Carmine J. Coscia - US grants

neurotransmitter biosynthesis; microsomes; cell membrane; Golgi apparatus; prosencephalon; catecholamines; synapses; electrophysiology; growth /development; newborn animals; endoplasmic reticulum; neurotransmitter metabolism; autoradiography; histochemistry /cytochemistry; density gradient ultracentrifugation; electron microscopy; high performance liquid chromatography;

It has been estimated that maternal drug abuse and its treatment is placing epidemic-proportions of offspring at considerable risk each year in the United States alone. Exposure of pregnant humans and laboratory animals to opiates can result in a range of problems for their fetuses including developmental retardation. Since the etiology of many of the developmental abnormalities is unknown, it is important that we understand the types and functions of opioid systems in normal developing mammalian brain. Use of this knowledge to elucidate the mechanism by which opiate drugs influence opioid receptor mediated actions during early brain development may allow us to determine, prevent or reverse pathological effects of these drugs. The objectives of this proposal are to continue the characterization of opioid receptors in rodent brain focusing on the 55 kDa molecular weight mu and the kappa opioid binding sites that we have detected in the perinatal period. By affinity labeling with [125I]-beta-endorphin the glycoprotein nature and subcellular compartmentation of the 55 kDa mu site will be examined to determined whether it is an incompletely processed precursor or a transiently expressed functional receptor. Using homologous and heterologous competition binding assays followed by a computer analysis, the subtype specificity of mouse perinatal mu and kappa sites will be probed. The sequence of appearance of opioid receptor subtypes will be correlated with that of the endogenous opioid peptides, G protein subunits and the neurotrophic functions described below that are prevalent at this stage of development. The role of individual receptor subtypes and their mechanism of action in opioid receptor mediated inhibition of brain DNA synthesis and ornithine decarboxylase activity will be investigated in cultured rat fetal brain cell aggregates and in vivo. Mixed agonist-antagonists such as buprenorphine will be administered to pregnant rats or cultured rat fetal brain cell aggregates and the consequences to normal expression of opioid receptors and of their neurotrophic effects assessed. Since buprenorphine has recently been demonstrated to be a good candidate for pharmacotherapy in cocaine and /or heroin abuse, these results may be of significance to the treatment of maternal drug abuse.

This is a Shannon Award providing partial support for research projects that fall short of the assigned institute's funding range but are in the margin of excellence. The Shannon award is intended to provide support to test the feasibility of the approach; develop further tests and refine research techniques; perform secondary analysis of availability data sets; or conduct discrete projects that can demonstrate the PI's research capabilities or lend additional weight to an already meritorious application. Further scientific data for the CRISP System are unavailable at this time.

APPLICANT'S ABSTRACT: The broad goal of our research is to delineate the mechanisms involved in the neurotrophic actions of opioids. The particular focus of this proposal involves identifying elements in the receptor-dependent signalling system mediating opioid inhibition of DNA synthesis in rat brain cell cultures. There are four specific aims: AIM 1) determine whether the neurotrophic action of opioids is dependent on modulation of the synthesis, activity or turnover of protein kinase c; AIM 2) determine whether phospholipase D (PLD) has a role in the mechanism of opioid action; AIM 3) determine whether the neurotrophic action of opioids is dependent on modulation of the synthesis, activity or turnover of mitogen- activated protein (MAP) kinase; AIM 4) establish the nature of the transducer involved in the pertussis toxin- insensitive, mu receptor-mediated action of opioids. A variety of biochemical, immunological, cell biological and molecular biological techniques will be used taking advantage of fetal rat brain cell aggregate, rat C6BU-1 glioma and opioid receptor-transfected CHO cell culture systems, all of which are responsive to opioids. Since maternal drug abuse and its treatment places hundreds of thousands of offspring at risk each year in the United States alone and infants exposed to opiates in utero are afflicted with a range of problems including developmental delays, the answers sought in these studies both have fundamental significance, and bear on an important public health problem.

DESCRIPTION (Applicant's abstract reproduced verbatim): Maternal drug abuse places hundreds of thousands of progeny at risk each year in the United States. This problem has become more severe recently, due to the greater availability of purer heroin and an increase in young abusers. The effect of opioids on mitogenic pathways in the fetus of drug-addicted mothers may contribute to the physiological and psychological delays observed in their offspring. Glial cell proliferation plays an integral role in normal brain development. Opioids modulate mitogenic signaling in astrocytes from developing brain. The broad goal of our research is to delineate mechanisms involved in opioid modulation of mitogen-mediated glial cell growth and differentiation. Our previous studies disclosed major features of the mechanism of the mitogenic action of an agonist of the endogenous kappa OR in C6 glioma cells, an astrocytic model system. Evidence for both receptor endocytosis-dependent and -independent opioid mitogenic signaling has been discovered. The particular focus of this application is to delineate how and where in the cell opioid and RTK signaling converge to modulate proliferation of primary astrocytes from perinatal brain of various ages. There are three specific aims: AIM 1) Identify the point of convergence between opioid and mitogenic signaling pathways upstream of the MAP kinase cascade in primary astrocytes. AIM 2) Test the hypothesis that opioid receptor internalization is required to "switch" opioid signal transduction to a MAP kinase-modulating mode for mitogens. AIM 3) Test the hypothesis that mitogenic signaling component(s) are desensitized upon chronic exposure to opioids that attenuate mitogen-induced proliferation of astrocytes. We will use biochemical, immunological, recombinant protein and other molecular biological techniques to study opioid signaling in primary astrocytes. The answers sought in these mechanistic studies have fundamental significance, and also bear on the important public health problem of maternal drug abuse. They may also have implications for understanding some aspects of AIDS dementia.

DESCRIPTION (provided by applicant): Heroin abuse has increased significantly in recent years especially among the young. The statistics emphasize the need for new approaches to the treatment of maternal heroin abuse. Currently, the only drug approved for treating opiate dependence in pregnant U.S. women is methodone. Successful clinical trials suggest that buprenorphine will soon become a useful addition to the armamentarium of heroine addiction pharmacotherapy. Buprenorphine is an opiate alkaloid that acts as a mu opiate partial agonist, a nociceptin/orphanin receptor full agonist and a kappa antagonist. In comparison to methadone, buprenorphine has a lower abuse liability, a longer lasting action, a less intense withdrawal state and less physical dependence. Our previous findings demonstrate that in utero buprenorphine administration induces mu and kappa opioid receptor adaptation in developing rat brain. By acting at mu, kappa and nociceptin/orphanin receptors, buprenorphine may exert a blockade of mesolimbic reward mechanisms, thereby accounting for its ability to antagonize the reinforcing effects of heroine. Our focus will be to test the hypothesis that gestational opiates induce changes in opioid receptor binding density, phosphorylation and G protein coupling, an estimate of signaling capacity, thereby altering opioid receptor function in mesolimbic regions of offspring. We will determine whether correlative changes occur to these 3 signaling parameters. Discrete mesolimbic structures of developing rats exposed to gestational buprenorphine plu and minus naloxone will be compared to those treated with methadone and heroine. There are 3 specific aims: AIM 1. Measure gestational buprenorphine-induced opioid receptor adaptation in mesolimbic brain regions of developing offspring and dams by receptor autoradiography and semi-quantitative immunofluorescence confocal microscopy. AIM 2. Measure buprenorphine-induced mu opioid receptor phosphorylation in selected mesolimbic brain regions of developing offspring and dams by immunoblotting. AIM 3. Measure changes in mesolimbic opioid receptor activation of G proteins induced by gestational buprenorphine. This is an exploratory/developmental grant proposal (R21) to develop semi-quantitative immunological and autoradiographic techniques with anatomical and cellular resolution in neonatal mesolimbic brain regions. They will be used to determine whether gestational opiate exposure alters opioid receptor adaptation, receptor phosphorylation and/or coupling to G proteins in the mesolimbic regions of offspring brain.