Animals ; Aquaporin 5 ; physiology ; Cytokines ; physiology ; Gene Expression Regulation ; Humans ; Intracellular Signaling Peptides and Proteins ; physiology ; Membrane Proteins ; physiology ; Mucins ; genetics ; Mucus ; secretion ; Myristoylated Alanine-Rich C Kinase Substrate ; Pulmonary Disease, Chronic Obstructive ; metabolism ; Respiratory Mucosa ; secretion ; Signal Transduction

OBJECTIVE: To construct phosphorylation sites domain (PSD) mutant of myristoylated alaninerich C kinase substrate (MARCKS) and explore the role of transient receptor potential melastatin 8 cation channels (TRPM8) and MARCKS in cold-induced synthesis and exocytosis of mucin (MUC) 5AC. METHODS: Human placental cDNA was used as a template to amplify the full coding region of MARCKS cDNA by PCR. Ser159, Ser 163, Ser 167, Ser 170 in the PSD were mutated to aspartic acids by an overlap PCR method. The resultant PSD mutant cDNA and the wild-type MARCKS cDNA were each subcloned into a mammalian expression vector pcDNA3.0. Recombinant constructs were confirmed by restriction enzyme digestion analysis and DNA sequencing. In intervention experiments, cells were pretreated with the TRPM8 channel antagonist BCTC and transfected with MARCKS-PSD mutant cDNA, and thereafter cold stimulation was applied. The levels of MUC5AC were measured by immunofluorescence and ELISA to clarify the roles of TRPM8 and PSD mutant on the synthesis and secretion of MUC5AC induced by cold, respectively. RESULTS: Restriction enzyme digestion analysis and DNA sequencing revealed that the pcDNA3.0- MARCKS and pcDNA3.0-MARCKS-PSD mutants were successfully constructed. The levels of intracellular and secreted MUC5AC of cold treated group were significantly higher than those of control group (P<0.05). BCTC attenuated the cold-induced synthesis and secretion of MUC5AC when compared with cold treated group (P<0.05). Transfection of 16HBE cells with the MARCKS-PSD mutant cDNA resulted in significant inhibition of mucin secretion in response to cold, and significantly higher level of intracellular MUC5AC than that of control group (P<0.01), whereas transfection with the vector DNA or the wild-type MARCKS cDNA had no effect on the mucin synthesis and secretion in response to cold (P>0.05). CONCLUSION TRPM8 and phosphorylation of MARCKS-PSD mediates the cold-induced exocytosis of MUC5AC by airway epithelial cells.
Base Sequence ; Cell Line ; Cold Temperature ; Epithelial Cells ; cytology ; metabolism ; Exocytosis ; physiology ; Humans ; Intracellular Signaling Peptides and Proteins ; genetics ; metabolism ; Membrane Proteins ; genetics ; metabolism ; Molecular Sequence Data ; Mucin 5AC ; metabolism ; Mutation ; Myristoylated Alanine-Rich C Kinase Substrate ; Phosphorylation ; TRPM Cation Channels ; metabolism ; Trachea ; cytology ; metabolism

OBJECTIVETo observe the contribution of Qingnao drop pilula to the alteration of myristoylated alanine-rich C kinase substrate (MARCKS) mRNA expression in acute multi-infarction hippocampus.

METHODRat models of acute multi-infarction were established by injecting the embolus of blood powder through the right external carotid arteryinto the internal carotid artery, rats were randomly divided into five groups (n = 12 in each): normal, sham operation, model, Chinese medicine treatment, and Western medicine treatment. Qingnao drop pilula (133.28 mg x kg(-1)), nimodipine (7.25 mg x kg(-1)) were administered respectively to Chinese medicine treatment group and Western medicine treatment group by gavage, equal volume of normal saline were given to three groups. Rats were treated with drugs starting at 3rd day before the operation, one time per day. Observing morphologic changes in hippocampus by optical microscope and electron microscope. Detecting expression level of MARCKS mRNA in hippocampus by semi-quantification PCR method.

RESULTHippocampus cells arrange tidy, administrative levels were compactness in normal group, which cells differentially impaired in model group, Chinese medicine treatment group and Western medicine treatment group. Hippocampus cells damage of Chinese medicine treatment group have more reckless than the model group in histopathology. The MARCKS mRNA were expressioned in model group vs medication treatment groups, in Chinese medicine treatment group vs the model group.

CONCLUSIONQingnao drop pilula can alleciate histomorphology lesion of hippocampus when occurring acute multi-infarction, to turn slower MARCKS mRNA expression, may play a neuroprotective effect role through accommodating PKC-MARCKS signal transduction system.

Acute Disease ; Animals ; Brain Ischemia ; drug therapy ; genetics ; metabolism ; Drugs, Chinese Herbal ; administration & dosage ; Gene Expression Regulation ; drug effects ; Hippocampus ; drug effects ; metabolism ; Humans ; Intracellular Signaling Peptides and Proteins ; genetics ; metabolism ; Male ; Membrane Proteins ; genetics ; metabolism ; Myristoylated Alanine-Rich C Kinase Substrate ; Random Allocation ; Rats ; Rats, Wistar