IDENTIFYING NOVEL MECHANISMS OF ACTION OF ALLOPREGNANOLONE, A NEUROSTEROID
Carlos Martinez1, Synthia Mellon2.
1University of California, Berkeley, Berkeley, CA, 2University of California, San Francisco, San Francisco, CA.
Allopregnanolone (ALLO), a neurosteroid derived from progesterone, is a naturally-occurring anxiolytic and anticonvulsant compound that activates GABAA receptors. ALLO also affects neuronal proliferation and inflammation, suggesting that ALLO may activate additional receptors. We hypothesize that ALLO may activate the G-protein coupled receptor, TGR5, since ALLO’s structure is similar to lithocholic acid (LCA), a TGR5 ligand. We used mouse enteroendocrine STC1 cells to determine if ALLO could mimic LCA’s actions at TGR5 by assessing calcium uptake and glucagon-like-peptide-1 (GLP-1) secretion. Immunocytochemistry demonstrated the presence of the TGR5 receptor in STC1 cells. To study intracellular events triggered by TGR5 activation, STC1 cells were loaded with Fura-2, a calcium indicator. While both 30 µM LCA or ALLO caused similar increases in Fura-2 fluorescence (i.e.,increased intracellular calcium), dose-dependent effects were inconclusive. TGR5-stimulation of STC1 cells also causes secretion of GLP-1, measured by ELISA. Stimulation of STC1 cells with 30 µM LCA or ALLO increased GLP-1 secretion, but effects were not dose-dependent. Our results demonstrate that STC1 cells express the TGR5 receptor, whose stimulation by LCA or ALLO increases calcium entry and GLP-1 secretion, but the dose-dependent effect of either compound was not ascertained. Future studies will determine maximally effective doses of both LCA and ALLO, and will indicate whether ALLO may be another natural ligand for the TGR5 receptor.