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.
