Hirohisa Toga

Stimulation of alveolar epithelial fluid clearance in human lungs by exogenous epinephrine

Objectives:Because several experimental studies have demonstrated that cyclic adenosine monophosphate generation following β-adrenoceptor activation can markedly stimulate alveolar fluid clearance, we determined whether the endogenous levels of catecholamines that occur in the pulmonary edema fluid and plasma of patients with acute lung injury are high enough to stimulate alveolar fluid clearance in the human lung. Design:Observational clinical study. Setting:Academic university hospital and laboratory. Patients:Twenty-one patients with acute pulmonary edema plus ex vivo human lungs. Interventions:Measurements of catecholamine levels in patient samples and controlled laboratory studies of the effects of these catecholamine levels on the rates of alveolar fluid clearance in ex vivo human lungs. Measurements and Main Results:The concentrations of both epinephrine and norepinephrine in the pulmonary edema fluid and plasma were ∼10−9 M (range of 1–8 × 10−9 M) in hydrostatic pulmonary edema (n = 6) and acute lung injury patients (n = 15). We therefore tested whether 10−9 M epinephrine or norepinephrine stimulated alveolar fluid clearance in isolated human lungs and found that these epinephrine or norepinephrine concentrations did not stimulate alveolar fluid clearance. However, higher concentrations of epinephrine (10−7 M), but not norepinephrine (10−7 M), significantly stimulated alveolar fluid clearance by 84% above control. Glibenclamide (10−5 M) and CFTRinh-172 (10−5 M), cystic fibrosis transmembrane conductance regulator inhibitors, completely inhibited the epinephrine-induced stimulation of alveolar fluid clearance. Conclusions:These results indicate that endogenous catecholamine concentrations in pulmonary edema fluid are probably not sufficient to stimulate alveolar fluid clearance. In contrast, administration of exogenous catecholamines into the distal airspaces can stimulate alveolar fluid clearance in the human lung, an effect that is mediated in part by cystic fibrosis transmembrane conductance regulator. Therefore, exogenous cyclic adenosine monophosphate-dependent stimulation will probably be required to accelerate the resolution of alveolar edema in the lungs of patients with pulmonary edema.

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.

Effects of endothelin, platelet activating factor and thromboxane A2 in ferret lungs

We have compared the effects of three vasoactive agents, endothelin, platelet activating factor and thromboxane A2 analogue, U 46,619, in the pulmonary circulation of ferrets. Lungs of nine adult ferrets, body weight 1.06 +/- 0.27 kg, were isolated and perfused with sheep red blood cells suspended in Krebs solution with 2 g% Dextran 70 (hematocrit 33 +/- 6%), under conditions of constant flow in zone 3. Endothelin-1 (ET: 0.1-1.35 micrograms/kg), platelet activating factor (PAF: 0.5-5.5 micrograms/kg) or thromboxane A2 analogue (U 46,619: 1 microgram/kg), was infused into the pulmonary artery and the pressure response determined. To locate the site of action of the agents, the pulmonary circulation was partitioned into arteries, microvessels and veins by measuring pressures in 20-50 microns diameter subpleural arterioles and venules by micropuncture, both during baseline and after the peak response to each vasoactive drug. We found that the ferret pulmonary circulation constricted in response to ET, PAF and U 46,619, but the magnitude of constriction varied. Ferret lungs were most sensitive to U 46,619 and least sensitive to PAF. The major site of action also differed among the agents; U 46,619 and ET predominantly constricted veins whereas PAF predominantly constricted arteries. We conclude that the ferret pulmonary circulation demonstrates differential responsiveness to vasoactive agents, that venous constriction is a common feature and that the predominant site of action varies with the specific agent.

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.

Pharyngeal Cross-Sectional Area and Pharyngeal Compliance in Normal Males and Females

Obstructive sleep apnea syndrome is ascribed to pharyngeal dysfunction, but there are only a few reports about the normal morphological values in this anatomical region. We measured the pharyngeal cross-sectional area and the compliance (collapsibility), using the acoustic reflection technique with air breathing, in 181 healthy subjects (age 21–69 years). We assessed their sex-related differences, and the effects of age, body size and body postures on these parameters. The pharyngeal cross-sectional area, defined as the region from the fauces to the glottis, posturally changed with successive decreases in the sitting, left lateral decubitus and supine positions. The area was significantly greater in male than in female subjects in the sitting position (p < 0.01), but no difference was present in the recumbent positions. The pharyngeal cross-sectional area did not correlate with either age or body size. The specific pharyngeal compliance was greater in the males than in the females (p < 0.01) and increased with age only in the male subjects.

Altered lung surfactant system in a Rab38-deficient rat model of Hermansky-Pudlak syndrome

Several Long-Evans rat substrains carrying the phenotype of oculocutaneous albinism and bleeding diathesis are a rat model of Hermansky-Pudlak syndrome (HPS). The mutation responsible for the phenotype (Ruby) was identified as a point mutation in the initiation codon of Rab38 small GTPase that regulates intracellular vesicle transport. As patients with HPS often develop life-limiting interstitial pneumonia accompanied by abnormal morphology of alveolar type II cells, we investigated lung surfactant system in Long-Evans Cinnamon rats, one strain of the Ruby rats. The lungs showed conspicuous morphology of type II cells containing markedly enlarged lamellar bodies. Surfactant phosphatidylcholine and surfactant protein B were increased in lung tissues and lamellar bodies but not in alveolar lumen. Expression levels of mRNA for surfactant proteins A, B, C, and D were not altered. Isolated type II cells showed aberrant secretory pattern of newly synthesized [(3)H]phosphatidylcholine, i.e., decreased basal secretion and remarkably amplified agonist-induced secretion. [(3)H]phosphatidylcholine synthesis and uptake by type II cells were not altered. Thus Rab38-deficient type II cells appear to carry abnormality in lung surfactant secretion but not in synthesis or uptake. These results suggest that aberrant lung surfactant secretion may be involved in the pathogenesis of interstitial pneumonia in HPS.

A Mutation in Rab38 Small GTPase Causes Abnormal Lung Surfactant Homeostasis and Aberrant Alveolar Structure in Mice

The chocolate mutation, which is associated with oculocutaneous albinism in mice, has been attributed to a G146T transversion in the conserved GTP/GDP-interacting domain of Rab38, a small GTPase that regulates intracellular vesicular trafficking. Rab38 displays a unique tissue-specific expression pattern with highest levels present in the lung. The purpose of this study was to characterize the effects of Rab38-G146T on lung phenotype and to investigate the molecular basis of the mutant gene product (Rab38(cht) protein). Chocolate lungs exhibited a uniform enlargement of the distal airspaces with mild alveolar destruction as well as a slight increase in lung compliance. Alveolar type II cells were engorged with lamellar bodies of increased size and number. Hydrophobic surfactant constituents (ie, phosphatidylcholine and surfactant protein B) were increased in lung tissues but decreased in alveolar spaces, consistent with a malfunction in lamellar body secretion and the subsequent cellular accumulation of these organelles. In contrast to wild-type Rab38, native Rab38(cht) proteins were found to be hydrophilic and not bound to intracellular membranes. Unexpectedly, recombinant Rab38(cht) proteins retained GTP-binding activity but failed to undergo prenyl modification that is required for membrane-binding activity. These results suggest that the genetic abnormality of Rab38 affects multiple lysosome-related organelles, resulting in lung disease in addition to oculocutaneous albinism.

Expression and Localization of a Novel Rab Small G Protein (Rab38) in the Rat Lung

The Rab small G protein family participates in intracellular vesicle transport, including exocytosis and endocytosis. The cDNA encoding a novel Rab-related small G protein (Rab38) has been cloned from rat lung cDNA library and recorded in GenBank (accession no. M94043). However, the expression and localization of the protein in the lung remains primarily unknown. We produced polyhistidine-tagged recombinant Rab38 and a polyclonal antibody with a synthetic peptide. Immunohistochemistry demonstrated that the protein is specifically localized in alveolar type II cells and in bronchial epithelial cells. In situ hybridization using a digoxygenin-labeled RNA riboprobe clearly showed that the mRNA of the protein is localized in alveolar type II cells and bronchial epithelial cells, especially terminal airway epithelial cells. Western blot and reverse transcriptase-polymerase chain reaction showed distinct expression of the protein and mRNA in isolated alveolar type II cells, but not in alveolar macrophages. The native protein was predominantly hydrophobic and was enriched in a high-density vesicle fraction but was barely detectable in nuclear and lamellar body fractions in alveolar type II cells. Immunofluorescence cytochemistry performed on cultured alveolar type II cells showed that Rab38 distributed extensively in the cytoplasm with a distribution pattern similar to endoplasmic reticulum rather than other subcellular organelles. These results suggest that this novel rab small G protein (Rab38) mediates vesicular transport in terminal airway epithelium.

Tone dependent nitric oxide production in ovine vessels in vitro

We have determined the role of endogenous nitric oxide (NO) in regulation of vasomotor tone in ovine intrapulmonary and mesenteric vessels with resting tension and elevated vasomotor tone. Third generation intrapulmonary vessel rings and mesenteric vessel rings, 2-3 mm in diameter, were isolated from 20 sheep. NO production in the vessels was assessed by the change in tension induced by NG-nitro-L-arginine methyl ester (L-NAME), a competitive inhibitor of NO synthase. In vessels under resting tension, 10(-4) to 10(-3) M L-NAME induced a significant increase in tension only in veins but not in arteries. When tone was elevated with phenylephrine or U 46,619, a thromboxane A2 analogue, there was now a significant increase in tension in arteries with 10(-4) M L-NAME and in veins with 10(-5) M L-NAME. The increase in tension induced by L-NAME in veins was greater than that in arteries and greater when tone was elevated than under resting tension. Responses of pulmonary and mesenteric vessels were similar. Our data suggest that NO may play a role in regulating venous tone under baseline conditions and that the role of NO in regulation of vasomotor tone becomes more significant in the presence of nonspecific elevation of vasomotor tone in both arteries and veins. We speculate that endogenous NO production may be one mechanism by which pulmonary and systemic vessels counter the effects of vasoconstrictive agents.

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