Biochemical Pharmacology,

Vol. 26. pp. 1489-1493. Pergamon Press. 1977. Printed in Great Britain

STUDIES

ON PHAGOCYTOSIS-III.

TRICARBOXYLIC ACID CYCLE AND THE CYTOCHROME SYSTEM AS ENERGY SOURCES FOR PHAGOCYTOSIS IN RABBIT PERITONEAL EXUDATE POLYMORPHONUCLEAR LEUKOCYTES

Department of Medicine/Pharmacology,

YI-HAN CHANG UCLA School of Medicine, Los Angeles, CA 90024, U.S.A.

(Received 16 August 1976; accepted 29 October 1976)

Abstract-The phagocytosis of starch granules by rabbit peritoneal exudate polymorphonuclear leukocytes measured by the uptake of ‘311-labeled human serum albumin was found to be suppressed by anaerobiosis, iodoacetate, malonate, cyanide and ATPase inhibitors. The suppressive effect of iodoacetate can be partially relieved by pyruvate. The results suggest that the tricarboxylic acid cycle and the cytochrome system play important roles in supplying energy for the phagocytic process.

It has been well established that phagocytosis by polymorphonuclear leukocytes (PMN) is accompanied by increases in respiration, glycolysis, and the hexose monophosphate shunt (HMP) activity [l-9]. However, the relationship between these metabolic changes and the phagocytic process per se is not yet clearly understood. Glycolysis is commonly regarded as the metabolic pathway in polymorphonuclear leukocytes that provides energy for the phagocytic process, and the oxidative metabolism via the tricarboxylit acid (TCA) cycle is believed to be of minor importance in these cells [2,4, 10,il). This view has been questioned recently by Patriarca et al. [12], who made the observation that PMN phagocytosis in vitro is not influenced by glucose, but lactate production in PMN is higher in the presence of glucose than in its absence. It has also been reported that mild degrees of physical injury augment glycolysis in PMN and cause a decrease in respiration [13,14]. The role of the tricarboxylic acid cycle and of the cytochrome system in supplying energy for PMN phagocytosis was re-investigated employing a recently developed quantitative and sensitive method for measuring phagocytosis which forms the subject of this report.

MATERIALS AND METHODS Rabbits. New Zealand albino rabbits were used to provide PMN. PMN. Rabbit peritoneal leukocytes were obtained according to the procedure of Cohn and Morse [lS]. The cells were sedimented at 1000 rev/min (125 g) for 15 min in a Sorvall centrifuge at 4”. After removing the supematant, the cells were gently resuspended in Krebs-Ringer phosphate buffer to a concentration of 5 x 10’ cells/ml. The final leukocyte suspension was chilled in ice and used within 20 min. Repeated observations on stained smears showed that better than 98 per cent of the leukocytes were polymorphonuclear. All exudates employed were essentially free of red blood cells. In any given experiment, only cells from a single animal were used.

Starch. The starch used was isolated from the seed of Saponaria vaccaria or Montana “cow soapwart” [16]. It is made up of extremely small granules with diameters distributed between 0.5 and 1.6 m. Bu@. Krebs-Ringer phosphate buffer, pH 7.4, containing one-half of the usual amount of CaCl,, was filtered through a treated Millipore filter [17] (Millipore Filter Corp., Bedford, MA) and used within 3 days of preparation. Chemicals. Radio-iodinated human serum albumin ([I 3’ I-JHSA), (65-90 @i/mg) was purchased from Abbott Laboratories (North Chicago, IL). [14C-l]glucase (16.3 &i/mg) was purchased from the NuclearChicago Instrument and Chemical Co. Measurement of phagocytosis. The degree of phagocytosis was determined by measuring the uptake of [1311]HSA as previously described [18]. Briefly, a suspension of PMN, rabbit serum and drug solution or buffer was placed in a 25-ml serum vial. The mixture was incubated with shaking at 37” for 30min. A suspension of starch, containing 1311-labeled albumin and glucose, was .then added to the serum vial. The incubation was resumed for an additional 30 min and then terminated by the addition of 2.0ml of icecold sodium ethylenediamine tetra-acetic acid (NaEDTA) and the serum vial was then placed immediately on ice. The contents were transferred to a precooled centrifuge tube. After centrifugation, the supematant was discarded and the pellet was washed three times with NaEDTA saline. The radioactivity associated with the final pellet was determined by liquid scintillation after treatment with hydroxide of hyamine. In any given experiment, only cells from a single animal were used. Measurement of [‘4C-l]-glucose utilization. [‘4C-l]glucose utilization by PMN was determined according to a previously reported procedure [ 181. RESULTS Effect of ATPase inhibitors on phagocytosis of starch granules by PMN. Each experiment was performed

five times. Chlorpromazine

14,89

and ouabain

suppressed

YI-HAN CHANC

1490

Table I. Effect of ATPase inhibitors on the [13’IJHSA uptake by rabbit peritoneal exudate PMN during phagocytosis Compound

Concn (M)

Radioactivity* @pm)

Control Chlorpromazine Quinine Ouabain

0 1 x 10-4 1 x 1o-4 1 x 1o-4

9829 f 126 3884 + 151 6032 rl: 201 8061&- 69

Pt

Studies on phagocytosis--III. Tricarboxylic acid cycle and the cytochrome system as energy sources for phagocytosis in rabbit peritoneal exudate polymorphonuclear leukocytes.

Biochemical Pharmacology, Vol. 26. pp. 1489-1493. Pergamon Press. 1977. Printed in Great Britain STUDIES ON PHAGOCYTOSIS-III. TRICARBOXYLIC ACID C...
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