Smokeless tobacco potentiates VIP-induced DNA synthesis and inactivates NEP 24.11 in oral keratinocytes

The purpose of this study was to determine whetherexposure of cultured chemically transformed hamster oral keratinocytes (HCPC-1) to an aqueous extract of smokeless tobacco (STE) potentiates DNA synthesis elicited by vasoactive intestinal peptide (VIP), anautocrine neuropeptide, and, if so, whether this response is associatedwith inactivation of neutral endopeptidase 24.11 (NEP 24.11), anectoenzyme that cleaves and inactivates VIP very effectively, in thesecells. I found that STE and VIP each elicited a modest, albeitsignificant, increase in DNA synthesis in cultured HCPC-1 cells(P < 0.05). However, incubation of HCPC-1 cells with STE together with VIP evoked a significant, concentration- dependent increase in DNA synthesis that was mediated by VIP receptors. Theeffects of STE and VIP were synergistic. Maximal response was observedafter a 48-h incubation. STE significantly attenuated NEP 24.11 activity in HCPC-1 cells at a time when VIP-induced DNA synthesis wasmaximal. Collectively, these data indicate that STE potentiatesVIP-induced DNA synthesis in cultured oral keratinocytes, and that thisresponse is temporally related to STE-induced inactivation of NEP 24.11 in these cells. I suggest that NEP 24.11 modulates the mitogeniceffects of smokeless tobacco in the oral epithelium, in part, byinactivating VIP.

     

The effect of dexamethasone on the cytotoxic and enzymatic response of cultured endothelial cells to radiation

Experiments were conducted to determine (1) whether glucocorticoids directly protected endothelial cells (EC) from radiation and (2) if angiotensin converting enzyme (ACE) activity, known to be increased by glucocorticoid, played a role in the EC response to radiation. Confluent monolayers of EC cultured from bovine aorta EC were treated with dexamethasone (10(-6) M); after irradiation (5.0 Gy, 60Co gamma), ACE and lactate dehydrogenase (LDH) activities, DNA and protein contents, and nuclei number were measured. Twenty-four hours after 5 Gy, there was increased cell loss (-40%, P less than 0.001), greater LDH release (greater than 100%, P less than 0.001), more LDH activity per cell (+40%, P less than 0.001), and unchanged ACE activity compared to sham-irradiated control EC. However, 48 hr after 5 Gy, ACE activity per cell was decreased (-24%, P less than 0.005). A 48-hr exposure to dexamethasone alone was accompanied by a slight cell loss (-10%, P less than 0.001) and increased cellular ACE activity (+40-140%, P less than 0.001), but a 24-hr dexamethasone exposure was not cytotoxic and did not change ACE activity. Dexamethasone exposure for 48 hr before and after irradiation did not attenuate cell loss or LDH release. However, combined dexamethasone treatment and radiation increased cellular ACE activity at a time when neither agent alone had an effect (24-hr dexamethasone exposure before 5 Gy and assayed 24 hr after 5 Gy). This interaction between radiation and dexamethasone treatment suggests that the glucocorticoid modifies the cell's response to injury. Although this interaction does not ameliorate radiation cytotoxicity, maintenance of ACE levels in injured vessels by hormones may have physiological significance in the hemodynamics of irradiated tissues.     

Direct inhibition of neutral endopeptidase in vasopeptidase inhibitor-mediated amelioration of cardiac remodeling in rats with chronic heart failure

Vasopeptidase inhibitors possess dual inhibitory actions on neutral endopeptidase (NEP) and angiotensin-converting enzyme (ACE) and have beneficial effects on cardiac remodeling. However, the contribution of NEP inhibition to their effects is not yet fully understood. To address the role of cardiac NEP inhibition in the anti-remodeling effects of a vasopeptidase inhibitor, we examined the effects of omapatrilat on the development of cardiac remodeling in rats with left coronary artery ligation (CAL) and those on collagen synthesis in cultured fibroblast cells. In vivo treatment with omapatrilat (30 mg/kg/day for 5 weeks) inhibited cardiac NEP activity in rats with CAL, which was associated with a suppression of both cardiac hypertrophy and collagen deposition. In cultured cardiac fibroblasts, omapatrilat (10^–7~10^–5 M) inhibited NEP activity and augmented the ANP-induced decrease in [³H]-proline incorporation. ONO-BB, an active metabolite of the NEP selective inhibitor ONO-9902, also augmented the ANP-induced response, whereas captopril, an ACE inhibitor, did not. The angiotensin I-induced increase in [³H]-proline incorporation was prevented by omapatrilat and captopril, but not by ONO-BB. The results suggest that vasopeptidase inhibitor suppressed cardiac remodeling in the setting of chronic heart failure, possibly acting through the direct inhibition of cardiac NEP. Vasopeptidase inhibitors may have therapeutic advantages over the classical ACE and NEP inhibitors alone with respect to the regression of cardiac fibrosis.     

Angiotensin and bradykinin metabolism by peptidases identified in cultured human skeletal muscle myocytes and fibroblasts

Angiotensin (ANG) and kinin metabolizing enzymes, angiotensin-converting enzyme (ACE; EC 3.4.15.1), neutral endopeptidase-24.11 (NEP-24.11; EC 3.4.24.11), and aminopeptidase M (AmM; EC 3.4.11.2), have recently been identified in a purified skeletal muscle glycoprotein fraction. We have analyzed the cellular localization of these enzymes. In cultured human skeletal muscle adult myoblasts, myotubes, and fibroblasts, kinins and angiotensins were metabolized by NEP-24.11 and AmM but not by ACE. NEP-24.11 degraded ANG II, ANG III, and bradykinin (BK) and converted ANG I to the active metabolite ANG(1–7). ANG III was converted to the novel ANG IV metabolite [des-Arg¹]ANG III by AmM. These data suggest that, due to their abundance in the body, skeletal muscle myocytes and fibroblasts may play a major role in modulation of the systemic and local effects of angiotensins and kinins. This role could be particularly important in individuals receiving treatment with ACE inhibitors.     

Expression and polarized apical secretion in Madin-Darby canine kidney cells of a recombinant soluble form of neutral endopeptidase lacking the cytosolic and transmembrane domains.

Neutral endopeptidase-24.11 (NEP; EC 3.4.24.11) is an abundant metalloendopeptidase of the brush border membrane of kidney proximal tubules. We have recently shown that NEP is delivered directly to the apical domain of the plasma membrane when expressed in polarized Madin-Darby canine kidney (MDCK) cells in culture (Jalal, F., Lemay, G., Zollinger, M., Berteloot, A., Boileau, G., and Crine, P. (1991) J. Biol. Chem. 266, 19826-19832). Here, a soluble form of NEP consisting of the signal peptide of pro-opiomelanocortin fused in-frame with the ectodomain of NEP has been expressed in MDCK cells. Enzymatic assays performed on apical and basolateral culture media of MDCK cells grown on semi-permeable supports indicated that the recombinant enzyme was predominantly released at the apical surface. In contrast, when the chimeric protein was expressed in NIH 3T3 cells or when pro-opiomelanocortin was expressed in MDCK cells, non-polarized secretion was observed into both the apical and basolateral compartments of the culture chamber. Our results suggest that the ectodomain of NEP is sufficient for directing the targeting of this protein to the apical membrane of polarized MDCK epithelial cells.     

Interaction of vasoactive intestinal peptide (VIP) with a mouse adrenal cell line (Y-1): specific binding and biological effects.

Y-1 cells specifically bind radiolabelled vasoactive intestinal peptide (VIP) with a dissociation constant of about 10^?9 M. [¹²⁵I]-VIP bound was not displaced by ACTH. VIP stimulates both steroid and cAMP production, with half-maximal stimulation at 10^?9 and 10^?8 M, respectively. At maximal concentration VIP produces the same stimulation of steroidogenesis as ACTH, but induced three times lower production of cAMP than ACTH. Y-1 DNA synthesis is inhibited by VIP in a dose-dependent manner with half-maximal inhibition at 10^?8 M. At submaximal concentrations the effects of VIP and ACTH on cAMP and steroid production and on inhibition of DNA synthesis are additive. Similar additive effects on cAMP production and on inhibition of DNA synthesis were observed at submaximal ACTH and maximal VIP concentration, but the phenomenon was no longer seen at maximal concentrations of both peptides. These data suggest that in Y-1 cells VIP stimulates, through its own distinct receptors, only a part of the pool of adenylate cyclase sensitive to ACTH.     

Antagonists of epithelial chloride channels inhibit cAMP synthesis.

In past studies we observed that the chloride channel blocker, diphenylamine-2-carboxylate (DPC) and chemically related drugs (Hoechst compounds 131, 143, 144) inhibited cAMP formation in mouse pituitary tumor cells. The object of this study was to determine whether these drugs inhibited chloride transport in human T-84 colonic carcinoma cells through an effect on cAMP metabolism. Chloride secretion (measured as 125I efflux from isotope-preloaded cells) was stimulated in a concentration-dependent manner by vasoactive intestinal polypeptide (VIP) (EC50 = 1.5 x 10(-10) M) which similarly increased cAMP synthesis (EC50 = 1.6 x 10(-8) M). The cAMP response to VIP was inhibited 17, 52, 55, and 78% maximally by DPC and compounds 144, 143, and 131, respectively. In untreated T-84 cells, 125I secretion fell by 66% after 3 min; VIP (10(-7) M) increased secretion about fivefold over the same period. Both basal and VIP-stimulated 125I secretion were inhibited up to 60% by compound 131. Pretreatment of cells with pertussis toxin did not attenuate the inhibitory effect of channel blockers on either VIP-stimulated cAMP synthesis or 125I secretion. The cationophore, A-23187, which had no effect on cAMP formation, and 8-Br-cAMP both stimulated 125I secretion from T-84 cells. These secretory responses were inhibited by compound 131. The mechanism by which phenylanthranilic acids antagonize cAMP synthesis and its significance is not known; however, the data suggest that this family of drugs may inhibit chloride transport by both cAMP-dependent and independent mechanisms.     

Angiotensin converting enzyme (ACE) and neprilysin hydrolyze neuropeptides: a brief history, the beginning and follow-ups to early studies

Our investigations started when synthetic bradykinin became available and we could characterize two enzymes that cleaved it: kininase I or plasma carboxypeptidase N and kininase II, a peptidyl dipeptide hydrolase that we later found to be identical with the angiotensin I converting enzyme (ACE). When we noticed that ACE can cleave peptides without a free C-terminal carboxyl group (e.g., with a C-terminal nitrobenzylamine), we investigated inactivation of substance P, which has a C-terminal Met(11)-NH(2). The studies were extended to the hydrolysis of the neuropeptide, neurotensin and to compare hydrolysis of the same peptides by neprilysin (neutral endopeptidase 24.11, CD10, NEP). Our publication in 1984 dealt with ACE and NEP purified to homogeneity from human kidney. NEP cleaved substance P (SP) at Gln(6)-Phe(7), Phe(7)[see text]-Phe(8), and Gly(9)-Leu(10) and neurotensin (NT) at Pro(10)-Tyr(11) and Tyr(11)-Ile(12). Purified ACE also rapidly inactivated SP as measured in bioassay. HPLC analysis showed that ACE cleaved SP at Phe(8)-Gly(9) and Gly(9)-Leu(10) to release C-terminal tri- and dipeptide (ratio = 4:1). The hydrolysis was Cl(-) dependent and inhibited by captopril. ACE released only dipeptide from SP free acid. ACE hydrolyzed NT at Tyr(11)-Ile(12) to release Ile(12)-Leu(13). Then peptide substrates were used to inhibit ACE hydrolyzing Fa-Phe-Gly-Gly and NEP cleaving Leu(5)-enkephalin. The K(i) values in microM were as follows: for ACE, bradykinin = 0.4, angiotensin I = 4, SP = 25, SP free acid = 2, NT = 14, and Met(5)-enkephalin = 450, and for NEP, bradykinin = 162, angiotensin I = 36, SP = 190, NT = 39, Met(5)-enkephalin = 22. These studies showed that ACE and NEP, two enzymes widely distributed in the body, are involved in the metabolism of SP and NT. Below we briefly survey how NEP and ACE in two decades have gained the reputation as very important factors in health and disease. This is due to the discovery of more endogenous substrates of the enzymes and to the very broad and beneficial therapeutic applications of ACE inhibitors.     

Neutral endopeptidase 24.11 and angiotensin converting enzyme like activity in CALLA positive and CALLA negative lymphoid cells

Taking advantage of the recently demonstrated identity of common acute lymphoblastic leukemia antigen (CALLA) and neutral endopeptidase EC.24.11 (NEP) the presence of this ectoenzyme on lymphoid cells has been reassessed using highly sensitive assays (cleavage of [3H]-D-Ala2-leucine-enkephalin and binding of the inhibitor [3H]HACBO-Gly. NEP activity was found not only on already classified CALLA + ve cells but also on numerous cells (including mature B and polyclonal T cells) previously considered as CALLA-ve. This suggests that CALLA/NEP is expressed all along the differentiation pathway in B and T cell lineage. Moreover substantial ACE-like activity was also detected in three tested cells, all of the pre-B phenotype. The availability of specific inhibitors for these enzymes should help clarify their role in cell-differentiation.     

Do Secretin and Vasoactive Intestinal Peptide Have Independent Receptors on Striatal Neurons and Glial Cells in Primary Cultures?

Vasoactive intestinal peptide (VIP) and secretin are two related peptides that activate adenylate cyclase on membranes of striatal neurons and glial cells from embryonic mouse brain grown in primary culture. On the two cell types, the maximal activation that could be induced by secretin was only 40% above basal activity, which represented less than 15% of the maximal effect obtainable with VIP. From competition experiments performed on glial cells and the neuroblastoma X glioma hybrid, NG 108-15, a cell line known to possess both VIP and secretin sensitive-adenylate cyclase, we demonstrate that secretin does not activate VIP receptors. Furthermore, secretin has an apparent high affinity (EC50 10(-8) M) for its receptors on striatal neurons and NG 108-15 whereas an apparent low affinity (EC50 7 X 10(-6) M) was found on striatal glial cells. This suggests the existence of either two distinct secretin receptors or a desensitized form.     

The 38-amino acid form of pituitary adenylate cyclase-activating polypeptide stimulates dual signaling cascades in PC12 cells and promotes neurite outgrowth.

Vasoactive intestinal peptide (VIP) activates adenylylcyclase in sympathoadrenal cells at concentrations greater than 10(-6) M. We demonstrate here that two forms of a newly discovered peptide with homology to VIP named pituitary adenylate cyclase-activating polypeptide (PACAP) are much more potent activators of signal transduction in PC12 cells. Both the 27- and 38-amino acid forms of PACAP elevate cAMP levels in PC12 cells and stimulate adenylylcyclase in PC12 membranes, with an EC50 near 10(-9) M. PACAP38 additionally is a potent activator of the inositol lipid cascade in PC12 cells, elevating the content of inositol phosphates by 8-fold at 10(-8) M (EC50 = 7 x 10(-9) M). PACAP38 and PACAP27 have been thought to have essentially identical actions, but PACAP27 is 2-3 logs less potent in increasing inositol lipid levels. Moreover, PACAP38 at 10(-8) M is an effective inducer of neuronal morphology in PC12 cells, whereas PACAP27 is much less active in promoting neurite outgrowth. In contrast to the PACAP-preferring receptors on PC12 cells, another class of PACAP-binding sites with equal high affinities for VIP, PACAP38, and PACAP27 has been identified on several other cell types. We find that the cAMP content of rat CH3 pituitary cells, known to have high affinity VIP receptors, is in fact potently elevated by PACAP27 and PACAP38 as well as by VIP. However, PACAP38, even at 10(-6) M, is not capable of significant activation of inositol lipid turnover via these VIP/PACAP nondiscriminating sites.     

In vivo properties of thiol inhibitors of the three vasopeptidases NEP, ACE and ECE are improved by introduction of a 7-azatryptophan in P2' position.

Three zinc metallopeptidases are implicated in the regulation of fluid homeostasis and vascular tone and represent interesting targets for the treatment of chronic heart failure. We have previously reported the synthesis of a triple inhibitor able to simultaneously inhibit neprilysin (NEP, EC 3.4.24.11), angiotensin-converting enzyme (ACE, EC 3.4.15.1) and endothelin-converting enzyme (ECE-1, EC 3.4.24.71) with nanomolar potency towards NEP and ACE and a lesser affinity for ECE. Here, we report the optimization and biological activities of analogs derived from lead compound 1 (2S)-2-[(2R)-2-((1S)-5-bromo-indan-1-yl)-3-mercapto-propionylamino]-3- (1H-indol-3-yl)-propionic acid by a structural approach. Among several inhibitors, compound 21, (2S)-2-[(2R)-2-((1S)-5-bromo-indan-1-yl)-3-mercapto-propionylamino]-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)-propionic acid was selected by taking into account its good molecular adaptation with the recently published structures of the three vasopeptidases. This optimization procedure led to an improved pharmacologic activity when compared with 1.     

A fragment of vasoactive intestinal peptide, VIP(10-28), is an antagonist of VIP in the colon carcinoma cell line, HT29

The 19 amino acid carboxyl terminus fragment of vasoactive intestinal peptide (VIP), VIP(10-28), inhibits [125I]VIP binding in intact HT29 colonic adenocarcinoma cells and in membranes from these cells. However, VIP(10-28) alone has no effect on adenylate cyclase activity (membranes) or cyclic AMP synthesis (intact cells) in HT29 cells although VIP receptor agonists are markedly stimulatory. The indicated antagonist character of VIP(10-28) was confirmed by rightward parallel shifts of VIP dose response curves in the presence of VIP(10-28) in adenylate cyclase and cyclic AMP synthesis experiments. The equilibrium dissociation constant values for VIP(10-28) from these experiments agree with values from inhibition binding studies. The lack of effect of VIP(10-28) on forskolin dose response curves in HT29 adenylate cyclase assays indicates the specificity of the VIP(10-28) antagonism, thus suggesting that VIP(10-28) may be a useful tool in studying VIP receptor regulation and other aspects of the mechanisms of VIP action. The potential regulatory role of a proteolytically generated fragment of VIP acting antagonistically at VIP receptors is discussed.     

Angiotensin-converting enzyme activity in ovine bronchial vasculature.

Angiotensin-converting enzyme (ACE) plays a major role in the metabolism of bradykinin, angiotensin, and neuropeptides, which are all implicated in inflammatory airway diseases. The activity of ACE, which is localized on the luminal surface of endothelial cells (EC), has been well documented in pulmonary EC; however, few data exist regarding the relative activity of ACE in the airway vasculature. Therefore, we measured ACE activity in cultured EC from the sheep bronchial artery and bronchial mucosa (microvascular) and compared it with pulmonary artery EC. The baseline level of total ACE activity (cellular plus secreted) was significantly greater in bronchial microvascular EC (1.24 +/- 0.24 mU/106 cells) compared with bronchial artery EC (0.59 +/- 0.15 mU/106 cells; P < 0.05) and comparable to pulmonary artery EC (1.12 +/- 0.14 mU/106 cells; P > 0.05). Measured ACE activity secreted into culture medium for each cell type was 64-74% of total activity and did not differ among the three EC types (P = 0.17). Hydrocortisone (10 microg/ml; 48-72 h) treatment resulted in a significant increase in ACE activity in bronchial EC. Likewise, TNF-alpha (0.1 ng/ml) treatment markedly increased ACE activity in all cell lysates (P < 0.05). We confirmed the importance of ACE activity in vivo since, at the highest dose of bradykinin studied (10-8 M), bronchial artery pressure at constant flow showed a greater decrease after captopril treatment (36% before vs. 60% after; P = 0.05). These results demonstrate high ACE expression of the bronchial microvasculature and suggest an important regulatory role for ACE in the metabolism of kinin peptides known to contribute to airway pathology.     

Vasoactive intestinal peptide and forskolin regulate proliferation of the HT29 human colon adenocarcinoma cell line.

Although several lines of evidence implicate cAMP in the regulation of intestinal cell proliferation, the precise role of this second messenger in the control of the human colon cancer cell cycle is still unclear. In order to investigate the role of cAMP in HT29 cell proliferation, we have tested the effect of vasoactive intestinal peptide (VIP) and forskolin on DNA synthesis and cell number, focusing on the time-dependent efficacy of the treatment. The cells were arrested in G0/G1 phase by incubation for 24 h in serum-free medium and proliferation was re-initiated by addition of either 85 nM insulin or 0.5% fetal calf serum. In the presence of fetal calf serum, G1/S transition was found to occur earlier than with insulin. Exposure of the HT29 cells to 10(-5) M forskolin in the early stages of growth induction (within 12 h from FCS addition or within 14 h from insulin treatment) resulted in a significant inhibition of DNA synthesis and a delayed entry in the S phase. By contrast, VIP (10(-7) M) was inhibitory only when added within a narrow window (10 to 12 h or 12 to 14 h following FCS or insulin addition, respectively). The difference in efficiency of forskolin and VIP to inhibit cell proliferation may be correlated with their own potency to promote long-lasting cAMP accumulation. The combination of VIP plus forskolin had synergistic effects on both cAMP accumulation and cell-growth inhibition. Taken together, our data indicate that cAMP may act at a step in the late G1 or G1/S transition.     

四味镇痛中药对内腓肽降解酶作用的实验研究

目的 :研究四味镇痛中药对内腓肽降解酶的作用。方法 :从大鼠的肾脏制备含有内腓肽降解酶的物质 ,建立内腓肽降解酶活性检测模型 ;并用此模型观察四味镇痛中药提取物对内腓肽降解酶特别是对中性内肽酶活性 (NEP2 4 .11)的抑制作用。结果 :(1)钩藤和羌活的水提物显示很强的NEP2 4 11酶抑制作用 ;(2 )无论是有机提取物还是水提物 ,延胡索和川芎对NEP2 4 11的抑制作用都弱于钩藤和羌活 ;(3)钩藤和羌活不仅是氨肽酶和中性内肽酶的双重抑制剂 ,而且是氨肽酶、中性内肽酶和血管紧张素转化酶的三重抑制剂。结论 :四味镇痛中药具有不同的镇痛机制 ,其中 ,钩藤和羌活通过抑制氨肽酶和中性内肽酶而发挥镇痛作用     

Time and concentration dependence of the action of cortisol on fibroblasts in vitro

The effect of cortisol on cultured fibroblasts from human skin were studied. After 0–84-h preincubations in the presence of cortisol the cells were labeled for 12 h with [³H]thymidine, [³H]proline or [³H]glucosamine and the radioactivity incorporated into DNA, collagen, total proteins, hyaluronic acid and sulphated glycosaminoglycans was determined.Cortisol (1 · 10^?5 M) caused a rapid, progressive decrease in the synthesis of hyaluronic acid when compared to the controls. Similarly, it decreased the synthesis of sulphated glycosaminoglycans and DNA, but this was seen first after 12- and 24-h preincubations, respectively. The synthesis of collagen and other proteins was significantly increased when the preincubation time was 0–24 h. This stimulation, however, turned to inhibition when an 84-h preincubation was used. It was found that 1 · 10^?7 M cortisol was the lowest concentration which caused the early inhibition in hyaluronate synthesis, while even 1 · 10^?8 M was sufficient after an 84-h preincubation. The syntheses of sulphated glucosaminoglycans and DNA were significantly inhibited by 1 · 10^?8 and 1 · 10^?7 M cortisol, after an 54-h preincubation, respectively. Thus, the studies of cortisol effects on fibroblast functions may result in quite variable conclusions unless the time sequence and the steroid concentration effects are taken into account.     

Peptidase modulation of noncholinergic vagal bronchoconstriction and airway microvascular leakage

We investigated whether inhibition of neutral endopeptidase 24.11 (NEP) and/or angiotensin-converting enzyme (ACE) modifies vagally induced nonadrenergic noncholinergic (NANC) airflow obstruction and airway microvascular leakage as measured by extravasation of Evans blue dye (intravenous) in anesthetized guinea pigs. We gave phosphoramidon to inhibit NEP and enalapril maleate or captopril to inhibit ACE. Animals pretreated with inhaled phosphoramidon (7.5 or 75 nmol), enalapril maleate (87 or 870 nmol), or captopril (350 nmol) reached higher peak lung resistance (RL) values (14.3 +/- 2.7, 15.7 +/- 3.8, 16.7 +/- 3.8, 11.4 +/- 1.6, and 24.6 +/- 3.5 cmH2O.ml-1.s, respectively) than saline-treated animals (5.9 +/- 1.1; P less than 0.05) after bilateral vagus nerve stimulation (5 Hz, 10 V, 10 ms, 150 s). Intravenous phosphoramidon (1 mg/kg), but not intravenous captopril (6 mg/kg), potentiated peak RL (22.9 +/- 6.9 and 7.1 +/- 1.5 cmH2O.ml-1.s, respectively). Vagal nerve stimulation (1 and 5 Hz) increased the extravasation of Evans blue dye in tracheobronchial tissues compared with sham-stimulated animals, but this was not potentiated by inhaled enzyme inhibitors or intravenous captopril. However, intravenous phosphoramidon significantly augmented the extravasation of Evans blue dye in main bronchi and intrapulmonary airways. We conclude that degradative enzymes regulate both NANC-induced airflow obstruction and airway microvascular leakage.