Camostat attenuates airway epithelial sodium channel function in vivo through the inhibition of a channel-activating protease.

Inhibition of airway epithelial sodium channel (ENaC) function enhances mucociliary clearance (MCC). ENaC is positively regulated by channel-activating proteases (CAPs), and CAP inhibitors are therefore predicted to be beneficial in diseases associated with impaired MCC. The aims of the present study were to 1) identify low-molecular-weight inhibitors of airway CAPs and 2) to establish whether such CAP inhibitors would translate into a negative regulation of ENaC function in vivo, with a consequent enhancement of MCC. To this end, camostat, a trypsin-like protease inhibitor, provided a potent (IC(50) approximately 50 nM) and prolonged attenuation of ENaC function in human airway epithelial cell models that was reversible upon the addition of excess trypsin. In primary human bronchial epithelial cells, a potency order of placental bikunin > camostat > 4-guanidinobenzoic acid 4-carboxymethyl-phenyl ester > aprotinin >> soybean trypsin inhibitor = alpha1-antitrypsin, was largely consistent with that observed for inhibition of prostasin, a molecular candidate for the airway CAP. In vivo, topical airway administration of camostat induced a potent and prolonged attenuation of ENaC activity in the guinea pig trachea (ED(50) = 3 microg/kg). When administered by aerosol inhalation in conscious sheep, camostat enhanced MCC out to at least 5 h after inhaled dosing. In summary, camostat attenuates ENaC function and enhances MCC, providing an opportunity for this approach toward the negative regulation of ENaC function to be tested therapeutically.

Inhaled porcine pancreatic elastase causes bronchoconstriction via a bradykinin-mediated mechanism.

Neutrophil elastase has been linked to inflammatory lung diseases such as chronic obstructive pulmonary disease, adult respiratory distress syndrome, emphysema, and cystic fibrosis. In guinea pigs, aerosol challenge with human neutrophil elastase causes bronchoconstriction, but the mechanism by which this occurs is not completely understood. Our laboratory previously showed that human neutrophil elastase releases tissue kallikrein (TK) from cultured tracheal gland cells. TK has been identified as the major kininogenase of the airway and cleaves both high- and low-molecular weight kininogen to yield lysyl-bradykinin. Because inhaled bradykinin causes bronchoconstriction and airway hyperresponsiveness in asthmatic patients and allergic sheep, we hypothesized that elastase-induced bronchoconstriction could be mediated by bradykinin. To test this hypothesis, we measured lung resistance (RL) in sheep before and after inhalation of porcine pancreatic elastase (PPE) alone and after pretreatment with a bradykinin B(2) antagonist (NPC-567), the specific human elastase inhibitor ICI 200,355, the histamine H(1)-antagonist diphenhydramine hydrochloride, the cysteinyl leukotriene 1 receptor antagonist montelukast, or the cyclooxygenase inhibitor indomethacin. Inhaled PPE (125-1,000 microg) caused a dose-dependent increase in RL. Aerosol challenge with a single 500 microg dose of PPE increased RL by 132 +/- 8% over baseline. This response was blocked by pretreatment with NPC-567 and ICI-200,355 (n = 6; P < 0.001), whereas treatment with diphenhydramine hydrochloride, montelukast, or indomethacin failed to block the PPE-induced bronchoconstriction. Consistent with pharmacological data, TK activity in bronchial lavage fluid increased 134 +/- 57% over baseline (n = 5; P < 0.02). We conclude that, in sheep, PPE-induced bronchoconstriction is in part mediated by the generation of bradykinin. Our findings suggest that elastase-kinin interactions may contribute to changes in bronchial tone during inflammatory diseases of the airways.

Budesonide affects allergic mucociliary dysfunction.

Airway inflammation characterized by neutrophils and free elastase contributes to allergic mucociliary dysfunction. Glucocorticosteroids are the most important anti-inflammatory agents used in the treatment of asthma, but their effect on allergic mucociliary dysfunction is not known. Therefore, we assessed both the prophylactic and therapeutic effects of the glucocorticosteroid budesonide on antigen-induced mucociliary dysfunction in sheep. Tracheal mucus velocity (TMV), a marker of mucociliary clearance, was measured by using a roentgenographic technique. When budesonide was administered either 30 min before or 1 h after airway challenge with Ascaris suum, the antigen-induced fall in TMV at 6 h was prevented. The effects on TMV at 8 and 24 h after challenge were also determined when budesonide and, for comparative purposes, alpha1-protease inhibitor were given 6 h after antigen challenge. Budesonide treatment improved TMV at 8 h, but TMV was not significantly different from antigen alone at 24 h. Treatment with alpha1-protease inhibitor, however, caused only a significant reversal of the antigen-induced fall in TMV at 24 h after challenge; this indicates a more prolonged effect than budesonide. Our results suggest that antiproteases may have a potential role as a therapeutic approach to mucociliary dysfunction in asthma and provide evidence for another means by which glucocorticosteroids contribute to the control of the disease.

Mechanism of pyocyanin- and 1-hydroxyphenazine-induced lung neutrophilia in sheep airways.

Pyocyanin (Pyo) and 1-hydroxyphenazine (1-HP) are extracellular products of Pseudomonas aeruginosa. To test whether these products were capable of producing an inflammatory response in the airways, combinations of Pyo and 1-HP at concentrations of 10(-4) and 10(-5) M were instilled into sheep airways, and indexes of inflammation were assessed by bronchoalveolar lavage (BAL) 24 h later. Challenge with the phenazines caused a significant dose-dependent increase in the number of cells and neutrophils recovered by BAL. Control challenges produced no such changes. The lung neutrophilia was accompanied by an increased concentration of albumin in BAL. The increases in BAL neutrophils and albumin could be blocked by treating the sheep with the 5-lipoxygenase inhibitor zileuton. Neither 1-HP nor Pyo was chemotactic to neutrophils when tested in vitro, but when alveolar macrophages (AM) were cultured in vitro in the presence of both Pyo and 1-HP (1 microM), the supernatants caused neutrophil chemotaxis. Analysis of AM culture supernatants incubated with the combination of pigments showed significant increases in leukotriene B4 and interleukin-8, and blocking these mediators separately or together reduced AM supernatant-induced neutrophil chemotaxis. We conclude that local instillation of Pyo and 1-HP can initiate an inflammatory response in the airways of sheep in vivo. This effect can be explained, in part, by the release of chemotactic factors produced by AM.

Aerosolization of P2Y(2)-receptor agonists enhances mucociliary clearance in sheep.

The purpose of this study was to determine whether aerosolized INS316 (UTP) stimulates lung mucociliary clearance (MCC) in sheep and, if so, to compare its effects with INS365, a novel P2Y(2)-receptor agonist. In the first series of studies, we used a previously described roentgenographic technique to measure tracheal mucus velocity (TMV), an index of MCC, before and for 4 h after aerosolization of INS316 (10(-1) M and 10(-2) M) and INS365 (10(-1) M and 10(-2) M), or normal saline in a randomized crossover fashion (n = 6). In a second series of studies, we compared the ability of these agents to enhance total lung clearance. For these tests, the clearance of inhaled technetium-labeled human serum albumin was measured serially over a 2-h period after aerosolization of 10(-1) M concentration of each agent (n = 7). Aerosolization of both P2Y(2)-receptor agonists induced significant dose-related increases in TMV (P < 0.05) compared with saline. The greatest increase in TMV was observed between 15 and 30 min after drug treatment. The highest dose (10(-1) M) of INS316 produced a greater overall stimulation of TMV than did INS365 (10(-1) M). Both compounds, compared with saline, induced a significant increase in MCC (P < 0.05) within 20 min of treatment. This enhancement in MCC began to plateau at 60 min. Although the response to INS316 started earlier, there was no significant difference between the clearance curves for the two compounds. We conclude that inhaled P2Y(2)-receptor agonists can increase lung MCC in sheep and that for P2Y(2)-receptor stimulation TMV accurately reflects changes in whole lung MCC.

Effects of unilateral bronchoconstriction on distribution of aerosols in ovine airways.

The purpose of the study was to assess the effect of unilateral bronchoconstriction on the deposition patterns of aerosolized particles in a sheep model. Unilateral bronchoconstriction was induced in intubated conscious sheep by placing a protective, obstructing balloon catheter in either main bronchus, prior to administration of aerosolized carbachol at a dose that increased pulmonary resistance by 200-400% above baseline. The catheter was then removed and the animals were positioned under a gamma camera. An equilibrium image was obtained with xenon (133Xe), to determine a lung outline that was used to calculate the proportion of counts in each lung. Aerosols, labeled with technetium (99mTc) and generated by two jet nebulizers, were inhaled tidally by the sheep in serial experiments. (For nebulizer A, mass median aerodynamic diameter [MMAD] = 0.39 microm; for nebulizer B, MMAD = 1.1 microm.) For nebulizer A, percentage deposition in the treated and untreated lungs was not significantly different (50.8% versus 49.2%, respectively), while for nebulizer B, the median deposition in the carbachol treated lung was significantly greater than in the untreated lung (55.8% versus 44.2% respectively; p = 0.005). There was a more central pattern of deposition in the treated lung than in the untreated lung for both nebulizers, but the degree of central deposition was significantly greater with nebulizer B. The findings of the present study suggest that regional obstruction does not preclude the delivery of therapeutic aerosols to the airways in such a region, and may, depending on the size of the aerosol, result in enhanced airway deposition relative to less obstructed regions.

Endothelin-1 depresses tracheal mucus velocity in ovine airways via ET-A receptors.

Endothelin-1 (ET-1) is a potent constrictor of bronchial smooth muscle, but there is limited information on its actions on the airway mucociliary clearance in vivo. The purpose of this study was to determine (1) the effect of aerosolized ET-1 on tracheal mucus velocity (TMV), a marker of mucociliary clearance, in sheep and (2) if the ET-1-induced effects were mediated by ET-A or ET-B receptors. To measure TMV, radiopaque teflon particles were insufflated into six intubated, spontaneously breathing, adult sheep, and the velocity at which these particles traveled up the trachea was measured using a previously reported roentgenographic technique. After baseline TMV measurements, 50 breaths of either ET-1 (10(-7) M) or vehicle (phosphate-buffered saline) were aerosolized into the airways. TMV measurements were then obtained over a 2-h period. After exposure to ET-1, mean TMV decreased significantly as compared with vehicle, the effects being most marked within 30 min after administration (54%, p < 0.05). On subsequent days, animals were pretreated with an aerosolized ET-A receptor antagonist (BQ-123) or an ET-B receptor antagonist (BQ-788) before exposure to ET-1. When ET-1 was given after BQ-123, no significant drop in TMV was noted. In contrast, pretreatment with BQ-788 exhibited no protective effect on the decrease in TMV. The ET-1 effects were not influenced by pretreatment with either the cyclo-oxygenase inhibitor indomethacin or the leukotriene receptor antagonist MK-571, indicating that ET-1-induced depression in TMV does not involve the activation of prostanoids or peptide leukotrienes. Thus, exogenous ET-1 reduces TMV, an in vivo effect that is mediated through stimulation of ET-A receptors.

Comparative effects of a fixed combination of reproterol hydrochloride and disodium cromoglycate with each agent alone on antigen-induced airway responses in sheep.

The purpose of this study was to compare the effectiveness of an eight day treatment with clinically relevant doses of a fixed combination of the beta 2 mimetic reproterol hydrochloride and disodium cromoglycate with each agent given alone against antigen-induced early (EAR) and late airway responses (LAR) as well as post-antigen-induced airway hyperresponsiveness (AHR) in allergic sheep. Animals were treated in a randomized fashion with either the inhaled combination (n = 6), reproterol hydrochloride alone (n = 6), disodium cromoglycate alone (n = 6), or placebo (n = 8). Treatments (two puffs from a metered dose inhaler) were given three times a day for 7 days and once on the 8th day 1 h before airway challenge with Ascaris suum antigen. In the placebo trial, antigen challenge resulted in EAR and LAR as measured by increases in specific lung resistance; these changes were followed 24h later by AHR to inhaled carbachol. With respect to the placebo trial, treatment with reproterol hydrochloride reduced the EAR (P < 0.05) and blocked the LAR (P < 0.05), but had no effect on the post-challenge AHR. Treatment with disodium cromoglycate also reduced the EAR (P < 0.05), blocked the LAR (P < 0.05), and blocked the post-antigen-induced AHR (P < 0.05). Treatment with the fixed combination reduced the EAR (P < 0.05), blocked the LAR (P < 0.05), and blocked the post-antigen-induced AHR (P < 0.05). Comparison of the different agents indicated that the fixed combination gave significantly increased protection against the EAR than either agent alone, gave slightly better (P < 0.05) protection against the late response than cromolyn sodium and gave better protection against post-antigen-induced AHR than reproterol hydrochloride alone. These results suggest that a fixed combination of a beta 2-mimetic and disodium cromoglycate provides some increased protection against antigen-induced airway responses when compared to either agent alone in a controlled laboratory setting.

Pharmacology of INS37217 [P(1)-(uridine 5')-P(4)- (2'-deoxycytidine 5')tetraphosphate, tetrasodium salt], a next-generation P2Y(2) receptor agonist for the treatment of cystic fibrosis.

INS37217 [P(1)-(uridine 5')-P(4)-(2'-deoxycytidine 5')tetraphosphate, tetrasodium salt] is a deoxycytidine-uridine dinucleotide with agonist activity at the P2Y(2) receptor. In primate lung tissues, the P2Y(2) receptor mRNA was located by in situ hybridization predominantly in epithelial cells and not in smooth muscle or stromal tissue. The pharmacologic profile of INS37217 parallels that of UTP, leading to increased chloride and water secretion, increased cilia beat frequency, and increased mucin release. The combined effect of these actions was confirmed in an animal model of tracheal mucus velocity that showed that a single administration of INS37217 significantly enhanced mucus transport for at least 8 h after dosing. This extended duration of action is consistent with the ability of INS37217 to resist metabolism by airway cells and sputum enzymes. The enhanced metabolic stability and resultant increased duration of improved mucociliary clearance may confer significant advantages to INS37217 over other P2Y(2) agonists in the treatment of diseases such as cystic fibrosis.

Selectin blockade prevents antigen-induced late bronchial responses and airway hyperresponsiveness in allergic sheep.

Antigen challenge can elicit an allergic inflammatory response in the airways that involves eosinophils, basophils, and neutrophils and that is expressed physiologically as a late airway response (LAR) and airway hyperresponsiveness (AHR). Although previous studies have suggested that E-selectin participates in these allergic airway responses, there is little information concerning the role of L-selectin. To address this question, we examined the effects of administering an L-selectin-specific monoclonal antibody, DU1-29, as well as three small molecule selectin binding inhibitors, on the development of early airway responses (EAR), LAR and AHR in allergic sheep undergoing airway challenge with Ascaris suum antigen. Sheep treated with aerosol DU1-29 before antigen challenge had a significantly reduced LAR and did not develop postchallenge AHR. No protective effect was seen when sheep were treated with a nonspecific control monoclonal antibody. Treatment with DU1-29 also reduced the severity of the EAR to antigen. Similar results were obtained with each of the three small molecule selectin inhibitors at doses that depended on their L-, but not necessarily E-selectin inhibitory capacity. The inhibition of the EAR with one of the inhibitors, TBC-1269, was associated with a reduction in histamine release. Likewise, treatment with TBC-1269 reduced the number of neutrophils recovered in bronchoalveolar lavage (BAL) during the time of LAR and AHR. TBC-1269, given 90 min after antigen challenge also blocked the LAR and the AHR, but this protection was lost if the treatment was withheld until 4 h after challenge, a result consistent with the proposed time course of L-selectin involvement in leukocyte trafficking. These are the first data indicating that L-selectin may have a unique cellular function that modulates allergen-induced pulmonary responses.