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.

Memory conformity: exploring misinformation effects when presented by another person.

Two experiments demonstrate that post-event information, when delivered by another person, can affect people's memory reports. In the first experiment participants were shown several cars, and later, in pairs, given an 'old'/'new' recognition test on these cars plus several lures. There was a small but reliable effect of memory conformity. When the person was given misinformation this lowered accuracy, while presenting accurate information increased accuracy. In the second experiment participants, in pairs, viewed an identical crime except that half saw an accomplice with the thief and half did not. Initial memories were very accurate, but after discussing the crime with the other person in the pair (who saw a slightly different sequence), most pairs conformed. Confidence ratings strongly predicted which person in the pair persuaded the other. Parallels with eyewitness testimony in the Oklahoma bombing case and implications for police interviewing more generally are discussed.

Vascular surgical society of great britain and ireland: audit of surgical technique during carotid endarterectomy by intraoperative duplex ultrasonography: trainees compared with consultant

BACKGROUND: There has been increasing interest in audit of outcome following surgery. A previous study reported a significant difference in clinical outcome between consultant and trainees but there is no information on technical factors. Duplex ultrasonography before wound closure was used to compare clinical and technical outcome for consultant and trainees. METHODS: Patients underwent endarterectomy (89 by consultant, 60 by six trainees). In the consultant group nine vessels were patched and 36 patients underwent shunting compared with seven and 31 respectively for trainees. RESULTS: In the consultant group there were two deaths, one stroke, one transient ischaemic attack (TIA) and two cranial nerve injuries. The neurological event rate was 2 per cent, and overall stroke and death rate 3 per cent. There were ten residual flaps (11 per cent) (three re-explored) and eight kinks (9 per cent). The residual stenosis rate was 10 per cent (nine of 89) and following re-exploration this reduced to 7 per cent. In the trainee group there was one death, two strokes, one TIA and one cranial nerve injury. The neurological event rate was 5 per cent, and the overall stroke and death rate 3 per cent. There were nine flaps (15 per cent) (four re-explored) and 13 kinks (22 per cent). The residual stenosis rate was 22 per cent (13 of 60) which reduced to 15 per cent (nine of 60) following re-exploration. There was no significant difference in clinical outcome between consultant and trainees but there was an increased incidence of technical problems among the trainees (t = 2.12, P < 0.05). CONCLUSION: Intraoperative duplex ultrasonography is a valuable method for assessing surgical technique; it gives immediate feedback to the surgeon, enables corrective measures to be taken and may facilitate training.

Root production and turnover in an upland grassland subjected to artificial soil warming respond to radiation flux and nutrients, not temperature.

Root demographic processes (birth and death) were measured using minirhizotrons in the soil warming experiments at the summit of Great Dun Fell, United Kingdom (845 m). The soil warming treatment raised soil temperature at 2 cm depth by nearly 3°C. The first experiment ran for 6 months (1994), the second for 18 (1995-1996). In both experiments, heating increased death rates for roots, but birth rates were not significantly increased in the first experiment. The lack of stimulation of death rate in 1996 is probably an artefact, caused by completion of measurements in late summer of 1996, before the seasonal demography was concluded: root death continued over the winter of 1995-1996. Measurements of instantaneous death rates confirmed this: they were accelerated by warming in the second experiment. In the one complete year (1995-1996) in which measurements were taken, net root numbers by the end of the year were not affected by soil warming. The best explanatory environmental variable for root birth rate in both experiments was photosynthetically active radiation (PAR) flux, averaged over the previous 5 (first experiment) or 10 days (second experiment). In the second experiment, the relationship between birth rate and PAR flux was steeper and stronger in heated than in unheated plots. Death rate was best explained by vegetation temperature. These results provide further evidence that root production acclimates to temperature and is driven by the availability of photosynthate. The stimulation of root growth due to soil warming was almost certainly the result of changes in nutrient availability following enhanced decomposition.

Intraoperative high resolution duplex imaging during carotid endarterectomy: which abnormalities require surgical correction?

OBJECTIVES: This study evaluates high resolution, duplex ultrasound imaging for quality control of carotid endarterectomy in order to determine which technical factors were linked to residual stenosis and to define duplex criteria for re-exploration. DESIGN, MATERIAL AND METHODS: A consecutive series of 100 patients undergoing carotid endarterectomy were evaluated. Duplex imaging was performed prior to wound closure and repeated at 6-8 weeks postoperatively. Stenoses were classified as non-significant, moderate or severe based on duplex criteria. Intimal flaps, shelves, kinks, clamp damage and fronds were identified by ultrasound imaging. RESULTS: Five moderate stenoses were noted in the proximal endarterectomy site (PES), and at follow-up three had resolved. Adherent fronds were detected in 83% of vessels and resolved in all but three cases. At the distal endarterectomy site there were 10 severe and 12 moderate stenoses. Intimal flaps were associated with an increased incidence of residual stenosis (p = 0.010). CONCLUSIONS: We conclude that severe stenoses with an intimal flap should be corrected immediately. Further data is required to establish the significance of kinks. Residual intimal flaps in the PES appear to remodel. The role of completion duplex may lie in the modification of surgical technique to eradicate anatomical and haemodynamic imperfections.

Root production, turnover and respiration under two grassland types along an altitudinal gradient: influence of temperature and solar radiation.

We have measured the rates of root production and death and of root respiration in situ under two grasslands along an altitudinal gradient in the northern Pennines, UK, represented by a lowland site at 171 m in an agricultural setting, and three upland sites between 480 and 845 m. One grassland was dominated by Festuca ovina and was on a brown earth soil; the other was dominated by Juncus squarrosus and Nardus stricta and occurred on a peaty gley. The natural altitudinal gradient was extended by transplantation. Although root biomass and root production (estimated using minirhizotrons) both showed pronounced seasonal peaks, there was no simple altitudinal gradient in either variable, and neither root production nor root death rate was a simple function of altitude. Increased root accumulation in summer was a function of change in the length of the growing season, not of soil temperature. Root populations in winter were similar at all sites, showing that increased production at some sites was accompanied by increased turnover, a conclusion confirmed by cohort analyses. Respiration rate, measured in the field by extracting roots and measuring respiration at field temperature in an incubator, was unrelated to temperature. The temperature sensitivity of respiration (expressed as the slope of a plot of log respiration rate against temperature) showed no simple seasonal or altitudinal pattern. Both root growth (under Festuca) and respiration rate were, however, closely related to radiation fluxes, averaged over the previous 10 days for growth and 2 days for respiration. The temperature sensitivity of respiration was a function of soil temperature at the time of measurement. These results show that root growth and the consequent input of carbon to soil in these communities is controlled by radiation flux not temperature, and that plants growing in these upland environments may acclimate strongly to low temperatures. Most carbon cycle models assume that carbon fluxes to soil are powerfully influenced by temperature, but that assumption is based largely on short-term studies and must be reassessed.

Root production and turnover and carbon budgets of two contrasting grasslands under ambient and elevated atmospheric carbon dioxide concentrations.

Monoliths of two contrasting vegetation types, a species-rich grassland on a brown earth soil over limestone and species-poor community on a peaty gley, were transferred to solardomes and grown under ambient (350 µ 1-1 ) and elevated (600 µ11-1 ) CO2 for 2 yr. Shoot biomass was unaltered but root biomass increased by 40-50% under elevated CO2 . Root production was increased by elevated CO2 in the peat soil, measured both as instantaneous and cumulative rates, but only the latter measure was increased in the limestone soil. Root growth was stimulated more at 6 cm depth than at 10 cm in the limestone soil. Turnover was faster under elevated CO2 in the peat soil, but there was only a small effect on turnover in the limestone soil. Elevated CO2 reduced nitrogen concentration in roots and might have increased mycorrhizal colonization. Respiration rate was correlated with N concentration, and was therefore lower in roots grown at elevated CO2 . Estimates of the C budget of the two communities, based upon root production and on net C uptake, suggest that C sequestration in the peat soil increases by c. 0.2 kg C m -2 yr-1 (= 2 t ha yr-1 ) under elevated CO2 .

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.

Comparison of UW solution and St. Thomas' solution in the rat: importance of potassium concentration.

BACKGROUND: University of Wisconsin solution (UW) is in limited clinical use for heart transplantation, but there are doubts about its efficacy and concerns about the effect of its high K+ concentration on endothelium. St. Thomas' solution with or without aspartate is widely used and is of proven efficacy. METHODS: Using a modified (starch-free) variant of UW (MUW) we studied: (1) recovery of function with UW compared with aspartate-containing St. Thomas' solution; (2) effect of elevation of K+ in St. Thomas' solution to the level in UW; and (3) effect of reduction of K+ in UW and addition of Ca2+ or aspartate. Isolated rat hearts underwent 7 hours of arrest at 1 degrees C using MUW with or without 20 mmol/L aspartate or using aspartate-containing St. Thomas' solution. RESULTS: Functional recovery with MUW (51.8% +/- 2.5%) was superior to that with aspartate-containing St. Thomas' solution (37.1% +/- 4.3%; p < 0.01). Addition of aspartate to MUW had no effect. During 6 hours of arrest, lowering the K+ in MUW from 125 mmol/L to 20 mmol/L reduced functional recovery from 59.9% +/- 4.2% to 42.3% +/- 4.3% (p < 0.01). The addition of 1 mmol/L Ca2+ had no effect. Elevation of K+ in St. Thomas' solution produced more rapid arrest but no improvement in recovery. CONCLUSIONS: The protective effect of starch-free UW is greater (+13%) than that of aspartate-enriched St. Thomas' solution. Reduction of K+ in UW to lessen possible deleterious effects would decrease its protective effect by about 30% to a level comparable with that of St. Thomas' solution.

Endothelin-1 receptor density, distribution, and function in human isolated asthmatic airways.

The potent bronchoconstrictor and mitogenic actions of the peptide endothelin-1 (ET-1) on airway smooth muscle may contribute significantly to the bronchial obstruction observed in asthma. However, the status of the receptor-effector systems that mediate these actions of ET-1 in asthmatic airways is currently unknown. Thus, we have used quantitative autoradiographic and isometric-tension recording techniques to evaluate the density, distribution, and function of the specific receptors that mediate the actions of ET-1 in both asthmatic and nonasthmatic airways. Here, we report that similar numbers of specific binding sites for [125I]-ET-1 exist in asthmatic and nonasthmatic airways, with the greatest densities located in airway smooth muscle in both tissue types. The ETB-receptor subtype constituted approximately 82% and 88% of these receptors for ET-1 in asthmatic and nonasthmatic human bronchial smooth muscle, respectively, and mediated contraction in response to this peptide. In addition, a component of ET-1-induced contraction appeared to be mediated by a non-ETB, BQ-123-resistant mechanism. Furthermore, a small population of ETA sites was identified that did not mediate contraction, but which may have a role in ET-1-induced prostanoid release and airway smooth-muscle proliferation. Interestingly, bronchial smooth muscle from asthmatic lung was significantly less sensitive to the contractile effects of ETB receptor activation, consistent with desensitization of this receptor subtype in response to the increased production and release of ET-1 that occurs in this disease.

Predominance of endothelinA (ETA) receptors in ovine airway smooth muscle and their mediation of ET-1-induced contraction.

1. Autoradiographic studies were conducted to investigate the receptor subtypes for endothelin-1 (ET-1) that were present in the ovine respiratory tract. In addition, the receptor subtypes mediating contraction of airway smooth muscle and the possible involvement of extracellular Ca2+ and inositol phosphate generation in intracellular signal transduction were assessed. 2. Specific [125I]-ET-1 binding in ovine trachea increased in a time- and concentration-dependent manner. Autoradiographic studies demonstrated that significant binding was associated with airway smooth muscle, although higher densities of specific binding were associated with submucosal glands and with cells immediately below the epithelial basement membrane (lamina propria). The ETA receptor-selective antagonist, BQ 123 (1 microM), virtually abolished specific binding to airway smooth muscle. Quantitative analyses of autoradiographic data describing the time-dependence of specific [125I]-ET-1 binding in ovine airway smooth muscle in the presence and absence of BQ 123 or sarafotoxin S6c, revealed a homogeneous population of ETA receptors. BQ 123 (1 microM) also abolished specific binding to structures associated with submucosal glands, whereas the ETB receptor selective agonist, sarafotoxin S6c (100 nM) had little effect on this binding, indicating the predominance of ETA receptors at these sites. In contrast, ETB receptors predominated in the lamina propria, since sarafotoxin S6c abolished specific binding in this tissue. 3. High levels of specific [125I]-ET-1 binding were also detected in the alveoli and in the walls of blood vessels and small airways in ovine peripheral lung. Specific binding associated with alveoli was reduced to similar extents by BQ 123 (1 MicroM; 54%) and sarafotoxin S6c (100 nM; 40%), suggesting the coexistence of both ETA and ETB receptors in approximately equal proportions in this tissue. In contrast,specific binding to blood vessels and to peripheral bronchial smooth muscle was abolished in the presence of BQ 123 (1 MicroM), but was unaffected by sarafotoxin S6c, indicating the presence of only ETA receptors at these sites.4. ET-1 caused concentration-dependent contractions of ovine tracheal smooth muscle which were inhibited in the presence of BQ 123 (1 MicroM). ET-1 also caused concentration-dependent contraction of ovine lung parenchyma strips. In contrast, the ETB receptor-selective agonists, sarafotoxin S6c and BQ 3020, were virtually inactive as spasmogens in both tracheal smooth muscle and lung strip preparations.Thus contraction was mediated by ETA receptors in ovine tracheal smooth muscle and this is consistent with binding and autoradiographic data demonstrating a homogeneous population of these binding sites in this tissue. Contraction of parenchymal lung strip preparations to ET-1 was mediated via non-ETB receptors, presumably ETA receptors, with contributions to this response perhaps coming from airway and vascular smooth muscle and from alveolar wall contractile cells.5. ET-1-induced contraction of tracheal smooth muscle was not significantly altered in the presence of indomethacin (5 MicroM), indicating that cyclo-oxygenase metabolites of arachidonic acid were not involved in this response. Contraction induced by ET-1 was virtually abolished in Ca2+-free medium containing 0.1 mM EGTA, indicating that this response was dependent upon the influx of extracellular Ca2 .Contraction was inhibited by about 50% in the presence of nicardipine (1 MicroM), indicating that a significant component of this response was mediated via the activation of L-type Ca2+ channels.6. ET-1 caused poorly defined increases in the accumulation of intracellular inositol phosphates in ovine tracheal smooth muscle. The maximal response to ET-1 was less than 20% of that to the cholinoceptor agonist, carbachol. Furthermore, sarafotoxin S6c was inactive. These data, when taken together with the results of autoradiographic and contraction studies, indicate that ovine airway smooth muscle contraction in response to ET-1 is mediated via ETA receptors which are linked to the influx of extracellular Ca2+, partly through voltage-dependent channels. ETB receptors also exist in the lamina propria of ovine trachea and in peripheral alveoli, perhaps residing in vascular endothelial cells.

Exogenously administered alpha-bungarotoxin binds to embryonic chick spinal cord: implications for the toxin-induced arrest of naturally occurring motoneuron death.

Administration of alpha-bungarotoxin and other curare-like drugs during embryogenesis arrests motoneuron death which normally occurs in the spinal cord from day 6 to day 10 of embryogenesis. The accepted explanation is that such motoneuron rescue is mediated by inhibition of neuromuscular transmission following the blockade of nicotinic cholinoceptors at the neuromuscular junction. In this study we investigated a further possibility, namely that motoneuron rescue might also involve the blockade of alpha-bungarotoxin-sensitive sites within the spinal cord. The kinetic profile of [125I]alpha-bungarotoxin binding was examined in the brachial and lumbar regions of chick spinal cord at embryonic day 15. Binding was specific and apparently saturable within the range 1-34 nM reaching a maximum after 45 min. Specific binding involved a single class of non-interacting sites with a KD of 8.0 nM and a Bmax of 106 +/- 12 fmol/mg of protein. Nicotine displaced specific [125I]alpha-bungarotoxin binding in a concentration-dependent manner. Furthermore, specific binding dissociated slowly in the absence of nicotine. Autoradiographs localizing [125I]alpha-bungarotoxin binding in embryonic spinal cord revealed that, at embryonic day 15, specific toxin binding sites could be detected throughout the gray matter. In contrast, at embryonic day 6, the ventral horn contained the majority of specific binding sites. Exogenously administered [125I]alpha-bungarotoxin reached and bound to nicotine-sensitive sites in the spinal cord at embryonic day 7. To conclude, these data demonstrate that central nicotine-sensitive sites which bind [125I]alpha-bungarotoxin in a saturable and specific manner were present at the beginning of the critical motoneuron death phase of neurogenesis and that they were accessible to exogenously administered toxin. It is proposed that the [125I]alpha-bungarotoxin binding characterized here is to a class of putative alpha-bungarotoxin-sensitive nicotinic cholinoceptors. These studies raise the possibility that alpha-bungarotoxin blockade of such putative nicotinic cholinoceptors within the spinal cord may contribute to toxin-induced arrest of naturally occurring motoneuron death.

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.

Relationship between endothelin-1 binding site densities and constrictor activities in human and animal airway smooth muscle.

1. Endothelin-1 (ET-1) binding site densities and constrictor activities were compared in airway smooth muscle preparations of human, guinea-pig, rat and mouse. 2. The mean contractile response to 0.3 microM ET-1 (measured as the % maximum response to 10 microM carbachol, % Cmax +/- s.e.mean) and the mean concentration of ET-1 producing 30% Cmax (95% confidence limits) were respectively; 85.9 +/- 5.4% and 3.4 nM (2.4-5.0) for mouse trachea (n = 11), 88.8 +/- 4.7% and 18.2 nM (11.2-25.2) for rat trachea (n = 6), 71.0 +/- 7.1% and 35.2 nM (5.4-231) for human bronchus (n = 3), and 32.3 +/- 3.0% and 241 nM (125-460) for guinea-pig trachea (n = 6). 3. Light microscopic autoradiography revealed specific [125I]-ET-1 binding sites localized to the smooth muscle band, with very low levels of binding associated with cartilage, submucosal and epithelial cells. 4. Quantitative autoradiographic analyses of the concentration-dependence of specific [125I]-ET-1 binding (0.1-2 nM) to smooth muscle revealed similar dissociation constants but markedly different specific binding site densities for the various animal species. The order of densities of specific [125I]-ET-1 binding sites was rat trachea (69.0 +/- 11.2 amol mm-2) greater than human bronchus (42.7 +/- 17.5 amol mm-2) greater than mouse trachea (28.7 +/- 2.6 amol mm-2) greater than guinea-pig trachea (8.3 +/- 1.8 amol mm-2). 5. A positive relationship between [125I]-ET-1 binding site density and ET-1 constrictor activity was observed in airway smooth muscle preparations from rat, human and guinea-pig. The greater sensitivity of mouse trachea to the constrictor actions of ET-1 was not dependent on the release of cyclo-oxygenaseor epithelium-derived constrictor substances, but may have been due to an inter-species difference in the receptor-effector system for ET-1.