Rat Lung Homogenates Research Articles

Degradation of a novel tripeptide, tert-butoxycarbonyl-Tyr-Leu-Val-CH2Cl, with inhibitory effect on human leukocyte elastase in aqueous solution and in biological fluids.

The stability of tert-butoxycarbonyl-Tyr-Leu-Val-CH2Cl (YLV) with inhibitory effect on human leukocyte elastase was investigated in aqueous solution, alpha-chymotrypsin solution and biological media. In all cases studied here, the degradation was observed as a pseudo-first order reaction. The half-life for the degradation of YLV in an aqueous solution of pH 7.4 at 37 degrees C was 35.9 h. YLV was most stable at about pH 3.8-5.8 and the effect of temperature was explained by the Arrhenius equation. The activation energies of the degradation in aqueous solutions at pH 2.0, 4.8, and 7.4 were 24.6, 22.1 and 23.4 kcal/mol, respectively. The degradation products in aqueous solution were analyzed by HPLC-MS and were estimated as Boc-Tyr-Leu-Val-CH2OH at pH 7.4 and H2N-Tyr-Leu-Val-CH2Cl at pH 2.0. In a bovine pancreas alpha-chymotrypsin solution at 37 degrees C, the half-life of YLV was 15 min at 25.6 micrograms/ml of alpha-chymotrypsin solution. In the rat plasma, the half-life of YLV was 42.4 min (YLV 26.7 micrograms/ml plasma), and in rat liver, lung and spleen homogenates, the degradation rate constants of YLV were 37.6, 10.3 and 23.5 times larger than that in plasma solution, respectively (all fluids containing 5 mg protein/ml). YLV was less stable than nafarelin acetate, secretin, adrenocorticotropic hormone (ACTH) and gonadorelin in an aqueous solution of pH 7.4.

Rapid enrichment of hematopoietic stem cells from human vertebral body marrow

The objective of the present study was to evaluate the effect of protease inhibitors on the pulmonary absorption of therapeutic peptides and proteins with varying molecular weights. Dry powder formulations of leuprolide (1.2 kD), salmon calcitonin (3.4 kD), human insulin (5.8 kD), human leptin (16.0 kD), and human chorionic gonadotropin (HCG) (36.5 kD) were prepared with or without protease inhibitors; aprotinin and bestatin. The formulations were administered intrapulmonary to anesthetized rats. The pharmacokinetics of these proteins were assessed by measuring serum drug concentrations. In addition, in vitro stability of these proteins in rat lung homogenate was assessed using the trifluoroacetic acid method. Bioavailability of leuprolide following pulmonary administration was 75% higher compared to subcutaneously administered leuprolide. Protease inhibitors had little or no effect on the pulmonary bioavailability of leuprolide. However, protease inhibitors (1 mg/kg) increased the bioavailability of calcitonin by more than 50%. Similarly, the bioavailabilities of leptin and HCG in the presence of bestatin were increased by 1.9 and 2.1-fold, respectively. Leuprolide was stable both in the lung cytosol and subcellular pellets with about 10% degradation at the end of the study period (4 h). In contrast, calcitonin, insulin, leptin and HCG were significantly degraded in the lung cytosol and subcellular pellets. Presence of protease inhibitors in formulation could improve the stability of protein drugs. The results of this study demonstrate that the pulmonary absorption of proteins may be enhanced by the selection of optimal concentration and type of protease inhibitor.

The use of panel of monoclonal antibodies against different components of crude collagenase to study the function of collagenase in the digestion of the human pancreas

The objective of the present study was to evaluate the effect of protease inhibitors on the pulmonary absorption of therapeutic peptides and proteins with varying molecular weights. Dry powder formulations of leuprolide (1.2 kD), salmon calcitonin (3.4 kD), human insulin (5.8 kD), human leptin (16.0 kD), and human chorionic gonadotropin (HCG) (36.5 kD) were prepared with or without protease inhibitors; aprotinin and bestatin. The formulations were administered intrapulmonary to anesthetized rats. The pharmacokinetics of these proteins were assessed by measuring serum drug concentrations. In addition, in vitro stability of these proteins in rat lung homogenate was assessed using the trifluoroacetic acid method. Bioavailability of leuprolide following pulmonary administration was 75% higher compared to subcutaneously administered leuprolide. Protease inhibitors had little or no effect on the pulmonary bioavailability of leuprolide. However, protease inhibitors (1 mg/kg) increased the bioavailability of calcitonin by more than 50%. Similarly, the bioavailabilities of leptin and HCG in the presence of bestatin were increased by 1.9 and 2.1-fold, respectively. Leuprolide was stable both in the lung cytosol and subcellular pellets with about 10% degradation at the end of the study period (4 h). In contrast, calcitonin, insulin, leptin and HCG were significantly degraded in the lung cytosol and subcellular pellets. Presence of protease inhibitors in formulation could improve the stability of protein drugs. The results of this study demonstrate that the pulmonary absorption of proteins may be enhanced by the selection of optimal concentration and type of protease inhibitor.

Prevention of the Expression of Inducible Nitric Oxide Synthase by Aminoguanidine or Aminoethyl-Isothiourea in Macrophages and in the Rat

An enhanced formation of nitric oxide (NO) following the induction of the inducible isoform of NOS (iNOS) has been implicated in the pathogenesis of circulatory shock and inflammation. This study elucidates the effects of the NOS inhibitors aminoethyl-isothiourea (AE-ITU), aminoguanidine (AG), NG-methyl-L-arginine (L-NMMA) or Nω-Nitro-L-arginine methyl ester (L-NAME) on the expression of iNOS protein lipopolysaccharide (LPS) in macrophages and the rat (lung homogenates). The expression of iNOS protein was detected by Western blot analysis using a specific iNOS antibody. In the absence of LPS, the iNOS protein was expressed neither in macrophages nor in the lung. LPS (1 μg·ml−1) resulted in the expression of iNOS protein in macrophages, which was significantly inhibited by pretreatment of cells with AE-ITU or AG, but not by L-NMMA or L-NAME. In addition, LPS (10 mg·kg−1, i.v.) also caused an increase in the expression of iNOS protein in lungs obtained from rats at 6 h after LPS, which was significantly reduced by treatment of LPS-rats with AE-ITU or AG, but not with L-NMMA or L-NAME. Thus, AE-ITU or AG inhibit not only iNOS activity, but also the induction of iNOS proteinin vitroandin vivocaused by endotoxin.

Role of phospholipase A2 enzymes in degradation of dipalmitoylphosphatidylcholine by granular pneumocytes

The role of phospholipase A2 (PLA2) enzymes in the degradation of internalized dipalmitoylphospharidylcoline (DPPC) by rat granular pneumocytes was evaluated with cells in 24 h primary culture on microporous membranes. In cell sonicates and rat lung homogenates, the transition state analogue MJ33 inhibited acidic (pH 4), Ca(2+)-independent PLA2 (aiPLA2) while p-bromophenacylbromide (pBPB) inhibited alkaline (pH 8.5), Ca(2+)-dependent PLA2 and phospholipase C activities. With intact cells, degradation of [3H]methylcholine-labeled DPPC during 2 h incubation was inhibited 48% by MJ33, 20% by pBPB, and 69%by the combination. The inhibitors (20 microM pBPB, 3 mol% MJ33) had no effect on cellular dye exclusion, adherence to membranes, or uptake of DPPC. Arachidonyl trifuoromethylketone, a cytoplasmic PLA2 inhibitor, had no effect on cellular degradation of DPPC. Degradation was depressed approximately 20% by the addition of NH4Cl or methylamine to the medium, suggesting a role for an acidic intracellular compartment in DPPC metabolism. Subcellular fractions prepared by differential centrifugation of rat lung homogenates showed highest specific activity of aiPLA2 in the lamellar body and lysosomal fractions, lower activity in cytosol, and essentially no activity in mitochondria, microsomes, or plasma membranes. The results of this study indicate that aiPLA2 has the major role in the degradation of internalized DPPC by granular pneumocytes and they are compatible with participation of lysosomes/lamellar bodies in DPPC metabolism.

ADP-ribosylation of an approximately 70-kilodalton protein of Klebsiella pneumoniae

An approximately 70-kDa protein in the culture supernatant of a human pathogenic strain of Klebsiella pneumoniae was labeled in the presence of [32P-adenylate]NAD. Labeling was significantly increased by the addition of dithiothreitol ( > 1 mM) but prevented by treatment of the culture supernatant for 3 min at 56 degrees C. The addition of unlabeled NAD, but not of ADP-ribose, blocked labeling of the approximately 70-kDa protein. The radioactive label was released by formic acid but not by HgCl2 (1 mM) or neutral hydroxylamine (0.5 M). The addition of homogenates of human platelets, human neutrophils, rat brain, rat lung, or rat spleen tissues to the culture supernatant did not induce labeling of eukaryotic proteins. The data indicate that the K. pneumoniae strain produces ADP-ribosyltransferase which modifies an endogenous protein.

Clinical surfactant preparations mediate SOD and catalase uptake by type II cells and lung tissue.

Pulmonary surfactant mixtures are rapidly taken up by alveolar type II cells and thus may serve as vectors for the pulmonary delivery of antioxidant enzymes to the alveolar epithelium. We prepared emulsions of Survanta with superoxide dismutase (CuZn-SOD) and catalase and quantified their cellular uptake both in vitro and in vivo. Incubations of fetal lung epithelial cells with an emulsion of Survanta plus SOD and catalase mixtures resulted in significant augmentation of SOD and catalase activities (12.8 +/- 4.6 U SOD/microgram DNA; 7.49 +/- 2.21 U catalase/microgram DNA). These numbers were significantly greater than those obtained in controls (1.8 U SOD/microgram DNA; 0.55 +/- 0.52 U catalase/microgram DNA, Survanta alone (0.43 U SOD/microgram DNA; 0.16 U catalase/microgram DNA), and SOD and catalase alone (3.47 +/- 5.2 U SOD/microgram DNA; 4.24 +/- 3.0 U catalase/microgram DNA). Intratracheal instillation of the Survanta plus SOD and catalase mixture resulted in significant augmentation of enzymes by the rat lung homogenates. Confocal microscopic analysis revealed the presence of antioxidant enzymes in the cytoplasm of epithelial cells. We concluded that Survanta supplementation, in addition to replenishing surfactant stores, can also enhance the delivery of antioxidant enzymes to alveolar epithelium both in vitro and in vivo.

Effect of oxygen concentration on production of ethane and thiobarbituric acid‐reactive substances by peroxidizing lung and liver homogenates and formation of ethanol by peroxidizing docosahexaenoic acid preparations under hyperoxic conditions

The oxygen dependence of ethane formation was investigated in rat lung and liver homogenates, incubated in sealed flasks, in which the peroxidation was stimulated by the addition of ferrous ions. For both tissues, the production of ethane was maximal under a 20% oxygenated gas phase, while hyperoxic conditions led to a decreased ethane in the gas phase. The formation of thiobarbituric acid-reactive substances (TBA-RS), another marker of the lipid peroxidation process, in the homogenates of lung and liver was strongly stimulated at 100% compared to 20% oxygen. Experiments were also carried out on iron-stimulated peroxidation of pure docosahexaenoic acid preparations, which under air led to a large production of ethane. As for tissue homogenates, the TBA-RS content was increased in the presence of 100% oxygen. Those conditions, however, did not induce an increase in ethane production but led to the formation of ethanol. Therefore, the quenching of ethyl radical by molecular oxygen seems to be a very attractive hypothesis to explain the lack of increased ethane production in favor of ethanol when iron-induced lipid peroxidation was stimulated by oxygen.

CIC-2: a developmentally dependent chloride channel expressed in the fetal lung and downregulated after birth.

Growth and differentiation of the fetal lung are dependent on chloride and fluid secretion, yet the specific molecular identities of fetal chloride channels have not been fully determined. In this study, we demonstrate mRNA expression of the volume-activated chloride channel, CIC-2, in fetal rat lung using reverse-transcriptase polymerase chain reaction (RT-PCR) and ribonuclease (RNase) protection assay. By RNase protection assay, CIC-2 mRNA expression is most abundant in fetal lung and diminishes after birth until it is almost undetectable in adult rat lung. To confirm this result at the protein level, a C-terminal fragment of CIC-2 cDNA derived from 19-day fetal rat lung was cloned into an expression plasmid. The truncated 33-kD CIC-2 protein was expressed in Escherichia coli and purified by column chromatography. Polyclonal antibodies to this antigen were raised in chickens, and the antisera detected a 94-kD protein in fetal rat lung homogenates by Western blotting. Protein expression of CIC-2 was most abundant in mid and late gestation and decreased significantly shortly after birth, as would be predicted by the RNase protection data. CIC-2 protein was localized along the apical surface of fetal airway epithelium by immunocytochemistry. The abundant fetal expression of CIC-2 RNA and protein supports the hypothesis that CIC-2 is important to fetal lung development, and its apical location suggests that it may be involved in fluid secretion during normal lung morphogenesis.

Pulmonary and blood enzyme kinetics as predictors of pulmonary metabolism of esters

The enzymatic hydrolysis of four aliphatic esters of biphenylacetic acid by rat lung homogenate and blood was investigated. The log V max K m ratios derived in both tissues were parabolically related to lipophilicity, similar trends were also observed for log V max values against lipophilicity. K m values could not be related to lipophilicity. The pulmonary and blood intrinsic clearances were linearly related, suggesting blood could be used to predict the pulmonary metabolism of esters. Pulmonary extraction ratios, predicted from the intrinsic clearance using the 'well-stirred' model, were negligible.

EFFECTS OF VARIOUS FACTORS ON PULMONARY ABSORPTION OF PEPTIDE DRUGS USING <I>IN VITRO</I> EXPERIMENTAL SYSTEMS

We examined the in vitro penetration characteristics of FITC-dextrans (FDs) with various molecular weights and peptides such as calcitonin and insulin across human lung carcinoma A-549 cell monolayer. The permeability coefficients of FDs through the A-549 monolayers (type-II like cells) were much larger than those in the rat alveolar epithelial cell monolayers (RAEMs: type-I like cells), which were established by Kim and coworkers. The high permeability characteristics of A-549 monolayers may be attributed to their morphological differences as compared with the RAEMs.On the other hand, we studied the degradation characteristics of peptide drugs such as calcitonin and insulin using rat lung and A-549 cell homogenates. Calcitonin was rapidly disappeared than insulin in both homogenates. The apparent degradation clearance (Km/Vmax) values of these peptides were higher in the cytosol fraction than membrane fraction. In both homogenates, chymotrypsin activity was the highest among the six protease activities such as trypsin, elastase, chymotrypsin, Leu-aminopeptidase, aminopeptidase B and aminopeptidase N examined in this study. These results confirmed our previous report that the degradation of insulin in the rat lung homogenate was suppressed by the addition of soybean trypsin inhibitor.Furthermore, the penetration characteristics of water soluble compounds, such as phenol red and FDs across the Xenopus isolated pulmonary membrane were examined with the modified Ussing chamber system. The permeability coefficients of FDs with various molecular weights through the Xenopus isolated pulmonary membrane were similarto those through the small and large intestine. A good correlation was observed between the penetrated amounts of drugs across the Xenopus isolated pulmonary membrane with the in vitro Ussing chamber system and their absorbed amounts calculated by deconvolution method in an in situ rat intrapulmonary absorption experiment. Three types of absorption enhancers, such as laurylmaltoside, mixed micelle and Na-caprate, increased the penetration of phenol red across the Xenopus isolated pulmonary membrane, while EDTA and Na-salicylate did not affect its penetration across the Xenopus isolated pulmonary membrane. These results were in agreement with our previous report of an in situ rat intrapulmonary experiment.

Susceptibility of insulin to proteolysis in rat lung homogenate and its protection from proteolysis by various protease inhibitors.

The objective of this study was to determine the stability of insulin in rat lung homogenate and to confirm the effectiveness of various protease inhibitors for insulin delivery from the lung. The stability of insulin in rat lung homogenate was compared with that in the small intestine. Insulin was rapidly degraded in lung homogenate similarly to degradation in the small intestinal homogenate. The effects of various protease inhibitors on the degradation of insulin were studied in the lung. Protease inhibitors such as Na-glycocholate (Na-GC; 10 mM), aprotinin (10 mg/ml), soybean trypsin inhibitor (STI; 10 mg/ml) and bacitracin (20 mM) effectively reduced insulin degradation in lung homogenate. The rank order of effectiveness for decreasing the insulin hydrolysis in lung homogenate was bacitracin > aprotinin > STI > Na-GC. In addition, a slight correlation was observed between the in situ pulmonary absorption of insulin and its stability in vitro in the lung homogenate in the presence of various protease inhibitors. These findings suggest that the coadministration of protease inhibitors would be useful for improving the stability of insulin in the lung.

Pulmonary O2 toxicity: role of endogenous tumor necrosis factor.

The role of endogenous tumor necrosis factor (TNF) in the pathogenesis of pulmonary O2 toxicity was investigated. Intratracheal insufflation of anti-TNF antibodies prolonged the survival of rats exposed to 100% O2. No TNF bioactivity or immunoreactive protein was detectable in the alveolar lavage fluid or lung homogenate of rats exposed to normoxia or hyperoxia. However, levels of pulmonary TNF mRNA were markedly enhanced in rats exposed to hyperoxia. These results suggest that hyperoxia may cause the production of low level TNF, which in turn enhances O2 toxicity.

Purification and characterization of a calcium-independent acidic phospholipase A2 from rat lung.

Several phospholipase A2 (PLA2) activities have been identified in rat lung homogenate and shown to be important in metabolism of lung phospholipids. One PLA2 activity is Ca(2+)-independent, active in vitro at pH 4, and inhibited by a substrate analogue, 1-hexadecyl-3-trifluoroethylglycero-sn-2-phosphomethanol (MJ33). Purification of this rat lung PLA2 by approx. 550-fold was carried out by sequential column chromatographies using DE-52, Sephacryl-100, heparin-Sepharose, and phenyl-Sepharose columns. The purified activity had an acidic pH optimum, was Ca(2+)-independent, was inhibited by MJ33 in a dose-dependent manner (50% inhibition at 3 mol%), was unaffected by treatment with p-bromophenacyl bromide or mercaptoethanol, and had a unique N-terminal amino acid sequence. The apparent molecular mass was 15 kDa on gel electrophoresis and activity was recovered in part by renaturation of protein from the gel. The properties of this enzyme suggest a new class of PLA2.

Inhibition of lung calcium-independent phospholipase A2 by surfactant protein A.

The effect of lung surfactant protein A (SP-A) on lung phospholipase A2 (PLA2) activity was investigated. SP-A was purified from bovine surfactant obtained by lung lavage. PLA2 was assayed using radiolabeled 1,2-dipalmitoyl phosphatidylcholine (DPPC) in surfactant-like unilamellar liposomes with Ca(2+)-free acidic (pH 4) or 10 mM Ca2+, alkaline (pH 8.5) buffer. SP-A significantly inhibited Ca(2+)-independent acidic PLA2 of rat lung homogenate or isolated lamellar bodies but had no effect on the Ca(2+)-dependent alkaline enzyme. Lamellar body PLA2 was inhibited by 50% with 0.25 micrograms SP-A/microgram lamellar body protein. Similar inhibition by SP-A was observed when 1-palmitoyl,2-oleoyl PC (POPC) was the substrate. Binding assay showed binding of 125I-labeled SP-A to DPPC but not to POPC, indicating that removal of substrate was not the mechanism for inhibition of the enzyme by SP-A. Chemical reduction or alkylation of SP-A abolished its inhibitory effect on PLA2 activity. Inactivation of endogenous SP-A in isolated lamellar bodies or surfactant increased Ca(2+)-independent PLA2 activity in these fractions. The presence of SP-A in liposomes stimulated the uptake of DPPC by isolated granular pneumocytes in primary culture but significantly inhibited its degradation. These results indicate that the Ca(2+)-independent acidic PLA2 has a role in the metabolism of internalized surfactant phospholipid and that SP-A can modulate the activity of this enzyme.

Synthesis of type II cell lamellar body lysozyme-15 kD protein (lbl-15) by perfused rat lung.

Isolated alveolar type II pneumocytes of the rat have been shown to secrete a 14 to 15 kD protein that has some sequence homology and immunoreactivity with lysozyme. Using immunochemical analyses of rat lung subcellular fractions and 35S metabolic labeling of isolated perfused lung preparations, we studied the subcellular distribution and synthetic pathway for this protein. SDS-PAGE and Western blotting of lamellar bodies (LB) using a polyclonal anti-human lysozyme (anti-HLZ) demonstrated a single band at 15 kD that was significantly enriched over rat lung homogenates, isolated lysosomes, and type II cell lysates. This 15 kD protein isolated from LB by immunoprecipitation with anti-HLZ also demonstrated functional lysozyme activity and was termed lamellar body lysozyme (lbl-15). Analysis of LB and surfactant (SF) isolated from 12 separate perfused lung preparations labeled for 6 h with 35S-labeled amino acids demonstrated that lbl-15 represented a significant portion of the radiolabeled LB proteins (5.9% of total LB radioactivity). Lamellar bodies and an extracellular fraction (surfactant) obtained from rat lungs perfused with 35S-methionine-cysteine for varying times both showed time-dependent appearance of lbl-15. At all time points, the specific activity of lbl-15 was greater in LB than in SF. The kinetics for appearance of lbl-15 in LB and SF was similar to that for surfactant protein A. These results indicate that in the rat lung, type II cells synthesize a 15 kD protein (lbl-15) that is secreted into the alveolar space via an organellar pathway involving LB.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of non-conventional opioid binding sites in rat and human lung

Indirect evidence suggests that nebulized morphine relieves dyspnoea and bronchoconstriction via opioid receptors within the lung. This study used equilibrium binding studies to characterize opioid binding sites in lung membrane preparations. [ 3H]Morphine and [ 3H]naloxone were incubated separately with homogenates of Wistar rat brain and lung, and human lung. Binding affinities for both morphine and naloxone in rat and human lung were two orders of magnitude lower than those in brain. However, opioid binding site densities in lung were up to 100 times greater than that in brain. The addition of Na + or GTP to lung homogenate preparations caused atypical effects on opioid binding. Na + (50 mM) decreased the specific binding of [ 3H]naloxone 50% viz-à-vis a 20% increase in binding in the brain. GTP (100 μM) caused a 200% increase in the apparent capacity of morphine binding in the lung compared with a marked decrease in binding in the brain.

Genetic diversity of Pneumocystis carinii derived from infected rats, mice, ferrets, and cell cultures.

The degree of strain and/or species diversity among Pneumocystis carinii isolates is unknown. As a first approach to the study of P. carinii genetic relatedness, we compared the pulsed field gel electrophoretic karyotypes of P. carinii derived from lung homogenates of three immunosuppressed host animals: rats transtracheally inoculated with P. carinii-infected mouse lung; and ferrets which developed reactivated latent P. carinii pneumonia. Rat P. carinii propagated on HEL299 cells was also examined. Karyotypes of P. carinii DNA from both rat lung homogenate and cell culture were identical (14 bands, 315-680 kb). In contrast, mouse and ferret P. carinii DNA karyotypes were each distinctly different from the rat P. carinii samples (mouse P. carinii 15 bands, 315-610 kb; ferret P. carinii nine bands, 410-760 kb). Three distinct rat P. carinii gene probes reacted with both Southern-transferred rat and mouse P. carinii DNA but not with ferret P. carinii DNA. Thus, P. carinii from rat, mouse, and ferret are genetically diverse. The results are consistent with recently reported antigenic and nucleic acid sequence differences among P. carinii isolates recovered from different hosts.

Determination of angiotensins by capillary electrophoresis

A protocol to separate ten peptides of the angiotensin family by capillary electrophoresis was described. The experiment was carried out using the Waters Quanta 4000 Electrophoresis system and the steps taken to determine the optimum electrophoretic conditions include (i) the use of different electrolytes, (ii) variation of ionic composition and pH of the electrolytes, (iii) variation of applied voltage and the wavelength of ultraviolet detection. Successful separation of the ten angiotensin peptides was obtained using a voltage of 10 kV, 0.1 M phosphoric acid (pH 1.95) as electrolyte and ultraviolet detection at 185 nm. The protocol was then used to follow the metabolism of exogenous angiotensin I (ANG I) in rat lung homogenate and the separation and identification of an angiotensin peptide in human plasma. In addition to these two applications, the protocol can be use to separate and identify angiotensins and other peptides for which specific antibodies have yet to be developed.

Improvement of the pulmonary absorption of (Asu1,7)-eel calcitonin by various protease inhibitors in rats.

The effects of protease inhibitors, Na-glycocholate, bacitracin, bestatin, nafamostat mesilate and soybean trypsin inhibitor (STI) on the pulmonary absorption of (Asu1,7)-eel calcitonin (ECT, molecular weight 3363) were investigated in rats. The pulmonary absorption of ECT was estimated by measuring its hypocalcemic effect. When ECT alone was administered into the lung, the pharmacological availability of ECT was 2.7%. Co-administration with STI or bestatin did not change the pharmacological effect of ECT. However, Na-glycocholate, bacitracin and nafamostat mesilate caused a significant hypocalcemic effect following the pulmonary absorption of ECT and a maximal effect was noted in the presence of 20 mM bacitracin, approaching the effect after intravenous administration of ECT. Bacitracin and Na-glycocholate reduced the degradation of 111In-ECT in rat lung homogenate. Therefore, protease inhibitors effectively improved the pulmonary absorption of ECT.