Corticotropin-releasing Hormone Stimulating Corticotropin-releasing Hormone MRNA Expression Regulated By PKA Signal Pathway In Rat's Hypothalamic

The main characteristics of stress response are a series of physiological and psychological changes from activation of hypothalamus-pituitary-adrenal (HPA) axis. It is very complicated to control the activation of HPA axis in central nervous system. Synthesis and release of corticotropin-releasing hormone (CRH) from hypothalamus plays a key role in the regulation of the HPA axis. CRH controls the stimulating level of HPA axis. Stress-induced activation of the hypothalamus-pituitary-adrenal (HPA) axis and the consequent increase in plasma adrenocorticotropic hormone(ACTH) and glucocorticoid levels are dependent upon CRH.Apart from its effect on the pituitary,it has been suggested that CRH acts within the central nervous system(CNS) and influence a number of behavioral,neuroendocrine,and autonomic responses to stress. The existence of ultrashort positive feedback control of CRH secretion during stress, first suggested by Ono et al in 1985, was confirmed later by many studies, but its mechanism remains to be clarified.In this study, the model of hypothalamic slices in rats was established. After CRH stimulated CRH1R of hypothalamic slices in rats in vitro, the relation between the changes of activity of PKA signal passway and those of CRHmRNA expression observed by immunocytochemistry, Western blotting and RT-PCR means to clarify the regulatory mechanism of signal passway in ultrashort positive feedback control of CRH secretion in hypothalamus, providing theoretical and experimental data for the studies of the stress due to severe traumas.The main results and conclusions are as follows:l. The model of hypothalamic slices of rat in vitro was established. This model with the advantages of the experimental ones in vivo and in vitro and satisfactory repeatability remains the specificity of histocytes and is an ideal animal model for the studies of stress due to severe traumas.2. CRH may cause the remarkable increase in phosphorylated PKA (P-PKA) content in hypothalamic slices in rats. However, CPl54526 could have significant inhibitory effect on the synthesis of P-PKA, but CRF9-41 had slight effect, The concentration ofphosphorylated PKA increased, indicating that binding of CRH to its cognate receptors was associated with the activation of PKA signal pathway.3. CRH may cause the remarkable increase in phosphorylated CREB (P-CREB) content in hypothalamic slices in rats. However, CPl54526 or H89 could have significant inhibitory effect on the synthesis of P-CREB, but CRF9-41 had slight effect, suggesting that CRH can activate the PKA signal passway by stimulating neuronal CRHlR in hypothalamus, Combining with CRH may cause the increase of activated PKA production in the neuron of hypothalamus, indicating the signal pathway may increase the content of P-CREB ,thus regulate transcription activation of the neuron in hypothalamus.4. CRH may cause marked increase of CRHmRNA content in cultured hypothalamic neuron, while CP-154526, H89 may greatly inhibit the increase of CRHmRNA content, indicating PKA signal pathway plays an important role in combination of CRH with CRH1R and stimulating hypothalamic neuron to cause its self gene expression. It suggested that CRH can regulate expression of CRH in hypothalamic neuron by the PKA signal passway.5. An increased intracellular concentration of CRH and CRH1R in the hypothalamus was observed through CRH stimulating hypothalamic slices in rats., while CP-154526 or H89 significantly inhibit the expression of CRH and CRH1R.It suggested expression of CRH in hypothalamic neuron may exist regulatory mechanism of in ultrashort positive feedback.