Biochimica et biophysica acta · 2011
Effect of bitter compounds on amylase secretion in murine submandibular glands: Signaling pathway mechanisms.
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Dasso Maximiliano, Pagotto Romina, Pignataro Omar P, Diez Roberto A, Sales María E
Аннотация
BACKGROUND: Amylase is synthesized in submandibular glands (SMG) and released into the oral cavity to degrade carbohydrates in the mouth. Bitter taste receptors (T2R) belong to the G-protein coupled receptor (GPCR) family and are expressed in the taste cells and also in the digestive tract.
METHODS: The activity of amylase secreted by murine SMG was measured, detecting maltose by Bernfeld's method. Amylase and T2R6 were detected by imunohistochemistry and Western blot. The expression of Ggustducin, Gi, and phospholipase Cβ2 was also studied by Western blot. cAMP levels were measured by radioimmunoassay and inositol monophosphate production was quantified by ELISA.
RESULTS: Theophylline, denatonium and cycloheximide exerted a dose-dependent inhibition on amylase secretion. This effect was reverted by preincubating SMG with an anti-Gαi antibody. cAMP production was increased by the same compounds, an effect that was also abrogated by an anti-Gαi antibody. Bitter compounds reduced inositol monophosphate formation in SMG and H-89, a protein kinase A inhibitor, reverted this action, revealing that this protein kinase down regulates phospholipase C activity.
GENERAL SIGNIFICANCE: We demonstrated that theophylline, denatonium and cycloheximide inhibit salivary amylase secretion, activating an intracellular signaling pathway that involves cAMP and phospholipase C, that cross talks via protein kinase A.
METHODS: The activity of amylase secreted by murine SMG was measured, detecting maltose by Bernfeld's method. Amylase and T2R6 were detected by imunohistochemistry and Western blot. The expression of Ggustducin, Gi, and phospholipase Cβ2 was also studied by Western blot. cAMP levels were measured by radioimmunoassay and inositol monophosphate production was quantified by ELISA.
RESULTS: Theophylline, denatonium and cycloheximide exerted a dose-dependent inhibition on amylase secretion. This effect was reverted by preincubating SMG with an anti-Gαi antibody. cAMP production was increased by the same compounds, an effect that was also abrogated by an anti-Gαi antibody. Bitter compounds reduced inositol monophosphate formation in SMG and H-89, a protein kinase A inhibitor, reverted this action, revealing that this protein kinase down regulates phospholipase C activity.
GENERAL SIGNIFICANCE: We demonstrated that theophylline, denatonium and cycloheximide inhibit salivary amylase secretion, activating an intracellular signaling pathway that involves cAMP and phospholipase C, that cross talks via protein kinase A.