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IMMUNOPHARMACOLOGY AND IMMUNOTOXICOLOGY, 14( 3), 515-521 (1992)

INTERACTION BETWEEN (-)NALOXONE AND MORPHINE IN MODIFYING SUPEROXIDE GENERATION FROM HUMANGRANULOCYTES G. Ricevuti, A. Mazzone, D. Pasotti, M. Marcoli, F. De Ponti, and G.M. Frigo

S.Lecchini,

Department of Internal Medicine and Therapeutics and Znd Faculty of Medicine, University of Pavia, Viale Luigi Borri 57, 1-21100 Varese, Italy ABSTRACT The effect of the opioid agonist morphine and of the (-) and (+) stereoisomers of the antagonist naloxone were studied on superoxide generation from human granulocytes. Morphine or naloxone had no effect on basal or phorbol 12my rist at e 13-ace t ate (PMA) -st imuI at ed supe rox ide g enerat ion. Cornbined equimolar (-)naloxone and morphine concentrations (0.1 pM - 0.1 pM) inhibited PMA-stimulated superoxide generation, while combined (+)naloxone and morphine had no effect. The stereospecific effect of naloxone suggests the involvement of opioid receptors. Thus, inhibition of superoxide generation by combined (-)naloxone and morphine could result from the unmasking of non opioid effects of morphine. It is suggested that the opioid control of oxidative metabolism in human granulocytes could involve multiple receptors mediating opposite effects. INTRODUCTION There is a large body of evidence that morphine and naturally occurring opioid peptides can affect granulocyte function (1, 2). However, the pattern of cell response to opioids and the role of specific opioid receptors is still largely unknown. While opioids are likely to modulate granulocyte motility, adherence and aggregation through the activation of specific receptors (2-5), there is a great deal of controversy regarding their ability to regulate a key cell function such as the oxidative metabolism. Morphine increased basal 02- generation (6), while inhibition of stimulated 02' generation, seemingly unrelated to specific

515

516

R I C E V U T I ET A L .

receptor occupancy, was reported following exposure to morphine or opioid antagonists (7). On the other hand, the naturally occurring opioid peptides can stimulate, reduce or leave unaffected basal or stimulated 0 2 - generation (6, 8Immunopharmacology and Immunotoxicology Downloaded from informahealthcare.com by University of Sydney on 12/31/14 For personal use only.

11) from human granulocytes. Recently no effect of opioid receptor agonists or antagonists on basal or stimulated respiratory burst was found in human granulocytes (12). In the present paper, the opioid regulation of oxidative metabolism in human granulocytes has been investigated by measuring the effect of morphine, and of the active (-) and the inactive (+) stereoisomers of the opioid antagonist naloxone on the basal and phorbol 12-myristate 13-acetate (PMA)l-stimulated 0 2 - generation. MATERIALS AND METHODS

n of Granulocv-

Granulocytes were prepared from heparinized (2

U/ml heparin) peripheral blood taken from twelve healthy male donors aged 2140 years (30.17 k 1.64, mean f s.e.m.), taking no medication and checked for haematological and serum immunoglobulin abnormalities as well as for plasma lipids, lipoproteins and serum electrophoresis. Samples were taken between 8.30 and 9.30 a.m. after a 12-hour fasting period. The subjects were invited to remain in bed for 30 min before blood samples were taken. Granulocytes were separated on a lymphoprep (Nyegaard C., Norway) density gradient, according to Boyum method (13). The granulocyte fraction was obtained by sedimentation of the pellet in a 1.5 (w/v) dextran (mol. wt. 177,000) phosphate-buffered saline (PBS) solution. Contaminating erythrocytes were removed by gentle shaking for 10 min at 37" C in ammonium chloride solution containing 20 mM tris-HCI buffer (final pH 7.4) and 0.25% autologous plasma. This preparation led to 9599% pure granulocytes. Cell viability was tested by trypan blue exclusion and

was greater than 96%.

--

Generat ion, Generation of 0 2 - radicals was measured as

superoxide dismutase-inhibitable reduction of ferricytochrome C type VI according to Babior method (14). Reduced ferricytochrome C was determined by using a Perkin-Elmer spectrophotometer at wavelengths of 550 nm and 468 nm. The production of 0 2 - (nmol/107 cells per 15 min) was obtained by the

GRANULOCYTE SUPEROXIDE GENERATION following equation: A(550

-

517

468) - B (550 - 468) where A indicates the

absorbance values in the absence and B in the presence of superoxide dismutase,

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multiplied by a correction factor - (2.510.0245) related to the final volume and to the coefficient of extinction of iron. In the stimulated 02' generation test, oxidative burst activation was obtained by a 15-min exposure of the cells to PMA (1.6 pM ). Each experiment was carried out in triplicate. Samples obtained from the same subjects for 6-7 consecutive days gave a variation of less than 10% for the tests performed.

-2-

Generat i o a Concentration-effect relationships of

morphine and of the (-) and (+) stereoisomers of naloxone in modifying granulocyte basal and PMA-stimulated 0 2 - generation were studied by adding the drugs at various concentrations to the cells 20 min before processing the cells for 0 2 - generation. When the ability of (-) or (+) naloxone to modify the effect of morphine was tested, cells were preincubated for a further 20 min with the naloxone stereoisomer before adding morphine. In separate experiments designed

to test whether the incubation time was responsible for the effect of (-)naloxone when added together with morphine, (-)naloxone alone was added 40 min before processing the cells for 0 2 - generation. Drug effects were measured as percentage variations of basal or stimulated 0 2 - generation with respect to the control values. D u a s and ChernicalL Ferricytochrome C type VI, (-)naloxone, phorbol 12myristate 13-acetate, superoxide dismutase were purchased from Sigma Chemicals (USA) and morphine hydrochloride from SIFAC (Italy). (+) Naloxone was a generous gift from dr. A. North (Oregon Health Science University , USA). &&tical

A r d g *& Significance of the difference between means was evaluated

by applying the Student's t-test for unpaired data. RESULTS Basal and PMA-stimulated 0 2 - generation were (mean f s.e.m., n=12) 1.97

_+

0.25

nmol 02-/ l o 7 cells per 15 min and

88.37 f 2.85 nmol 0 2 -

/ l o 7 cells per 15 min, respectively. Morphine and (+) or (-)naloxone per se

518

RICEVUTI ET AL. TABLE 1.

Effect of Morphine and of (-) or (+)Naloxone on Basal and PMA-Stimulated 0 2 -

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Generation from Human Granulocytes. PMA-Stimulated 0 2 - a

Basal 02- a Control

1.97 k 0.25b (12)c

88.37 k 2.85 (12)

Morphine (0.1 pM)

1.89 k 0.43 (9)

86.72 k 3.44 (9)

(-)Naloxone (0.1 pM )

2.02 k 0.41 (9)

89.00 k 4.31 (9)

(+)Naloxone (0.1 pM)

1.98 k 0.32 (9)

87.52 k 5.19 (9)

(-)Naloxone (0.1 pM)

1.87 k 0.40 (12)

63.18 k 3.01 (12) d

2.01 f 0.31 (9)

81.20 k 5.81 (9)

and Morphine (0.1 pM) (+)Naloxone (0.1 pM) and Morphine (0.1 pM)

a nrnol/lO7 cells per 15 min. b Means k s.e.rn.

C

Number of experiments in

triplicate. d p c 0.05 vs control.

were ineffective on basal

or PMA-stimulated 0 2 - generation up to the

concentration of 0.1 pM (Table 1). Even a 40-rnin incubation with (-)naloxone alone did not affect 02- generation (data not shown). A significant inhibition of PMA-stimulated 0 2 - generation was found when

cells were incubated with combined equirnolar (-)naloxone and morphine in the 0.1 pM - 0.1 pM concentration range (Table 1 and Figure 1). The effect of

combined morphine and (-)naloxone at equimolar concentrations was not concentration-dependent in the 0.1 pM - 0.1 pM concentration range (Figure 1). However, a concentration-dependent response was observed when varying

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GRANULOCYTE SUPEROXIDE GENERATION

z

*

F2 20

L

?1 I

519

E

Figure 1. Concentration-response relationships of combined morphine and (-)naloxone in modifying PMA (1.6 pM)-stimulated 02- generation from human granulocytes. Percentage inhibitions of PMA-stimulated 02-generation from cells preincubated with combined equimolar increasing concentrations of (-)naloxone and morphine ( ) or with (-)naloxone increasing concentrations with a fixed concentration (0.1 pM) of morphine (A) are shown. Each point represents the mean (+s.e.m., n=9 form, n=3 for A). * = p < 0.05 vs control.

n

(-)naloxone concentrations were added to a fixed morphine concentration (0.1 pM) (Figure 1). (+)Naloxone was devoid of any effect when added to an equimolar morphine concentration (Table 1).

We were unable to find any effect of morphine or naloxone on basal or PMAstimulated 0 2 - generation, confirming the report by Seifert et at. (12). Unexpectedly, adding increasing concentrations of (-)naloxone to a fixed concentration of morphine revealed a concentration-dependent decrease of PMAstimulated 0 2- generation. Decrease of stimulated 02- generation by combined morphine and (-)naloxone cannot be easily explained. However, blockade of specific opioid receptors by (-)naloxone might be involved, since the effect of

520

RICEVUTI E T AL.

naloxone was stereospecific. If the inability of morphine to alter 02- generation were due to opposite effects following the activation of different receptors,

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blockade of

opiate

receptors by (-)naloxone

could

unmask an effect of

morphine at non-opiate sites. As a matter of fact, opioid peptides such as pendorphin and y-endorphin have been shown to inhibit PMA-stimulated 02generation by interacting with non-opiate receptors (10). Interestingly, morphine1 was proven to impair leukotriene 84 (LTB4) production from granulocytes in a naloxone-insensitive way (15). LTB4 synthesis seems to play a role in granulocyte activation (16) and its inhibition could be involved in the hereby reported effect of morphine. The hypothesis that morphine may interact with different opioid receptor subtypes to modify 02- is only speculative. Unfortunately, no data are available on the effects of selective agonists for opioid receptor subtypes on 0 2 generation. In conclusion, specific opioid receptors might be involved in the control of

02-generation from human granulocytes, as suggested by the stereospecific effect of (-)naloxone. Understanding the mechanism(s) of regulation by opioids of oxidative metabolism requires further study. However, our finding could help to explain discrepancies in the literature suggesting that the opioid end effects on 0 2 - generation probably result from a complex interaction of different opioid

and possibly non opioid-mediated effects.

Abbreviations used: PMA, phorbol 12-myristate 13-acetate; LTB4, leukotriene B4. REFERENCES

E.G.and Falke, N.E., in Enkephalins and Endorphins - Stress and the Immune System., edited by N.F. Plotnikoff, A.E. Faith, A.J. Murgo and R.A. Good, p. 263, Plenum, New York, 1986.

1 ) Fisher,

2 ) Van Epps, D.E. and Kutvirt, S.L., Modulation of human neutrophil adherence by P-endorphin and met-enkephalin, J. Neuroimmunol., 15:219, 1987.

3 ) Simpkins, B.M., Dickey, C.A., and Fink, M.P., Human neutrophil migration is enhanced by beta-endorphin, Life Sci., 34:2251, 1984.

GRANULOCYTE SUPEROXIDE GENERATION

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4 ) Marcoli, M., Ricevuti, G., Mazzone, A., Bekkering, M., Lecchini, S., and Frigo, G.M., Opioid-induced modification of granulocyte function, Int. J. Immunopharmacol., 10:425, 1988. 5 ) Marcoli, M., Ricevuti, G., Mazzone, A., Pasotti D., Lecchini, S., and Frigo, G.M., A stereoselective blockade by naloxone of opioid and non-opioid induced granulocyte activation, Int. J. Immunopharmacol., 11:57, 1989.

6 ) Sharp, B.M., Keane, W.F., Gekker, G., Tsukayama, D., and Peterson, P.K., Opioid peptides rapidly stimulate superoxide production by human polymorphonuclear leukocytes and macrophages, Endocrinology 117:793, 1985. 7 ) Simpkins, C.O., Ives, N., Tate, E., and Johnson, M., Naloxone inhibits superoxide release from neutrophils, Life Sci., 37:1381, 1985. 8 ) Sharp, C.O., Keane, W.F., Gekker, G., Tsukayama, D., and Peterson, P.K. , pendorphin stimulates human polymorphonuclear leukocyte superoxide production via stereo- selective opiate receptors, J. Immunol., 242:579, 1985. 9 ) Nagy, J.T., Foris, G., Fulop, T., Paragh, G., and Plotnikoff, N.P., Activation of the lipoxygenase pathway in the methionine enkephalin induced respiratory burst in human polymorphonuclear leukocytes, Life Sci., 42:2299, 1988. 10) Diamant, M., Henricks, P.A.J., Nijkamp, F.P., and de Wied, D., Endorphin and related peptides suppress phorbol myristate acetate-induced respiratory burst in human polymorphonuclear leukocytes, Life Sci., 4 9 1 537, 1989. 11) Marotti, T., Sverko, V., and Hrsak, I., Modulation of superoxide anion release from human polymorphonuclear cells by met- and leu-enkephalin, Brain Behav. Immunol., 4:13, 1990. 12) Seifert, R., Burde, R., and Schultz, G., Lack of effect of opioid peptides, morphine and naloxone on superoxide formation in human neutrophils and HL-60 leukemic cells, Naunyn-Schmiedeberg's Arch. Pharmacol., 340:101, 1989. 13) Boyum, A., Isolation of mononuclear cells and granulocytes from human blood, Scand. J. Clin. Lab. Invest., 21 (Suppl 97):77, 1968. 14) Babior, B.M., Oxygen-dependent microbial killing by phagocytes, N. Engl. J. Med., 298:659, 1978. 15) Mazzone, A., Ricevuti, G., Pasotti, D., Fioravanti, A., Marcoli, M., Lecchini, S., Notario, A., and Frigo G.M., Peptide opioids and morphine effects on inflammatory process, Inflammation, 14:717, 1990. 16) Weissmann, G., Serhan, C., Korchak, M., and Smolen, J.E., Neutrophils: release of mediators of inflammation with special reference to rheumatoid arthritis, Ann. N. Y. Acad. Sci., 389:11, 1982.

Interaction between (-)naloxone and morphine in modifying superoxide generation from human granulocytes.

The effect of the opioid agonist morphine and of the (-) and (+) stereoisomers of the antagonist naloxone were studied on superoxide generation from h...
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