Effects of human rhinovirus on epithelial barrier integrity and function in children with asthma.

BACKGROUND: Bronchial epithelial tight junctions (TJ) have been extensively assessed in healthy airway epithelium. However, no studies have yet assessed the effect of human rhinovirus (HRV) infection on the expression and resultant barrier function in epithelial tight junctions (TJ) in childhood asthma. OBJECTIVES: To investigate the impact of HRV infection on airway epithelial TJ expression and barrier function in airway epithelial cells (AECs) of children with and without asthma. Furthermore, to test the hypothesis that barrier integrity and function is compromised to a greater extent by HRV in AECs from asthmatic children. METHODS: Primary AECs were obtained from children with and without asthma, differentiated into air-liquid interface (ALI) cultures and infected with rhinovirus. Expression of claudin-1, occludin and zonula occluden-1 (ZO-1) was assessed via qPCR, immunocytochemistry (ICC), in-cell western (ICW) and confocal microscopy. Barrier function was assessed by transepithelial electrical resistance (TER; RT ) and permeability to fluorescent dextran. RESULTS: Basal TJ gene expression of claudin-1 and occludin was significantly upregulated in asthmatic children compared to non-asthmatics; however, no difference was seen with ZO-1. Interestingly, claudin-1, occludin and ZO-1 protein expression was significantly reduced in AEC of asthmatic children compared to non-asthmatic controls suggesting possible post-transcriptional inherent differences. HRV infection resulted in a transient dissociation of TJ and airway barrier integrity in non-asthmatic children. Although similar dissociation of TJ was observed in asthmatic children, a significant and sustained reduction in TJ expression concurrent with both a significant decrease in TER and an increase in permeability in asthmatic children was observed. CONCLUSION: This study demonstrates novel intrinsic differences in TJ gene and protein expression between AEC of children with and without asthma. Furthermore, it correlates directly the relationship between HRV infection and the resultant dissociation of epithelial TJ that causes a continued altered barrier function in children with asthma.

Impaired airway epithelial cell responses from children with asthma to rhinoviral infection.

BACKGROUND: The airway epithelium forms an effective immune and physical barrier that is essential for protecting the lung from potentially harmful inhaled stimuli including viruses. Human rhinovirus (HRV) infection is a known trigger of asthma exacerbations, although the mechanism by which this occurs is not fully understood. OBJECTIVE: To explore the relationship between apoptotic, innate immune and inflammatory responses to HRV infection in airway epithelial cells (AECs) obtained from children with asthma and non-asthmatic controls. In addition, to test the hypothesis that aberrant repair of epithelium from asthmatics is further dysregulated by HRV infection. METHODS: Airway epithelial brushings were obtained from 39 asthmatic and 36 non-asthmatic children. Primary cultures were established and exposed to HRV1b and HRV14. Virus receptor number, virus replication and progeny release were determined. Epithelial cell apoptosis, IFN-ß production, inflammatory cytokine release and epithelial wound repair and proliferation were also measured. RESULTS: Virus proliferation and release was greater in airway epithelial cells from asthmatics but this was not related to the number of virus receptors. In epithelial cells from asthmatic children, virus infection dampened apoptosis, reduced IFN-ß production and increased inflammatory cytokine production. HRV1b infection also inhibited wound repair capacity of epithelial cells isolated from non-asthmatic children and exaggerated the defective repair response seen in epithelial cells from asthmatics. Addition of IFN-ß restored apoptosis, suppressed virus replication and improved repair of airway epithelial cells from asthmatics but did not reduce inflammatory cytokine production. CONCLUSIONS: Collectively, HRV infection delays repair and inhibits apoptotic processes in epithelial cells from non-asthmatic and asthmatic children. The delayed repair is further exaggerated in cells from asthmatic children and is only partially reversed by exogenous IFN-ß.

Toward a comprehensive evaluation of the impact of electronic aids to daily living: evaluation of consumer satisfaction.

PURPOSE: It is generally accepted that electronic aids to daily living (EADLs) play an important role in the lives of many people with severe disabilities by providing the means to access and control devices for daily living activities. Despite this, little proof exists to support the contention that consumers are satisfied with relevant aspects of these assistive devices. The purpose of this study was to explore consumer satisfaction with EADLs and investigate the value that people with degenerative neuromuscular conditions place on these technologies. METHOD: Interviews were conducted with 40 EADL users and non-users to compare their views about these devices and their daily life experiences. Users were interviewed twice, six months apart, to establish the stability of their views and experiences with EADLs. The Functional Independence Measure (FIM instrument), the personal profile and Quebec User Evaluation of Satisfaction with assistive Technology (QUEST) were administered to determine functional levels of participants, gather personal data pertinent to the study of device utility and explore user satisfaction with EADLs. RESULTS: Results suggest that overall consumers were quite satisfied with their EADLs and that this was relatively stable over time. However, some consumers expressed concerns regarding the cost of these technologies and their associated services. Both users and non-users rated EADLs similarly in relation to relative degree of importance ascribed to them. CONCLUSIONS: Combining the QUEST with outcome measurement tools that explore other important dimensions such as the effect on quality of life and psychosocial impact will help service providers to justify the costs associated with the prescription of sophisticated, costly assistive devices such as EADLs.

The impact of inflammation on bronchial neuronal networks.

It is well-recognized that the activities of airway neuronal systems can be modulated by various agonist molecules. This brief review examines some of the evidence that inflammation and some of the mediators relevant to the expression of inflammatory processes can also significantly alter the function and activities of airway nerves. The concept of neuronal plasticity and phenotype switching induced by inflammation is also examined, with particular emphasis on sensory airway nerves.

Differential modulation of endothelin ligand-induced contraction in isolated tracheae from endothelin B (ET(B)) receptor knockout mice.

The role of endothelin B (ET(B)) receptors in mediating ET ligand-induced contractions in mouse trachea was examined in ET(B) receptor knockout animals. Autoradiographic binding studies, using [(125)I]-ET-1, confirmed the presence of ET(A) receptors in tracheal and bronchial airway smooth muscle from wild-type (+/+) and homozygous recessive (-/-) ET(B) receptor knockout mice. In contrast, ET(B) receptors were not detected in airway tissues from (-/-) mice. In tracheae from (+/+) mice, the rank order of potencies of the ET ligands was sarafotoxin (Stx) S6c>ET-1>ET-3; Stx S6c had a lower efficacy than ET-1 or ET-3. In tissues from (-/-) mice there was no response to Stx S6c (up to 0.1 microM), whereas the maximum responses and potencies of ET-1 and ET-3 were similar to those in (+/+) tracheae. ET-3 concentration-response curve was biphasic in (+/+) tissues (via ET(A) and ET(B) receptor activation), and monophasic in (-/-) preparations (via stimulation of only ET(A) receptors). In (+/+) preparations SB 234551 (1 nM), an ET(A) receptor-selective antagonist, inhibited the secondary phase, but not the first phase, of the ET-3 concentration-response curve, whereas A192621 (100 nM), an ET(B) receptor-selective antagonist, had the opposite effect. In (-/-) tissues SB 234551 (1 nM), but not A192621 (100 nM), produced a rightward shift in ET-3 concentration-response curves. The results confirm the significant influence of both ET(A) and ET(B) receptors in mediating ET-1-induced contractions in mouse trachea. Furthermore, the data do not support the hypothesis of atypical ET(B) receptors. In this preparation ET-3 is not an ET(B) receptor-selective ligand, producing contractions via activation of both ET(A) and ET(B) receptors.

Influence of endothelin-1(1-31) on smooth muscle tone and cholinergic nerve-mediated contraction in rat isolated trachea.

Endothelin-1(1-21) (ET-1(1-21)) is a strong candidate as a significant mediator in asthma, in part because of its powerful spasmogenic actions and its ability to enhance cholinergic nerve-mediated contraction in human and animal airway smooth muscle. In the study reported here, we have demonstrated that [125I]ET-1(1-31) binds specifically to BQ-123-sensitive sites (presumably ET(A)-receptors) and to sarafotoxin S6c (S6c)-sensitive sites (presumably ET(B)-receptors) in rat tracheal and pulmonary airways, as well as in lung alveoli. These sites coexist in tracheal airway smooth muscle and in alveolar tissue in approximately equal proportions. ET-1(1-21) and ET-1(1-31) were equipotent and approximately equally active as spasmogens in rat tracheal smooth muscle. Importantly, both peptides were shown to potentiate cholinergic nerve-mediated rat tracheal contraction, although ET-1(1-31) was less active in this regard. These data are consistent with the idea that ET-1(1-31) could play a significant mediator role in obstructive airway diseases such as asthma.

Ageing and epithelial integrity as modulators of airway smooth muscle responsiveness to endothelin-1.

This study has examined the effects of animal age on the contractile responsiveness and inositol phosphate-generating capacities of guinea-pig (0-104 weeks) and rat (4-104 weeks) isolated tracheal smooth muscle in response to endothelin-1 (ET-1). The influence of animal age on the specific binding of [125I]ET-1 to guinea-pig and rat isolated tracheal tissue was also examined. The potency (pD2) of ET-1 was three to four times greater in tracheal tissue taken from 4-week-old rats than in similar tissue from 12- to 32-week-old animals, although maximum response (Emax) was not significantly altered. Neither pD2 nor Emax were influenced by ageing in epithelium-intact guinea-pig tracheal preparations. In contrast, removal of the airway epithelium significantly increased the contractile potency of ET-1 by two- to three-fold in tissue from animals of 6-20 weeks of age, but not in tissue from newborn animals. Significant falls in specific [125I]ET-1 grain density with ageing were demonstrated during the maturation phase in both species. In the rat, the decrease between 4 and 12 weeks was reflected in the fall in ET-1 potency at 12 weeks. However, the age-associated reduction in airway smooth muscle ET receptor number in the guinea-pig was not mirrored by significant changes in sensitivity to ET-1, suggesting the presence of a functional receptor reserve. ET-1 (1 nM) caused significant increases in intracellular inositol phosphates, with levels generally higher in rat than in guinea-pig trachea. ET-1-induced inositol phosphate accumulation decreased significantly with respect to animal age in both guinea-pig and rat isolated tracheal tissue. However, this was not correlated with changes in contractile pD2 or Emax. For example, in both rat and guinea-pig, the smallest ET-1-induced increases in intracellular inositol phosphates were measured in airway smooth muscle from the oldest animals tested, although tissue sensitivity to ET-1 was stable in both species after 12 weeks of age. These data suggest that relatively low levels of inositol phosphates were required to elicit Emax, consistent with the presence of more than one signal transduction process.

The influence of animal age on beta-adrenoceptor density and function in tracheal airway smooth muscle.

We examined the influence of animal age on the functional response of guinea-pig (0-156 weeks) and rat (4-136 weeks) isolated tracheal tissue to beta-adrenoceptor agonists. In addition, the binding density and affinity of [125I]iodocyanopindolol ([125I]CYP) binding to tracheal tissue was examined with respect to animal age. Significant age-related changes in isoprenaline potency were observed in tracheal ring preparations taken from animals during the early maturation phase of animal growth in the guinea-pig and rat. In addition, in rat isolated tracheal tissue, age-related decreases in fenoterol potency were observed during senescence, but not maturation. The changes in the functional responsiveness of tracheal tissue were not reflected by changes in the binding density or affinity for [125I]cyanopindolol ([125I]CYP) of beta-adrenoceptors, or in changes in specific autoradiographic grain density over smooth muscle tissue. In both guinea-pig and rat, no significant age-related changes in the influence of catechol-O-methyl transferase (COMT) or of extraneuronal uptake inhibition were detected. This study has demonstrated significant age-related changes in the responsiveness of guinea-pig and rat isolated tracheal tissue to beta-adrenoceptor agonists that were not related to changes in the density or affinity of the beta-adrenoceptor population or in the activity of COMT or extraneuronal uptake. The possibility of age-related changes in receptor-signal transduction coupling should be explored.

Confocal microscopy in biomedical research.

Confocal microscopy has allowed a major advance in biological imaging, since it represents a rapid, cost effective means of ecamining thick tissue specimens. In most cases, this involves fluorescence imaging and it is increasingly being used as a basic tool in biomedical research. Confocal microscopy allows the collection of thin optical sections, without the need for physical sectioning of the tissue. Additionally, confocal microscopes can usually produce images with greater sensitivity, contrast and resolution than those produced with normal light microscopes. We attempt to explain how this technology might be better used as a routine research tool. Since high quality, in-focus optical sections of thick tissue preparations can be generated quickly, confocal microscopy, in combination with immunofluorescence histochemistry, can now be used to examine complex three-dimensional distributions of distinct structures within tissues such as nerves within airways. Additionally, ultraviolet confocal microscopy allows the assessment of both dynamic and static phenomena in living cells and tissues. Thus, in addition to the imaging of fluorescence associated with structural elements, confocal microscopes can be used to quantitatively evaluate the distribution and fluxes of intracellular ions like calcium. Rapid, line-scanning confocal microscopes can be used in the assessment of dynamic events. For example, the in vivo imaging of microvascular permeability in airways becomes possible for the first time. By providing examples of some of our uses for confocal microscopy, we might encourage others to explore this relatively new and important texhnology for examining events and structures in single cells, tissue samples and in intact animals.

The influence of post-mortem conditions on contractile and relaxant responsiveness of guinea-pig isolated tracheal smooth muscle.

Responsiveness to various contractile and relaxant agonists was assessed in tracheal preparations from guinea-pigs that had been incubated in situ at 4-37 degrees C for 0-168 h post-mortem. The potencies of histamine and acetylcholine were increased up to 168 h at 4 degrees C post-mortem and up to 24 h post-mortem at 22 degrees C. Histamine potency also increased with increasing post-mortem time at 37 degrees C. After 48 h at 22 degrees C and 8 h at 37 degrees C, responses to all spasmogens were abolished. Increases in histamine and acetylcholine potencies were similarly observed in tracheal tissue that had been removed at death and then incubated at 4 degrees C in oxygenated Krebs-bicarbonate solution for 0-168 h. The increased potency of these drugs may be explained by epithelial damage and/or loss of an epithelium-derived inhibitory factor (EpDIF). Both basal and spasmogen-stimulated increases in intracellular phosphoinositides fell with increasing time and ambient temperature post-mortem, despite the fact that contraction in response to these agonists could still be evoked. This suggests the selective failure of this signal transduction pathway and the maintenance of responsiveness via other mechanisms. The potencies and maximum effects of relaxant agonists remained unaltered in tracheal tissue with increasing time post-mortem, suggesting little change in the function of the appropriate receptor-signal transduction processes. This study has therefore demonstrated that at 4 degrees C. contractile and relaxant responses were preserved for up to 168 h post-mortem, although the modulatory influence of the epithelium on histamine and acetylcholine responses was rapidly lost.

Age and region-dependent contraction to alpha-adrenoceptor agonists in rat and guinea-pig isolated trachea.

1. The influence of age and of region on alpha-adrenoceptor-mediated contraction to (-)-adrenaline and (-)-noradrenaline was examined in rat (4-136 weeks) and guinea-pig (2-156 weeks) isolated tracheal ring preparations with particular emphasis on the early (up to 12 weeks) maturation phase. 2. In rat tracheal rings, significant regional variation was observed with respect to maximal (-)-adrenaline-induced contraction, such that the greatest activity was seen in ring preparations from the laryngeal end of the trachea. Tracheal rings from the carinal end responded very poorly or were unresponsive to (-)-adrenaline, depending on animal age. These regional differences were seen across the age range. The potencies of (-)-adrenaline and (-)-noradrenaline remained unchanged with respect to animal age, but the maximum contractile tension that developed in response to these agonists increased with increasing animal age in all regions of the trachea. 3. In guinea-pig isolated tracheal tissue, maximum contractile responses (Emax) to (-)-adrenaline and (-)-noradrenaline remained unchanged with increasing animal age. In addition, there was no evidence for a region-dependence in the responsiveness of tracheal tissue to alpha-adrenoceptor-mediated contraction in this species. 4. In both guinea-pig and rat isolated tracheal tissue, alpha-adrenoceptor-mediated contraction appeared to involve the activation of alpha1-adrenoceptors.

Influence of respiratory tract viral infection on endothelin-1-induced modulation of cholinergic nerve-mediated contractions in murine airway smooth muscle.

The effects of endothelin-1 (ET-1) and sarafotoxin S6c (S6c) on cholinergic contractions elicited by electrical field stimulation (EFS) were examined in mouse tracheal preparations from healthy animals and from animals infected with parainfluenza-1 (P-1) virus. S6c (an ETB-selective agonist) and ET-1 caused marked ETA and/or ETB receptor-mediated potentiation of EFS-induced contraction in tracheal tissue from both groups. Despite the fact that such infection is known to markedly alter ET receptor density and function in mouse tracheal smooth muscle, no evidence for modulated neuronal ET receptor function was obtained. The reason for this differential sensitivity of smooth muscle and neuronal ET receptors to P-1 infection is unknown.

Influence of parainfluenza-1 respiratory tract viral infection on endothelin receptor-effector systems in mouse and rat tracheal smooth muscle.

1. In this study we have compared the effects of parainfluenza-1 respiratory tract viral infection on the density and function of ETA and ETB receptors in rat and mouse tracheal airway smooth muscle. 2. The bronchoconstrictor effect of inhaled methacholine was significantly enhanced in virus-infected rats, at both 4 and 12 days post-inoculation. That is, the concentration of methacholine causing an increase in resistance of 100% (PC100 methacholine) was significantly lower in virus-infected animals at both 4 and 12 days post-inoculation (n = 6-8; P < 0.05). 3. Total specific binding of [125I]-endothelin-1 and the relative proportions of ETA and ETB binding sites for [125I]-endothelin-1 were assessed in tracheal airway smooth muscle in parainfluenza-1-infected rats and mice at days 2, 4 and 12 post-inoculation using the ligands BQ-123 (1 microM; ETA receptor-selective) and sarafotoxin S6c (100 nM; ETB receptor-selective). Total specific binding in mice was significantly reduced at day 2 post-inoculation (n = 5; P < 0.05) but not at days 4 and 12 post-inoculation (n = 5). In control mice, the proportions of ETA and ETB binding sites were 53%:47% at day 2 and 43%:57% at day 4 and these were significantly altered by parainfluenza-1 infection such that, the ratios were 81%:19% at day 2 and 89%:11% at day 4 (P < 0.05). By day 12 post-inoculation, the proportion of ETA and ETB binding sites in tracheal smooth muscle from mice infected with parainfluenza-1 was not significantly different from control. In rat tracheal airway smooth muscle, neither total specific binding nor the ETA and ETB binding site ratio (64%:36%) were significantly altered in virus-inoculated rats at days 2, 4 or 12 post-inoculation (n = 5). 4. Parainfluenza-1 infection in mice had no effect on the sensitivity or maximal contractile effect of endothelin-1 in tracheal smooth muscle at days 2, 4 or 12 post-inoculation (n = 4). In contrast, contraction in response to the ETB receptor-selective agonist sarafotoxin S6c was attenuated by 39% at day 2 and by 93% at day 4 post-inoculation (P < 0.05). However, by day 12 post-inoculation, contractions to sarafotoxin S6c were not significantly different between control and virus-infected mice. In parainfluenza-1-infected rats, there were small but significant reductions in the sensitivity to carbachol, endothelin-1 and sarafotoxin S6c whilst the maximal responses to the highest concentrations of these agonists were not significantly altered by virus infection (n = 8). 5. BQ-123 (3 microM) had no significant effect on cumulative concentration-effect curves to endothelin-1 in tracheal preparations from control mice (n = 4) or parainfluenza-1-infected rats (n = 8). In contrast, in tissues taken from virus-infected mice at day 4 post-inoculation, BQ-123 caused a marked 9.6 fold rightward shift in the concentration-effect curve to endothelin-1 (n = 4). 6. In summary, we have demonstrated that parainfluenza-1 infection in mice transiently reduced the density of tracheal airway smooth muscle ETB receptors and this was reflected in reduced responsiveness to the ETB receptor-selective agonist sarafotoxin S6c. In contrast, whilst parainfluenza-1 infection in rats was associated with the pathological features and bronchial hyperresponsiveness common to respiratory tract viral infection, there was no selective down-regulation of ETB receptor expression or functional activity. The reasons for these species differences are not clear, but may relate to differences in the airway inflammatory response to parainfluenza-1 virus.

EndothelinB (ETB) receptor-activated potentiation of cholinergic nerve-mediated contraction in human bronchus.

In human isolated bronchial preparations, the endothelinB (ETB) receptor-selective agonist, sarafotoxin S6c (Stx6c; 1 nM) increased nerve-mediated contraction in response to electrical field stimulation (EFS) at 0.5-1 Hz from 19 +/- 4% to 42 +/- 7% (n = 9). This effect was blocked in the presence of the ETB receptor-selective antagonist, BQ-788 (10 microM). These data are consistent with findings in some animal species that ET-1 and related peptides have marked neuromodulatory influences on the cholinergic system. Furthermore, they provide additional support for the concept that ET-1 may have a mediator role in bronchial obstruction in asthma.

Endothelin-1-induced potentiation of human airway smooth muscle proliferation: an ETA receptor-mediated phenomenon.

1. In this study the mitogenic effects in human cultured tracheal smooth muscle cells of endothelin-1 (ET-1), ET-3, and sarafotoxin S6c (S6c), the ETB receptor-selective agonist, were explored either alone or in combination with the potent mitogen, epidermal growth factor (EGF). 2. In confluent, growth-arrested human airway smooth, neither ET-1 (0.01 nM-1 microM) nor ET-3 (0.001 nM-1 microM) or S6c (0.01 nM-1 microM) induced cell proliferation, as assessed by [3H]-thymidine incorporation. In contrast, EGF (1.6 pM-16 nM) produced concentration-dependent stimulation of DNA synthesis (EC50 of about 0.06 nM). The maximum increase of about 60 fold above control, elicited by 16 nM EGF, was similar to that obtained with 10% foetal bovine serum (FBS). EGF (0.16-16 nM) also produced a concentration-dependent increase in cell counts, whereas ET-1 (1-100 nM) was without effect on this index of mitogenesis. 3. ET-1 (1-100 nM) potentiated EGF-induced proliferation of human tracheal smooth muscle cells. For example, ET-1 (100 nM), which alone was without significant effect, increased by 3.0 to 3.5 fold the mitogenic influence of EGF (0.16 nM). The potentiating effect of ET-1 on EGF-induced proliferation was antagonized by BQ-123 (3 microM), the ETA receptor antagonist, but was unaffected by the ETB receptor antagonist BQ-788 (10 microM). 4. Neither ET-3 (1-100 nM) nor S6c (1-100 nM) influenced the mitogenic effects of EGF (0.16-1.6 nM). 5. [125I]-ET-1 binding studies revealed that on average the ratio of ETA to ETB receptors in human cultured tracheal smooth muscle cells was 35:65 ( +/- 3; n = 4), confirming the predominance of the ETB receptor subtype in human airway smooth muscle. 6. These data indicate that ET-1 alone does not induce significant human airway smooth muscle cell proliferation. However, it potently potentiated mitogenesis induced by EGF, apparently via an ETA receptor-mediated mechanism. These findings suggest that ET-1, a mediator detected in increased amounts in patients with acute asthma, may potentiate the proliferative effects of mitogens and contribute to the airway smooth muscle hyperplasia associated with chronic severe asthma.

The distribution and density of receptor subtypes for endothelin-1 in peripheral lung of the rat, guinea-pig and pig.

1. Quantitative autoradiographic studies were conducted to determine the distributions and densities of endothelin-A (ETA) and ETB receptor subtypes in peripheral lung alveolar wall tissue of the rat, guinea-pig and pig, with a view to assessing the potential suitability of these tissues as models for investigations of ET receptor function in human alveolar tissue. 2. High levels of specific [125I]-ET-1 binding were detected in peripheral lung components from all three species tested. In mature porcine alveolar wall tissue, specific binding increased in a time-dependent manner to a plateau, consistent with the previously described pseudo-irreversible binding of this ligand to a finite population of specific binding sites. 3. [125I]-ET-1 was associated specifically with both ETA and ETB binding site subtypes in alveolar wall tissue of foetal pig lung as early as 36 days gestation, raising the possibility of a functional role for ET-1 in lung development. In addition, both ETA and ETB binding site subtypes were detected in alveolar wall tissue and in peripheral airway smooth muscle of mature lung parenchyma from all three species. However, the binding subtype proportions differed in these tissues. For example, in porcine peripheral bronchial smooth muscle, ETA sites apparently predominated, whereas ETB sites constituted the major subtype detected in alveolar wall in this species. These data suggest significant shifts in ET receptor subtype expression at different levels in the respiratory tract. 4. ET binding site subtype proportions in the alveolar wall also differed markedly between species. In rat lung alveoli, ETA and ETB sites were detected in similar proportions (52 +/- 3% and 43 +/- 5% respectively). In contrast, in guinea-pig peripheral lung, ETB binding sites clearly predominated, constituting approximately 80% of total specific binding, with ETA sites accounting for only 12%. Porcine alveolar wall tissue also contained a mixture of these ET receptor subtypes, with ETA and ETB binding comprising 23 +/- 3% and 65 +/- 1% respectively of the total population of specific binding sites detected. These latter proportions are similar to values previously obtained in human peripheral lung tissue, suggesting that porcine lung might be a useful model of the human peripheral lung in subsequent studies of the functions of these pulmonary ET receptor subtypes.

Influence of regional differences in ETA and ETB receptor subtype proportions on endothelin-1-induced contractions in porcine isolated trachea and bronchus.

1. Quantitative autoradiographic studies were conducted to determine the distributions and densities of ETA and ETB binding site subtypes in porcine tracheal and bronchial smooth muscle. In addition, the roles of ETA and ETB receptors in endothelin-1-mediated contraction of these tissues were assessed. 2. Quantitative autoradiographic studies revealed that both ETA and ETB binding sites for [125I]-endothelin-1 were present in both bronchial and tracheal airway smooth muscle. However, the proportions of these sites were markedly different at these two levels within the respiratory tract. In tracheal smooth muscle, the proportions of ETA and ETB sites were 30 +/- 1% and 70 +/- 1% respectively, whereas in bronchial smooth muscle, these proportions were virtually reversed, being 73 +/- 2% and 32 +/- 8% respectively. 3. Endothelin-1 induced concentration-dependent contraction of porcine tracheal and bronchial airway smooth muscle. Endothelin-1 had similar potency (concentration producing 30% of the maximum carbachol contraction, Cmax) in trachea (22 nM; 95% confidence limits (c.l.), 9-55 nM; n = 9) and bronchus (22 nM; c.l., 9-55 nM; n = 6). Endothelin-1 also produced comparable maximal contractions in trachea (59 +/- 5% Cmax; n = 9) and bronchus (65 +/- 4% Cmax, n = 6). 4. In trachea, endothelin-1 induced contractions were not significantly inhibited by either the ETA receptor-selective antagonist, BQ-123 (3 microM) or the ETB receptor-selective antagonist, BQ-788 (1 microM). However, in the combined presence of BQ-123 and BQ-788, the concentration-effect curve to endothelin-1 was shifted to the right by 3.7 fold (n = 8; P = 0.01). 5. In bronchus, concentration-effect curves to endothelin-1 were shifted to the right by BQ-123 (3 microM; 4.3 fold; P < 0.05), but not by BQ-788 (1 microM). In the presence of both antagonists, concentration-effect curves to endothelin-1 were shifted by at least 6.7 fold (n = 6; P = 0.01). 6. Sarafotoxin S6c induced contraction in both tissue types, although the maximum contraction was greater in trachea (53 +/- 7% Cmax; n = 6) than in bronchus (21 +/- 5% Cmax; n = 6). BQ-788 (1 microM) markedly reduced sarafotoxin S6c potency in both trachea and bronchus (e.g. by 50 fold in trachea; c.l., 14-180; n = 6; P < 0.05). 7. These data demonstrate that the proportions of functional endothelin receptor subtypes mediating contraction of airway smooth muscle to endothelin-1, vary significantly at different levels in the porcine respiratory tract.

Eosinophil peroxidase and 125I- ion binding in guinea-pig trachea.

The present study further characterizes ascorbic acid-dependent 125I- ion binding in guinea-pig trachea. Binding of 125I- ion in the presence of ascorbic acid was detected at the epithelial/submucosal interface, apparently involving individual cells containing peroxidase enzyme. A similar binding pattern was observed when hydrogen peroxide was substituted for ascorbic acid. Binding could be inhibited by the addition of the H2O2 degrading enzyme catalase or the peroxidase inhibitor thiourea. Results demonstrated that this binding was dependent upon the addition of, or endogenous production of hydrogen peroxide and its metabolism via airway eosinophil peroxidase. The relevance of this anomalous iodine binding phenomenon to radioiodinated ligand binding studies is discussed.

Endothelin-1-induced [3H]-inositol phosphate accumulation in rat trachea.

1. The effects of endothelin-1 (ET-1) and of the muscarinic cholinoceptor agonist, carbachol, on [3H]-inositol phosphate ([3H]-InsP) accumulation and smooth muscle contraction were determined in rat isolated tracheal tissue. 2. ET-1 (1 microM) and carbachol (10 microM) induced significant accumulation of [3H]-InsPs in myo-[2-3H]-inositol-loaded rat tracheal segments. Several components of the tracheal wall including the airway smooth muscle band, the cartilaginous region and the intercartilaginous region generated significant levels of [3H]-InsPs in response to ET-1 and carbachol. Following stimulation with ET-1, a greater proportion of tracheal [3H]-InsPs were generated in the intercartilaginous region (49%) than in either the airway smooth muscle band (25%) or cartilaginous region (26%). However, when the respective weights of these regions is taken into account, ET-1-induced accumulation of [3H]-InsPs was greatest in the airway smooth muscle band. The tracheal epithelium did not appear to generate [3H]-InsPs in response to ET-1 or modulate either basal or ET-1-induced accumulation of [3H]-InsPs in rat tracheal segments. 3. In the rat tracheal smooth muscle band, ET-1 caused a time- and concentration-dependent accumulation of [3H]-InsPs. Concentrations of ET-1 as low as 10 nM produced significant accumulation of [3H]-InsPs (1.23 +/- 0.10 fold increase above basal levels of 295 +/- 2 d.p.m. mg-1 wet wt., n = 3 experiments). At 10 microM, the highest concentration ?tsed, ET-1 produced similar levels of [3H]-InsP accumulation (7.03 +/- 0.55 fold above basal levels, t = 5) to that produced by a maximally effective concentration of carbachol (10 microM; 7.97 +/- 0.31 fold increase above basal levels, n = 4). ET-1-induced accumulation of [3H]-InsPs was not significantly affected by indomethacin (5 microM), nordihydroguaiaretic acid (NDGA, 10 microM), WEB 2086 (10 microM) or phosphoramidon (10 microM).4. ET-1 also produced concentration-dependent contractions of epithelium-denuded rat tracheal ring preparations. The mean concentration of ET-1 producing 50% of the maximum contractile response to carbachol (EC50) was 31 nm (95% confidence limits, 20-49 nM, n = 12). The presence of an intact tracheal epithelium, indomethacin (5 microM), WEB 2086 (10 microM) and phosphoramidon (10 microM) had no significant effect on the mean EC50 for ET-1-induced contraction (n = 5). In contrast, NDGA (10 microM) inhibited ET-1- induced contractions (4.0 fold increase in mean EC50, P < 0.001, n = 5). However, this effect of NDGA did not appear to be related to inhibition of leukotriene synthesis via lipoxygenase since the leukotriene antagonist SKF 104353 did not affect ET-1-induced contractions (n = 5) and moreover, leukotriene C4 and leukotriene D4 did not contract rat isolated tracheal smooth muscle preparations (n = 4).5. The threshold concentrations of ET-1 that produced increases in smooth muscle contraction and [3H]-InsPs accumulation were similar, although the EC50 for [3H]-InsP accumulation was 2.9 fold greater than that for smooth muscle contraction. For carbachol, the EC50 for [3H]-InsP accumulation (mean ECQO = 5.0 microM, 1.2-21 microM, n = 4) was 25 fold greater than that for smooth muscle contraction(mean EC50 = 0.20 miicroM, 0.17-0.24 microM, n = 12).6. It seems likely that ET-1 has a direct effect on InsP generation in rat tracheal smooth muscle and that this is largely responsible for the spasmogenic actions of this peptide.