This proposal considers the general hypothesis that the cAMP and phosphoinositide-dependent pathways play coordinate roles in signal transduction leading to the activation of salivary amylase secretion. These studies will be performed in intact and saponin permeabilized rat parotid acinar cells, prelabeled with radioactive precursors (e.g. [14C]arachidonic acid, [3H]inositol to measure [14C]diacylglycerol (DAG) and [3H]inositol triphosphate ([3H]InsP3), respectively. The mass of DAG will also be determined by enzymatic assay using diglyceride kinase; and its metabolic fate will be probed by tlc analysis and the use of putative enzyme inhibitors. Ca2+ mobilization will be assessed using fura-2, and cAMP will be measured by protein binding. We will determine whether agents that raise DAG levels do so by stimulating phospholipases C and/or D. The concentration response and kinetic relationships between DAG formation, cAMP production, [Ca2+]i, and amylase secretion in response to the differential and simultaneous activation of alpha and beta adrenoceptors will be investigated to determine the relative importance of each biochemical parameter in exocytotic secretion. We will examine the comparative abilities of protein kinase C activators (phorbol dibutyrate, dioctanoylglycerol) to alter Ca2+ availability in intact cells and enhance the secretory response to Ca2+ in permeable cells. We will also compare the relative abilities of Ca2+,l ionomycin, and NE to elevate DAG levels and trigger secretion in intact and permeable cells. Also, stable guanine nucleotides will be utilized in permeable cells to determine whether amylase secretion is coupled to DAG production by a G protein. These studies should render strong support for the concept that cAMP and phosphoinosotide-derived messengers underlie the integrative actions of alpha- and beta-adrenoceptor agonists on amylase secretion by the parotid gland. These findings should also provide salient information concerning the role of salivary secretion in dental health.
Showing the most recent 10 out of 24 publications
