Heroin modulates the expression of inducible nitric oxide synthase

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Abstract

The use of heroin (diacetylmorphine) is associated with a high incidence of infectious disease, and the immunologic alterations responsible for heroin-induced changes in resistance to infection have not been well characterized. The present study tests the hypothesis that expression of inducible nitric oxide synthase (iNOS) is modulated by the administration of heroin. The initial study using rats showed that heroin administration (0, 0.01, 0.1, or 1.0 mg/kg s.c.) results in a pronounced reduction in lipopolysaccharide (LPS)-induced expression of iNOS mRNA in spleen, lung, and liver tissue as measured by RT-PCR. Heroin also produced a reduction in the level of plasma nitrite/nitrate, the more stable end-product of nitric oxide degradation. In a subsequent study, administration of the opioid receptor antagonist, naltrexone (0.1 mg/kg) prior to the injection of heroin (1.0 mg/kg) blocked the heroin-induced reduction of iNOS expression and plasma nitrite/nitrate levels indicating that the effect is mediated via the opioid-receptor. This study provides the first evidence that heroin induces an alteration of iNOS expression, and suggests that a reduction in nitric oxide production may be involved in the increased incidence of infectious diseases amongst heroin users.

Introduction

The high incidence of bacterial, viral, and fungal infections among long-term heroin users indicates that the use of heroin alters resistance to infectious disease (Luttgens, 1949; Hussey and Katz, 1950; Louria et al., 1967). Several investigators have suggested that the increase in susceptibility to diseases among intravenous heroin users may be directly related to opioid-induced alterations of immune function (Brown et al., 1974; McDonough et al., 1980; Donahoe et al., 1986; Novick et al., 1989; Ochshorn et al., 1990). In fact, several studies report immunological abnormalities associated with heroin use in humans, including decreased numbers of circulating lymphocytes, decreased natural killer cell activity and antibody-dependent cellular cytotoxicity (e.g., Nair et al., 1986; Govitrapong et al., 1998).

There are very few controlled animal studies investigating the effects of heroin on immune status. There are, however, extensive investigations of the effect of morphine, one of the major active metabolites of heroin that is commonly used clinically in the treatment of pain, on immune status. Morphine administration is associated with alterations in a number of immune parameters, such as natural-killer cell activity (Shavit et al., 1986; Bayer et al., 1990), proliferation of lymphocytes (Bryant et al., 1988; Bayer et al., 1990), antibody production (Lefkowitz and Chiang, 1975; Lockwood et al., 1994), and the production of interferon (Hung et al., 1973). Studies in our laboratory have shown that acute morphine treatment in rats suppresses splenic lymphocyte proliferative responses to both T- and B-cell mitogens, splenic natural-killer cell activity, blood lymphocyte mitogenic responsiveness to T-cell mitogens, and the in vitro production of the cytokines interleukin-2 and interferon-γ (Fecho et al., 1993, Fecho et al., 1994, Fecho et al., 1996a, Fecho et al., 1996b; Lysle et al., 1993). The immune alterations induced by morphine are dose-dependent and antagonized by the opioid-receptor antagonist, naltrexone (e.g., Lysle et al., 1993). Morphine administration also has been shown to alter the production of nitric oxide by Con A stimulated splenocytes (Fecho et al., 1994).

Although the study of morphine may contribute to our understanding of heroin effects, there is evidence showing that heroin and morphine act through different pathways. For example, differences in potency between heroin and morphine are well-documented with heroin as much as 16 times more potent than morphine in producing reinforcing effects in animals (Harrigan and Downs, 1978; Van Ree et al., 1978) and subjective effects in humans (Seevers and Pfeiffer, 1936; Martin and Fraser, 1961; Jasinski and Nutt, 1972; Kaiko et al., 1981). Similar potency relations have been obtained in a number of different analgesic assays using animals (Shemano and Wendel, 1964; Switzman et al., 1981; Umans and Inturrisi, 1981; Tasker and Nakatsu, 1984) and humans (Seevers and Pfeiffer, 1936; Reichle et al., 1962; Dundee et al., 1966; Kaiko et al., 1981). There also are reports that animals made tolerant to morphine do not show cross-tolerance to heroin (Lange et al., 1980; Bolger et al., 1988; Rossi et al., 1996). Molecular studies using antisense probes indicate that heroin produces analgesia through a different mu-receptor subtype than that involved in morphine-induced analgesia (Rossi et al., 1996). Interesting recent work shows that in vivo administration of 3-methoxynaltrexone selectively antagonizes the analgesic actions of heroin, but does not interfere with morphine-induced analgesia (Brown et al., 1997). Collectively, these several lines of investigation indicate important qualitative differences between heroin and morphine. Given the major health issues surrounding heroin use and the important pharmacological and behavioral differences between heroin and morphine, it is important to directly examine the impact of heroin on immune status.

The present investigations evaluated the effect of heroin on the expression of inducible nitric oxide synthase (iNOS), the enzyme responsible for nitric oxide formation. Nitric oxide has been shown to have a critical role in immune processes including resistance to infectious disease. The generation of nitric oxide by cells of the immune system, particularly the macrophage, provides a substantial degree of microbial resistance (James and Glaven, 1989; Vincendeau et al., 1992; Green and Nacy, 1993; Green et al., 1990). Indeed, mice lacking the gene for iNOS have markedly reduced resistance to parasitic and bacterial infections (MacMicking et al., 1995; Wei et al., 1995). In addition to the impact of nitric oxide directly upon infectious organisms, nitric oxide also has important regulatory functions in the immune system. For example, evidence supports a role for nitric oxide in the suppression of antibody formation to tetanus toxoid and sheep red blood cells after immunization with Salmonella typhimurium (Al-Ramadi et al., 1992; Eisenstein et al., 1994). Furthermore, numerous studies have shown that nitric oxide limits the proliferative activity of lymphocytes (e.g., Albina and Henry, 1991; Pascual et al., 1992).

Lipopolysaccharide (LPS) treatment in vivo has been extensively used to induce a very rapid, wide spread, expression of mRNA for iNOS (Liu et al., 1993; Liu et al., 1997). In our prior work, we showed that the injection of LPS produces dose- and time-dependent effects on the expression of iNOS mRNA in the spleen, as well as the accumulation of nitrite/nitrate, the more stable end-products of nitric oxide degradation, in plasma (Lysle and How, 1999). The present studies test whether heroin (0.01–1.0 mg/kg) administration alters LPS-induced iNOS mRNA expression in the spleen, lung, and liver, of rats using RT-PCR. The effect of heroin on the level of plasma nitrite/nitrate was also determined. In a follow-up study, the effect of administration of the opioid receptor antagonist, naltrexone prior to the injection of heroin was evaluated to determine whether the effect of heroin is mediated via the opioid-receptors. The doses of heroin employed in the present investigation are within the range of those commonly employed in animal models of human drug abuse such as drug self administration (Martin et al., 1996; Martin et al., 1998) and drug discrimination studies (e.g., Lamas et al., 1998). In these models of human drug abuse, the relative potency estimates of opioids is highly correlated with those of comparable procedures with humans (Woods et al., 1982; Dykstra et al., 1997).

Section snippets

Animals

Male Lewis rats 225–250 g in weight, were purchased from Charles Rivers Laboratories (Raleigh, NC). Upon arrival, animals were individually caged in a colony room where a reversed light–dark (12-h) cycle was maintained through artificial illumination. Food and water were provided ad libitum during both a two-week acclimation period and the experimental procedure.

Drug administration

To evaluate the effect of heroin, rats received a subcutaneous injection of saline or heroin at doses of 0.01, 0.1, or 1.0 mg/kg (n=4)

Effect of heroin on INOS expression

Fig. 1 shows the effect of heroin on LPS-induced expression of iNOS mRNA in the spleen, lung, and liver, as determined by RT-PCR. The results showed a distinct product of the expected size (496 bp) for iNOS, and a distinct product of the expected size (764 bp) for β-actin. The analysis of the ratio of iNOS/β-actin expression showed a significant effect of heroin on iNOS expression in the spleen, lung, and liver, F(3,12)=8.06, p<0.01; F(3,12)=23.08, p<0.001; and F(3,12)=40.76, p<0.001,

Discussion

The present study provides the first data showing that heroin induces a pronounced reduction of iNOS expression that is wide-spread with the effect being found in spleen, lung, and liver, as well as in the accumulation of nitrite/nitrate in the plasma. Moreover, the opioid antagonist naltrexone blocked heroin-induced modulation of iNOS expression indicating that the effect of heroin was mediated via opioid receptors.

An interesting question is whether heroin and morphine have the same effect on

Acknowledgements

This research was supported by grants from the National Institute on Drug Abuse (DA10167; DA07481). Donald T. Lysle is the recipient of a Research Scientist Development Award (DA00334) from the National Institute on Drug Abuse.

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