Masanobu Ishigaki

Expression and characterization of Rab38, a new member of the Rab small G protein family.

Abstract Rab38 is a new member of the Rab small G protein family that regulates intracellular vesicle trafficking. Rab38 is expressed in melanocytes and it has been clarified that a point mutation in the postulated GTP-binding domain of Rab38 is the gene responsible for oculocutaneous albinism in chocolate mice. However, basic information regarding recombinant protein production, intracellular location, and tissue-specific expression pattern has not yet been reported. We produced recombinant Rab38 using a baculovirus/insect cell-protein expression system. A combination of Triton X-114 phase separation and nickel-affinity chromatography yielded exclusively prenylated Rab38 that bound [α-32P]-GTP. The mRNA and the native protein were expressed in a tissue-specific manner, e.g., in the lung, skin, stomach, liver, and kidney. Freshly isolated rat alveolar type II cells were highly positive for the mRNA signal, but the signal was rapidly lost over time. Immunofluorescence staining demonstrated that expressed GST-tagged Rab38 was mainly co-localized with endoplasmic reticulum-resident protein and also partly with intermittent vesicles between the endoplasmic reticulum and the Golgi complex. These results indicate that Rab38 is expressed non-ubiquitously in specific tissues and regulates early vesicle transport relating to the endoplasmic reticulum, and hence suggest that Rab38 abnormality may cause multiple organ diseases as well as oculocutaneous albinism.

Lung deflation impairs alveolar epithelial fluid transport in ischemic rabbit and rat lungs

BACKGROUND Because the fluid transport capacity of the alveolar epithelium after lung ischemia with and without lung deflation has not been well studied, we carried out experimental studies to determine the effect of lung deflation on alveolar fluid clearance. METHODS After 1 or 2 hr of ischemia, we measured alveolar fluid clearance using 125I-albumin and Evans blue-labeled albumin concentrations in in vivo rabbit lungs in the presence of pulmonary blood flow and in ex vivo rat lungs in the absence of any pulmonary perfusion, respectively. RESULTS The principal results were: (1) lung deflation decreased alveolar fluid clearance while inflation of the lungs during ischemia preserved alveolar fluid clearance in both in vivo and ex vivo studies; (2) alveolar fluid clearance was normal in the rat lungs inflated with nitrogen (thus, alveolar gas composition did not affect alveolar fluid clearance); (3) amiloride-dependent alveolar fluid clearance was preserved when the lungs were inflated during ischemia; (4) terbutaline-simulated alveolar fluid clearance was preserved in the hypoxic rat lungs inflated with nitrogen; (5) lecithinized superoxide dismutase, a scavenger of superoxide anion, and N(omega)-nitro-L-arginine methyl ester, an inhibitor of nitric oxide, preserved normal alveolar fluid clearance in the deflated rat lungs. CONCLUSION Lung deflation decreases alveolar fluid clearance by superoxide anion- and nitric oxide-dependent mechanisms.

BETA1-ADRENOCEPTOR STIMULATION BY HIGH-DOSE TERBUTALINE DOWNREGULATES TERBUTALINE-STIMULATED ALVEOLAR FLUID CLEARANCE IN EX VIVO RAT LUNG

Because high-dose terbutaline and isoproterenol (10-3

Inducible nitric oxide synthase expression and nuclear factor-κB activation in alveolar type II cells in lung injury

Alveolar type II cells (type II cells)play a crucial role in the progression and repair of lung inflammation and injury. We investigated whether inducible nitric oxide synthase (iNOS) was expressed and nuclear factor- κ B (NF- κ B)was activated in type IIcells in lung injury. After injecting lipopolysaccharide (LPS)or saline in the rat, the lungs were excised and type II cells were isolated. iNOS and its mRNAwere expressed both in lung tissue and isolated type II cells in response to LPS. The lungs from saline-treated rats showed only minimal expression ofiNOS. Electrophoretic mobility shift assay revealed that expression of NF- κ B in the nuclear extracts was augmented by LPS, and p50/NF- κ B was expressed in type II cells in LPS-treated rats. Intraperitoneal dexamethasone almost completely inhibited the iNOS expression and attenuated the activation ofNF- Bin the LPS-treated lung. These findings suggest that type II cells can be a source of NO production in lung injury, and that the effects ofcorticosteroids may be in part through inhibition of both iNOS expression and NF- κ B activation.