Search

The pulmonary route has recently attracted attention as a noninvasive administration route for peptide and protein drugs, and an insulin powder for inhalation was approved by authorities in Europe and the USA. The present study examined usefulness of insulin and gene powders for systemic and local inhalation therapy. We prepared several dry insulin powders by spray drying to examine the effect of additives on insulin absorption. Citric acid appears to be a safe and potent absorption enhancer for insulin in dry powder. However, in the powder with citric acid (MIC0.2 SD) insulin was unstable compared with the other powders examined. To improve insulin stability, a combination of insulin powder and citric acid powder was prepared (MIC Mix). MIC Mix showed hypoglycemic activity comparable to MIC0.2 SD while the insulin stability was much better than that of MIC SD. Next, dry insulin powders with mannitol were prepared with supercritical carbon dioxide (SCF); the powder thus prepared reduced blood glucose level rapidly and was more effective than that prepared by spray drying. Chitosan-pDNA complex powders as a pulmonary gene delivery system were also prepared with SCF and their in vivo activity was evaluated. The addition of chitosan suppressed the degradation of pCMV-Luc during preparation and increased the storage stability. The luciferase activity in mouse lung was evaluated after pulmonary administration of the powders. The chitosan-pDNA powder with an N/P ratio=5 increased the luciferase activity to 27 times that of the pCMV-Luc solution. These results suggest that gene powder with chitosan is a useful pulmonary gene delivery system.

The lung ball is a special type of pulmonary aspergillosis (PA) occurring often after chemotherapy for leukemia. Histologically the ball, with air crescent sign on roentgenogram, is compatible with necrotizing lung tissue admixed with Aspergilli. The lung ball differs entirely from the common "fungus ball" in its quality, though they are similar in roentgenological appearances. The present two cases were leukemia which showed pulmonary findings in their therapeutic course, resulting in lung resection. In both cases the lung ball was confirmed histopathologically. Immunostaining of the lung tissue for neutrophil elastase showed elastase in various sites in the bronchial wall, pulmonary blood vessels (artery, vein) and cavitary wall. In our previous studies, much importance was attached to the disturbance of the pulmonary circulation caused by fibrin deposition as a factor in the developmental course of the fungus ball type aspergillosis (semi-invasive type). The circulatory disturbance of the lung was recognized also in the present cases. This two-way destruction of the pulmonary tissue, resulting from both neutrophil elastase activities and pulmonary circulatory disturbances, were regarded as the most important factor for the development of the lung ball. There are few studies on aspergillar lung ball with regard to the above respects.

The pathogenesis of the influenza and Sendai viruses is primarily determined by host cellular trypsin-type processing proteases that activate viral fusion activity and infectivity. We isolated three secretory trypsin-type proteases from rat lungs, such as tryptase Clara, mini-plasmin, and ectopic anionic trypsin, candidates for the processing proteases of viral envelope glycoproteins. These enzymes specifically cleave the precursor of fusion glycoprotein hemagglutinin (HA) of influenza virus at Arg(325) and the F(0) of Sendai virus at Arg(116) in the consensus cleavage motif, Gln(Glu)-X-Arg, resulting in the induction of infectivity of these viruses. These proteases show different localization in the airway and susceptibility for the processing of various subtypes of influenza virus HA, suggesting that these processing proteases determine the viral pathogenicity. Influenza virus readily infects and replicates in the airway epithelial cells but occasionally replicates in the central nervous system, particularly in children below 5-6 years of age and Reye's syndrome patients. We found an invasion by a non-neurovirulent influenza virus in cerebral capillaries with progressive brain edema of mice having impaired mitochondrial fatty acid metabolism congenitally or posteriorly in the newborn period. In the brain of these mice, mini-plasmin, which potentiates viral-multiplication in vivo and destroys the blood-brain barrier, accumulated with virus antigen in the brain capillaries but only a little in the control mice without impaired mitochondrial fatty acid metabolism.

Recently it has been demonstrated a role of fat-cell SSAO on glucose transport and GLUT4 translocation to the cell surface. Many compounds have been identified as relatively selective SSAO inhibitors, but those currently available also inhibit monoamine oxidase (MAO, EC 1.4.3.4). In this study, inhibitory properties of a haloamine, 2-bromoethylamine (2-BEA), to guinea pig and rat lung-bound SSAO have been studied. 2-BEA could not inhibit both forms of MAO, but it competitively inhibited rat lung SSAO activity with a Ki value of 2.5 microM without preincubation and after preincubation, the mode of inhibition changed to be non-competitive. Dialysis and dilution experiments with 2-BEA-pretreated preparations resulted in no recovery of SSAO activity. A decreased rate of SSAO inhibition under N2 atmosphere to that obtained under O2 was produced after 2-BEA treatment, suggesting that oxidised intermediate was necessary for its inhibition. The plot of 1/k' vs 1/2-BEA intersected on the y-axis indicate that the inhibition by 2-BEA is not affinity-labeling agent, but a suicide inhibitor of SSAO.

To test whether a potent cGMP-specific phosphodiesterase inhibitor, E 4021, is a selective pulmonary vasodilator in pulmonary hypertension, we studied its acute hemodynamic effects in conscious, chronically hypoxic pulmonary hypertensive rats. Chronically hypoxic pulmonary hypertension was induced by keeping adult Sprague-Dawley rats in a hypobaric chamber for 3 weeks. Two days after catheterization. E 4021 was injected intravenously at doses of 3, 10, 30, 100, 300, and 1,000 micrograms/kg at 10-min intervals. E 4021 caused significant decreases in mean pulmonary arterial pressure of 11 +/- 5, 12 +/- 6, and 18 +/- 5% at doses of 100, 300, and 1,000 micrograms/kg, respectively. In contrast to its depressor effect on mean pulmonary arterial pressure, E 4021 decreased mean systemic arterial pressure significantly (by 9 +/- 2%) at a dose of 1,000 micrograms/kg only. Heart rate and cardiac output were unchanged after the administration of E 4021. Tissue cGMP-specific phosphodiesterase activity was markedly higher in lung than in aorta. These results indicate that E 4021 is a relatively selective pulmonary vasodilator in chronically hypoxic pulmonary hypertensive rats. We conclude E 4021 may be useful for the treatment of pulmonary hypertension.

To test the long-term effect of a beta-adrenergic agonist on Na+/K(+)-ATPase activity in rat lung explants, we measured the activity in lung explants kept continuously in the presence or absence of 0.1 mM terbutaline for 5 days. Once it was clear that this agent increased Na+/K(+)-ATPase activity, we then performed a similar measurement in cultured alveolar type II cells from rats. Continuous exposure to 0.1 mM terbutaline enhanced the enzyme activity in those cells, and this enhancement was completely blocked by 0.01 mM of the beta-antagonist propranolol. Western blotting showed that this increase in Na+/ K(+)-ATPase activity in the cells could involve modulation of protein expression. We conclude that beta-adrenergic agonists can increase Na+/K(+)-ATPase activity in alveolar type II cell in lung explants by modulating expression of the enzyme in a long period of time.

Nitric oxide (NO), discovered as an endothelium derived relaxing factor, is generated during conversion of L-arginine to L-citrulin by nitric oxide synthase (NOS). Now, genes for three isoforms of NOS (nNOS, eNOS, and iNOS) have been cloned and pulmonary expressions of these NOS isoforms have been studied extensively. Localization of these NOS isoforms in the lung and their functional roles are discussed.

The nitric oxide (NO) levels in exhaled air and the population of nitric oxide synthase (NOS) synthesizing epithelium in airways are reported to be increased in patients with asthma. NO may be implicated in the pathophysiology of asthma. In this study, we examined the inducible NOS (iNOS) mRNA levels in the lung of a guinea pig model of experimental asthma which exhibits both the immediate and late asthmatic responses (IAR/LAR), following challenge with antigen, using the methods of Northern blot analysis. After challenge with antigen, iNOS mRNA were increased in a time-dependent manner (before challenged < IAR < LAR). Furthermore, we estimated NO productions in the trachea using bioassay by 5-HT-induced tracheal smooth muscle relaxation which are involved in NO. The 5-HT-induced relaxations were accelerated in a time-dependent manner after challenge with antigen, reflecting increased NO production (before challenged < IAR < LAR). These observations suggest that NO would be involved in the pathophysiology of asthma.

To identify the molecular nature of nitric oxide synthase (NOS) and its regulation and function in the airway, NOS activity and NOS mRNA expression were measured in cultured lung epithelial cell. All epithelial cells exhibited constitutive NOS activity that was calcium-dependent, and inducible, lesser calcium-dependent activity in the presence of interferon-gamma, interleukin-1 beta, tumor necrosis factor-alpha, and lipopolysaccharide. Two distinct NOS mRNA species (neuronal and inducible) were found with the reverse transcription polymerase chain reaction. The co-existence of constitutive and inducible NOS in human alveolar and bronchial epithelial cells indicates that nitric oxide may have many roles in normal and diseased lungs.

Glutathione S-transferases (GSTs) are a family of multifunctional detoxifying enzymes that catalyse the conjugation of glutathione with large number of compounds bearing an electrophilic center, including carcinogens, and also bind a variety of nonsubstrate ligands. The transferases are widely distributed in the mammalian species and can be grouped into three classes on the basis of subunit composition: alpha (basic), mu (neutral), and pi (acidic). The liver is an organ possessing abundant GST-alpha. GST-mu is also present in the liver and lymphocytes, but this is absent in approximately 50% of the human population. GST-pi (originally found in the placenta) is widely located in the lung, kidney, GI tract, erythrocytes and cancer cells. The present review describes a new nomenclature of human GST isoenzymes and recent investigations of this enzymes, including ours. Implications of each isoenzyme are also discussed.

"At first glance the lungs may seem uncomplicated, but many wise men have gone astray in their labyrinths." These words were written by Dr. A.A. Liebow, a famous pathologist, in a foreword to the first edition of Pathology of the Lung by H. Spencer. This same thought can also be applied to the field of medicolegal autopsies. 1. The gross appearance of the lungs in medicolegal autopsies Plucks consisting of the lungs, neck organs, the esophagus and the aorta were removed from human cadavers and after taking photos of the frontal and rear view, the lungs were carefully examined to reveal whether the lung shows characteristic morphological changes depending on causes of death. Based on their appearance, the lungs were classified into the 3 following types: a collapsed, a non-collapsed and an inflated type, each of these types reflecting the probable cause of death. The collapsed type of lung was seen in cases of death from exanguination, and the lung falling into shrinkage due to traumatic pneumo- and/or hemo-thorax was also classified into the collapsed type. The non-collapsed type of lung was seen in cases whose lungs were thermo-coagulated and in a case of death from a pulmonary embolism. Also, the deflating lungs of drowning victims before falling into collapse, were classified into a non-collapsed type. The inflated type of lung consisted of lungs that showed ballooning soon after death by drowning, and lungs that had inflated due to emphysema or edema from various causes. This lung study has reconfirmed that the lungs show hypostatic changes more clearly than any other organs of the body, and in the absence of skin color changes reflecting hypostasis, the settling of the blood in the lung could be detected in most cases. 2. Early histopathological lung changes induced by shock One hundred and thirty medicolegal cases were reviewed to detect early histopathological changes of the lung induced by shock. In many cases of death from various causes, pulmonary edema and hemorrhage were noted, but the incidence of such changes did not reveal any significant differences among the causes of death. When death had resulted from a hemorrhage or occurred during a state of shock, megakaryocytes in the pulmonary vessels tended to increase. However, if death from such causes had occurred shortly after the event, no increase in megakaryocytes was noted.(ABSTRACT TRUNCATED AT 400 WORDS)

Enkephalinase exists in airway epithelial cells, smooth muscle, and submucosa near glands, and cleaves tachykinins to inactive metabolites, thereby reducing there effects. To study the role of enkephalinase in asthmatic response, we measured its activity in guinea pig model of asthma. When compared with the control values, the enkephalinase activity was reduced during in immediate asthmatic response (IAR) and late asthmatic response (LAR). Compared with the control values (100%), each value was 79.7%, 73.4% in the trachea and 74.3%, 55.7% in the lung respectively. Tracheal muscle preparation taken from the control, IAR, and LAR groups were made and mounted in oxygenated modified Krebs-Ringer solution. The response was monitored by isometric transducer. Concentration response curves to NKA with or without phosphoramidon were obtained. The contractile responses of the LAR groups were enhanced in potency and efficiency. Phosphoramidon potentiated the NKA induced contraction of control and the IAR groups but was less potent in enhancing the contractile response in the LAR group, showing less enkephalinase activity in the LAR. These results suggest that the enkephalinase plays an important role in LAR. In LAR, the enkephalinase activity may be inhibited and the responsiveness of the smooth muscle to some bronchoconstrictor, such as tachykinins, may be increased.

Imidapril is a newly synthesized non-sulfhydryl-containing angiotensin I converting enzyme (ACE) inhibitor. The present study describes the inhibitory effects of imidapril and its active metabolite 6366A on ACEs from various tissues and compares its effects to those of captopril, enalapril and enalaprilat in vitro. 6366A inhibited swine renal and human serum ACEs with an inhibition constant (Ki) of 0.067 nM and 0.04 nM, respectively. These values were 3 to 18 times more potent than those of the other inhibitors. The kinetic study showed that 6366A exerted competitive type inhibition. The ACE inhibition (IC50 values) of 6366A, enalaprilat and the structurally related compounds (6366DM and 6366PY) were compared in homogenates of lung, aorta, heart, brain and kidney from spontaneously hypertensive rats (SHRs) and Wistar Kyoto rats (WKYs). The inhibitory effects of 6366A on all tissue ACEs from SHRs and WKYs were the most potent among these compounds. And the inhibitory potencies of these compounds were correlated with their chemical structure. The present results suggest that 6366A may show a strong inhibitory effect on ACEs from several tissues and species due to its chemical characteristics.

Non-plasmin fibrinolysis enzyme was extracted from the lung and spleen of conventional rats (Thrombos. Haemostas., 1979), although the enzyme was not found in germfree rats, suggesting the possibility that the enzyme may participate in the defence mechanism of the body. The present study was made in an attempt to determine the behavior of non-plasmin fibrinolysis enzyme of the lung tissue in the DIC model of conventional rats induced by a single injection of bacterial endotoxin. The plasminogen-activator activity of the lung tissue, and the fibrinogen level, platelet count, urea nitrogen and plasminogen-activator activity in the blood were also measured. Examination of the lung tissue in the DIC rats indicated a remarkable increase in non-plasmin fibrinolysis activity and a disappearance of plasminogen-activator activity. Inhibitor studies using t-AMCHA and DFP demonstrated that the increased non-plasmin fibrinolysis activity was not derived from activated plasmin, but from serine protease. The disappearance of plasminogen-activator activity in the lung and increase of plasminogen-activator activity in the blood suggested a release of the activator from the lung into the blood due to the endotoxin injection.

Neuron-specific enolase (NSE) as a tumor marker for carcinoma of the lung was evaluated by immunostaining or enzyme immunoassay. NSE productions were immunocytochemically detected in most of small cell carcinoma and a few cases of other histological type of carcinoma. The data obtained in this study suggested that NSE is a marker for lung carcinoma which exhibit the properties of neuroendocrine tumors and is not a marker for histogenesis of small cell carcinoma of the lung. Serum NSE analysis is suggested to be useful for the evaluation of disease extent, monitoring the clinical course and prediction of sensitivity to cytotoxic treatments.

N-alpha-[(S)-1-carboxy-3-phenylpropyl]-L-lysyl-L-proline (MK-0521) is a new non-sulfhydryl-containing angiotensin converting enzyme (ACE) inhibitor. The present investigation describes its ACE and other enzymes inhibitory properties and compares it to those of captopril, MK-421 and MK-422 in vitro. MK-0521 inhibited rat pulmonary ACE by 50% (IC50) at a concentration of 3 nM and was 6.13 times more potent than captopril. The IC50 values of MK-421 and MK-422 against ACE were 2,000 nM and 3.5 nM, respectively. MK-0521 had practically no inhibitory activities against carboxypeptidase A, carboxypeptidase B, leucine aminopeptidase, papain, pepsin and trypsin. The kinetic study on the inhibitory activity of M-0521 against ACE using Lineweaver-Burk plots indicated that MK-0521 exerted competitive ACE inhibition. The dialysis study conducted on the ACE-MK-0521 complex revealed that the inhibitory effect of MK-0521 against ACE was reversible. In the guinea pig ileum, MK-0521 potentiated the contractile effect of bradykinin and depressed the contractile effect of angiotensin I. These effects on bradykinin and angiotensin I were 33.11 and 2.63 times more potent than that of captopril, respectively. The present results suggest that MK-0521 may show a potent hypotensive effect in vivo.

The angiotensin converting enzyme (ACE) activity in tissues and plasma renin activity (PRA) were measured in 2-kidney, 1-clip renal hypertensive rats (2K-RHR) and normotensive rats after a single and 3-weeks oral administrations of ACE inhibitors such as MK-421 and captopril. In the single dose study, MK-421 (1 and 3 mg/kg) and captopril (3 and 10 mg/kg) inhibited the ACE activities in kidney, aorta and plasma in a dose-dependent fashion. The inhibition of ACE activity in kidney or aorta was observed for a longer time than that in plasma. PRA took a time course reversal to that of plasma ACE activity. In the 3-weeks repeated dose study, the ACE activity in kidney and aorta was strongly inhibited after the administration of each ACE inhibitor, while there was no significant change in lung ACE activity at any time point examined. The plasma ACE activity markedly elevated after the administration of each agent. PRA significantly increased after the administration of either agent, while the plasma angiotensin II level was significantly inhibited. These results indicate that the inhibition of the ACE activity in blood vessel or kidney correlate well with the antihypertensive activity in 2K-RHR after a single and repeated administration of both ACE inhibitors, but not well with the inhibition of plasma ACE activity.

Serum amylase level was examined in 129 cases (225 episodes) of chronic respiratory failure at acute exacerbation, and in 59 cases (62 episodes) of pneumonia without respiratory failure as a control. Cases accompanying diseases, such as acute pancreatitis, parotiditis, ileus, and renal dysfunction, which were expected to develop hyperamylasemia were excluded. The 225 episodes were divided according to the cause of acute exacerbation into 4 groups: pneumonia, bronchitis, right heart failure without infection, and others (e.g. hemoptysis). Hyperamylasemia (greater than 400 S-U) was observed in groups of pneumonia (15/40 = 35.5%) and of bronchitis (12/95 = 12.6%) respectively, but not in those of right heart failure without infection (0/73 = 0%) and others (0/17 = 0%). As a result, hyperamylasemia was found only under conditions of inflammation of lung parenchyma and bronchi with acute exacerbation of respiratory failure. On the other hand no hyperamylasemia was observed in 62 episodes of only pneumonia without respiratory failure. It was concluded that both respiratory tract infection and acute respiratory failure are necessary factors for development of hyperamylasemia originating from lung or bronchi.

A manganese-containing superoxide dismutase (Mn-SOD) was isolated from human liver mitochondria by the procedures including heat treatment, ammonium sulfate precipitation, ion exchange chromatography, gel filtration and isoelectric focusing. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of the purified enzyme showed a single band with an approximate molecular weight of 20,000. Judging from the molecular weight of an approximate 80,000 estimated by gel filtration and the above electrophoretic result, the native Mn-SOD exists as a tetramar. Ouchterlony test using a monospecific goat antiserum against the purified liver Mn-SOD indicated that the enzymes from normal lung as well as lung cancer were immunochemically identical. A solid phase enzyme immunoassay (EIA) was established to detect this Mn-SOD enzyme protein in the tissue homogenates. The working range by this assay was from 10 ng to 1,000 ng. The enzymatic activities and the immunological contents of the Mn-SOD in human lung carcinoma and uninvolved lung were compared. The enzyme contents determined by EIA were significantly elevated in carcinoma tissues as compared to the normal uninvolved tissues (37.4 micrograms/mg protein and 17.9 micrograms/mg protein, respectively). However no significant difference was seen in the enzyme activities. Among histological types of lung carcinoma, adenocarcinoma had a significantly high Mn-SOD content. These results suggest that the carcinoma tissues contain an immunologically active, but enzymatically inactive form of Mn-SOD enzyme.

Experimental allergy was induced in animals: asthma, a Type I allergy, was induced in guinea pigs by sensitizing them with alpha-amylase (inhalation), and experimental allergic encephalomyelitis (EAE), a Type IV allergy, was induced in rats. Pulmonary, brain and serum lysosomal enzyme activities were measured in normal and allergic conditions. beta-Glucuronidase (beta-G) and arylsulfatase (AS) activities were determined by the fluorescent technique. During the asthmatic attack, pulmonary lysosomal enzyme activities were not different from that in the normal state in guinea pigs. However, brain lysosomal enzyme activities were elevated markedly on the 1 st day of EAE induction. Brain beta-G activity was elevated on the 2nd day, and AS activity had a tendency to be increased. On the other hand, serum lysosomal enzyme activity was not altered significantly. In the experimental allergy, lysosomal enzyme activity was altered in Type IV, but not in Type I.