Metabotropic Glutamatergic Signaling Contributes To Hypoxia-induced Chemoreflex Plasticity Of Carotid Body

BackgroundPatients with obstructive sleep apnea can induce hypertension due to recurrent intermittent hypoxemia,but the mechanism is not clear.The carotid body is a chemoreceptor that senses changes in arterial partial pressure of oxygen,reflexively regulating the respiratory system and the cardiovascular system.Glutamate receptors are divided into metabotropic glutamate receptor（mGluR）and ionotropic glutamate receptor（iGluR）,both of which have a coordinating role in synaptic plasticity.Our previous study found that cyclic intermittent hypoxia regulates carotid body plasticity through ionotropic glutamatergic signaling pathways,activates sympathetic nerves,and induces hypertension.We speculate that the metabotropic glutamatergic signaling pathway exists in the carotid body and participates in the regulation of carotid body plasticity.Objective1.To elucidate the expression of glutamate metabolic signaling pathway molecules in the carotid body.2.To determine the localization of mGluR1/5 in carotid body.3.To elucidate the regulation of persistent hypoxia on downstream signal molecules of mGluR1/5 signaling pathway in isolated carotid body.4.To elucidate the effect of cyclic intermittent hypoxia on the expression of mGluR1/5 mRNA in the carotid body.Method1.Detection of mRNA expression of metabotrapic glutamate receptor（mGluR） subunits in human and rat carotid bodies by RT-PCR technology.2.Immunofluorescence double labeling technique was used to identify the localization of mGluR1 and mGluR5 in the carotid body type I cells and type II type cells by staining with type I cell marker,tyrosine hydroxylase（TH）and type II cell marker,glial fibrillary acidic protein（GFAP）.Detecting the localization of mGluR1/5 in neuron and nerve fiber by co-stainning with neurofilament（NF）.3.Carotid body culture and Western blot analysis:1)Carotid bodies were cultured under normoxic environment to detect the regulation of mGluR1/5 downstream signal pathway molecular proteins by DHPG,JNJ16259685 and MPEP.2)The effects of JNJ162599685 and MPEP on the molecular protein level of mGluR1 and mGluR5 downstream signal pathway responsed to hypoxia（5%O₂-5% CO₂-90%N₂）were detected with western blot analysis.DHPG:3,5-dihydroxyphenylglycine,nonselective mGluR1/5 agonist.JNJ16259685:（3,4-Dihydro-2H-pyrano[2,3-b]quinolin-7-yl）-（cis-4-methoxycycloh-exyl）-methanone,selective mGluR1 antagonist.MPEP:2-methyl-6-（2-phenylethynyl）-pyridine,monohydrochloride,selective mGluR5 antagonist.4.Animal model:adult male SD rats were exposed to cyclic intermittent hypoxia (3min of 10%O₂ followed by 1min of 21%O₂;10 episodes h^-1;8h d^-1;for 14 days).RT-PCR technique was used to detect the effect of cyclic intermittent hypoxia on the expression of mGluR1/5 mRNA in the carotid body of rats.Result1.Through RT-PCR,we found that multiple mGluR subunits mRNA expressed in human and rat carotid bodies:1)Expressions of mGluR1,mGluR2,mGluR3,mGluR4,mGluR5,mGluR6,mGluR7 and mGluR8 mRNA were existed in the rat carotid body.2)Human carotid body expressed mGluR1,mGluR5,mGluR6,mGluR7 and mglur8 Mrna.However,there were no visible amplified bands of mGluR3 and mGluR4 mRNA on the human carotid body.2.The results of double immunofluorescence staining showed that:1)mGluR1 co-localized with TH but not GFAP,indicated that the localization of mGluR1 was constrained within type I cells of carotid body.2)In contrast,the immunostaining of mGluR5 had a fine appearance and was distributed widely in the carotid body included type I and type II cell,as evidenced by an overlap with the localization of TH and GFAP.Furthermore,it may also be expressed in capillary endothelial cells according to the immunoblotting of mGluR5 and the morphology of carotid body.Both mGluR1 and mGluR5 expressed in neuron but not nerve fiber.3.The results form in vitro culture of rat carotid body show that:1)In normoxic condition,DHPG increased the phosphorylation level of CaMKII and Akt compared with the control group,and this activation inhibited by JNJ16259685 （mGluR1 antagonist）and MPEP（mGluR5 antagonist）,indicating that mGluR1/5 signaling pathway existed in carotid body.2)Compared with the control group,hypoxia increased the phosphorylation level of Akt and ERK,and JNJ16259685 inhibited hypoxia-induced the phosphorylation of Akt and ERK,but MPEP had no effect on it.It suggests that mGluR1 signaling pathway may be involved in the regulation of carotid body response to hypoxia.3)Compared with control group,continuous hypoxia increased the expression level of TH,and JNJ16259685 faciliated CH-induced TH activation.However,On the contrary,MPEP could inhibit TH expression and make it significantly lower than that in control group.These results suggested that mGluR1/5 is related to TH activation induced by continuous hypoxia,and mGluR1 and mGluR5 have the opposite effect on it.It seemed that mGluR1 has inhibitory effect on TH,while mGluR5 can activate TH.4.By establishing animal models,it demonstrated that rats exposed to cyclic intermittent ypoxia for 14 days could up-regulate the expression of mGlur5 mRNA,but had no ffect on the expression of mGluR1 mRNA,it suggesting that cyclic intermittent hypoxia may regulate carotid body plasticity through mGluR5 signaling pathway.Conclusion1.Both human and rat catotid body expressed multiple mGluR subtypes mRNA.2.The localizations of mGluR1 and mGluR5 are different in rat carotid body.mGluR1 ocalized in type I cell of carotid body and neuron.However,mGluR5 was widely xpressed in type I cell,type II cell,neuron and capillary endothelial cell of carotid ody.3.CIH regulated plasticity of carotid body by upregulatd mGluR5 expression.4.mGluR1/5 signaling pathway may involved in hypoxia-induced plasticity of carotid ody.