The effect of denopamine, a selective beta(1)-adrenergic agonist, on alveolar fluid clearance was determined in both ex vivo rat and guinea pig lungs. Alveolar fluid clearance was measured by the progressive increase in the concentration of Evans blue-labeled albumin over 1 h at 37 degrees C. Denopamine (10(-6) to 10(-3) M) increased alveolar fluid clearance in a dose-dependent manner in ex vivo rat lungs. Denopamine also stimulated alveolar fluid clearance in guinea pig lungs. Atenolol, a selective beta(1)-adrenergic antagonist, and amiloride, a sodium channel inhibitor, inhibited denopamine-stimulated alveolar fluid clearance. The potency of denopamine was similar to that of similar doses of isoproterenol or terbutaline. Short-term hypoxia (100% nitrogen for 1-2 h) did not alter the stimulatory effect of denopamine. Denopamine (10(-4), 10(-3) M) increased intracellular adenosine 3',5'-cyclic monophosphate levels in cultured rat alveolar type II cells. In summary, denopamine, a selective beta(1)-adrenergic agonist, stimulates alveolar fluid clearance in both ex vivo rat and guinea pig lungs.

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 membranebinding activity. These results suggest that the genetic abnormality of Rab38 affects multiple lysosome-related organelles , resulting in lung disease in addition to oculocutaneous albinism. (Am J

We examined the synthesis, transport, and localization of surfactant protein A (SP-A) in primary cultures of alveolar type II cells. In type II cells maintained in culture for 6 h, 39% of the SP-A pool detected with an enzyme-linked immunosorbent assay (ELISA) was found in lamellar bodies (LBs). After 24 h in culture, 53% of the cellular SP-A pool was found in LBs. The absolute amount of SP-A in the LB compartment was almost identical at 6 and 24 h of culture. In contrast to the results obtained with ELISA, 35S labeling of newly synthesized SP-A revealed that less than 7% of the cellular SP-A pool was in LBs at either 6 or 24 h of culture. In the 6-h cultures, 17% of the total (i.e., cells and media) [35S]SP-A pool was extracellular. In the 24-h cultures, 70% of the [35S]SP-A pool was extracellular. The secretion of [35S]SP-A was blocked by brefeldin A at all times. When medium containing newly secreted [35S]SP-A was incubated with alveolar type II cells maintained in culture for 24 h, the protein was taken up and incorporated into the LB fraction. More than 80% of the internalized SP-A was associated with the LB compartment after a 6 h incubation. The uptake of [35S]SP-A was blocked at 4 degrees C and was promoted by addition of unlabeled SP-A at 37 degrees C. These findings support a pathway of extracellular routing of SP-A prior to its accumulation in LBs in cultured type II cells.

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.Ruby; Long-Evans Cinnamon rat; alveolar type II cells; lamellar body HERMANSKY-PUDLAK SYNDROME (HPS) is a rare autosomal recessive disorder resulting from abnormal trafficking relating to lysosome-related organelles such as melanosomes, platelet dense granules, and lamellar bodies in alveolar type II cells (10,29). HPS is clinically characterized by oculocutaneous albinism, bleeding diathesis, and, based on its genetic background, additional abnormalities such as progressive interstitial pneumonia. Interstitial pneumonia is the most serious complication arising in patients with HPS and has no effective therapy and is generally fatal by middle age. The lung histology show interstitial pneumonia characterized by enlarged alveolar type II cells that are fulfilled with giant lamellar bodies containing prominently increased surfactant phospholipids (16). It is possible that lung surfactant abnormalities may be closely related to lung pathology in HPS lungs.Several Long-Evans (LE) substrains including Fawn-hooded (FH) and Tester-Moriyama (TM) rats show oculocutaneous albinism and bleeding diathesis and hence are considered to be rat models of human HPS. The responsible gene Ruby was recently identified as Rab38 (18), a member of the Rab small GTPase family that regulates intracellular vesicle transport. A point mutation found in the initiation codon of Rab38 has been assumed to cause null translation of the protein. LE Cinnamon (LEC) rats, which carry ATP7B gene mutation and have been used as a rat model of Wilson disease (31), have also been identified as Ruby rats that harbor the Rab38 mutation (18). Thus Rab38 has been nominated...

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 [alpha-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.