Halothane Enhances Pulmonary Artery Endothelial Eicosanoid Release Steve D. Barnes,
MD,
Lynn D. Martin,
MD,
and Randall C. Wetzel,
MB, BS, FCCM
Departments of Anesthesiology/CriticalCare Medicine and Pediatrics, The Johns Hopkins Medical Institutions, Baltimore, Maryland
To determine whether anesthetics alter endothelial eicosanoid release, cultured bovine pulmonary artery endothelial cells were studied during constant flow and pressure perfusion at two oxygen tensions (hypoxia, 50 * 2 mm Hg; normoxia, 144 & 5 mm Hg; mean ? SEM) with and without 1%halothane. Endothelialized microcaniers containing -5 x lo6 cells were loaded into cartridges and perfused (3 mL/min) with Krebs' solution (pH 7.4, at 37°C) equilibrated with each gas mixture. Eicosanoids (6-keto prostaglandin Fla,thromboxane B2, and total peptidoleukotrienes [C,, D,, E,, F,]) were measured by radioimmunoassay and quantified per gram of cellular protein per minute. Eicosanoid release did not vary over time. The 6-keto prostaglandin F,, release increased during hypoxia (normoxia 291 & 27 vs hy-
A
nesthetics alter vascular tone and inhibit hypoxic pulmonary vasoconstriction (HPV) (1,2). This has been attributed to many factors, including alterations in autonomic activity and direct anesthetic effects on vascular smooth muscle. Because the endothelium is intimately involved in the regulation of both systemic and pulmonary vascular resistance (PVR), a further explanation for the effect of anesthetics could be either the potentiation or inhibition of the release of endothelial-derived vasodilators, such as prostacyclin or nitric oxide, or both, or endothelial-derived vasoconstrictors, such as thromboxane, leukotrienes, or endothelin, or all of these (3-5). Modulation of endothelial function by inhaled anesthetics could, in part, mediate their vascular effects (1,2,6-10). Although the anesthetic modulation of endothelial-derived relaxing factor has recently been studied (lo), the effect of anesthetics on endothelial eicosanoid release has received little attention. A relationship between endothelial eiAccepted for publication July 28, 1992. Address correspondence to Dr.Wetzel, Division of Pediatric Anesthesia, The Johns Hopkins Hospital, 600 North Wolfe Street, CMSC 7-110, Baltimore, MD 21287-3711. 61992 by the International Anesthesia Research Soaety 0003-2999/92/$5.00
poxia 395 & 35 ng-min-'.g protein-'; P < 0.01). Halothane (H) increased release of each eicosanoid during both normoxia and hypoxia: 6-keto prostaglandin F,,-normoxia 291 2 27 versus normoxia + H 356 ? 32 ngmin-'.g protein-', hypoxia 395 & 35 versus hypoxia + H 464 2 40 ngmin-'.g protein-', P < 0.05; thromboxane B,-normoxia 19 2 2 versus normoxia + H 26 f 2 ngmin-'.g protein-', hypoxia 20 f 2 versus hypoxia + H 38 f 5 ng.min-'.g protein-', P < 0.001; leukotriene-normoxia 363 & 35 versus normoxia + H 489 & 52 ng.mh-'.g protein-', hypoxia 329 2 29 versus hypoxia + H 455 -+ 39 ngmin-'.g protein-', P = 0.001. We conclude that alteration of endothelial eicosanoid release by halothane and hypoxia could modulate pulmonary vascular tone. (Anesth Analg 1992;75:1007-13)
cosanoid release and inhaled anesthetics-mediated vasodilation was suggested by Stone and Johns (lo), but whether volatile anesthetics directly affect endothelial eicosanoid release is not known. Pulmonary vascular resistance and its relation to oxygen tension is influenced by many factors, including volatile and intravenous anesthetics (1,2,11,12). Despite numerous investigations to define how anesthetics and oxygen interact to influence PVR, unanswered questions remain. Pulmonary artery endothelium produces numerous eicosanoids, some potent vasoconstrictors (thromboxane, prostaglandin F,, [PGF,,]), and other potent vasodilators (prostacyclin, prostaglandin El [PGE,]) (13,14). Studies in intact animals and isolated lungs have demonstrated eicosanoid involvement in the pulmonary vascular response to hypoxia (11). In vitro studies in static endothelial cells have failed to clearly demonstrate altered eicosanoid release by hypoxia (15-17). These studies, however, did not include the important physiologic factor, shear stress, which is ever present in perfused, in vivo endothelium (18,19). This study was designed to determine whether halothane alters the release of prostacyclin, thromboxane, and total peptidoleukotriene (C4, D,, E,, F4) Anesth Analg 1992;75:1007-13
1007
1008
BARNES ET AL. HALOTHANE ENHANCES ENDOTHELIAL EICOSANOID RELEASE
Krebs’ reservoir
Gas Bubbling flask
ANESTH ANALG 1992;751007-13
Cell cartridge
Waste flask
Figure 1. The perfusion circuit. Krebs’ solution bubbled with 100%N, was pumped via a multichannel variable-speed roller-head pump to the gas-bubbling flask. Krebs’ perfusate oxygen tension and halothane concentration was controlled by bubbling gas (1 Umin) of known oxygen tension (normoxia, 21% 0,; hypoxia, 4% 0,; normoxia + 1% halothane; hypoxia + 1%halothane) through the perfusate. The perfusate was pumped through the cell cartridge containing 5 x lo6 pulmonary artery endothelial cells on microcarrier beads to the C,, Sep-Pak extraction cartridge and into a vacuum (VAC)-evacuated waste flask. Temperature was maintained at 37°C by immersion of the perfusion circuit components in a servoregulated water bath.
by perfused bovine pulmonary artery endothelial cells during normoxia and hypoxia.
Methods Cell Culture Bovine pulmonary artery endothelial cells (CCL-209 CPAE; American Type Culture Collection, Rockville, Md.) >95% pure, as determined by morphology, the presence of factor VIII antigen, angiotensinconverting enzyme activity, and diacyl low-density lipoprotein uptake, were cultured according to the method of Ryan et al. (20). Endothelial cells were suspended in M-199 containing 20% fetal bovine serum, peniallin (50 U/mL), streptomycin (50 pg/mL), and amphotericin (0.25 pg/mL) and initidy seeded in 75-cm2 flasks (Fisher, Orangeburg, N.Y .). Culture flasks were incubated at 37°C in 95% air-5% CO, and the medium changed at 24 h and then every 3 days until confluence was reached (5-8days). The cells were scraped from the flask with a sterile rubber policeman, resuspended, and seeded onto 2 x 105 microcarrier beads (Cytodex-3, Sigma Chemical Co., St. Louis, Mo.) hydrated in Dulbecco’s phosphate-buffered saline (PBS) and placed in a 100-mL siliconized roller bottle (Gibco, Grand Island, N.Y.). The roller bottle was gassed with 95% &-5% CO,, sealed, and rotated at 2 rpm for 2 min every 30 min for 3 h and then continuously rotated at 1.5 rpm at 37°C. When cells were confluent on the microcarrier beads, as seen on phase-contrast microscopy, additional microcarrier beads (1 x 105) were added to the roller bottle (every 2-3 days) until 5 x 105
confluent miaocamer beads were obtained (approximately 3 4 wk). The medium was changed approximately every 2 days and roller bottles were gassed with 95% air-5% CO,. Twenty-four hours before the experiment, the cell medium in the roller bottles was changed to standard medium enriched with 20 pM arachidonic acid to ensure adequate substrate for eicosanoid release. This supplementation with arachidonate does not alter either prostacyclin release or the proportions of eicosanoids released (15,21,22).
Perfusion Systern The perfusion system was designed to perfuse four identical circuits simultaneously with the same pressure and flow characteristics (Figure 1).Each circuit contained a similar number of cells from a single roller bottle. For each experiment (n = 8), cells from a single population were exposed to four conditions (normoxia, normoxia + halothane, hypoxia, hypoxia + halothane) while being identically perfused. Perfusate oxygen tension was controlled by bubbling gas (1 Limin) of known oxygen concentration (normoxia-21% O,, 5% CO,, 74% N,; hypoxia-4% 0,, 5% CO,, 91% N,) through the perfusate in a closed, 250-mL vacuum-evacuated Erlenmeyer flask until equilibration occurred (
MD,
Lynn D. Martin,
MD,
and Randall C. Wetzel,
MB, BS, FCCM
Departments of Anesthesiology/CriticalCare Medicine and Pediatrics, The Johns Hopkins Medical Institutions, Baltimore, Maryland
To determine whether anesthetics alter endothelial eicosanoid release, cultured bovine pulmonary artery endothelial cells were studied during constant flow and pressure perfusion at two oxygen tensions (hypoxia, 50 * 2 mm Hg; normoxia, 144 & 5 mm Hg; mean ? SEM) with and without 1%halothane. Endothelialized microcaniers containing -5 x lo6 cells were loaded into cartridges and perfused (3 mL/min) with Krebs' solution (pH 7.4, at 37°C) equilibrated with each gas mixture. Eicosanoids (6-keto prostaglandin Fla,thromboxane B2, and total peptidoleukotrienes [C,, D,, E,, F,]) were measured by radioimmunoassay and quantified per gram of cellular protein per minute. Eicosanoid release did not vary over time. The 6-keto prostaglandin F,, release increased during hypoxia (normoxia 291 & 27 vs hy-
A
nesthetics alter vascular tone and inhibit hypoxic pulmonary vasoconstriction (HPV) (1,2). This has been attributed to many factors, including alterations in autonomic activity and direct anesthetic effects on vascular smooth muscle. Because the endothelium is intimately involved in the regulation of both systemic and pulmonary vascular resistance (PVR), a further explanation for the effect of anesthetics could be either the potentiation or inhibition of the release of endothelial-derived vasodilators, such as prostacyclin or nitric oxide, or both, or endothelial-derived vasoconstrictors, such as thromboxane, leukotrienes, or endothelin, or all of these (3-5). Modulation of endothelial function by inhaled anesthetics could, in part, mediate their vascular effects (1,2,6-10). Although the anesthetic modulation of endothelial-derived relaxing factor has recently been studied (lo), the effect of anesthetics on endothelial eicosanoid release has received little attention. A relationship between endothelial eiAccepted for publication July 28, 1992. Address correspondence to Dr.Wetzel, Division of Pediatric Anesthesia, The Johns Hopkins Hospital, 600 North Wolfe Street, CMSC 7-110, Baltimore, MD 21287-3711. 61992 by the International Anesthesia Research Soaety 0003-2999/92/$5.00
poxia 395 & 35 ng-min-'.g protein-'; P < 0.01). Halothane (H) increased release of each eicosanoid during both normoxia and hypoxia: 6-keto prostaglandin F,,-normoxia 291 2 27 versus normoxia + H 356 ? 32 ngmin-'.g protein-', hypoxia 395 & 35 versus hypoxia + H 464 2 40 ngmin-'.g protein-', P < 0.05; thromboxane B,-normoxia 19 2 2 versus normoxia + H 26 f 2 ngmin-'.g protein-', hypoxia 20 f 2 versus hypoxia + H 38 f 5 ng.min-'.g protein-', P < 0.001; leukotriene-normoxia 363 & 35 versus normoxia + H 489 & 52 ng.mh-'.g protein-', hypoxia 329 2 29 versus hypoxia + H 455 -+ 39 ngmin-'.g protein-', P = 0.001. We conclude that alteration of endothelial eicosanoid release by halothane and hypoxia could modulate pulmonary vascular tone. (Anesth Analg 1992;75:1007-13)
cosanoid release and inhaled anesthetics-mediated vasodilation was suggested by Stone and Johns (lo), but whether volatile anesthetics directly affect endothelial eicosanoid release is not known. Pulmonary vascular resistance and its relation to oxygen tension is influenced by many factors, including volatile and intravenous anesthetics (1,2,11,12). Despite numerous investigations to define how anesthetics and oxygen interact to influence PVR, unanswered questions remain. Pulmonary artery endothelium produces numerous eicosanoids, some potent vasoconstrictors (thromboxane, prostaglandin F,, [PGF,,]), and other potent vasodilators (prostacyclin, prostaglandin El [PGE,]) (13,14). Studies in intact animals and isolated lungs have demonstrated eicosanoid involvement in the pulmonary vascular response to hypoxia (11). In vitro studies in static endothelial cells have failed to clearly demonstrate altered eicosanoid release by hypoxia (15-17). These studies, however, did not include the important physiologic factor, shear stress, which is ever present in perfused, in vivo endothelium (18,19). This study was designed to determine whether halothane alters the release of prostacyclin, thromboxane, and total peptidoleukotriene (C4, D,, E,, F4) Anesth Analg 1992;75:1007-13
1007
1008
BARNES ET AL. HALOTHANE ENHANCES ENDOTHELIAL EICOSANOID RELEASE
Krebs’ reservoir
Gas Bubbling flask
ANESTH ANALG 1992;751007-13
Cell cartridge
Waste flask
Figure 1. The perfusion circuit. Krebs’ solution bubbled with 100%N, was pumped via a multichannel variable-speed roller-head pump to the gas-bubbling flask. Krebs’ perfusate oxygen tension and halothane concentration was controlled by bubbling gas (1 Umin) of known oxygen tension (normoxia, 21% 0,; hypoxia, 4% 0,; normoxia + 1% halothane; hypoxia + 1%halothane) through the perfusate. The perfusate was pumped through the cell cartridge containing 5 x lo6 pulmonary artery endothelial cells on microcarrier beads to the C,, Sep-Pak extraction cartridge and into a vacuum (VAC)-evacuated waste flask. Temperature was maintained at 37°C by immersion of the perfusion circuit components in a servoregulated water bath.
by perfused bovine pulmonary artery endothelial cells during normoxia and hypoxia.
Methods Cell Culture Bovine pulmonary artery endothelial cells (CCL-209 CPAE; American Type Culture Collection, Rockville, Md.) >95% pure, as determined by morphology, the presence of factor VIII antigen, angiotensinconverting enzyme activity, and diacyl low-density lipoprotein uptake, were cultured according to the method of Ryan et al. (20). Endothelial cells were suspended in M-199 containing 20% fetal bovine serum, peniallin (50 U/mL), streptomycin (50 pg/mL), and amphotericin (0.25 pg/mL) and initidy seeded in 75-cm2 flasks (Fisher, Orangeburg, N.Y .). Culture flasks were incubated at 37°C in 95% air-5% CO, and the medium changed at 24 h and then every 3 days until confluence was reached (5-8days). The cells were scraped from the flask with a sterile rubber policeman, resuspended, and seeded onto 2 x 105 microcarrier beads (Cytodex-3, Sigma Chemical Co., St. Louis, Mo.) hydrated in Dulbecco’s phosphate-buffered saline (PBS) and placed in a 100-mL siliconized roller bottle (Gibco, Grand Island, N.Y.). The roller bottle was gassed with 95% &-5% CO,, sealed, and rotated at 2 rpm for 2 min every 30 min for 3 h and then continuously rotated at 1.5 rpm at 37°C. When cells were confluent on the microcarrier beads, as seen on phase-contrast microscopy, additional microcarrier beads (1 x 105) were added to the roller bottle (every 2-3 days) until 5 x 105
confluent miaocamer beads were obtained (approximately 3 4 wk). The medium was changed approximately every 2 days and roller bottles were gassed with 95% air-5% CO,. Twenty-four hours before the experiment, the cell medium in the roller bottles was changed to standard medium enriched with 20 pM arachidonic acid to ensure adequate substrate for eicosanoid release. This supplementation with arachidonate does not alter either prostacyclin release or the proportions of eicosanoids released (15,21,22).
Perfusion Systern The perfusion system was designed to perfuse four identical circuits simultaneously with the same pressure and flow characteristics (Figure 1).Each circuit contained a similar number of cells from a single roller bottle. For each experiment (n = 8), cells from a single population were exposed to four conditions (normoxia, normoxia + halothane, hypoxia, hypoxia + halothane) while being identically perfused. Perfusate oxygen tension was controlled by bubbling gas (1 Limin) of known oxygen concentration (normoxia-21% O,, 5% CO,, 74% N,; hypoxia-4% 0,, 5% CO,, 91% N,) through the perfusate in a closed, 250-mL vacuum-evacuated Erlenmeyer flask until equilibration occurred (
