Budesonide affects allergic mucociliary dysfunction

Airwayinflammation characterized by neutrophils and free elastase contributesto allergic mucociliary dysfunction. Glucocorticosteroids are the mostimportant anti-inflammatory agents used in the treatment of asthma, buttheir effect on allergic mucociliary dysfunction is not known.Therefore, we assessed both the prophylactic and therapeutic effects ofthe glucocorticosteroid budesonide on antigen-induced mucociliarydysfunction in sheep. Tracheal mucus velocity (TMV), amarker of mucociliary clearance, was measured by using aroentgenographic technique. When budesonide was administered either 30 min before or 1 h after airway challenge with Ascarissuum, the antigen-induced fall in TMV at 6 h wasprevented. The effects on TMV at 8 and 24 h after challenge were alsodetermined when budesonide and, for comparative purposes,₁-protease inhibitor were given6 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 ₁-proteaseinhibitor, however, caused only a significant reversal of theantigen-induced fall in TMV at 24 h after challenge; this indicates amore prolonged effect than budesonide. Our results suggest thatantiproteases may have a potential role as a therapeutic approach tomucociliary dysfunction in asthma and provide evidence for anothermeans by which glucocorticosteroids contribute to the control of thedisease.

     

Effects of leukotriene D4 on mucociliary and respiratory function in allergic and nonallergic sheep

We determined the effect of aerosol challenge with leukotriene D4 (LTD4) on specific lung resistance (sRL) and tracheal mucous velocity (TMV) in conscious sheep with (allergic) and without (nonallergic) Ascaris suum hypersensitivity. In allergic sheep LTD4 in concentrations of 50, 100, and 150 micrograms/ml produced dose-dependent increases in mean sRL by 44 (P = NS), 154 (P less than 0.05), and 233% (P less than 0.05), respectively. The increase in sRL produced by 150 micrograms/ml LTD4 was prevented by FPL 55712, an antagonist of slow-reacting substance of anaphylaxis. In nonallergic sheep 150 micrograms/ml LTD4 failed to elicit a significant change in sRL. In contrast to the changes in airway mechanics, concentrations of LTD4 as low as 25 micrograms/ml produced significant decreases in TMV in allergic sheep. The maximum decrease in TMV at this dose occurred 2 h after challenge; with larger doses of LTD4 (100 and 150 micrograms/ml) the maximum effect was observed 3 h after challenge. Furthermore, 150 micrograms/ml LTD4 reduced TMV in nonallergic sheep (mean decrease 43%, P less than 0.05). FPL 55712 only had a minor effect on the LTD4-induced decreases in TMV. We conclude that allergic sheep exhibit greater airway responsiveness to inhaled LTD4 than nonallergic sheep but that this difference is not evident for the concomitant changes in mucociliary transport. This suggests that the allergic state is associated with an increased responsiveness to LTD4 in tissues controlling airway caliber but not in those contributing to mucociliary function.     

Developmental changes in the tracheal mucociliary system in neonatal sheep

We studied the postnatal development of the tracheal epithelium and mucociliary system in neonatal sheep. Secretion of macromolecules (radiolabeled with 35SO4 and [3H]-threonine), unidirectional fluxes of Cl-, Na+, and water (measured with radioactive tracers), and ciliary beat frequency (CBF) were measured in tracheal tissues in vitro. Tracheal mucus transport velocity (TMV) was measured in vivo. Sheep were studied at 0, 2, 4, 8, and greater than 24 (adult) wk after birth. In newborn sheep trachea, secretion of macromolecules was significantly elevated (cf. adults), and there was basal net secretion of Cl- under short-circuit and open-circuit conditions. This induced open-circuit secretion of Na+. Secretion of macromolecules decreased rapidly by 2 wk (by 40-50%) and was not different from adult values by 4 wk. Active Na+ absorption developed rapidly, and from 2 wk onward it predominated under open-circuit conditions, inducing net Cl- absorption. These changes in secretory function were associated with an age-related increase in TMV, whereas inherent tracheal CBF was unchanged. In sheep, therefore, the newborn's trachea has elevated secretion of macromolecules and secretes Cl- and liquid under basal conditions. Normal secretory function (a reduction in secretion of macromolecules coupled with net absorption of ions and presumably of liquid also) approaches adult function by 2-4 wk of age.     

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.     

The phospholipase activity of sheep pancreatic juice.

Sheep pancreatic juice was found to contain at least two enzymes which hydrolysed biliary lecithin. One enzyme was heat and acid labile and hydrolysed the fatty acid from position 1 (phospholipase A1); the other was heat and acid stable hydrolysing the fatty acid at position 2 (phospholipase A2). Lysophospholipase activity was also present. The phospholipases were active at pH values greater than 4.2, and would therefore function in the acid conditions (pH 3-6) of the sheep small intestine. The activity of the pancreatic phospholipases, and A2 in particular, was dramatically stimulated by the presence of the secretions of Brunner's glands which could be important in accelerating the hydrolysis of biliary lecithin in the lumen of the intestine. Phospholipase A1 was sensitive to acid in the range pH 2.5-3.5 and could therefore be partially inactivated by abomasal digesta; but phospholipase A2 was resistent to acid treatment.     

Late-phase bronchial vascular responses in allergic sheep

Sheep were classified on the basis of their airway response to Ascaris suum antigen aerosols as allergic or nonsensitive. Allergic sheep were classed as acute or dual responders. Acute responders had only an immediate increase in mean airflow resistance after antigen, whereas dual responders had an immediate and late-phase (6-8 h after antigen challenge) increase in mean airflow resistance; nonsensitive sheep had minimal airway responses to antigen (less than 30% increase from base line). The sheep were anesthetized 2 wk later and, after a left thoracotomy, were challenged with antigen to determine bronchial vascular responses; bronchial artery blood flow was measured with an electromagnetic flow probe. Airway responses to antigen aerosol challenge were similar in the anesthetized and conscious animals. The mean fall in bronchial vascular resistance (BVR) immediately after antigen challenge was similar in acute and dual responders (41 +/- 7 and 47 +/- 9% of base line, respectively). In dual responders, late-phase airway responses were preceded by a significant increase from base line in Qbr and a fall in bronchovascular resistance (BVR). The mean fall in BVR 6-8 h after antigen challenge in documented dual responders was significantly different from bronchial vascular responses in acute responders (59 +/- 3 vs. 89 +/- 10%, respectively). Sheep without airway responses to A. suum had no significant changes in bronchial hemodynamics or airways mechanics. Late-phase-associated changes in BVR are a specific response to antigen challenge and may be a sensitive index of mediators being released.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solution structure of porcine pancreatic phospholipase A2.

The lipolytic enzyme phospholipase A2 (PLA2) is involved in the degradation of high-molecular weight phospholipid aggregates in vivo. The enzyme has very high catalytic activities on aggregated substrates compared with monomeric substrates, a phenomenon called interfacial activation. Crystal structures of PLA2s in the absence and presence of inhibitors are identical, from which it has been concluded that enzymatic conformational changes do not play a role in the mechanism of interfacial activation. The high-resolution NMR structure of porcine pancreatic PLA2 free in solution was determined with heteronuclear multidimensional NMR methodology using doubly labeled 13C, 15N-labeled protein. The solution structure of PLA2 shows important deviations from the crystal structure. In the NMR structure the Ala1 alpha-amino group is disordered and the hydrogen bonding network involving the N-terminus and the active site is incomplete. The disorder observed for the N-terminal region of PLA2 in the solution structure could be related to the low activity of the enzyme towards monomeric substrates. The NMR structure of PLA2 suggests, in contrast to the crystallographic work, that conformational changes do play a role in the interfacial activation of this enzyme.     

Recombinant alpha 1-proteinase inhibitor blocks antigen- and mediator-induced airway responses in sheep.

Alpha(1)-proteinase inhibitor (alpha(1)-PI) is a natural serine protease inhibitor. Although mainly thought to protect the airways from neutrophil elastase, alpha(1)-PI may also regulate the development of airway hyperresponsiveness (AHR), as indicated by our previous findings of an inverse relationship between lung alpha(1)-PI activity and the severity of antigen-induced AHR. Because allergic stimulation of the airways causes release of elastase, tissue kallikrein, and reactive oxygen species (ROS), all of which can reduce alpha(1)-PI activity and contribute to AHR, we hypothesized that administration of exogenous alpha(1)-PI should protect against pathophysiological airway responses caused by these agents. In untreated allergic sheep, airway challenge with elastase, xanthine/xanthine oxidase (which generates ROS), high-molecular-weight kininogen, the substrate for tissue kallikrein, and antigen resulted in bronchoconstriction. ROS and antigen also induced AHR to inhaled carbachol. Treatment with 10 mg of recombinant alpha(1)-PI (ralpha(1)-PI) blocked the bronchoconstriction caused by elastase, high-molecular-weight kininogen, and ROS, and the AHR induced by ROS and antigen. One milligram of ralpha(1)-PI was ineffective. These are the first in vivo data demonstrating the effects of ralpha(1)-PI. Our results are consistent with and extend findings obtained with human plasma-derived alpha(1)-PI and suggest that alpha(1)-PI may be important in the regulation of airway responsiveness.     

DNA-protein interactions. Destabilizing activity of sheep pancreatic RNAase]

Evidence is presented that ovine pancreatic ribonuclease, a protein strictly homologous to bovine RNAase A but with one positive charge less, has a definite 'destabilizing' activity (quite similar to that of the bovine enzyme) on double-stranded DNA. This action of sheep pancreas RNAase has been measured by differential spectrophotometry and determining the thermal-transition profiles of the protein-DNA complexes.     

Angiotensin II blocks ATP-sensitive K+ channels in porcine coronary artery smooth muscle cells.

ATP-sensitive K+ channels with small conductance (30 pS in symmetrical K(+)-rich solutions) in porcine coronary artery smooth muscle cells were highly active at physiological concentrations of Ca2+ (greater than 10(-4) M) even in the presence of physiological ATP levels, suggesting that these channels contribute to the generation of the resting membrane potential in vascular smooth muscle cells and their modulation is important in controlling vasomotor tone. Angiotensin II, applied from outside the membrane, blocked these channels in a concentration-dependent manner. This would be expected to cause depolarization and result in vasoconstriction.     

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.     

Physical parameters and chemical composition of porcine pancreatic elastase II.

Some molecular properties of the elastase II preparation, homogenous in ultracentrifugation, have been determined. The molecular weight is 25 000, the sedimentation coefficient and the diffusion coefficient are 3.69-10(-13) s(-1) and 12.09-10(-7) cm2/s, respectively. The partial specific volume was 0.716 g/cm3, and the axial ratio is 1.95. Elastase II exhibited a considerably lower content of arginine, tyrosine, and valine, and a higher content of proline, serine and conjugated carbohydrates than elastase I. The N-terminal amino acid of the enzyme is leucine, and its isoelectric point was 10.7.     

Substrate-selective activation of histidine-modified porcine pancreatic alpha-amylase by chloride ion.

Porcine pancreatic alpha-amylase (1,4-alpha-D-glucan glucanohydrolase) [EC 3.2.1.1] has both amylase activity (hydrolysis of alpha-1,4-D-glucoside bond of starch) and maltosidase activity (hydrolysis of p-nitrophenyl-alpha-D-maltoside to p-nitrophenol and maltose). By the modification of histidine residues of porcine pancreatic alpha-amylase with diethylpyrocarbonate (DEP), both amylase and maltosidase activities were decreased in the absence of chloride ion. In the presence of chloride ion, however, maltosidase activity of the modified enzyme was increased to more than 260% of that of the native enzyme, whereas amylase activity was decreased to less than 15% of the native enzyme. Since the chloride ion binding site is part of the active site loop [Buisson et al. (1987) Food Hydrocolloids 1,399-406 and Buisson et al. (1987) EMBO J. 6, 3909-3916], the special arrangements of both catalytic and modified histidine residues induced by the chloride ion binding would enhance only the maltosidase activity of the histidine-modified enzyme.     

Soluble ST2 blocks interleukin-33 signaling in allergic airway inflammation

The ST2 gene produces a soluble secreted form and a transmembrane form, referred to as soluble ST2 and ST2L, respectively. A recent study has reported that interleukin (IL)-33 is a specific ligand of ST2L and induces production of T helper type 2 (Th2) cytokines. Although soluble ST2 is highly produced in sera of asthmatic patients and plays a critical role for production of Th2 cytokines, the function of soluble ST2 in relation to IL-33 signaling remains unclear. Here we show antagonistic effects of soluble ST2 on IL-33 signaling using a murine thymoma EL-4 cells stably expressing ST2L and a murine model of asthma. Soluble ST2 directly bound to IL-33 and suppressed activation of NF-kappaB in EL-4 cells stably expressing ST2L, suggesting that the complex of soluble ST2 and IL-33 fails to bind to ST2L. In a murine model of asthma, pretreatment with soluble ST2 reduced production of IL-4, IL-5, and IL-13 from IL-33-stimulated splenocytes. These results indicate that soluble ST2 acts as a negative regulator of Th2 cytokine production by the IL-33 signaling. Our study provides a molecular mechanism wherein soluble ST2 modulates the biological activity of IL-33 in allergic airway inflammation.     

Studies on the role of methionine in porcine pancreatic phospholipase A2.

The unique methionine-15 residue located at the N-terminal site of iso- or beta-phospholipase A2 from porcine pancrease has been specifically carboxymethylated with iodoacetic acid. The modification results in a complete inactivation of the enzymatic activity toward micellar and monomeric substrates. Spectroscopic measurements reveled that the carboxymethylated protein still binds Ca2+ and monomeric substrates with comparable affinities as the native enzyeme. The active site histidine-54 residue in the modified enzyme shows a reactivity toward the active site-directed irreversible inhibitor p-bromophenacylbromide which is identical to that of the native enzyme. The alkylated protein, however, has lost its ability to bind to lipid-water interfaces. Although circular dichroic spectra of the carboxymethylated enzyme display some changes in the tertiary structure as compared with the native enzyme, the alpha-helix content remains rather constant. It is concluded that carboxymethylation of methionine-15 destroys the interface recognition site but has only limited influence on the active site of the molecule. Therefore, it seems that methionine-15 is not involved in the catalytic events but that this residue is part of the interface recognition site which embraces the N-terminal hydrophobic part of the enzyme: Ala-Leu-Trp-Gln-Phe-Arg-Ser-Met.