Oxidant damage to endothelial cells occurs during inflammation and reperfusion after ischemia, mediated in part by endogenously produced hydrogen peroxide (H2O2). Previous studies have established a role for increased cytosolic calcium in the mechanism of endothelial oxidant injury, and have suggested that volatile anesthetics may exacerbate oxidant injury in pulmonary endothelium. However, the effect of volatile anesthetics on oxidant injury to systemic arterial endothelial cells, and their effect on oxidant-related changes in cytosolic calcium homeostasis, have not been reported previously.
Primary cultures of human aortic and pulmonary arterial endothelial cells were studied. The rate of cell death after H2O2 exposure was determined in cell suspension by propidium iodide fluorimetry and lactate dehydrogenase release. The final extent of cell death 24 h after H2O2 exposure was determined in monolayer cultures by methyl thiazolyl tetrazolium reduction. Cytosolic calcium and cell death were determined in single cells using fura-2 and propidium iodide imaging with digitized, multiparameter, fluorescent video microscopy.
In aortic endothelial cells, clinical concentrations of halothane (1.0%) and isoflurane (1.5%) decreased both the rate of cell death and the final extent of cell death after H2O2 exposure, with halothane being more protective. Supraclinical concentrations of halothane (2.7%) and isoflurane (4.0%) were less protective. In pulmonary arterial endothelial cells, halothane and isoflurane had essentially no effect on H2O2-mediated cell death. The protective effect of anesthetic in aortic endothelial cells was not due to an enhanced removal of H2O2 by endogenous enzymes. Hydrogen peroxide exposure caused a large increase in cytosolic calcium well before cell death, and this was moderated by anesthetic treatment.
The effect of volatile anesthetics on oxidant injury to endothelial cells may differ between cells derived from systemic and pulmonary vascular beds. Halothane, and to a lesser extent, isoflurane, protects against oxidant injury in aortic endothelial cells. The mechanism of protection may involve modulation of the interaction of H2O2 with vital cellular constituents, and/or amelioration of the toxic increase in cytosolic calcium that follows such interaction.
THE integrity and viability of the vascular endothelium are essential for homeostasis of all vital organs, [1–3]and of obvious importance to anesthesia for a surgical procedure affecting any vital organ. Viable endothelial cells produce locally active vasoregulatory compounds, particularly nitric oxide and prostaglandins, which optimize vascular supply. An uninjured endothelial monolayer provides a luminal surface that is itself resistant to thrombosis, and also shields the highly thrombogenic collagen of the vessel wall from the lumen. In some organs, the intact, uninjured endothelium provides a selectively permeable barrier. Conversely, endothelial injury can not only allow entry of undesirable substances into the parenchymal cells of the organ, but through edema can cause microvascular occlusion and critical reduction in oxygen delivery.
Endothelial cells are quite resistant to hypoxia per se. [4–6]They are much more susceptible to oxidant injury, and hydrogen peroxide (H2O2) is a major endogenously produced oxidant causing endothelial cell injury and death. [1,4,7–11](Superoxide will yield H2O2whether it decomposes by enzymatic or nonenzymatic dismutation. ) Vascular superoxide, and hence H2O2, is produced by activated neutrophils and other inflammatory cells, [9–11,13]as well as by parenchymal cells and endothelial cells during reperfusion after ischemia. [12,14–17]Both sources may combine to injure coronary endothelium during cardiopulmonary bypass. [18,19]Endothelial oxidant injury has been documented in heart, lung, and liver transplantation.
A previous investigation demonstrated that volatile anesthetics do not affect the production of toxic oxygen-derived species from activated neutrophils, but reported that isoflurane, and to a lesser extent halothane, exacerbated the toxicity of H2O2in cultured pulmonary artery endothelial cells. However, the effect of anesthetics on susceptibility of systemic artery endothelial cells has not been examined heretofore, despite the crucial importance that these endothelial cells can play in the outcome of anesthesia for neurovascular, coronary vascular, and renovascular procedures. The first two goals of this study therefore were:(1) to study the effect of isoflurane and halothane on H2O2toxicity in cultured human aortic endothelial cells; and (2) to compare their response with that of cultured human pulmonary artery endothelial cells.
The mechanism of systemic artery endothelial H2O2toxicity is also of interest in the attempt to understand the effects of volatile anesthetics on such toxicity. Increased cytoplasmic free calcium (Calcium2+i) has been shown to precede cell death from H2O2in umbilical vein [1,23]and coronary artery endothelial cells. One documented mechanism of toxicity from increased Calcium2+iafter H2O2exposure is the activation of calcium-dependent proteases. There are multiple other potential sites for increased Calcium2+ito exert a toxic effect; for example, activating Calcium2+-dependent phospholipases and endonucleases, and altering membrane ion transport. Volatile anesthetics are known to perturb Calcium2+ihomeostasis in multiple cell types. [26–28]The final goal of this study, therefore, was (3) to determine, using multiparameter digitized video fluorescence microscopy of single aortic endothelial cells exposed to H2O2: whether a large increase in Calcium2+ipreceded cell death, and whether isoflurane and halothane affected any observed changes in Calcium sup 2+ibefore cell death.
Materials and Methods
Chemicals and Reagents
Fura-2 and fura-2-acetoxymethyl ester (fura-2 AM) were obtained from Molecular Probes, Inc. (Eugene, OR). Trypsin was obtained from Gibco (Grand Island, NY). Fetal bovine serum was obtained from Hyclone (Logan, UT). Albumin (bovine serum, fraction V), catalase (bovine liver), horseradish peroxidase (type X), hydrogen peroxide (30%), propidium iodide (PI), methylthiazole tetrazolium (MTT), 2-deoxyglucose, and digitonin were obtained from Sigma Chemical Co. (St. Louis, MO). All other reagents were of analytical grade and were obtained from established chemical suppliers. All buffers were HEPES-buffered Krebs-Ringers solution (KRH)containing 5.0 mM D-glucose, with additions or omissions as specified.
Primary cultures of human aortic and pulmonary arterial endothelial cells were obtained from Clonetics (San Diego, CA), and cultured in Clonetics Endothelial Growth Medium-UV containing 2% fetal bovine serum. Each culture was shown to take up diacetylated low density lipoprotein, and to contain Factor VIII, by the supplier. Cells for microscope imaging experiments were from passages 4–8, replated sparsely on glass coverslips to allow imaging of individual cells. Cells for suspension and tetrazolium experiments were from passages 5–9, grown to confluence. Because there can be significant variability in susceptibility to H2O2(and other toxins) among different cultures and even different passages within the same culture, [25,30,31]all experiments were performed with simultaneous+/-anesthetic and+/-H2O2controls, and analyzed and reported as separate, independent experiments.
Cell Suspension Assays of the Rate of Cell Death
The fluorimetric assay for cell death in suspension was adapted from that described previously. Propidium iodide exhibits a large increase in fluorescence when it binds to DNA. Hence, when cell death occurs with PI in the extracellular buffer, plasma membrane integrity is lost, PI gains access to intracellular DNA, and its fluorescence increases. Because the assay is nondestructive, multiple readings over time can be obtained from a single sample.
Confluent monolayers of cells were washed with phosphate-buffered saline, then treated with 0.1% trypsin for 2 min at room temperature. All buffers used subsequently were prepared with 2 micro meter filtration to reduce particulate-derived fluorescent noise in the final cell suspension. A volume of 5.0% albumin in KRH-glucose equal to the trypsin volume was then added, the cells were suspended by gentle aspiration and expulsion through a 1-ml micropipette tip, then centrifuged at 220g for 5 min. The pellet was washed, recentrifuged, and finally suspended in 1.0% albumin in KRH-glucose, at twice (for clinical anesthetic concentrations) or five times (for supraclinical anesthetic concentrations) the desired final cell concentration (4–10 x 104cells/ml; > 93% viable initially) and PI concentration (1.0 micro Meter). Aliquots of the cell suspension were placed in 12 x 75 mm borosilicate glass tubes (Fisher Scientific, Pittsburgh, PA). Diluent was prepared by bubbling 100 ml 1.0% albumin in KRH-glucose in a 2 1 glass bottle at 37 degrees C with 200 ml/min 100% Oxygen2+/-the desired anesthetic concentration for 30 min, then tightly sealing the bottle with a glass stopper until use within the next 30 min. For clinical anesthetic concentrations (1.0% halothane or 1.5% isoflurane, final), 0.75 ml diluent equilibrated with Oxygen2+/-2.0% halothane or 3.0% isoflurane was added to 0.75 ml cell suspension, the tube was immediately flushed with 100% Oxygen2+/-1.0% halothane or 1.5% isoflurane at 100 ml/min for 20 s, then capped with a thick rubber stopper taken from an unused blood collection tube (Venoject, Terumo Medical Corp., Elkton, MD; or Monoject, Sherwood Medical, St. Louis, MO). For supra-clinical anesthetic concentrations (2.7% halothane or 4.0% isoflurane, final), 1.2 ml diluent equilibrated with 3.3% halothane or 5.0% isoflurane was added to 0.3 ml cell suspension, the tube was flushed with 100% Oxygen2+/-2.7% halothane or 4.0% isoflurane for 20 s, then capped.
Capped tubes were equilibrated in a gently shaking 37 degrees Celsius water bath for 15 min, then H2O2(50 micro liter of a freshly prepared 30 mM stock in KRH-glucose) was added with a gas-tight Hamilton syringe (Reno, Nevada) through the rubber cap to give 1.0 mM final [H2O2]. Tubes were kept in the same gently shaking 37 degrees C water bath during the experiment except for periods of < 1 min when fluorescence readings were taken. Fluorescence was measured in a Turner model 450 fluorimeter (Curtin Matheson Scientific, Inc., Houston, TX), excitation with 520-nm narrow band filter, emission with > 605 nm filter. At the end of the experiment, 37.5 micro liter 15 mM digitonin was added to kill all cells, the tubes were shaken at 37 degrees C for 20 min, then a final fluorescence reading was taken. Percent cell death for each tube at each time point was calculated from the fraction of digitonin-induced fluorescence present, as described. .
Samples prepared in this manner (1.0% albumin) using clinical anesthetic concentrations (1.0% halothane or 1.5% isoflurane, nominal vol/vol % gas phase) and analyzed by gas chromatography as described, contained 0.68+/-0.04 mM halothane or 0.46+/-0.02 mM isoflurane. Using supraclinical anesthetic concentrations (2.7% halothane or 4.0% isoflurane, nominal vol/vol % gas phase), samples contained 1.34 +/-0.04 mM halothane or 1.16+/-0.07 mM isoflurane.
In one experiment in which lactate dehydrogenase (LDH) release was also determined as a measure of cell death, a second set of capped cell suspension tubes were prepared and treated, except that PI was omitted. At the time point selected, the tubes were centrifuged at 220g for 5 min, and the supernatant and pellet were separated and assayed for LDH activity as described. Percent cell death was determined from the LDH activity present in the supernatant, as a percentage of the total LDH activity.
Cell Monolayer Assay for Final Extent of Cell Death
The mitochondrial reduction of MTT is a well established, quantitative assay for cell viability, [33,34]and was used to determine the effect of anesthetic on the extent of cell death 24 h after exposure to H2O2. The medium over confluent cultures in Falcon Primaria 35 mm x 10 mm tissue culture dishes (Curtin Matheson Scientific, Inc., Houston, TX) was changed to 2.0 ml KRH-glucose. Cultures were then equilibrated for 60 min at 37 degrees C with 100% Oxygen2+/-halothane or isoflurane in an exposure apparatus identical to that described previously, except that a third limb identical to the other two was added, so that simultaneous exposures to three different gas phases (no anesthetic, halothane, and isoflurane) could be performed.
Samples prepared in this manner (KRH-glucose, no albumin) using clinical anesthetic concentrations (1.0% halothane or 1.5% isoflurane, nominal vol/vol % gas phase) and analyzed by gas chromatography as described, contained 0.35+/-0.02 mM halothane or 0.31 +/-0.08 mM isoflurane. Using supraclinical anesthetic concentrations (2.7% halothane or 4.0% isoflurane, nominal vol/vol % gas phase), samples contained 0.86+/-0.05 mM halothane or 0.69 +/-0.08 mM isoflurane.
After culture dishes were equilibrated with the gas phase for 60 min, the desired concentration of H2O2was then added as a 50–100- micro liter bolus from a freshly prepared stock solution, using a concentration determined to give < 100% toxicity by the previous day's preliminary dose-response curve on sibling cultures, with a continuous gas flow over the opened exposure chamber during H2O2addition. Exposure to the three gas phases at 37 degrees C was then continued for 120 min, after which the cultures were removed from the exposure chambers and placed in a regular incubator (94% air, 6% CO2) at 37 degrees C overnight.
At 24 h after H2O2exposure, 0.1 ml 104units/ml catalase in KRH-glucose was added to each culture dish to destroy any residual H2O2, and incubation in the regular incubator continued for 15 min. Methylthiazole tetrazolium, 0.2 ml 5 mg/ml in KRH-glucose, was then added to each culture dish and incubation in the regular incubator continued for 3 h. During this time, mitochondria in viable cells reduced MTT to an insoluble, intracellular, blue formazan, so that the final color produced was proportional to the number of viable cells present. After 3 h, the buffer on each culture plate was removed and centrifuged to recover any viable but nonadherent cells, and 1.0 ml isopropanol added to the pellet. An additional 2.0 ml isopropanol was added to the cells adhering to the culture dish. After 10 min incubation at room temperature to completely dissolve the blue formazan, the isopropanol solutions were combined, and A570-690 (absorbance at 570 nm minus that at 690 nm) was determined in a Beckman (Palo Alto, CA) DU-7 spectrophotometer.
There was no effect of anesthetic on A570-690 in the absence of H2O2. There was also < 10% cell death for cells treated with this protocol in the absence of H2O2, using a double fluorescent stain technique with fluorescein diacetate and PI and direct fluorescent microscopic examination to directly count dead and live cells. Therefore, percent cell death in each culture plate was estimated from MTT color development as follows:Equation 1.
The concentration of hydrogen peroxide was determined by its oxidation of scopoletin in the presence of peroxidase, with a corresponding change in fluorescence. A 100- micro liter aliquot of the sample to be assayed was added to 900 micro liter 0.1 N hydrochloric acid, to inactivate cellular enzymes and stabilize H2O2; 100 micro liter 0.9 N sodium hydroxide was added to this mixture just before assay. The assay solution contained 2.5 ml KRH-glucose buffer with 0.2 units/ml horseradish peroxidase and 2 micro Meter scopoletin. Using an Aminco-Bowman (SLM, Urbana, IL) spectrophotofluorimeter in ratio mode, with 350 nM excitation and 460 emission, baseline fluorescence versus time was recorded. A 25–50- micro liter aliquot of the acidified-neutralized sample was then added, and the fluorescence was recorded versus time until a new baseline was reached within 3 min. The amount of H2O2in the sample was determined from the difference in fluorescence before and after addition of the sample to the assay, compared to a standard curve prepared using known amounts of H2O2. The standard curve was linear from 0 to at least 2.5 nmol H2O2, with r2= 0.983.
Digitized, Multiparameter, Fluorescent Video Microscopy for Single Cell Assays
The Attofluor Ratio Vision system (Atto Instruments, Rockville, MD) with filters, calibration, and temperature control at 37 degrees C as described previously for fura-2 (Calcium2+i) and PI (cell death) imaging was used. Because of interculture variability in susceptibility to H2O2, experiments were performed and are reported as matched controls without and with a single anesthetic, performed on the same day with sibling cultures of identical culture history.
Cells subcultured on a glass coverslip were incubated with 5 micro Meter fura-2 AM in KRH + 10 mM glucose + 10% fetal bovine serum at 37 degrees C in air for 20 min, washed thrice with KRH + 10 mM glucose, incubated in KRH + 10 mM glucose at 37 degrees C in air for 15 min, washed thrice more with KRH + 10 mM glucose, mounted on the Attofluor polycarbonate coverslip chamber (total volume = 1.2 ml), and covered with 0.5 ml KRH + 10 mM glucose + 100 nM PI. After acquiring baseline images, 0.5 ml KRH + 10 mM glucose, equilibrated with 100% Oxygen sub 2+/-2.0% halothane or 3.0% isoflurane, was added (to yield 1.0% halothane or 1.5% isoflurane), the chamber was covered on top with another glass coverslip, and additional baseline images were acquired. With the top coverslip briefly removed, H2O2was then added as 110 micro liter of freshly prepared 10 mM stock solution in KRH + 10 mM glucose, the top of the chamber again was covered, and images were acquired every 2–4 min.
Statistical Analysis *RF 35*.
All error bars in figures, and+/-numbers given in the text or table, are standard deviations. Statistical significance was considered to be P < 0.05. To compare survival curves of cell suspension experiments, with 3–4 replicates for each exposure condition, a two-way analysis of variance was performed at each time point, with H2O2and anesthetic as the two independent factors, and cell death as the dependent variable. When analysis of variance indicated a significant difference, individual means were compared using the least significant difference correction for multiple comparisons. For clarity in the figures, only the effect of anesthetic is indicated; the effect of H2O2was always highly significant. Cell death of monolayers, assayed by MTT, was similarly analyzed for the single time point at which data were obtained, at the end of the experiment. The effect of anesthetic on the rate of disappearance of H2O2was analyzed by one-way analysis of variance, with anesthetic as the independent variable and H2O2concentration as the dependent variable. The effect of anesthetic on Calcium2+iwas analyzed by paired, two-tailed t test, using the mean of Calcium2+ifor each cell before and after anesthetic addition. The effect of anesthetic on time required for Calcium2+ito double after addition of H2O2was determined by unpaired, two-tailed t test. All of the preceding were calculated using the algorithms in Stat-Packets 1.0 (StatPac Inc., Minneapolis, MN).
Survival curves for microscopy experiments were created using a tabulation of the time of death for each cell in an experiment. From this, a frequency distribution was generated for the time of death with a resolution of 3-min intervals. A plot of survival versus time was prepared by subtracting the number of cells dying in each 3-min interval from the number of cells alive in the preceding time interval, then converting number of cells alive to percent survival. To analyze the effect of anesthetic on survival curves for microscopy experiments, where two fields of single cells were observed after H2O2addition, one with and one without anesthetic, the Mantel-Haenszel log-rank test was performed, using the algorithm in the SURVIVAL 1.0 module of SYSTAT 5.03 (Evanston, IL).
Sensitivity of Endothelial Cells to Hypoxia and Hydrogen Peroxide
The viability of cultured human aortic endothelial cells in suspension was initially determined using PI fluorimetry as an assay for cell death. As shown in Figure 1, these cells were much more resistant to metabolic inhibition by cyanide and 2-deoxyglucose, simulating ischemia, than to 1 mMH2O2, a concentration that can be achieved in vivo locally. [13,36]There also was a lag period of about 2 h before the toxic effect of H2O2became apparent. These qualities are consistent with results reported for endothelial cells from other sources. [7,11,29,37].
Effect of Anesthetic on the Rate of Hydrogen Peroxide-induced Cell Death
As shown in Figure 2, the rate of aortic endothelial cell death caused by H2O2in cell suspension, measured by PI fluorimetry, was significantly reduced by both 1.0% halothane and 1.5% isoflurane, with slightly more protection by halothane than isoflurane at later time points. A similar protective effect of halothane and isoflurane also was seen when identically prepared samples were assayed for LDH release (Figure 2), ruling out an artifactual effect of anesthetic to increase permeability of the viable cell membrane to PI. When similar experiments with supraclinical anesthetic concentrations were performed on aortic endothelial cells, 2.7% halothane and 4.0% isoflurane, the protective effect was much less, and statistically significant only at one time point (Figure 3).
In contrast, when pulmonary arterial instead of aortic endothelial cell suspensions were studied, the rate of H2O2-induced cell death was minimally affected by either clinical or supraclinical concentrations of halothane and isoflurane (Figure 4and Figure 5). Where an effect was seen, it was mildly protective (Figure 5).
Effect of Anesthetic on Final Extent of Hydrogen Peroxide-induced Cell Death
The preceding studies in cell suspension showed the effect of anesthetic on the rate of H2O2-mediated cell death. The concentration of H2O2that was used, 1.0 mM, was eventually toxic to most cells with or without anesthetic. This dose was used so that the effect of anesthetic addition could be determined within a reasonable time frame (less than or equal to 4 h) with the PI-cell suspension technique. To more rigorously determine whether halothane and isoflurane affected the final extent of endothelial cell death, survival at 24 h after H2O2treatment was determined using the tetrazolium dye MTT. Five separate experiments were performed for both aortic and pulmonary artery endothelial cells, using simultaneous controls to compensate for culture-to-culture variability in H2O2susceptibility, and testing both clinical and supraclinical concentrations of halothane and isoflurane. For aortic endothelial cells (Figure 6), halothane was protective in all five experiments, whereas isoflurane was protective in four of the five experiments. Halothane was more protective than isoflurane in four experiments, and this was statistically significant in two experiments. The difference between halothane and isoflurane was far less than the difference between halothane and no anesthetic. It is possible that a small true difference between halothane and isoflurane was sometimes not detected because of its small magnitude relative to the experimental error with the number of replications used. In contrast, pulmonary artery endothelial cells (Figure 7) were not protected by halothane or isoflurane.
Effect of Anesthetic on the Rate of Disappearance of Hydrogen Peroxide Exposed to Aortic Endothelial Cells
One potential mechanism for the anesthetic protection seen with aortic endothelial cells is an increase in the ability of cells to detoxify H2O2, i.e., by somehow augmenting the endogenous catalase and peroxidase activities already present in the cell. Many cytochrome P-450 enzymes are able to use peroxides, in place of nicotinamide adenine dinucleotide phosphate and O2, to hydroxylate their substrates. Given that halothane can be metabolized by multiple cytochrome P-450 isozymes, [38–40]that some cytochrome P-450 isozymes are widely distributed in many cell types, and that the more easily metabolized halothane was more protective than isoflurane in the models we tested, it was reasonable to hypothesize that halothane was accelerating the detoxification of H2O2by using it as an electron donor and oxygen source via an endogenous aortic endothelial cytochrome P-450. If this were the case, then halothane would cause H2O2to disappear more rapidly from suspensions of aortic endothelial cells.
We tested this hypothesis by assaying the concentration of H sub 2 O230 min after addition of 1.0 mMH2O2to aliquots of an aortic endothelial cell suspension, 1 x 105cells/ml, prepared identically to the regular cell death assays described earlier, except that PI was omitted. There was no significant effect on the rate of H2O2disappearance: after 30 min, [H2O2]= 0.56+/- 0.01 mM (no anesthetic); 0.56+/-0.03 mM (1.0% halothane), and 0.55+/-0.05 mM (1.5% isoflurane); N = 3 for each anesthetic condition; P > 0.7 for all anesthetic comparisons. In the absence of cells, < 5% of initial H2O2disappeared under the same conditions.
Effect of Anesthetic on Cytoplasmic Free Calcium during Hydrogen Peroxide-induced Aortic Endothelial Cell Death, Determined by Single Cell Assay
Volatile anesthetics perturb Calcium2+ihomeostasis in multiple cell types. [26–28]Hence, one potential mechanism of the anesthetic protection observed in aortic endothelial cells would be to ameliorate the H2O2-induced rise in Calcium2+ithat has been reported in other endothelial cells. [1,23,24,42]To study this, Calcium2+iassays must be performed on single cells with a simultaneous assay for viability to exclude dead cells from the analysis. Dead cells have a vastly increased intracellular Calcium2+ as a consequence, not a cause, of cell death and loss of plasma membrane integrity, which allows influx of extracellular Calcium2+ along the approximately 104concentration gradient between extracellular and cytoplasmic Calcium2+ in the living cell. We therefore used digitized, multiparameter, fluorescent video microscopy to simultaneously assay, in single aortic endothelial cells, both Calcium2+iby means of fura-2, and cell death by means of PI. Pulmonary artery endothelial cells were not studied in this manner, because they were not protected by volatile anesthetics from H2O2toxicity.
In initial experiments, the mean of Calcium2+ivalues for resting cells, before addition of anesthetic or H2O2, was 112+/-39 nM (N = 60 cells, each cell imaged every 2 min for 14 min). Addition of 1.0% halothane caused a small but statistically significant increase of 11.5+/-2.1 nM Calcium2+i(P = 0.0001; N = 30 cells, each cell imaged every 2 min for 14 min). Similarly, 1.5% isoflurane caused an increase of 9.5+/-2.8 nM Calcium2+ sub i (P = 0.002; N = 30 cells, each cell imaged every 2 min for 14 min). Typical survival curves obtained from PI microscopy during H2O2exposure are shown in Figure 8and Figure 9, confirming the protective effect of halothane and, to a lesser extent, isoflurane, on single aortic endothelial cells.
Simultaneous assay of Calcium2+iand aortic endothelial cell viability after addition of 1.0 mMH2O2showed that in every case, with or without anesthetic, a large increase in Calcium2+ipreceded cell death by at least 30 min, similar to that previously reported in umbilical vein endothelial cells. This is shown qualitatively for a single cell by the pseudocolor digitized microscope images in Figure 10, and quantitatively for the same cell by the graph of calculated Calcium2+iand PI fluorescence in Figure 11. Although the presence of clinical concentrations of halothane or isoflurane did not prevent the large increase in Calcium2+ibefore cell death, it did seem to delay it. To analyze this quantitatively, the time required for Calcium2+ito double from its baseline value (average of last 5 values before addition of H2O sub 2), was determined for each individual cell. The results, summarized in Table 1, demonstrate that 1.0% halothane unequivocally delayed the rise in Calcium2+icaused by H2O2. The effect of isoflurane (1.5%) was similar in magnitude, but not significant (P = 0.08).
A similar experiment with greater temporal resolution was performed with 2.7% halothane, as shown in Figure 12. Even though this supraclinical concentration of halothane was less protective against H2O2toxicity (above), it markedly suppressed the increase in Calcium sup 2+iassociated with H2O2exposure.
This is the first report of a protective effect of halothane and, to a lesser extent, isoflurane on H2O2toxicity in aortic endothelial cells. It is unlikely that this is an artifact of any particular assay system, because it was seen in single cells with PI assay, in cell suspension with PI and LDH assays, and in monolayer cultures with MTT reduction as an assay of cell viability. The protective effect of halothane affected both the initial rate of cell death after a fully toxic dose, and the final extent of cell death after a sublethal dose of H2O2.
The mechanism by which halothane protects aortic endothelial cells against H2O2is of obvious interest. Our data exclude an effect on the direct detoxification of H2O2via endogenous catalase or peroxidase. While our results show that halothane slows the rate of Calcium2+iincrease after H2O2, our data do not allow us to distinguish between a direct protective effect of halothane on Calcium2+ihomeostasis, and an earlier effect, e.g., slowing the transformation of H2O2to a more toxic metabolite, which would also indirectly result in a moderation of the rate of Calcium2+iincrease. Both potential mechanisms are plausible, and are consistent with the known biochemistry of halothane and isoflurane, the greater protection seen with halothane, and the known mechanisms of H2O2toxicity.
Our data confirm that in aortic endothelium, as in venous and coronary artery endothelium, [1,23,24,42]the toxic effect of H2O2is preceded by a large increase in Calcium2+i, and the rate of increase in Calcium2+iis correlated with the rate of cell death. The source and mechanism of this large increase in Calcium2+iare not fully elucidated, although in other endothelial systems both influx of Calcium2+ifrom the extracellular buffer, [1,42,43]and release of Calcium2+ifrom intracellular stores have been implicated. Volatile anesthetics have been shown, in multiple cell types, to limit the increase in Calcium2+iduring normal physiologic reactions, with halothane generally more potent in this regard than isoflurane. [26–28,44]To the extent that H2O2-mediated injury uses normal avenues of Calcium2+imetabolism in an exaggerated manner, these previous data would predict our current data, that volatile anesthetics protect against H2O2-mediated injury, with halothane being more protective.
It is also possible that halothane exerts its protective effect in the system examined here by affecting the transformation of H2O sub 2 to a more toxic metabolite. The toxicity of H2Oxygen2is associated with its reduction, e.g., by intracellular iron, to the far more reactive and toxic hydroxyl radical OH *symbol*, which disrupts Calcium2+ihomeostasis more potently than H2O2alone. [17,31,45–48]Halothane's chlorobromocarbon moiety, unique among the clinically used anesthetics, interacts with and accepts an electron readily from a variety of different biologic forms of reduced iron, including cytochrome P-450 isozymes, other heme proteins, and protein-free hemin. Halothane also is a potent inhibitor or inactivator of some cytochrome P-450 isozymes. [49,50]A testable hypothesis consistent with this information and our data is that halothane interferes with the intracellular generation of hydroxyl radical from H2O2by reduced iron. Endothelial cells exposed to iron-adenosine diphosphate as a hydroxyl radical generator showed increased lipid peroxidation in the presence of several chlorinated aliphatic hydrocarbons (e.g., carbon tetrachloride). However, the effect of any brominated or fluorinated hydrocarbon was not tested. Furthermore, those experiments were performed in buffer containing 120 mM KCl and no added calcium, which would depolarize the cells and significantly alter intracellular ionic homeostasis.
Our data suggest a difference in anesthetic effect between systemic and pulmonary artery endothelium, in terms of response to an endogenous oxygen-derived toxin (H2O2), although more experiments with a greater number of independent clones would be necessary to better define the apparent difference. On consideration, this is not surprising given the different physiologic roles the systemic and pulmonary vascular beds play, the different detoxification mechanisms for H2O2that they possess, the different concentrations of oxygen to which they are usually exposed, and their different pressor responses to changes in oxygen tension in vivo. [10,30,52]Pulmonary artery and umbilical vein endothelial cells often are used experimentally because of their abundance and relative ease of isolation and culture. Our findings suggest that, while data obtained with these cells are valuable in regard to those specific vascular beds, caution should be exercised in extrapolating results from pulmonary or venous endothelium to systemic endothelium. Our data also suggest that a separate examination of endothelium from other clinically important vascular beds (e.g., coronary, renal, hepatic, and basilar or carotid) might be worthwhile.
The difference between aortic and pulmonary artery endothelium does not fully account for the difference between our observation of essentially no harmful anesthetic effect in aortic or pulmonary cultures, up to 2.7% halothane and 4.0% isoflurane, and that of Shayevitz et al., who reported a harmful effect of 2.8% and 5.0% isoflurane, and of 2.8% halothane, on H2O2toxicity in rat pulmonary artery endothelial cell cultures. Lower, clinically useful concentrations were not harmful in that study. It is of interest that despite the differences, both studies found that where an effect was seen, halothane was more beneficial than isoflurane, and that clinical concentrations were more beneficial than supraclinical concentrations. A species difference may explain some of the discrepancies between the two studies. Another difference between the two studies is the use of a simultaneous control with sibling cultures, of the same culture history and passage, for every experiment reported here, which was not reported by Shayevitz et al. They also have reported recently that susceptibility of umbilical vein endothelial cells to H2O2varies markedly from passage to passage, due at least in part to variation in endogenous iron. It is also possible that the cytotoxicity assay used by Shayevitz et al., replating efficiency, was affected by factors in addition to cell death.
Cell viability after H2O2exposure in that study was determined by removing the exposure medium and nonadherent cells, trypsinizing and washing cells, plating in complete medium overnight, washing nonadherent cells off, then retrypsinizing and counting cells. Although an effect of anesthetic on this assay was not seen with healthy cells, there was no control in place to exclude an effect of anesthetic on cell adhesion and division in injured cells. Volatile anesthetics have been reported to affect protein kinase C and protein synthesis, which in turn may affect cell adhesion in culture. [55,56]Although these effects might lead to lower cell counts in a replating assay, they do not necessarily indicate increased cell death. In contrast, the PI and LDH assays reported in this study are direct measures of cell death. In the MTT assay, where cells were incubated overnight after H2O2exposure, a glucose-containing buffer without medium supplements or fetal calf serum was used to allow cell survival but minimize cell growth and division, and both adherent and nonadherent cells were included.
Shayevitz et al. have also reported that halothane is harmful to the ex vivo perfused rabbit lung treated with the oxidant tert-butyl-hydroperoxide. Those studies were performed using Calcium2+-free perfusion buffer to eliminate pulmonary arterial pressor responses. However, the omission of extracellular Calcium2+ is a well-documented method of decreasing Calcium2+i, and by itself protects against endothelial cell injury and death from peroxide oxidants. [1,23,24,42,43]If the protective effect of halothane is mediated in part by its moderation of increases in Calcium2+iafter oxidant exposure, as suggested by our data, then the protective effect would be modified by perfusion with Calcium2+-free buffer. In fact, Shayevitz et al. reported that earlier experiments with physiologic levels of Calcium2+ in the perfusate demonstrated a protection by halothane during oxidant injury. Given that Calcium2+-free perfusion would be lethal in the whole animal, it is more relevant to clinical application to determine the effect of volatile anesthetics in the presence of a normal extracellular Calcium2+. Our data are consistent with a number of reports of a protective effect of halothane, more than isoflurane, on pathologic responses in whole organ or animal models that may be mediated in part by increased Calcium2+i: myocardial injury during ischemia-reperfusion injury, [28,58]myocardial rigor during anoxia, myocardial arrhythmias after infarction, free radical mediated reduction in coronary blood flow, and platelet activation after vascular injury. Cardiac myocytes exposed to 20–200 micro Meter H2O2were not adversely affected by 1 mM halothane (approximately 3%); lower concentrations of halothane were not tested. .
The protective effects of anesthetics have been observed primarily in their normal clinical dose range. An attractive hypothesis is that anesthetics exert both protective and harmful effects during oxidant stress, with different dose-response relationships, so that the protective effects predominate at lower concentrations, whereas the harmful effects become apparent at higher concentrations. This is consistent with our finding that 2.7% halothane, although more effective than 1% halothane in preventing increased Calcium2+i, was less effective than 1% halothane in preventing cell death. (Another possible explanation is that changes in Calcium2+iduring H2O2exposure are an epiphenomenon unrelated to cell death, although this is less likely. [1,23–25]) For example, in simple defined solutions, halothane has been shown to compete successfully with Oxygen2for hydrated electrons generated by radiation, which should result in decreased superoxide and hence hydroxyl radical formation. However, the resultant halothane-derived free radical also reacts rapidly with Oxygen2to form a peroxyl radical, which should be capable of initiating lipid peroxidation and other harmful reactions. Certainly other free radical interactions are also probable in the more complex milieu of the cell. This might account for some of the differences between our results and those Shayevitz et al., if the different experimental conditions affected the protective and harmful effects in ways we do not yet appreciate. While consistent with our data, this hypothesis requires further research into the mechanisms by which anesthetics affect oxidant toxicity.
In conclusion, clinically useful concentrations of volatile anesthetic protected human aortic, but not pulmonary arterial, endothelial cells from H2O2toxicity. H2O2-mediated cell death was preceded by a large increase in Calcium2+i, which was moderated by volatile anesthetic. Halothane was more protective than isoflurane against H2O2cytotoxicity.