/ . Biochem., 77, 567-573 (1975)

Effect of Cyclic Nucleotides on the Cyanide-insensitive Respiration of Polymorphonuclear Leucocytes1 Akira NAKAGAWARA, Koichiro TAKESHIGE, and Shigeki MINAKAMI Department of Biochemistry, Kyushu University School of Medicine, Higashi-ku, Fukuoka, Fukuoka 812 Received for publication, August 17, 1974

The effects of N6,0!'-dibutyryladenosine 3', 5'-monophosphate (Buj-cyclic AMP) and N ! , O!'-dibutyrylguanosine 3', 5'-monophosphate (Bui-cyclic GMP) on the cyanideinsensitive respiration of guinea pig peritoneal exudate polymorphonuclear leucocytes were studied. Buj-cyclic AMP inhibited the respiration induced both by phagocytosis of E. coli and by the interaction with trypsin-digested rat liver microsomes. The addition of theophylline gave rise to an inhibitory pattern similar to that with Bur cyclic AMP against both the respirations induced. On the other hand, Bui-cyclic GMP did not affect the respiration induced by phagocytosis whereas it inhibited the respiration induced by trypsin-digested microsomes. The cyanide-insensitive respiration induced by the addition of myristic acid was inhibited by Bui-cyclic AMP, which was similar to that with E. coli. The respiration induced by methylene blue was inhibited neither by Buj-cyclic AMP nor by Bus-cyclic GMP. These observations suggest that the cyanide-insensitive respiration of polymorphonuclear leucocytes may be classified into at least three types from the inhibitory pattern of cyclic AMP and cyclic GMP.

T h e involvement of cyclic nucleotides in the phagocytotic reaction of polymorphonuclear leucocytes has recently been reported by several investigators. The increase in the intra•cellular level of cyclic AMP induced by the •addition of N8, C-dibutyryladenosine 3', 5'monophosphate (Buj-cyclic AMP), theophylline •or prostaglandin Ei has been shown to depress the ingestion of particles into the cells and the formation of u COi from [l-uC]glucose (7, 1

This study was supported in part by the Yamanouchi Foundation of Metabolism and Diseases and a Research Grant from the Ministry of Education. "Vol. 77, No. 3, 1975

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2). Zurier el al. {3) have indicated that the increase of intracellular cyclic AMP inhibits the release of lysosomal enzymes during zymosan particle ingestion by cytochalasin B treated cells but cyclic GMP stimulates enzyme release. An important metabolic characteristic of the leucocytes is the appearance of cyanideinsensitive respiration during phagocytosis (4, 5). In a previous communication (6), we reported that trypsin-digested microsomes induced cyanide-insensitive respiration without being ingested into the cells. In connection with the mechanism of the metabolic change,

A. NAKAGAWARA, K. TAKESHIGE, and S. MINAKAMI

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we are interested in two points: 1) Are the effects of cyclic AMP and cyclic GMP different, as reported in other tissues, and 2) Do the digested microsomes induce cyanide-insensitive respiration by a similar mechanism to that of the phagocytotic reaction. The cyanideinsensitive respiration induced either by phagocytosis of E. coli or by trypsin-digested microsomes was inhibited by cyclic AMP, though only the latter was inhibited by cyclic GMP. EXPERIMENTAL PROCEDURE Procedures to obtain the leucocyte preparation, the trypsin-digested microsomes and the measurement of oxygen uptake were as described in a previous publication (6), except that the final volume for oxygen measurement was 2.0 ml. 5'AMP, cyclic AMP, Buj-cyclic AMP, and N1, O2'-dibutyrylguanosine 3', 5'-monophosphate (Bu2-cyclic GMP) were obtained from Bohringer u. Sohne, Mannheim, Germany. RESULTS Effect of Nucleotides on the Cyanide-insensitive Respiration Induced by Trypsin-digested Microsomes — When trypsin-digested microsomes were added to a leucocyte suspension in the presence of glucose and cyanide, a biphasic respiratory pattern was observed: rapid oxygen uptake continued for about 2 min, followed by slow respiration as described in a previous paper (6). The initial rapid respiration was inhibited by the addition of cyclic AMP, though the inhibition was slight (about 15— 20%) when 1.5 mM cyclic AMP was added 3 min before the addition of the microsomes. Other nucleotides, namely ATP, ADP, and 5'AMP, did not inhibit respiration under these conditions.2 The inhibition by cyclic AMP was more noticeable if the cells were preincubated with the cyclic nucleotide for a longer time-interval. The respiratory rate became about 60% of the control rate when the cells were preincubated for 40 min (Fig. IB). Both the initial rate and the amount of oxygen-up' Addition of adenosine depressed respiration slightly (about 15%).

9J>jM(Vmln

B min

0,-0

Fig. LA. Effects of cyclic AMP(cAMP) and 5'-AMP on cyanide-insensitive respiration induced by trypsindigested microsomes (Try-Ms). Oxygen uptake was measured by a Clark-type oxygen electrode at 37° in the presence of glucose and cyanide. Leucocytes (5 x 107 cells) were suspended in 2 ml of modified Krebs Ringer phosphate solution (CaClj: 0.9 mM) and preincubated with 1.5 mM cyclic AMP or 5'-AMP for 3 min before the addition of trypaindigested microsomes (0.66 mg as protein). The dotted lines represent control experiments. The initial respiratory rates induced by trypsin-digested microsomes are indicated. Try-Ms O.Amg

I

3 min

Fig. IB. Effect of long preincubation with cyclic AMP on cyanide-insensitive respiration. Leucocytes (5 x 107 cells) were incubated in 2.0 ml of modified Krebs Ringer phosphate solution containing 1.5 mM cyclic AMP for 40 min at 37°. After centrifugation at 80 x (7 for 6 min, the precipitated cells were resuspended in a freshly prepared solution containing 1.5 mM cyclic AMP. Oxygen consumption was measured as stated in the legend to Fig. 1A. A control experiment without the addition of cyclic AMP is shown as a dotted line.

take due to the initial rapid respiration were decreased. Since the above results indicate the pos/ . Biochem.

CYCLIC NUCLEOTIDES ON LEUCOCYTES

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Bui-cGMP E.COliOimg

2.25 mM

-») 5 mbi (*• 0j«0

Fig. 1C. Effect of Bus-cyclic AMP and Bu,-cyclic GMP on respiration. Conditions were the same as stated in the legend to Fig. 1A.

sibility that the inhibition is exerted by the cyclic AMP inside the cells and that the penetration rate of the nucleotide is limiting the inhibition, Bu2-cyclic AMP, a membrane permeable derivative of the nucleotide, was used; As shown in Fig. 1C, Buj-cyclic AMP inhibited the cyanide-insensitive respiration: both the initial rate after addition of the microsomes and the amount of oxygen uptake due to the first initial phase decreased. The inhibition was about 50% with a preincubation time of 3 min. When another dibutyryl cyclic nucleotide, Bui-cyclic GMP, was added to the cells, the inhibition was more noticeable even with a short preincubation period (3 min): at a concentration of 1.5 mM, the nucleotide almost completely inhibited the initial cyanide-insensitive respiration induced by the digested microsomes (Fig. 1C). Titration by Bui-cyclic AMP— Leucocytes were preincubated with various concentrations of Bu2-cyclic AMP for 40 min and then trypsfn [EC 3.4.21.4]-digested microsomes were added. When the initial respiratory burst was over, heat-killed E. coli was added to measure the oxygen uptake due to phagocytosis, as shown in Fig. 2A. Cyanide-insensitive respiration induced both by trypsin-digested microsomes and by phagocytosis was inhibited by preincubation with the cyclic nucleotide. The degrees of inhibition were essentially parallel for both inducers: this can be shown by the titration curves of the respirations induced by Vol. 77, No. 3, 1975

Oj«0

Fig. 2A. Effect of But-cyclic AMP on the cyanideinsensitive respiration induced by trypsin-digested microsomes and E. coli. Cells (5xlO7) were preincubated for 40 min with various concentrations of Bui-cyclic AMP as specified on the right-hand side of the figure. The procedures for preincubation and resuspension were as indicated in the legend for Fig. IB. The oxygen uptake was measured as stated in the legend to Fig. 1A.

Bu,-cAMP ( mM Fig. 2B. Titration of the cyanide-insensitive respiration induced either by trypsin-digested microsomes or by E. coli with Bu2-cyclic AMP. After the leucocytes (5xlO 7 cells) were preincubated with the nucleotide for 3 min, as in the legend to Fig. 1A, either E. coli (0.6 mg dry weight) or trypsin-digested microsomes (0.66 mg protein) was added. The initial linear respiratory rate after induction was measured and the inhibition was expressed as a percent of the control. O, E. coli; • , trypsindigested microsomes.

heat-killed E. coli and the digested microsomes, to which Bui-cyclic AMP was added 3 min before the addition of the inducers (Fig. 2B). Titration with Theophylline—ln order to verify the effect of cyclic AMP on cyanide-

A. NAKAGAWARA, K. TAKESHIGE, and S. MINAKAMI

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c

150

c

1 120

o 4.0 mM

z3 k >.

90

.

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60

'o

O

>

—| 5 mln

30 0 0

0.5

1.0

1.5

Try-Ms ( mg protein)

Fig. 3A. Effect of theophylline on the cyanideinsensitive respiration induced by either trypsindigested microsomes or by E. coli. The cells were suspended in a modified Krebs Ringer phosphate solution at 37° containing various concentrations of theophylline, as specified on the right-hand side of the figure. After about 6min, trypsin-digested microsomes (0.5 mg) were added to the suspension and then E. coli was added. The conditions for the measurement of Oj-uptake were as stated in the legend to Fig. 1A.

Fig. 3C. Dose-dependent increase of the initial respiratory rate induced by trypsin-digested microsomes and its inhibition by theophylline. The experimental conditions were the same as stated in the legend to Fig. 3A, except that the amount of trypsin-digested microsomes varied. Concentrations of theophylline, • , 3.0 mM; • , 1.5 mM, • , control without theophylline. Bu,-c6MP

E.COdOSmg

KCNlrtl

Try-Ms Sjmg _L

to

2.0

3.0

4.0

R E.COli OBmg

5.0

Th*ophyllin« ( mM )

Fig. 3B. Titration of the respirations with theophylline. The initial linear respiratory rates were measured from the oxygen electrode traces, carried out as stated in the legend to Fig. 3A. O, E. coli; • , trypsin-digested microsomes.

insensitive respiration, theophylline, which is known to increase intracellular cyclic AMP by inhibiting the phosphodiesterase, was used. Leucocytes were suspended in a medium containing various concentrations of theophylline

Effect of cyclic nucleotides on the cyanide-insensitive respiration of polymorphonuclear leucocytes.

/ . Biochem., 77, 567-573 (1975) Effect of Cyclic Nucleotides on the Cyanide-insensitive Respiration of Polymorphonuclear Leucocytes1 Akira NAKAGAWAR...
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