Regulation Of The Cell Signaling Pathway Of The Rat CRFR1Receptor Under Hypoxia

Background and ObjectiveHypoxia is one of the main stresses that creatures may encounter in their natural activities, the ability that creatures including human beings response to hypoxia are essential for postnatal development, behavior and health. It is a non-specific stressor; it may induce a stress response. Hypothalamus-pituitary-adrenal axis (Hypothalamus-pituitary-adrenal axis, HPA) is an important body fluid regulation system and stress response system, which makes a variety of stress responses to the new steady-state regulation, to adapt to the unfavorable environment. Hypothalamic corticotropin-releasing hormone (Corticotropin releasing factor, CRF) peptide is brain central regulation of the HPA axis, which regulates the neural and humoral systems. CRFR1is one of G-protein coupled receptors (the G-protein coupled receptor GPCRs), which widely distributed in the central nervous system and peripheral tissues. The CRFR1takes part in hypoxic stress physiological functions regulation, and plays a central regulatory role in hypoxia target genes and proteins which in nerve-endocrine-immune network system. CRF plays an important role with CRFR1 (Corticotropin-releasing factor receptor,1) and CRFR2(Corticotropin-releasing factor receptor,2) that two major receptors.P-Arrestin is an important negative regulator of GPCRs signaling pathway. p-Arrestin and G protein-coupled receptor kinase make G-protein coupled receptors (the G-protein coupled receptor, GPCRs) decrease sensitivity to agonists, the receptor desensitization reaction, and adjustment by bodyendocytosis, signal transduction and cell apoptosis. First, when GPCRs that actived by agonist binded with GRK, phosphorylation occurs. This prompted p-Arrestin from the non-activation crystal structure into structure that the receptor with high affinity. And it combined with the phosphorylation of activated GPCRs to form a trimer, which stopped receptor and G-protein go on.Our laboratory used low pressure cabin altitude hypoxia and studied the high altitude hypoxic stress, to adapt and its mechanism systematicly in the overall and cellular level. The purpose of this experiment is to study the effects on rat brain cortex about the CRF, CRFR1, CRFR2, GRK3and p-Arrestin1/2gene expression though acute and continuous chronic hypoxia. And analysis of the hypoxic regulation of the CRFR1receptor intracellular signaling pathways. To study neuropeptide CRF on cultured rat primary astrocytes regulate the expression of intracellular P-Arrestin1/2, Gas, cAMP, PKC isoforms.Design and MethodsUse of low-oxygen chamber simulated high altitude hypoxia, the rats were exposed to hypoxia7km (8.0%O2,41.83kPa)8h and24h,5km (10.8%O2,54.02kPa) continuous5d and15d. Animals were sacrificed in the end, and the brain was prepared for the experimental study. Real-time quantitative PCR studies the hypoxia on rat brain cortex CRF, CRHR1, CRHR2, GRK3and β-Arrestin1/2gene expressions. Research neuropeptide CRF, β-Arrestin1/2, Gas, cAMP, PKC isoforms expression regulation on cultured primary rat astrocytes.Results1. Acute hypoxia7km for8h increased rat CRF mRNA in cortex, and hypoxia7km for24h did not change CRF mRNA in cortex. Acute hypoxia7km for8h increased rat CRFR1mRNA in cortex, but decreased CRFR1mRNA after hypoxia7km for24h. Acute hypoxia7km for8h decreased rat CRFR2mRNA in cortex, and hypoxia7km for24h did not change CRFR2mRNA in cortex. Chronic hypoxia5km for5d and15d did not change CRF, CRFR1and CRFR2mRNA in cortex.2. Acute hypoxia7km for8h increased rat GRK3mRNA in cortex, but decreased GRK3mRNA after hypoxia7km for24h; chronic hypoxia5km for5d and15d did not change GRK3mRNA in cortex.3. Hypoxia7km for8h decreased rat p-Arrestinl mRNA in cortex, and hypoxia7km for24h did not change β-Arrestinl mRNA in cortex; but hypoxia5km for5d and15d increased rat β-Arrestinl mRNA in cortex. Hypoxia7km for8h did not change β-Arrestin2mRNA in cortex, and Hypoxia7km for24h decreased rat p-Arrestin2mRNA in cortex; hypoxia5km for5d and15d did not change β-Arrestin2mRNA in cortex.4. lnmol, lOnmol CRF30min increased active Gas protein and cAMP in astrocyte and CRF2h and8h did not change active Gas protein and cAMP in astrocyte. CRF8h increased PKCs protein expression, but did not change PKCβⅡ protein expression. CRF8h decreased β-Arrestinl mRNA, but did not change β-Arresting mRNA.ConclusionsAcute hypoxia7km8h increase GRK3, CRF and CRFR1mRNA, and decrease CRFR2mRNA expression.7km24h severe hypoxia decrease GRK3, CRF, CRFR1 and CRFR2mRNA expression and also decrease P-Arrestin2mRNA. Chronic continuous hypoxia increase β-Arrestinl mRNA to decrease CRFR1mRNA expression. This is one of the mechanisms of the CRFR1receptor desensitization. Reduction the body CRF secretion, and increase β-Arrestinl mRNA may be CRFR1receptor desensitization that reduce the basis of the body exposed to chronic hypoxia.In primary astrocytes, the CRF regulates PKA signaling pathway though CRFR1cell receptor that active Gas and cAMP in the short time. And the CRF regulates PKC signaling pathway though PKCε subtype is not PKCβⅡ in the long time. β-Arrestin1mRNA down regulation after CRF incubated8h is consistent with the results of the acute hypoxia7km. This suggests that severe hypoxia or exposure to high levels of CRF decrease β-Arrestinl mRNA and CRFR1mRNA.