Abstracts

Studies have shown the endogenous tripeptide TRH (pGlu-His-ProNH2) and specific analogs to be effective against seizures in several animal models and in certain intracatable epilepsies. However, its mechanism of action is unknown. Overactivation of Glutamate (Glu) receptors is implicated in these and other neurodegenerative disorders. We presented that TRH enhances cell survival when added to pituitary adenoma cells (GH3), neuronal pheochromacytoma cells (PC12), and cultured fetal hippocampal neurons before, during or after toxic Glu exposure (Endocrine Soc. Abs., 2001; 2003). Herein we utilized the superfused slice preparation in vitro to more closely mimic in vivo neural networks. We used dentate gyrus slices as this subfield exhibits the highest TRH receptor density in the hippocampus and is the major subfield of perforant path input. We tested the hypothesis that TRH is neuroprotective via downregulation of its signal transduction cascade.
Slice integrity was determined in hippocampal slices (500 [micro]m) equilibrated for 2 hours in low Ca++ (1.0 mM) KRB. Slices were superfused in normal KRB and effluent samples taken at 0, 12, 24, 36, 48 & 60 hours for LDH assay while tissue was assayed for Caspase activity. Next, dentate gyrus slices or anterior pituitaries were equilibrated for 2 hours in low Ca++ (1.0 mM) KRB, then superfused for 12 hours in either normal Ca++ (2.5 mM) KRB with 10 [micro]M TRH, 3-Me-His-TRH (high affinity analog), or Free Acid-TRH (inactive metabolite) -containing KRB. After superfusion, slices were homogenized and equal proteins were subjected to Western blotting separately with antibodies against G[alpha]q/11 and [beta]-amyloid precurser protein (APP). Blots were scanned and protein band densities were determined.
LDH in the superfusate and tissue caspase activity was not significantly different from controls over time or treatment after 12 hours. Morphology via cresyl violet staining revealed no apparent difference between slices superfused over time or with drugs. Slices superfused with TRH or 3-Me-His-TRH showed significant (P[lt]0.05) reductions (79 & 93%, respectively) in G[alpha]q/11 compared to controls. Superfusion with Free Acid-TRH showed no significant change. Similar results were seen with anterior pituitaries. No change in APP was evident with any of the TRH analogs.
These results indicate that prolonged exposure to TRH results in considerable downregulation of Gaq/11 in the hippocampus. Since Group 1 metabotropic glutamate receptors and TRH receptors share the same signal transduction cascade in dentate gyrus granule cells, we suggest that the anticonvulsant/neuroprotective role of TRH in part may involve heterologous receptor downregulation of metabotropic glutamate receptors.
[Supported by: Binational Science Foundation (Israel & US) and NIH grants]