Anesth Analg 2001;93:641-644
© 2001 International Anesthesia Research Society
ANESTHETIC PHARMACOLOGY
A Comparison of Atenolol, Labetalol, Esmolol, and Landiolol for Altering Human Neutrophil Functions
Kahoru Nishina, MD*,
Hirohiko Akamatsu, MD ,
Katsuya Mikawa, MD*,
Makoto Shiga, MD*,
Hidefumi Obara, MD*, and
Yukie Niwa, MD
*Department of Anesthesiology and Intensive Care Unit, Kobe University School of Medicine, Kobe, Japan; Department of Dermatology, Fujita Health University School of Medicine, Aichi, Japan; and Niwa Institute for Immunology, Kochi, Japan
Address correspondence and reprint requests to Dr. K. Mikawa, Department of Anesthesiology, Kobe University School of Medicine, Kusunoki-cho 7, Chuo-ku, Kobe 650-0017, Japan.
 |
Abstract
|
|---|
IMPLICATIONS: Neutrophils play a pivotal role in the antibacterial host defense system. Atenolol, labetalol, esmolol, and landiolol at clinically relevant concentrations failed to change neutrophil functions. Our findings indicate that we may be able to use these ß-antagonists without great caution in clinical settings.
|
Introduction
|
|---|
Neutrophils play a crucial role in the antibacterial host defense system by releasing reactive oxygen species (ROS) (1). Neutrophils have a ß-adrenergic receptor on their cell surface (2). Propranolol, the most common ß-antagonist, suppresses superoxide (O2-) production (3) and phagocytic capacity of neutrophils (4). When propranolol is used to treat hypertension, tachycardia, or arrhythmia in patients undergoing surgery that inhibits several neutrophil functions (5), the ß-antagonist may enhance the neutrophil dysfunction, leading to an increase in postoperative infection. The use of propranolol in critically ill patients for the same purpose may further compromise an already depressed host defense system. Thus, although it is important to determine the effects of ß-antagonists often used in these clinical settings on neutrophil functions, little information is available as to whether atenolol and esmolol (common ß-antagonists) modulate ROS production. Published data concerning the effects of labetalol, a useful ß-blocker with  1-antagonistic activity for perioperative hemodynamic stability, on neutrophil O2- production are conflicting (6,7). Furthermore, the effects of these ß-antagonists on chemotaxis and phagocytosis, both of which are other important functions of neutrophils, remain to be determined. In this study, therefore, we investigated whether atenolol, labetalol, esmolol, and landiolol [a new ß-antagonist (8)] alter chemotaxis, phagocytosis, O2-, and hydrogen peroxide (H2O2) production of human neutrophils, by using an ex vivo system. H2O2 is an important oxygen derivative because it has more potent oxidative activity than O2- (9). We also assessed the scavenging effects of these ß-blockers on the excess of the ROS generated by using the cell-free system. To elucidate the mechanism underlying the effects on ROS production by neutrophils, we determined whether the ß-antagonist changes protein kinase C (PKC) activity. PKC is an important pathway by which extracellular stimuli are transmitted to the O2--producing enzyme of neutrophils (nicotinamide adenine dinucleotide phosphate-oxidase).
|
Methods
|
|---|
Isolation of neutrophils and assays for neutrophil functions used in this study were described in detail elsewhere (10). Heparinized venous blood was obtained from 12 healthy volunteers after institutional approval and informed consent. The neutrophils were collected by centrifugation of the blood over a Ficoll-Hypaque (Pharmacia, Piscataway, NJ) gradient after sedimentation at unit gravity in plasma containing 1% of dextran 170. The contaminating erythrocytes were lysed by NH4Cl. The neutrophils were resuspended in media appropriate for their subsequent use (10). Atenolol (Sigma, St. Louis, MO), labetalol hydrochloride (Sigma), esmolol hydrochloride (WelFide, Osaka, Japan), or landiolol hydrochloride (Ono, Osaka, Japan) was diluted with distilled water. Each drug was prepared at concentrations 10-fold larger than the final concentrations specified in Table 1. Microscope observations revealed that 95%–98% of the cells were neutrophils and that more than 95% of the cells were viable by the trypan blue exclusion test. Chemotaxis was determined by using a modified Boyden chamber with some modification (10). Neutrophils (0.2 x 106 cells per 0.2 mL) with the drugs tested were placed in the upper compartment, and N-formyl-L-methionyl-L-leucil-L-phenylalanine (FMLP) 10-7 M was added to the lower compartment. The chamber was incubated at 37°C for 45 min. The number of cells reaching the bottom surface of the filter was expressed as the average number of cells per field after we counted five fields. For assessment of phagocytosis, neutrophils (2 x 107 cells per 0.9 mL Krebs-Ringer phosphate buffer) preincubated for 5 min with the drugs tested were added to 0.1 mL of the opsonized emulsion paraffin oil containing oil red O. After an additional 5 min incubation, the reaction was stopped with 9 mL ice-cold Krebs-Ringer phosphate. Paraffin oil ingested by the cells was extracted with chloroform and methanol. The optical density of oil red O in the chloroform layer was determined at wavelengths of 525 nm. To determine O2- formation, the reduced cytochrome c was measured. Neutrophils (1 x 106) were preincubated with 1 mg/mL opsonized zymosan, and the drugs were tested at 37°C for 10 min. The neutrophils were incubated with ferricytochrome c (0.1 mM; Sigma) for another 30 min. After centrifugation, 0.1 mL of the supernatant was assayed for reduced cytochrome c by measuring the absorbance at 550 nm in 2 mL of 100 mM potassium phosphate buffer (pH 7.8) containing 0.1 mM EDTA. The H2O2 generation was determined by rate of the decrease in fluorescence intensity of scopoletin (Sigma). After incubation of 2.5 x 106 neutrophils with the drug tested and 1 mg/mL opsonized zymosan for 10 min at 25°C, 0.1 mL of 50 mM scopoletin and 0.05 mL of 1 mg/mL horseradish peroxidase (Sigma) were added. The H2O2 concentration was calculated assuming that 1 mol H2O2 oxidizes 1 mol scopoletin.
The mixture of 0.1 mM hypoxanthine, 1.25 mM EDTA, and the drugs tested was incubated in 125 mM phosphate buffer for 2 min at 37°C. Dialyzed xanthine oxidase (0.006 U/mL) was added to the mixture to start a reaction. The O2- and H2O2 generation was determined by the method described previously.
Neutrophils isolated from another set of 12 healthy volunteers were incubated with labetalol in the presence or absence of phorbol 12-myristate 13-acetate (11). Then, the neutrophils were sonicated in ice-cold sample preparation buffer (2 x 107 cells/mL) consisting of 50 mM Tris/HCl pH 7.5, 5 mM EDTA, 10 mM EGTA, 50 mM 2-mercaptoethanol, 1 mM phenylmethyl-sulfonyl fluoride, and 10 mM benzamidine. Cytosol fraction was separated by centrifugation at 100,000g for 1 h at 4°C. The cytosol PKC activity was measured by using a protein kinase assay kit (MESACUP; MBL, Nagoya, Japan) according to the manufacturer’s protocol.
Raw data were statistically analyzed by the Friedman rank test, followed by Dunnett test for post hoc comparison. P < 0.05 was deemed significant.
|
Results
|
|---|
Table 2 shows absolute control values of any variable, indicating similarity between groups. No drug altered chemotaxis and phagocytosis of neutrophils (Table 1). Labetalol produced an inhibitory effect on production of O2- and H2O2 by human neutrophils, whereas the other three drugs failed to do so (Table 1). The four ß-adrenoceptor antagonists did not scavenge O2- and H2O2 generated in the cell-free system (Table 1). Labetalol had no effect on cytosolic PKC activity (Table 1).
|
Discussion
|
|---|
Our major findings are that 1) atenolol, esmolol, and landiolol had no effect on neutrophils’ chemotaxis, phagocytosis, or O2- and H2O2 production; 2) labetalol at clinically relevant concentrations did not alter the ROS production, although larger concentrations of the drug inhibited the ROS production; 3) labetalol did not suppress chemotaxis, phagocytosis, or PKC activity; and 4) these four ß-antagonists failed to scavenge ROS generated in the acellular system.
One laboratory demonstrated inhibition of phorbol myristate acetate-induced O2- production with labetalol (6), and the other failed to do so (7). These conflicting data may be due to different experimental protocols, including stimulants (with or without cytochalasin B), incubation time, and the number of neutrophils.
Because not all the ß-antagonists failed to impair neutrophil functions, inhibition of the ROS by labetalol would not be associated with direct ß-adrenoceptor blockade. Calcium mobilization and PKC activation in neutrophils are among the most important pathways of O2- production (1). In this study, we could not measure intracellular calcium levels in the presence of labetalol because the drug per se inhibited dual wavelength fura-2 fluorescence. Labetalol seems to suppress the ROS produced from neutrophils through mechanisms unrelated to PKC activity, because the drug did not reduce PKC activity. Lipid solubility and membrane-stabilizing activity (MSA) may explain additional possible mechanisms unrelated to ß-adrenoceptor blockade. Drugs with high lipid solubility may be likely to accumulate in the lipid membranes, leading to alteration of their physical properties. This may change conformation of nicotinamide adenine dinucleotide phosphate-oxidase, consequently suppressing O2- generation (12). The ability of propranolol to inhibit O2- generation seems to be related to MSA rather than ß-receptor-directed effects (4). Unlike atenolol and esmolol, labetalol is a lipophilic drug with MSA (13). These mechanisms may contribute to labetalol-induced reduction of ROS. However, the precise mechanism underlying impairment of the ROS production with labetalol remains to be elucidated.
Labetalol inhibited ROS production from neutrophils, but the drug failed to impair chemotaxis and phagocytosis. Although we are unable to give a satisfactory explanation for this discrepancy, it may be due to the difference in stimulants used: opsonized zymosan and FMLP. The two stimulants activate phospholipase C through guanosine triphosphate-binding protein. Stimulation with FMLP 10-7 M for chemotaxis may have been potent enough not to be blocked by labetalol.
Labetalol, at a clinically relevant concentration, did not attenuate ROS production from neutrophils. The drug even at 10 times clinically relevant concentrations reduced ROS only by 8%–9%. The biological significance of the decrease may be minimal, because this reduction seems to be within a range of normal variability, although it is statistically significant. The clinical implications of our findings remain unknown because they are based on acute in vitroexperiments with neutrophils obtained from healthy volunteers and may differ from in vivo effects when given chronically. Use of neutrophils from patients with various diseases exhibit a low level of continuing inflammation (e.g., sepsis). The membrane potential of neutrophils in such a primed or hyperreactive state may be modified sufficiently to alter ROS production (14).(14)
|
Acknowledgments
|
|---|
We would like to express our gratitude to Ono and Yoshitomi for a generous supply of landiolol and esmolol.
|
References
|
|---|
-
Minakami S, Kakinuma K, Takeshige K. Leukocytes and biological defense. Tokyo: Kodansha, 1990.
-
Lee TP, Szefler S, Ellis EF. Beta-adrenergic receptors of human polymorphonuclear leukocytes. Res Commun Chem Pathol Pharmacol 1981; 31: 453–62.[Medline]
-
Anderson R, Ramafi G, Theron AJ. Membrane stabilizing, anti-oxidative interactions of propranolol and dexpropranolol with neutrophils. Biochem Pharmacol 1996; 52: 341–9.[Web of Science][Medline]
-
Straussberg-Djaldetti R, Fishman P, Bessler H, Djaldetti M. The effect of propranolol on protein synthesis and phagocytosis by polymorphonuclear leukocytes from newborn, children and elderly individuals. Biomed Pharmacother 1986; 40: 265–9.[Medline]
-
Khan FA, Kamal RS, Mithani CH, et al. Effect of general anaesthesia and surgery on neutrophil function. Anaesthesia 1995; 50: 769–75.[Web of Science][Medline]
-
Jaboureck-Bouttier R, Gressier B, Dine T, et al. Effects of two antihypertensive agents, labetalol and metoprolol, on the production of reactive oxygen species by normal polymorphonuclear leukocytes in vitro. Hypertens Pregnancy 1999; 18: 239–49.[Medline]
-
Yue TL, McKenna PJ, Ruffolo RR Jr, Feuerstein G. Carvedilol, a new beta-adrenoceptor antagonist and vasodilator antihypertensive drug, inhibits superoxide release from human neutrophils. Eur J Pharmacol 1992; 214: 277–80.[Web of Science][Medline]
-
Yoshiya I. Landiolol hydrochloride, a new sympathetic beta blocker. Jpn J Anesth 1998; 47 (Suppl): S126–32.
-
Miyachi Y, Yoshioka A, Imamura S, Niwa Y. Anti-oxidant effects of gold compounds. Br J Dermatol 1987; 116: 39–46.[Web of Science][Medline]
-
Mikawa K, Akamatsu H, Nishina K, et al. The effect of phosphodiesterase III inhibitors on human neutrophil function. Crit Care Med 2000; 28: 1001–5.[Medline]
-
Mikawa K, Akamatsu H, Nishina K, et al. Inhibitory effects of pentobarbital and phenobarbital on human neutrophil functions. J Intensive Care Med 2001; 16: 79–87.
-
Mikawa K, Maekawa N, Hoshina H, et al. Inhibitory effect of barbiturates and local anaesthetics on protein kinase C activation. J Int Med Res 1990; 18: 153–60.[Web of Science][Medline]
-
Hoffman BB, Lefkowitz RJ. Catecholamines, sympathomimetic drugs, and adrenergic receptor antagonists. In: Hardman JG, Limbird LE, Molinoff PB, Ruddon RW, Gilman AG, eds. Goodman & Gilman’s The pharmacological basis of therapeutics. 9th ed. New York: McGraw-Hill, 1996: 199–248.
-
Tennenberg SD, Solomkin JS. Neutrophil activation in sepsis: the relationship between fmet-leu-phe receptor mobilization and oxidative activity. Arch Surg 1988; 123: 171–5.[Abstract/Free Full Text]
Accepted for publication May 1, 2001.
|
Top
Abstract
Introduction
