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D W Hay

Publications and source records attributed to D W Hay.

At least 55 records · Page 3Linked to original sources
PubMed1997

Characterization of NK3 receptors in rabbit isolated iris sphincter muscle.

1. Tachykinin NK3 receptors were characterized in the rabbit isolated iris sphincter muscle by use of autoradiography and in vitro functional studies. 2. [125I]-[MePhe7]-neurokinin B (NKB) (1nM), a selective NK3 receptor agonist, specifically labelled a population of NK3 receptors that were uniformly distributed throughout the rabbit iris sphincter muscle. This labelling was inhibited by unlabelled [MePhe7]-NKB (1 microM) but not by the NK1 receptor antagonist CP 99994 (1 microM). 3. In the presence of CP 99994 (1 microM), the selective NK3 receptor agonists senktide (n = 14) and [Pro7]-NKB (n = 4), and the natural preferred ligand for the NK3 receptor, NKB (n = 8), were potent contractile agents in the rabbit iris sphincter muscle. They all produced monophasic concentration-effect curves with pD2 values of 9.53 +/- 0.08, 8.56 +/- 0.09 and 9.75 +/- 0.09, and nH values of 0.93 +/- 0.03, 1.53 +/- 0.17 and 0.76 +/- 0.06, respectively. [MePhe7]-NKB (n = 12) was also a potent agonist, but produced shallow concentration-effect curves which appeared biphasic (nH = 0.45 +/- 0.04). 4. Contractile responses to senktide were surmountably antagonized in a concentration-dependent manner by the selective non-peptide NK3 receptor antagonist, SR 142801 (3-30 nM; pA2 = 8.9; slope = 0.99) and the non-peptide NK2/NK3 receptor antagonist, SR 48968 (3-30 microM; pA2 = 6.1; slope = 1.5). These pA2 values were consistent with functional rabbit NK3 receptors more closely resembling guinea-pig and human NK3 receptors, than rat NK3 receptors. SR 142801 (10-100 nM) and SR 48968 (3 and 30 microM) inhibited responses to low (< or = 1 nM) but not higher (> 1 nM) concentrations of [MePhe7]-NKB, and concentration-effect curves to [MePhe7]-NKb became steeper and monophasic in the presence of either antagonist. 5. SR 142801 (3-30 nM) and SR 48968 (3-30 microM) also surmountably antagonized concentration-effect curves to [Pro7]-NKB and NKB, although results were more difficult to interpret, since the relationship between log concentration-ratios and the concentration of antagonist used did not adhere to the Schild equation. However, analysis of data with the lowest concentration of SR 142801 (3 nM) tested against NKB, and SR 48968 (3 microM) tested against [Pro7]-NKB and NKB, yielded apparent pA2 estimates of 9.3, 6.8 and 6.4, respectively, consistent with blockade of NK3 receptors. 6. SR 142801 (100 nM) had no effect on contractions induced by transmural nerve stimulation (2 Hz, 0.3 ms, 20 V for 30 s), whereas CP 99994 (1 microM) abolished these responses. 7. Phenoxybenzamine pretreatment (20 microM, 10 min) markedly reduced maximum responses to [MePhe7]-NKB (from 101 +/- 6.2% to 38 +/- 9.5% reference contraction, n = 4) and induced a marked (10 fold) rightward shift in the concentration-effect curve. The residual responses to [MePhe7]-NKB after phenoxybenzamine pretreatment were unaffected by 1 microM CP 99994 (maximum response = 41 +/- 9.4%, n = 4). 8. These results demonstrate autoradiographically and functionally, the presence of NK3 receptors in rabbit iris sphincter muscle that mediate contractile responses to NK3 receptor agonists, but not to sensory trigeminal nerve simulation. The present data with senktide and selective NK3 receptor antagonists suggest that functional rabbit NK3 receptors more closely resemble human and guinea-pig NK3 receptors than rat NK3 receptors. However, the pharmacological profiles of [MePhe7]-NKB, SR 142801 and SR 48968 suggest the presence of an 'atypical' NK3 receptor or a heterogeneous population of NK3 receptors in this tissue.

Adrenergic alpha-Antagonists↗
PubMed1997

In vitro and in vivo characterization of NK3 receptors in the rabbit eye by use of selective non-peptide NK3 receptor antagonists.

1. Inhibition of NK3 receptor agonist-induced contraction in the rabbit isolated iris sphincter muscle was used to assess the in vitro functional activity of three 2-phenyl-4-quinolinecarboxamides, members of a novel class of potent and selective non-peptide NK3 receptor antagonists. In addition, an in vivo correlate of this in vitro response, namely NK3 receptor agonist-induced miosis in conscious rabbits, was characterized with some of these antagonists. 2. In vitro senktide (succinyl-[Asp9,MePhe8]-substance P (6-11) and [MePhe7]-neurokinin B ([MePhe7]-NKB) were potent contractile agents in the rabbit iris sphincter muscle but exhibited quite different profiles. Senktide produced monophasic log concentration-effect curves with a mean pD2=9.03+/-0.06 and mean nH=1.2+/-0.02 (n=14). In contrast, [MePhe7]-NKB produced shallow log concentration-effect curves which often appeared biphasic (nH=0.54+/-0.04, n=8), preventing the accurate determination of pD2 values. 3. The contractile responses to the NK3 receptor agonist senktide were antagonized in a surmountable and concentration-dependent manner by SB 223412 ((-)-(S)-N-(alpha-ethylbenzyl)-3-hydroxy-2-phenylquinoline-4-ca rboxamide; 3-30 nM, pA2=8.4, slope=1.8+/-0.3, n=4). SB 222200 ((-)-(S)-N-(alpha-ethylbenzyl)-3-methyl-2-phenylquinoline-4-car box amide; 30-300 nM, pA2=7.9, slope=1.4+/-0.06, n=4) and SB 218795 ((-)-(R)-N-(alpha-methoxycarbonylbenzyl)-2-phenylquinoline-4-carboxamide; 0.3 and 3 microM apparent pKB=7.4+/-0.06, n=6). 4. Contractile responses to the NK3 receptor agonist [MePhe7]-NKB in the rabbit iris sphincter muscle were unaffected by SB 218795 (0.3 and 3 microM, n=8). In contrast, SB 223412 (30 and 300 microM n=4) and SB 222200 (0.3 and 3 microM, n=4) inhibited responses to low concentrations (< or = 1 nM), to a greater extent than higher concentrations (> 1 nM) of [MePhe7]-NKB. Furthermore, log concentration-effect curves to [MePhe7]-NKB became steeper and monophasic in the presence of each antagonist. 5. SB 218795 (3 microM, n=4) had no effect on contractions induced by transmural nerve stimulation (2 Hz) or substance P, exemplifying the selectivity of this class of antagonist for functional NK3 receptors over NK1 receptors in the rabbit. 6. In vivo, senktide (1, 10 and 25 microg i.v., i.e. 1.2, 11.9 and 29.7 nmol, respectively) induced concentration-dependent bilateral miosis in conscious rabbits (maximum pupillary constriction=4.25+/-0.25 mm; basal pupillary diameter 7.75+/-0.48 mm; n=4). The onset of miosis was within 2-5 min of application of senktide and responses lasted up to 30 min. Responses to two i.v. administrations of 25 microg senktide given 30 min apart revealed no evidence of tachyphylaxis. Topical administration of atropine (1%) to the eye enhanced pupillary responses to 25 microg senktide. This was probably due to the mydriatic effect of atropine since it significantly increased baseline pupillary diameter from 7.0+/-0.4 mm to 9.0+/-0.7 mm (n=4), thereby increasing the maximum capacity for miosis. Senktide-induced miosis was inhibited by SB 222200 (1 and 2 mg kg[-1], i.v., i.e. 2.63 and 5.26 micromol kg[-1]; maximum inhibition 100%; n=3-4), SB 223412 (0.5 and 1 mg kg[-1], i.v., i.e. 1.31 and 2.61 micromol kg[-1]; maximum inhibition 100%; n=3), SB 218795 (0.5 and 1 mg kg[-1] i.v., i.e. 1.26 and 2.52 micromol kg-1; maximum inhibition 78%; n=3), and the structurally distinct NK3 receptor antagonist SR 142801 ((S)-(N)-(1-(3-(1-benzoyl-3-(3,4-dichlorophenyl)piperidin-3-yl)propyl)-4-phenylepipiperidin-4-yl)-N-methylacetamide; 1.5mg kg-1, i.v., i.e. 2.47micromol kg-1, maximum inhibition 92%; n=3). 7. Topical administration of senktide (25microg; 29.7nmol) to the eye induced unilateral miosis in the treated eye only. At this dose there was no significant difference (P<0.05) between pupillary constriction obtained by topical or i.v. senktide, and topically administered atropine had no significant effect on responses to topical senktide (n=4). 8. [MePhe7]-NKB (125, 250 and 500microg, i.v., i.e. 98.31, 196.62 and 393.24nmol, respectively) also induced bilateral miosis in conscious rabbits (maximum pupillary constriction=4.13+/-0.30mm; n=4), but in contrast to in vitro studies this agonist was approximately 100 fold less potent than senktide. [MePhe7]-NKB-induced miosis was inhibited by SB 222200 (5mg kg-1, i.v., i.e. 13.14micromol kg-1; maximum inhibition 69%; n=3). 9. In summary, SB 223412, SB 222200 and SB 218795 are potent and selective antagonists of NK3 receptor-mediated contraction in the rabbit isolated iris sphincter muscle. In addition, NK3 receptor agonist-induced miosis in conscious rabbits is a good in vivo correlate of the in vitro rabbit iris sphincter muscle preparation and appears to be a useful model for characterizing the pharmacodynamic profile and efficacy of structurally distinct NK3 receptor antagonists, such as SB 222200, SB 223412, SB 218795 and SR 142801.

Animals↗
PubMed1997

Hypoxia-induced pulmonary hypertension in an optimized environment for the guineapig.

Prolonged exposure to hypoxia elicits a variety of time-related morphologic and physiologic changes in the pulmonary vasculature of mammals, including humans. The study of hypoxia-induced changes in rodents generally requires a prolonged exposure to 9% oxygen for a minimum of 10 days in an airtight chamber, which has only been generally described in the literature as large (200-400 l), sealed acrylic chambers. To assist in the search for better therapies for diseases associated with chronic hypoxia using animal models, we have custom-built an airtight chamber for hypoxic exposure of rodents, and characterized the effect of chronic hypoxia on functional and morphologic changes in the pulmonary vasculature of the guineapig using this system. This chamber has been designed to alleviate any unnecessary stress related to food or water intake, cleanliness and excess illumination to the animals during the hypoxic-exposure period. Chronic exposure of the guineapig to hypoxia (0-21 days) produced time-related physiologic, morphologic, and haematologic changes. For example, after 10 days in hypoxia (9% oxygen), pulmonary artery pressure was significantly increased from 13 +/- 1 mmHg in normoxic controls (day 0, n = 6) to 26 +/- 0 mmHg (day 10, n = 4, P < 0.01). Right ventricular hypertrophy in hypoxic animals, presented as a ratio of right ventricle free wall weight to body weight, showed a significant increase from 0.054 +/- 0.004 (day 0) to 0.069 +/- 0.004 on day 10 (P < 0.05), while age-matched normoxic animals showed no changes in right ventricular weight (day 0 = 0.059, day 10 = 0.058; P > 0.05). Red blood cell count significantly increased over the same time period, from 5.9 +/- 0.1 (day 0) to 6.4 +/- 0.1 (day 10, P < 0.05), as did haematocrit, 48 +/- 0.7 (day 0) to 61 +/- 0.9 (day 10, P < 0.05), and haemoglobin, 16 +/- 0.2 (day 0) to 20 +/- 0.1 (day 10, P < 0.05). It is concluded that considerations for the well-being of the test animals (i.e. continuous water, ample food supplies, burrow-like hiding places, sanitation and protection from excess illumination) can easily be incorporated into a hypoxic chamber. The purpose of the present study was to explore modifications that may provide the animal with an optimized environment which will reduce anxiety and stress, as seen in their behaviour when inside the chambers, and to thoroughly characterize the morphologic and physiologic changes associated with chronic hypoxia which develop in a consistent time-related manner.

Animals↗
PubMed1997

Pharmacology of leukotriene receptor antagonists. More than inhibitors of bronchoconstriction.

The cysteinyl leukotrienes (LTs) are chemical mediators that are thought to contribute to the pathophysiologic condition of asthma and other inflammatory diseases. The biological effects of the cysteinyl LTs in the lung are pleiotropic, including both bronchoconstrictor and a growing list of nonbronchoconstrictor activities that extend to inflammatory cell recruitment, vascular leakage, mucus production, neuronal dysfunction, and airways remodeling. This spectrum of effects of cysteinyl LTs is consistent with an expanded view of asthma that extends beyond simply bronchoconstriction and inflammation. Consequently, the clinical efficacy of cysteinyl LT receptor antagonists (LTRAs) in asthma may be related to antagonism of more than cysteinyl LT-induced bronchoconstriction. The relationship of antagonism of the multiple effects of cysteinyl LTs by cysteinyl LTRAs to their utility in the therapy of asthma is addressed, and the preclinical and clinical pharmacology of cysteinyl LTRAs is reviewed.

Animals↗
PubMed1997

Nonpeptide endothelin receptor antagonists. VIII: attentuation of acute hypoxia-induced pulmonary hypertension in the dog.

It has been proposed that endothelin-1 (ET-1), a potent endogenous vasoactive peptide, may play an important role in the regulation of pulmonary blood flow. The purpose of the present study was to characterize the effects of ET-1 and a nonpeptide mixed ET(A) and ET(B) receptor antagonist, SB 209670, in isolated segments of the canine pulmonary artery and to examine the effects of SB 209670 in a canine model of acute hypoxia-induced pulmonary hypertension. In isolated segments of the pulmonary artery, SB 209670 (3-300 nM) produced a concentration-dependent antagonism of contraction elicited by ET-1 (pA2 = 8.9; slope = 0.9) and had no effect on phenylephrine responses. In addition, SB 209670 antagonized the small, endothelium-dependent relaxation induced by sarafotoxin 6c in phenylephrine (10 microM)-precontracted vessels (pKB = 8.6). In anesthetized dogs, the driving pressure across the pulmonary circulation increased approximately 100% during the hypoxic period (area under the curve [AUC] = 267.1 +/- 25.3 mm Hg x min). SB 209670 treatment (3 and 30 microg/kg/min i.v.) reduced pulmonary vascular resistance and produced a profound dose-related inhibition of hypoxia-induced pulmonary hypertension (AUC = 158.3 +/- 22.7 mm Hg x min and 50.1 +/- 4.9 mm Hg x min, respectively). None of the other hemodynamic or arterial blood gas parameters differed significantly in the vehicle and treatment groups. In addition, SB 209670 produced a significant reversal of hypoxia-induced pulmonary hypertension (AUC = 267.1 +/- 25.3 mm Hg x min vs. 167.8 +/- 23.4 mm Hg x min) when administered at the plateau of the hypoxic response. It was found that SB 209670 administration significantly elevated plasma levels of ET-1-LI (> or = 25-fold). These results suggest that ET-1 is an important mediator of hypoxia-induced pulmonary hypertension in the dog and that SB 209670, a potent and selective mixed ET(A) and ET(B)receptor antagonist in the pulmonary circulation, may represent an important therapeutic approach to the treatment of pulmonary hypertension.

Animals↗
PubMed1997

Nonpeptide endothelin receptor antagonists. IX. Characterization of endothelin receptors in guinea pig bronchus with SB 209670 and other endothelin receptor antagonists.

In this study the endothelin (ET) receptors mediating contractions produced by ET-1, ET-3 and the selective ET(B) ligands sarafotoxin 6c (S6c) and BQ-3020 in guinea pig bronchus were investigated using SB 209670, a nonpeptide, mixed ET(A)/ET(B) receptor antagonist, and the peptide ET receptor antagonists BQ-123 (ET(A) receptor-selective), BQ-788 (ET(B) receptor-selective) and RES-701 (ET(B) receptor-selective). SB 209670 (10 microM) antagonized concentrations induced by ET-1 (pK(B) = 6.1). In contrast, BQ-788 (10 microM) and BQ-123 (10 microM), either alone or in combination, were without significant effect on ET-1 concentration-response curves. SB 209670 (10 microM) and BQ-788 (10 microM) antagonized S6c concentration-response curves with pKB values of 6.6 and 5.5, respectively, whereas RES-701 (10 microM) and BQ-123 (10 microM) were without effect. SB 209670 (10 microM) was about a 10-fold less potent antagonist of contractions produced by ET-3 (pK(B) = 5.4) than of those elicited by S6c. BQ-788 (10 microM), RES-701 (10 microM) and BQ-123 (10 microM) were without effect on ET-3 concentration-response curves. BQ-788 (10 microM) had similar potencies for inhibition of contractions induced by S6c (pK(B) = 5.8) and BQ-3020 (pK(B) = 6.25). These data indicate that contractions induced by ET-1, ET-3, S6c and BQ-3020 in guinea pig bronchus appear to be mediated predominantly via stimulation of ET(B) receptors. However, these receptors are not very sensitive to the standard ET(B) receptor antagonists BQ-788 and RES-701, which suggests that responses produced by these ligands in this tissue involve activation not of the classical ET(B) receptor, but rather of an atypical ET receptor population. The results also provide additional evidence that the potencies of ET receptor antagonists depend upon the specific ET agonist.

Animals↗
PubMed1997

Nonpeptide tachykinin receptor antagonists: I. Pharmacological and pharmacokinetic characterization of SB 223412, a novel, potent and selective neurokinin-3 receptor antagonist.

The in vitro and in vivo pharmacological profile of SB 223412 [(S)-(-)-N-(alpha-ethylbenzyl)-3-hydroxy-2-phenylquinoline-4-carbo xamide], a novel human NK-3 (hNK-3) receptor antagonist, is described. SB 223412 demonstrated enantioselective affinity for inhibition of [125I][MePhe7]neurokinin B (NKB) binding to membranes of CHO cells expressing the hNK-3 receptor (CHO hNK-3). SB 223412, the (S)-isomer, (Ki = 1.0 nM), has similar affinity as the natural ligand, NKB (Ki = 0.8 nM) and another nonpeptide NK-3 receptor antagonist, SR 142801 (Ki = 1.2 nM). SB 223412 was selective for hNK-3 receptors compared with hNK-1 (>10,000-fold selective) and hNK-2 receptors (>140-fold selective), and selectivity was further demonstrated by its lack of effect, in concentrations up to 1 or 10 microM, in >60 receptor, enzyme and ion channel assays. SB 223412 enantioselectively inhibited the NKB-induced Ca++ mobilization in HEK 293 cells stably expressing the hNK-3 receptor. SB 223412 (10-1,000 nM) produced concentration-dependent rightward shifts in NKB-induced Ca++ mobilization concentration-response curves with a Kb value of 3 nM. In addition, SB 223412 antagonized senktide-induced contraction in the isolated rabbit iris sphincter muscle (Kb = 1.6 nM). In mice, oral administration of SB 223412 produced dose-dependent inhibition of behavioral responses induced by the NK-3 receptor-selective agonist, senktide (ED50 = 12.2 mg/kg). Pharmacokinetic evaluation of SB 223412 in rat and dog indicated low plasma clearance, oral bioavailability and high and sustained plasma concentrations after 4 to 8 mg/kg oral dosages. The preclinical profile of SB 223412 (high affinity, selectivity, reversibility and oral activity) suggests that it will be a useful tool compound to define the physiological and pathophysiological roles of NK-3 receptors.

Animals↗
PubMed1997

Nonpeptide endothelin receptor antagonists. X. Inhibition of endothelin-1- and hypoxia-induced pulmonary pressor responses in the guinea pig by the endothelin receptor antagonist, SB 217242.

This study investigated the effects of the nonpeptide endothelin (ET) receptor antagonist, SB 217242, against ET-1-induced pulmonary pressor responses and in a model of hypoxia-induced pulmonary hypertension in the guinea pig. In guinea pig isolated pulmonary artery rings, SB 217242 (3-300 nM) produced a concentration-dependent inhibition of ET-1-induced contractions, with a pA2 of 8.1. SB 217242 (1 or 3 mg/kg i.v.) elicited a dose-related inhibition of ET-1-induced increases in pulmonary artery and airway insufflation pressure responses in anesthetized guinea pigs. Chronic exposure to hypoxia (9% O2 for 0-14 days) produced a time-dependent increase in mean pulmonary artery pressure. After a 10-day exposure to hypoxia there was about a 100% elevation in pulmonary artery pressure, and right ventricular mass and plasma irET levels increased 3-fold compared with normoxic animals. SB 217242, administered by continuous intraperitoneal infusion via mini osmotic pump (0.36, 3.6 or 10.8 mg/day), significantly reduced (by about 50%) hypoxia-induced pulmonary artery pressure increases at all three doses used. The hypoxia-induced right ventricular hypertrophy was significantly attenuated by the 3.6 and 10.8 mg/day doses. Based on hematocrit, hemoglobin and red blood cell counts, SB 217242 did not affect the normal physiological erythropoietic response to hypoxia. There were no appreciable differences in the maximum contractile effects of ET-1 or the potency of SB 217242 (pKB values, 8.3 and 8.0, respectively) versus ET-1-induced responses in isolated pulmonary arteries from hypoxic versus normoxic guinea pigs. However, there was a marked reduction in endothelium-dependent relaxation of precontracted pulmonary artery isolated from hypoxic compared with normoxic animals. The results of the present study provide further preclinical evidence for a pathophysiological role of ET-1 and the potential therapeutic utility of ET receptor antagonists, such as SB 217242, in pulmonary hypertension.

Animals↗
PubMed1996

Expression and function of voltage-dependent potassium channel genes in human airway smooth muscle.

Patch clamp and RNA-polymerase chain reaction methods were used to determine the expression of voltage-dependent potassium channel currents and mRNAs in human airway smooth muscle cells, and tension measurements were used to examine the functional role of specific potassium channel gene products in human bronchial smooth muscle. RNA from airway smooth muscle tissue revealed the presence of Kv1.2 (11 kilobases (kb)) and Kv1.5 (3.5 and 4.4 kb) transcripts, as well as Kv1.1 mRNA (9.5 kb), which has not previously been reported in smooth muscle; transcripts from other gene families were not detected. RNA-polymerase chain reaction from cultured human myocytes confirmed that the identified transcripts were expressed by smooth muscle cells. The available voltage-dependent potassium current in human airway myocytes was insensitive to charybdotoxin (200 nM) but blocked by 4-aminopyridine. Dendrotoxin (1-300 nM; inhibits Kv1.1 and Kv1.2 channels), charybdotoxin (10 nM to 1 microM; inhibits KCa and Kv1.2 channels), and glybenclamide (0.1-100 microM; inhibits KATP channels) had no effect on resting tone. Conversely, 4-aminopyridine increased resting tension with an EC50 (1.8 mM) equivalent to that observed for current inhibition (1.9 mM). Human airway myocytes express mRNA from several members of the Kv1 gene family; the channel that underlies the predominate voltage-dependent current and the regulation of basal tone appears to be Kv1.5.

Base Sequence↗
PubMed1996

The endothelins in the pulmonary system.

Although initial research interest in the ETs was focused on their cardiovascular effects, it is now clearly established that these peptides have wide-ranging activities in the respiratory track. Importantly, ET-1 is synthesized, stored, released and metabolized in the lung, suggesting that these activities may be relevant to both physiological function and pathophysiological processes in the lung. To the present time, only two ET receptor subtypes have been definitively characterized in the lung, namely ETA and ETB receptors, both of which have been shown to mediate contraction and mitogenesis in airway smooth muscle from humans and animals. However, the possibility that further receptor subtypes might be defined from functional, biochemical, and molecular biological studies cannot be dismissed. It is apparent that the ETs may have a role to play in the pathogenesis of several pulmonary diseases. However, most of the current evidence for this falls short of establishing convincing causal associations. Perhaps the most convincing data relate to pulmonary hypertension. Although it is too early to ascribe a role for the ETs in the pathophysiology of asthma, the preliminary data are intriguing. Thus far, research has centered largely on the bronchoconstrictor effects of ET-1 in vitro and in vivo. In relation to asthma, it is important that future studies should focus increasingly on the effects of the ETs in nerves and inflammatory cells. In addition, the effects of chronic airway exposure to ET-1 on smooth muscle and fibroblast proliferation is an important area for future research. The unequivocal testing of the pathophysiological role of the ETs in asthma requires the clinical evaluation of potent and selective receptor antagonists for the various ET receptor subtypes. It seems likely that these studies will be conducted in the not-too-distant future, as compounds which possess the appropriate pharmacological profile become available for clinical evaluation. Additionally, ECE-1 and ECE-2 present as potentially important targets for therapeutic intervention, although the development of selective nonpeptide inhibitors may be some years away.

Amino Acid Sequence↗
PubMed1996

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.

Amino Acid Sequence↗
PubMed1996

Comparison of endothelin B (ETB) receptors in rabbit isolated pulmonary artery and bronchus.

1. To explore potential differences between endothelin (ET) receptors in airway versus vascular smooth muscle from the same species, the ETB receptors mediating contractions produced by ET-1, ET-3 and the selective ETB ligands, sarafotoxin S6c (S6c) and BQ-3020, in rabbit bronchus and pulmonary artery were investigated by use of peptide and non-peptide ET receptor antagonists. 2. In rabbit pulmonary artery SB 209670 (10 microM), a mixed ETA/ETB receptor antagonist, was a more potent antagonist of contractions produced by S6c (pKB = 7.7; n = 9; P < 0.05), than those elicited by ET-1 (pKB = 6.7; n = 6) or ET-3 (pKB = 6.7; n = 5). BQ-788 (10 microM), an ETB receptor antagonist, inhibited responses produced by ET-3 (pKB = 5.1; n = 8), BQ-3020 (pKB = 5.2; n = 4) or S6c (pKB = 6.2; n = 9; P < 0.05 compared to potency versus ET-3- or BQ-3020-induced contractions), but was without inhibitory effect on ET-1-induced contractions (n = 5). RES-701 (10 microM), another selective ETB receptor antagonist, was without effect on contractions produced by S6c (n = 4) or ET-1 (n = 4), and potentiated ET-3- (n = 5) or BQ-3020-induced responses (n = 4). 3. The combination of BQ-788 (10 microM) and BQ-123 (10 microM), an ETA-selective receptor antagonist, antagonized contractions produced by lower concentrations of ET-1 (1 and 3 nM) in rabbit pulmonary artery, but was without effect on responses elicited by higher concentrations of ET-1 (n = 5). The combination of RES-701 (10 microM) and BQ-123 (10 microM) potentiated responses elicited by ET-1, producing a 3.7 fold shift to the left in the agonist concentration-response curve (n = 5). 4. In rabbit bronchus SB 209670 (3 microM) had similar potency for antagonism of contractions produced by ET-1 (pKB = 6.3; n = 6), ET-3 (pKB = 6.5; n = 6) or S6c (pKB = 6.1; n = 8). BQ-788 (3 microM) was without effect on responses elicited by ET-1, ET-3 or S6c (n = 6) but antagonized BQ-3020-induced contractions (pKB = 6.4; n = 4). RES-701 (3 microM) was without effect on contractions produced by S6c (n = 6) or BQ-3020 (n = 4), and potentiated rather than antagonized ET-1- or ET-3-induced responses (n = 6), reflected by a significant (about 6 fold) shift to the left in ET-1 or ET-3 concentration-response curves. The combination of BQ-788 (3 microM) and BQ-123 (3 microM) was without effect on contractions produced by ET-1 in rabbit bronchus (n = 6). The combination of RES-701 (3 microM) and BQ-123 (3 microM) potentiated responses elicited by ET-1, producing a 5.2 fold shift to the left in the agonist concentration-response curve (n = 5). 5. BQ-123 (3 or 10 microM), an ETA-selective receptor antagonist, was without effect on ET-1, ET-3 or S6c concentration-response curves (n = 3-6) in rabbit pulmonary artery or rabbit bronchus. 6. These data indicate that contractions induced by ET-1, ET-3, S6c and BQ-3020 in rabbit pulmonary artery or rabbit bronchus appear to be mediated predominantly via stimulation of ETB receptors. However, the qualitative and quantitative differences in the relative profiles of the various structurally diverse peptide and non-peptide antagonists examined suggests that responses produced by the ET ligands may not be mediated by a homogeneous ETB receptor population. In addition, the results suggest that differences exist in the ETB receptors mediating contraction in pulmonary vascular versus airway tissues in the same species. These receptors are not very sensitive to the standard ETB receptor antagonists, BQ-788 and RES-701. Furthermore, the results also provide further evidence that the potencies of ET receptor antagonists depend upon the ET agonist.

Animals↗
PubMed1996

Persistent airway eosinophilia after leukotriene (LT) D4 administration in the guinea pig: modulation by the LTD4 receptor antagonist, pranlukast, or an interleukin-5 monoclonal antibody.

Aerosolized cysteinyl leukotrienes (CysLTs) elicit migration of eosinophils into guinea pig lungs and the airways of patients with asthma. The present studies were designed to analyze the concentration-response relationship, time course, and pharmacologic and histologic characteristics of leukotriene D4 (LTD4)-induced eosinophil influx into the airways of conscious guinea pigs. Animals were exposed to aerosols of 0.3 to 30 microg/ml LTD4 for 1 min, during which specific airway conductance (sGaw) was monitored. Bronchoalveolar lavages (BALs) of guinea pig airways were conducted at selected times from 4 h to 4 wk after LTD4 challenge. LTD4 produced maximal decreases in sGaw (70 to 90% reduction) at all concentrations tested and concentration-related increases in eosinophil levels in BALs, assessed 24 h after challenge. Increased numbers of eosinophils in the bronchial epithelium and subepithelium were confirmed histologically. Significant eosinophilia was maintained for up to 4 wk postchallenge. Pretreatment with the LTD4 receptor antagonist, pranlukast (ONO-1078, SB 205312) (20 mg/kg, intragastrically), significantly inhibited both the bronchoconstriction and the eosinophilia at 24 h, whereas the cyclooxygenase inhibitor, meclofenamic acid (5 mg/kg, intragastrically), had no effect on either parameter. Histologic observations were consistent with BAL results. Pretreatment with the rat anti-mouse antibody to interleukin-5 (IL-5), TRFK-5 (10-300 microg, intraperitoneally), produced dose-related inhibition of LTD4-induced eosinophilia, measured in 24 h or 3 wk BAL, but did not affect the acute bronchoconstriction. These results indicate that LTD4 elicits airway eosinophil influx in guinea pigs which persists as long as 4 wk after a single exposure, and provide the first evidence that IL-5 may have a role in LTD4-induced airways inflammation. This and other previously reported proinflammatory effects of LTD4 may contribute significantly to its overall influential role in the pathophysiology of asthma, and may underlie the therapeutic benefit of CysLT receptor antagonists, such as pranlukast, in this disorder.

Animals↗
PubMed1996

Is endothelin-1 a mediator in asthma?

There is a growing understanding of the effects and potential pathophysiological role of the ETs in asthma and several of the criteria required to strongly implicate ET-1 as a mediator in this disease have been fulfilled. However, some of the information is preliminary, equivocal and requires confirmation. Furthermore, the most important and most difficult criteria, the clinical testing of potent and selective ET receptor antagonists in these diseases has not been conducted and will probably not be completed for several years. A critical area of research continues to be the elucidation and classification of the ET receptor subtypes mediating the diverse effects of ET-1 in the lung. The unequivocal determination of the pathophysiological role of the ETs in asthma awaits the results of clinical trials with appropriate compounds. Only after these results will we know whether ET-1 has fulfilled its promise as a key player in lung pathophysiology, rather than being added to the list of purported important mediators with unfulfilled expectations.

Asthma↗
PubMed1996

Nonpeptide endothelin receptor antagonists. VI: Pharmacological characterization of SB 217242, a potent and highly bioavailable endothelin receptor antagonist.

This study describes the pharmacological characterization of SB 217242, a highly potent orally bioavailable nonpeptide antagonist of both endothelin type A (ETA) and endothelin type B (ETB) receptors. In human cloned ETA and ETB receptors, SB 217242 produced concentration-dependent displacement of [125]I-endothelin-1 (ET-1) in both receptor subtypes with Ki values of 1.1 and 111 nM, respectively. SB 217242 produced concentration-dependent, parallel rightward shifts in the ET-1 concentration-response curves in rat isolated aorta and human isolated pulmonary artery (ETA receptor-mediated vascular contraction) with Kb values of 4.4 and 5.0 nM, respectively. SB 217242 was 4-, 62- and 125-fold more potent as an ETA receptor antagonist than the previously reported compounds BQ-123, PD 142893 and Ro 46-2005, respectively. SB 217242 (10 microM) did not produce significant effects against contraction produced by other vasoactive agents. SB 217242 produced concentration-dependent antagonism of responses produced by ETB receptor activation as demonstrated by antagonism of sarafotoxin S6c-mediated contraction in the rabbit isolated pulmonary artery with a Kb value of 352 nM. In vitro cell monolayers of Caco-2 cells had high permeability to SB 217242. In vivo pharmacokinetics in the rat confirmed that SB 217242 was rapidly absorbed from the gastrointestinal tract with a bioavailability of 66%. The SB 217242 plasma half-life in rats after intraduodenal administration was 3.3 hr, with a systemic clearance of 27.3 ml/min/kg. Orally administered SB 217242 (0.3-30 mg/kg) produced dose-dependent inhibition of the pressor response to exogenous ET-1 in conscious rats; with a dose of 30 mg/kg p.o., inhibition was observed for at least 5.5 hr. The present study demonstrates that SB 217242 is a highly potent antagonist of both ETA and ETB receptors. In addition, SB 217242 has high in vitro permeability and high oral bioavailability. SB 217242 represents a new orally active pharmacological tool that should assist in the elucidation of the chronic role of endothelin in pathophysiology.

Animals↗
PubMed1995

Cysteinyl leukotrienes in asthma: old mediators up to new tricks.

The cysteinyl leukotrienes have long been suspected to play a role in the pathogenesis of asthma. This speculation was based largely on their release in human lung following antigen challenge as well as their potent bronchoconstrictor activity. However, there is increasing evidence that the cysteinyl leukotrienes also produce several pro-inflammatory effects and alter the activity of neuronal pathways in the airways. Douglas Hay, Theodore Torphy and Bradley Undem review these recent data and discuss the therapeutic possibilities of cysteinyl leukotriene receptor antagonists and 5-lipoxygenase inhibitors.

Animals↗
PubMed1995

Receptors for endothelin-1 in asthmatic human peripheral lung.

[125I]-endothelin-1 ([125I]-ET-1) binding was assessed by autoradiography in peripheral airway smooth muscle and alveolar wall tissue in human non-asthmatic and asthmatic peripheral lung. Levels of specific binding to these structures were similar in both non-asthmatic and asthmatic lung. The use of the receptor subtype-selective ligands, BQ-123 (ETA) and sarafotoxin S6c (ETB), demonstrated the existence of both ETA and ETB sites in airway smooth muscle and in alveoli. In airway smooth muscle from both sources, the great majority of sites were of the ETB subtype. Quantitative analyses of asthmatic and non-asthmatic alveolar wall tissue demonstrated that 29-32% of specific [125I]-ET-1 binding was to ETA sites and 68-71% was to ETB sites. Thus, asthma was not associated with any significant alteration in the densities of ETA and ETB receptors in peripheral human lung.

Adolescent↗