Evaluating Associations between Average Pain Intensity and Genetic Variation in People with Sickle Cell Disease: An Exploratory Study.

BACKGROUND: Pain is one of the most common and deleterious symptoms experienced by individuals with sickle cell disease (SCD). There is a paucity of studies identifying potential genetic mechanisms of pain in this population. AIM: Examine associations between 11 functional single nucleotide polymorphisms in 9 candidate genes with reports of average pain intensity in individuals with sickle cell disease. METHOD: Cross-sectional analyses were performed on data and blood samples collected through the Duke SCD Implementation Consortium Registry. Participants were asked to rate their pain "on the average" using an 11-point numeric rating scale (0 = no pain; 10 = pain as bad as you can imagine). We genotyped 11 single nucleotide polymorphisms in 9 pain-related genes using TaqMan® Genotyping Assays. Associations between each polymorphism and reports of average pain were evaluated. RESULTS: The 86 participants (mean age: 28.7 years; 64% female) included in this study reported moderate pain on average (Mean = 4, Standard Deviation = 2.4). ICAM1 rs1799969 was the only genetic polymorphism that was significantly associated with pain (p = .01). Individuals with one or more minor alleles had lower average pain (Mean = 1.25, Standard Deviation = 1.50) than individuals without a minor allele (Mean = 4.13, Standard Deviation = 2.25). The effect size for ICAM1 rs1799969 was 1.30, which is considered large. The effect sizes for all other single nucleotide polymorphisms ranged from small to medium (range: 0-0.3). CONCLUSIONS: Our findings provide preliminary evidence that the minor allele in ICAM1 rs1799969 had protective effects against experiencing more severe pain in sickle cell disease.

Exogenous leptin enhances markers of airway fibrosis in a mouse model of chronic allergic airways disease.

BACKGROUND: Asthma patients with comorbid obesity exhibit increased disease severity, in part, due to airway remodeling, which is also observed in mouse models of asthma and obesity. A mediator of remodeling that is increased in obesity is leptin. We hypothesized that in a mouse model of allergic airways disease, mice receiving exogenous leptin would display increased airway inflammation and fibrosis. METHODS: Five-week-old male and female C57BL/6J mice were challenged with intranasal house dust mite (HDM) allergen or saline 5 days per week for 6 weeks (n = 6-9 per sex, per group). Following each HDM exposure, mice received subcutaneous recombinant human leptin or saline. At 48 h after the final HDM challenge, lung mechanics were evaluated and the mice were sacrificed. Bronchoalveolar lavage was performed and differential cell counts were determined. Lung tissue was stained with Masson's trichrome, periodic acid-Schiff, and hematoxylin and eosin stains. Mouse lung fibroblasts were cultured, and whole lung mRNA was isolated. RESULTS: Leptin did not affect mouse body weight, but HDM+leptin increased baseline blood glucose. In mixed-sex groups, leptin increased mouse lung fibroblast invasiveness and increased lung Col1a1 mRNA expression. Total lung resistance and tissue damping were increased with HDM+leptin treatment, but not leptin or HDM alone. Female mice exhibited enhanced airway responsiveness to methacholine with HDM+leptin treatment, while leptin alone decreased total respiratory system resistance in male mice. CONCLUSIONS: In HDM-induced allergic airways disease, administration of exogenous leptin to mice enhanced lung resistance and increased markers of fibrosis, with differing effects between males and females.

A deep learning approach to student registered nurse anesthetist (SRNA) education.

OBJECTIVES: This manuscript describes the application of deep learning to physiology education of Student Registered Nurse Anesthetists (SRNA) and the benefits thereof. A strong foundation in physiology and the ability to apply this knowledge to challenging clinical situations is crucial to the successful SRNA. Deep learning, a well-studied pedagogical technique, facilitates development and long-term retention of a mental knowledge framework that can be applied to complex problems. Deep learning requires the educator to facilitate the development of critical thinking and students to actively learn and take responsibility for gaining knowledge and skills. METHODS: We applied the deep learning approach, including flipped classroom and problem-based learning, and surveyed SRNA students (n=127) about their learning experience. RESULTS: Survey responses showed that the majority of students favored the deep learning approach and thought it advanced their critical thinking skills. CONCLUSIONS: SRNAs reported that their physiology knowledge base and critical thinking benefited from the use of the deep learning strategy.

ß-Arrestin-2-Dependent Signaling Promotes CCR4-mediated Chemotaxis of Murine T-Helper Type 2 Cells.

Allergic asthma is a complex inflammatory disease that leads to significant healthcare costs and reduction in quality of life. Although many cell types are implicated in the pathogenesis of asthma, CD4+ T-helper cell type 2 (Th2) cells are centrally involved. We previously reported that the asthma phenotype is virtually absent in ovalbumin-sensitized and -challenged mice that lack global expression of ß-arrestin (ß-arr)-2 and that CD4+ T cells from these mice displayed significantly reduced CCL22-mediated chemotaxis. Because CCL22-mediated activation of CCR4 plays a role in Th2 cell regulation in asthmatic inflammation, we hypothesized that CCR4-mediated migration of CD4+ Th2 cells to the lung in asthma may use ß-arr-dependent signaling. To test this hypothesis, we assessed the effect of various signaling inhibitors on CCL22-induced chemotaxis using in vitro-polarized primary CD4+ Th2 cells from ß-arr2-knockout and wild-type mice. Our results show, for the first time, that CCL22-induced, CCR4-mediated Th2 cell chemotaxis is dependent, in part, on a ß-arr2-dependent signaling pathway. In addition, we show that this chemotactic signaling mechanism involves activation of P-p38 and Rho-associated protein kinase. These findings point to a proinflammatory role for ß-arr2-dependent signaling and support ß-arr2 as a novel therapeutic target in asthma.

Targeted HAS2 Expression Lessens Airway Responsiveness in Chronic Murine Allergic Airway Disease.

Hyaluronan (HA), a major component of the extracellular matrix, is secreted by airway structural cells. Airway fibroblasts in allergic asthma secrete elevated levels of HA in association with increased HA synthase 2 (HAS2) expression. Thus, we hypothesized that HA accumulation in the airway wall may contribute to airway remodeling and hyperresponsiveness in allergic airways disease. To examine this hypothesis, transgenic mice in which the α-smooth muscle actin (α-SMA) promoter drives HAS2 expression were generated. Mixed male and female α-SMA-HAS2 mice (HAS2+ mice, n = 16; HAS2- mice, n = 13) were sensitized via intraperitoneal injection and then chronically challenged with aerosolized ovalbumin (OVA) for 6 weeks. To test airway responsiveness, increasing doses of methacholine were delivered intravenously and airway resistance was measured using the forced oscillation technique. HA, cytokines, and cell types were analyzed in bronchoalveolar lavage fluid, serum, and whole lung homogenates. Lung sections were stained using antibodies specific for HA-binding protein (HABP) and α-SMA, as well as Masson's trichrome stain. Staining of lung tissue demonstrated significantly increased peribronchial HA, α-SMA, and collagen deposition in OVA-challenged α-SMA-HAS2+ mice compared with α-SMA-HAS2- mice. Unexpectedly, OVA-challenged α-SMA-HAS2+ mice displayed significantly reduced airway responsiveness to methacholine compared with similarly treated α-SMA-HAS2- mice. The total numbers of inflammatory cell types in the bronchoalveolar lavage fluid did not differ significantly between OVA-challenged α-SMA-HAS2+ mice and α-SMA-HAS2- mice. We conclude that allergen-challenged mice that overexpress HAS2 in myofibroblasts and smooth muscle cells develop increased airway fibrosis, which lessens airway hyperresponsiveness to bronchoconstrictors.

Phosphodiesterase 4 Inhibitors Attenuate the Asthma Phenotype Produced by ß2-Adrenoceptor Agonists in Phenylethanolamine N-Methyltransferase-Knockout Mice.

Mice lacking the endogenous ß2-adrenoceptor (ß2AR) agonist epinephrine (phenylethanolamine N-methyltransferase [PNMT]-knockout mice) are resistant to developing an "asthma-like" phenotype in an ovalbumin sensitization and challenge (Ova S/C) model, and chronic administration of ß2AR agonists to PNMT-KO mice restores the phenotype. Based on these and other studies showing differential effects of various ß2AR ligands on the asthma phenotype, we have speculated that the permissive effect of endogenous epinephrine and exogenous ß2AR agonists on allergic lung inflammation can be explained by qualitative ß2AR signaling. The ß2AR can signal through at least two pathways: the canonical Gαs-cAMP pathway and a ß-arrestin-dependent pathway. Previous studies suggest that ß-arrestin-2 is required for allergic lung inflammation. On the other hand, cell-based assays suggest antiinflammatory effects of Gαs-cAMP signaling. This study was designed to test whether the in vitro antiinflammatory effects of phosphodiesterase 4 inhibitors, known to increase intracellular cAMP in multiple airway cell types, attenuate the asthma-like phenotype produced by the ß2AR agonists formoterol and salmeterol in vivo in PNMT-KO mice, based on the hypothesis that skewing ß2AR signaling toward Gαs-cAMP pathway is beneficial. Airway inflammatory cells, epithelial mucus production, and airway hyperresponsiveness were quantified. In Ova S/C PNMT-KO mice, formoterol and salmeterol restored the asthma-like phenotype comparable to Ova S/C wild-type mice. However, coadministration of either roflumilast or rolipram attenuated this formoterol- or salmeterol-driven phenotype in Ova S/C PNMT-KO. These findings suggest that amplification of ß2AR-mediated cAMP by phosphodiesterase 4 inhibitors attenuates the asthma-like phenotype promoted by ß-agonists.

Specificity of arrestin subtypes in regulating airway smooth muscle G protein-coupled receptor signaling and function.

Arrestins have been shown to regulate numerous G protein-coupled receptors (GPCRs) in studies employing receptor/arrestin overexpression in artificial cell systems. Which arrestin isoforms regulate which GPCRs in primary cell types is poorly understood. We sought to determine the effect of ß-arrestin-1 or ß-arrestin-2 inhibition or gene ablation on signaling and function of multiple GPCRs endogenously expressed in airway smooth muscle (ASM). In vitro [second messenger (calcium, cAMP generation)], ex vivo (ASM tension generation in suspended airway), and in vivo (invasive airway resistance) analyses were performed on human ASM cells and murine airways/whole animal subject to ß-arrestin-1 or -2 knockdown or knockout (KO). In both human and murine model systems, knockdown or KO of ß-arrestin-2 relative to control missense small interfering RNA or wild-type mice selectively increased (40-60%) ß2-adrenoceptor signaling and function. ß-arrestin-1 knockdown or KO had no effect on signaling and function of ß2-adrenoceptor or numerous procontractile GPCRs, but selectively inhibited M3 muscarinic acetylcholine receptor signaling (∼50%) and function (∼25% ex vivo, >50% in vivo) without affecting EC50 values. Arrestin subtypes differentially regulate ASM GPCRs and ß-arrestin-1 inhibition represents a novel approach to managing bronchospasm in obstructive lung diseases.

Genetic Deletion of ß-Arrestin-2 and the Mitigation of Established Airway Hyperresponsiveness in a Murine Asthma Model.

ß-Arrestin-2 (ßarr2) is a ubiquitously expressed cytosolic protein that terminates G protein-coupled receptor signaling and transduces G protein-independent signaling. We previously showed that mice lacking ßarr2 do not develop an asthma phenotype when sensitized to, and challenged with, allergens. The current study evaluates if an established asthma phenotype can be mitigated by deletion of ßarr2 using an inducible Cre recombinase. We sensitized and challenged mice to ovalbumin (OVA) and demonstrated that on Day (d) 24 the allergic asthma phenotype was apparent in uninduced ßarr2 and wild-type (WT) mice. In a second group of OVA-treated mice, tamoxifen was injected on d24 to d28 to activate Cre recombinase, and OVA aerosol challenge was continued through d44. The asthma phenotype was assessed using lung mechanics measurements, bronchoalveolar lavage cell analysis, and histological assessment of mucin and airway inflammation. Compared with their respective saline-treated controls, OVA-treated WT mice and mice expressing the inducible Cre recombinase displayed a significant asthma phenotype at d45. Whereas tamoxifen treatment had no significant effect on the asthma phenotype in WT mice, it inhibited ßarr2 expression and caused a significant reduction in airway hyper-responsiveness (AHR) in Cre-inducible mice. These findings suggest that ßarr2 is actively required for perpetuation of the AHR component of the allergic asthma phenotype. Our finding that ßarr2 participates in the perpetuation of AHR in an asthma model means that targeting ßarr2 may provide immediate and potentially long-term relief from daily asthma symptoms due to AHR irrespective of inflammation.

ß-Arrestin-2 mediates the proinflammatory effects of proteinase-activated receptor-2 in the airway.

Proteinase-Activated receptor-2 (PAR(2)), a G-protein-coupled Receptor, activated by serine proteinases, is reported to have both protective and proinflammatory effects in the airway. Given these opposing actions, both inhibitors and activators of PAR(2) have been proposed for treating asthma. PAR(2) can signal through two independent pathways: a ß-arrestin-dependent one that promotes leukocyte migration, and a G-protein/Ca(2+) one that is required for prostaglandin E(2) (PGE(2)) production and bronchiolar smooth muscle relaxation. We hypothesized that the proinflammatory responses to PAR(2) activation are mediated by ß-arrestins, whereas the protective effects are not. Using a mouse ovalbumin model for PAR(2)-modulated airway inflammation, we observed decreased leukocyte recruitment, cytokine production, and mucin production in ß-arrestin-2(-/-) mice. In contrast, PAR(2)-mediated PGE(2) production, smooth muscle relaxation, and decreased baseline airway resistance (measures of putative PAR(2) "protective" effects) were independent of ß-arrestin-2. Flow cytometry and cytospins reveal that lung eosinophil and CD4 T-cell infiltration, and production of IL-4, IL-6, IL-13, and TNFα, were enhanced in wild-type but not ß-arrestin-2(-/-) mice. Using the forced oscillation technique to measure airway resistance reveals that PAR(2) activation protects against airway hyperresponsiveness by an unknown mechanism, possibly involving smooth muscle relaxation. Our data suggest that the PAR(2)-enhanced inflammatory process is ß-arrestin-2 dependent, whereas the protective anticonstrictor effect of bronchial epithelial PAR(2) may be ß-arrestin independent.

GPCRs and arrestins in airways: implications for asthma.

The obstructive lung disease asthma is treated by drugs that target, either directly or indirectly, G protein-coupled receptors (GPCRs). GPCRs coupled to Gq are the primary mediators of airway smooth muscle (ASM) contraction and increased airway resistance, whereas the Gs-coupled beta-2-adrenoceptor (ß2AR) promotes pro-relaxant signaling in and relaxation of ASM resulting in greater airway patency and reversal of life-threatening bronchoconstriction. In addition, GPCR-mediated functions in other cell types, including airway epithelium and hematopoietic cells, are involved in the control of lung inflammation that causes most asthma. The capacity of arrestins to regulate GPCR signaling, via either control of GPCR desensitization/resensitization or G protein-independent signaling, renders arrestins an intriguing therapeutic target for asthma and other obstructive lung diseases. This review will focus on the potential role of arrestins in those GPCR-mediated airway cell functions that are dysregulated in asthma.

ß2-Adrenoceptor agonists are required for development of the asthma phenotype in a murine model.

ß(2)-Adrenoceptor (ß2AR) agonists are the most effective class of bronchodilators and a mainstay of asthma management. The first potent ß2AR agonist discovered and widely used in reversing the airway constriction associated with asthma exacerbation was the endogenous activator of the ß2AR, epinephrine. In this study, we demonstrate that activation of the ß2AR by epinephrine is paradoxically required for development of the asthma phenotype. In an antigen-driven model, mice sensitized and challenged with ovalbumin showed marked elevations in three cardinal features of the asthma phenotype: inflammatory cells in their bronchoalveolar lavage fluid, mucin over production, and airway hyperresponsiveness. However, genetic depletion of epinephrine using mice lacking the enzyme to synthesize epinephrine, phenylethanolamine N-methyltransferase, or mice that had undergone pharmacological sympathectomy with reserpine to deplete epinephrine, had complete attenuation of these three cardinal features of the asthma phenotype. Furthermore, administration of the long-acting ß2AR agonist, formoterol, a drug currently used in asthma treatment, to phenylethanolamine N-methyltransferase-null mice restored the asthma phenotype. We conclude that ß2AR agonist-induced activation is needed for pathogenesis of the asthma phenotype. These findings also rule out constitutive signaling by the ß2AR as sufficient to drive the asthma phenotype, and may help explain why chronic administration of ß2AR agonists, such as formoterol, have been associated with adverse outcomes in asthma. These data further support the hypothesis that chronic asthma management may be better served by treatment with certain "ß-blockers."

Vertical sleeve gastrectomy associates with airway hyperresponsiveness in a murine model of allergic airway disease and obesity.

Introduction: Asthma is a chronic airway inflammatory disease marked by airway inflammation, remodeling and hyperresponsiveness to allergens. Allergic asthma is normally well controlled through the use of beta-2-adrenergic agonists and inhaled corticosteroids; however, a subset of patients with comorbid obesity experience resistance to currently available therapeutics. Patients with asthma and comorbid obesity are also at a greater risk for severe disease, contributing to increased risk of hospitalization. Bariatric surgery improves asthma control and airway hyperresponsiveness in patients with asthma and comorbid obesity, however, the underlying mechanisms for these improvements remain to be elucidated. We hypothesized that vertical sleeve gastrectomy (VSG), a model of metabolic surgery in mice, would improve glucose tolerance and airway inflammation, resistance, and fibrosis induced by chronic allergen challenge and obesity. Methods: Male C57BL/6J mice were fed a high fat diet (HFD) for 13 weeks with intermittent house dust mite (HDM) allergen administration to induce allergic asthma, or saline as control. At week 11, a subset of mice underwent VSG or Sham surgery with one week recovery. A separate group of mice did not undergo surgery. Mice were then challenged with HDM or saline along with concurrent HFD feeding for 1-1.5 weeks before measurement of lung mechanics and harvesting of tissues, both of which occurred 24 hours after the final HDM challenge. Systemic and pulmonary cytokine profiles, lung histology and gene expression were analyzed. Results: High fat diet contributed to increased body weight, serum leptin levels and development of glucose intolerance for both HDM and saline treatment groups. When compared to saline-treated mice, HDM-challenged mice exhibited greater weight gain. VSG improved glucose tolerance in both saline and HDM-challenged mice. HDM-challenged VSG mice exhibited an increase in airway hyperresponsiveness to methacholine when compared to the non-surgery group. Discussion: The data presented here indicate increased airway hyperresponsiveness in allergic mice undergoing bariatric surgery.

Severe Persistent Pain and Inflammatory Biomarkers in Sickle Cell Disease: An Exploratory Study.

BACKGROUND: Severe pain is among the most common and deleterious symptoms experienced by individuals with sickle cell disease (SCD), of whom more than 50% report chronic pain. Despite this, the understanding of the biological contributors to persistent severe SCD pain is limited. This exploratory study sought to describe pain phenotypes based on frequency of severe pain experienced over 6 months and identify inflammatory biomarkers associated with pain phenotypes among individuals with SCD. METHODS: This study used self-report and electronic health record data collected from 74 individuals enrolled in the Duke Sickle Cell Disease Implementation Consortium Registry. Plasma from previously collected blood specimens was used to generate inflammatory biomarker data using the Inflammation 20-plex ProcartaPlexTM panel. Descriptive statistics were used to describe the occurrence of severe pain over the past 6 months, and bi-variate analyses were used to evaluate the relationship between inflammatory biomarkers and pain phenotypes. RESULTS: Among the 74 participants included in this study, 33.8% reported severe pain occurring never or rarely, 40.5% reported severe pain occurring sometimes, and 25.7% reported severe pain occurring often or always. Soluble E-selectin (sE-selectin) was the only inflammatory biomarker significantly associated with the pain phenotype groups (p = 0.049). Post hoc comparisons identified that participants in the often/always severe pain group had significantly higher plasma concentrations of sE-selectin compared to those in the sometimes severe pain group (p = 0.040). CONCLUSIONS: Our findings provide preliminary evidence of the frequent occurrence of severe pain and that sE-selectin may be an objective biomarker for the frequent occurrence of severe pain in this population.

Nitric oxide mediates relative airway hyporesponsiveness to lipopolysaccharide in surfactant protein A-deficient mice.

Surfactant protein A (SP-A) mediates innate immune cell responses to LPS, a cell wall component of gram-negative bacteria that is found ubiquitously in the environment and is associated with adverse health effects. Inhaled LPS induces lung inflammation and increases airway responsiveness (AR). However, the role of SP-A in mediating LPS-induced AR is not well-defined. Nitric oxide (NO) is described as a potent bronchodilator, and previous studies showed that SP-A modulates the LPS-induced production of NO. Hence, we tested the hypothesis that increased AR, observed in response to aerosolized LPS exposure, would be significantly reduced in an SP-A-deficient condition. Wild-type (WT) and SP-A null (SP-A(-/-)) mice were challenged with aerosolized LPS. Results indicate that despite similar inflammatory indices, LPS-treated SP-A(-/-) mice had attenuated AR after methacholine challenge, compared with WT mice. The attenuated AR could not be attributed to inherent differences in SP-D concentrations or airway smooth muscle contractile and relaxation properties, because these measures were similar between WT and SP-A(-/-) mice. LPS-treated SP-A(-/-) mice, however, had elevated nitrite concentrations, inducible nitric oxide synthase (iNOS) expression, and NOS activity in their lungs. Moreover, the administration of the iNOS-specific inhibitor 1400W completely abrogated the attenuated AR. Thus, when exposed to aerosolized LPS, SP-A(-/-) mice demonstrate a relative airway hyporesponsiveness that appears to be mediated at least partly via an iNOS-dependent mechanism. These findings may have clinical significance, because recent studies reported associations between surfactant protein polymorphisms and a variety of lung diseases.

Both hematopoietic-derived and non-hematopoietic-derived {beta}-arrestin-2 regulates murine allergic airway disease.

Allergic asthma, a major cause of morbidity and leading cause of hospitalizations, is an inflammatory disease orchestrated by T helper cells and characterized by the lung migration of eosinophils, which are important asthma effector cells. Lung migration of inflammatory cells requires, among other events, the chemokine receptor transduction of lung-produced inflammatory chemokines. Despite the widespread prevalence of this disease, the molecular mechanisms regulating chemokine production and receptor regulation in asthma are poorly understood. Previous work from our laboratory demonstrated that beta-arrestin-2 positively regulates the development of allergic airway disease in a mouse model, partly through positive regulation of T-lymphocyte chemotaxis to the lung. However, beta-arrestin-2 is expressed in many cell types, including other hematopoietic cells and lung structural cells, which are involved in the development and manifestation of allergic airway disease. To determine the cell types required for beta-arrestin-2-dependent allergic inflammation, we generated bone marrow chimera mice. Using the ovalbumin murine model of allergic airway disease, we show that eosinophilic and lymphocytic inflammation is restored in chimeric mice, with expression of beta-arrestin-2 exclusively on hematopoietic-derived cell types. In contrast, airway hyperresponsiveness is dependent on the expression of beta-arrestin-2 in structural cells. Our data demonstrate that the expression of beta-arrestin-2 in at least two divergent cell types contributes to the pathogenesis of allergic airway disease.

Beta-arrestins specifically constrain beta2-adrenergic receptor signaling and function in airway smooth muscle.

Chronic use of inhaled beta-agonists by asthmatics is associated with a loss of bronchoprotective effect and deterioration of asthma control. Beta-agonist-promoted desensitization of airway smooth muscle beta-2-adrenergic receptors, mediated by G protein-coupled receptor kinases and arrestins, is presumed to underlie these effects, but such a mechanism has never been demonstrated. Using in vitro, ex vivo, and in vivo murine models, we demonstrate that beta-arrestin-2 gene ablation augments beta-agonist-mediated airway smooth muscle relaxation, while augmenting beta-agonist-stimulated cyclic adenosine monophosphate production. In cultures of human airway smooth muscle, small interfering RNA-mediated knockdown of arrestins also augments beta-agonist-stimulated cyclic adenosine monophosphate production. Interestingly, signaling and function mediated by m2/m3 muscarinic acetylcholine receptors or prostaglandin E(2) receptors were not affected by either beta-arrestin-2 knockout or arrestin knockdown. Thus, arrestins are selective regulators of beta-2-adrenergic receptor signaling and function in airway smooth muscle. These results and our previous findings, which demonstrate a role for arrestins in the development of allergic inflammation in the lung, identify arrestins as potentially important therapeutic targets for obstructive airway diseases.

Methods to Investigate the Roles for ß-Arrestin-2 in Allergic Inflammatory Airway Disease.

Spatial and temporal control of gene expression using cre/loxP technology is a major methodological advance for biomedical research. The ability to alter gene expression after an in vivo disease model has been established and allows researchers the opportunity to examine the impact of that gene on the perpetuation of a disease, a mechanistic insight that is arguably more therapeutically relevant than developmental mechanisms.We used the cre/LoxP technology in mice to show that ß-arrestin-2, a gene previously shown to be required for the development of the asthma phenotype, is also required for the perpetuation of, at least, the airway hyperresponsiveness characteristic of that phenotype. Here we describe stepwise methods for the activation of the cre-loxP technology and induction of murine allergic inflammatory airway disease. We comment on the unanticipated problems encountered, which we speculate were a result of interactions between the allergen and ß-arrestin-2 gene (Arrb2) regulation and the effect of tamoxifen on the asthma phenotype. The issues encountered here may be generally applicable to cre/loxP utilization in inflammatory disease models, especially if the gene of interest is associated with the inflammatory cascade.

Therapeutic Potential of Targeting ß-Arrestin.

ß-arrestins are multifunctional proteins that modulate heptahelical 7 transmembrane receptors, also known as G protein-coupled receptors (GPCRs), a superfamily of receptors that regulate most physiological processes. ß-arrestin modulation of GPCR function includes termination of G protein-dependent signaling, initiation of ß-arrestin-dependent signaling, receptor trafficking to degradative or recycling pathways, receptor transactivation, transcriptional regulation, and localization of second messenger regulators. The pleiotropic influence ß-arrestins exert on these receptors regulates a breadth of physiological functions, and additionally, ß-arrestins are involved in the pathophysiology of numerous and wide-ranging diseases, making them prime therapeutic targets. In this review, we briefly describe the mechanisms by which ß-arrestins regulate GPCR signaling, including the functional cellular mechanisms modulated by ß-arrestins and relate this to observed pathophysiological responses associated with ß-arrestins. We focus on the role for ß-arrestins in transducing cell signaling; a pathway that is complementary to the classical G protein-coupling pathway. The existence of these GPCR dual signaling pathways offers an immense therapeutic opportunity through selective targeting of one signaling pathway over the other. Finally, we will consider several mechanisms by which the potential of dual signaling pathway regulation can be harnessed and the implications for improved disease treatments.

A murine model of hyperdopaminergic state displays altered respiratory control.

The dopamine transporter (DAT) protein plays an important role in the termination of dopamine signaling. We addressed the hypothesis that loss of DAT function would result in a distinctive cardiorespiratory phenotype due to the significant role of dopamine in the control of breathing, especially with respect to chemical control, metabolism, and thermoregulation. The DAT knockout mouse (DAT-/-) displays a state of functional hyperdopaminergia characterized by marked novelty driven hyperactivity. Certain behavioral and drug responses in these mice are reminiscent of endophenotypes of individuals with attention deficit hyperactivity disorders (ADHD). We performed experiments on conscious, unrestrained DAT-/- mice (KO) and littermate DAT+/+ wild-type (WT) controls. Ventilation was measured by the barometric technique during normoxia, hypoxia, or hypercapnia. We measured core body temperature and CO2 production as an index of metabolism. DAT-/- mice displayed a significantly lower respiratory frequency than WT mice, reflecting a prolonged inspiratory time. DAT-/- mice exhibited a reduced ventilatory response to hypoxia characterized by an attenuation of both the respiratory frequency and tidal volume responses. Both groups showed similar metabolic responses to hypoxia. Circadian measurements of body temperature were significantly lower in DAT-/- mice than WT mice during inactive periods. We conclude that loss of the DAT protein in this murine model of altered dopaminergic neurotransmission results in a significant respiratory and thermal phenotype that has possible implications for understanding of conditions associated with altered dopamine regulation.