A Study On Relaxation Effect Of Bronchial Epithelium Relaxing Factor On Pulmonary Artery And On Its Signal Pathway

Pulmonary artery and bronchus has the same origin during embryogenesis; anatomically, they are close, this lay the foundation of signal transduction. Pulmonary vessel resistance control is a complex and unclear process. Although the relationship between airway and pulmonary vessel bed is reported, the bronchial signal transduction and the mechanism of its effect on pulmonary artery are rarely studied. It is reported about trachea effect on pulmonary artery which indicated that smooth muscle of pulmonary artery relaxes likely through nitric oxide (NO) release. There are three know niitric oxide synthase (NOS), includes endothelial NOS (eNOS), neuronal NOS (nNOS) and inducible NOS (iNOS). The three isoforms of NOS are abundantly present in the lung. In pulmonary vasulature, eNOS is presented soley in the endothelial cells. Smooth muscle cells express nNOS constitutively, and iNOS is only expressed following stimulation (e.g. cytokines). It is proved that eNOS can phosphorylate phosphatidylinositol 3-kinase (PI3-kinase) through which produce amount of NO, and NO is expired by neonate is reported, too. But under physical condition, bronchial epithelium derived relaxing factor (BrEpDRF) can relaxes pulmonary artery and the effect of nNOS on NO synthesis during this process is rarely reported.PI3-kinase is a multifunctional, hereodimer enzyme composed of a catalytic and a regulatory subunit. The PI3-kinase family is subdivided into three classes. ClassⅠPI3-kinases consist of either aα-,β- orγ-catalytic subunit. This class is present in smooth muscle cells and through which to modulate contraction. Inhibitors of PI30kinase (Wotmannin and LY294002) work predominantly by inhibiting the p110γ-catalytic subunit. Akt (protein kinase B) is a known effector signaling by factors that activate the PI3-kinase. Three Akt isoforms are known: Akt1, Akt2, Akt3. All 3 isofroms are widely expressed in various tissues, but Akt1 is the one most abundant in the lung. In ustimulated cell, Akt protein exists in cytoplasm and the tow regulatory phosphorylation sites at threonine at 308 and serine at 473 are in an unphosphorylated state. Following stimulation, Akt is reruited to plasma membrane and sequentially phosphoralated at T308 and S 473 by upstream kinases referred to as 3-PI-dependent protein kinase 1 (PKD1) and PKD2, respectively, to yield a fully activated kinase. This pathway can be activated by growth and other tissue secreted factors. Activation of Akt can directly phospholate guanylate cyclase inhibitor ODQ through which improve NO production. Rarely known function of nNOS, but its isoforms is activated by PI3-kinase/Akt pathway.Methacholine (Mch) is an agonist of Ach which can stimulate M1 subtype of muscarinic receptor via which to induce smooth muscle contraction of airway. Similar to Mch can also relax pulmonary artery of adult rat through stimulating M1, M3 phenotype of muscarinic receptor which is endothelium dependent. It is reported firstly by Flavahan et al. that airway epithelium derived relaxing factor is capable of relaxing of bronchus smooth muscle in cyon, in that Mch or histamine relaxes aorta smooth muscle in endothelial denuded adult rat, which a proved observation on basis of the existence of airway epithelium later.U466196 is an analogue of thromboxane A2 (TXA2) which can produce smooth muscle contraction; and methacholine (Mch) can relaxes smooth muscle of pulmonary artery; L-NIO, 7NINA and 400W are inhibitor of eNOS, nNOS and iNOS; LY294002 is a PI3-kinnase inhibitor and ODQ is a NO-cGMP pathway inhibitor, respectively. To study on BrEpDRF and the way of synthesis of NO and the signal pathway in neonate rat, we employed male Sprague-Dawley of 4-8 days. After sacrifice them by pentobarbital overdose i.p. injection, free stage 4 pulmonary artery with/without bronchi attach. Measure contractile force of pulmonary artery attached epithelium denuded bronchi is stimulated by U46619; observe vessel relaxation rate of pulmonary artery with bronchus attached stimulated by Mch under different types of NOS inhibitors, L-NIO, 7NINA and 1400W after pre–stimulated by U46619 to study function of different NOS during the process of NO production. Inhibit relaxation effect of Mch by LY294002 and ODQ is employed to observe if signal is conducted by PI3-kinase/Akt and NO-cGMP pathway. To observe their inhibitory effect, Wormannin, LY294002 and ODQ with Mch are stainned by DAF to compare with the function of Mch alone.Results: Stimulated by U46619, pulmonary artery with epithelium denuded bronchus contract force is averagely 0.36 mN/mm2,artery alone is averagely 1.89 mN/mm2 under stimulation, (p<0.01). After stimulated by U46619, the relax rate of pulmonary artey with bronchus attached is 40.04% stimulated by Mch, and the relax rate is 23.30% stimulated by Mch with eNOS and nNOS inhibitors, (p<0.01); the relax rate is 38.59% stimulated by Mch with eNOS inhibitor, which is higher than that of caused by Mch with both inhibitors (p<0.01); the relax rate is 37.63% stimulated by Mch with iNOS and eNOS inhibitor together, which is not less than that of stimulated by Mch alone, (p>0.01), respectively. After stimulated by U46619, the relax rate of pulmonary artery with bronchus attached is 39.37% stimulated by Mch, and the relax rate is 11.72% stimulated by Mch with PI3/Akt inhibitor LY294002, compared significantly (p<0.01); the relax rate is 2.15% stimulated by Mch with NO-GMP inhibitor ODQ, which is less than that of stimulated by Mch alone statistically (p<0.01), respectively. Both are stained to observe. U46619, an analogue of thromboxane A2, stimulates pulmonary artery bronchus epithelium denuded/intact group differed significantly implicate that BrEpDRF is associated with pulmonary relaxation under physiological condition, which supports the existence of BrEpDRF. It is found in this study that this effect abolished when the epithelium of bronchus was denuded. It is found this is true even when bronchus floated in Kresb-Henseleit buffer and is nNOS independent by others. Thus implicate BrEpDRF is soluble.Mch decreases relax effect of Pulmonary artery when inhibit by L-NINO and 7NINA, a inhibitor of eNOS and nNOS respectively, indicateds endothelial and neuronal phenotype acted. Inhibitory effect of both is compared with eNOS alone, which shows nNOS is required, and Mch is eNOS independent, and iNOS do not participate in NO synthesis under physiological condition, which is different from inhibitory effect by others, but supports no changes in pulmonary smooth muscle contaction. L-NINO and 1400W cannot inhibit relaxation of pulmonary artery by Mch denotes effect of nNOS again.Activation of Akt phosphorates eNOS directly enhances NO production. Isoforms of NOS is presumably activated by PI3/Akt pathway, which is inhibited by LY294002. LY294002 and ODQ inhibits pulmonary artery relaxation induced by Mch in this study, respectively, implicates BrEpDRF is regulated by PI3-kinase and NO-cGMP pathway.This study denotes bronchial epithelium is associated with pulmonary artery relaxation, supports BrEpDRF exists. BrEpDRF is associated with relaxation via NO synthesis; eNOS and nNOS participate in NO synthesis, but iNOS did not found functional; under Mch stimulation, BrEpDRF is PI3-kinase/Akt pathway dependent and regulated via NO-cGMP pathway.LY294002, a PI-3 kinase inhibitor, is first employed to show BrEpDRF decreases note to adjacent pulmonary artery smooth muscle via NOS phosphoralation mechanism. We concluded in this study after compared different groups, that bronchus promotes pulmonary relaxation; approve NO production is participated by nNOS; BrEpDRF is possibly exist through which relax pulmonary dilation via NO production. These contribute to further research on pulmonary hypertension and ischemia-reperfuse injury in transplantation. BrEpDRF is signaled by PI3-kinase/Akt pathway and NO-cGMP pathway. The nature of BrEpDRF is to be further evaluated.