Inhibition Of Pulmonary Surfactant Lipid Synthesis By Glutamate And The Protective Role Of Calcitonin Gene-related Peptide

Glutamate (Glu) is one of the major excitatory neurotransmitter in the central nervous system. The excitotoxicity resulting from overactivation of glutamate receptors is one of the most extensively studied processes of neuronal cell death, and plays an important role in many central nervous system diseases. Recent observations suggest that several subtypes of glutamate receptors are distributed in lung and airway. However there is no report about the effect of Glu on alveolar epithelium. This study is aimed to investigate the modulation of Glu on synthesis of PS and illustrate the biological significance of Glu and its receptor in lung in order to offer new ideas for the prevention and treatment of pulmonary diseases. ?Methods: In cultured lung explants without serum, the lipid component synthesis of pulmonary surfactant was evaluated in [3H]-choline incorporation; mRNA content of phosphocholine cytidylyltransferase (CCT) in lung explants was investigated in RT-PCR; the changes of the ultrastructure of the AT II cells were observed with electron microscope; the expression of NMDAR1 subtype was observed in immunohistochemistry staining ; nitric oxide synthase(NOS) activity , nitric oxide (NO) content , superoxide dismutase (SOD) level , malondialdehyde (MDA) content and lactae dehydroase (LDH) level were determined by biochemistry methods.Results:1. Influence of glutamate on synthesis of the lipid component of pulmonary surfactant?with L-arginine, Glu inhibited [3H]-choline incorporation with good dose-dependence and time-dependence ;(2) mRNA content of CCT of the Glu treatment groups was decreased; (3) Glu increases the release of LDH in cultured lung explants ; (Dwith electron microscope histochemistry , Glu induced the changes of the ultrastruture of AT IIIVcells .2. Mechanism of glutamate-induced inhibition of pulmonary surfactantlipid synthesis㎞MDA receptor (NMDAR) antagonist MK-801 could reverse glutamate-induced inhibition of [3H]-choline incorporation in lung explants; ﹖he expression of NMDAR1 subtype existed in alveolar type II epithelial cells; ﹖he effect of Glu on PC synthesis required L-arginine in cultured lung explants, NOS activity and NO production were increased significantly in lung explants with Glu treatment ; and MK-801 and NOS inhibitor L-NNA could block the increase of NOS activity and NO production induced by Glu; L-NNA also could reverse the inhibition of [3H]-choline incorporation induced by Glu; (4)MDA content increased and SOD level decreased in cultured lung explants of Glu treament groups, MK-801 and L-NNA could reverse these effects; (3) MK-801, L-NNA could block the release of LDH induced by Glu. 3. Calcitonin gene-related peptide attenuates glutamate-inducedinhibition of pulmonary surfactant lipid synthesis(D CGRP could reverse the decrease of [3H]-choline incorporation induced by Glu with dose-dependence; (2) CGRP could block the decrease of the content of CCT a mRNA induced by Glu in lung tissue; (3}CGRP could reduce the impairment of the ultrastructure of AT II cells induced by Glu; (4) CGRP could reverse the increase of MDA content and decrease of SOD level induced by Glu in cultured lung explants, respectively;〤GRP had no significant effect on NOS activity and increase of NO production induced by Glu.Conclusions: It was firstly demonstrated that Glu has toxic effect on alveolar epithelial cells and inhibits the synthesis of PC; By activating NMDA receptor, Glu could activate NOS, induce the production of both NO and free radical, reduce the function of anti-oxidation defense system in lung, induce the injury of AT II cells, and inhibit the synthesis of PC. It was firstly observed that CGRP plays a protective role on lung injury and attenuates glutamate-induced inhibition of pulmonary surfactant lipid synthesis. The protective mechanisms of CGRP are not mediated by blocking NO production.