Mast cell-dependent IL-33/ST2 signaling is protective against the development of airway hyperresponsiveness in a house dust mite mouse model of asthma.

Interleukin-33 (IL-33) and its receptor ST2 have been influentially associated with the pathophysiology of asthma. Due to the divergent roles of IL-33 in regulating mast cell functions, there is a need to further characterize IL-33/ST2-dependent mast cell responses and their significance in the context of asthma. This study aimed to investigate how IL-33/ST2-dependent mast cell responses contribute to the development of airway hyperresponsiveness (AHR) and airway inflammation in a mouse model of house dust mite (HDM)-induced asthma. Mast cell-deficient C57BL/6-KitW-sh (Wsh) mice engrafted with either wild-type (Wsh + MC-WT) or ST2-deficient bone marrow-derived mast cells (Wsh + MC-ST2KO) were exposed to HDM delivered intranasally. An exacerbated development of AHR in response to HDM was seen in Wsh + MC-ST2KO compared with Wsh + MC-WT mice. The contribution of this IL-33/ST2-dependent mast cell response to AHR seems to reside within the smaller airways in the peripheral parts of the lung, as suggested by the isolated yet marked effect on tissue resistance. Considering the absence of a parallel increase in cellular inflammation in bronchoalveolar lavage fluid (BALF) and lung, the aggravated AHR in Wsh + MC-ST2KO mice seems to be independent of cellular inflammation. We observed an association between the elevated AHR and reduced PGE2 levels in BALF. Due to the protective properties of PGE2 in airway responses, it is conceivable that IL-33/ST2-dependent mast cell induction of PGE2 could be responsible for the dampening effect on AHR. In conclusion, we reveal that IL-33/ST2-dependent mast cell responses can have a protective, rather than causative role, in the development of AHR.

Attenuated airway hyperresponsiveness and mucus secretion in HDM-exposed Igf1r-deficient mice.

BACKGROUND: Asthma is a common chronic lung disease characterized by airflow obstruction, airway hyperresponsiveness (AHR), and airway inflammation. IGFs have been reported to play a role in asthma, but little is known about how the insulin-like growth factor 1 receptor (IGF1R) affects asthma pathobiology. METHODS: Female Igf1r-deficient and control mice were intranasally challenged with house dust mite (HDM) extract or PBS five days per week for four weeks. Lung function measurements, and bronchoalveolar lavage fluid (BALF), serum, and lungs were collected on day 28 for further cellular, histological, and molecular analysis. RESULTS: Following HDM exposure, the control mice responded with a marked AHR and airway inflammation. The Igf1r-deficient mice exhibited an increased expression of the IGF system and surfactant genes, which were decreased in a similar manner for control and Igf1r-deficient mice after HDM exposure. On the other hand, the Igf1r-deficient mice exhibited no AHR, and a selective decrease in blood and BALF eosinophils, lung Il13 levels, collagen, and smooth muscle, as well as a significant depletion of goblet cell metaplasia and mucus secretion markers after HDM exposure. The Igf1r-deficient mice displayed a distinctly thinner epithelial layer than control mice, but this was not altered by HDM. CONCLUSIONS: Herein, we demonstrate by the first time that the Igf1r plays an important role in murine asthma, mediating both AHR and mucus secretion after HDM exposure. Thus, our study identifies IGF1R as a potential therapeutic target, not only for asthma but also for hypersecretory airway diseases.

The interleukin-33 receptor ST2 is important for the development of peripheral airway hyperresponsiveness and inflammation in a house dust mite mouse model of asthma.

BACKGROUND: Several clinical and experimental studies have implicated IL-33 and its receptor ST2 in the development of asthma. However, the effect of IL-33/ST2 signalling on airway responses and inflammation in allergic asthma is not well established. OBJECTIVE: To investigate the role of IL-33/ST2 signalling in promoting allergen-induced airway hyperresponsiveness (AHR), airway inflammation, antigen-specific IgE production and mast cell activity in a mouse model of asthma. METHODS: ST2-deficient (ST2(-/-)) mice and control BALB/c mice were given house dust mite (HDM) extract over a 6-week period. Forty-eight hours after the final HDM administration, lung function and airway inflammation were evaluated. Airway responsiveness was determined in the central airways and peripheral lung. Cellular infiltration and mast cell protease mMCP-1 levels were quantified in bronchoalveolar lavage fluid (BALF). Recruitment of inflammatory cells and inflammatory cytokine profiles were assessed in pulmonary tissue, and HDM-specific IgE was measured in serum. RESULTS: ST2 deficiency diminished HDM-induced AHR in the peripheral lung, while AHR in the central airways was unaffected. Inflammatory responses to HDM were also reduced in ST2(-/-) mice as reflected by the lower induction of HDM-specific serum IgE, inhibition of HDM-induced eosinophilia and reduced macrophage count in BALF, and a diminished influx of inflammatory cells and reduced goblet cell hyperplasia around the peripheral airways. Furthermore, the levels of the inflammatory cytokines IL-1ß, IL-5, IL-13, IL-33, GM-CSF, thymic stromal lymphopoietin and mast cell protease mMCP-1 were reduced in HDM-treated ST2(-/-) mice compared with wild-type controls. CONCLUSIONS: In addition to promoting Th2 inflammation, we now suggest a role for the IL-33/ST2 pathway for the induction of peripheral inflammation and mucus production that causes AHR in the peripheral lung. This mechanism for inducing AHR at distal parts of the lung may be of specific importance as asthma is considered as a small airway disease.

Interleukin-33 exacerbates allergic bronchoconstriction in the mice via activation of mast cells.

BACKGROUND: Interleukin-33 (IL-33) is implicated as an epithelium-derived danger signal promoting Th2-dependent responses in asthma. We hypothesized that IL-33 might also have direct effects on mast cell-driven allergic airway obstruction. METHODS: The effects of IL-33 on allergic responses in the airways of sensitized mice were assessed both in vivo and ex vivo, as well as on cultured mast cells in vitro. RESULTS: In vivo, the allergen-induced increase in resistance in the conducting airways was enhanced in mice pretreated with IL-33. Also, in the isolated airways, the allergen-induced contractions were increased in preparations from animals subjected to intranasal IL-33 pretreatment. These effects in vivo and ex vivo were blocked by the 5-HT2A receptor antagonist ketanserin and absent in mice without mast cells. Likewise, the IL-33-induced enhancement of the allergen response was absent in isolated airways from mice lacking the IL-33 receptor. Moreover, exposure to IL-33 increased secretion of serotonin from allergen-challenged isolated airways. In cultured mast cells, IL-33 enhanced the expression of tryptophan hydroxylase 1, serotonin synthesis, and storage, as well as the secretion of serotonin following IgE receptor cross-linking. CONCLUSION: These results demonstrate that IL-33 exacerbates allergic bronchoconstriction by increasing synthesis, storage, and secretion of serotonin from the mast cell. This mechanism has implications for the development of airway obstruction in asthma.

Corticosteroid treatment selectively decreases mast cells in the smooth muscle and epithelium of asthmatic bronchi.

BACKGROUND: Mast cells are important in the pathophysiology of airway inflammation and evidence suggests their sub-localisation within the airway is altered in asthma. Little is known about the effect of corticosteroids on mast cell localisation within the bronchi. METHODS: We therefore performed an immunohistochemical analysis of mast cell numbers within the smooth muscle, epithelium and submucosa of healthy subjects (n = 10) and well-characterised asthmatic patients, using either ß(2)-agonists alone (n = 10) or ß(2)-agonists and inhaled corticosteroids (n = 10). RESULTS: Patients using inhaled corticosteroids displayed significantly lower numbers of mast cells within their epithelium and smooth muscle compared to those not treated with inhaled corticosteroids. Submucosal mast cells were not affected by corticosteroid treatment. Numbers of smooth muscle mast cells correlated with bronchial responsiveness and epithelial mast cells with exhaled NO. CONCLUSION: We demonstrate that glucocorticosteroids differentially affect mast cell numbers within specific airway sub-locations highlighting the importance of mast cell and smooth muscle/epithelial interactions in asthma pathogenesis.

Systemic up-regulation of TLR4 causes lipopolysaccharide-induced augmentation of nasal cytokine release in allergic rhinitis.

BACKGROUND: Allergic rhinitis is a systemic disorder, and it is clinically well recognized that it can be aggravated by infection. Activation of the innate immune system constitutes a critical element in the process. Toll-like receptors (TLRs) comprise a part of the innate immune system, and lipopolysaccharide (LPS)-induced activation of TLR4 represents bacterial-induced interactions in various model systems. The present study examines how TLR2 and TLR4 expression is affected by symptomatic allergic rhinitis, and if LPS added upon allergen affects nasal cytokine release. METHODS: In patients with pollen-induced allergic rhinitis and healthy non-allergic volunteers, nasal lavage (NAL), peripheral blood and bone marrow were sampled before and during the pollen season. TLR2 and TLR4 expression was determined flow cytometrically. Changes in the TLR receptor expression pattern were evaluated by a nasal challenge with allergen followed by LPS, or vice versa. Symptoms along with cells and cytokines in NAL were analyzed. RESULTS: TLR4 expression increased in leukocytes in NAL, peripheral blood and bone marrow during symptomatic allergic rhinitis. A similar increase was seen for TLR2 in neutrophils in blood. Nasal challenge with allergen followed by LPS augmented the release of IL-4, IL-5, IL-10, IL-13, IFN-γ and TNF-α. CONCLUSION: A systemic up-regulation of TLR4 in symptomatic allergic rhinitis may explain why LPS preceded by allergen increases nasal cytokine release.

Nod1, Nod2 and Nalp3 receptors, new potential targets in treatment of allergic rhinitis?

BACKGROUND: Recently, a new set of pattern-recognition receptors, the nucleotide-binding oligomerization domain (Nod)-like receptors (NLRs), have emerged. Their activation, either by allergens or microbes, triggers an inflammatory response. The knowledge about NLRs in human airways is limited. AIM OF THE STUDY: To investigate presence of NLRs in the human nose of healthy individuals and patients with intermittent allergic rhinitis outside and during pollen season. METHODS: The expression of Nod1, Nod2, and Nalp3 in nasal biopsies was determined with real-time RT-PCR and immunohistochemistry. Cultured primary human nasal epithelial cells (HNECs) were analyzed using real-time RT-PCR and flow cytometry to further verify the presence of NLRs in the epithelium. RESULTS: Immunohistochemical analysis revealed presence of Nod1, Nod2, and Nalp3 in the nasal epithelium. This was corroborated in cultured HNECs. Patients suffering from symptomatic allergic rhinitis exhibited lower Nod1 and Nalp3 mRNA levels than both controls and patients during pollen season. Nod2 expression was found in all specimens tested, but no differences were seen between the three groups. CONCLUSION: Nod1, Nod2, and Nalp3 receptors were found to be present in the human nose. The expression of Nod1 and Nalp3 were down-regulated during pollen season among patients with allergic rhinitis. This opens up for new insights and novel therapeutic strategies in inflammatory airway disease.

Leukocyte phenotype changes induced by specific immunotherapy in patients with birch allergy.

BACKGROUND: The underlying mechanisms of allergen-specific immunotherapy (SIT) are not fully understood. OBJECTIVES: The present study aimed to investigate how leukocyte phenotypes are affected by SIT. METHODS: Blood samples were taken from 10 patients with birch pollen--induced allergic rhinitis before, during, and immediately after SIT. Further samples were obtained after 1 year and 3 years. All samples were analyzed by flow cytometry and leukocyte differentiation. RESULTS: SIT caused a decrease in cell-bound immunoglobulin (Ig) E on granulocytes, along with a corresponding increase in the high-affinity IgG receptor. Accordingly, a lower level of allergen-specific IgE was found after 3 years. The treatment induced a decrease in neutrophil CD1 1b levels, a shift in monocyte subsets, and an increase in the number of activated T lymphocytes, manifested as an upregulation of CD69 and CD98, and an expansion of the CD4+CD25+ T-cell pool. CONCLUSION: The present study shows that the clinical effects of SIT are mirrored by systemic changes in cellular events and in antibodies, and offers new targets for immunomodulation.

Dissociation of airway inflammation and hyperresponsiveness by cyclooxygenase inhibition in allergen challenged mice.

The aim of the current study was to define how cyclooxygenase (COX)-activity affects airway hyperresponsiveness (AHR) and inflammation using interventions with COX inhibitors at different time points during allergen challenge and/or prior to measurement of AHR in an eosinophil-driven allergic mouse model. Inflammatory cells were assessed in bronchioalveolar lavage (BAL) and AHR was evaluated as the total lung resistance to methacholine (MCh) challenge. Administration of FR122047 (COX-1 inhibitor) during ovalbumin (OVA) challenge and prior to MCh challenge enhanced AHR without affecting the inflammatory cell response. In contrast, administration of lumiracoxib (COX-2 inhibitor) during the same time period had no effect on AHR but reduced the inflammatory cells in BAL. Nonselective COX inhibition with diclofenac both enhanced the AHR and reduced the inflammatory cells. Administration of diclofenac only during OVA challenge reduced the cells in BAL without any changes in AHR, whereas administration of diclofenac only prior to MCh challenge enhanced AHR but did not affect the cells in BAL. The present study implicates distinct roles of prostanoids generated along the COX-1 and COX-2 pathways and, furthermore, that inflammatory cells in BAL do not change in parallel with AHR. These findings support the fact that AHR and the inflammatory response are distinct and, at least in part, uncoupled events.

Nasal CpG oligodeoxynucleotide administration induces a local inflammatory response in nonallergic individuals.

BACKGROUND: We have previously demonstrated the presence of toll-like receptor 9 in the nasal mucosa of both healthy and allergic individuals. CpG motifs, found in bacterial and viral DNA, elicit strong immunostimulatory effects via this receptor. CpG is known to skew the immune system towards a T helper 1 (Th1) profile, thereby suppressing Th2-driven allergic responses. This study was designed to examine the effects of CpG administration in the human nose. METHODS: Twenty subjects, of whom 10 suffered from seasonal allergic rhinitis (AR), were challenged intranasally with CpG outside pollen season. Symptom scores, nasal airway resistance (NAR), and nasal and pulmonary nitric oxide (NO) levels were assayed prior to challenge and 30 min, 6, 24 and 48 h post challenge. The presence of leukocytes and various cytokines were analyzed in nasal lavage (NAL) fluids before and after CpG exposure. RESULTS: Increased NAR, nasal NO production and secretion of interleukin (IL)-1beta, IL-6, and IL-8 were seen after CpG exposure. Further analysis revealed that this inflammatory response was more marked in healthy subjects than among patients with AR, although a higher basal inflammatory response was recorded in the allergic group. In vitro experiments suggest that the effects induced by CpG are mediated by epithelial cells and neutrophils. CONCLUSION: Nasal administration of CpG induces a local airway inflammation, more distinct among healthy than allergic individuals. The reduced responsiveness to CpG in allergic patients might be related to the ongoing minimal persistent inflammation. Results from cytokine analyses reflect the ability of CpG to induce a pro-inflammatory Th1-like immune response.

Genes regulating molecular and cellular functions in noninfectious nonallergic rhinitis.

BACKGROUND: Chronic noninfectious, nonallergic rhinitis (NINAR) is a complex syndrome with a principally unknown pathophysiology. New technology has made it possible to examine differentially expressed genes and according to network theory, genes connected by their function that might have key roles in the disease. METHODS: Connectivity analysis was used to identify NINAR key genes. mRNA was extracted from nasal biopsies from 12 NINAR patients and 12 healthy volunteers. Microarrays were performed using Affymetrix chips with 54 613 genes. Data were analysed with the Ingenuity Pathway System for organization of genes into annotated biological functions and, thereafter, linking genes into networks due to their connectivity. The regulation of key genes was confirmed with reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: In all, 43 genes were differentially expressed. The functional analysis showed that these genes were primarily involved in cellular movement, haematological system development and immune response. Merging these functions, 10 genes were found to be shared. Network analysis generated three networks and two of these 'shared genes' in key positions, c-fos and cell division cycle 42 (Cdc42). These genes were upregulated in both the array and the RT-PCR analysis. CONCLUSION: Ten genes were found to be of pathophysiological interest for NINAR and of these, c-fos and Cdc42 seemed to be of specific interest due to their ability to interact with other genes of interest within this context. Although the role of c-fos and Cdc42 in upper airway inflammation remains unknown, they might be used as potential disease markers.

Lipopolysaccharide administration to the allergic nose contributes to lower airway inflammation.

BACKGROUND: Allergic rhinitis (AR) is an inflammatory reaction not confined to a single local compartment, but rather involving the whole airway system. Allergens known to induce AR are not always the sole trigger of the inflammatory reaction as infections and organic dust might also cause exacerbations of rhinitis and associated conditions. OBJECTIVE: To examine the effects of intranasal lipopolysaccharide (LPS) exposure, as a surrogate for upper airway bacterial infections, in patients with symptomatic AR. METHODS: Fourteen patients with a history of moderate to severe pollen-induced AR were challenged intranasally with LPS. After 3-6 weeks, the same patients were challenged again, first with allergen and 24 h later with LPS. Nasal symptom scores, nasal lavage leucocyte counts and nasal airway resistance were assessed at 6-24 h after each provocation along with measurements of nitric oxide (NO) levels in the nose and lung. RESULTS: Six hours after the LPS challenge, an increased level of leucocytes could be obtained in the lavage fluid, but no symptoms were experienced and no increase in nasal resistance could be recorded. The NO production in the upper and lower airways was similar before and 6 h after the provocation. In contrast, in patients exposed to pollen before the LPS challenge, both the nasal and the pulmonary NO levels were enhanced. This was accompanied by an increase in leucocytes. CONCLUSION: The present study demonstrates a priming effect of allergen on the nasal response to LPS as well as the presence of a systemic link between airway mucosal sites in the upper and lower airways. This suggests that exogenously derived signals, like upper airway infections, can interfere with the initiation, maintenance and progression of asthma.

Interleukin 33 exacerbates antigen driven airway hyperresponsiveness, inflammation and remodeling in a mouse model of asthma.

Interleukin 33 (IL-33) represents a potential link between the airway epithelium and induction of Th2-type inflammatory responses associated with the development of asthma. This study investigated the potential of IL-33 to exacerbate antigen driven asthma responses. An ovalbumin (OVA) asthma model was used in which sensitized C57BL/6 mice were exposed to IL-33 before each OVA challenge. IL-33 given to sensitized mice acted synergistically with antigen and aggravated airway inflammation, hyperresponsiveness and remodeling compared with mice that were only OVA sensitized and challenged and mice that were only exposed to IL-33. Elevated levels of local and systemic mast cell protease mMCP-1, as well as antigen-specific IgE production, were observed following IL-33 administration to sensitized mice. Similarly, exposing OVA-sensitized mice to IL-33 increased the Th2 cytokine levels, including IL-4, IL-5 and IL-13. Furthermore, IL-33 and OVA administration to OVA-sensitized mice increased ILC2s in the lung, suggesting a role for ILC2s in IL-33-mediated exacerbation of OVA-induced airway responses. Collectively, these findings show that IL-33 aggravates important features of antigen-driven asthma, which may have implications for asthma exacerbations.

Regional variation in appearance of vascular contractile endothelin-B receptors following organ culture.

OBJECTIVE: The aim of this study was to investigate the appearance of contractile endothelin (ET)-B receptors following organ culture in different vascular regions. METHOD: The contractile responses of vascular smooth muscle induced by ET-1 and the selective ETB receptor agonist sarafotoxin 6c (S6c) were investigated in circular segments representing eight vascular regions in the rat (aorta, femoral artery, mesenteric artery, branch of the mesenteric artery, proximal and distal parts of the caudal artery, femoral and mesenteric veins). To allow the ETB receptor to be expressed, the segments were placed in organ culture for 1 to 5 days. Pharmacological characterisation of the ET receptors was performed in mesenteric arterial segments. All contractile responses were measured in percentage of K(+)-induced contraction. RESULTS: ET-1 induced strong concentration-dependent contractions of all fresh (not cultured) segments. S6c had negligible effects on all fresh vessels with the exception of the mesenteric vein, where a small contraction was seen. After 1 day of organ culture all tested segments, with the exception of aorta and the proximal part of the caudal artery, showed concentration-dependent contractile responses to S6c which were further augmented after 5 days of culture. The ET-1-induced responses were only slightly affected by organ culture. Contractions induced by S6c were more enhanced in small arteries and veins than in larger arteries. Furthermore, the S6c-induced response was more pronounced in the mesenteric region as compared to the hindlimb. In fresh mesenteric arterial segments FR139317 (ETA receptor antagonist) and bosentan (ETA/ETB receptor antagonist) but not IRL 2500 (ETB receptor antagonist) shifted the ET-1-induced concentration-response curve in parallel to the right. In contrast, after organ culture the S6c-induced concentration-response curves were shifted parallel to the right in the following potency order: IRL 2500 > bosentan > FR139317. CONCLUSION: During normal conditions, the ETA receptor is the dominating mediator of endothelin-induced contraction in eight different vascular regions. Furthermore, this study indicates that most of the vessels have the ability to develop contractile ETB receptors and that this plasticity differs in vascular regions.

Characterization of endothelin receptors in the cerebral vasculature and their lack of effect on spreading depression.

The changes in cerebral blood flow that accompany spreading depression are well-described, as are parallel changes in cellular activity, with a wave of hyperemia followed by a prolonged oligemic phase. In this study, a cat model of the CBF changes associated with spreading depression and in vitro pharmacology were used to determine if there is a role for the powerful peptide vasoconstrictor endothelin in this response. For the pharmacological studies, the middle cerebral artery was harvested from cats postmortem. For the physiological studies, cats were anesthetized with halothane induction and alpha-chloralose (60 mg/kg, intraperitoneal loading; 20 mg/kg i.v. 2-h maintenance). CBF was monitored continuously in the parietal cortex using laser Doppler flowmetry (CBFLDF) after exposure of the dura mater. The in vitro work demonstrated that endothelin-1 (ET-1) mediates a strong and potent contraction of cerebral vessels. Both the selective ETA receptor antagonist FR139317 and the combined ETA and ETB receptor antagonist Bosentan caused a rightward shift of the concentration-response curve without attenuation of the maximum effect. The calculated pA2 values were 6.28 and 6.90, respectively. The slope did not differ from unity, suggesting that the ET-1-mediated contraction is evoked by a single population of ETA receptors, which were effectively antagonized by these compounds. Spreading depression was induced with a needle stick injury to the cortex. Local administration of the endothelin antagonists FR139317 (10 microM) and Bosentan (10 microM) did not affect resting blood flow in the cortex. Induction of spreading depression following local administration of FR139317 and Bosentan resulted in responses no different from those in control cortex. These data demonstrate that endothelin does not play a significant role in the vasoconstrictor portion of the CBF change seen in spreading depression, nor does it affect resting flow. Since it is widely held that spreading depression, or a very similar mechanism, underlies the aura phase of migraine, it may be suggested from these studies that endothelin antagonists are unlikely to be useful in migraine.

Evidence that ET-1, but not ET-3 and S6b, ET(A)-receptor mediated contractions in isolated rat mesenteric arteries are modulated by co-activation of ET(B) receptors.

The effects of agonists with endothelin (ET) ET(A)-receptor activity have been analysed in relation to their interaction with ET(B) receptors in rat mesenteric arteries. ET-1, sarafotoxin 6b (S6b) and ET-3 induced large, slow-onset and sustained contractions whereas S6c induced weak transient contractions. However, following pre-contraction with U46619 and subsequent relaxation with forskolin, the effect of S6c was amplified, indicating a potential for powerful ET(B)-receptor mediated contraction. The selective ET(A)-receptor antagonist, FR139317, produced parallel rightward shifts of ET-1, S6b and ET-3 concentration-effect curves indicating that the contractions were mediated by ET(A) receptors. However, the corresponding FR139317 pK(B) values were significantly different between the agonists. As expected FR139317 had no effect on S6c responses. Pre-treatment with S6c to desensitize ET(B) receptors, increased ET-1 potency and the pK(B) value for FR139317. In contrast, neither the potency of S6b and ET-3 nor the pK(B) values for FR139317 estimated using these agonists were affected by ET(B)-receptor desensitization. Segments pre-contracted with submaximal concentrations of S6b and ET-3, but not ET-1, rapidly relaxed following wash-out or FR139317 administration. The results indicate that the small contractile response to selective ET(B) receptor activation, barely detectable under standard bioassay conditions, is greatly amplified when adenylate cyclase activity is elevated. Moreover, the response to ET(A) receptor activation by ET-1, but not ET-3 and S6b, is significantly modified by co-activation of ET(B) receptors. This interaction has a significant effect on the apparent affinity of ET(A)-receptor selective antagonists when ET-1 is used as agonist and decreases the potency of ET-1.

Effects of selective ETB-receptor stimulation on arterial, venous and capillary functions in cat skeletal muscle.

1. This paper describes, in quantitative terms, the in vivo effects of two selective ETB-receptor agonists (IRL 1620 and BQ 3020) on vascular resistance (tone) in the following consecutive sections of the vascular bed of sympathectomized cat skeletal muscle: large-bore arterial resistance vessels (> 25 microns), small arterioles (< 25 microns) and the veins. The effects on capillary pressure transcapillary fluid exchange were also recorded. 2. Both IRL 1620 and BQ 3020, infused i.a. to the muscle preparation, evoked an initial transient dilator response followed by a moderate dose-dependent constrictor response, both being preferentially confined to the small arterioles. The dilator response was associated with a transient increase, and the constrictor response with a sustained decrease, in capillary pressure, the latter causing net transcapillary fluid absorption. The capillary filtration coefficient decreased during the constrictor response, indicating constriction of terminal arterioles/precapillary sphincters. 3. The vascular responses to the ETB-receptor agonists were unaffected by blockade of endothelium-derived nitric oxide (NG-nitro-L-arginine methyl ester) and by selective ETA-receptor blockade (FR139317). However, blockade of prostacyclin production with indomethacin decreased the amplitude of the dilator response, and decreased the time required to reach a steady-state vasoconstrictor response to the ETB-receptor agonists. 4. The effect of ETB-receptor stimulation on vascular tone was also evaluated in vitro on the cat femoral artery and vein. IRL 1620 had no effect on the femoral artery but caused a weak dose-dependent relaxation in the femoral vein. This large vein relaxation response seemed to be mediated by endothelium-derived nitric oxide and not by prostacyclin. 5. It may be concluded that ETB-receptor stimulation is responsible for the dilator response, and can contribute to the constrictor response, elicited by endothelins in cat skeletal muscle in vivo.

Plasticity of contractile endothelin-B receptors in human arteries after organ culture.

1. The pharmacology and mRNA expression of endothelin (ET) receptors in human omental arteries were characterized by use of functional contractile assays and the reverse transcriptase-polymerase chain reaction (RT-PCR). 2. In freshly obtained segments of human omental arteries, ET-1 and ET-3 induced concentration-dependent contractions which were normalized to the response produced by 60 mM K+. ET-1 produced a maximum contraction (Emax) amounting to 151 +/- 17% of the K+ response. The pEC50 for this agonist was 8.64 +/- 0.17. The effect of ET-3 was less pronounced (Emax: 71 +/- 22% and pEC50: 6.69 +/- 0.17) than that of ET-1. The ET receptors involved were characterized with FR139317 (a selective ETA receptor antagonist), PD 145065 (a mixed ETA and ETB receptor antagonist) and BQ 788 (an ETB receptor antagonist). A high concentration of these antagonists (10 microM) abolished the contractile responses to ET-3, and produced a parallel rightward shift of the ET-1 concentration-response curve without changing the maximal effect. FR139317 and PD 145065 were equally effective while BQ 788 was much less effective. This is consistent with ETA receptors mediating contraction in human omental arteries. 3. Arterial segments cultured for 5 days in serum-free Dulbecco's medium at 37 degrees C under sterile and humidified conditions retained contractility although responses to 60 mM K+ were somewhat reduced. ET-3 was significantly more potent in the cultured arteries (pEC50: 8.56 +/- 0.15) and achieved a greater maximum effect (Emax: 116 +/- 19%). Responses were not antagonised by FR139317 but were competitively blocked by PD 145065 and BQ 788 with the latter antagonist being the more potent. In contrast Emax (179 +/- 17%) and pEC50 (8.66 +/- 0.23) values for ET-1 were not significantly different from those obtained with fresh arteries. PD 145065 still demonstrated a rightward shift of the ET-1-induced concentration-response curve, whereas FR139317 and BQ 788 caused non-significant shifts. These findings suggest that functional ETB receptors contribute significantly to the endothelin contractile response in cultured arteries. 4. Two-site analysis of the ET-1 induced concentration-response curve from cultured arteries suggests that ETB receptors, at the high potency component, and ETA receptors, at the low potency component, contribute both to the contractile response in relative proportion of 70% and 30%, respectively. Further analysis suggested that the ETA receptor would be capable of evoking at least 75% of the ET-1 contraction in the absence of ETB receptors, although with a lower potency as compared to fresh arteries. 5. Electrophoresis of RT-PCR products from the smooth muscle layer of freshly obtained human arteries indicated the presence of mRNA for both ETA and ETB receptors. Arteries cultured for 1 and 5 days demonstrated an increase of mRNA for the ETB receptor as compared to the ETA receptor. The identities of the PCR products were verified by restriction enzyme digestion. 6. In freshly obtained human omental arteries, the contractile effects of endothelins appear to be mediated predominantly by the ETA receptor subtype, with a negligible contribution by ETB receptors. Cultured arterial segments, however, exhibited a substantial ETB receptor mediated contractile response and an increase in ETB receptor mRNA content, consistent with an upregulation of functional ETB receptors. These in vitro data suggest plasticity in the smooth muscle cell expression of contractile ETB receptors.

The stable pyrimidines UDPbetaS and UTPgammaS discriminate between the P2 receptors that mediate vascular contraction and relaxation of the rat mesenteric artery.

The contractile and relaxant effects of the different P2 receptors were characterized in the rat isolated mesenteric artery by use of extracellular nucleotides, including the stable pyrimidines uridine 5'-O-thiodiphosphate (UDPbetaS) and uridine 5'-O-3-thiotriphosphate (UTPgammaS). The selective P2X receptor agonist, alphabeta-methylene-adenosine triphosphate (alphabeta-MeATP) stimulated a potent (pEC(50)=6.0) but relatively weak contraction (E:(max)=57% of 60 mM K(+)). The contractile concentration-response curve of adenosine triphosphate (ATP) was biphasic when added in single concentrations. The first part of the response could be desensitized by alphabeta-MeATP, indicating involvement of P2X receptors, while the second part might be mediated by P2Y receptors. The contractile P2Y receptors were further characterized after P2X receptor desensitization with 10 microM alphabeta-MeATP. Uridine diphosphate (UDP), uridine triphosphate (UTP) and ATP stimulated contraction only in high concentrations (1 - 10 mM). The selective P2Y(6) agonist, UDPbetaS, and the P2Y(2)/P2Y(4)-receptor agonists UTPgammaS and adenosine 5'-O-3-thiotriphosphate (ATPgammaS) were considerably more potent and efficacious (E:(max) approximately 250% of 60 mM K(+)). Adenosine 5'-O-thiodiphosphate (ADPbetaS) was inactive, excluding contractile P2Y(1) receptors. After precontraction with 1 microM noradrenaline, UTP, ADP and ATP induced relaxations with similar potencies (pEC(50) approximately 5.0). UTPgammaS, ADPbetaS and ATPgammaS were approximately one log unit more potent indicating the presence of endothelial P2Y(1) and P2Y(2)/P2Y(4) receptors. The P2Y(6) receptor agonist, UDPbetaS, had no effect. UDPbetaS and UTPgammaS are useful tools when studying P2 receptors in tissue preparations with ectonucleotidase activity. Contractile responses can be elicited by stimulation of P2Y(6) and, slightly less potently, P2Y(2)/P2Y(4) receptors. The P2X response was relatively weak, and there was no P2Y(1) response. Stimulation of P2Y(1) and P2Y(2)/P2Y(4) receptors elicited relaxation, while P2Y(6) did not contribute.

Enhanced endothelin-1-induced contractions in mesenteric arteries from rats with congestive heart failure: role of ET(B) receptors.

Studies of congestive heart failure (CHF) in man and in experimental CHF have demonstrated elevated circulating levels of endothelin (ET). In order to examine whether there are concomitant ET receptor alterations, the vasomotor effects of endothelin-1 (ET-1) and sarafotoxin 6c (S6c) were examined in endothelium-intact and -denuded isolated mesenteric arteries from rats with CHF. CHF was induced by ligation of the left anterior descending coronary artery. Vasomotor responses were studied using small mesenteric arteries (approx. 250 microm in diameter, determined after normalisation). The antagonists IRL2500 and FR139317 were used in order to characterise the ET-1-induced response. In mesenteric arteries with intact endothelium, ET-1-induced contractions were more potent in CHF as compared to sham (pEC(50) 9.6+/-0.2 and 9.1+/-0.1, respectively, P<0.01). In endothelium-denuded arteries, there was no difference in potency of ET-1 between CHF and sham arteries, or in maximum contraction. In the presence of IRL2500, a selective ET(B)-receptor antagonist, ET-1 was more potent in endothelium-denuded arteries of CHF rats, while this difference was not seen in sham arteries. S6c had no consistent contractile or dilatory effect in CHF and sham rats. The results indicate that the enhanced contractile effects of ET-1 noted in CHF might be due to an attenuated endothelial function and that inhibition of smooth muscle cell ET(B) receptors increase the effects of contractile ET(A) receptors in CHF rats.