Saliva is important for maintaining oral health. The long term goal of this proposal is to elucidate the molecular mechanisms which regulate the synthesis and secretion of salivary proteins. Previous studies have established that beta adrenergic, alpha adrenergic and cholinergic receptors modulate these functions of salivary glands; however, the underlying molecular mechanisms of action are not fully understood. Mechanistic studies are difficult to perform on short- term primary cultures of salivary glands because of the paucity of homogeneous cultures. The development of immortalized clones of rat and human salivary gland specific cell types should permit one to study the molecular mechanisms by which the synthesis and secretion of salivary gland proteins are regulated.
The specific aims of this proposal are: 1) To develop immortalized clones of rat and human parotid epithelial cells and then characterize the serous clones with respect to ultrastructures, karyotype, tumorigenicity and differentiated functions; 2) to establish requirements for maximal growth and differentiation; and 3) to test the hypothesis that alpha or beta adrenergic and muscarinic cholinergic receptor mechanisms modulate the synthesis and secretion of amylase and proline-rich proteins (PRPs) in immortalized serous cells by changing protein kinases. Plasmid and retroviral vectors carrying the specific DNA sequences from viruses, SV40, polyoma or adenoma type 5, which have been successfully employed to immortalize other epithelial cells will be used to immortalize partoid cells. An immortalized serous clone will be selected for further studies. The levels of mRNA coding for amylase and PRPs, the intra- and extra-cellular levels of PRPs and amylase activity will be measured as a function of time after treatment with alpha- or beta- adrenoceptor or cholinergic agonists or activators of protein kinase C. These changes will be correlated with the activities of beta-adrenoceptor linked adenylate cyclase and alpha-adrenoceptor or cholinergic receptor linked activation of guanylate cyclase; PKA, PKB and PKC activities, and the levels of protein phosphorylation mediated by these kinases. These results will serve as a basis to test another hypothesis, viz. that the stimulation of alpha- or beta-adrenergic or muscarinic-cholinergic receptors modulates the interaction of transcriptional factors with cis- acting DNA regions that code for amylase and PRPs via protein kinases.
