Involvements Of G Protein-coupled Corticotropin Releasing Factor Receptor-1 In Hypobaric Acute Hypoxia-induced Rat Brain Edema And Pulmonary Edema

The Qinghai-Tibet Plateau in west China, the most significant geographical feature is the low oxygen level. Hypoxia not only affects people health, but also leads to serious altitude sickness. High altitude cerebral edema (HACE) and high altitude pulmonary edema (HAPE), the most serious altitude sickness, often occur in severe hypoxia environment, which would endanger life if improper handling. Its pathogenesis remains obscure, and the mechanism would be important in both theory and clinical practice.As a non-specific stressor, hypoxia can induce stress response generated. The hypothalamus-pituitary-adrenal axis (HPA axis) is the humoral regulation system and the stress response system, making a variety of stress response to adapt the harmful environment into a new homeostasis. Hypothalamic corticotropin releasing factor (CRF) peptide is the central regulator of HPA axis, which regulates both body fluid system and nervous system. Previous studies in our lab found that hypoxia activated CRF release, CRF mRNA and CRFR1 receptor express in hypothalamic PVN, which led to stimulate HPA axis cascade. Corticotropin releasing factor receptor-1 is a member of G-protein coupled receptor family, widely distribute in the central nervous system and periphery system. We also found that CRFR1 participate hypoxia-induced stress responses, playing a central role in regulation of important hypoxia target genes and proteins in neuro-endocrine-immune network system. Aquaporins (AQPs) are a family of membrane channels that serve as selective pores through which water flow cross the plasma membranes in many cell types. AQP4 is the major water channel in brain tissue, strongly enriched in astrocytes end-feet. AQP4 plays an important role both in the cerebral water balance in normal physiological condition and in the formation of brain edema during pathological injury. Therefore, we assume that:severe hypoxia stress could stimulate CRF secretion from hypothalamus to activate CRFR1, driving intracellular signaling pathways to increase AQP4 water channel permeability, involving in the formation of brain edema at last. Furthermore, CRFR1 and transcriptor NF-kB may play a role in hypoxia-induced pulmonary edema. Therefore, the paper is to explore whether CRFR1 involves in HACE and HAPE as well as what is the intracellular signaling mechanism.In this study, we developed a hypoxia model for brain edema and pulmonary edema. The rats were exposed to hypoxia (7.8% O2, equal to 7000m altitude) in a hypobaric chamber for 8 hours. Magnetic resonance imaging was applied to measure the ADC value for brain cell water. The water content in brain and lung was assessed by dry/wet ratio. The AQP4 and AQP5 protein expressed in rat brain cortex and in lung tissue respectively were determined by Western blot. ET-1 in cortex and corticosterone in plasma was detected by ELISA kit. ET-1 in lung was detected by RIA. Coexistence of CRFR1 with AQP4 protein was showed by confocal immunofluorescence in brain cortex and in primary cortex astrocytes cell. The AQP4 and AQP5 phosphorylation was detected by protein immunoprecipitation. Intracellular [Ca2+]i was determined by Fluo3/AM calcium indicator. The water permeability was measured using laser scanning confocal microscopy. And the level of nitric oxide and nitric oxide synthase active unit in the pulmonary tissue were measured by colorimetric method.This study will address whether the CRFR1 contributes to cerebral and pulmonary edema, and what the intracellular signaling pathway driven from CRFR1 is linked to AQPs, making its phosphorylation and altering its permeability to water, then induced cell swelling and the brain and pulmonary edema. Part One1 G-protein coupled CRFR1 mediates acutely simulated altitude hypoxia-induced rat brain edemaAcutely simulated hypoxia (7.8% O2,8 h) significantly increased water content in brain and the ADC value for water permeability, leading brain edema, and the effects were blocked by i.p. preinjection with CRFR1 antagonist CP154,526 (30mg/kg).2 G-protein coupled CRFR1 mediates AQP4 and ET-1 expression induced by acutely simulated altitude hypoxia in rat brainConfocal immunofluorescence image showed coexistence of CRFR1 protein and AQP4 channel protein in rat prefrontal cortex.Western blot results showed that AQP4 channel protein was enhanced in the hypoxic rat prefrontal cortex (P<0.01). The effect was significantly blocked by i.p. preinjection of CRFR1 antagonist CP154,526 (P<0.05 vs. hypoxia), but not by i.p. preinjection of NF-kB inhibitor PDTC(pyrrolidinedithio carbamate ammonium), or combination of CP154,526 plus PDTC. These antagonists had no effect on AQP4 expression in control rat.The hypoxia significantly increased ET-1 expression in rat prefrontal cortex, which was blocked by i.p. preinjection of CP154,526, PDTC, or combination of CP154,526 plus PDTC.The hypoxia significantly enhanced plasma corticosterone (P<0.01), which was blocked by i.p. preinjection of CRFR1 antagonist CP154,526 and combination of CP154,526 plus PDTC, partly blocked by PDTC. And these antagonists had no effect on plasma corticosterone.3 Modulations of intracellular pathway driven by G-protein coupled CRFR1 in AQP4 channel protein in primarily cultured rat astrocytes under the hypoxiaConfocal immunofluorescence image showed that coexistence of CRFR1 protein and AQP4 channel protein in the primary cortical astrocytes.In cultured primary astrocytes, CRF induced intracellular [Ca2+]i fast increase, following water permeability enhancement, which was blocked by i.p. preinjection with CRFR1 antagonist CP154,526 for 30min.Incubation of rat primary cortical astrocytes with CRF for 10 or 30min increased cAMP accumulation in the cells, which was blocked by preincubation with CRFR1 antagonist for 30min. In CRF treated astrocytes for 30min, but not 10min, induced AQP4 protein phosphorylation at PKA site of AQP4 channel protein. Again CRF induced for 30 min AQP4 protein phosphorylation occured at PKC site of the AQP4 protein, which was blocked by preincubation with PKC antagonist (Bisindolylmaleimide) for 30min. PKG sites appeared to be not involved in CRF challenged astrocytes, and the PKG antagonist KT-5823 had no effect.In cultured primary astrocytes treated with 10nM CRF for 8h, cAMP levels increased significantly in these cells, which were blocked by preincubation of CRFR1 antagonist. Exposed to CRF for 8h or hypoxia 8h had no effect on AQP4 protein phosphorylation at PKA site. Western blot showed CRF for 8h induced AQP4 protein expression.Part Two1 Effects of G-protein coupled CRFR1 and NF-kB on rat pulmonary edema induced by acutely simulated hypoxiaAcutely simulated hypoxia (7.8% O2,8 h) significantly increased water content in rat lung, which was blocked by i.p. preinjection of NF-kB inhibitor PDTC, but not by i.p. preinjection of CP154,526, or combination of PDTC plus CP154,526.2 Effects of G-protein coupled CRFR1 and NF-kB on rat pulmonary ET-1 upregulation induced by acutely simulated hypoxiaNF-kB mediated rat pulmonary ET-1 upregulation. Acute hypoxia (7.8% O2,8 h) significantly induced ET-1 expression increase in rat lung tissue, the effect was not blocked by i.p. preinjectiont of CRFR1 antagonist CP154,526 (15mg/kg), whereas enhanced by i.p. pretreatment of antagonist CP154,526 (30mg/kg) or combination of PDTC plus CP154,526 (30mg/kg). However the effect was blocked by single pretreatment of PDTC.3 Effects of G-protein coupled CRFR1 on downregulation of rat pulmonary NO and NOS activity induced by acutely simulated hypoxiaCRFR1 antagonist induced further downregultion of the NO content and NOS activity. Acute hypoxia (7.8% O2,8h) induced NO level decrease in rat lung, which was further reduced by i.p. pretreatment of antagonist CP154,526 (30mg/kg), not by 15mg/kg. Acute hypoxia downregulated NOS activity and the effect was further reduced by i.p. pretreatment of antagonist CP154,526 15 or 30mg/kg. But hypoxia or CRFR1 antagonist had no effect on inducible NOS activity.4 G-protein coupled CRFR1 is not involved in the regulation of hypoxia-induced upregulation of AQP5 channel proteinAcute hypoxia (7.8% O2,8h) upregulated the expression of AQP5 channel protein in rat lung, the effect was not blocked by i.p. pretreatment of CRFR1 antagonist CP 154,526.Conclusion:1 Acute simulated hypoxia (7.8% O2,8 h) induces rat brain edema, which is associated with G-protein coupled CRFR1 triggering, CRFR1 triggering mediates hypoxia-induced ET-1 and AQP4 upregulation in the brain. ET-1 upregulation contributes to brain edema via NF-kB transcription2 In the primary cultured rat cortical astrocytes, CRFR1 and AQP4 co-exist. CRF stimulates CRFR1 on the astrocytes, elevating [Ca2+]i concentration, activating the cAMP/PKA and PKC pathways, inducing AQP4 phosphorylation at the PKA and PKC site of AQP4 channel protein which lead to increase AQP4 channel permeability and make the astrocyte swelling.3 The acute hypoxia (7.8% O2,8 h) increased water content in rat lung and pulmonary ET-1 expression, as well as decreased NO level, total NOS activity, and upregulated AQP5 channel expression, which all associate with hypoxia-induced pulmonary edema. Nuclear transcription factor NF-kB may contribute to pulmonary edema by upreguating ET-1 expression. And CRFR1 may potentially be involved in altitude hypoxic pulmonary edema through the NO/NOS pathways and the ET-1 expression.