Anesth Analg 2003;97:430-435
© 2003 International Anesthesia Research Society
ANESTHETIC PHARMACOLOGY
The Volatile Anesthetic Isoflurane Inhibits the Histamine-Induced Ca2+ Influx in Primary Human Endothelial Cells
Piet W. L. Tas, PhD,
Christiane Stößel, MD, and
Norbert Roewer, MD
Department of Anesthesiology, University of Würzburg, Würzburg, Germany
Address correspondence and reprint requests to Piet Tas, PhD, Klinik für Anaesthesiologie, Der Universität Würzburg, Josef-Schneider-Strasse 2, 97080 Würzburg, Germany. Address e-mail to ptas{at}anaesthesie.uni-wuerzburg.de
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Abstract
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Although isoflurane is a known vasodilator, the mechanism of isoflurane-induced vasodilation is not clear. One of the most important systems in this context is the nitric oxide (NO)-mediated vasodilation. The activity of this system is regulated by the agonist-induced Ca2+ influx rather than Ca2+ release from internal stores. A number of reports have studied the effect of volatile anesthetics on the cytoplasmic calcium concentration signaling in mammalian endothelial cells. However, similar studies using human endothelial cells are lacking. In this study, therefore, we investigated whether isoflurane affects the histamine-induced Ca2+ influx in primary cultures of human endothelial cells. Using confocal laser scanning microscopy and cells loaded with the Ca2+ indicator Fluo-3, we studied the effect of isoflurane on the plateau phase of the histamine-induced Ca2+ influx, which is considered to be due to capacitative Ca2+ entry. In addition, we measured the ion flux through capacitative Ca2+ channels directly by using Mn2+ ions, which, on entering the cell, quench the Fura-2 fluorescence. The results of these two methods were in close agreement and showed a dose-dependent inhibition of the capacitative Ca2+ entry by isoflurane. Isoflurane apparently depresses NO-mediated vasodilation when the observed inhibition is not compensated for downstream of the endothelial NO synthase activation.
IMPLICATIONS: In response to vasoactive agents, endothelial cells produce nitric oxide (NO), which relaxes the underlying smooth muscle cells. Inhaled anesthetics inhibit this system by an unknown mechanism. Using primary human endothelial cells, we showed that the anesthetic isoflurane depresses a Ca2+ influx, which is responsible for the activation of the endothelial NO synthase.
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Introduction
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Research in several laboratories has shown that volatile anesthetics inhibit endothelium-dependent nitric oxide (NO)-mediated vasodilation (1–4). This is a complex pathway involving endothelial and smooth muscle cells. Vasoactive substances, such as histamine, acetylcholine, and adenosine triphosphate (ATP), increase the cytoplasmic calcium concentration ([Ca2+]i) of endothelial cells, which leads to activation of endothelial NO synthase (eNOS). The NO produced by this enzyme diffuses to the underlying smooth muscle and activates soluble guanylate cyclase resulting in an increase in cyclic guanosine monophosphate (cGMP) and relaxation of the smooth muscle cells (5). Possible mechanisms by which volatile anesthetics may interfere with the NO-dependent relaxation are an inhibition of endothelial receptor activation, a decrease in the availability of calcium for the activation of eNOS, direct or indirect inhibition of eNOS, accelerated decay of NO by generation of superoxide, and inhibition of the activation of soluble guanylate cyclase.
Investigation of NO-mediated vasodilation in different mammalian systems suggests that inhibition by inhaled anesthetics occurs downstream of receptor activation in endothelial cells and upstream of NO-mediated activation of guanylate cyclase in smooth muscle cells (4). The intracellular Ca2+ transient [Ca2+]i could therefore be a likely target for the volatile anesthetics. The Ca2+ transient induced by vasoactive substances such as histamine, acetylcholine, or ATP is characterized by a biphasic change in [Ca2+]i, consisting of an initial transient increase due to the release of Ca2+ from intracellular stores followed by a sustained increase of [Ca2+]i caused by Ca2+ influx from the extracellular medium, also called capacitative calcium influx (6).
It has been observed that the agonist-induced NO formation and subsequent vasodilation are abolished by removal of Ca2+ from the extracellular medium (7,8). There are conflicting reports on the effects of volatile anesthetics on agonist-induced Ca2+ entry in mammalian endothelial cells. Pajewski et al. (9) showed that halothane and enflurane, but not isoflurane, inhibit bradykinin and ATP-stimulated Ca2+ transients in bovine aortic endothelial (BAE) cells. Isoflurane also did not affect bradykinin-induced [Ca2+]i transients in BAE cells (10). In contrast to the findings of Pajewski et al. (9), Loeb et al. (10) did not find an effect of halothane and isoflurane on Ca2+ transients induced by ATP. However, Simoneau et al. (11), also working with BAE cells, showed that both halothane and isoflurane affected the bradykinin-induced Ca2+ influx. Their experiments strongly suggest that inhaled anesthetics mediate their effect on Ca2+ influx by the inhibition of Ca2+-dependent K+ channels, which provide the driving force for Ca2+ entry in agonist-stimulated endothelial cells. Sevoflurane also seems to affect bradykinin-induced Ca2+ transients in porcine aortic endothelial cells (12).
No data are available on the effect of volatile anesthetics on the agonist-induced Ca2+ influx into human endothelial cells. Therefore, we studied the effect of isoflurane on the histamine-induced Ca2+ entry in primary human umbilical vein endothelial cells (HUVEC).
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Methods
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Fluo-3 AM and Fura-2 AM were obtained from Molecular Probes (Eugene, OR), and anti-human von Willebrand factor was obtained from Dako (Hamburg, Germany). Type I collagenase was purchased from Roche (Mannheim, Germany). SK&F 96365 was a kind gift of Dr. Janet Merritt (Smith-Kline-Beecham Pharmaceuticals, Welwyne, UK). All other chemicals were obtained from Merck (Darmstadt, Germany).
HUVEC were isolated from the vein of human umbilical cords, essentially as described previously (13), by using Type I collagenase (0.1%) and were cultured in M199 medium (Invitrogen, Karlsruhe, Germany) supplemented with 20% fetal calf serum, 1% retina-derived growth factor (14), 100 U/mL of penicillin, and 0.1 mg/mL of streptomycin. Cells were identified as endothelial cells by their typical cobblestone appearance and the presence of von Willebrand factor (by immunolabeling).
For the measurement of the [Ca2+]i passage, one or two of the HUVEC cells were seeded on gelatin-coated glass coverslips (4-cm diameter; Bioptechs, Butler, PA) and used in the experiments when the cells reached confluency. The cells were loaded with the Ca2+ indicator Fluo-3 AM (5 µM final concentration) for 20 min at 37°C in the dark in Buffer A (150 mM NaCl, 5 mM KCl, 2 mM CaCl2, 0.4 mM MgSO4, 25 mM glucose, and 25 mM HEPES, pH 7.0). After loading, the glass coverslips were inserted into a gas-tight, temperature-controlled perfusion chamber (FCS2; Bioptechs) and perfused with Buffer A at a flow rate of 1 mL/min. Additions of agonists or anesthetic are indicated in the figures. Fluorescence changes were examined by using a confocal laser scanning microscope (MRC 1024; Hemel Hempstead, Hertfordshire, England). Excitation was provided by the 488-nm laser line of a krypton/argon laser (15 mW), and the [Ca2+]i signals were monitored by using the LaserSharp acquisition software (Bio-Rad).
During perfusion, the FCS2 chamber was kept at 30°C by using the Bioptechs heater system. For all experiments, a flow rate of 1 mL/min was used. Rapid change between different solutions was obtained by using syringe pumps and a four-valve liquid switch (Harvard Apparatus, Holliston, MA). Isoflurane was dissolved in the perfusion buffer by bubbling the desired anesthetic concentration for 20 min by using an isoflurane vaporizer (Dräger, Lübeck, Germany). The concentration of isoflurane was determined by gas chromatography (15). During the experiment, the buffer containing dissolved isoflurane was kept in gas-tight 50-mL glass syringes (Hamilton). The loss of anesthetic during the perfusion at a flow rate of 1 mL/min was <10%.
Mn2+ ions can permeate capacitative Ca2+ channels. On entering the cell, these ions strongly quench the Fura-2 fluorescence. At an excitation wavelength of 360 nm, the Mn2+ quench is independent of the [Ca2+]i. The decrease in Fura-2 fluorescence at 360 nm is therefore a direct measure of the Mn2+ transport through capacitative Ca2+ channels (16,17). For these measurements, HUVEC cells were cultivated on 1% gelatin (cross-linked with 0.5% glutaraldehyde)-coated coverslips (1-cm diameter). After loading for 1 h with 5 µM Fura-2 AM, the coverslips were washed twice with Buffer A. At the start of the measurement, the coverslip with the cells was briefly washed in Ca2+-free Buffer A and mounted in an angled holder that minimizes reflections. The holder was then inserted into a 1-cm square cuvette, which was placed into a PerkinElmer (Rodgau-Jügesheim, Germany) LS50 B spectrofluorimeter. The solution was stirred and kept at a temperature of 30°C. After equilibration for 2 min, histamine was added at a concentration of 100 µM, and the decrease in fluorescence was monitored for 6 min. The initial rate of fluorescence quenching was assessed by measuring the slope of Fura-2 fluorescence decrease after addition of MnCl2 (corrected for the slope of the Fura-2 signal before Mn2+ application) (17). The data were corrected for the loss of anesthetic during the incubation period. Isoflurane was tested at 1.5, 2.5, and 5 vol%. The loss of anesthetic was 25.7% at 1.5 vol%, 15.8% at 2.5 vol%, and 9.6% at 5 vol% isoflurane.
Statistical evaluation was performed with the Spearman rho test. A P value of <0.05 was considered statistically significant.
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Results
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In HUVEC, histamine induces a biphasic Ca2+ signal consisting of an extensive Ca2+ release from internal Ca2+ stores and a long-lasting Ca2+ influx from the extracellular medium (Fig. 1). The two signals can be studied separately by stimulation of the cells in Ca2+-free medium. The first [Ca2+]i signal then represents Ca2+ release from internal stores. Subsequent supplementation of the medium with Ca2+ generates a second [Ca2+]i signal, which is caused by Ca2+ influx into the cell (Fig. 1B). This second Ca2+ signal is often designated as capacitative Ca2+ influx (6).
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Figure 1. Histamine-induced changes in the cytoplasmic calcium concentration ([Ca2+]i) in primary human umbilical vein endothelial cells. The cells on 4-cm glass coverslips were loaded with the Ca2+ indicator Fluo-3 AM and inserted into the FCS-2 perfusion chamber. The chamber was kept at 30°C, and the flow rate was 1 mL/min. A, Biphasic [Ca2+]i signal in Ca2+-containing medium after the addition of 100 µM histamine. B, Splitting up of the biphasic [Ca2+]i signal in Ca2+ release from internal stores (first signal) and Ca2+ influx from the extracellular medium (second signal).
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Figure 2, A and B shows the effect of 5 and 10 µM SK&F 96365 on the capacitative Ca2+ entry. Interestingly, Figure 2B shows a HUVEC cell that develops [Ca2+]i oscillations. These oscillations are generated by brief periods of Ca2+ release from intracellular stores (18). They were observed occasionally in some preparations. These oscillating cells were omitted from the calculations, although the patterns suggest that they show a similar behavior as the nonoscillating cells. The capacitative Ca2+ influx in HUVEC cells was also sensitive to nickel (1 mM inhibited the influx by 60% ± 16%; n = 19) (not shown).
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Figure 2. Effect of 5 µM (A) and 10 µM (B) of the capacitative Ca2+ entry blocker SK&F 96365 on the plateau phase of the histamine-induced Ca2+ transient. Shown are representative traces. An oscillating human umbilical vein endothelial cell is also shown in (B).
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We tested the effect of increasing concentrations of isoflurane on the sustained plateau phase 5 min after histamine addition. At this time, the Ca2+ release from internal stores is complete, and the increased [Ca2+]i then represents only the capacitative Ca2+ entry (Fig. 3A). The inhibition is reversed by removal of the anesthetic from the perfusion medium. At the end of the experiment, we changed to Ca2+-free buffer to determine the fluorescence level corresponding to the absence of capacitative Ca2+ influx. From the data points of these curves, the percentage inhibition of the capacitative Ca2+ influx by isoflurane was calculated. Figure 3B summarizes the data of several of these experiments. Isoflurane shows a dose-dependent inhibition of the plateau phase of the histamine-induced calcium influx (Spearman rho test; P < 0.001; n = 20). A clinically relevant concentration of 1 vol% isoflurane (equivalent to 0.87 minimum alveolar anesthetic concentration [MAC]) inhibits the plateau phase by 15.1% ± 2.5%.
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Figure 3. A, Effect of isoflurane (5 vol%) on the plateau phase of the histamine (100 µM)-induced Ca2+ transient. Shown are the original traces of 3 representative cells from a total of 140 cells. B, Dose-dependent depression of the plateau phase of the histamine-induced Ca2+ transient by isoflurane. The curves in (A) were used to determine the percentage depression of the plateau phase. Data are mean ± SEM of 3 to 4 experiments performed in duplicate (15–19 cells were analyzed for each concentration or experiment). The dose dependence was checked with the Spearman rho test (rs = 0.879; P < 0.001; n = 20).
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We used the Mn2+ quench of the Fura-2 fluorescence at an excitation wavelength of 360 nm as a measure of the ion flux through capacitative Ca2+ channels. Figure 4A shows the decrease of the Fura-2 fluorescence at 360 nm after the addition of 100 µM histamine, which activates capacitative Ca2+ entry. Using this method, we observed that increasing isoflurane concentrations slowed down the decrease in Fura-2 fluorescence (shown in Fig. 4A for 5% isoflurane). This is equivalent to a decreased Mn2+ entry into the HUVEC cells. Figure 4B summarizes the data from several experiments. Isoflurane at a concentration of 1.1 vol% inhibited the Mn2+ entry by 14.6% ± 3.9%. Using this direct measurement of the ion flux through capacitative Ca2+ channels, we observed a dose-dependent inhibition by isoflurane (Spearman rho test; P < 0.001; n = 29), which is very similar to the indirect measurement using the plateau phase of the histamine-induced Ca2+ entry (Fig. 3B).
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Figure 4. A, Capacitative Ca2+ entry measured by the Mn2+ quench of the Fura-2 fluorescence. Human umbilical vein endothelial cells on glass coverslips (1-cm diameter) were loaded with 5 µM Fura-2 AM. The experiment was started in Ca2+-free Buffer A containing 0.5 mM MnCl2. After 2 min ( ), histamine was added to a final concentration of 100 µM, and the decrease in fluorescence was measured for an additional 6 min. Shown are the original traces of the control and 5% isoflurane. B, Dose-dependent effect of isoflurane on the capacitative Ca2+ entry as measured by the Mn2+ quench of Fura-2 fluorescence. The isoflurane concentrations were corrected for losses during the incubation period (see Methods). The data are mean ± SEM of four experiments performed in duplicate. The dose dependence was checked with the Spearman rho test (rs = 0.65; P < 0.001; n = 29).
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Discussion
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Capacitative Ca2+ entry is important for the activation of eNOS and therefore plays a pivotal role in agonist-induced relaxation. In agreement with this view is the observation that lack of an endothelial store-operated Ca2+ current due to gene knockout impairs agonist-dependent vasorelaxation in mice (19). Because clinical concentrations of volatile anesthetics cause vasodilation, it was of interest to study their effect on the capacitative Ca2+ influx. As a test system, we used the histamine-induced Ca2+ entry in HUVEC and studied the effect of isoflurane on this ion flux. The plateau phase of this Ca2+ transient was dose-dependently depressed by isoflurane. At 1 MAC (1.15 vol%), isoflurane reduced the plateau phase by 17%. A somewhat stronger inhibition of 23.5% was obtained with 1 MAC halothane (0.75 vol%) in the same system (20). Unfortunately, there are no specific inhibitors for the capacitative Ca2+ influx. The imidazole derivative SK&F 96365 has been used as an inhibitor of the capacitative Ca2+ entry in a number of reports (21,22), although this substance can also inhibit voltage-dependent Ca2+ channels (21), which are not present in HUVEC cells. The plateau phase of the histamine-induced Ca2+ entry was dose-dependently depressed by SK&F 96365 with a 50% inhibitory concentration of 5 µM (20), in agreement with reports in the literature (21,22). Sensitivity to nickel was also as reported (23).
In the absence of anesthetic, the height of the plateau phase is a direct measure of the capacitative Ca2+ entry. However, when the anesthetic affects the reuptake of calcium into internal stores or the Ca2+ efflux from the cell, this is no longer the case.
To measure the effect of isoflurane on the capacitative Ca2+ entry directly, we used the unidirectional Mn2+ influx into HUVEC cells loaded with the Ca2+ indicator Fura-2 AM (16,17). The histamine-induced Mn2+ entry was sensitive to SK&F 96365, which proves that Mn2+ passes through capacitative Ca2+ channels. Isoflurane caused a dose-dependent inhibition of the Mn2+ entry (Fig. 4B), which was quite similar to the dose-dependent depression of the histamine-induced plateau phase.
Previous reports studied the effect of anesthetics on agonist-induced Ca2+ transients (9,10,12). These Ca2+ transients consist of Ca2+ release from internal stores and Ca2+ influx into the cell. Certain agonists, e.g., bradykinin, induce only a very minor Ca2+ influx in HUVEC cells (unpublished observation). The bulk of the bradykinin-induced Ca2+ transient, therefore, consists of Ca2+ release from internal stores, and only a minor part is due to Ca2+ influx through capacitative Ca2+ channels. With such a system, it will be very difficult to detect an effect of anesthetics on capacitative Ca2+ entry.
Only one report (11) directly studied the effect of the volatile anesthetic halothane on capacitative Ca2+ entry by using the Mn2+ quench of the Fura-2 fluorescence. They observed a 42%–64% inhibition of the capacitative Ca2+ influx in BAE cells by 2 mM halothane. The data presented here are the first report on the effect of the volatile anesthetic isoflurane on the capacitative Ca2+ entry in human endothelial cells.
The fact that anesthetics interfere with the capacitative Ca2+ influx is in agreement with the observation of Johns et al. (4), that inhaled anesthetics inhibit the [Ca2+]i 
NO guanylate cyclase cGMP pathway downstream of receptor activation and upstream of the activation of guanylate cyclase. Several reports show that Ca2+ influx from the extracellular space is required to activate the eNOS (7,8). It seems likely, therefore, that inhibition of capacitative Ca2+ entry will result in a reduced NO synthesis when this effect is not counteracted by a stimulating effect of the anesthetics on the eNOS enzyme itself.
The data provided here do not explain the vasodilation by volatile anesthetics observed in vivo. On the contrary, assuming that isoflurane has no other effects on endothelial or smooth muscle cells, it would generate a vasoconstriction. This indicates that other effects of isoflurane at the level of the endothelial or smooth muscle cells have to overcompensate for this effect by, e.g., increased production of prostacyclin or endothelial-derived hyperpolarizing factor or decreased Ca2+ influx into smooth muscle cells.
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Acknowledgments
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We thank Andrea Jahn for excellent technical assistance and Dr. Kai Schuh of the Cardiology Department for help with the spectrofluorimeter measurements.
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Accepted for publication March 10, 2003.
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Abstract
Introduction
