The effects of the morphine analogue levorphanol on leukocytes: Metabolic effects at rest and during phagocytosis Nancy Wurster, … , Penelope Pettis, Sharon Lebow J Clin Invest. 1971;50(5):1091-1099. https://doi.org/10.1172/JCI106580. Studies on bacteria have suggested that morphine-like drugs have effects on the cell membrane. To determine the effect of this class of drugs on a mammalian cell, we selected the rabbit peritoneal exudate granulocyte, which undergoes striking membrane changes during phagocytosis. We examined the effect in vitro of the morphine analogue, levorphanol on phagocytosis and metabolism by granulocytes incubated with and without polystyrene particles or live Escherichia coli. Levorphanol (1 or 2 mmoles/liter) decreased: (a) acylation of lysolecithin or lysophosphatidylethanolamine in the medium (which is stimulated about two-fold during phagocytosis) both at rest (40%) and during phagocytosis (60%); (b) uptake of latex particles and Escherichia coli, as judged by electron microscopy; (c) killing of live Escherichia coli (10-fold); (d) 14 14 + CO2 production from glucose-1- C during phagocytosis by at least 80%; (e) K content of granulocytes (35%); (f) oxidation of linoleate-1-14C by 50%, and its incorporation into triglyceride by more than 80%. However, levorphanol stimulated 2 to 3-fold the incorporation of linoleate-1-14C or palmitate-1-14C into several phospholipids. Glucose uptake, lactate production, and adenosine triphosphate (ATP) content are not affected by the drug. Thus, levorphanol does not appear to exert its effects through generalized metabolic suppression. Removal of levorphanol by twice resuspending the granulocytes completely reverses all inhibition. In line with observations on bacteria, it appears that the complex effects of levorphanol on […] Find the latest version: https://jci.me/106580/pdf The Effects of the Morphine Analogue Levorphanol on Leukocytes METABOLIC EFFECTS AT REST AND DURING PHAGOCYTOSIS NANcY WuRsTE, PETER ELSBACH, ERIc J. SIMON, PENELOPE PETris, and SHARON LEBOW From the Department of Medicine, New York University School of Medicine, New York 10016 A B S T R A C T Studies on bacteria have suggested that INTRODUCTION morphine-like drugs have effects on the cell membrane. During phagocytosis numerous biochemical and morpho- To determine the effect of this class of drugs on a logical changes take place that indicate that engulfment mammalian cell, we selected the rabbit peritoneal exu- and killing of microorganisms require a set of active date granulocyte, which undergoes striking membrane metabolic responses (1, 2). Recently we reported from changes during phagocytosis. We examined the effect in this laboratory that these responses include net synthesis vitro of the morphine analogue, levorphanol on phagocy- of new membrane lipids (3, 4). It was shown that a tosis and metabolism by granulocytes incubated with and quantitatively important source of membrane phospho- without polystyrene particles or live Escherichia coli. lipid of granulocytes is provided by lysocompounds in Levorphanol (1 or 2 mmoles/liter) decreased: (a) acyla- the extracellular environment. These compounds, spe- tion of lysolecithin or lysophosphatidylethanolamine in cifically lysolecithin (LPC)1 and lysophosphatidyletha- the medium (which is stimulated about two-fold during nolamine (LPE), are acylated to the major membrane phagocytosis) both at rest (40%) and during phagocy- phospholipids, phosphatidylcholine (PC), and phospha- tosis (60%); (b) uptake of latex particles and Esche- tidylethanolamine (PE) by granulocytes at rest. This richia coli, as judged by electron microscopy; (c) killing reaction is stimulated up to three-fold during engulfment of live Escherichia coli (10-fold); (d) 14C02 production of particles. from glucose-1-14C during phagocytosis by at least 80%; A series of observations has shown that levorphanol, (e) K' content of granulocytes (35%); (f) oxidation an analogue of morphine, exerts a profound inhibitory of and its into linoleate-l-C by 50%, incorporation effect on and on the of ribosomal triglyceride by more than 80%. However, levorphanol growth biosynthesis RNA in Escherichia More an ad- stimulated 2 to 3-fold the incorporation of linoleate-1-1'C coli, (5-7). recently, or palmitate-1-14C into several phospholipids. Glucose up- ditional effect of the drug on the bacterial membrane has take, lactate production, and adenosine triphosphate been suggested by the findings that levorphanol inter- (ATP) content are not affected by the drug. Thus, feres with the accumulation of various substances by levorphanol does not appear to exert its effects through E. coli (8, 9) and that levorphanol markedly alters the generalized metabolic suppression. labeling pattern of the phospholipids of this bacterial Removal of levorphanol by twice resuspending the species from radioactive precursors such as palmitate-1- granulocytes completely reverses all inhibition. 4C, glycerol-'4C, and 'Pt (unpublished observations). In line with observations on bacteria, it appears that Similar results have been reported for Staphylococcus the complex effects of levorphanol on granulocytes may aureus by Gale (10, 11). Changes in phospholipid metab- be due at least in part to an effect on the cell membrane. olism of brain tissue under the influence of narcotic drugs Dr. Wurster's present address is the Graduate School of have also been reported (12, 13). Nutrition, Cornell University, Ithaca, New York 14850. Dr. Elsbach and Dr. Simon are Career Scientists of the 1 Abbreviations used in this paper: LPC, lysolecithin; Health Research Council of the City of New York. LPE, lysophosphatidylethanolamine; PC, phosphatidylcho- Received for publication 12 June 1970 and in revised form line; PE, phosphatidylethanolamine; PMN, polymorphonu- 9 November 1970. clear; TEA, triethanolamine. The Journal of Clinical Investigation Volume 50 1971 1091 To explore further the possibility that levorphanol the fatty acid was washed before use according to the exerts an effect on mammalian cell membranes, we method of Dole and Meinertz (18) to remove any contami- chose polymorphonuclear These nating water-soluble radioactivity. In other experiments glu- leukocytes. cells can cose-1-YC' (260 ,Ci/mg) or glucose-6-"C6 (30 iCi/mg) engulf particles in vitro and thus can be examined while in ethanol were dissolved in Hanks' solution after evapora- engaged in a process during which the cellular mem- tion of the ethanol under nitrogen. branes undergo extensive changes. The results indicate Incubation mixtures in a total volume of 0.5 ml, contained that levorphanol indeed suppresses the acylation of lyso- 0.1 ml of the labeled albumin solution and granulocytes compounds, exerts a number of other metabolic (usually 5 X 107 cells) suspended in Hanks' solution. Other effects, additions, including the narcotic levorphanol,8 were as indi- and inhibits ingestion of particles and bacterial killing cated in the text or in the legends. by granulocytes. Phagocytosis in vitro was initiated by adding 0.1 ml of polystyrene latex particles' (1.1 a diameter, 3.6 X 109 par- METHODS ticles) suspended in physiological saline, or about 5 X 108 E. coli, in 0.1 ml of Hanks' solution, giving a 10: 1 ratio of Cells bacteria to granulocytes. In experiments involving killing of live bacteria, E. coli were treated with rabbit serum at a POLYMORPHONUCLEAR (PMN) LEUKOCYTES concentration of 10% by volume in Hanks' solution and PMN leukocytes were collected from sterile peritoneal incubated at 37'C for 20 min. The suspending medium was exudates produced in rabbits as described before (14). The removed by centrifugation, and the bacteria were resus- granulocytes, which comprised more than 90%o of the total pended in Hanks' solution alone. Unless stated otherwise, cell population, were resuspended in Hanks'2 solution plus drugs and bacteria were added at the same time. At ap- 4% bovine albumin3 to give a final concentration of from propriate intervals, the leukocyte suspension that contained 4 to 8 X 107 cells per 0.5 ml of complete incubation mixture. bacteria was thoroughly mixed, and a portion was trans- A cell number of 2 X 107 corresponds to approximately 1 mg ferred into cold TEA buffer. Suitable dilutions were made, of protein. and viable counts were determined by conventional techniques. Homogenates were prepared in 0.25 M sucrose in a glass Collection of '4COs. Suspensions of leukocytes were placed homogenizing tube, using a motor driven teflon pestle. in Erlenmeyer flasks containing polyethylene cups suspended Homogenization of granulocytes was carried out for at least from rubber stoppers.10 At 0 min., a portion of glucose-1-'C, three periods of 30 sec. glucose-6-"C. or linoleic acid-1-"C was added. As soon as Bacteria. A leucine auxotroph of E. coli B was grown the radioactivity was added, the flasks were stoppered and in minimal medium buffered with triethanolamine (TEA) incubated at 37'C. At the indicated time intervals, the reac- at a pH of about 7.9 as reported by Simon and Van Praag tion was stopped by injecting 0.2 ml of 10 N H2S04 through (6). Growth was followed by optical density measurements the rubber stopper into the main chamber. Hyamine was at 550 my in a Lumetron colorimeter and by viable cell added to the collection cups and incubation at 37°C was counts on nutrient agar plates. An OD of 1.0 is equivalent to continued for 1 hr. At this time, the cups that contained 3 x 108 bacteria. Stationary phase cultures were harvested hyamine were removed