Increase In Plasma Extravasation Research Articles

Development of solitary chemosensory cells in the distal lung after severe influenza injury.

H1N1 influenza virus infection induces dramatic and permanent alveolar remodeling mediated by p63+ progenitor cell expansion in both mice and some patients with acute respiratory distress syndrome. This persistent lung epithelial dysplasia is accompanied by chronic inflammation, but the driver(s) of this pathology are unknown. This work identified de novo appearance of solitary chemosensory cells (SCCs), as defined by the tuft cell marker doublecortin-like kinase 1, in post-influenza lungs, arising in close proximity with the dysplastic epithelium, whereas uninjured lungs are devoid of SCCs. Interestingly, fate mapping demonstrated that these cells are derived from p63-expressing lineage-negative progenitors, the same cell of origin as the dysplastic epithelium. Direct activation of SCCs with denatonium + succinate increased plasma extravasation specifically in post-influenza virus-injured lungs. Thus we demonstrate the previously unrecognized development and activity of SCCs in the lung following influenza virus infection, implicating SCCs as a central feature of dysplastic remodeling.

Dorsal Vagal Complex Modulates Neurogenic Airway Inflammation in a Guinea Pig Model With Esophageal Perfusion of HCl.

Neurogenic airway inflammation in chronic cough and bronchial asthma related to gastroesophageal reflux (GER) is involved in the esophageal–bronchial reflex, but it is unclear whether this reflex is mediated by central neurons. This study aimed to investigate the regulatory effects of the dorsal vagal complex (DVC) on airway inflammation induced by the esophageal perfusion of hydrochloric acid (HCl) following the microinjection of nuclei in the DVC in guinea pigs. Airway inflammation was evaluated by measuring the extravasation of Evans blue dye (EBD) and substance P (SP) expression in the airway. Neuronal activity was indicated by Fos expression in the DVC. The neural pathways from the lower esophagus to the DVC and the DVC to the airway were identified using DiI tracing and pseudorabies virus Bartha (PRV-Bartha) retrograde tracing, respectively. HCl perfusion significantly increased plasma extravasation, SP expression in the trachea, and the expression of SP and Fos in the medulla oblongata nuclei, including the nucleus of the solitary tract (NTS) and the dorsal motor nucleus of the vagus (DMV). The microinjection of glutamic acid (Glu) or exogenous SP to enhance neuronal activity in the DVC significantly potentiated plasma extravasation and SP release induced by intra-esophageal perfusion. The microinjection of γ-aminobutyric acid (GABA), lidocaine to inhibit neuronal activity or anti-SP serum in the DVC alleviated plasma extravasation and SP release. In conclusion, airway inflammation induced by the esophageal perfusion of HCl is regulated by DVC. This study provides new insight for the mechanism of airway neurogenic inflammation related to GER.

β₂-Adrenoceptor Blockade Deteriorates Systemic Anaphylaxis by Enhancing Hyperpermeability in Anesthetized Mice.

PurposePatients treated with propranolol, a nonselective β-adrenoceptor antagonist, develop severe anaphylaxis, but the mechanism remains unknown. We determined effects of β1- and β2-adrenoceptor antagonists on the anaphylaxis-induced increase in vascular permeability in mice.MethodsIn anesthetized ovalbumin-sensitized C57BL mice, mean arterial blood pressure (MBP) was measured, and Evans blue dye extravasation and hematocrit (Hct) were assessed at 20 minutes after antigen injection. The following pretreatment groups (n=7/group) were studied: (1) sensitized control (non-pretreatment), (2) propranolol, (3) the selective β2-adrenoceptor antagonist ICI 118,551, (4) the selective β1-adrenoceptor antagonist atenolol, (5) adrenalectomy, (6) the selective β2-adrenoceptor agonist terbutaline, and (7) non-sensitized groups.ResultsThe antigen injection decreased MBP, and increased Hct and vascular permeability in the kidney, lung, mesentery, and intestine, but not in the liver or spleen. Pretreatment with ICI 118,551, propranolol and adrenalectomy, but not atenolol, reduced the survival rate and augmented the increases in Hct and vascular permeability in the kidney, intestine, and lung as compared with the sensitized control group. Pretreatment with terbutaline abolished the antigen-induced alterations. Plasma epinephrine levels were increased significantly in the sensitize control mice.ConclusionsBlockade of β2-adrenoceptor can deteriorate systemic anaphylaxis by augmenting hyperpermeability-induced increase in plasma extravasation by inhibiting beneficial effects of epinephrine released from the adrenal glands in anesthetized mice.

Involvement of the TRPV1 receptor in plasma extravasation in airways of rats treated with an angiotensin-converting enzyme inhibitor

Angiotensin-converting enzyme inhibitors (ACEIs) are widely used in the treatment of hypertension, congestive heart failure and renal disease, and are considered relatively safe and generally well-tolerated drugs. However, adverse effects of ACEIs have been reported, including non-productive cough and angioedema, which can lead to poor adherence to therapy. The mechanisms by which ACEIs promote adverse effects are not fully elucidated, although increased bradykinin plasma levels following ACEI therapy seem to play an important role. Since bradykinin can sensitise the transient potential vanilloid receptor 1 (TRPV1), we investigated the role of TRPV1 in plasma extravasation in the trachea and bronchi of rats treated with the ACEI captopril. We observed that intravenous (i.v.) administration of captopril did not cause plasma extravasation in the trachea or bronchi of spontaneously breathing rats, but induced plasma extravasation in the trachea and bronchi of artificially ventilated rats. The intratracheal (i.t.) instillation of capsaicin or bradykinin also induced an increase in plasma extravasation in the trachea and bronchi of artificially ventilated rats. As expected, capsaicin-induced plasma extravasation was inhibited by i.t. pretreatment with the TRPV1 selective antagonist capsazepine (CPZ) while bradykinin-induced plasma extravasation was reduced by i.t. pretreatment with the selective B2 receptor antagonist Icatibant, originally known as HOE 140 (HOE). Interestingly, bradykinin-induced plasma extravasation was also inhibited by CPZ. The pretreatment with HOE and CPZ, singly or in combination and at doses which do not cause inhibitory effects per se, significantly inhibited the plasma extravasation induced by captopril treatment in artificially ventilated rats. In addition, treatment with a high dose of capsaicin in newborn rats, which induces degeneration of TRPV1-expressing sensory neurons, abolished both capsaicin and captopril-induced plasma extravasation in artificially ventilated rats. In conclusion, our study identified that captopril treatment promoted sensitisation of TRPV1, via B2 receptor activation, inducing plasma extravasation in the airways of mechanically ventilated rats. The present findings add a new view about the role of TRPV1 in the plasma extravasation induced by captopril and could to contribute to the elucidation of mechanisms by which ACEI induces adverse effects on airways.

523 Helicobacter pylori cag+ Strains Stimulate Expansion of Lrig1+ Progenitor Cells Within the Gastric Mucosa in a Strain-Specific Manner

Background & Aims. Despite the preponderance of stress-related diseases and inflammatory bowel disease (IBD) in women, the role of the corticotropin-releasing factor (CRF) system in mediating sex-specific effects on cellular signaling and immune function has not been systematically investigated. In this study, we determined the contribution of CRF receptor 2 (CRF2) in mediating sex-specific responses during colitis in mice. Methods. Colitis was induced by 2,4,6-trinitrobenzenesulfonic acid (TNBS) in wild-type, CRF2, and CRF2 mice of both sexes and treated with the CRF2 agonist urocortin1 (Ucn1) or saline. Changes in gastrointestinal permeability were detected using Evan's blue dye method to measure plasma extravasation. Changes in immune cell infiltration were determined using flow cytometry and changes in the MAPK signaling pathway were assessed by western blot analysis. Results. Baseline plasma extravasation in the gastrointestinal tract was ~1.4-1.74fold higher in females than in males in all three different genotypes. Colitis increased plasma extravasation in the distal colon in both sexes in all three different genotypes. Interestingly, the sex-specific difference in plasma extravasation was maintained in Wt, but lost in CRF2 and CRF2 mice during colitis. Colitis resulted in greater thymus atrophy, loss of spleen mass, adrenal hypertrophy, and neutrophil infiltration in the blood and in the colon in Wt female mice than in Wt males. Wt females showed a ~4-fold higher neutrophil infiltration in the colon than Wt males and this sex-specific difference was lost in CRF2 and CRF2 mice. Surprisingly, colitis-induced mortality was abolished in male CRF2 mice, but exceeded 25% in female CRF2 mice. Although Ucn1 intervention rescued colitis-induced mortality in male CRF2 mice, female CRF2 mice showed increased mortality and worsened inflammation after Ucn1 intervention. The p38 MAPK signaling pathway regulates inflammatory responses and is altered in IBD. We found dysregulated p38 MAPK signaling in CRF2 mice of both sexes, accompanied by decreased phosphorylation of downstream target heat shock protein 27 (Hsp27) during colitis. Ucn1 restored the phosphorylation levels of Hsp27 back to baseline levels in male, but not female CRF2 mice. Conclusions. Ucn1 and CRF2 mediate contextand sex-dependent inflammatory responses at the molecular, cellular and organ levels during IBD. Our observations suggest that Ucn1 intervention can rescue colitisinduced mortality in males alone. In males, Ucn1 ameliorates inflammation by mechanisms that involve the MAPK signaling pathway and phosphorylation of Hsp27 that regulates stabilization and formation of tight junctions in the gut epithelium.

Microcirculatory effects of physostigmine on experimental burn edema.

In order to further understand the role of the cholinergic anti-inflammatory pathway, the authors determined the effects of burn plasma from donor rats (DRs) on the microvascular circulation of healthy recipient rats and whether these could be altered by pretreatment with physostigmine (PT). DRs underwent thermal injury (100°C water, 12 seconds, 30% BSA) for positive controls. For negative controls DRs underwent sham burn (same procedure but water at 37°C). DR-plasma (harvested 4 hours posttrauma) was transferred to healthy rats. Bolus injection of PT (70 μg/kg body weight) was performed 15 minutes before starting the infusion of DR-plasma in the study group. Intravital microscopy was performed in mesenteric venules (0/60/120 minutes). Edema was assessed by fluorescein isothiocyanate (FITC)-albumin extravasation. Additionally, leukocyte rolling and sticking (cells/mm) as well as hemodynamic parameters were assessed. Burn plasma transfer significantly increases albumin extravasation in healthy individuals when compared with sham-burn treatment. Additional bolus administration of PT (70 μg/kg body weight) to burn plasma treatment reduces plasma extravasation to sham-burn levels. PT also attenuates leukocyte-endothelial interactions. After 120 minutes no significant changes in the systemic circulation (mean arterial pressure, heart rate, wall shear rate) were found between the groups. Burn plasma transfer results in significant increases in plasma extravasation and leukocyte-endothelial wall adherence, which are reversed by pretreatment with PT. These results suggest that the cholinergic anti-inflammatory pathway may play a role in the microcirculatory response to thermal injury.

ETB receptor activation as a mechanism of modulation of inflammatory pain and neurogenic inflammation in the temporomandibular joint of capsaicin-treated rats

Background: Endothelin (ET), a peptide best known for its vascular effects, also evokes pain and hyperalgesia, independently of its vascular actions. Data suggest that ET can have nociceptive effects, acting directly on receptors expressed in sensory neurons. As such, the aim this study was to investigate the direct effect of ET on hyperalgesia and edema, induced by carrageenan, on the temporomandibular joint (TMJ) of capsaicin-treated rats. Methods: Capsaicin was administered by subcutaneous injection to newborn, male Wistar rats. Inflammation was induced 60 days later by a single intra-articular injection of carrageenan into the left TMJ (control group received sterile saline). Inflammatory parameters, such as plasma extravasation, leukocyte influx and mechanical allodynia (measured as the head-withdrawal force threshold) were evaluated 4 h after edematogenic stimulus. ET-1 and ET-3, and the ET-B receptor (ETBR) antagonist were administered 3 min before edematogenic stimulus. ET and transient receptor potential vanilloid (TRPV1) mRNA expression was assessed by reverse-transcription polymerase chain reaction (RT-PCR). Edema formation was evaluated by measurement of the extravascular accumulation of injected 125I-human serum albumin into the TMJ soft tissues of anesthetized rats. Results: Capsaicin neonatal treatment significantly reduced edema formation, leukocyte influx and mechanical allodynia in TMJ, when compared to the control group, while the ETBR antagonist increased plasma extravasation and hyperalgesia in the capsaicin-treated group. ET-1 treatment reduced both plasma extravasation and myeloperoxidase activity. Capsular mRNA for ET-1 was significantly augmented in the TMJ of capsaicin-treated rats, when compared to controls. Conclusions: Our results suggest, for the first time, that ET-1, via ETBR activation, reduces plasma extravasation, leukocyte influx and inflammatory pain in the temporomandibular joint of capsaicin-treated rats.

Role of transient receptor potential vanilloid 4 in rat joint inflammation

To determine whether activation of transient receptor potential vanilloid 4 (TRPV-4) induces inflammation in the rat temporomandibular joint (TMJ), and to assess the effects of TRPV-4 agonists and proinflammatory mediators, such as a protease-activated receptor 2 (PAR-2) agonist, on TRPV-4 responses. Four hours after intraarticular injection of carrageenan into the rat joints, expression of TRPV-4 and PAR-2 in trigeminal ganglion (TG) neurons and in the TMJs were evaluated by real-time reverse transcription-polymerase chain reaction and immunofluorescence, followed by confocal microscopy. The functionality of TRPV-4 and its sensitization by a PAR-2-activating peptide (PAR-2-AP) were analyzed by measuring the intracellular Ca(2+) concentration in TMJ fibroblast-like synovial cells or TG neurons. Plasma extravasation, myeloperoxidase activity, and the head-withdrawal threshold (index of mechanical allodynia) were evaluated after intraarticular injection of selective TRPV-4 agonists, either injected alone or coinjected with PAR-2-AP. In the rat TMJs, TRPV-4 and PAR-2 expression levels were up-regulated after the induction of inflammation. Two TRPV-4 agonists specifically activated calcium influx in TMJ fibroblast-like synovial cells or TG neurons. In vivo, the agonists triggered dose-dependent increases in plasma extravasation, myeloperoxidase activity, and mechanical allodynia. In synovial cells or TG neurons, pretreatment with PAR-2-AP potentiated a TRPV-4 agonist-induced increase in [Ca(2+) ](i) . In addition, TRPV-4 agonist-induced inflammation was potentiated by PAR-2-AP in vivo. In this rat model, TRPV-4 is expressed and functional in TG neurons and synovial cells, and activation of TRPV-4 in vivo causes inflammation in the TMJ. Proinflammatory mediators, such as PAR-2 agonists, sensitize the activity of TRPV-4. These results identify TRPV-4 as an important signal of inflammation in the joint.

PAR2 and Temporomandibular Joint Inflammation in the Rat

The proteinase-activated receptor 2 (PAR2) is a putative therapeutic target for arthritis. We hypothesized that the early pro-inflammatory effects secondary to its activation in the temporomandibular joint (TMJ) are mediated by neurogenic mechanisms. Immunofluorescence analysis revealed a high degree of neurons expressing PAR2 in retrogradely labeled trigeminal ganglion neurons. Furthermore, PAR2 immunoreactivity was observed in the lining layer of the TMJ, co-localizing with the neuronal marker PGP9.5 and substance-P-containing peripheral sensory nerve fibers. The intra-articular injection of PAR2 agonists into the TMJ triggered a dose-dependent increase in plasma extravasation, neutrophil influx, and induction of mechanical allodynia. The pharmacological blockade of natural killer 1 (NK1) receptors abolished PAR2-induced plasma extravasation and inhibited neutrophil influx and mechanical allodynia. We conclude that PAR2 activation is pro-inflammatory in the TMJ, through a neurogenic mechanism involving NK1 receptors. This suggests that PAR2 is an important component of innate neuro-immune response in the rat TMJ.

Participation of peripheral tachykinin NK 1 receptors in the carrageenan-induced inflammation of the rat temporomandibular joint

Temporomandibular disorders represent one of the major challenges in dentistry therapeutics. This study was undertaken to evaluate the time course of carrageenan-induced inflammation in the rat temporomandibular joint (TMJ) and to investigate the role of tachykinin NK 1 receptors. Inflammation was induced by a single intra-articular (i.art.) injection of carrageenan into the left TMJ (control group received sterile saline). Inflammatory parameters such as plasma extravasation, leukocyte influx and mechanical allodynia (measured as the head-withdrawal force threshold) and TNFα and IL-1β concentrations were measured in the TMJ lavages at selected time-points. The carrageenan-induced responses were also evaluated after treatment with the NK 1 receptor antagonist SR140333. The i.art. injection of carrageenan into the TMJ caused a time-dependent plasma extravasation associated with mechanical allodynia, and a marked neutrophil accumulation between 4 and 24 h. Treatment with SR140333 substantially inhibited the increase in plasma extravasation and leukocyte influx at 4 and 24 h, as well as the production of TNFα and IL-1β into the joint cavity, but failed to affect changes in head-withdrawal threshold. The results obtained from the present TMJ-arthritis model provide, for the first time, information regarding the time course of this experimental inflammatory process. In addition, our data show that peripheral NK 1 receptors mediate the production of both TNFα and IL-1β in the TMJ as well as some of the inflammatory signs, such as plasma extravasation and leukocyte influx, but not the nociceptive component.

Retinal plasma extravasation in streptozotocin‐diabetic rats mediated by kinin B1 and B2 receptors

We investigated whether or not kinin receptors play a role in diabetic blood-retinal barrier breakdown, which is a leading cause of vision loss. Blood-retinal barrier breakdown was quantified using Evans blue, and expression of kinin B(1) receptor mRNA was measured using quantitative reverse transcrition-PCR. Diabetic rats (streptozotocin (STZ), 65 mg kg(-1)) received a single intraocular injection of bradykinin (BK) or des-Arg(9)-BK, alone, or in combination with antagonists for B(1) (des-Arg(10)-Hoe140, R-715) and/or B(2) (Hoe140) receptors, given intraocularly or intravenously (i.v.). In control rats, BK (0.1-10 nmol) dose-dependently increased plasma extravasation, which was inhibited by Hoe140 (0.2 nmol), whereas des-Arg(9)-BK (0.1 and 1 nmol) was without effect. B(1) receptor mRNA was markedly increased in retinas of diabetic rats, and this was prevented by N-acetyl-L-cysteine (1 g kg(-1) day(-1) for 7 days). Plasma extravasation in retinas of STZ-diabetic rats was higher than in controls and enhanced by des-Arg(9)-BK. Response to des-Arg(9)-BK was inhibited by intraocular or i.v. injection of B(1) receptor antagonists. Diabetes-induced plasma extravasation was inhibited only by a combination of des-Arg(10)-Hoe140 and Hoe 140 (100 nmol kg(-1), i.v. 15 min earlier) or by R-715 (1 micromol kg(-1), i.v.) injected daily for 7 days. Kinin B(1) receptors are upregulated in retinas of STZ-diabetic rats through a mechanism involving oxidative stress. Both kinin B(1) and B(2) receptors contribute to increased plasma extravasation in diabetic retinopathy. Chronic inhibition of both kinin receptors, possibly with antioxidant adjuvants, may be a novel therapeutic strategy for diabetic retinopathy.

Involvement of tumour necrosis factor‐α in Clostridium perfringensβ‐toxin‐induced plasma extravasation in mice

Clostridium perfringens beta-toxin, an important agent of necrotic enteritis, causes plasma extravasation due to the release of a tachykinin NK(1) receptor agonist in mouse skin. In this study, we investigated the role of cytokines in beta-toxin-induced plasma extravasation. Male Balb/c, C3H/HeN and C3H/HeJ mice were anaesthetized with pentobarbitone and beta-toxin was injected i.d. into shaved dorsal skin. SR140333, capsaicin, chlorpromazine and pentoxifylline were given as pretreatment when required before the injection of the toxin. Cytokines in the dorsal skin were measured by ELISA. Injection (i.d.) of beta-toxin induced a dose-dependent increase in dermal TNF-alpha and interleukin (IL)-1beta levels with a concomitant increase in plasma extravasation, but not the release of IL-6. SR140333 and capsaicin significantly inhibited the toxin-induced release of TNF-alpha and IL-1beta. The plasma extravasation and the release of TNF-alpha induced by beta-toxin were significantly inhibited by chlorpromazine and pentoxifylline which inhibit the release of TNF-alpha. The toxin-induced plasma extravasation in mouse skin was attenuated by pretreatment with a monoclonal antibody against TNF-alpha, but not anti-IL-1beta. Furthermore, the toxin caused an increase in plasma extravasation in both C3H/HeN (TLR4-intact) and C3H/HeJ (TLR4-deficient) mice. In C3H/HeN mice, the toxin-induced leakage was not inhibited by pretreatment with anti-TLR4/MD-2 antibody. These observations show that beta-toxin-induced plasma extravasation in mouse skin is related to the release of TNF-alpha via the mechanism involving tachykinin NK(1) receptors, but not via TLR4.

Blockade of airway inflammation and hyper-responsiveness by an angiopoietin-1 variant, COMP-Ang1

Inflammation of the asthmatic airway is usually accompanied by increased vascular permeability and plasma exudation. Angiopoietin-1 (Ang1) has potential therapeutic applications in preventing vascular leakage. Recently, we developed a soluble, stable, and potent Ang1 variant, COMP-Ang1. COMP-Ang1 is more potent than native Ang1 in phosphorylating the tyrosine kinase with immunoglobulin and epidermal growth factor homology domain 2 receptor in lung endothelial cells. We have used a mouse model for allergic airway disease to determine effects of COMP-Ang1 on allergen-induced bronchial inflammation and airway hyper-responsiveness. These mice develop the following typical pathophysiological features of allergic airway disease in the lungs: increased numbers of inflammatory cells of the airways, airway hyper-responsiveness, increased levels of Th2 cell cytokines (IL-4, IL-5, and IL-13), adhesion molecules (intercellular adhesion molecule-1 and vascular cell adhesion molecule-1), and chemokines (eotaxin and RANTES), and increased vascular permeability. Intravenous administration of COMP-Ang1 reduced bronchial inflammation and airway hyper-responsiveness. In addition, the increased plasma extravasation in allergic airway disease was significantly reduced by the administration of COMP-Ang1. These results suggest that COMP-Ang1 attenuates airway inflammation and hyper-responsiveness, prevents vascular leakage, and may be used as a therapeutic agent in allergic airway disease.

Ethanol Causes Neurogenic Vasodilation by TRPV1 Activation and CGRP Release in the Trigeminovascular System of The Guinea Pig

Ethanol stimulating transient receptor potential vanilloid 1 (TRPV1) on primary sensory neurons promotes neurogenic inflammation, including calcitonin gene-related peptide (CGRP)-mediated coronary dilation. Alcoholic beverages trigger migraine attacks and activation of trigeminal neurons plays a role in migraine. We have investigated in guinea pigs whether ethanol by TRPV1 stimulation causes neurogenic inflammation in the trigeminovascular system. Ethanol-evoked release of neuropeptides from slices of dura mater was abolished by Ca(2+) removal, capsaicin pretreatment and the TRPV1 antagonist, capsazepine. Intragastric ethanol increased plasma extravasation in dura mater, an effect abolished by capsazepine and the NK1 receptor antagonist, SR140333, and caused vasodilation around the middle meningeal artery, an effect abolished by capsazepine and the CGRP receptor antagonist, BIBN4096BS. Vasodilation of meningeal vessels by TRPV1 activation and CGRP release may be relevant to the mechanism by which alcohol ingestion triggers migraine attacks.

Mechanism of Vasopeptidase Inhibitor-Induced Plasma Extravasation: Comparison of Omapatrilat and the Novel Neutral Endopeptidase 24.11/Angiotensin-Converting Enzyme Inhibitor GW796406

We describe N-[(2S)-2-(mercaptomethyl)-3-methylbutanoyl]-4-(1H-pyrazol-1-yl)-L-phenylalanine (GW796406), a vasopeptidase inhibitor (VPI) that possessed approximately 3-fold selectivity for neutral endopeptidase 24.11 (NEP) versus angiotensin-converting enzyme (ACE) in in vitro assays using rat and human enzymes. In the same assays, omapatrilat, the most extensively studied VPI, displayed approximately 3-fold selectivity for ACE. The in vivo ACE and NEP inhibition profile and the liability of the compounds to increase plasma extravasation were compared at two (low and high) therapeutically equivalent intravenous doses in the rat. At the low dose, both agents inhibited ACE activity by approximately 85%. Consistent with their in vitro ACE/NEP selectivity, omapatrilat produced 49% inhibition, whereas GW796406 produced >95% inhibition of NEP. Neither compound increased plasma extravasation. When the low dose was administered to rats pretreated with the NEP inhibitor ecadotril to normalize NEP background to <5% of control, only omapatrilat significantly increased plasma extravasation. At the high dose, omapatrilat and GW796406 produced profound, nonselective inhibition of ACE (>90%) and NEP (>95%), and they significantly increased plasma extravasation. The activity of the agents as inhibitors of dipeptidylpeptidase IV (DPP IV) and aminopeptidase P (APP) was also investigated. Neither compound inhibited DPP IV. Interestingly, omapatrilat, but not GW796406, was a relatively potent inhibitor of APP (IC50 = 260 nM). We investigated whether APP inhibition increased the plasma extravasation liability of GW796406. The low dose of GW796406 administered with apstatin, an APP inhibitor, did not increase plasma extravasation. This finding inferred that APP inhibition is not involved in plasma extravasation in the rat and that APP inhibition does not explain the increased plasma extravasation produced by omapatrilat in NEP-inhibited rats.

Effects of a selective bradykinin B 1 receptor antagonist on increased plasma extravasation in streptozotocin-induced diabetic rats: Distinct vasculopathic profile of major key organs

Diffuse vasculopathy is a common feature of the morbidity and increased mortality associated with insulino-dependent type 1 diabetes. Increased vascular permeability leading to plasma extravasation occurs in surrounding tissues following endothelial dysfunction. Such micro- and macro-vascular complications develop over time and lead to oedema, hypertension, cardiomyopathy, renal failure (nephropathy) and other complications (neuropathy, retinopathy). In the present investigation, we studied the effect of a selective bradykinin B 1 receptor antagonist, R-954, on the enhanced vascular permeability in streptozotocin (STZ)-induced diabetic Wistar rats compared with age-matched controls. Plasma extravasation was determined using Evans blue dye in selected target tissues (left and right heart atria, ventricles, lung, abdominal and thoracic aortas, liver, spleen, renal cortex and medulla), at 1 and 4 weeks following STZ administration. The vascular permeability was significantly increased in the aortas, cortex, medulla, and spleen in 1-week STZ rats and remained elevated at 4 weeks of diabetes. Both atria showed an increased vascular permeability only after 4-week STZ-administration. R-954 (2 mg/kg, bolus, s.c.), given 2 h prior to Evans blue dye, to 1- and 4-week diabetic rats significantly inhibited (by 48–100%) plasma leakage in most tested tissues affected by diabetes with no effect in healthy rats. These results showed that the inducible bradykinin B 1 receptor subtype participates in the modulation of the vascular permeability in diabetic rats and suggest that selective bradykinin B 1 receptor antagonism could have a beneficial role in reducing diabetic vascular complications.

Urotensin II stimulates plasma extravasation in mice via UT receptor activation

The peptide urotensin II (U-II) is the cognate ligand of the G-protein coupled receptor UT (formerly GPR14). A role in the regulation of cardiovascular functions has been proposed for this novel peptide/receptor system. In the present study, we evaluated the ability of U-II to induce plasma extravasation in mice and attempted to characterize the receptor involved using the novel UT receptor ligand, [Orn(8)]U-II. The Evans blue technique was used to quantify plasma extravasation. U-II (0.01, 0.1, 1 and 10 nmol/kg) dose-dependently stimulated plasma extravasation in airways, gastrointestinal and urogenital tract tissues from mice, but not in the skin. In most tissues, the dose/response curves to U-II were bell shaped with the maximal effect induced by 1 nmol/kg. [Orn(8)]U-II at 30 nmol/kg was per se either inactive or produced a non-significant increase in plasma extravasation; in the presence of 30 nmol/kg [Orn(8)]U-II, the effects of 1 nmol/kg U-II were always reduced and, in some tissues, abolished. The present findings demonstrate that U-II promotes plasma extravasation in various mouse vascular regions via activation of UT receptors. The mouse plasma extravasation assay will be a useful tool in future studies aimed at characterizing the pharmacological features of novel UT receptor ligands in vivo.

The TRPV1: a possible role in GERD, asthma and migraine

The transient receptor potential vanilloid type 1 (TRPV1) is a non‐selective ion channel that belongs to the TRP family of channels that are activated by vanilloid molecules, including capsaicin. The cloned TRPV1 is a thermosensor, gated by temperature (43–52°C) and low pH. Additional putative endogenous activators of the TRPV1 are the cannabinoid receptor agonist, anandamide, N‐arachydonoil‐dopamine and certain lipoxigenase metabolites of arachidonic acid, as 12‐HPETE and LTB4. The TRPV1 is expressed selectively in a subpopulation of primary sensory neurons with C‐ and A‐delta fibres which also express NGF receptors, the neuropeptides, substance P (SP), neurokinin A and calcitonin gene‐related peptide (CGRP). These neurons being activated by different sensory (mechanichal, thermal and chemical) modalities are defined as polymodal nociceptors, and the peptides released from their peripheral terminals cause neurogenic inflammation. TRPV1 seems to be coupled with PLC and PIP2 hydrolysis results in channel activation. Additional modes of TRPV1 sensitization comprise PKC‐ and PKA‐dependent pathways. Activation of either G‐protein‐coupled receptors or tyrosine kinase receptors causes TRPV1 sensitization. Of pathophysiological interest is the finding that endogenous and exogenous molecules may also cause TRPV1. Recently, we found that exposure to 0.3–3% ethanol causes a remarkable increase in Ca2+ mobilization in capsaicin‐sensitive cultured DRG neurons of newborn rat, an effect that was inhibited by the TRPV1 antagonist capsazepine. Wild‐type human embryonic kidney (HEK) cells did not respond to ethanol, but transfection with the human TRPV1 conferred to these cells the ability to respond to ethanol (Ca2+ mobilization) in a capsazepine‐sensitive manner. Electrophysiological studies showed that ethanol dramatically potentiated currents activated by anandamide and protons, and that ethanol reduced the threshold temperature for TRPV1 activation by about 8°C. Thus, in the presence of ethanol, the physiological temperature of 37°C is a sufficient stimulus to activate TRPV1 on sensory neurons and activate their afferent and efferent functions. Ethanol can trigger attacks of asthma and migraine and aggravates the symptoms of GERD. Exposure to ethanol caused a Ca2+‐dependent release of neuropeptides (SP/CGRP) from slices of rat esophagus and dura mater and guinea‐pig airways and increased plasma extravasation in the rat oesophagus and dura mater and caused bronchoconstriction in vitro and in vivo in guinea pigs. All these responses were inhibited selectively by capsazepine. Activation of sensory neurons and neurogenic inflammatory responses via TRPV1 stimulation may contribute to the mechanism of attacks of migraine and to the worsening of GERD and asthma symptoms precipitated by alcohol ingestion.

Animal models of chronic pain: Effect of repetitive joint movement on inflammation and pain in an animal model of monoarthritis

Range of motion (ROM) exercises improve the function of arthritic joints and are thus often prescribed to patients suffering with rheumatoid arthritis. However, effects of ROM exercise on disease progression and pain have not been demonstrated. We investigated the effects of manipulating the arthritic joint on mechanical withdrawal threshold and inflammation in a CFA-induced animal model of monoarthritis. Repetitive daily joint movements for 5 days improved the range of joint movement. However, this also led to increased swelling of the ankle and increased plasma extravasation, a monitor of loss of vascular endothelial integrity. Sensory testing of the affected joint to mechanical stimulation demonstrated a profound and persisting decrease in withdrawal threshold to stimulation with von Frey hairs after repetitive joint movement. To determine the role of endogenous opioids in modulating this decrease in withdrawal threshold a non-specific opioid antagonist, naloxone, was administered and the effect of joint movement was determined. There was a marked decrease in withdrawal threshold on all days, indicating an anti-nociceptive role of endogenous opioids. Our results demonstrate an improvement in the range of movement of an arthritic joint after daily repetitive joint movement. Surprisingly, our study also revealed an exacerbation of inflammation and pain sensitivity in the arthritic joint following repetitive joint movement; endogenous opioid mechanisms appear to mask these changes. These data raise questions regarding the value of exercising arthritic joints in people with rheumatoid arthritis. Range of motion (ROM) exercises improve the function of arthritic joints and are thus often prescribed to patients suffering with rheumatoid arthritis. However, effects of ROM exercise on disease progression and pain have not been demonstrated. We investigated the effects of manipulating the arthritic joint on mechanical withdrawal threshold and inflammation in a CFA-induced animal model of monoarthritis. Repetitive daily joint movements for 5 days improved the range of joint movement. However, this also led to increased swelling of the ankle and increased plasma extravasation, a monitor of loss of vascular endothelial integrity. Sensory testing of the affected joint to mechanical stimulation demonstrated a profound and persisting decrease in withdrawal threshold to stimulation with von Frey hairs after repetitive joint movement. To determine the role of endogenous opioids in modulating this decrease in withdrawal threshold a non-specific opioid antagonist, naloxone, was administered and the effect of joint movement was determined. There was a marked decrease in withdrawal threshold on all days, indicating an anti-nociceptive role of endogenous opioids. Our results demonstrate an improvement in the range of movement of an arthritic joint after daily repetitive joint movement. Surprisingly, our study also revealed an exacerbation of inflammation and pain sensitivity in the arthritic joint following repetitive joint movement; endogenous opioid mechanisms appear to mask these changes. These data raise questions regarding the value of exercising arthritic joints in people with rheumatoid arthritis.

Ethanol causes inflammation in the airways by a neurogenic and TRPV1-dependent mechanism.

Ethanol (EtOH) stimulates peptidergic primary sensory neurons via the activation of the transient receptor potential vanilloid-1 (TRPV1). EtOH is also known to trigger attacks of asthma in susceptible individuals. Our aim was to investigate whether EtOH produces airway inflammation via a TRPV1-dependent mechanism and to verify whether this effect is produced via a mechanism distinct from that of acetaldehyde (AcH). EtOH caused a Ca(2+)-dependent release of neuropeptides from guinea pigs airways, an effect that was inhibited by both capsaicin pretreatment and the TRPV1 antagonist capsazepine (CPZ). Furthermore, EtOH contracted isolated guinea pig bronchi, showing efficacy similar to that of carbachol: this effect of EtOH was sensitive to capsaicin pretreatment, tachykinin receptor blockade, and TRPV1 antagonism. The EtOH metabolite AcH also contracted isolated guinea pig bronchi, but this action was not affected by capsaicin pretreatment, tachykinin receptor, or TRPV1 antagonism. EtOH by intravenous or intragastric route of administration caused bronchoconstriction and increased plasma extravasation in the guinea pig airways, effects that were abolished selectively by CPZ. In conclusion, we have demonstrated that EtOH stimulates peptidergic primary sensory neurons in the guinea pig airways by TRPV1 activation. This excitatory effect of EtOH, distinct from that of AcH, results in neurogenic inflammatory responses that may contribute to the mechanism of EtOH-induced asthma.