Michael Meredith - US grants

Conjugation of potentially toxic electrophilic compounds by glutathione (GSH) is a major function of the mammalian liver and a significant mechanism of cellular defense against many hepatotoxic environmental contaminants. Acute depletion of GSH presents a clear threat to the organism by alteration of the GSH/GSSG ratio, the NAD(P)+/NAD(P)H ratio and the cytotoxic events associated with oxidative challenge. Chronic depletion of GSH due to protracted exposure to GSH depleting agents through air and water contamination presents a different set of toxin related challenges induced by the continuous depletion of GSH and the presence of elevated intracellular levels of the GSH conjugate. Resynthesis of GSH is limited by cysteine concentration. The mechanism by which cysteine is supplied varies between cell types; some dependent on dietary absorbtion, some synthesizing cysteine from methionine, while others are proposed to depend on recovery of cysteine by degradation of GSH by Gamma-glutamyl transpeptidase. The proposed research will examine the effect of GSH depletion by diethylmaleate, ethacrynic acid, t-butylhydroperoxide, and 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD), chemicals representative of the major class of xenobiotic GSH depleting agents, or the thiol status of selected cell types. The cells to be examined are primary cultures of rat and human hepatocytes, hepatocarcinoma HR-35 cells, the nontumorigenic epithelial adult rat liver ARL-15C1 and the tumorigenic line ARL-16T2. This collection of cells presents a developmental and transformational gradient from normal to transformed lines with intermediate stages also represented. More importantly, an apparent range of cysteine recovery mechanisms is also represented, from primarily transsulfuration to a strict cysteine requirement for growth. The specific goals to be accomplished are: 1) Evaluation of the thiol status in ARL-15C1 and 16T2 cell lines. 2) Characterization of the mitochondrial pool of GSH in ARL-15C1 and 16T2 and the effect of pool depletion on toxicity. 3) Characterize the thiol status of HR-35 hepatocarcinoma cells. 4) Determine the effect of chronic GSH depletion on chemical toxicity in Gamma-glutamyl transpeptidase containing cells. 5) By cell fusion introduce Gamma-glutamyl transpeptidase into nontumorigenic cells to evaluate alterations in the thiol status. 6) Elucidate mechanism of acute toxicity of TCDD in ARL-15C1. 7) Investigate the regulation of Gamma-cystathionase activity in rat hepatocytes before and after GSH depletion. 8) Examine the thiol status of human hepatocytes and evaluate the importance of the mitochondrial GSH pool.

The nervus terminalis (NT) is a ganglionated cranial nerve which connects the brain and the peripheral olfactory structures. The NT has been found in most vertebrates, including adult humans and, although studied repeatedly with a variety of techniques, the NT remains the only cranial nerve for which the function is completely unknown in humans or in any species. Based on the connections of the nerve and the morphology of cells in the NT ganglion, anatomists have suggested that the nerve may serve a combined sensory and autonomic function. Perhaps one of the most significant recent findings concerning the NT is that some neurons in the nerve contain high levels of luteinizing hormone- releasing hormone (LHRH) immunoreactivity. Some of these LHRH-immunoreactive cells project to brain areas involved with reproduction and some appear to have direct connections with blood vessels. The presence of LHRH-immunoreactivity in the NT neurons and the central and peripheral connections of the nerve has led some researchers to suggest that the NT may be chemosensitive to pheromones and involved in reproductive functions in fish and mammals, possibly by releasing LHRH directly into the blood. However, there has been no electrophysiological evidence for either a chemosensory or a neuroendocrine function for the NT and it is not known if the circuitry of the NT ganglion is compatible with these suggested functions. Investigation of the physiological and morphological organization of the NT ganglion will provide a ground-plan for a more informed investigation of NT function. Elasmobranchs will be used in these studies, because it is only in these animals that the NT is separate from the brain and directly accessible for electrophysiological experiments. Experiments proposed here include: 1) electrophysiological, pharmacological and morphological investigations of NT ganglion cells, which will provide data on the afferent and/or efferent projections of physiologically characterized cells; 2) combined electrophysiology, dye-fills and immunocytochemistry will investigate the function(s) of peptide containing cells, including the possibility that such cells constitute the afferent or efferent component of the nerve; 3) integration of data from all experiments will provide the first coherent picture of the neuronal circuitry of the NT ganglion, including the synaptic interactions between elements within the ganglion.

This proposal requests support for a portion of the costs of the International Symposium on Olfaction and Taste (ISOT) to be held July 7 - 12 1997 in San Diego CA, hosted by the Association for Chemoreception Sciences (AChemS). ISOT meetings are now held every 4 years, hosted on a rotating basis by the chemosensory research organizations of Europe, Japan and the U.S. The ISOT meetings serve as a periodic review and rededication of research efforts in the chemical senses, and as a forum for the world's leading researchers in the field to disseminate the results of their research, and to exchange information and expertise. This interchange, vital to the success of any multidisciplinary research enterprise, is not just between experts but also with the junior investigators, postdoctoral scientists and graduate students that attend the meeting. As such it is a unique opportunity for younger researchers to make contacts and to present their work to an international audience. AChemS provides some assistance to graduate students, members of minority groups and clinical residents to attend. This ISOT meeting is combined with the 1997 annual meeting of AChemS, which affords all members of the chemosensory research community an opportunity to present their latest results as volunteer presentations. Some of these presentations, selected as innovative and important, will be assembled into mini-symposia associated with the fifteen main symposia which form the backbone of the ISOT meeting. This proposal is specifically for support of seven of the fifteen main symposia, in which leading experts will address current and emerging issues in chemical senses research, primarily at the molecular, cellular and systems level. Other symposia, supported from other sources, will address psychophysical, clinical, aging and industrial aspects. Several symposia will include junior researchers and several also include experts from other fields, such as molecular genetics and systems analysis, with techniques and insight to spark innovative research in the chemical senses. A program with so many symposia by outstanding chemosensory and other scientists, each bringing cutting edge insight into research questions, is what makes the ISOT meeting so valuable to chemical senses research. The intellectual chain reactions set off from this critical mass of scientific talent energize the field, contributing in unexpected ways by ideas, information, contacts and access to resources, to many individual research efforts. It is for the assembly of this critical mass that support is requested in this proposal. The symposia, and abstracts of the volunteer presentations, will be published as one of the series of "Olfaction and Taste" volumes, and will be widely disseminated.

The long-term goals of this research are to understand better the operation of a simple cortical circuit, the olfactory bulb. This knowledge will improve understanding of the mechanisms of olfactory discrimination, potentially important in some olfactory dysfunctions; and will contribute to understanding of cortex function in general. The specific aims involve tests of spatial extent and synaptic mechanisms for lateral inhibition between cells associated with regions of the bulb (glomeruli) that receive input from different types of olfactory sensory neurons. This input appears to create a spatial map of odors across the surface of the olfactory bulb. Lateral inhibition, by increasing contrast between weakly and strongly activated regions of the bulb, should therefore increase the brain's ability to distinguish between small differences in the location of activity within that map. One form of lateral inhibition between output neurons of the bulb is mediated by granule interneurons and is reasonably well understood. Another set of lateral connections, via periglomerular (PG) interneurons using GABA and dopamine as transmitters is thought to be inhibitory but is poorly understood. The spatial extent of these lateral influences and the mechanisms of PG cell action are investigated here. The electrical activity of neurons in thin slices of olfactory bulb maintained in vitro will be recorded using patch-clamp electrodes, in response to stimulation of individual glomeruli. Neurotransmitter mechanisms will be investigated using bath-applied drugs that stimulate or block transmitter action. Results of the recordings in main and accessory olfactory bulbs slices will be simulated in computer models to make testable predictions about the function of the intact systems.