Specific Inhibition of Orai1-mediated Calcium Signalling Resolves Inflammation and Clears Bacteria in an Acute Respiratory Distress Syndrome Model.

Rationale: Acute respiratory distress syndrome (ARDS) has an unacceptably high mortality rate (35%) and is without effective therapy. Orai1 is a Ca2+ channel involved in store-operated Ca2+ entry (SOCE), a process that exquisitely regulates inflammation. Orai1 is considered a druggable target, but no Orai1-specific inhibitors exist to date. Objectives: To evaluate whether ELD607, a first-in-class Orai1 antagonist, can treat ARDS caused by bacterial pneumonia in preclinical models. Methods: ELD607 pharmacology was evaluated in HEK293T cells and freshly isolated immune cells from patients with ARDS. A murine acute lung injury model caused by bacterial pneumonia was then used: mice were infected with Pseudomonas aeruginosa, Staphylococcus aureus, methicillin-resistant S. aureus, or multidrug-resistant P. aeruginosa and then treated with ELD607 intranasally. Measurements and Main Results: ELD607 specifically inhibited SOCE in HEK293T cells with a half-maximal inhibitory concentration of 9 nM. ELD607 was stable in ARDS airway secretions and inhibited SOCE in ARDS immune cells. In vivo, inhaled ELD607 significantly reduced neutrophilia and improved survival. Surprisingly, Orai1 inhibition by ELD607 caused a significant reduction in lung bacteria, including methicillin-resistant S. aureus. ELD607 worked as an immunomodulator that reduced cytokine levels, reduced neutrophilia, and promoted macrophage-mediated resolution of inflammation and clearance of bacteria. Indeed, when alveolar macrophages were depleted with inhaled clodronate, ELD607 was no longer able to resolve inflammation or clear bacteria. Conclusions: These data indicate that specific Orai1 inhibition by ELD607 may be a novel approach to reduce multiorgan inflammation and treat antibiotic-resistant bacteria.

Identification of redundancy between human FcεRIß and MS4A6A proteins points toward additional complex mechanisms for FcεRI trafficking and signaling.

BACKGROUND: Allergic diseases are triggered by signaling through the high-affinity IgE receptor, FcεRI. In both mast cells (MCs) and basophils, FcεRI is a tetrameric receptor complex comprising a ligand-binding α subunit (FcεRIα), a tetraspan ß subunit (FcεRIß, MS4A2) responsible for trafficking and signal amplification, and a signal transducing dimer of single transmembrane γ subunits (FcεRIγ). However, FcεRI also exists as presumed trimeric complexes that lack FcεRIß and are expressed on several cell types outside the MC and basophil lineages. Despite known differences between humans and mice in the presence of the trimeric FcεRI complex, questions remain as to how it traffics and whether it signals in the absence of FcεRIß. We have previously reported that targeting FcεRIß with exon-skipping oligonucleotides eliminates IgE-mediated degranulation in mouse MCs, but equivalent targeting in human MCs was not effective at reducing degranulation. RESULTS: Here, we report that the FcεRIß-like protein MS4A6A exists in human MCs and compensates for FcεRIß in FcεRI trafficking and signaling. Human MS4A6A promotes surface expression of FcεRI complexes and facilitates degranulation. MS4A6A and FcεRIß are encoded by highly related genes within the MS4A gene family that cluster within the human gene loci 11q12-q13, a region linked to allergy and asthma susceptibility. CONCLUSIONS: Our data suggest the presence of either FcεRIß or MS4A6A is sufficient for degranulation, indicating that MS4A6A could be an elusive FcεRIß-like protein in human MCs that performs compensatory functions in allergic disease.

Integrating digital inhalers into clinical care of patients with asthma and chronic obstructive pulmonary disease.

Modernizing inhaled medications through digital technology can help address persistent problems of non-adherence and poor inhaler technique in patients with obstructive lung diseases. With a growing body of supportive clinical studies, advances in digital inhaler sensors and platforms, greater support from payers and healthcare organizations, significant growth with these technologies is expected. While all digital (smart) inhalers record adherence, these are distinguished by their compatibility with commercial inhalers, capabilities to guide inhaler technique, use of patient-reported outcomes, and user-friendliness for both the healthcare professional (HCP) and patient. Due to the complexity and novelty of employing digital inhalers, collaboration with multiple entities within health systems is necessary and a well-planned integration is needed. For HCPs and patients, cybersecurity and privacy are critical, it will require review by each healthcare organization. In the US, some payers reimburse for remote monitoring using digital inhalers, but reimbursement is currently unavailable in other countries. There are several models for remote patient care, as employing an active, ongoing digital interface between the HCP and patient or they may choose to only review data at clinical encounters. Personalization of therapies and feedback are key to success. While digital inhaler malfunction uncommonly occurs, patient attrition over a year is significant. Some patients will be challenged to use digital platforms or have the necessary technology. Additional research is needed to address cost-effectiveness, in vivo accuracy of inspiratory measurement capable devices, ability to teach inhaler technique, their application for monitoring lung function, and lastly real-world adoption and implementation in routine clinical practice.

Neuropilin-2 regulates airway inflammation in a neutrophilic asthma model.

BACKGROUND: Asthma is a heterogenous disease that can be classified into eosinophilic (type 2-high) and noneosinophilic (type 2-low) endotypes. The type 2-low endotype of asthma can be characterized by the presence of neutrophilic airway inflammation that is poorly responsive to corticosteroids. Dysregulated innate immune responses to microbial products including Toll-like receptor (TLR) ligands have been associated with the pathogenesis of neutrophilic asthma. The key molecules that regulate inflammatory responses in individuals with neutrophilic asthma remain unclear. We previously reported that the immunoregulatory receptor neuropilin-2 (NRP2) is expressed by murine and human alveolar macrophage (AM) and suppresses lipopolysaccharide (LPS)-induced neutrophilic airway inflammation. METHODS: Here, we investigated the immunoregulatory role of NRP2 in a mouse model of neutrophilic asthma. RESULTS: We found that TLR ligands, but not T helper 2 (Th2)-promoting adjuvants, induced NRP2 expression by AM. Using an LPS-mediated model of neutrophilic asthma, we demonstrate that NRP2 was increased in AM and other lung antigen-presenting cells following airway challenge with antigen. Conditional deletion of NRP2 in myeloid cells exacerbated airway inflammation in a neutrophilic asthma model. In contrast, myeloid-specific ablation of NRP2 did not affect airway inflammation in a Th2-mediated eosinophilic asthma model. Myeloid-specific ablation of NRP2 did not affect Th1/Th17 responses to inhaled antigens or expression of neutrophil chemokines but rather resulted in impaired efferocytosis by AM, which is necessary for effective resolution of airway inflammation. CONCLUSION: Our findings suggest that NRP2 is a negative regulator of airway inflammation associated with neutrophilic asthma.

Identification of an ATP/P2X7/mast cell pathway mediating ozone-induced bronchial hyperresponsiveness.

Ozone is a highly reactive environmental pollutant with well-recognized adverse effects on lung health. Bronchial hyperresponsiveness (BHR) is one consequence of ozone exposure, particularly for individuals with underlying lung disease. Our data demonstrated that ozone induced substantial ATP release from human airway epithelia in vitro and into the airways of mice in vivo and that ATP served as a potent inducer of mast cell degranulation and BHR, acting through P2X7 receptors on mast cells. Both mast cell-deficient and P2X7 receptor-deficient (P2X7-/-) mice demonstrated markedly attenuated BHR to ozone. Reconstitution of mast cell-deficient mice with WT mast cells and P2X7-/- mast cells restored ozone-induced BHR. Despite equal numbers of mast cells in reconstituted mouse lungs, mice reconstituted with P2X7-/- mast cells demonstrated significantly less robust BHR than mice reconstituted with WT mast cells. These results support a model where P2X7 on mast cells and other cell types contribute to ozone-induced BHR.

Digital Inhalers for Asthma or Chronic Obstructive Pulmonary Disease: A Scientific Perspective.

Impressive advances in inhalation therapy for patients with asthma and chronic obstructive pulmonary disease (COPD) have occurred in recent years. However, important gaps in care remain, particularly relating to poor adherence to inhaled therapies. Digital inhaler health platforms which incorporate digital inhalers to monitor time and date of dosing are an effective disease and medication management tool, promoting collaborative care between clinicians and patients, and providing more in-depth understanding of actual inhaler use. With advances in technology, nearly all inhalers can be digitalized with add-on or embedded sensors to record and transmit data quantitating inhaler actuations, and some have additional capabilities to evaluate inhaler technique. In addition to providing an objective and readily available measure of adherence, they allow patients to interact with the device directly or through their self-management smartphone application such as via alerts and recording of health status. Clinicians can access these data remotely and during patient encounters, to better inform them about disease status and medication adherence and inhaler technique. The ability for remote patient monitoring is accelerating interest in and the use of these devices in clinical practice and research settings. More than 20 clinical studies of digital inhalers in asthma or COPD collectively show improvement in medication adherence, exacerbation risk, and patient outcomes with digital inhalers. These studies support previous findings about patient inhaler use and behaviors, but with greater granularity, and reveal some new findings about patient medication-taking behaviors. Digital devices that record inspiratory flows with inhaler use can guide proper inhaler technique and may prove to be a clinically useful lung function measure. Adoption of digital inhalers into practice is still early, and additional research is needed to determine patient and clinician acceptability, the appropriate place of these devices in the therapeutic regimen, and their cost effectiveness. Video: Digital Inhalers for Asthma or Chronic Obstructive Pulmonary Disease: A Scientific Perspective (MP4 74535 kb).

Adenosine A2A receptor and vascular response: role of soluble epoxide hydrolase, adenosine A1 receptor and angiotensin-II.

Previously, we have reported that the coronary reactive hyperemic response was reduced in adenosine A2A receptor-null (A2AAR-/-) mice, and it was reversed by the soluble epoxide hydrolase (sEH) inhibitor. However, it is unknown in aortic vascular response, therefore, we hypothesized that A2AAR-gene deletion in mice (A2AAR-/-) affects adenosine-induced vascular response by increase in sEH and adenosine A1 receptor (A1AR) activities. A2AAR-/- mice showed an increase in sEH, AI AR and CYP450-4A protein expression but decrease in CYP450-2C compared to C57Bl/6 mice. NECA (adenosine-analog) and CCPA (adenosine A1 receptor-agonist)-induced dose-dependent vascular response was tested with t-AUCB (sEH-inhibitor) and angiotensin-II (Ang-II) in A2AAR-/- vs. C57Bl/6 mice. In A2AAR-/-, NECA and CCPA-induced increase in dose-dependent vasoconstriction compared to C57Bl/6 mice. However, NECA and CCPA-induced dose-dependent vascular contraction in A2AAR-/- was reduced by t-AUCB with NECA. Similarly, dose-dependent vascular contraction in A2AAR-/- was reduced by t-AUCB with CCPA. In addition, Ang-II enhanced NECA and CCPA-induced dose-dependent vascular contraction in A2AAR-/- with NECA. Similarly, the dose-dependent vascular contraction in A2AAR-/- was also enhanced by Ang-II with CCPA. Further, t-AUCB reduced Ang-II-enhanced NECA and CCPA-induced dose-dependent vascular contraction in A2AAR-/- mice. Our data suggest that the dose-dependent vascular contraction in A2AAR-/- mice depends on increase in sEH, A1AR and CYP4A protein expression.

The FcεRIß homologue, MS4A4A, promotes FcεRI signal transduction and store-operated Ca2+ entry in human mast cells.

Members of the membrane spanning 4A (MS4A) gene family are clustered around 11q12-13, a region linked to allergy and asthma susceptibility. Other than the known functions of FcεRIß (MS4A2) and CD20 (MS4A1) in mast cell and B cell signaling, respectively, functional studies for the remaining MS4A proteins are lacking. We thus explored whether MS4A4A, a mast cell expressed homologue of FcεRIß, has related functions to FcεRIß in FcεRI signaling. We establish in this study that MS4A4A promotes phosphorylation of PLCγ1, calcium flux and degranulation in response to IgE-mediated crosslinking of FcεRI. We previously demonstrated that MS4A4A promotes recruitment of KIT into caveolin-1-enriched microdomains and signaling through PLCγ1. Caveolin-1 itself is an important regulator of IgE-dependent store-operated Ca2+ entry (SOCE) and promotes expression of the store-operated Ca2+ channel pore-forming unit, Orai1. We thus further report that MS4A4A functions through interaction with caveolin-1 and recruitment of FcεRI and KIT into lipid rafts. In addition to proximal FcεRI signaling, we similarly show that MS4A4A regulates Orai1-mediated calcium entry downstream of calcium release from stores. Both MS4A4A and Orai1 had limited effects with compound 48/80 stimulation, demonstrating some degree of selectivity of both proteins to FcεRI receptor signaling over Mas-related G Protein coupled receptor X2 signaling. Overall, our data are consistent with the conclusion that MS4A4A performs a related function to the homologous FcεRIß to promote PLCγ1 signaling, SOCE, and degranulation through FcεRI in human mast cells and thus represents a new target in the regulation of IgE-mediated mast cell activation.

Impaired bone marrow B-cell development in mice with a bronchiolitis obliterans model of cGVHD.

Chronic graft-versus-host disease (cGVHD) causes significant morbidity and mortality in patients after allogeneic bone marrow (BM) or stem cell transplantation (allo-SCT). Recent work has indicated that both T and B lymphocytes play an important role in the pathophysiology of cGVHD. Previously, our group showed a critical role for the germinal center response in the function of B cells using a bronchiolitis obliterans (BO) model of cGVHD. Here, we demonstrated for the first time that cGVHD is associated with severe defects in the generation of BM B lymphoid and uncommitted common lymphoid progenitor cells. We found an increase in the number of donor CD4+ T cells in the BM of mice with cGVHD that was negatively correlated with B-cell development and the frequency of osteoblasts and Prrx-1-expressing perivascular stromal cells, which are present in the B-cell niche. Use of anti-DR3 monoclonal antibodies to enhance the number of donor regulatory T cells (Tregs) in the donor T-cell inoculum ameliorated the pathology associated with BO in this model. This correlated with an increased number of endosteal osteoblastic cells and significantly improved the generation of B-cell precursors in the BM after allo-SCT. Our work indicates that donor Tregs play a critical role in preserving the generation of B-cell precursors in the BM after allo-SCT. Approaches to enhance the number and/or function of donor Tregs that do not enhance conventional T-cell activity may be important to decrease the incidence and severity of cGVHD in part through normal B-cell lymphopoiesis.

Neuropilin-2 regulates airway inflammatory responses to inhaled lipopolysaccharide.

Neuropilins are multifunctional receptors that play important roles in immune regulation. Neuropilin-2 (NRP2) is expressed in the lungs, but whether it regulates airway immune responses is unknown. Here, we report that Nrp2 is weakly expressed by alveolar macrophages (AMs) in the steady state but is dramatically upregulated following in vivo lipopolysaccharide (LPS) inhalation. Ex vivo treatment of human AMs with LPS also increased NRP2 mRNA expression and cell-surface display of NRP2 protein. LPS-induced Nrp2 expression in AMs was dependent upon the myeloid differentiation primary response 88 signaling pathway and the transcription factor NF-κB. In addition to upregulating display of NRP2 on the cell membrane, inhaled LPS also triggered AMs to release soluble NRP2 into the airways. Finally, myeloid-specific ablation of NRP2 resulted in increased expression of the chemokine (C-C motif) ligand 2 ( Ccl2) in the lungs and prolonged leukocyte infiltration in the airways following LPS inhalation. These findings suggest that NRP2 expression by AMs regulates LPS-induced inflammatory cell recruitment to the airways and reveal a novel role for NRP2 during innate immune responses in the lungs.

Reduced coronary reactive hyperemia in mice was reversed by the soluble epoxide hydrolase inhibitor (t-AUCB): Role of adenosine A2A receptor and plasma oxylipins.

Coronary reactive hyperemia (CRH) protects the heart against ischemia. Adenosine A2AAR-deficient (A2AAR-/-) mice have increased expression of soluble epoxide hydrolase (sEH); the enzyme responsible for breaking down the cardioprotective epoxyeicosatrienoic acids (EETs) to dihydroxyeicosatrienoic acids (DHETs). sEH-inhibition enhances CRH, increases EETs, and modulates oxylipin profiles. We investigated the changes of oxylipins and their impact on CRH in A2AAR-/- and wild type (WT) mice. We hypothesized that the attenuated CRH in A2AAR-/- mice is mediated by changes in oxylipin profiles, and that it can be reversed by either sEH- or ω-hydroxylases-inhibition. Compared to WT mice, A2AAR-/- mice had attenuated CRH and changed oxylipin profiles, which were consistent between plasma and heart perfusate samples, including decreased EET/DHET ratios, and increased hydroxyeicosatetraenoic acids (HETEs). Plasma oxylipns in A2AAR-/- mice indicated an increased proinflammatory state including increased ω-terminal HETEs, decreased epoxyoctadecaenoic/dihydroxyoctadecaenoic acids (EpOMEs/DiHOMEs) ratios, increased 9-hydroxyoctadecadienoic acid, and increased prostanoids. Inhibition of either sEH or ω-hydroxylases reversed the reduced CRH in A2AAR-/- mice. In WT and sEH-/- mice, blocking A2AAR decreased CRH. These data demonstrate that A2AAR-deletion was associated with changes in oxylipin profiles, which may contribute to the attenuated CRH. Also, inhibition of sEH and ω-hydroxylases reversed the reduction in CRH.

Targeting the Canonical Nuclear Factor-κB Pathway with a High-Potency IKK2 Inhibitor Improves Outcomes in a Mouse Model of Idiopathic Pneumonia Syndrome.

Idiopathic pneumonia syndrome (IPS) is a noninfectious inflammatory disorder of the lungs that occurs most often after fully myeloablative allogeneic hematopoietic stem cell transplantation (HSCT). IPS can be severe and is associated with high 1-year mortality rates despite existing therapies. The canonical nuclear factor-(NF) κB signaling pathway has previously been linked to several inflammatory disorders of the lung, including asthma and lung allograft rejection. It has never been specifically targeted as a novel IPS treatment approach, however. Here, we report that the IκB kinase 2 (IKK2) antagonist BAY 65-5811 or "compound A," a highly potent and specific inhibitor of the NF-κB pathway, was able to improve median survival times and recipient oxygenation in a well-described mouse model of IPS. Compound A impaired the production of the proinflammatory chemokines CCL2 and CCL5 within the host lung after transplantation. This resulted in significantly lower numbers of donor lung infiltrating CD4+ and CD8+ T cells and reduced pulmonary inflammatory cytokine production after allograft. Compound A's beneficial effects appeared to be specific for limiting pulmonary injury, as the drug was unable to improve outcomes in a B6 into B6D2 haplotype-matched murine HSCT model in which recipient mice succumb to lethal acute graft-versus-host disease of the gastrointestinal tract. Collectively, our data suggest that the targeting of the canonical NF-κB pathway with a small molecule IKK2 antagonist may represent an effective and novel therapy for the specific management of acute lung injury that can occur after allogeneic HSCT.

Identification of BPIFA1/SPLUNC1 as an epithelium-derived smooth muscle relaxing factor.

Asthma is a chronic airway disease characterized by inflammation, mucus hypersecretion and abnormal airway smooth muscle (ASM) contraction. Bacterial permeability family member A1, BPIFA1, is a secreted innate defence protein. Here we show that BPIFA1 levels are reduced in sputum samples from asthmatic patients and that BPIFA1 is secreted basolaterally from healthy, but not asthmatic human bronchial epithelial cultures (HBECs), where it suppresses ASM contractility by binding to and inhibiting the Ca2+ influx channel Orai1. We have localized this effect to a specific, C-terminal α-helical region of BPIFA1. Furthermore, tracheas from Bpifa1-/- mice are hypercontractile, and this phenotype is reversed by the addition of recombinant BPIFA1. Our data suggest that BPIFA1 deficiency in asthmatic airways promotes Orai1 hyperactivity, increased ASM contraction and airway hyperresponsiveness. Strategies that target Orai1 or the BPIFA1 deficiency in asthma may lead to novel therapies to treat this disease.

In vivo assessment of coronary flow and cardiac function after bolus adenosine injection in adenosine receptor knockout mice.

Bolus injections of adenosine and the A2A adenosine receptor (AR) selective agonist (regadenoson) are used clinically as a substitute for a stress test in people who cannot exercise. Using isolated tissue preparations, our lab has shown that coronary flow and cardiac effects of adenosine are mostly regulated by the AR subtypes A1, A2A, and A2B In this study, we used ultrasound imaging to measure the in vivo effects of adenosine on coronary blood flow (left coronary artery) and cardiac function in anesthetized wild-type, A1 knockout (KO), A2AKO, A2BKO, A3KO, A1, and A3 double KO (A1/3 DKO) and A2A and A2B double KO (A2A/2B DKO) mice in real time. Echocardiographic and Doppler studies were performed using a Visualsonic Vevo 2100 ultrasound system. Coronary blood flow (CBF) baseline data were obtained when animals were anesthetized with 1% isoflourane. Diameter (D) and velocity time integral (VTI) were measured on the left coronary arteries (CBF = ((π/4) × D(2) × VTI × HR)/1000). CBF changes were the highest within 2 min of injection (about 10 mg/kg). Heart rate, cardiac output, and stroke volume were measured by tracing the left ventricle long axis. Our data support a role for the A2 AR in CBF and further support our conclusions of previous studies from isolated tissues. Adenosine-mediated decreases in cardiac output and stroke volume may be A2B and/or A3 AR-mediated; however, the A1 and A2 ARs also play roles in overall cardiac function. These data further provide a powerful translational tool in studying the cardiovascular effects of adenosine in disease states.

Role of Adenosine Receptor(s) in the Control of Vascular Tone in the Mouse Pudendal Artery.

Activation of adenosine receptors (ARs) has been implicated in the modulation of renal and cardiovascular systems, as well as erectile functions. Recent studies suggest that adenosine-mediated regulation of erectile function is mainly mediated through A2BAR activation. However, no studies have been conducted to determine the contribution of AR subtype in the regulation of the vascular tone of the pudendal artery (PA), the major artery supplying and controlling blood flow to the penis. Our aim was to characterize the contribution of AR subtypes and identify signaling mechanisms involved in adenosine-mediated vascular tone regulation in the PA. We used a DMT wire myograph for muscle tension measurements in isolated PAs from wild-type, A2AAR knockout, A2BAR knockout, and A2A/A2BAR double-knockout mice. Real-time reverse transcription-polymerase chain reaction was used to determine the expression of the AR subtypes. Data from our pharmacologic and genetic approaches suggest that AR activation-mediated vasodilation in the PA is mediated by both the A2AAR and A2BAR, whereas neither the A1AR nor A3AR play a role in vascular tone regulation of the PA. In addition, we showed that A2AAR- and A2BAR-mediated vasorelaxation requires activation of nitric oxide and potassium channels; however, only the A2AAR-mediated response requires protein kinase A activation. Our data are complemented by mRNA expression showing the expression of all AR subtypes with the exception of the A3AR. AR signaling in the PA may play an important role in mediating erection and represent a promising therapeutic option for the treatment of erectile dysfunction.

Mast Cell Deficiency Limits the Development of Chronic Rhinosinusitis in Mice.

BACKGROUND: Chronic rhinosinusitis (CRS) is one of the most common chronic diseases in adults in both developing and developed countries. The etiology and pathogenesis of CRS remain poorly understood, and the disease is refractory to therapy in many patients. Mast cell activation has been demonstrated in the sinonasal mucosa of patients with CRS; however, the specific contribution of mast cells to the development and pathogenesis of this disease has not been established. OBJECTIVE: The objective of this study was to investigate the role of mast cells in the development of CRS. METHODS: C57BL/6 wild-type and C57BL/6-Kit(W-sh/W-sh) mast cell-deficient mice were immunized by intraperitoneal allergen injection and subsequent chronic low dose intranasal allergen challenges. The sinonasal phenotypes of these groups were then evaluated and compared to saline-treated controls using radiologic, histologic, and immunologic methods. RESULTS: Wild-type mice exposed to chronic intranasal allergen developed many features seen in human CRS, including mucosal thickening, cystic changes, polyp development, eosinophilia, goblet cell hyperplasia, and mast cell activation. In contrast, sinonasal pathology was significantly attenuated in mast cell-deficient mice subjected to the same chronic allergen protocol. Specifically, tissue eosinophilia and goblet cell hyperplasia were reduced by approximately 50% compared to wild-type levels. Surprisingly, none of the mast cell-deficient mice subjected to chronic allergen challenge developed cystic changes or polypoid changes in the nose or sinuses. CONCLUSIONS: These data identify a critical role for mast cells in the development of many features of a mouse model of eosinophilic CRS, suggesting that therapeutic strategies targeting mast cells be examined in humans afflicted with this disease.

Angiotensin II stimulation alters vasomotor response to adenosine in mouse mesenteric artery: role for A1 and A2B adenosine receptors.

BACKGROUND AND PURPOSE: Stimulation of the A1 adenosine receptor and angiotensin II receptor type-1 (AT1 receptor) causes vasoconstriction through activation of cytochrome P450 4A (CYP4A) and ERK1/2. Thus, we hypothesized that acute angiotensin II activation alters the vasomotor response induced by the non-selective adenosine receptor agonist, NECA, in mouse mesenteric arteries (MAs). EXPERIMENTAL APPROACH: We used a Danish Myo Technology wire myograph to measure muscle tension in isolated MAs from wild type (WT), A1 receptor and A2B receptor knockout (KO) mice. Western blots were performed to determine the expression of AT1 receptors and CYP4A. KEY RESULTS: Acute exposure (15 min) to angiotensin II attenuated the NECA-dependent vasodilatation and enhanced vasoconstriction. This vasoconstrictor effect of angiotensin II in NECA-treated MAs was abolished in A1 receptor KO mice and in WT mice treated with the A1 receptor antagonist DPCPX, CYP4A inhibitor HET0016 and ERK1/2 inhibitor PD98059. In MAs from A2B receptor KO mice, the vasoconstrictor effect of angiotensin II on the NECA-induced response was shown to be dependent on A1 receptors. Furthermore, in A2B receptor KO mice, the expression of AT1 receptors and CYP4A was increased and the angiotensin II-induced vasoconstriction enhanced. In addition, inhibition of KATP channels with glibenclamide significantly reduced NECA-induced vasodilatation in WT mice. CONCLUSIONS AND IMPLICATIONS: Acute angiotensin II stimulation enhanced A1 receptor-dependent vasoconstriction and inhibited A2B receptor-dependent vasodilatation, leading to a net vasoconstriction and altered vasomotor response to NECA in MAs. This interaction may be important in the regulation of BP.