Edward w. Westhead - US grants

Secretory vesicles of the adrenal medulla, and of the neurons of the peripheral catecholaminergic system, contain a mixed-function oxidase, dopamine-Beta-hydroxylase, and a b-type cytochrome. The former converts dopamine to norepinephrine, using molecular oxygen and a source of electrons. Ascorbate has been thought to be the physiological electron donor but we have previously found the vesicle membrane impermeable to ascorbate. The function of the cytochrome is unknown but it might serve to convey electrons from a cytoplasmic donor to dopamine Beta-hydroxylase. Njus et al. have recently shown that electrons can be transferred across the membranes of resealed secretory vesicles. To understand the function of this membrane better, we propose to locate possible sites of electron transfer on both sides of the membrane, using a method already demonstrated by others to be useful in studies of other cytochromes. Our approach will be to study the labeling patterns and reaction kinetics of chromous ion with electron-accepting proteins of the secretory vesicle membrane. The conversion of chromous ion to chromic ion due to electron transfer has been shown with cytochrome c to produce irreversible labeling of the cytochrome chromic ion. This approach is expected to identify all proteins capable of carrying electrons. The results should discriminate among those that are on the external face of the vesicle membrane, those on the internal face and those that functionally span the membrane. Determination of reaction stoichiometry (and in the case of cytochrome b, kinetic parameters) is expected to identify any proteins that transfer electrons as functional dimers or higher order complexes.

The hypothesis of this proposal is that substances presented to the erythrocyte by other cells of the circulatory system cause protein phosphorylation leading to an immediate increase in O2 release and an increase in deformability of the cell. The postulate that red cell oxygen affinity is regulatable through protein phosphorylation is novel. The postulate that deformability of the cell is readily alterable is not novel but is controversial. We predict that these events occur on a time-scale that will permit increased oxygen delivery and increased blood flow through a capillary bed at a time of special need--for example, during hypoxia. At a second level of detail our hypothesis states that increased O2 release is the result of increased concentrations of 2,3 diphosphoglycerate (2,3 DPG) caused by a phosphorylation-induced decrease of pyruvate kinase activity. Changes in red cell deformability are postulated to be dependent on reversible changes in the association of certain proteins with the membrane skeleton. Membrane skeletal protein association in turn is controlled in part by the state of phosphorylation of those proteins. Our approach will be to attack the problem at 3 levels simultaneously in the expectation that progress at one level will provide insights that will increase the efficiency of work on the other levels. The first level is to observe biochemial and physical changes in the red cell in response to externally added signal molecules. The second is to study the pathways within the cell which mediate the signal-response coupling. The third level of study is to mimic the passage of erythrocytes through a capillary bed in close contact with endothelial cells and to determine effects that secretions from endothelial cells have upon the red cell under simulated in vivo conditions.

This project focusses on control of the secretory response when cells from the adrenal modulla are stimulated. These catecholamina-secreting cells abovine chromuffin cells) have become a widely used as models for the study of neurosecretion. Two aspects of this project are novel. The first is based in our discovery that nucleotides and adenosine can modulate the secretary response. The cells necessarily secrete high concentrations of nucleotides together with catecholamines so it is very likely that the response to stimulation in vivo is regulated not only by homologous desensitization to acetylcholine, but also by feedback effects from secreted nucleotides. We have linked these two types of secretory modulation in an integrated study of the mechanisms through which they operate. The second new feature of this proposal is the technique we have developed for measuring cytosolic calcium levels and secretary output simultaneously and continuously. In this method, cultured cells are in a flowing stream of buffer which continuously washes away secreted substances. Stimulants and modulators are administrated by injection and by stream-switching. The method provides kinetic details of the secretory process not previously available. It allows us to determine both the time-course of cytosolic calcium concentration and secretory output during desensitization and modulation of the response. This is a critical step in determining that level it which control over secretion is exerted. Plans are described also for further dissecting the controls into those operating at the receptor level, action channels, and it the level of the calcium extrusion pump. "Second messengers" involved in the regulatory pathways will be determined. Based on the working hypothesis that these changes in secretary response operate through protein phosphorylation and dephosphorylation, our final goal is to identify those target proteins whose state of phosphorylation accurately reflects detailed changes in the response.

biomedical equipment purchase;

A common characteristic of nerve cells is that as they are repeatedly stimulated, their responses are diminished. Less commonly, nerve cells will respond to repeated stimulation with enhanced activity. Such changes can be observed at all levels from a single cell to a complex organism, and are thought to represent a form of memory. This behavior is being studied in cells isolated from the adrenal gland of the cow. These cells are widely used as models of nerve cell behavior since they can be isolated and grown in culture, are embryologically related to neurons, synthesize and secrete neurotransmitters and provide sufficient material for biochemical studies. A special technique has been developed by the investigator to stimulate these cells repeatedly and reproducibly and to measure neurotransmitter secretion in real time. By manipulating the cellular control mechanisms that regulate protein phosphorylation, the cells can be make to respond to repeated stimulation by a steady increase in secretion, rather than a decrease. To further understand the cellular mechanisms regulating this response, the role of intracellular calcium levels in the phosphorylation of specific proteins will be studied. These studies promise to increase our understanding of cellular processes underlying the formation of memory.