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From the Center for Anesthesiology Research, The Cleveland Clinic Foundation, Cleveland, Ohio.
Address correspondence to Paul A. Murray, PhD, Carl E. Wasmuth Endowed Chair and Director, Center for Anesthesiology Research, NE63, The Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195. Address e-mail to murrayp{at}ccf.org.
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Abstract |
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METHODS: The effects of these IV anesthetics on acetylcholine contraction were assessed in isolated canine pulmonary vein rings. Tension and [Ca2+]i were measured simultaneously in fura-2 loaded endothelium-denuded PVSM strips after being permeabilized with 
-toxin. The effects of the IV anesthetics on tension ([Ca2+]i remains constant) in the absence or the presence of muscarinic receptor activation (acetylcholine) were assessed. The immunofluorescence technique and confocal microscopy were used to localize the cellular distribution of protein kinase C (PKC) isoforms in PVSM cells before and after the addition of ketamine.
RESULTS: Ketamine, etomidate, and midazolam each attenuated acetylcholine contraction dose-dependently, whereas thiopental had no effect. None of the IV anesthetics alone had an effect on tension in strips at constant [Ca2+]i (i.e., they had no direct effect on myofilament Ca2+ sensitivity). Acetylcholine increased tension by 56% ± 7% at constant [Ca2+]i. In acetylcholine-stimulated strips, etomidate, midazolam, and thiopental had no additional effect on tension at constant [Ca2+]i, whereas ketamine decreased tension by 33% ± 3%. Activation with acetylcholine induced translocation of PKC from cytoplasm to membrane, and this effect was blocked by ketamine.
CONCLUSIONS: Ketamine, etomidate, and midazolam each attenuated acetylcholine-induced pulmonary venous contraction. Ketamine attenuates acetylcholine contraction by inhibiting the acetylcholine-induced increase in myofilament Ca2+ sensitivity and the acetylcholine-induced translocation of PKC.
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Introduction |
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TopAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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Vascular smooth muscle contractility is dependent not only on intracellular Ca2+ concentration ([Ca2+]i), but also on the Ca2+ sensitivity of the contractile apparatus 8. Agonist-mediated activation is generally associated with a higher Ca2+ sensitivity (greater maintained isometric force per unit increase in [Ca2+]i) than that observed for activation via depolarization 9. We have recently reported that the muscarinic receptor agonist, acetylcholine, causes dose-dependent contraction of canine pulmonary venous smooth muscle (PVSM) by increasing both intracellular Ca2+ concentration and myofilament Ca2+ sensitivity 10. Moreover, we observed that acetylcholine contraction is attenuated by protein kinase C (PKC) inhibition, and is associated with activation and translocation of PKC from cytoplasm to membrane in PVSM 11.
IV anesthetics (ketamine, etomidate, thiopental, and midazolam) are widely used in anesthetic practice as premedicants and induction drugs. In general, IV anesthetics have been shown to exert a pulmonary vasodilator influence 12,13. We recently reported that IV anesthetics inhibit capacitative Ca2+ entry in PV 14. In addition, we have observed that ketamine attenuates acetylcholine-induced contraction of PV, and our results provided indirect evidence that this effect was due to a decrease in myofilament Ca2+ sensitivity 15.
The goal of the present study was to directly assess the effects of IV anesthetics on myofilament Ca2+ sensitivity using permeabilized PV in which [Ca2+]i can be "clamped" at a constant value. Our hypothesis is that IV anesthetics decrease myofilament Ca2+ sensitivity of PV, and this effect involves the PKC signaling pathway.
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METHODS |
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TopAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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Preparation of Pulmonary Venous Rings
Healthy mongrel dogs weighing approximately 28 kg were anesthetized with IV pentobarbital sodium (30 mg/kg) and fentanyl citrate (15 µg/kg). After tracheal intubation and mechanical ventilation, a catheter was placed in the right femoral artery, and the dogs were exsanguinated. The heart and lungs were removed from the thorax en bloc. Intralobar PV (third generation, 1–2 mm ID) were carefully dissected and immersed in cold modified Krebs–Ringer bicarbonate (KRB) solution composed of 118.3 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 2.5 mM NaHCO3, 0.016 mM Ca-EDTA, and 11.1 mM glucose. The endothelium was intentionally removed by gently rubbing the intimal surface with a cotton swab, and verified by >90% decrease in the response to bradykinin (10–8 M).
Isometric Tension Experiments
PV were vertically mounted between two stainless steel hooks in organ baths filled with 25 mL KRB solution (37°C), gassed with 95% oxygen and 5% carbon dioxide. The rings were stretched at 5 min intervals in increments of 0.5 g to achieve optimal resting tension (1.5 g). After the rings had been stretched to their optimal resting tension, the contractile response to 60 mM KCl was assessed. Then, a concentration–response curve to acetylcholine (from 10–8 to 10–3 M) was performed in each ring under baseline tone conditions (i.e., no precontraction).
The effects of ketamine (10–5 to 10–3 M), etomidate (10–6 to 10–4 M), thiopental (10–6 to 10–4 M), and midazolam (10–6 to 10–4 M) on the acetylcholine concentration–response relationship were assessed in endothelium-denuded PV. Anesthetics were directly added to the organ bath 30 min before acetylcholine contraction. The contractile responses to acetylcholine in anesthetic-pretreated rings were compared with responses in matched untreated rings.
Preparation of Pulmonary Venous Strips
Intralobar PV (second generation, 2–4 mm ID) were dissected carefully and immersed in cold modified KRB solution. The PV were cleaned of connective tissue and cut into strips (2 x 6 mm). The endothelium was removed by gently rubbing the intimal surface with a cotton swab.
Permeabilization Procedure
PV strips were permeabilized by incubation in relaxing solution containing 2500 U/mL Staphylococcus aureus -toxin 16. S. aureus -toxin creates pores of approximately 26 Å in the cell membrane, thereby allowing substances of small molecular weight, such as Ca2+, to freely diffuse across the cell membrane, whereas proteins necessary for contraction are retained within the cell. Therefore, changes in isometric force induced by a contractile agonist or anesthetic are due entirely to changes in Ca2+ sensitivity, because [Ca2+]i is clamped and not allowed to change 17. After optimal length was set, subsequent experimental protocols were performed at room temperature (25°C) and without aeration of the solutions. Experiments were performed at room temperature to maintain the integrity of the permeabilized PV strips. PV strips were superfused for 20 min with a relaxing solution that contained 2500 U/mL of -toxin. The relaxing solution was made up using the algorithm of Fabiato and Fabiato 18 and was composed of: 7.5 mM MgATP, 4 mM ethylene glycol-bis([ß]-aminoethyl ether)-N,N,N'N'-tetraacetic acid, 20 mM imidazole, 1 mM dithiothreitol, 1 mM free Mg2+, 1 nM free Ca2+, 10 mM creatine phosphate, and 0.1 mg/mL creatine phosphokinase. After the permeabilization procedure, PV strips were superfused with the relaxing solution for 10 min to wash out the excess -toxin. The calcium ionophore, A23187 (10 µM), was added to the relaxing solution and all subsequent experimental solutions to deplete the sarcoplasmic reticulum Ca2+ stores 19. Solutions of varying free Ca2+ concentrations used in the subsequent experiment also were prepared using the above algorithm 18.
Simultaneous Measurement of Tension and [Ca2+]i
Intralobar PV strips without endothelium were loaded with 5 x 10–6 M acetoxylmethyl ester of fura-2 (fura-2/AM) solution. After fura-2 loading, the strips were mounted between two stainless steel hooks in a temperature-controlled (25°C) 3-mL cuvette. Fluorescence measurements were performed using a dual-wavelength spectrofluorometer (Deltascan RFK6002, Photon Technology International, Lawrenceville, NJ) at excitation wavelengths of 340 and 380 nm and an emission wavelength of 510 nm. Because calculations of absolute concentration of [Ca2+]i rely on a number of assumptions, the 340–380 fluorescence ratio (340/380 ratio) was used as a measure of [Ca2+]i. Fura-2 fluorescence signals (340 and 380 nm and 340/380 ratio) and tension were measured at a sampling frequency of 2 Hz, and were collected with a software package from Photon Technology International.
The Effects of Anesthetics on Ca2+ Sensitivity in Permeabilized Canine PVSM
Two -toxin permeabilized strips were prepared from the same dog for each experiment. Strips were first contracted maximally with 10 µM Ca2+. Strips were washed out with relaxing solution. Strips were then contracted with 1 µM Ca2+. After stable contractions were obtained, cumulative doses of either thiopental (10–6 to 10–4 M), ketamine (10–5 to 10–3 M), etomidate (10–6 to 10–4 M), or midazolam (10–6 to 10–4 M) were applied to one strip. The diluent for the anesthetics was applied to the other strip as a control. In another set of experiments, one strip was contracted with 0.3 µM Ca2+ for 10 min, and then stimulated with 100 µM acetylcholine and 10 µM guanosine 5'-triphosphate (GTP). After 10 min, one of the IV anesthetics was added to each strip for 15 min. The other strip was not exposed to drugs and served as a time control.
Cell Culture of PVSM Cells
Primary cultures of PVSM cells (PVSMCs) were prepared according to the method of Campbell and Campbell 20 with minor modifications. Briefly, the endothelium was removed by gently rubbing with a sterile cotton swab. The remaining portion of the media was cut into 1-mm2 pieces that were explanted on precleaned 22-mm2 glass coverslips placed individually in 6-well culture plates for immunofluorescence studies. The explants were nourished by Dulbecco's modified Eagle medium/F-12 containing 10% fetal bovine serum and 1% antibiotic mixture solution (10,000 U/mL penicillin and 10,000 µg/mL streptomycin). PVSMCs began to proliferate from explants after 7 days in culture. Cells were allowed to grow for an additional 10–14 days until subconfluence was achieved. Twenty-four hours before experimentation, the culture medium containing 10% fetal bovine serum was replaced with serum-free medium to arrest cell growth, and to allow for establishment of steady-state cellular events independent of cell division.
Immunofluorescence Labeling of PKC Isoforms
We have previously demonstrated that PKC , 
, µ, 
are expressed in PVSMCs and that activation with acetylcholine only induces translocation of PKC from cytoplasm to membrane 11. We also have reported that ketamine decreases an acetylcholine-induced increase in myofilament Ca2+ sensitivity via the PKC pathway 15. Therefore, in the present study, we investigated the effect of ketamine on acetylcholine-induced translocation of PKC. Primary cultures of PVSMCs were divided into five experimental groups. The first group of cells was untreated. The second group was treated with acetylcholine (10–4 M). The third group was exposed to ketamine (10–4 M). The fourth group was exposed to acetylcholine after pretreatment with ketamine. All treatments were for 15 min. Immediately after each treatment, the reaction was stopped by placing the coverslips in 1:1 (vol/vol) acetone–methanol at 20°C for 10 min to simultaneously fix the cells and permeabilize their plasma membranes. Fixed cells were then washed with 0.1 M phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA) for 10 min, and subsequently incubated with PKC antibody at a dilution of 10 µg/mL in PBS–BSA overnight at 4°C. After incubation with the primary antibody, the coverslips were thoroughly washed in PBS–BSA and incubated with fluorescein-isothiocyanate-conjugated goat anti-mouse immunoglobulin G (secondary antibody) diluted 1:400 in PBS–BSA for 60 min at 37°C. An immunocytochemical control for antibody specificity was performed by incubating the cells with the secondary antibody only. After a thorough washing in PBS–BSA, the coverslips were mounted on microscope slides. The specimens were viewed and photographed using confocal microscopy.
Solutions and Chemicals
Dulbecco's modified Eagle's medium-F-12, the antibiotic–antimycotic mixture, and BSA (fraction V) were purchased from GIBCO (Grand Island, NY). PKC was purchased from Upstate Cell Signaling Solutions (Lake Placid, NY). Fluorescein-isothiocyanate-labeled goat anti-mouse immunoglobulin G was purchased from Molecular Probes (Eugene, OR). All other drugs and chemicals were purchased from Sigma Chemical (St. Louis, MO).
Statistical Analysis
All summarized data are expressed as mean ± sd. Statistical analysis was performed using two-way repeated-measures ANOVA with SPSS for WINDOWS software (version 11.5; SPSS Inc., Chicago, IL). When differences between groups were detected, post hoc analysis used the LSD test. Statistical assessments were made using Student's t-test for paired comparison. P < 0.05 was considered to be statistically significant.
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RESULTS |
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TopAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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The Effect of Ketamine, Etomidate, Midazolam, and Thiopental on Myofilament Ca2+ Sensitivity in Permeabilized Pulmonary Venous Strips
In -toxin-permeabilized PV, 1 µM Ca2+ (pCa 6.0) induced stable contraction of 76% ± 4% of the maximal force caused by 10 µM Ca2+ (pCa 5.0). Neither ketamine (Fig. 3), etomidate (Fig. 4A), thiopental (Fig. 4B) nor midazolam (Fig. 4C) had an effect on tension in strips treated with pCa 6.0 at constant [Ca2+]i, indicating that these IV anesthetics alone had no direct effect on myofilament Ca2+ sensitivity.
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The Effect of Acetylcholine on Myofilament Ca2+ Sensitivity in Permeabilized Pulmonary Venous Strips
To directly assess the effects of acetylcholine on myofilament Ca2+ sensitivity, permeabilized pulmonary venous strips were perfused with 0.3 µM free Ca2+, and then with solutions containing 0.3 µM free Ca2+ and 100 µM acetylcholine plus 10 µM GTP 0.3 µM free Ca2+ (pCa 6.5), which caused a sustained increase in tension (Fig. 5). The subsequent addition of 100 µM acetylcholine plus 10 µM GTP caused a marked increase in tension (76% ± 16%) (Fig. 5). This result indicates that acetylcholine directly increased myofilament Ca2+ sensitivity in permeabilized pulmonary venous strips.
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The Effect of Ketamine, Etomidate, Midazolam, and Thiopental on Myofilament Ca2+ Sensitivity in the Presence of Acetylcholine
After the acetylcholine-induced increase in tension became stable, ketamine (10–4 M), etomidate (10–4 M), midazolam (10–4 M), or thiopental (10–5 M) was administrated. Etomidate, midazolam, and thiopental had no effect on tension at constant [Ca2+]i, indicating that they had no effect on myofilament Ca2+ sensitivity in the presence of acetylcholine (Fig. 6). In contrast, ketamine decreased tension by 67% ± 6%, indicating that ketamine decreased myofilament Ca2+ sensitivity in the presence of acetylcholine (Fig. 7).
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Immunolocalization of PKC
In unstimulated cells, PKC was detected in the cytoplasm (Fig. 8A). Treatment with acetylcholine caused translocation of PKC from cytoplasm to membrane (Fig. 8B). Ketamine alone had no effect on the subcellular distribution of PKC compared with unstimulated cells (Fig. 8C). However, pretreatment with ketamine (10–4 M) before acetylcholine administration inhibited the acetylcholine-induced translocation of PKC (Fig. 8D).
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DISCUSSION |
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TopAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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The Effects of IV Anesthetics on Pulmonary Venous Contraction
Many anesthetics can induce pulmonary arterial vasodilation 21–23. In general, these direct effects are caused by a decrease in [Ca2+]i, a decrease in the force maintained for a particular [Ca2+]i (i.e., Ca2+ sensitivity), or a combination of both mechanisms. It is clear that during submaximal contraction of pulmonary and other types of smooth muscle, anesthetic-induced relaxation is associated with a decrease in [Ca2+]i. This is true for volatile anesthetics 24 and for IV anesthetics 14,15,22,23. For IV anesthetics, decreases in [Ca2+]i are caused by inhibition of Ca2+ influx via capacitative Ca2+ entry 14 through L-type voltage-operated Ca2+ channels and inhibition of Ca2+ release 15,21,22. Although a great deal is known about the effect of anesthetics on pulmonary arterial vasomotor tone, comparatively little is known about the effect of anesthetics on pulmonary venous tone. In the present study, we demonstrated that ketamine, midazolam, and etomidate attenuated acetylcholine contraction in PV in a dose-dependent manner. Thiopental had no effect on acetylcholine contraction. IV anesthetics could attenuate acetylcholine contraction by decreasing [Ca2+]i or myofilament Ca2+ sensitivity or both, because acetylcholine contraction in PV is mediated by an increase in [Ca2+]i and myofilament Ca2+ sensitivity 10. We have previously shown that IV anesthetics decrease Ca2+ influx by decreasing capacitative Ca2+ entry in PVSMCs 14. Moreover, we have demonstrated that the IV anesthetics, propofol and thiopental, inhibit Ca2+ influx through L-type voltage-operated Ca2+ channels in PVSM in response to adenosine triphosphate-sensitive K+ channel activation 25. However, the direct effects of IV anesthetics on myofilament Ca2+ sensitivity in PV have not been previously investigated.
The Effects of IV Anesthetics on Myofilament Ca2+ Sensitivity in PV
In the present study, we evaluated Ca2+ sensitivity by using -toxin permeabilized PV. -Toxin-permeabilized smooth muscle preparations have been used as a tool to investigate the mechanisms regulating Ca2+ sensitivity 26–28. Our results indicate that ketamine, etomidate, thiopental, and midazolam alone had no direct effect on myofilament Ca2+ sensitivity in PV. We also investigated the effects of the IV anesthetics on myofilament Ca2+ sensitivity in the presence of muscarinic receptor activation with acetylcholine. It has been reported that muscarinic receptor agonists activate a GTP-dependent messenger cascade that increases myofilament Ca2+ sensitivity 29. Although the mechanism is not fully known, there is considerable evidence that the Ca2+- and lipid-dependent enzyme, PKC, plays a key role 30,31. Our previous studies have shown that acetylcholine causes a leftward shift in the [Ca2+]i–tension relationship (an indirect measure of myofilament Ca2+ sensitivity), and ketamine decreases this effect by inhibiting the PKC pathway 15. In the present study, we directly assessed the effects of the IV anesthetics on myofilament Ca2+ sensitivity by using permeabilized PV strips activated with acetylcholine in which [Ca2+]i is maintained at a constant value. Our results indicate that only ketamine decreased myofilament Ca2+ sensitivity in the presence of acetylcholine. Taken together with previous studies of the effect of these IV anesthetics on [Ca2+]i, we conclude that midazolam and etomidate relax canine PVSM by decreasing [Ca2+]i without altering Ca2+ sensitivity.
We are aware of only two studies that have directly investigated the effects of IV anesthetics on myofilament Ca2+ sensitivity. Akata et al. 32 reported that ketamine had no effect on myofilament Ca2+ sensitivity in either membrane-permeabilized or membrane-intact rat mesenteric resistance arterial strips. Hanazaki et al. 33 reported that the IV anesthetics (ketamine, propofol, and midazolam) had no effect on myofilament Ca2+ sensitivity in the presence or absence of muscarinic receptor stimulation (acetylcholine 10–5 M) in canine tracheal smooth muscle. Moreover, that same group demonstrated that PKC plays little or no role in regulating Ca2+ sensitivity during muscarinic stimulation (acetylcholine) in canine tracheal smooth muscle 34, which likely explains why ketamine had no effect on Ca2+ sensitivity in the presence of acetylcholine in their previous study 33. We have previously shown that ketamine alone had no effect on the [Ca2+]i–tension relationship in PV, although ketamine attenuated the acetylcholine-induced shift in the [Ca2+]i–tension relationship via an effect on the PKC pathway 15. Thus, canine tracheal smooth muscle and PV are distinct in terms of the role played by PKC in the response to acetylcholine. Ketamine only exerts an effect on the acetylcholine response when the PKC pathway is activated. Muscarinic receptor activation results in increased levels of 1,2-diacylglycerol via hydrolysis of membrane-associated phospholipase C, which in turn activates the Ca2+- and lipid-dependent enzyme, PKC 31. Once activated, PKC may directly or indirectly inhibit myosin light chain phosphatase 30,31, thereby increasing regulatory myosin light chain phosphorylation and force for a given [Ca2+]i 35. In the present study, we investigated the effect of ketamine on acetylcholine-induced translocation of PKC. Our results indicate that acetylcholine causes translocation of PKC from cytoplasm to membrane, and ketamine inhibits this effect. Taken together, we speculate that ketamine decreases the acetylcholine-induced increase in myofilament Ca2+ sensitivity by inhibiting the translocation of PKC.
The plasma concentration of ketamine after the IV administration of 2 mg/kg has been reported to be 1.1 x 10–4 M 36. Ketamine at a concentration of 10–4 M attenuated the acetylcholine contractile response in PV, and so this effect is apparent at a clinically relevant concentration. The clinical concentration of midazolam is 0.3–10 µM 37. The peak plasma concentration of etomidate during induction of general anesthesia is approximately 10–5 M 38, whereas the free plasma concentration is likely to be <10–5 M because 75% of etomidate is bound to plasma protein 39. The peak plasma concentration of thiopental during induction of general anesthesia has been estimated at 50–300 µM 40. Assuming the high degree of protein binding (83%–86%), the peak concentration of free thiopental would not normally exceed 50 µM. Thus, clinical concentrations of midazolam, etomidate, and thiopental had no effect on myofilament Ca2+ sensitivity in the presence or absence of acetylcholine.
In summary, thiopental, midazolam, and etomidate directly relax PVSM stimulated with a muscarinic agonist without altering myofilament Ca2+ sensitivity, whereas ketamine attenuates acetylcholine-induced contraction by decreasing myofilament Ca2+ sensitivity by inhibiting PKC translocation.
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Footnotes |
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This work was funded by HL 38291 from the National Heart, Lung and Blood Institute of the National Institutes of Health, Bethesda, MD, and by Postdoctoral Fellowship (0425317B) from the OH Valley Affiliate of the American Heart Association, St. Petersburg, FL.
Presented in part at the Annual meeting of the American Society of Anesthesiology, Atlanta, GA, October 27, 2005.
Reprints will not be available from the author.
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