- Email: [email protected]
The effect of methionine-enkephalin on nitric oxide release in mice is age and gender related
Pharmacological Research, Vol. 44, No. 4, 2001 doi:10.1006/phrs.2001.0866, available online at http://www.idealibrary.com on
THE EFFECT OF METHIONINE-ENKEPHALIN ON NITRIC OXIDE RELEASE IN MICE IS AGE AND GENDER RELATED ˇ ˇ TIHOMIR BALOG∗ , T. MAROTTI, V. MUSANI, S. SOBOCANEC and V. SVERKO Department of Molecular Medicine, Rud−er Boˇskovi´c Institute, 10000 Zagreb, Croatia Accepted 17 July 2001
Gender- and age-related differences in nitric oxide (NO) release and in response to drugs of abuse has been reported in both humans and experimental animals. So far, we have demonstrated in vivo methionine-enkephalin- (MENK-) modulated NO release in mice. However, no data on the influence of age and gender on this immunomodulatory effect of MENK have been reported. In this study we examined the influence of age (2, 4, 8 month old mice) and gender (male and female mice) on MENK-induced NO release of mouse peritoneal macrophages (PEMs) of the CBA strain of mice. NO release was not age but was gender related in that males generally produced more NO than females. The effect of MENK on NO release was age (demonstrated only in mature 4 and 8 month old mice) and gender related in that it could be observed only in female mice. Apoptotic cells that paralleled the increase of NO in MENK-treated female mice were, however, observed also in male mice although MENK was in males without effect. These data provide evidence that some immunomodulatory properties of MENK are age and gender related which may be relevant to the potential use of MENK in adjuvant therapy for c 2001 Academic Press immunocompromised status.
K EY WORDS : nitric oxide, methionine-enkephalin, gender, age.
INTRODUCTION Experimental studies have indicated that reactive oxygen species are the primary mediators in the pathogenesis of immune and non-immune injuries. Among oxygen species, NO over-production by inducible nitric oxide synthase (iNOS) occurs under pathophysiological conditions of chronic infection, inflammation and in septic patients. NOS activity is changed in diverse pathophysiological conditions including migraine headache, hypertrophic pyloric stenosis and male impotence. NO appears to play the major role in pathophysiology of stroke, Parkinson’s disease, Huntington’s disease and amyotrophic lateral sclerosis [1]. By the reaction of NO with superoxide − anion (O− 2 ), peroxynitrite (ONOO ), which represents a major pathway of NO-derived reactivity and toxicity, can be generated. Literature data of MENK-stimulated generation of biologically active oxygen species of rat peritoneal macrophages existed already in 1984 [2]. We ∗ Corresponding author. Department of Molecular Medicine, Rud −er Boˇskovi´c Institute, Bijeniˇcka c. 54, 10000 Zagreb, Croatia. E-mail: [email protected]
1043–6618/01/100287–06/$35.00/0
have shown [3] that MENK modulates O− 2 release from human neutrophils. Our recent work showed MENKmodulated NO release [4] from peritoneal macrophages of 4 month old CBA strain mice. However, the data about the effect of age on NO release in the literature are controversial. While some authors [5, 6] claimed to have age-dependent progressive and marked decline in NO production of mice or rat peritoneal macrophages, others [7] demonstrated that macrophages from young and old mice secreted similar levels of NO. Another point of interest is the observed difference in gender-related NO release of murine peritoneal macrophages [8] and exhaled NO and plasma nitrate in humans [9]. Such differences have been observed also in response to opioid peptides or psychostimulants [10, 11]. Recently, a gender-related difference in murine T- and B-lymphocyte proliferative ability in response to in vivo MENK administration was found [12]. The aim of the present study was to assess the possible influence of age and gender on MENK-induced modulation of NO release in mice and to correlate the effect with the induction of apoptosis. This was done by comparing the effect of MENK on the release of NO from c 2001 Academic Press
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peritoneal macrophages from 2, 4 or 8 month old male and female CBA strain mice.
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examined under a fluorescent microscope. They were counted as apoptotic if chromatin was condensed, nuclei fragmented and cells shrank. At least 300 cells were counted per sample.
MATERIALS AND METHODS
Analysis of DNA fragmentation in agarose gels Animals Male and female CBA/HZgr mice, aged 2, 4 or 8 months, raised in the breeding colony of the Rud−er Boˇskovi´c Institute, Zagreb, Croatia, were used. They were kept five to a cage, fed a standard diet and given water ad libitum. Mice of different age and of both sexes were divided into mice receiving i.p. phosphate buffered saline (PBS) (control) or 2.5, 5.0 or 10.0 mg kg−1 bw of MENK (Sigma, St Louis, USA). Sample preparations were performed 18 h after the injection of MENK.
Preparation of samples At a given time after drug injection (18 hours) the animals were killed by cervical dislocation. Peritoneal macrophages (PEM) were aseptically collected from the peritoneal cavities of mice 5 days after i.p. injection of 0.12% glycogen (Kemika, Zagreb, Croatia). Macrophages were resuspended in RPMI 1640 (without phenol red; Sigma, St Louis, USA) and red cells were removed by NH4 Cl lysis. The remaining cells were washed three times, resuspended in RPMI 1640 with antibiotics and 10% fetal calf serum (FCS; Sigma St Louis, USA) and adjusted to 2 × 106 ml−1 . This procedure typically yielded preparations containing more than 95% of viable peritoneal macrophages, as tested by trypan blue.
Measurement of nitrite production The measurement of NO was assayed by the colorimetric reaction of the Griess reagent [13]. Briefly, isolated PEMs were adjusted to 2 × 106 /well and (in duplicate) incubated in plastic 24-well flat-bottom microplates (Falcon, USA) for 48 h at 37 ◦ C and 5% CO2 . Aliquots (800 µl) of each supernatant were placed in tubes and mixed with 800 µl of GRIESS reagent (1% sulphanilamide in 2.5% phosphoric acid and 0.5% naphthylethylenediamine in 2.5% phosphoric acid, 1 : 1). The resulting colorimetric reaction was measured at 540 nm with a spectrophotometer. NO concentration was calculated from a standard curve using sodium nitrite (0–100 µM) as standard and was expressed as nmol/2 × 106 cells/well.
Fluorometric viability assay Fluorometric viability assay of macrophages from mice injected in vivo with PBS (control) or 2.5 and 10.0 mg kg−1 bw of MENK was performed according to the method of London et al. [14]. Briefly, the kinetics of apoptosis was followed in control macrophages and macrophages of MENK-treated mice 18 h after treatment. Macrophages (10 µl) were mixed with 4 µl of 1% propidium iodide in water and 3 µl of 1% fluorescein diacetate in acetone in 1 ml of PBS. The cells were
3 × 106 adherent cells were incubated for 10 min in 500 µl of lysis buffer (20 mM Tris-HCl pH = 7.4, 10 mM EDTA, 0.2% Triton X-100) and centrifuged at 10 000 g for 10 min. The supernatant was incubated overnight at 50 ◦ C with 100 µg ml−1 proteinase K. The DNA was extracted with 1 vol of chloroform/phenol (1 : 1). The DNA was precipitated from the aqueous phase with 1 vol of isopropanol, 500 mM NaCl at −20 ◦ C overnight and collected by centrifugation. Pellets were air dried and incubated in 10 µl of 10 mM Tris pH = 7.5 and 1 mM EDTA for 1 h at 37 ◦ C with 1 µg ml−1 RNAse A. Horizontal electrophoresis was performed for 1 h at 80 V in 1% agarose gel containing 1 µg ml−1 ethidium bromide with 90 mM Tris, 90 mM boric acid and 2 mM EDTA, pH = 8.0, as running buffer. Samples were loaded after suspension in 1 µl of loading buffer (10 mM Tris, 1 mM EDTA) and heating at 65 ◦ C for 10 min. DNA was visualized in UV light.
Statistical analysis Data are presented as mean ± standard deviation. The significance of the differences between groups was assessed by Student’s t-test. P-values below 0.05 were considered significant.
RESULTS
Nitric oxide release and apoptosis—the effect of various MENK doses NO release of macrophage from peritoneal cavity of female mice injected with 2.5, 5.0 or 10.0 mg kg−1 bw of MENK is presented in Fig. 1. In the corresponding groups some mice received 10.0 mg kg−1 bw naloxone 30 minutes before the injection of MENK. Control macrophages were obtained from mice receiving either Hanks balanced salt solution (HBSS) or naloxone (10.0 mg kg−1 bw). MENK modulated NO release in a concentration-dependent manner. It increased NO release at 2.5 mg and 10.0 mg kg−1 bw (P = 0.001 or P = 0.03, respectively) and decreased NO release at 5.0 mg kg−1 bw (P < 0.05). While naloxone per se had no effect and was ineffective in abrogating the suppressive effect of 5.0 mg kg−1 bw of MENK, it efficiently blocked the stimulatory effect of MENK at doses of 2.5 and 10.0 mg kg−1 bw. Apoptotic macrophages were examined under fluorescent microscope in cultures of macrophages of control mice or mice treated in vivo with 2.5 or 10.0 mg kg−1 bw of MENK (5.0 mg kg−1 bw was not examined since it was not opioid receptor mediated). As demonstrated in Fig. 2, only 2.5 mg kg−1 bw MENK induced apoptosis
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Fig. 1. NO release of PEM of 4 month old female mice injected with 2.5, 5.0 or 10.0 mg kg−1 bw of MENK or/and naloxone (NAL) (10 mg kg−1 bw). Control mice were injected with HBSS. Data are expressed as mean ± SD of six animals per group; the experiments were repeated two or three times. **P = 0.001, *P = 0.03, NP < 0.05. ( Control, 2 MENK, 2 NAL, 2 MENK + NAL.)
visualized by condensed chromatin, nuclei fragmentation and shrunk cells. We have chosen to examine the involvement of age and gender in MENK-modulated NO release of a single dose of 2.5 mg kg−1 bw, since 5.0 mg kg−1 bw of MENK was not opioid receptor mediated and the 10.0 mg kg−1 bw of MENK did not induce apoptosis. To test whether the effect of MENK was age and/or gender related, we injected 2, 4 and 8 month old male and female mice with 2.5 mg kg−1 bw of MENK and the respective control mice with HBSS.
NO release in controls—the effect of age and gender The results of NO release in control mice of different age and gender are presented in Fig. 3. NO release in control HBSS injected mice was not age related either in male or in female mice. However, NO release of control mice was gender related since it was significantly higher in 4 month (P < 0.05) and 8 month old male mice (P = 0.032) than in the respective control of female mice.
The effect of age and gender on MENK-stimulated NO As demonstrated in Fig. 3, MENK had no effect on NO release in male mice. In contrast, in female mice MENK significantly elevated NO release in 4 month (P = 0.036) and 8 month old mice (P < 0.001) as compared with control mice of identical age. The effect of MENK on NO release in female mice was more expressed with age since it was significantly higher in 8 than in 4 month old mice (P = 0.035).
MENK-induced NO release and apoptosis are not gender related The stimulatory effect of MENK on NO release in female mice was accompanied by an increased
Fig. 2. Detection of apoptotic cells as chromatin condensation and DNA fragmentation (arrows) in macrophages injected in vivo with 2.5 mg kg−1 bw of MENK (B). Macrophages from control mice injected with HBSS (A) or from mice injected with 10.0 mg kg−1 bw of MENK exhibited a normal nuclear morphology (C). Macrophages were stained and fixed as outlined in Materials and Methods.
percentage of apoptotic macrophages in 4 and 8 month old female mice injected with MENK (14% of apoptotic macrophages in MENK treated mice compared to 3–4% in control samples) (Fig. 4). However, male mice injected with MENK although not expressing increased NO
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Fig. 4. Gender- (male, female) and age- (2, 4 and 8 month old mice) related DNA fragmentation in macrophages from mice injected with HBSS ( control) or 2.5 mg kg−1 bw of MENK (2 ). The results are representative of three independent experiments.
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Fig. 3. Gender- (male, female) and age- (2, 4 and 8 month old mice) related NO release of macrophages injected with HBSS ( control) or 2.5 mg kg−1 bw of MENK (N, 2 ). Data are expressed as mean ± SD of six animals per group; the experiments were repeated two or three times. a vs a1 P < 0.05; b vs b1 P = 0.032.
release had a high percentage of apoptotic macrophages (2 months 27% and 4 months 19%) as compared to control samples. In spite of the differences in MENKinduced NO release between male and female mice (especially at 2 and 4 months of age) no difference in onset of DNA fragmentation was observed concerning age or gender (Fig. 5).
DISCUSSION According to the results of our study NO release was age related neither in males nor in females. Literature data about age-related NO production are controversial; i.e. decline with age [6], increase with age [15] or no change [7] has been observed. These contradictory results might be due to the use of different strains of mice (Balb/C, B6JC3J/Nia or C57Bl6, respectively) or CBA mice in our study. Beside that, in the literature changes in NO release have been reported in mice older than 12 months and/or activated by different stimuli. In contrast, NO release was gender related, being, irrespective of age, higher in male than in female mice. The gender-related NO production in our study is in accordance with the results in the literature [8] where castration in male mice had a greater influence on NO release than ovariectomy in females. However, sex hormones, depending on the concentration, can either stimulate or inhibit NO release by regulating the production of TNF [16].
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Fig. 5. Gel electrophoresis of DNA from macrophages isolated from 2 and 4 month old male (A) or female (B) mice treated in vivo with HBSS (C) or 2.5 mg kg−1 of MENK (M). A DNA size standard is shown to the left (S). Results are representative of three similar experiments.
Opioid peptides in addition to their neurotransmitter function have significant immunomodulatory activity [2, 10, 27]. A gender-related response of opiod peptides was observed by Gabrilovac et al. [12] in mice Tlymphocyte proliferation; enhancement by doses of 2.5 and 5 mg kg−1 bw and no effect with 10 mg kg−1 bw of MENK. The role of sex steroids in the antinociceptive effects of morphine is contradictory; castration and ovariectomy have been found to increase, decrease and leave unchanged opioid-mediated antinociception [17, 18]. A gender-related difference in the immunomodulatory response of rats to cocaine was reported also [11]. In our study the effect of MENK was age and gender related in that it could be observed only in mature (4 or 8 month old) female mice. Thus, gonadal steroid seems to play an important role in immunomodulation by opioids. Sex differences might be the result of proenkephalin level as indicated by the results of Segarra et al. [19] who found gender-related total proenkephalin level in the medial preoptic area of rats and estrogenic regulation of proenkephalin mRNA.
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Although males contain a higher level of proenkephalin mRNA, females demonstrated greater response to estrogen than males. Another point of sex difference might be the sex-related dimorphism of the nervous system [20] which may imply differences in the opioid receptor expression and/or sensitivity related to steroid hormones [21]. Also, the binding ability to µ opioid receptors in certain brain regions is gender related [22]. Nitric oxide is a pleiotropic mediator that, among other biological effects, causes apoptosis in a variety of cell types including macrophages, lymphocytes, T-cells and neurons [23]. In our study 2.5 mg kg−1 bw of MENK that stimulated NO release of 4 and 8 month old female mice was associated with nuclear and cytoplasmic alterations characteristic of apoptosis. To our surprise such induction of apoptosis in macrophages from MENK-treated 2 and 4 month old male mice was not associated with increased NO release. The reason for this might lie in the fact that NO is not a universal inducer of apoptosis as stated by Albina et al. [24]. In fact, as demonstrated by Chun et al. [25] Il-1β even suppresses apoptosis by increasing nitric oxide production. Sandau et al. [26] recently claimed that coincubation of NO· and O− 2 could be cross-protective in NO induction; the maximum protection would require the existence of balanced NO· /O− 2 ratio. Since MENK has been defined in numerous studies as the suppressor or inducer of oxygen species, the controversy observed might be the result of a reactive oxygen species imbalance [3, 27, 28]. In conclusion, this study has shown that in the potential use of MENK as adjuvant therapy of various types of immunocompromised status, one has to take into account age and gender of the recipient. REFERENCES 1. Bredt DS. Endogenous nitric oxide synthesis: biological functions and pathophysiology. Free Radic Res 1999; 31: 577–96. 2. Foris G, Medgyesi A, Gyimesi E, Hauck M. Met-enkephalin induced alterations of macrophage functions. Mol Immunol 1984; 21: 747–50. ˇ 3. Marotti T, Sverko V, Hrˇsak I. Modulation of superoxide anion release from human polymorphonuclear cells by Met- and Leuenkephalin. Brain Behav Immun 1990; 4: 13–22. 4. Marotti T, Balog T, Maˇzuran R, Roˇci´c B. The role of cytokines in Met-enkephalin-modulated nitric oxide release. Neuropeptides 1998; 32: 57–62. 5. Alvarez E, Machado A, Sobrino F, Santa Maria C. Nitric oxide and superoxide anion production decrease with age in resident and activated rat peritoneal macrophages. Cell Immunol 1996; 169: 152–5. 6. Kissin E, Tomasi M, McCartney-Francis N, Gibbs CL, Smith PD. Age-related decline in murine macrophage production of nitric oxide. J Infect Dis 1997; 175: 1004–7. 7. Rhoades ER, Orme IM. Similar responses by macrophages from young and old mice infected with Mycobacterium tuberculosis. Mech Ageing Dev 1998; 106: 145–53. 8. Rai S, Rai U. Sex steroid hormones modulate the activation of murine peritoneal macrophages: receptor mediated modulation. Comp Biochem Phys 1998; 119C: 199–204. 9. Jilma B, Kastner I, Mensik C, Vondrovec B, Hildebrandt J, Krejcy K, Wagner OF, Eichler HG. Sex differences in concentration of exhaled nitric oxide and plasma nitrate. Life Sci 1996; 58: 469–76.
292 10. Brummitt CF, Sharp BM, Gekker G, Keane WF, Peterson PK. Modulatory effects of β-endorphin on interferon-γ production by cultured peripheral blood mononuclear cells: heterogeneity among donors and the influence of culture medium. Brain Behav Immun 1988; 2: 187–97. 11. Matulka RA, Jordan SD, Stanulis ED, Holsapple MP. Evaluation of sex- and strain-dependency of cocaine-induced immunosuppression in B6C3F1 and DBA/2 mice. J Pharmacol Exp Ther 1996; 279: 12–7. 12. Gabrilovac J, Marotti T. Gender-related differences in murine T- and B-lymphocyte proliferative ability in response to in vivo [Met5 ]enkephalin administration. Eur J Pharmacol 2000; 392: 101–8. 13. Naslund PK, Miller WC, Granger DL. Cryptococcus neoformans fails to induce nitric oxide synthase in primed murine macrophagelike cells. Infect Immun 1995; 63: 1298–304. 14. London NJM, Contractor H, Lake SP, Aucott GC, Bell PRF, James JFR. A fluorimetric viability assay for single human and rat islets. Horm Metab Res 1989; 25: 82–7. 15. Chorinchath BB, Kong LY, Mao L, McCallum RE. Ageassociated differences in TNF-alpha and nitric oxide production in endotoxic mice. J Immunol 1996; 156: 1525–30. 16. Chao TC, Van Alten PJ, Greager JH, Walter RJ. Steroid sex hormones regulate the release of tumour necrosis factor by macrophages. Cell Immunol 1995; 160: 43–9. 17. Kepler KL, Kest B, Kiefel JM, Cooper ML, Bodnar RJ. Roles of gender, gonadectomy and estrous phase in the analgesic effects of intracerebroventricular morphine in rats. Pharmacol Biochem Behav 1989; 34: 119–27. 18. Islam AK, Cooper ML, Bodnar RJ. Interaction among ageing, gender and gonadectomy effects upon morphine antinociception
Pharmacological Research, Vol. 44, No. 4, 2001 in rats. Physiol Behav 1993; 54: 45–53. 19. Segarra AC, Acosta AM, Gozales JM, Angulo JA, McEwen BS. Sex differences in estrogenic regulation of preproenkephalin mRNA levels in the medial preoptic area of prepubertal rats. Mol Brain Res 1998; 60: 133–9. 20. Breedlove SC. Sexual dimorphism in the vertebrate nervous system. J Neurosci 1992; 12: 4133–42. 21. Kawata M, Yuri K, Morimoto M. Steroid hormone effects on gene expression, neuronal structure, and differentiation. Horm Behav 1994; 28: 477–82. 22. Rimanoczy A, Vathy I. Prenatal exposure to morphine alters brain µ opioid receptor characteristics. Brain Res 1995; 690: 245–8. 23. Filep JG, Baron C, Lachance S, Perreault C, Chan JSD. Involvement of nitric oxide in target-cell lysis and DNA fragmentation induced by murine natural killer cells. Blood 1996; 87: 5136–43. 24. Albina JE, Cui S, Mateo RB, Reicher JS. Nitric oxide-mediated apoptosis in murine peritoneal macrophages. J Immunol 1993; 150: 5080–985. 25. Chun SY, Eisenhauer KM, Kubo M, Hsueh AJW. Interleukin-1β suppresses apoptosis in rat ovarian follicles by increasing nitric oxide production. Endocrinology 1995; 136: 3120–7. 26. Sandau K, Pfeilschifter J, Brune B. The balance between nitric oxide and superoxide determines apoptotic and necrotic death of rat mesangial cells. J Immunol 1997; 158: 4938–46. 27. Peterson PK, Sharp B, Gekker G, Brummitt C, Keane WF. Opioidmediated suppression of cultured peripheral blood mononuclear cell respiratory burst activity. J Immunol 1987; 138: 3907–12. 28. Sharp B, Keane VF, Suh HJ, Gekker G, Tsukayama D, Peterson PK. Opioid peptides rapidly stimulate superoxide production by human polymorphonuclear leukocytes and peritoneal macrophages. Endocrinology 1985; 117: 793–7.
THE EFFECT OF METHIONINE-ENKEPHALIN ON NITRIC OXIDE RELEASE IN MICE IS AGE AND GENDER RELATED ˇ ˇ TIHOMIR BALOG∗ , T. MAROTTI, V. MUSANI, S. SOBOCANEC and V. SVERKO Department of Molecular Medicine, Rud−er Boˇskovi´c Institute, 10000 Zagreb, Croatia Accepted 17 July 2001
Gender- and age-related differences in nitric oxide (NO) release and in response to drugs of abuse has been reported in both humans and experimental animals. So far, we have demonstrated in vivo methionine-enkephalin- (MENK-) modulated NO release in mice. However, no data on the influence of age and gender on this immunomodulatory effect of MENK have been reported. In this study we examined the influence of age (2, 4, 8 month old mice) and gender (male and female mice) on MENK-induced NO release of mouse peritoneal macrophages (PEMs) of the CBA strain of mice. NO release was not age but was gender related in that males generally produced more NO than females. The effect of MENK on NO release was age (demonstrated only in mature 4 and 8 month old mice) and gender related in that it could be observed only in female mice. Apoptotic cells that paralleled the increase of NO in MENK-treated female mice were, however, observed also in male mice although MENK was in males without effect. These data provide evidence that some immunomodulatory properties of MENK are age and gender related which may be relevant to the potential use of MENK in adjuvant therapy for c 2001 Academic Press immunocompromised status.
K EY WORDS : nitric oxide, methionine-enkephalin, gender, age.
INTRODUCTION Experimental studies have indicated that reactive oxygen species are the primary mediators in the pathogenesis of immune and non-immune injuries. Among oxygen species, NO over-production by inducible nitric oxide synthase (iNOS) occurs under pathophysiological conditions of chronic infection, inflammation and in septic patients. NOS activity is changed in diverse pathophysiological conditions including migraine headache, hypertrophic pyloric stenosis and male impotence. NO appears to play the major role in pathophysiology of stroke, Parkinson’s disease, Huntington’s disease and amyotrophic lateral sclerosis [1]. By the reaction of NO with superoxide − anion (O− 2 ), peroxynitrite (ONOO ), which represents a major pathway of NO-derived reactivity and toxicity, can be generated. Literature data of MENK-stimulated generation of biologically active oxygen species of rat peritoneal macrophages existed already in 1984 [2]. We ∗ Corresponding author. Department of Molecular Medicine, Rud −er Boˇskovi´c Institute, Bijeniˇcka c. 54, 10000 Zagreb, Croatia. E-mail: [email protected]
1043–6618/01/100287–06/$35.00/0
have shown [3] that MENK modulates O− 2 release from human neutrophils. Our recent work showed MENKmodulated NO release [4] from peritoneal macrophages of 4 month old CBA strain mice. However, the data about the effect of age on NO release in the literature are controversial. While some authors [5, 6] claimed to have age-dependent progressive and marked decline in NO production of mice or rat peritoneal macrophages, others [7] demonstrated that macrophages from young and old mice secreted similar levels of NO. Another point of interest is the observed difference in gender-related NO release of murine peritoneal macrophages [8] and exhaled NO and plasma nitrate in humans [9]. Such differences have been observed also in response to opioid peptides or psychostimulants [10, 11]. Recently, a gender-related difference in murine T- and B-lymphocyte proliferative ability in response to in vivo MENK administration was found [12]. The aim of the present study was to assess the possible influence of age and gender on MENK-induced modulation of NO release in mice and to correlate the effect with the induction of apoptosis. This was done by comparing the effect of MENK on the release of NO from c 2001 Academic Press
288
peritoneal macrophages from 2, 4 or 8 month old male and female CBA strain mice.
Pharmacological Research, Vol. 44, No. 4, 2001
examined under a fluorescent microscope. They were counted as apoptotic if chromatin was condensed, nuclei fragmented and cells shrank. At least 300 cells were counted per sample.
MATERIALS AND METHODS
Analysis of DNA fragmentation in agarose gels Animals Male and female CBA/HZgr mice, aged 2, 4 or 8 months, raised in the breeding colony of the Rud−er Boˇskovi´c Institute, Zagreb, Croatia, were used. They were kept five to a cage, fed a standard diet and given water ad libitum. Mice of different age and of both sexes were divided into mice receiving i.p. phosphate buffered saline (PBS) (control) or 2.5, 5.0 or 10.0 mg kg−1 bw of MENK (Sigma, St Louis, USA). Sample preparations were performed 18 h after the injection of MENK.
Preparation of samples At a given time after drug injection (18 hours) the animals were killed by cervical dislocation. Peritoneal macrophages (PEM) were aseptically collected from the peritoneal cavities of mice 5 days after i.p. injection of 0.12% glycogen (Kemika, Zagreb, Croatia). Macrophages were resuspended in RPMI 1640 (without phenol red; Sigma, St Louis, USA) and red cells were removed by NH4 Cl lysis. The remaining cells were washed three times, resuspended in RPMI 1640 with antibiotics and 10% fetal calf serum (FCS; Sigma St Louis, USA) and adjusted to 2 × 106 ml−1 . This procedure typically yielded preparations containing more than 95% of viable peritoneal macrophages, as tested by trypan blue.
Measurement of nitrite production The measurement of NO was assayed by the colorimetric reaction of the Griess reagent [13]. Briefly, isolated PEMs were adjusted to 2 × 106 /well and (in duplicate) incubated in plastic 24-well flat-bottom microplates (Falcon, USA) for 48 h at 37 ◦ C and 5% CO2 . Aliquots (800 µl) of each supernatant were placed in tubes and mixed with 800 µl of GRIESS reagent (1% sulphanilamide in 2.5% phosphoric acid and 0.5% naphthylethylenediamine in 2.5% phosphoric acid, 1 : 1). The resulting colorimetric reaction was measured at 540 nm with a spectrophotometer. NO concentration was calculated from a standard curve using sodium nitrite (0–100 µM) as standard and was expressed as nmol/2 × 106 cells/well.
Fluorometric viability assay Fluorometric viability assay of macrophages from mice injected in vivo with PBS (control) or 2.5 and 10.0 mg kg−1 bw of MENK was performed according to the method of London et al. [14]. Briefly, the kinetics of apoptosis was followed in control macrophages and macrophages of MENK-treated mice 18 h after treatment. Macrophages (10 µl) were mixed with 4 µl of 1% propidium iodide in water and 3 µl of 1% fluorescein diacetate in acetone in 1 ml of PBS. The cells were
3 × 106 adherent cells were incubated for 10 min in 500 µl of lysis buffer (20 mM Tris-HCl pH = 7.4, 10 mM EDTA, 0.2% Triton X-100) and centrifuged at 10 000 g for 10 min. The supernatant was incubated overnight at 50 ◦ C with 100 µg ml−1 proteinase K. The DNA was extracted with 1 vol of chloroform/phenol (1 : 1). The DNA was precipitated from the aqueous phase with 1 vol of isopropanol, 500 mM NaCl at −20 ◦ C overnight and collected by centrifugation. Pellets were air dried and incubated in 10 µl of 10 mM Tris pH = 7.5 and 1 mM EDTA for 1 h at 37 ◦ C with 1 µg ml−1 RNAse A. Horizontal electrophoresis was performed for 1 h at 80 V in 1% agarose gel containing 1 µg ml−1 ethidium bromide with 90 mM Tris, 90 mM boric acid and 2 mM EDTA, pH = 8.0, as running buffer. Samples were loaded after suspension in 1 µl of loading buffer (10 mM Tris, 1 mM EDTA) and heating at 65 ◦ C for 10 min. DNA was visualized in UV light.
Statistical analysis Data are presented as mean ± standard deviation. The significance of the differences between groups was assessed by Student’s t-test. P-values below 0.05 were considered significant.
RESULTS
Nitric oxide release and apoptosis—the effect of various MENK doses NO release of macrophage from peritoneal cavity of female mice injected with 2.5, 5.0 or 10.0 mg kg−1 bw of MENK is presented in Fig. 1. In the corresponding groups some mice received 10.0 mg kg−1 bw naloxone 30 minutes before the injection of MENK. Control macrophages were obtained from mice receiving either Hanks balanced salt solution (HBSS) or naloxone (10.0 mg kg−1 bw). MENK modulated NO release in a concentration-dependent manner. It increased NO release at 2.5 mg and 10.0 mg kg−1 bw (P = 0.001 or P = 0.03, respectively) and decreased NO release at 5.0 mg kg−1 bw (P < 0.05). While naloxone per se had no effect and was ineffective in abrogating the suppressive effect of 5.0 mg kg−1 bw of MENK, it efficiently blocked the stimulatory effect of MENK at doses of 2.5 and 10.0 mg kg−1 bw. Apoptotic macrophages were examined under fluorescent microscope in cultures of macrophages of control mice or mice treated in vivo with 2.5 or 10.0 mg kg−1 bw of MENK (5.0 mg kg−1 bw was not examined since it was not opioid receptor mediated). As demonstrated in Fig. 2, only 2.5 mg kg−1 bw MENK induced apoptosis
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Fig. 1. NO release of PEM of 4 month old female mice injected with 2.5, 5.0 or 10.0 mg kg−1 bw of MENK or/and naloxone (NAL) (10 mg kg−1 bw). Control mice were injected with HBSS. Data are expressed as mean ± SD of six animals per group; the experiments were repeated two or three times. **P = 0.001, *P = 0.03, NP < 0.05. ( Control, 2 MENK, 2 NAL, 2 MENK + NAL.)
visualized by condensed chromatin, nuclei fragmentation and shrunk cells. We have chosen to examine the involvement of age and gender in MENK-modulated NO release of a single dose of 2.5 mg kg−1 bw, since 5.0 mg kg−1 bw of MENK was not opioid receptor mediated and the 10.0 mg kg−1 bw of MENK did not induce apoptosis. To test whether the effect of MENK was age and/or gender related, we injected 2, 4 and 8 month old male and female mice with 2.5 mg kg−1 bw of MENK and the respective control mice with HBSS.
NO release in controls—the effect of age and gender The results of NO release in control mice of different age and gender are presented in Fig. 3. NO release in control HBSS injected mice was not age related either in male or in female mice. However, NO release of control mice was gender related since it was significantly higher in 4 month (P < 0.05) and 8 month old male mice (P = 0.032) than in the respective control of female mice.
The effect of age and gender on MENK-stimulated NO As demonstrated in Fig. 3, MENK had no effect on NO release in male mice. In contrast, in female mice MENK significantly elevated NO release in 4 month (P = 0.036) and 8 month old mice (P < 0.001) as compared with control mice of identical age. The effect of MENK on NO release in female mice was more expressed with age since it was significantly higher in 8 than in 4 month old mice (P = 0.035).
MENK-induced NO release and apoptosis are not gender related The stimulatory effect of MENK on NO release in female mice was accompanied by an increased
Fig. 2. Detection of apoptotic cells as chromatin condensation and DNA fragmentation (arrows) in macrophages injected in vivo with 2.5 mg kg−1 bw of MENK (B). Macrophages from control mice injected with HBSS (A) or from mice injected with 10.0 mg kg−1 bw of MENK exhibited a normal nuclear morphology (C). Macrophages were stained and fixed as outlined in Materials and Methods.
percentage of apoptotic macrophages in 4 and 8 month old female mice injected with MENK (14% of apoptotic macrophages in MENK treated mice compared to 3–4% in control samples) (Fig. 4). However, male mice injected with MENK although not expressing increased NO
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Fig. 4. Gender- (male, female) and age- (2, 4 and 8 month old mice) related DNA fragmentation in macrophages from mice injected with HBSS ( control) or 2.5 mg kg−1 bw of MENK (2 ). The results are representative of three independent experiments.
8 6 a1
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Fig. 3. Gender- (male, female) and age- (2, 4 and 8 month old mice) related NO release of macrophages injected with HBSS ( control) or 2.5 mg kg−1 bw of MENK (N, 2 ). Data are expressed as mean ± SD of six animals per group; the experiments were repeated two or three times. a vs a1 P < 0.05; b vs b1 P = 0.032.
release had a high percentage of apoptotic macrophages (2 months 27% and 4 months 19%) as compared to control samples. In spite of the differences in MENKinduced NO release between male and female mice (especially at 2 and 4 months of age) no difference in onset of DNA fragmentation was observed concerning age or gender (Fig. 5).
DISCUSSION According to the results of our study NO release was age related neither in males nor in females. Literature data about age-related NO production are controversial; i.e. decline with age [6], increase with age [15] or no change [7] has been observed. These contradictory results might be due to the use of different strains of mice (Balb/C, B6JC3J/Nia or C57Bl6, respectively) or CBA mice in our study. Beside that, in the literature changes in NO release have been reported in mice older than 12 months and/or activated by different stimuli. In contrast, NO release was gender related, being, irrespective of age, higher in male than in female mice. The gender-related NO production in our study is in accordance with the results in the literature [8] where castration in male mice had a greater influence on NO release than ovariectomy in females. However, sex hormones, depending on the concentration, can either stimulate or inhibit NO release by regulating the production of TNF [16].
Pharmacological Research, Vol. 44, No. 4, 2001
Fig. 5. Gel electrophoresis of DNA from macrophages isolated from 2 and 4 month old male (A) or female (B) mice treated in vivo with HBSS (C) or 2.5 mg kg−1 of MENK (M). A DNA size standard is shown to the left (S). Results are representative of three similar experiments.
Opioid peptides in addition to their neurotransmitter function have significant immunomodulatory activity [2, 10, 27]. A gender-related response of opiod peptides was observed by Gabrilovac et al. [12] in mice Tlymphocyte proliferation; enhancement by doses of 2.5 and 5 mg kg−1 bw and no effect with 10 mg kg−1 bw of MENK. The role of sex steroids in the antinociceptive effects of morphine is contradictory; castration and ovariectomy have been found to increase, decrease and leave unchanged opioid-mediated antinociception [17, 18]. A gender-related difference in the immunomodulatory response of rats to cocaine was reported also [11]. In our study the effect of MENK was age and gender related in that it could be observed only in mature (4 or 8 month old) female mice. Thus, gonadal steroid seems to play an important role in immunomodulation by opioids. Sex differences might be the result of proenkephalin level as indicated by the results of Segarra et al. [19] who found gender-related total proenkephalin level in the medial preoptic area of rats and estrogenic regulation of proenkephalin mRNA.
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Although males contain a higher level of proenkephalin mRNA, females demonstrated greater response to estrogen than males. Another point of sex difference might be the sex-related dimorphism of the nervous system [20] which may imply differences in the opioid receptor expression and/or sensitivity related to steroid hormones [21]. Also, the binding ability to µ opioid receptors in certain brain regions is gender related [22]. Nitric oxide is a pleiotropic mediator that, among other biological effects, causes apoptosis in a variety of cell types including macrophages, lymphocytes, T-cells and neurons [23]. In our study 2.5 mg kg−1 bw of MENK that stimulated NO release of 4 and 8 month old female mice was associated with nuclear and cytoplasmic alterations characteristic of apoptosis. To our surprise such induction of apoptosis in macrophages from MENK-treated 2 and 4 month old male mice was not associated with increased NO release. The reason for this might lie in the fact that NO is not a universal inducer of apoptosis as stated by Albina et al. [24]. In fact, as demonstrated by Chun et al. [25] Il-1β even suppresses apoptosis by increasing nitric oxide production. Sandau et al. [26] recently claimed that coincubation of NO· and O− 2 could be cross-protective in NO induction; the maximum protection would require the existence of balanced NO· /O− 2 ratio. Since MENK has been defined in numerous studies as the suppressor or inducer of oxygen species, the controversy observed might be the result of a reactive oxygen species imbalance [3, 27, 28]. In conclusion, this study has shown that in the potential use of MENK as adjuvant therapy of various types of immunocompromised status, one has to take into account age and gender of the recipient. REFERENCES 1. Bredt DS. Endogenous nitric oxide synthesis: biological functions and pathophysiology. Free Radic Res 1999; 31: 577–96. 2. Foris G, Medgyesi A, Gyimesi E, Hauck M. Met-enkephalin induced alterations of macrophage functions. Mol Immunol 1984; 21: 747–50. ˇ 3. Marotti T, Sverko V, Hrˇsak I. Modulation of superoxide anion release from human polymorphonuclear cells by Met- and Leuenkephalin. Brain Behav Immun 1990; 4: 13–22. 4. Marotti T, Balog T, Maˇzuran R, Roˇci´c B. The role of cytokines in Met-enkephalin-modulated nitric oxide release. Neuropeptides 1998; 32: 57–62. 5. Alvarez E, Machado A, Sobrino F, Santa Maria C. Nitric oxide and superoxide anion production decrease with age in resident and activated rat peritoneal macrophages. Cell Immunol 1996; 169: 152–5. 6. Kissin E, Tomasi M, McCartney-Francis N, Gibbs CL, Smith PD. Age-related decline in murine macrophage production of nitric oxide. J Infect Dis 1997; 175: 1004–7. 7. Rhoades ER, Orme IM. Similar responses by macrophages from young and old mice infected with Mycobacterium tuberculosis. Mech Ageing Dev 1998; 106: 145–53. 8. Rai S, Rai U. Sex steroid hormones modulate the activation of murine peritoneal macrophages: receptor mediated modulation. Comp Biochem Phys 1998; 119C: 199–204. 9. Jilma B, Kastner I, Mensik C, Vondrovec B, Hildebrandt J, Krejcy K, Wagner OF, Eichler HG. Sex differences in concentration of exhaled nitric oxide and plasma nitrate. Life Sci 1996; 58: 469–76.
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