Effect of tecastemizole on pulmonary and cutaneous allergic inflammatory responses.

BACKGROUND: Tecastemizole, a major metabolite of astemizole, is a potent and selective H1 receptor antagonist. Evidence suggests that this and certain other H1 receptor antagonists may possess anti-inflammatory effects that are, in some cases, independent of H1 receptor antagonism. Objective The aim of this study was to investigate the anti-inflammatory effects of tectastemizole in models of allergic inflammation. METHODS: Effects of tecastemizole were assessed in a murine model of allergic lung inflammation, in passive cutaneous anaphylaxis (PCA) responses in guinea-pig skin and in in vitro assays measuring endothelial adhesion molecule expression and leucocyte-endothelial adhesion. RESULTS: Tecastemizole inhibited antigen-induced eosinophil recruitment to the lungs of allergic mice in a dose-dependent manner. Furthermore, combination of a sub-effective dose of tecastemizole, combined with a sub-effective dose of dexamethasone inhibited eosinophil accumulation in this model. Plasma extravasation in PCA reactions was inhibited by tecastemizole, although by a mechanism that would appear to be H1 receptor-dependent. Cytokine-induced endothelial intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 expression, as well as mononuclear cell adhesion to human umbilical vein endothelial cells was inhibited by tecastemazole in a manner independent of H1 receptor antagonism. CONCLUSION: These data suggest that tecastemizole may have H1 receptor-independent effects in inhibiting late-phase inflammatory responses, while acute responses appear to be inhibited in a H1 receptor-dependent manner. Furthermore, our data suggest an important potential steroid-sparing role for such drugs in the treatment of allergic inflammatory conditions.

Molecular expression and pharmacological identification of a role for K(v)7 channels in murine vascular reactivity.

BACKGROUND AND PURPOSE: This study represents a novel characterisation of KCNQ-encoded potassium channels in the vasculature using a variety of pharmacological and molecular tools to determine their role in contractility. EXPERIMENTAL APPROACH: Reverse transcriptase polymerase chain reaction (RT-PCR) experiments were undertaken on RNA isolated from mouse aorta, carotid artery, femoral artery and mesenteric artery using primers specific for all known KCNQ genes. RNA isolated from mouse heart and brain were used as positive controls. Pharmacological experiments were undertaken on segments from the same blood vessels to determine channel functionality. Immunocytochemical experiments were performed on isolated myocytes from thoracic aorta. KEY RESULTS: All blood vessels expressed KCNQ1, 4 and 5 with hitherto 'neuronal' KCNQ4 being, surprisingly, the most abundant. The correlated proteins K(v)7.1, K(v)7.4 and K(v)7.5 were identified in the cell membranes of aortic myocytes by immunocytochemistry. Application of three compounds known to activate K(v)7 channels, retigabine (2 -20 microM), flupirtine (20 microM) and meclofenamic acid (20 microM), relaxed vessels precontracted by phenylephrine or 1 mM 4-aminopyridine but had no effect on contractions produced by 60 mM KCl or the K(v)7 channel blocker XE991 (10 microM). All vessels tested contracted upon application of the K(v)7 channel blockers XE991 and linopirdine (0.1-10 microM). CONCLUSIONS AND IMPLICATIONS: Murine blood vessels exhibit a distinctive KCNQ expression profile with 'neuronal' KCNQ4 dominating. The ion channels encoded by KCNQ genes have a crucial role in defining vascular reactivity as K(v)7 channel blockers produced marked contractions whereas K(v)7 channel activators were effective vasorelaxants.

A regression model using sediment chemistry for the evaluation of marine environmental impacts associated with salmon aquaculture cage wastes.

This study was undertaken to develop an approach for modelling changes of sediment chemistry related to the accumulation of aquaculture waste. Metal composition of sediment Al, Cu, Fe, Li, Mn, and Zn; organic carbon and < 63 microm particles were used to determine the extent of detectable effects around the cage. This study showed marked differences in the sediment chemistry between aquaculture sites and the natural background: (1) negative correlations between sediment Cu and Zn with Al, (2) poor correlations between metals and Li, and (3) concentrations of Fe and Mn decreased with increased accumulation of organic carbon. There is a trend among normalised metals, organic carbon and particles related to normal, hypoxic and anoxic sediment conditions. The trends are useful for detecting and assessing the cumulative effects from aquaculture wastes to the marine environment. Lithium is less interactive with other metals in aquaculture sediments compared with the natural background sediments. Principal components analysis (PCA) was carried out on the metals, organic carbon, and particles to cluster the similarities of the variables so as to establish the predicted or adjusted environmental monitoring program (EMP) ratings. This approach, using the adjusted EMP rating based on sediment chemistry, yields a regression model with R2 = 0.945 compared to R2= 0.653 for the regression model using unadjusted EMP for assessing the environmental conditions.

Distribution of contaminants in biota and sediments in the Musquash Estuary, Atlantic Canada, marine protected area site initiative and contaminant exclusion zone.

The Musquash Estuary, one of the last ecologically intact estuaries in New Brunswick, has been designated an area of interest for a marine protected area (MPA) under the Oceans Act. The area has been assessed for contaminant background levels as required for establishing MPA environmental quality. American lobster (Homarus americanus), blue mussel (Mytilus edulis) and sediments were collected for assessing contaminant levels and distribution in the harbour. Levels of contaminants from the indicator species and the abiotic component have shown: (1) two extremes of high and low Cu and Ag in lobster from the area; and (2) lower metal levels in inner Musquash Harbour sediments and mussels than in those from the harbour mouth. These suggest that deposition of contaminants into the Musquash MPA site was due to transport of contaminants by coastal currents from upstream coastal industrial activities. This reverse trend with higher contaminant levels in the biotic and abiotic components in the outer harbour than in the inner harbour differs from a contaminated harbour and suggests that a contaminant exclusion zone should be considered for controlling contamination from nearby coastal and estuarine industrial sites to protect the sensitive habitats within the marine protected area.

Selection of bioindicators for monitoring marine environmental quality in the Bay of Fundy, Atlantic Canada.

Distribution of metals, PAH's and PCB's in lobsters, mussels, and sediments were used to assess marine environmental quality of the Bay of Fundy. This study demonstrates that the lobster (Homarus americanus) is a better bioindicator for monitoring contaminants in the marine environment and has a greater capacity for the uptake and accumulation of contaminants than the mussel (Mytilus edulis) and sediments. A definite pattern in the spatial distribution of lobster Cu, Cd, and Ag was evident. The distribution of organic contaminants for both mussels and lobsters in the Bay of Fundy lacked a spatial trend, and organic contaminants were undetectable in sediments from all sites. The Gulf Watch Programme, which monitors chemicals in mussels in the Bay of Fundy, did not indicate a problem with high levels of Cu, Cd, and Zn in the ecosystem. Analytes below the detection limit, such as in mussels and sediments, increase the difficulties of chemical analysis and detection for environmental monitoring. Deficiencies of mussels in monitoring the Bay of Fundy were discussed.

Metal contaminants for modelling lobster (Homarus americanus) migration patterns in the Inner Bay of Fundy, Atlantic Canada.

A method, which uses metal compositions in lobster digestive glands as natural environmental tags, has been developed to trace lobster movements. Lobsters were collected from three selected sites, Minas Channel, Minas Basin, and Cobequid Bay, Inner Bay of Fundy, New Brunswick, Canada, that were known to be contaminated with Cu. Five metal variables (Ag, Cd, Cu, Mn and Zn) were processed for principal component analysis (PCA). Metal concentration and burden models were investigated and PCA was able to differentiate lobsters from the respective catch sites. The method was applied to investigate the May and June lobsters collected at the three sites to determine the migration rate during this period of the fishing season. The results show a high level of mixing at Minas Basin and Cobequid Bay in June, and lobster movement inward toward the inner reaches of the bay, with very limited movement outward from the inner bay.

Aquaculture-related trace metals in sediments and lobsters and relevance to environmental monitoring program ratings for near-field effects.

The study was undertaken to assess the marine environmental effects from feed and waste associated with aquaculture activities. Metal compositions of sediment, lobster, and feed were used to evaluate the extent of detectable effects at 0 m (under the cage) and 50 m distance. Sediments that were collected under the cages and were characterised as hypoxic or anoxic, showed elevated levels of Cu, Zn, organic carbon, and % <63 microm particles, and low Mn and Fe. At 50 m there was a major reduction in waste chemical impact. Using lobster, a bioindicator species, as a tool for detecting near-field impacts, showed accumulations of high Cu associated with active aquaculture sites. Chemical compositions and metal ratios normalised with organic carbon, were used to assess the sediment conditions associated with environmental monitoring program ratings (EMP--normal, hypoxic, and anoxic). Principal component analysis (PCA) was used to explore chemical data at all sites for differentiating normal, hypoxic and anoxic sediment conditions. Selected variables (Cu, Zn, Fe, Mn, organic carbon, and particles <63 microm) were sufficient for the PCA approach with >90% explainable variance of first two components. The groupings based on PCA and cluster analysis were similar to EMP classifications with some exceptions of mis-identification by EMP. The sediment chemistry components were valid indicators for evaluating marine environmental conditions and for assessing aquaculture operating sites. The developed techniques, using chemical variables in combination with EMP and the statistical approach should be useful to predict the effects of aquaculture practices and the suitability of aquaculture operations.

Protease-activated receptor (PAR) 1 but not PAR2 or PAR4 mediates endothelium-dependent relaxation to thrombin and trypsin in human pulmonary arteries.

Endothelial protease-activated receptors (PARs) may be important sensors of vascular inflammation and injury. Activation of endothelial PAR1 and PAR2 causes nitric oxide-mediated arterial smooth muscle relaxation in a number of species and PAR4 activation causes similar responses in isolated rat aorta. However, it is unclear whether these receptors mediate such responses in human arteries because the most potent activators of PAR1, PAR2, and PAR4, thrombin and trypsin, cause endothelium-dependent relaxation of human coronary arteries through a common PAR1-like receptor. This study aimed to determine whether this unique pharmacology of PARs in human coronary arteries extends to human pulmonary arteries. PAR1 and PAR2 mRNA and protein were detected in human pulmonary arteries via reverse transcription polymerase chain reaction and immunohistochemistry, respectively. PAR4 mRNA was also detected in human pulmonary arteries. Contracted human pulmonary artery ring segments suspended for isometric tension measurement relaxed in a concentration- and endothelium-dependent manner to thrombin (0.001-0.1 U/ml), trypsin (0.01-1 U/ml), and the PAR1-activating peptide, SFLLRN (0.1-10 microM). By contrast, the PAR2- and PAR4-activating peptides, SLIGKV and GYPGQV, respectively, caused neither contraction nor relaxation of precontracted human pulmonary arteries. Relaxations to thrombin and trypsin cross-desensitized, while tachyphylaxis to SFLLRN abolished subsequent relaxations to both thrombin and trypsin. We conclude that human pulmonary arteries express PAR1, PAR2, and PAR4, but that only PAR1, or a PAR1-like receptor, is coupled to endothelium-dependent relaxation.

Protease-activated receptor-2 (PAR2) in the airways.

Protease-activated receptors (PARs) act as sensors for active extracellular serine proteases. Since serine proteases like mast cell tryptase are associated with inflammatory processes, PARs may represent novel pharmacological targets in airway diseases like asthma and chronic obstructive pulmonary disease. However, our present understanding of the physiological roles of PARs is in its infancy. In this review we highlight evidence for the involvement of PARs in airway disease and propose that these novel receptors may play mainly protective roles.

Protease-activated receptors: sentries for inflammation?

Cell-surface protease-activated receptors (PARs) appear to have evolved to detect extracellular enzymatically active serine proteases such as trypsin and thrombin. The predominant location of PARs on endothelia and epithelia and the discovery of enzymes such as trypsin within these tissues, together with the linkage of PARs to cytoprotective pathways, provide new information on autocrine and paracrine signalling within these critical barriers. In this article, the ways in which the distribution and function of PARs could be harnessed by pharmacologists as novel anti-inflammatory therapeutic strategies are discussed.

Effect of protease-activated receptor (PAR)-1, -2 and -4-activating peptides, thrombin and trypsin in rat isolated airways.

Mechanisms of relaxation and contraction to protease-activated receptor- (PAR) tethered ligand peptides (SFLLRN/TFLLR, SLIGRL and GYPGKF (all C-terminally amidated) for PAR1, PAR2 and PAR4, respectively) and enzymes (thrombin and trypsin) were investigated in isolated segments of rat trachea, main and first order intrapulmonary bronchi. In airway segments previously exposed to SLIGRL, SFLLRN caused contractions that were potentiated by indomethacin, but were independent of mast cell degranulation. Contractions to TFLLR in the intrapulmonary bronchi were similarly potentiated by indomethacin. SLIGRL caused epithelium-dependent relaxations which were unaffected by N(G)-nitro-L-arginine, 1-H-oxodiazol-[1,2,4]-[4,3-a]quinoxaline-1-one or zinc-protoporphyrin-IX but were abolished by haemoglobin in all three regions of the airways. Relaxations to SLIGRL were markedly attenuated by indomethacin only in the main and intrapulmonary bronchi. GYPGKF caused epithelium-dependent relaxations in all three regions of the airway which were only significantly inhibited by indomethacin in the intrapulmonary bronchi. In general, thrombin and trypsin failed to cause any response in the airways tested. Intense PAR2-immunoreactivity was observed on airway epithelium. PAR1-immunoreactivity was faint on airway epithelium and smooth muscle, but was prevalent in mast cells. These findings indicate that PAR2 and possibly PAR4 present on rat airway epithelia mediate smooth muscle relaxation via cyclo-oxygenase-dependent and -independent mechanisms. PAR1-mediated contractions were most likely due to activation of smooth muscle receptors. The general failure of thrombin and trypsin to cause responses which may have been due to endogenous protease inhibitors, highlights the need for caution in assessing pathophysiological roles for PARs if only enzymes are used to activate PARs.

A simple co-precipitation inductively coupled plasma mass spectrometric method for the determination of uranium in seawater.

Inductively coupled plasma mass spectrometry (ICP-MS) was used in the determination of 238uranium in seawater after concentration by a simplified co-precipitation with iron hydroxide. Ocean water and reference seawater were used in the study. The co-precipitation method required a smaller sample volume (10 fold less), and less column separation to recover the uranium from the seawater matrix, compared to the original iron hydroxide method. The direct seawater dilution technique requires only a small seawater volume (0.5 mL) and offers a rapid, reliable method for uranium analysis in seawater compared to traditional methods. Comparison of the results for simple co-precipitation, direct dilution of seawater, and theoretical uranium values based on salinity concentrations, yielded negligible differences. Data from this work show that the certified value for NASS-4 is low.

The role of protease-activated receptor-2 (PAR2) in the modulation of beating of the mouse isolated ureter: lack of involvement of mast cells or sensory nerves.

1 The localization of protease-activated receptor-2 (PAR2) and the effects of PAR2 activators were investigated in the mouse isolated ureter in order to test the hypothesis that PAR2 activation may initiate neuropeptide release from sensory nerve fibres and hence contribute to inflammation. 2 PAR2 was localized by fluorescence immunohistochemistry to both the smooth muscle and epithelium of the ureter. Macrophage-like cells in the adventitia of the ureter were also PAR2-immunoreactive. PAR2-immunoreactivity was not observed in mast cells or nerve fibres. 3 In circular muscle preparations of the ureter in which continuous rhythmic beating was induced by KCl (20 mM) and the thromboxane A2 mimetic U46619 (0.3 microM), trypsin (0.3 U ml-1) reduced beat frequency to 84.6+/-2.0% of control rates. The PAR2-selective peptide agonist SLIGRL-NH2 concentration-dependently (0.1-3.0 microM) slowed beat frequency to a maximum of 72.7+/-2.0%. 4 Histamine (1-300 microM) was more efficacious than SLIGRL-NH2 in inhibiting ureter beat frequency in a concentration-dependent manner to a maximum (at 300 microM) of 7.9+/-2.5% of the control rate. 5 Pretreatment of preparations with capsaicin (10 microM for 30 min) markedly attenuated the inhibitory effect of histamine, but not that of SLIGRL-NH2, indicating a role for sensory nerves in the inhibitory effect of histamine only. 6 The inhibitory effect of SLIGRL-NH2 on ureter beat frequency was unaffected by the nitric oxide (NO) synthase inhibitor, L-NOARG (100 microM) or the cyclo-oxygenase inhibitor, indomethacin (3 microM). 7 In conclusion, PAR2 activation causes inhibition of beating in the mouse ureter that is not mediated by axon reflex release of inhibitory neuropeptides. This inhibitory effect of PAR2 appears to be mediated directly on smooth muscle cells, although the contribution of non-NO, non-prostanoid epithelium-derived factors cannot be ruled out.

Characterization of non-adrenergic, non-cholinergic inhibitory responses of the isolated guinea-pig trachea: differences between pre- and post-ganglionic nerve stimulation.

1 Differences in the mechanism of non-adrenergic, non-cholinergic (NANC) inhibitory responses to preganglionic- and post-ganglionic nerve stimulation were investigated in the guinea-pig isolated trachea. 2 Stimulation of the vagus nerve at frequencies above 4 Hz elicited NANC relaxation of the trachealis muscle. Responses to low frequencies of stimulation (4-8 Hz) were abolished by the nitric oxide (NO) synthase inhibitor L-NOARG (10 microM), while a L-NOARG resistant component was observed at higher stimulus frequencies. The L-NOARG-resistant component of NANC inhibitory responses to higher frequencies of vagus nerve stimulation were significantly attenuated by the proteinase alpha-chymotrypsin (2 U/ml), suggesting that a neuropeptide such as VIP may contribute to NANC responses. 3 When postganglionic nerves were stimulated by electrical field stimulation (EFS), responses were readily elicited at frequencies below 4 Hz. Like responses to vagus nerve stimulation, responses to low frequency (<4 Hz) EFS were abolished by L-NOARG while a L-NOARG-resistant component was apparent at higher stimulus frequencies. 4 The L-NOARG-resistant component of NANC inhibitory responses to EFS was sensitive to alpha-chymotrypsin only if stimuli were delivered in either long trains at a low frequency (4 Hz for 10-30 s) or short trains of high frequency (16 Hz for 2.5-7.5 s). 5 Responses to preganglionic nerve stimulation were approximately 35% of the amplitude of responses to EFS in the same preparations. 6 In conclusion, responses to preganglionic and postganglionic NANC inhibitory nerve stimulation in the guinea-pig trachea differ in maximum amplitude, frequency-response characteristics and the contributions of cotransmitters. We suggest that these differences may be explained by filtering of preganglionic input to postganglionic NANC neurons. These results have implications in all studies where EFS is considered to be representative of physiological stimulation of post-ganglionic nerve stimulation.

Evidence for selective effects of chronic hypertension on cerebral artery vasodilatation to protease-activated receptor-2 activation.

BACKGROUND AND PURPOSE: Protease-activated receptor-2 (PAR-2) can be activated after proteolysis of the amino terminal of the receptor by trypsin or by synthetic peptides with a sequence corresponding to the endogenous tethered ligand exposed by trypsin (eg, SLIGRL-NH(2)). PAR-2 mediates nitric oxide (NO)-dependent dilatation in cerebral arteries, but it is unknown whether PAR-2 function is altered in cardiovascular diseases. Since hypertension selectively impairs NO-mediated cerebral vasodilatation in response to acetylcholine and bradykinin, we sought to determine whether PAR-2-mediated vasodilatation is similarly adversely affected by this disease state. METHODS: We studied basilar artery responses in Wistar-Kyoto rats (WKY) (normotensive) and spontaneously hypertensive rats (SHR) in vivo (cranial window preparation) and in vitro (isolated arterial rings). The vasodilator effects of acetylcholine, sodium nitroprusside, and activators of PAR-2 and protease-activated receptor-1 (PAR-1) were compared in WKY versus SHR. Immunohistochemical localization of PAR-2 was also assessed in the basilar artery. RESULTS: Increases in basilar artery diameter in response to acetylcholine were 65% to 85% smaller in SHR versus WKY, whereas responses to sodium nitroprusside were not different. In contrast to acetylcholine, vasodilatation in vivo to SLIGRL-NH(2) was largely preserved in SHR, and SLIGRL-NH(2) was approximately 3-fold more potent in causing vasorelaxation in SHR versus WKY in vitro. In both strains, responses to SLIGRL-NH(2) were abolished by N(G)-nitro-L-arginine, an inhibitor of NO synthesis. Activators of PAR-1 had little or no effect on the rat basilar artery. PAR-2-like immunoreactivity was observed in both the endothelial and smooth muscle cells of the basilar artery in both strains of rat. CONCLUSIONS: These data indicate that NO-mediated vasodilatation to PAR-2 activation is selectively preserved or augmented in SHR and may suggest protective roles for PAR-2 in the cerebral circulation during chronic hypertension.

A protective role for protease-activated receptors in the airways.

The protection of cells in the upper intestine against digestion by pancreatic trypsin depends on the prostanoid prostaglandin E2 (PGE2) and is mediated by protease-activated receptors in the epithelium. As the airway epithelium is morphologically similar and also expresses one of these receptors, PAR2, and is a major source of PGE2, we reasoned that bronchial epithelial PAR2 might also participate in prostanoid-dependent cytoprotection in the airways. Here we show that activation of PAR2, which co-localizes immunohistochemically with trypsin(ogen) in airway epithelium, causes the relaxation of airway preparations from mouse, rat, guinea-pig and humans by the release of a cyclooxygenase product from the epithelium. This physiological protective response in isolated airways also occurred in anaesthetized rats, where activation of PAR2 caused a marked and prolonged inhibition of bronchoconstriction. After desensitization of PAR2, the response to trypsin recovered rapidly by mechanisms dependent on de novo synthesis and trafficking of proteins. Our results indicate that trypsin released from the epithelium can initiate powerful bronchoprotection in the airways by activation of epithelial PAR2.

Protease-activated receptors mediate apamin-sensitive relaxation of mouse and guinea pig gastrointestinal smooth muscle.

BACKGROUND & AIMS: Protease-activated receptor (PAR)-1 and PAR-2 are expressed on gastrointestinal smooth muscle, but knowledge of their functionality is limited. The aim of this study was to determine if PAR-1 and PAR-2 mediate gastrointestinal smooth muscle relaxation and to clarify the underlying mechanisms. METHODS: Responses to PAR activation using the serine proteases thrombin and trypsin and the peptide agonists for PAR-1 and PAR-2, SFLLRN-NH2 and SLIGRL-NH2, respectively, were investigated in submaximally contracted longitudinal strips of mouse gastric fundus and guinea pig taenia coli. RESULTS: In mouse gastric fundus, both thrombin and trypsin caused relaxations followed by contractions. SFLLRN-NH2 and SLIGRL-NH2 caused similar biphasic responses, the relaxation components of which were eliminated by apamin or ryanodine. For SFLLRN-NH2, apamin and ryanodine revealed contractions. Nifedipine inhibited both relaxations and contractions to each peptide. In guinea-pig taenia coli, thrombin but not trypsin caused relaxation, whereas SFLLRN-NH2 and SLIGRL-NH2 caused concentration-dependent relaxations that were eliminated by apamin but were unaffected by ryanodine. CONCLUSIONS: The mouse gastric fundus and guinea pig taenia coli contain functional PAR-1 and PAR-2 that mediate relaxations via ryanodine-sensitive and -insensitive activation of small-conductance, Ca2+-activated K+ channels. We propose that smooth muscle PARs act as sensors for inflammatory signals in gut and respond by inhibiting gut motility during peritoneal infections or tissue damage.