1) A beta agonist stimulated Na+ transport and decreased the intracellular Cl concentration ([Cl]c) associated with cell shrinkage via an increase in cytosolic cAMP level by activating adenylate cyclase in rat fetal distal lung epithelial (FDLE) cells. 2) Lowering [Cl-]c activated a 28-pS nonselective cation (NSC) channel by elongating the open time of the channel. 3) cAMP signals were converted to a protein tyrosine kinase (PTK)-mediated signal. 4) The PTK-mediated signal was involved in the cAMP-stimulated Na+ transport in rat FDLE cells.
Adrenergic beta-Agonists/pharmacology* ; Animal ; Biological Transport/physiology ; Biological Transport/drug effects ; Cell Size/physiology ; Chlorides/metabolism* ; Cyclic AMP/metabolism ; Cytosol/metabolism ; Enzyme Inhibitors/pharmacology ; Female ; Fetus/cytology ; Forskolin/pharmacology ; Nitrobenzoates/pharmacology ; Pregnancy ; Protein-Tyrosine Kinase/metabolism* ; Rats ; Rats, Wistar ; Respiratory Mucosa/enzymology* ; Respiratory Mucosa/embryology ; Respiratory Mucosa/cytology ; Sodium/metabolism* ; Tyrphostins/pharmacology

Sulforaphane (SFN) is a naturally occurring compound which is known to induce the phase II antioxidant genes via Nrf2 activation, although the underlying mechanism has not been fully elucidated. In this study, we investigated Nrf2 induction in response to SFN in human bronchial epithelial BEAS-2B cells and determined the signaling pathways involved in this process. SFN treatment reduced cell viability. Prior to cell death, intracellular reactive oxygen species (ROS) were generated at a high rate within a minute of commencing SFN treatment. Pretreatment with antioxidant N-acetylcysteine (NAC) blocked SFN-induced decrease in cell growth. Erk1/2 was activated within 30 min of SFN addition, whereas Akt phosphorylation did not significantly change until the first 8 hr after SFN treatment but then became substantially low until 48 hr. Inhibition of Erk1/2 phosphorylation attenuated SFN-induced loss of cell viability. Nrf2 protein levels in both nuclear and whole cell lysates were increased by SFN treatment, which was dependent on ROS production. Knockdown of Nrf2 with siRNA attenuated SFN-induced heme oxygenase-1 (HO-1) up-regulation. Induction of the Nrf2/HO-1 after SFN treatment was potently suppressed by pretreatment with NAC. Overall, our results indicate that SFN mediates antioxidative and antiproliferative responses by generating ROS in BEAS-2B cells.
Acetylcysteine/pharmacology ; Anticarcinogenic Agents/pharmacology ; Antioxidants/*pharmacology ; Bronchi/cytology/*drug effects/metabolism ; Cell Line ; Cell Proliferation/*drug effects ; Epithelial Cells/drug effects/metabolism ; Extracellular Signal-Regulated MAP Kinases/metabolism ; Free Radical Scavengers/pharmacology ; Heme Oxygenase-1/biosynthesis ; Humans ; NF-E2-Related Factor 2/biosynthesis/genetics ; Oxidative Stress/drug effects ; Proto-Oncogene Proteins c-akt/metabolism ; RNA Interference ; RNA, Small Interfering ; Reactive Oxygen Species/*metabolism ; Respiratory Mucosa/cytology/*drug effects/metabolism ; Signal Transduction/drug effects ; Thiocyanates/*pharmacology

The EGFR plays an essential role in goblet cell hyperplasia and mucus hypersecretion. EGFR has an intrinsic tyrosine kinase activity that, when activated, induces the production of MUC5AC through the signaling kinase cascade in the airway epithelium. We have investigated the effects of an EGFR tyrosine kinase inhibitor, gefitinib, on ovalbumin (OVA)-induced, allergic inflammation in airway epithelia of mice. OVA-sensitized mice were pretreated with gefitinib at two different doses (12.5 and 50 mg/kg) and then challenged with OVA. The OVA challenge increased the total cell count and eosinophil count in bronchoalveolar lavage fluid (BALF), as well as the concentrations of T-helper2 (Th2) cytokines, such as IL-4 and IL-13, overall eosinophil recruitment in the lung tissue and airway hyperresponsiveness (AHR). Pretreatment with gefitinib reduced the inflammatory cell counts and released cytokine concentrations (IL-4 and IL-13) in BALF, as well as eosinophil recruitment in the lungs and AHR, in a dose-dependent manner. This was associated with decreased EGFR and Akt phosphorylation. We showed that gefitnib inhibits EGFR and phosphoinositol 3'-kinase (PI3K)/Akt activation which were activated in OVA sensitized mice. These findings suggest that inhibitors of the EGFR cascade may have a role in the treatment of asthma.
Animals ; Antineoplastic Agents/*therapeutic use ; Bronchoalveolar Lavage Fluid/cytology ; Cytokines/biosynthesis ; Enzyme Activation ; Eosinophils/cytology ; Goblet Cells/pathology ; Inflammation/drug therapy/metabolism ; Male ; Mice ; Mice, Inbred BALB C ; Ovalbumin ; Phosphorylation ; Proto-Oncogene Proteins c-akt/metabolism ; Quinazolines/*therapeutic use ; Receptor, Epidermal Growth Factor/*antagonists & inhibitors/metabolism ; Respiratory Hypersensitivity/*drug therapy/etiology/metabolism ; Respiratory Mucosa/drug effects/pathology

BACKGROUNDAirway mucus hypersecretion is an important pathophysiological feature of chronic obstructive pulmonary disease, which is closely associated with cigarette smoking. However, the signal transduction pathway from the cell surface to the nucleus through which cigarette smoke causes upregulation of mucin gene expression is not well known. This study was designed to investigate the role of extracellular signal-regulated Kinase 1/2 (ERK 1/2) in airway mucus hypersecretion induced by cigarette smoke in rats.

METHODSA rat model of airway mucus hypersecretion was induced by exposure to cigarette smoke for 4 weeks.Rats exposed to inhalation of cigarette smoke or normal saline were given an intraperitoneal injection of U0126, a specific MEK1 kinase inhibitor, at doses of 0.25 mg/kg, 0.5 mg/kg and 1 mg/kg for 14 days. Expression of MUC5AC mRNA and protein, ERK 1/2 and phosphorylated-ERK 1/2 (p-ERK 1/2) were detected by RT-PCR, immunohistochemistry and Western blotting.

RESULTSCigarette smoke significantly increased airway goblet cells metaplasia, induced the overexpression of MUC5AC mRNA and protein in bronchial epithelia, and increased the ratio of p-ERK 1/2 and ERK 1/2. U0126 significantly attentuated the expression of MUC5AC mRNA and protein induced by cigarette smoke (P < 0.05). Moreover, there was a significant positive correlation between the ratio of p-ERK1/2 to ERK1/2 and the expression of MUC5AC mRNA and protein (P < 0.05).

CONCLUSIONSInhibition of ERK 1/2 by U0126 decreased the ratio of p-ERK 1/2 to ERK 1/2 and expression of MUC5AC mRNA and protein. ERK 1/2 may play an essential role in cigarette smoke-induced mucus hypersecretion in vivo.

Animals ; Blotting, Western ; Bronchi ; cytology ; metabolism ; Goblet Cells ; drug effects ; metabolism ; Immunohistochemistry ; Lung ; drug effects ; metabolism ; Mitogen-Activated Protein Kinase 1 ; genetics ; metabolism ; Mitogen-Activated Protein Kinase 3 ; genetics ; metabolism ; Mucin 5AC ; genetics ; metabolism ; Phosphorylation ; drug effects ; Rats ; Respiratory Mucosa ; secretion ; Reverse Transcriptase Polymerase Chain Reaction ; Smoking ; adverse effects

No abstract available.
Animal ; Biological Transport/physiology ; Biological Transport/drug effects ; Cholera/metabolism ; Diglycerides/pharmacology ; Epithelial Cells*/virology ; Epithelial Cells*/microbiology ; Epithelial Cells*/metabolism ; Escherichia coli ; Escherichia coli Infections/metabolism ; Estrenes/pharmacology ; In Vitro ; Indoles/pharmacology ; Influenza/metabolism ; Intestinal Mucosa/cytology ; Maleimides/pharmacology ; Mice ; Phosphodiesterase Inhibitors/pharmacology ; Pyrrolidinones/pharmacology ; Respiratory Mucosa/cytology ; Sodium Channels/metabolism* ; Staurosporine/pharmacology ; Vibrio cholerae

OBJECTIVETo study the alterations of FHIT gene and P16 gene in malignant transformed human bronchial epithelial cells induced by crystalline nickel sulfide using an immortal human bronchial epithelial cell line, and to explore the molecular mechanism of nickel carcinogenesis.

METHODS16HBE cells were treated 6 times with different concentrations of NiS in vitro, and the degree of malignant transformation was determined by assaying the anchorage-independent growth and tumorigenicity. Malignant transformed cells and tumorigenic cells were examined for alterations of FHIT gene and P16 gene using RT-PCR, DNA sequencing, silver staining PCR-SSCP and Western blotting.

RESULTSNiS-treated cells exhibited overlapping growth. Compared with that of negative control cells, soft agar colony formation efficiency of NiS-treated cells showed significant increases (P < 0.01) and dose-dependent effects. NiS-treated cells could form tumors in nude mice, and a squamous cell carcinoma was confirmed by histopathological examination. No mutation of exon 2 and exons 2-3, no abnormal expression in p16 gene and mutation of FHIT exons 5-8 and exons 1-4 or exons 5-9 were observed in transformed cells and tumorigenic cells. However, aberrant transcripts or loss of expression of the FHIT gene and Fhit protein was observed in transformed cells and tumorigenic cells. One of the aberrant transcripts in the FHIT gene was confirmed to have a deletion of exon 6, exon 7, exon 8, and an insertion of a 36 bp sequence replacing exon 6-8.

CONCLUSIONSThe FHIT gene rather than the P16 gene, plays a definite role in nickel carcinogenesis. Alterations of the FHIT gene induced by crystalline NiS may be a molecular event associated with carcinogen, chromosome fragile site instability and cell malignant transformation. FHIT may be an important target gene activated by nickel and other exotic carcinogens.

Acid Anhydride Hydrolases ; chemistry ; genetics ; metabolism ; Animals ; Base Sequence ; Bronchi ; cytology ; Cell Line ; Cell Transformation, Neoplastic ; chemically induced ; DNA Damage ; Exons ; Gene Deletion ; Genes, p16 ; Humans ; Mice ; Mice, Nude ; Molecular Sequence Data ; Mutagenicity Tests ; Neoplasm Proteins ; chemistry ; genetics ; metabolism ; Nickel ; toxicity ; RNA, Messenger ; metabolism ; Respiratory Mucosa ; cytology ; drug effects ; Reverse Transcriptase Polymerase Chain Reaction ; Sequence Analysis, DNA