Jack Brooks, Ph.D - US grants

We have used the adrenal chromaffin cell as a model catecholaminergic neuron to study the biochemical events which occur during neurosecretion. Considerable evidence suggests that protein phosphorylation plays a role in catecholamine secretion by chromaffin cells. Permeabilized cells were treated with an ATP analog, ATPgammaS (adenosine-5'-(3-thiotriphosphate) in order to "lock" phosphorylation reactions in the (thio) phosphorylated state. Our premise has been that this approach would then permit us to relate the various events of secretion to the phosphorylation reaction. ATPgammaS treatment of the cells irreversibly inhibits secretion and results in thiophosphorylation of several proteins. One of these, a 47 kilodalton (kDa) protein may be either a nucleotide translocator in chromaffin vesicles or be thiophosphorylated as a result of nucleotide translocation. The current proposal has two major goals. The first is to determine the intracellular location and function of this protein. An antibody to the 47 kDa protein will be used to help determine its location in subcellular fractions prepared from cultured cells. The function of the protein will be identified by determining if its thiophosphorylation is causally related to nucleotide transport in chromaffin vesicles isolated from cultured cells. The results of this work will provide the identify for a major phosphoprotein in chromaffin cells. The second goal is to extend our observations with thiophosphorylation of permeabilized cells to intact cells. These studies will determine if thiophosphorylation of intact cells produces the same effect on secretion and protein thio phosphorylation as it does in permeabilized cells. They will also provide evidence that the permeabilized cell model faithfully represents the role of phosphorylation in the function of intact cells.

DESCRIPTION (adapted from Investigator's Abstract): Although salivary secretions are critical to the maintenance of normal oral health, relatively little is known about the mechanisms which control secretion by salivary gland cells. Parotid cells exocytotically secrete the starch digesting enzyme amylase in response to the activation of several cellular receptors. We have devised a procedure for isolation of porcine parotid cells in quantities sufficient for biochemical experiments. The physiological behavior of the cells is similar to that of cells from other sources. These cells are to be studied as a model to provide insight into the role of protein phosphorylation in the control of amylase secretion. Evidence favors the involvement of protein phosphorylation/dephosphorylation reactions in control of the parotid secretory mechanism. However, these are rapid reactions and difficult to restrain long enough to evaluate their role in secretion. A new approach to studying these reactions in porcine parotid cells is proposed.This will involve treatment of isolated cells with inorganic thiophosphate in place of the normal medium phosphate. The cells will use this to make an ATP analog, ATPgS, that will be used in phosphorylation reactions. Since the thiophosphorylation of proteins is nearly irreversible, this will freeze the reactions in the phosphorylated state. The relationship of the phosphorylation reaction to the secretory process is to be evaluated by systematically determining the points at which inhibition occurs and relating this to the thiophosphoprotein profile.The hypothesis is that thiophosphorylation of proteins critical to secretion will result in a cessation of the secretory response.This should be reversible if the cells are given time to replace the modified proteins. For the hypothesis to be correct, the thiophosphoprotein profile should be tightly coupled to technical manipulations which alter cellular secretory response. The investigators expect this proposed work to determine if phosphorylation of cell proteins plays a role in excitation-secretion coupling in parotid amylase secretion.