Improved spatial resolution of matrix-assisted laser desorption/ionization imaging of lipids in the brain by alkylated derivatives of 2,5-dihydroxybenzoic acid.

RATIONALE: Matrix-assisted laser desorption/ionization (MALDI) is one of the major techniques for mass spectrometry imaging (MSI) of biological systems along with secondary-ion mass spectrometry (SIMS) and desorption electrospray mass spectrometry (DESI). The inherent variability of MALDI-MSI signals within intact tissues is related to the heterogeneity of both the sample surface and the matrix crystallization. To circumvent some of these limitations of MALDI-MSI, we have developed improved matrices for lipid analysis based on structural modification of the commonly used matrix 2,5-dihydroxybenzoic acid (DHB). METHODS: We have synthesized DHB containing -C6H13 and -C12H25 alkyl chains and applied these matrices to rat brain using a capillary sprayer. We utilized a Bruker Ultraflex II MALDI-TOF/TOF mass spectrometer to analyze lipid extracts and tissue sections, and examined these sections with polarized light microscopy and differential interference contrast microscopy. RESULTS: O-alkylation of DHB yields matrices, which, when applied to brain sections, follow a trend of phase transition from crystals to an oily layer in the sequence DHB → DHB-C6H13 → DHB-C12H25 . MALDI-MSI images acquired with DHB-C12H25 exhibited a considerably higher density of lipids than DHB. CONCLUSIONS: Comparative experiments with DHB and DHB-C12H25 are presented, which indicate that the latter matrix affords higher lateral resolution than the former.

Bleomycin: a pharmacologic tool in the study of the pathogenesis of interstitial pulmonary fibrosis.

Bleomycin is a unique DNA-interactive antitumor agent that has become a popular tool in studies of the pathogenesis of interstitial pulmonary fibrosis. The biochemical and morphological changes seen in the lungs of many species after bleomycin simulate those seen in humans. The availability of these animal models of interstitial pulmonary fibrosis also provides the opportunity to investigate novel pharmacological approaches to preventing this disease.

The inhibitory role of CpG immunostimulatory motifs in cationic lipid vector-mediated transgene expression in vivo.

We have previously reported that intravenous administration of cationic lipid-protamine-DNA complexes (LPD) induces production of large quantities of proinflammatory cytokines that are toxic and cause inhibition of transgene expression. Cytokine induction appears to be mediated by the unmethylated CpG sequences since methylation of plasmid DNA significantly decreases the cytokine levels. In this study, the inhibitory role of CpG in lipid-mediated gene transfer was further investigated using chemically well-defined, CpG-containing oligodeoxynucleotides (ODNs). Injection (intravenous) of ODNs formulated in LPD into mice triggered production of proinflammatory cytokines including interferon gamma and TNF-alpha. The potency of CpG-containing ODNs in cytokine induction was affected by its flanking sequences and was significantly reduced when CpG was methylated. Preinjection of ODN-containing LPD led to inhibition of transgene expression in lungs after a subsequent injection of LPD containing plasmid expression vector with luciferase gene. The degree of inhibition correlated with the levels of ODN-triggered cytokines. Finally, intraperitoneal injection of dexamethasone suppressed LPD-induced cytokine production, and led to significantly higher levels of transgene expression on both first and second injection. These studies suggest that mutation of potent CpG motifs in plasmid DNA together with the use of immune suppression agent may represent an effective approach to improve cationic lipid-mediated gene transfer to the lung.

Bifunctional anti/prooxidant potential of metallothionenin: redox signaling of copper binding and release.

Metallothioneins (MTs) are cysteine-rich metal-binding proteins that exert cytoprotection during metal exposure and oxidative stress. The roles of MT in copper (Cu) binding and release and modulation of redox cycling are unresolved. We hypothesized that Cu-binding to MT renders Cu redox inactive, but that oxidation of free thiols critical for metal binding can reduce MT/Cu interactions and potentiate Cu redox cycling. Overexpression of MT in cells by cadmium pretreatment or ectopic overexpression by gene transfer confers protection from Cu-dependent lipid oxidation and cytotoxicity. Using a chemically defined model system (Cu/ascorbate/H2O2) to study Cu/MT interactions, we observed that MT inhibited Cu-dependent oxidation of luminol. In the absence of H2O2, MT blocked Cu-dependent ascorbyl radical production with a stoichiometry corresponding to Cu/MT ratios < or = 12. In the presence of H2O2, Cu-dependent hydroxyl radical formation was inhibited only up to Cu/MT ratios < or = 6. Using low-temperature EPR of free Cu2+ to assess Cu/MT physical interactions, we observed that the maximal amount of Cu1+ bound to MT corresponded to 12 molar equivalents of Cu/MT with Cu and ascorbate alone and was reduced in the presence of H2O2. 2,2'-Dithiodipyridine titration of MT SH-groups revealed a 50% decrease after H2O2, which could be regenerated by dihydrolipoic acid (DHLA). DHLA regeneration of thiols in MT was accompanied by restoration of MT's ability to inhibit Cu-dependent oxidation of ascorbate. Thus, optimum ability of MT to inhibit Cu-redox cycling directly correlates with its ability to bind Cu. Some of this Cu, however, appears releasable following oxidation of the thiolate metal-binding clusters. We speculate that redox-dependent release of Cu from MT serves both as a mechanism for physiological delivery of Cu to specific target proteins, as well as potentiation of cellular damage during oxidative stress.

Effect of sodium arsenite on iNOS expression and vascular hyporeactivity associated with cecal ligation and puncture in the rat.

Induction of the heat shock response protects animals from either endotoxemia or peritonitis. In endotoxemia, heat shock protein (HSP) induction is associated with reversal of vascular hyporeactivity and inhibition of iNOS expression. Recent studies suggest differences in the inflammatory mechanisms during endotoxemia and peritonitis animal models and their response to therapeutic interventions. We therefore studied the effect of the HSP inducer sodium arsenite (SA) on vascular reactivity and iNOS expression in rats undergoing cecal ligation and puncture (CLP). CLP resulted in suppression of the pressor effect of norepinephrine (NE) in vivo (measured by changes in blood pressure in response to NE boluses) and ex vivo (changes in contraction force in isolated mesenteric arteries in response to NE concentrations), and in the expression of iNOS protein. Pretreatment of the rats with SA resulted in reversal of CLP-induced vascular hyporeactivity in vivo and ex vivo, and inhibition of iNOS expression after 22 h. SA pretreatment improved 7-day survival after CLP from 18.2% to 70% (P < 0.005). Glucocorticoid receptor inhibition did not affect the effect of HSP induction on iNOS expression. The similarity of the effect of HSP on vascular reactivity and iNOS expression in two distinct sepsis models suggests that this effect may be clinically important and that a causative relationship between HSP induction, iNOS inhibition, and reversal of vascular reactivity is likely.

Induced nitric oxide inhibits IL-6-induced stat3 activation and type II acute phase mRNA expression.

Inducible nitric oxide synthase (iNOS) can be coexpressed with acute phase reactants in hepatocytes; however, it is unknown if NO can regulate the acute phase response. We tested the hypothesis that iNOS-derived nitric oxide (NO) attenuates the acute phase response by inhibiting IL-6-enhanced Stat3 DNA-binding activity and type II acute phase mRNA expression. iNOS was overexpressed in cultured rat hepatocytes via transduction with a replication defective adenovirus containing cDNA for human iNOS (AdiNOS), and Stat3 DNA-binding activity was determined by electrophoretic mobility shift assay (EMSA). EMSAs demonstrated that AdiNOS inhibits IL-6-induced Stat3 activation and that this inhibition is reversible in the presence of the NOS inhibitor N(G)-monomethyl-L-arginine (L-NMA). The induction of beta-fibrinogen mRNA by IL-6, a Stat3 dependent process, is attenuated in AdiNOS-transduced cells and partially reversed by L-NMA. Thus, iNOS overexpression suppresses IL-6-induced Stat3 activation and type II acute phase mRNA expression in cultured hepatocytes. This suppression may represent a mechanism by which NO down-regulates the acute phase response.

Effects of drugs on mucociliary transport in the trachea and bronchial airways.

Pharmacologically active agents may change transport rates regionally within the airways of the lung, as well as affect the overall magnitude of the clearance of inhaled deposited radioaerosols. To investigate these possibilities the response of ethanol on pulmonary retention was determined and the responses of both the trachea and bronchial airways were measured after either oral administration of metaproterenol or inhalation of sulfuric acid mist. In the healthy nonchallenged lung, the velocity of mucociliary transport in the trachea was related to the percentage of activity cleared from the lung in two hours. Indices representing different portions of the pulmonary retention curve were also correlated. Changes in this interdependence of mucociliary transport within airways were produced by all agents. Metaproterenol increased tracheal mucus velocity but not lung clearance. Alcohol changed pulmonary retention in both magnitude and direction depending on the individual, resulting in an increase in variability of pulmonary mucociliary clearance between persons. Thus, to evaluate the effects of drugs or pollutants on the lower respiratory tract, measurements of mucociliary transport should be made in both the trachea and the bronchial airways.

Gene therapy and endothelial cell targeting for cancer.

The endothelium represents a potentially critical target for gene therapy because of its anatomical location and its importance in the viability in both normal and malignant tissues. Protecting the endothelium of normal tissues, such as the lungs, from the toxic effects of current antineoplastic agents and the destruction of the tumor vasculature are reasonable goals. As a target, however, the endothelium continues to represent a significant challenge. While gene delivery to cultured endothelial cells is possible, improved delivery systems are required, as well as cell-specific promoters, before in vivo gene therapy to important endothelial populations can be accomplished.

Adenoviral transfer of the inducible nitric oxide synthase gene blocks endothelial cell apoptosis.

BACKGROUND: We have previously reported that vascular inducible nitric oxide synthase (iNOS) gene transfer inhibits injury-induced intimal hyperplasia in vitro and in vivo. One mechanism by which NO may prevent intimal hyperplasia is by preserving the endothelium or promoting its regeneration. To study this possibility we examined the effect of iNOS gene transfer on endothelial cell (EC) proliferation and viability. METHODS: An adenoviral vector (AdiNOS) containing the human iNOS cDNA was constructed and used to infect cultured sheep arterial ECs. NO production was measured, and the effects of continuous NO exposure on EC proliferation, viability, and apoptosis were evaluated. RESULTS: AdiNOS-infected ECs produced 25- to 100-fold more NO than control (AdlacZ) infected cells as measured by nitrite accumulation. This increased NO synthesis did not inhibit EC proliferation as reflected by tritiated thymidine incorporation. Chromium 51 release assay revealed that EC viability was also unaffected by AdiNOS infection and NO synthesis. In addition, prolonged exposure to NO synthesis did not induce EC apoptosis. Instead, NO inhibited lipopolysaccharide-induced apoptosis in these cells by reducing caspase-3-like protease activity. CONCLUSIONS: Vascular iNOS gene transfer, while inhibiting smooth muscle cell proliferation, does not impair EC mitogenesis or viability. Augmented NO synthesis may also protect ECs against apogenic stimuli such as lipopolysaccharide. Therefore iNOS gene transfer may promote endothelial regeneration and can perhaps accelerate vascular healing.

Antisense oligodeoxynucleotide to inducible nitric oxide synthase inhibits nitric oxide synthesis in rat pulmonary artery smooth muscle cells in culture.

BACKGROUND: We recently demonstrated the induced expression of the inducible nitric oxide synthase (iNOS) gene in cultured rat pulmonary artery smooth muscle (RPASM) in response to lipopolysaccharide and cytokines, using a complementary DNA probe to murine macrophage iNOS. Because nitric oxide (NO) can be cytotoxic, iNOS in the pulmonary vasculature may contribute to lung injury in sepsis. We designed an antisense oligodeoxynucleotide complementary to the iNOS messenger RNA (mRNA) sequence to determine whether the probe prevented iNOS translation. METHODS: RPASM, preincubated in the presence of antisense and sense oligodeoxynucleotide to the first 18 bases after the initiation codon of iNOS mRNA, was exposed to interferon-gamma and tumor necrosis factor-alpha to induce NO production (as measured by NO2-, the stable end product of NO formation). RESULTS: Interferon-gamma and tumor necrosis factor-alpha induced NO production in RPASM: The antisense probe caused up to a 36% decrease in cytokine-induced NO2- production in a concentration-dependent manner (1 to 10 mumol/L). The sense probe had no effect. CONCLUSIONS: Increased transcription of iNOS mRNA is an essential step in the induced production of NO by RPASM: Antisense probes partially inhibit iNOS expression in vitro, suggesting its use to inhibit iNOS expression under pathologic conditions.

Nitric oxide produced by cytokine-activated pulmonary artery smooth muscle cells is cytotoxic to cocultured endothelium.

BACKGROUND: We recently demonstrated that rat pulmonary artery smooth muscle (RPASM) generates maximal nitric oxide (NO) when exposed to inflammatory cytokines, such as tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma. Our hypothesis is that NO produced by cytokine-stimulated RPASM has local cytotoxic effects on endothelium. Accordingly, we designed a pulmonary smooth muscle and endothelial coculture experiment in which the effects of NO on endothelium can be distinguished from the direct effects of cytokines. METHODS: RPASM cells were incubated with a mixture of TNF-alpha (500 units/ml) and IFN-gamma (100 units/ml) for 24 hours. This cytokine mixture was then removed and the NO-producing smooth muscle cells were incubated in a coculture transwell system with rat pulmonary artery endothelial (RPAE) cells. Subsequent NO production (as measured by nitrite concentration in cell supernatants), and the number of viable attached endothelial cells were then measured at 48 hours. RESULTS: RPASM continued to produce large amounts of NO, in the absence of further cytokine stimulation, after a 24-hour exposure to TNF-alpha and IFN-gamma. This RPASM-generated NO decreased the number of viable attached endothelial cells after 24 hour RPASM-RPAE coculture by 57%. The competitive stereospecific inhibitor of inducible NO synthase (iNOS), NG-monomethyl-L-arginine (NMA), returned the inducible NO production to basal levels and reversed the cytotoxic effects on endothelial cells. The number of viable attached endothelial cells returned to control levels. CONCLUSIONS: The NO produced by cytokine-activated RPASM has local cytotoxic effects on RPAE in coculture. Such NO produced in the vasculature may be a factor in the origin of acute lung injury under conditions of trauma and sepsis.

Selective effect of phorbol ester on serotonin removal and ACE activity in rabbit lungs.

The effect of phorbol myristate acetate (PMA) on pulmonary removal of [14C]serotonin (5-[14C]HT) and metabolism of [3H]benzoyl-phenylalanyl-alanyl-proline (BPAP), a synthetic substrate for angiotensin-converting enzyme (ACE), was evaluated in isolated rabbit lungs perfused in situ with Krebs-albumin. Metabolic functions were assessed before, during, and after perfusion with 80 nM PMA (n = 11), or PMA plus 133 microM papaverine (n = 10) or PMA diluent (dimethyl sulfoxide, n = 11). Organ kinetic parameters (apparent Vmax, Km) were calculated by use of indicator-dilution techniques and by a mathematical model of whole-organ metabolism. PMA treatment resulted in a significant decline in Vmax for BPAP metabolism (from 52 +/- 4 to 30 +/- 4 nmol/s) and 5-HT removal (from 2.1 +/- 0.2 to 1.1 +/- 0.1 nmol/s). Km for BPAP was not significantly altered, whereas Km for 5-HT removal was higher after treatment (before treatment, 1.1 +/- 0.1 microM; after treatment, 2.3 +/- 0.6 microM). Coperfusion with papaverine, which attenuated the pressor response to PMA, abolished PMA-induced changes in Vmax for BPAP metabolism and in Km for 5-HT removal but left PMA-induced changes in Vmax for 5-HT removal intact. We conclude that PMA alters endothelial metabolic function by both hemodynamic and biochemical mechanisms that are independent of circulating blood cells. Pulmonary capacity for BPAP metabolism may largely reflect perfused surface area, and capacity for 5-HT removal may be more sensitive to frank endothelial cell dysfunction in this model.

Effect of flow and surface area on angiotensin-converting enzyme activity in rabbit lungs.

Pulmonary angtiotensin-converting enzyme (ACE) is located on the luminal surface of pulmonary microvasculature. Multiple indicator-dilution techniques have been used to measure pulmonary ACE activity in vivo and in isolated lungs. These studies suggest that ACE activity is depressed in several forms of acute lung injury. Depression of ACE activity may reflect impaired substrate delivery to enzyme sites because of flow-related reduction of perfused surface area. To assess the role of altered microvascular flow and surface area in the measurement of ACE activity, we utilized similar techniques to estimate the apparent Km and Vmax of pulmonary ACE in isolated, Krebs-perfused rabbit lungs. Km is an estimate of the affinity of a synthetic ACE substrate, [3H]benzoyl-phenyl-alanyl-alanyl-proline ([3H]BPAP), for ACE and should not be influenced by the rate of substrate delivery to luminal enzyme sites. Conversely, Vmax is an index of the number of ACE sites and should be influenced by perfusion changes that alter the number of perfused sites (recruitment or derecruitment). When isolated lungs were subjected to physiological maneuvers designed to increase or decrease perfused surface area, apparent Vmax increased or decreased respectively. Apparent Km was not altered by these maneuvers. Km and Vmax were independent of changes in perfusion rate when surface area was held constant. Thus these parameters should be useful in evaluating perfusion changes in normal and injured lungs.

Pulmonary angiotensin-converting enzyme kinetics after acute lung injury in the rabbit.

Depression of lung endothelial cell metabolic function may be an early and sensitive indicator of lung damage. When such functions are measured in vivo, substrates injected usually must be limited to "trace" doses due to the significant hemodynamic effects of high doses of substrate. Under first-order conditions (i.e., trace doses) the enzyme or transport system rate constant Vmax/Km may be calculated, but independent estimates of each variable (Vmax and Km) are not available. We therefore used multiple indicator-dilution methods and higher substrate concentrations to apply a mathematical model, based on saturable kinetics that yield independent estimates of the apparent kinetic parameters Vmax and Km for pulmonary angiotensin-converting enzyme (ACE). We used the ACE substrate, [3H]benzoyl-phenylalanyl-alanyl-proline ([3H]BPAP) and made these measurements and also estimates of serotonin [5-hydroxytryptamine (5-HT)] removal, before and after acute lung injury induced by intratracheal administration of phorbol myristate acetate (PMA). PMA significantly depressed the percent 5-HT removal (62 +/- 3 to 44 +/- 4%) and BPAP percent metabolism (74 +/- 2 to 66 +/- 2), when trace amounts of either compound were injected as a bolus.(ABSTRACT TRUNCATED AT 250 WORDS)

Alveolar transfer of prostaglandin E2 in isolated perfused guinea pig lungs.

Alveolar transfer of prostaglandin E2 (PGE2) was characterized in isolated perfused guinea pig lungs (n = 19) by measuring radioactivity appearing in the venous effluent during 30 min after intratracheal instillation of [3H]PGE2, [14C]-mannitol, and [125I]iodoantipyrine. Recovery of lipid-soluble [125I]iodoantipyrine [91 +/- 3% (SE)] after 30 min was used to estimate total 3H and 14C delivered to the exchanging region of lung at time 0. In seven control lungs, 58 +/- 4% of [14C]mannitol and 16 +/- 4% of [3H]PGE2 was retained 10 min after instillation. Neither perfusion with diphloretin phosphate (10 micrograms/ml; n = 4) nor hypothermia (5 degrees C; n = 5) significantly affected the amount of [14C]mannitol retained; however, [3H]PGE2 remaining in these lungs increased significantly to 36 +/- 4 and 53 +/- 2%, respectively. Addition of unlabeled PGE2 (200 micrograms) to the instilled solution (n = 3) increased retention of both [14C]mannitol (80 +/- 3%) and [3H]PGE2 (65 +/- 4%). Alveolar transfer of [3H]PGE2 was calculated as the difference in percent retention of [14C]mannitol and [3H]PGE2 and normalized to that of [14C]mannitol. After 10 min, alveolar transfer of [3H]PGE2 was 71 +/- 8% in control lungs but was decreased to 26 +/- 7, 10 +/- 5, and 19 +/- 6% by diphloretin phosphate, hypothermia, or unlabeled PGE2, respectively. These data suggest that alveolar clearance of PGE2 involves a saturable drug- and temperature-sensitive process.

Functional assessment of pulmonary capillary surface area in the 2-mo-old lamb.

We recently reported that measurements of the maximal velocity of pulmonary endothelial angiotensin-converting enzyme (Vmax) in vivo provide information regarding microvascular surface area in the developing lamb. To obviate any subtle influences of development on Vmax aside from simple increases in surface area, we correlated Vmax with postmortem stereological assessments of alveolar surface area in the relatively mature lung of the 2-mo-old lamb (n = 14). We attempted to increase the range of surface area beyond its normal variability by injecting nine of the lambs with bleomycin, an antineoplastic agent with significant pulmonary toxicity in other species. Vmax, measured shortly after birth and then weekly, increased monotonically in all lambs. Despite their wide dispersion, Vmax and the stereological determinations correlated strongly at 2 mo of age, confirming that Vmax is a robust indicator of the surface area of the air-blood barrier. There was no significant difference in either measurement between the control lambs and those treated with bleomycin, suggesting that the newborn lamb is resistant to the effect of this agent.

Correlation of pulmonary ACE activity and capillary surface area during postnatal development.

Although a considerable amount of information is available regarding the remodeling and growth of the pulmonary arterial circulation, relatively little is known regarding postnatal development of the pulmonary microcirculation. We hypothesized that the maximal velocity (Vmax) of pulmonary angiotensin-converting enzyme (ACE) activity, measured from indicator-dilution outflow curves using a synthetic substrate, 3H-labeled benzoyl-phenylalanyl-alanyl-proline (BPAP), is directly related to the capillary endothelial cell surface area in the lungs of developing lambs. Accordingly we measured apparent kinetics of pulmonary ACE activity in 22 anesthetized ventilated lambs (2-171 days old) and compared our functional assessment to simultaneous in vivo determinations of CO diffusing capacity (DLCO) and postmortem structural assessment of alveolar septal dimensions using stereology and electron microscopy. There was a progressive increase in Vmax of ACE in this age group, with little change in apparent affinity for BPAP. Similar functional manifestation of growth was noted by an age-dependent increase in DLCO. Neither Vmax nor DLCO was significantly affected by an increase in left atrial pressure to 19 Torr (via inflation of a balloon in the left atrium), suggesting little recruitment of vessels under conditions of the present protocol. A close correlation was observed when either Vmax for ACE activity or DLCO was plotted vs. capillary endothelial cell surface area. Double logarithmic transformation of capillary endothelial cell surface area, Vmax-ACE and DLCO vs. lung volume revealed power functions with slopes all greater than that predicted from isotropic growth, suggesting selective differential postnatal development of the endothelium of the alveolar septum in lambs from 2-171 days of age.

Rapid increases in respiratory epithelial permeability occur after intratracheal instillation of PMA.

We measured pulmonary epithelial permeability by quantifying the disappearance of two water-soluble compounds, [14C]mannitol and [3H]inulin, after their instillation, with and without phorbol myristate acetate (PMA), into gas-filled perfused (50 ml/min) rabbit lungs in situ. Both tracers disappeared in a monoexponential fashion over 30 min with calculated first-order rate constants (control; n = 11) of 0.0008 +/- 0.0002 and 0.0027 +/- 0.0008 min-1 for inulin and mannitol, respectively. The ratio of the rate constants (3.1 +/- 0.5) was not significantly different from the ratio of diffusivities of mannitol:inulin (3.7). Addition of PMA (250 micrograms) significantly (n = 9, P less than 0.05) increased the rate constants for both inulin and mannitol to 0.0024 +/- 0.0007 and 0.0087 +/- 0.0025 min-1, respectively, while not affecting their ratio (4.3 +/- 0.5). Addition of human leukocytes (4-8 X 10(8)/l) to the perfusate did not exacerbate the effect of 250 micrograms PMA (n = 3). The addition of catalase (n = 7) completely inhibited the effect of 250 micrograms PMA. PMA (250 micrograms) did not significantly affect perfusion pressure but increased wet-to-dry weight ratios. Light microscopic histology showed damage to epithelial and endothelial cells after 250 micrograms PMA which was not seen after coinstillation of catalase. Catalase sensitivity of functional and structural effects of PMA suggests that the effect was secondary to production of hydrogen peroxide. Since this effect was noted in lungs not perfused with neutrophils and addition of leukocytes did not exacerbate the increase in permeability, we hypothesize that an undetermined pulmonary cell type was the source of hydrogen peroxide. Finally, we found no evidence for restrictive pores with radii of 0.4-1.4 nm.

In-line measurement of pulmonary metabolic function in the anesthetized rabbit.

Quantitative assessment of lung metabolic function is thought to provide biochemical information reflecting integrity of the pulmonary microcirculation. Although multiple indicator-dilution techniques are useful in such pharmacokinetic studies, the need for fractionation and subsequent processing of blood samples greatly prolongs data generation. Accordingly, we designed and tested an in-line system which rapidly can quantify single-pass disposition of photon-emitting substances in the pulmonary circulation of intact animals. The nuclear detection system consisted of a phoswich scintillation probe optically coupled to a photomultiplier tube. Pulses were discriminated for height and shape and counts recorded in a counter-timer, the output of which was interfaced with a personal computer. A mixture of an intravascular reference substance (99mTc-sulfur colloid) and an inhibitor of angiotensin-converting enzyme, N-[1(S)-carboxy-(4-OH-3-[125I]-phenyl) ethyl]-L-alanyl-L-proline (125I-CPAP), was injected as a bolus in the right heart of anesthetized ventilated rabbits and arterial blood was diverted through a flow-cell cuvette directly apposed to the phoswich detector. Single-pass extraction of 125I-CPAP was 39 +/- 3% (mean +/- SE; n = 20) and was depressed in a dose-dependent fashion by the addition of unlabeled CPAP (1-10 micrograms/kg) to the injection. These data indicate that we can now quantify, in the intact animal, saturable binding of an inhibitor to angiotensin-converting enzyme expressed on the surface of the pulmonary microvascular endothelium. Furthermore, such data can be obtained rapidly.