Burt M. Sharp

Morphine promotes the growth of HIV-1 in human peripheral blood mononuclear cell cocultures.

Because morphine has been shown to alter the function of human T lymphocytes and monocytes, we postulated that morphine would promote the growth of HIV-1 in these cells. To test this hypothesis, a coculture assay was used consisting of phytohemagglutinin (PHA)-activated peripheral blood mononuclear cells (PBMC) from normal donors and PBMC which had been infected with a viral isolate from an asymptomatic patient, HIV-1AT. The growth of HIV-1AT, as reflected by the concentration of p24 antigen in coculture supernatants, was markedly increased in cocultures that contained morphine. A bell-shaped dose-response curve was observed with three- to fourfold increased growth at a morphine concentration of 10(-12) M. Augmentation of HIV-1AT growth by morphine required an interaction with the PHA-activated donor PBMC. Furthermore, potentiation of HIV-1AT growth by morphine was stereospecific and was antagonized by naloxone and beta-funaltrexamine indicating involvement of an opiate receptor mechanism. These findings provide an additional explanation of how opiates could act as a cofactor in the pathogenesis of HIV-1 in intravenous drug users.

Evidence for opioid receptors on cells involved in host defense and the immune system

Although the role of opiates and opioids in the physiological and pathological function of the immune system is only beginning to be unraveled, converging lines of evidence indicate that the opioid receptors expressed by immune cells are often the same or similar to the neuronal subtypes, particularly delta and kappa. Recent studies also point to the existence of novel opioid receptors and/or binding sites on immune cells that are selective for morphine. Opioids and their receptors, particularly those with high affinity for delta agonists, appear to function in an autocrine/paracrine manner. Thus, opioid peptides generated from immune-derived proenkephalin A act as cytokines, capable of regulating myriad functions of both granulocytes and mononuclear cells. Further identification and characterization of receptors and signal transduction pathways that account for some of the unique properties of opiate binding and immunomodulation (e.g., dose-dependent effects of morphine that occur at exceptionally low concentrations relative to the Kds of the neuronal mu receptor or the morphine binding site reported on activated human T-cells) represents one of the major research challenges ahead. Elucidating mechanisms, such as these, may provide unique therapeutic opportunities through the application of opioid immunopharmacology to disorders involving immune responses in peripheral organs and the central nervous system.

Response of the hypothalamo-pituitary-adrenal axis to nicotine.

Nicotine has been shown to be a potent stimulus for the secretion of the stress-responsive hormones, adrenocorticotropin (ACTH) and prolactin. This paper reviews the findings by our laboratory and others that demonstrate the polysynaptic pathways involved in the neuroendocrine responses to systemic nicotine. It will focus primarily on the hypothalamo-pituitary-adrenal (HPA) axis and the effect of nicotine on ACTH secretion, with supplementary information on prolactin secretion, where relevant. Data are presented demonstrating that nicotine acts via a central mechanism to stimulate indirectly the release of ACTH from the anterior pituitary corticotropes. Nicotine does not appear to act directly at the hypothalamic paraventricular nucleus (PVN), the site of the corticotropin-releasing hormone (CRH) neurons crucial to the regulation of ACTH. However, brainstem catecholaminergic regions projecting to the PVN showed a regionally selective and dose-dependent sensitivity to nicotine, particularly the noradrenergic/adrenergic nucleus tractus solitarius (NTS). A reduction in the modulatory effect of these catecholamines (by neurotoxic lesion, synthetic enzyme inhibitors or adrenergic receptor antagonists) resulted in an inhibition of nicotine-stimulated ACTH secretion. In addition, blockade of nicotinic cholinergic receptors (NAchRs) in the brainstem by the antagonist, mecamylamine, resulted in a dose-dependent reduction in norepinephrine (NE) release from terminals in the PVN, and a concomitant reduction in plasma ACTH. The differential sensitivity of these receptors to the nicotinic agonists, cytisine and nicotine, reflects the heterogeneity of the NAchR subtypes involved. The desensitization characteristics of the neuroendocrine responses to both acute and chronic nicotine exposure are indicative of an alteration in these NAchRs.

Stress and Pathogenesis of Infectious Disease

Abstract Despite inherent difficulties in defining and measuring stress, a scientific framework has been provided in recent years for understanding how disruptive life experiences might be translated into altered susceptibility to infectious diseases. Studies of the effects of stress on pathogenesis of infectious disease are highly relevant to assessment of the biological importance of the immune impairments that have been associated with stress. With a few notable exceptions, investigations of viral infections in humans and in animal models support the hypothesis that stress promotes the pathogenesis of such infections. Similar conclusions can be drawn from studies of bacterial infections in humans and animals and from a small number of studies of parasitic infections in rodent models. While many of these studies have substantial limitations, the data nonetheless suggest that stress is a potential cofactor in the pathogenesis of infectious disease. Given recent unprecedented advances in the neurosciences, in immunology, and in the field of microbial pathogenesis, the relationship between stress and infection should be a fruitful topic for interdisciplinary research.

Detection of basal levels and induction of delta opioid receptor mRNA in murine splenocytes

Activation of delta opioid receptors (DOR) modulates calcium mobilization, interleukin-2 production, chemotaxis and proliferation of T-lymphocytes. Recent reports indicate that lymphocytes and mononuclear cells may express mRNA transcripts for DOR. The investigations reported herein show that low levels of DOR were consistently detected by RT-PCR amplification of RNA from freshly obtained Balb/c murine splenocytes, both weanling and adult. Culturing cells without stimulation increased DOR levels and concanavalin A apparently reduced this; DOR was preferentially expressed in a T-cell-enriched fraction. Thus, the expression of DOR mRNA by unactivated splenocytes is modulated by culture and con A in the T-cell fraction.

Delta opioid receptors expressed by stably transfected jurkat cells signal through the map kinase pathway in a ras-independent manner

Delta opioid receptors (DOR) are G-protein coupled 7-transmembrane receptors (GPCR), expressed by thymic and splenic T cells, that modulate interleukin (IL)-2 production and proliferation in response to concanavalin A or crosslinking the TCR. Mitogen-activated protein kinases (MAPKs) are involved in mediating intracellular responses to TCR crosslinking. In addition, MAPKs can be activated by signaling cascades that are initiated by the release of G-proteins from GPCRs. To determine whether DORs expressed by T cells signal through the MAPKs, extracellular-regulated kinases (ERKs) 1 and 2, two delta opioid peptides, deltorphin and [D-Ala2,D-Leu5]-enkephalin (DADLE), were studied in Jurkat cells that had been stably transfected with DOR (DOR-Ju.1). These peptides rapidly and dose-dependently induced ERK phosphorylation; pretreatment with naltrindole (NTI), a selective DOR antagonist, abolished this. Pertussis toxin (PTX) also inhibited phosphorylation, indicating the involvement of the Gi/o proteins. Herbimycin A, a protein tyrosine kinase (PTK) inhibitor, reduced the DADLE-induced ERK phosphorylation by 68%. ERK phosphorylation was inhibited by Bisindolylmaleimide 1 (GF109203X), an inhibitor of PKC, and by pretreatment with PMA prior to DADLE. A GTP/GDP exchange assay was used to assess the potential role of Ras in the pathway leading to ERK phosphorylation; DADLE failed to stimulate GTP/GDP exchange in comparison to PMA. Additional studies showed that DADLE stimulated an increase in cfos mRNA; this was reduced by the inhibitor of MAPK/ERK kinase (MEK), PD98059. Therefore, in DOR-Ju.1 cells, DOR agonists stimulate ERK phosphorylation in a Ras independent and PKC-dependent manner; PTKs appear to be involved. MAPKs mediate the increase in cfos mRNA induced by DOR agonists.

The Brain Immune Axis and Substance Abuse

Proceedings from the second annual symposium on the above-titled topic held in June 1994 in Palm Beach, Florida Papers address the direct effects of drugs of abuse on various components of the immune system, as well as those mediated indirectly by the central nervous system and the neuroendocrine sy

Nicotine regulates nicotinic cholinergic receptors and subunit rnRNAs in PC 12 cells through protein kinase A

To understand the up-regulation of neuronal nicotinic cholinergic receptors (nAcChRs) that results from chronic in vivo treatment with nicotine, we studied the effect of nicotine on [3H]nicotine binding sites on PC 12 cells. PC 12 cells were grown in nicotine hemisulfate (10(-6) to 10(-3) M) or vehicle for 7 days, and specific [3H]nicotine binding was measured. Nicotine (10(-6) to 10(-4) M) dose-dependently increased specific binding by up to 2.6-fold over basal levels in 5-7 days, whereas a 10(-3) M concentration failed to do so. In contrast, [3H]nicotine binding to PC 12 cell mutants (A126.1B2 and A123.7), deficient in cAMP-responsive protein kinase A Types I and/or II, was unaffected by nicotine. Northern gel analysis of nAcChR subunit mRNAs from wild type PC 12 cells showed that the mRNA encoding the dominant agonist-binding subunit, alpha 3, was significantly reduced by nicotine, as early as 4 h after treatment, whereas mRNA for the structural beta 2 subunit was slightly increased. In contrast, the alpha 3 subunit mRNA from the PC 12 cell mutant A123.7 was not significantly decreased after 4 h and 7 days of nicotine treatment. These studies indicate that nicotine up-regulates expression of nAcChRs on wild type PC 12 cells and reduces the content of alpha 3 subunit mRNA; these effects require an intact protein kinase A system. The divergent effects of nicotine on the nAcChR compared to its alpha 3 subunit mRNA suggests that enhanced expression of nicotinic receptors may not involve synthesis of new receptor subunit proteins.

Nicotine activates NPY and catecholaminergic neurons in brainstem regions involved in ACTH secretion.

Nicotine rapidly and potently stimulates ACTH secretion via a centrally mediated mechanism. The purpose of the current study was to identify the phenotype of nicotine-sensitive neurons in brainstem catecholaminergic regions previously shown to be responsive to nicotine. Immunocytochemical double-labeling was used to detect c-Fos expression in neurons positive for activin, galanin, or neuropeptide Y (NPY), in comparison to those containing tyrosine hydroxylase (TH, catecholaminergic biosynthetic enzyme). These neuropeptides were chosen because (1) each is located in nicotine-sensitive brainstem regions, (2) neurons containing each of these peptides project to the hypothalamic paraventricular nucleus, and (3) each has been shown to affect ACTH secretion. Freely moving, adult, male rats received an intravenous (i.v.) infusion of saline or nicotine (0.045 mg/kg over 30 s or 0.135 mg/kg over 90 s) and were cardiac perfused 60 min thereafter. Nicotine significantly increased c-Fos expression in a dose-dependent manner in the brainstem regions examined. In nucleus tractus solitarius (NTS)-A2 and NTS-C2, both NPY+ and TH+ neurons responded to the lower dose of nicotine, whereas the activin and galanin neurons in these regions were unresponsive to either dose of nicotine. In contrast, the higher dose of nicotine was required to activate NPY+ neurons in the A1 region and both NPY+ and galanin+ neurons in the locus coeruleus; the C1 region was unresponsive to nicotine. Since plasma ACTH is elevated by the low dose of nicotine and only NTS neurons are activated by this dose, NPY projections from the NTS are likely to contribute to nicotine-stimulated ACTH secretion, in addition to the previously described catecholaminergic neurons.

Distribution of nicotinic binding sites with respect to CRF and neurophysin immunoreactive perikarya within the rat hypothalamus

These studies determined the differential autoradiographic distribution of [125I]alpha-bungarotoxin versus [3H]nicotine relative to the histochemically defined perikarya for neurophysin and corticotropin releasing factor (CRF). Specific [3H]nicotine binding sites occurred in relatively greater density within the neuropil surrounding PVN and SON compared to within the nuclei. In contrast, the highest density of [125I]alpha-BTX sites codistributed with neurophysin immunoreactive perikarya within these nuclei.