ABSTRACT
Beta-adrenergic-stimulated parotid secretion is believed to be mediated by activation of the cAMP-dependent protein kinase (PK-A). However, the relative roles of the type I and II PK-A isoenzymes are still unclear. Combinations of site-selective, lipophilic cAMP analogues that synergistically activate each PK-A were used to investigate this problem. The selectivity of synergistic activation with these combinations was verified with the partially purified parotid PK-A isoenzymes, using kemptide as a substrate. Synergism in activation of PK-AII was only seen with 8-thiomethyl cAMP (8-TM) and N-6-benzoyl cyclic AMP (N6B), while PK-AI was only synergistically activated by 8-(6-aminohexyl) amino cyclic AMP (AHA) and N6B. Additive activation of each isoenzyme was observed for the combination of 8-TM and AHA. Rates of amylase secretion from dispersed parotid acini in response to secretagogues were determined with a coupled enzyme assay for amylase activity, which was adapted for use in a microplate reader. Cells were stimulated to secrete during 30 min with different doses (0.1-1.0 mM) and combinations of the cyclic nucleotide analogues. Alone, N6B was most effective in stimulating amylase secretion. The basal amylase secretory rate was stimulated by these secretagogues (0.44 mM) to the following extent: 53-fold (N6B), 8-fold (8-AHA), 2-fold (8-TM). In combination at a series of concentrations, only 8-TM + N6B produced synergistic stimulation of secretion, while AHA + N6B and 8-TM + AHA did not.(ABSTRACT TRUNCATED AT 250 WORDS)
Subject(s)
Cyclic AMP/analogs & derivatives , Isoenzymes/drug effects , Parotid Gland/metabolism , Protein Kinases/drug effects , Amylases/drug effects , Amylases/pharmacokinetics , Animals , Cells, Cultured , Cyclic AMP/pharmacology , Drug Synergism , Enzyme Activation/drug effects , Isoenzymes/metabolism , Male , Parotid Gland/drug effects , Parotid Gland/enzymology , Protein Kinases/metabolism , Rats , Rats, Inbred Strains , Thionucleotides/pharmacologyABSTRACT
A coupled enzyme assay for measuring alpha-amylase activity was adapted for analysis with a microplate reader. Activity was quantified by monitoring the cleavage of p-nitrophenol from a chemically defined substrate at 405 nm. Features of this assay method include: low sample volume (10 microliters); economical use of reagent (200 microliters); increased precision due to kinetic nature of assay; linearity with amylase content to 2600 U/liter; capability of processing up to 96 samples within 10 min; facilitated data analysis using readily available software. The ability to rapidly measure amylase content of a large number of samples permitted sophisticated analysis of alpha-amylase secretion patterns from dispersed rat parotid acinar cells. Examination of the dose-dependent increase in the rate of amylase secretion stimulated by carbachol revealed a biphasic response at higher concentrations, where a rapid and transient increase in amylase release was consistently observed during the first 10 min. This initial phase of amylase release was additive with the slower, sustained secretion which was stimulated by carbachol in a dose-dependent manner (Kd = 2.5 microM). Such a biphasic release pattern was not seen with other potent secretagogues (isoproterenol, dibutyryl cAMP) that are not thought to act via a Ca-dependent mechanism. These results suggest that parotid secretion patterns should be studied at a number of time points, which is feasible using the method reported herein.
