 10-5 M) did not affect relaxations to nitric oxide donors, whereas mexiletine augmented relaxations to sodium nitroprusside despite the presence of BaCl2 (10-5 M), 4-aminopyridine (10-3 M), iberiotoxin (5 x 10-8 M) and apamin (5 x 10-8 M). Relaxations to sodium nitroprusside were abolished by ODQ (5 x 10-6 M), whereas these relaxations were augmented by mexiletine (3 x 10-5 M) in arteries treated with ODQ (5 x 10-6 M). 相似文献
Methods: Volatile anesthetic (0.05-1 mm) action on stimulated [Ca2+]cyt transients were monitored in suspensions of SH-SY5Y cells loaded with fura-2. Potassium chloride (KCl; 100 mm) was used to depolarize and activate Ca2+ entry through voltage-dependent calcium channels; 1 mm carbachol was used to activate muscarinic receptor-mediated inositol triphosphate (IP3)-dependent intracellular Ca2+ release. Sequential stimulations, KCl followed by carbachol and vice versa, were used to investigate interactions between intracellular Ca2+ stores.
Results: Halothane and isoflurane in clinically relevant concentrations enhanced the K+-evoked [Ca2+]cyt transient whether intracellular Ca2+ stores were full or partially depleted. In contrast, halothane and isoflurane reduced the carbachol-evoked [Ca2+]cyt transient when the intracellular Ca2+ stores were full but had no effect when the Ca2+ stores were partially depleted by KCl stimulation. 相似文献
Methods: Experiments were conducted using the outside-out configuration of the patch-clamp method applied to enzymatically prepared peripheral nerve fibers of Xenopus laevis. Half-maximal inhibiting concentrations were determined for Na+ channels and different K+ channels by nonlinear least-squares fitting of concentration-inhibition curves, assuming a one-to-one reaction.
Results: Externally applied meperidine reversibly blocked all investigated channels in a concentration-dependent manner, i.e., voltage-activated Na+ channel (half-maximal inhibiting concentration, 164 [mu]M), delayed rectifier K+ channels (half-maximal inhibiting concentration, 194 [mu]M), the calcium-activated K+ channel (half-maximal inhibiting concentration, 161 [mu]M), and the voltage-independent flicker K+ channel (half-maximal inhibiting concentration, 139 [mu]M). Maximal block in high concentrations of meperidine reached 83% for delayed rectifier K+ channels and 100% for all other channels. Meperidine blocks the Na+ channel in the same concentration range as the local anesthetic agent lidocaine (half-maximal inhibiting concentration, 172 [mu]M) but did not compete for the same binding site as evaluated by competition experiments. Low concentrations of meperidine (1 nM to 1 [mu]M) showed no effects on Na+ channels. The blockade of Na+ and delayed rectifier K+ channels could not be antagonized by the addition of naloxone. 相似文献
Methods: Cytosolic [Ca2+] was measured by fluorescence at the left ventricular wall of guinea pig isolated hearts using indo-1 dye. Sarcoplasmic reticular Ca2+-cycling proteins, i.e., Ca2+ release channel (ryanodine receptor [RyR2]), sarcoplasmic reticular Ca2+-pump adenosine triphosphatase (SERCA2a), and phospholamban were measured by Western blots. Hearts were assigned to seven groups (n = 8 each): (1) time control; (2) ischemia; (3, 4) 10 [mu]m Na+-Ca2+ exchange inhibitor KB-R7943 (KBR) or 1 [mu]m SEA0400 (SEA), given during the first 10 min of reperfusion; (5) APC initiated by sevoflurane (2.2%, 0.41 +/- 0.03 mm) given for 15 min and washed out for 15 min before ischemia-reperfusion; (6, 7) APC plus KBR or SEA.
Results: The authors found that APC reduced the increase in systolic [Ca2+], whereas KBR and SEA both reduced the increase in diastolic [Ca2+] on reperfusion. Each intervention improved recovery of left ventricular function. Moreover, APC plus KBR or SEA afforded better functional recovery than APC, KBR, or SEA alone (P < 0.05). Ischemia-reperfusion-induced degradation of major sarcoplasmic reticular Ca2+-cycling proteins was attenuated by APC, but not by KBR or SEA. 相似文献
Methods: Brain slices from rat cerebral cortex were prepared and superfused with artificial cerebrospinal fluid, including normal (Pco2 = 40 mmHg; pH = 7.4), hypercapnic (Pco2 = 50 mmHg; pH = 7.3), and hypercapnic normal pH (Pco2 = 50 mmHg; pH = 7.4) solutions. The ID of a cerebral parenchymal arteriole (5-9.5 [mu]m) was monitored using computerized videomicroscopy.
Results: During contraction to prostaglandin F2[alpha] (5 x 10-7 m), hypercapnia, but not hypercapnia under normal pH, induced marked vasodilation, which was completely abolished by the selective ATP-sensitive K+ channel antagonist glibenclamide (5 x 10-6 m). However, the selective Ca2+-dependent K+ channel antagonist iberiotoxin (10-7 m) as well as the nitric oxide synthase inhibitor NG-nitro-l-arginine methyl ester (10-4 m) did not alter vasodilation. A selective ATP-sensitive K+ channel opener, levcromakalim (3 x 10-8 to 3 x 10-7 m), induced vasodilation, whereas this vasodilation was abolished by glibenclamide. 相似文献
Methods: N-type Ba2+ currents were measured in the human neuronal SH-SY5Y cell line by using the whole cell voltage-clamp method.
Results: Isoflurane was found to have two effects on N-type Ba2+ currents. First, isoflurane reduced the magnitude of N-type Ba2+ currents to a similar extent (IC50 ~ 0.28 mm) in the absence and presence of GDP[beta]S (a nonhydrolyzable GDP analog). Interestingly, GTP[gamma]S (a nonhydrolyzable GTP analog and G-protein activator) in a dose-dependent manner reduced the isoflurane block; 120 [mu]m GTP[gamma]S completely eliminated the block of 0.3 mm isoflurane and reduced the apparent isoflurane potency by ~ 2.4 times (IC50 ~ 0.68 mm). Pretreatment with pertussis toxin or cholera toxin did not eliminate the GTP[gamma]S-induced protection against the isoflurane block. Furthermore, isoflurane reduced the magnitude of voltage-dependent G-protein-mediated inhibition of N-type Ba2+ currents, and this effect was eliminated by pretreatment with pertussis toxin or cholera toxin. 相似文献
Methods: IK, SK1, and chimera channels were expressed in Xenopus laevis oocytes. Currents of expressed channels were measured in the presence of 10 [mu]m Ca2+ by excised patch clamp analysis. Time constants of inhibition by halothane were compared between inside-out and outside-out patch configurations.
Results: Currents from chimera channels possessing the pore domain derived from IK were inhibited by halothane, whereas those possessing the SK1 pore domain were insensitive. Time constants of inhibition by halothane were significantly smaller in the outside-out patches than in the inside-out patches of both wild-type IK and a chimera with pore domain of IK. 相似文献
Methods: Perfused attached human neuroblastoma SH-SY5Y cells were exposed to carbachol (1 mm, 2 min) in the absence and presence of isoflurane (1 mm) and in the absence and presence of extracellular Ca2+ (1.5 mm). The authors studied the effect of the nonspecific cationic channel blocker La3+ (100 [mu]m), of the L-type Ca2+ channel blocker nitrendipine (10 [mu]m), and of the N-type Ca2+ channel blocker [omega]-conotoxin GVIA (0.1 [mu]m) on isoflurane modulation of the carbachol-evoked [Ca2+]cyt transient. [Ca2+]cyt was detected with fura-2 and experiments were carried out at 37[degrees]C.
Results: Isoflurane reduced the peak and area of the carbachol-evoked [Ca2+]cyt transient in the presence but not in the absence of extracellular Ca2+. La3+ had a similar effect as the removal of extracellular Ca2+. [omega]-Conotoxin GVIA and nitrendipine did not affect the isoflurane sensitivity of the carbachol response although nitrendipine reduced the magnitude of the carbachol response. 相似文献
Methods: Rat Nav1.2, Nav1.4, or Nav1.5 [alpha] subunits heterologously expressed in Chinese hamster ovary cells were analyzed by whole cell voltage clamp recording. The effects of isoflurane on Na+ current activation, inactivation, and recovery from inactivation were analyzed.
Results: The cardiac isoform Nav1.5 activated at more negative potentials (peak INa at -30 mV) than the neuronal Nav1.2 (0 mV) or skeletal muscle Nav1.4 (-10 mV) isoforms. Isoflurane reversibly inhibited all three isoforms in a concentration- and voltage-dependent manner at clinical concentrations (IC50 = 0.70, 0.61, and 0.45 mm, respectively, for Nav1.2, Nav1.4, and Nav1.5 from a physiologic holding potential of -70 mV). Inhibition was greater from a holding potential of -70 mV than from -100 mV, especially for Nav1.4 and Nav1.5. Isoflurane enhanced inactivation of all three isoforms due to a hyperpolarizing shift in the voltage dependence of steady state fast inactivation. Inhibition of Nav1.4 and Nav1.5 by isoflurane was attributed primarily to enhanced inactivation, whereas inhibition of Nav1.2, which had a more positive V1/2 of inactivation, was due primarily to tonic block. 相似文献
Methods: Neonatal rat cerebellar granule neurons were isolated and cultured on coverslips and studied at 37 [degree sign]C. Spectrofluorometric assays were used during identical conditions to monitor intracellular Ca2+ with the Ca2+-sensitive fluorophore fura-2 and glutamate release by a glutamate dehydrogenase-coupled assay, which produced the reduced form of nicotinamide-adenine dinucleotide phosphate in proportion to the amount of glutamate released. Neurons were depolarized by a rapid increase in external [K+] from 5 to 55 mM. Control responses were compared with those in the presence of 10, 30, and 100 [micro sign]M thiopental; 3, 10, and 30 [micro sign]M methohexital; decreased external [Ca2+]; or voltage-gated calcium channel blockers.
Results: Thiopental and methohexital depressed the intracellular Ca2+ transient peak and plateau in a dose-dependent manner, as did decreased Ca (2+). The intermediate dose of either drug caused [almost equal to] 50% decrease in peak intracellular Ca2+ and 60% decrease in glutamate release. In the presence of specific L-and/or N-type voltage-gated calcium channel blockade by nicardipine or [Greek small letter omega]-conotoxin-GVIA, respectively, 30 [micro sign]M thiopental further decreased the intracellular Ca2+ transient. Thiopental caused a dose-dependent decrease in glutamate release, which was proportional to the decreased peak intracellular Ca2+. 相似文献
Methods: Freshly isolated adult rat ventricular myocytes were used for the study. Cardiac myofibrils were extracted for assessment of myofibrillar actomyosin adenosine triphosphatase (ATPase) activity. Myocyte shortening (video edge detection) and pHi (2',7'-bis-(2-carboxyethyl)-5(6')-carboxyfluorescein, 500/440 ratio) were monitored simultaneously in individual cells field-stimulated (0.3 Hz) and superfused with HEPES-buffered solution (pH 7.4, 30[degrees]C).
Results: Propofol (100 [mu]m) reduced the Ca2+ concentration required for activation of myofibrillar actomyosin ATPase from pCa 5.7 +/- 0.01 to 6.6 +/- 0.01. Increasing pHi (7.05 +/- 0.03 to 7.39 +/- 0.04) with NH4Cl increased myocyte shortening by 35 +/- 12%. Washout of NH4Cl decreased pHi to 6.82 +/- 0.03 and decreased myocyte shortening to 52 +/- 10% of control. Propofol caused a dose-dependent increase in pHi but reduced myocyte shortening. The propofol-induced increase in pHi was attenuated, whereas the decrease in myocyte shortening was enhanced after pretreatment with ethylisopropyl amiloride, a Na+-H+ exchange inhibitor, or bisindolylmaleimide I, a protein kinase C inhibitor. Propofol also attenuated the NH4Cl-induced intracellular acidosis, increased the rate of recovery from acidosis, and attenuated the associated decrease in myocyte shortening. Propofol caused a leftward shift in the extracellular Ca2+-shortening relation, and this effect was attenuated by ethylisopropyl amiloride. 相似文献
Methods: We studied the effects of caffeine on myoplasmic free calcium concentration ([Ca2+]i) in MHN and MHS swine muscle fibers by means of Ca2+-selective microelectrodes before and after K+-induced partial depolarization.
Results: [Ca2+]i in untreated MHN fibers was 123 +/- 8 nm versus 342 +/- 33 nm in MHS fibers. Caffeine (2 mm) caused an increase in [Ca2+]i in both groups (296 +/- 41 nm MHN vs. 1,159 +/- 235 nm MHS) with no change in resting membrane potential. When either MHN or MHS, muscle fibers were incubated in 10 mm K+ [Ca2+]i transiently increased to 272 +/- 22 nm in MHN and 967 +/- 38 nm in MHS for 6-8 min. Exposure of MHN fibers to 2 mm caffeine while resting [Ca2+]i was elevated induced an increment in [Ca2+]i to 940 +/- 37 nm. After 6-8 min of exposure to 10 mm K+, [Ca2+]i returned to control levels in all fibers, and the effect of 2 mm caffeine on resting [Ca2+]i returned to control, despite continued partial membrane depolarization. 相似文献
Methods: The dose-dependent effects of sevoflurane on angiotensin II (10-7 m)-induced contraction, the increase in intracellular Ca2+ concentration, and PKC phosphorylation of rat aortic smooth muscle were measured using an isometric force transducer, a fluorometer, and Western blotting, respectively.
Results: Angiotensin II induced a transient increase in intracellular Ca2+ concentration, phosphorylation of Ca2+-dependent PKC (cPKC)-[alpha], and consequently, a transient contraction of rat aortic smooth muscle. Phosphorylation of the Ca2+-independent PKC-[epsilon] was not detected. The angiotensin II-induced contraction was almost completely abolished by removing extracellular Ca2+ and was significantly inhibited by the selective cPKC inhibitor Go 6976 (10-5 m) but was not inhibited by the nonselective PKC inhibitor Ro 31-8425 (10-5 m). Sevoflurane dose-dependently inhibited the angiotensin II-induced contraction, with reductions of 14.2 +/- 5.2% (P > 0.05), 26.7 +/- 8.9% (P < 0.05), and 38.5 +/- 12.8% (P < 0.01) (n = 10) in response to 1.7, 3.4, and 5.1% sevoflurane, respectively. The angiotensin II-elicited increase in intracellular Ca2+ concentration was not significantly influenced by 3.4, 5.1, or 8.5% sevoflurane. However, cPKC-[alpha] phosphorylation induced by angiotensin II was inhibited dose dependently by 1.7, 3.4, and 5.1% sevoflurane, with depressions of 20.5 +/- 14.2% (P > 0.05), 37.0 +/- 17.8% (P < 0.05), and 62.5 +/- 12.2% (P < 0.01) (n = 4), respectively. 相似文献
Methods: Freshly isolated rat ventricular myocytes were loaded with the Ca2+ indicator, fura-2, and placed on the stage of an inverted fluorescence microscope in a temperature-regulated bath. [Ca2+]i (340/380 ratio) and myocyte shortening (video-edge detection) were monitored simultaneously in individual cells field-stimulated at 0.3 Hz. Amplitude and timing of myocyte shortening and [Ca2+]i were compared before and after addition of thiopental. Intracellular pH was measured with the pH indicator, BCECF (500/440 ratio). Real-time uptake of Ca2+ into isolated sarcoplasmic reticulum vesicles was measured using fura-2 free acid in the extravesicular compartment. One hundred thirty-two cells were studied.
Results: Field stimulation increased [Ca2+]i from 85 +/- 10 nM to 355 +/- 22 nM (mean +/- SEM). Myocytes shortened by 10% of resting cell length (127 +/- 5 [micro sign]m). Times to peak [Ca2+]i and shortening were 139 +/- 6 and 173 +/- 7 msec, respectively. Times to 50% recovery for [Ca2+]i and shortening were 296 +/- 6 and 290 +/- 6 ms, respectively. Addition of thiopental (30-1,000 [micro sign]M) resulted in dose-dependent decreases in peak [Ca2+]i and myocyte shortening. Thiopental altered time to peak and time to 50% recovery for [Ca2+]i and myocyte shortening and inhibited the rate of uptake of Ca2+ into isolated sarcoplasmic reticulum vesicles. Thiopental did not, however, alter the amount of Ca2+ released in response to caffeine in sarcoplasmic reticulum vesicles or intact cells. Thiopental (100 [micro sign]M) increased intracellular pH and caused an upward shift in the dose-response curve to extracellular Ca2+ for shortening, with no concomitant effect on peak [Ca2+]i. These effects were abolished by ethylisopropyl amiloride, an inhibitor of Na+ -H+ exchange. 相似文献