Influence of vasoactive intestinal peptide on expression of pulmonary surfactant associated protein A in lung explants / 中国应用生理学杂志

<p><b>AIM</b>To study the influence of VIP on the expression of SP-A and its intracellular signal transduction pathway.</p><p><b>METHODS</b>The influence of VIP on the expression of SP-A was studied by immunohistochemistry and RT-PCR. The intracellular signal transduction pathway was further investigated by using receptor antagonist, protein kinase inhibitor and antisense oligonucleotides.</p><p><b>RESULTS</b>(1) VIP(10(-8) mol/L) enhanced SP-A protein expression in alveolar type II cells (ATII) and increased the content of SP-A mRNA in lung tissue. (2) VIP receptor antagonist [D-P-C1-Phe (6)-Leu (17)]-VIP (10(-6) mol/L) could suppress the VIP-induced expression of SP-A protein and SP-A mRNA. (3) c-fos antisense oligonucleotides (9 x 10(-6) mol/L) could inhibit the VIP-induced expression of SP-A protein and SP-A mRNA. (4) Protein kinase C(PKC) inhibitor H7 (10(-5) mol/L) could also depress the V1P-induced SP-A protein and SP-A mRNA.</p><p><b>CONCLUSION</b>VIP can up-regulate the expression of SP-A through its receptor. PKC and c-fos protein play important roles in the intracellular signal transduction pathway through which VIP induces the expression of SP-A.</p>

Temporal and spatial distribution of VIP, CGRP and their receptors in the development of airway hyperresponsiveness in the lungs / 生理学报

To explore the role of intrapulmonary neuropeptides in the development of airway hyperresponsiveness, we established an animal model of airway hyperresponsiveness (AHR) in rabbits by using ozone exposure. With the model, after test of the mechanics of respiration and bronchoalveolar lavage assay, the levels of vasoactive intestinal peptide (VIP) and calcitonin gene-related peptide (CGRP) in the lungs were determined by radioimmunoassay, and the expression of mRNA coding receptors of these two neuropeptides was evaluated by reverse transcriptional-polymerase chain reaction (RT-PCR). At the same time, the distribution of VIP receptor-1 (VIPR1) and CGRP receptor-1 (CGRPR1) in lung tissues and its time-course were examined by in situ hybridization. The results showed: (1) in ozone-stressing groups, airway resistance increased significantly and typical inflammatory pathological changes were observed in pulmonary tissue slides, including neutrophil and eosinophil infiltration, mucus exudation and bronchial epithelial cells (BECs) shedding; (2) with elongation of ozone exposure, the levels of VIP and CGRP in the lungs increased at first, reaching a peak on d 2 to 4, then decreased slowly, and CGRP peaked somewhat earlier than VIP; (3) mRNA expression of the two neuropeptide receptors in the lungs changed in a similar manner like VIP and CGRP, but the high level of mRNA expression of VIPR1 lasted longer than that of CGRPR1; and (4) in situ hybridization for neuropeptide receptors demonstrated that, in unstressed control, VIPR1 and CGRPR1 positive cells appeared in the airway epithelium, pulmonary interstitial and focal areas of airway and vascular smooth muscles. With the elongation of ozone exposure, hybridization stained deeper and the majority of positive cells were located around the vessels and bronchus except a few in the alveoli. At 8 d, only a small number of positive cells were seen in the lungs. From the results, it is concluded that ozone-stressing can induce the development of AHR, in which VIP and CGRP may play important roles. That implies, through binding to CGRPR1, CGRP stimulates an early inflammation response which contributes in cleaning up of irritants, while VIP exerts a later dampening of pulmonary inflammation response. These two neuropeptides may play sequential and complementary roles in the development of AHR.

Fibronectin upregulates catalase gene expression in rabbit bronchial epithelial cells / 生理学报

We have previously shown that the binding of integrins with extracellular matrix component fibronectin (Fn) can improve the ability of bronchial epithelial cells (BECs) in resisting oxidant injury by up-regulating the activity of catalase and increasing the content of GSH. However, the molecular mechanism or its signaling pathway of this protection is still unclear. In order to examine the intracellular signaling mechanism activated by Fn-integrin binding reaction, the present study investigated the mRNA expression of catalase in primary cultured rabbit BECs using RT-PCR based on a cell-injury model made with ozone exposure. The product bands of target gene CAT were checked with Southern blot and oligonucleotide probe hybridization. The results showed that Fn (10 microg/ml) promoted the catalase mRNA transcription (P<0.01). This effect was abolished either by protein-tyrosine kinase inhibitor genistein or calmodulin inhibitor W(7) (P<0.01). These results indicate that the promotion of catalase activity induced by Fn-integrin reaction is partly due to the elevation of catalase mRNA transcription, and that its signalling are possibly relevant to tyrosine phosphorylation or calmodulin pathway.

Regulatory peptides modulate ICAM-1 gene expression and NF-kappaB activity in bronchial epithelial cells / 生理学报

Intercellular adhesion molecule-1 (ICAM-1) is an important adhesion molecule leading to adhesion between cells; NF-kappaB, being universally distributed in the organism, is an important nuclear transcription factor leading to a rapid response to the stimuli. Line of evidence have shown that ICAM-1 transcription and NF-kappaB activation is an important step of inflammatory reaction. To testify that intrapulmonary regulatory peptides modulate inflammatory lesion of bronchial epithelial cells (BECs) through their effect on ICAM-1 expression and nuclear factor kappaB (NF-kappaB) activation, we used immunocytochemistry, RT-PCR, and electrophoretic mobility-shift assay (EMSA) to determine the ICAM-1 expression and NF-kappaB activity in BECs. The effects of NF-kappaB inhibitor MG-132 on ICAM-1 expression were also observed. The results showed that vasoactive intestinal peptide (VIP) and epidermal growth factor (EGF) decreased ICAM-1 expression in O(3)-stressed BECs, while endothelin-1 (ET-1) and calcitonin gene-related peptides (CGRP) increased ICAM-1 expression in resting BECs. MG-132 blocked ICAM-1 expression induced by O(3), ET-1 and CGRP. The results obtained by using EMSA confirmed that VIP and EGF restrained the activation of NF-kappaB in O(3)-stressed BECs; CGRP and ET-1 promoted activation of NF-kappaB. These observations indicate that VIP and EGF abated the injury by means of down-regulatory effects on ICAM-1 transcription and NF-kappaB activation, while ET-1 and CGRP enhanced the inflammation reaction by an up-regulatory effect. It is suggested that a developing and intensive airway inflammation correlates closely with a persistent expression of ICAM-1 and repeated activation of NF-kappaB.

Protective effect of low concentration endothelin-1 on the reactive oxygen-induced inhibition of pulmonary surfactant lipid synthesis / 生理学报

The effects of endothelin-1 (ET-1) at low concentration (1-100 pmol/L) on the reactive oxygen-induced inhibition of both pulmonary surfactant (PS) lipid synthesis and the activity of CTP: phosphorylcholine cytidylyltransferase (CCT), a rate-limiting enzyme in biosynthesis of phosphoatidylcholine (PC), were studied in cultured lung explants without serum. The xanthine-xanthine oxidase superoxide anion generating system decreased (3)H-choline incorporation into PC in a dose-dependent manner in cultured lung explants. ET-1 reduced both the reactive oxygen-induced decrease in (3)H-choline incorporation and the increase in malondialdehyde (MDA) content of lung tissues, but did not change the levels of antioxidant enzymes superoxide dismutase (SOD), catalase (CAT) and the total antioxidant capability in the lung explants. ET-1 enhanced microsomal CCT activity of the lung tissues, while it decreased cytosolic CCT activity of lung tissues. ET-1 also prevented the inhibitive effect of reactive oxygen on microsomal CCT activity in the lung explants. These results suggest that ET-1 at low concentration can protect the microsomal CCT activity and reduce the inhibition of PS lipid synthesis induced by oxidant lung injury. The protective mechanism of ET-1 is not relative to the pulmonary endogenous antioxidant defense system.

Effects of vasoactive intestinal peptide on chemotaxis of bronchial epithelial cells / 生理学报

To investigate the influence of vasoactive intestinal peptide (VIP) on chemotaxis of bronchial epithelial cells (BECs). Rabbit chemotactic migration of primary BEC was assessed in a blind-well Boyden chamber. Radioimmunoassay and radio-ligand affinity analysis were used for determining VIP secretion and vasoactive intestinal peptide receptor (VIPR) expression. The results showed: (1) the method for determining chemotaxis of BECs by using insulin as chemotactic factor was stable and reproducible (r=0.9703, P<0.01). (2) VIP (0.001-1 micromol/L) elicited chemotaxis of BECs which was substantial and concentration-dependent. The effects of VIP were inhibited by W-7 and H-7 (P<0.01). (3) Heat stress enhanced the secretion of VIP (P<0.01) and upregulated the expression of VIPR on BECs (P<0.05). These results indicate that VIP in the lungs may play an important role in the repair of damaged epithelium, accelerating restoration of the airway to its normal state. Calmodulin and protein kinase C may be involved in the signal transduction of VIP effects.