Airway smooth muscle photorelaxation via opsin receptor activation.

Nonvisual opsin (OPN) receptors have recently been implicated in blue light-mediated photorelaxation of smooth muscle in various organs. Since photorelaxation has not yet been demonstrated in airway smooth muscle (ASM) or in human tissues, we questioned whether functional OPN receptors are expressed in mouse and human ASM. mRNA, encoding the OPN 3 receptor, was detected in both human and mouse ASM. To demonstrate the functionality of the OPN receptors, we performed wire myography of ex vivo ASM from mouse and human upper airways. Blue light-mediated relaxation of ACh-preconstricted airways was intensity and wavelength dependent (maximum relaxation at 430-nm blue light) and was inhibited by blockade of the large-conductance calcium-activated potassium channels with iberiotoxin. We further implicated OPN receptors as key mediators in functional photorelaxation by demonstrating increased relaxation in the presence of a G protein receptor kinase 2 inhibitor or an OPN chromophore (9- cis retinal). We corroborated these responses in peripheral airways of murine precision-cut lung slices. This is the first demonstration of photorelaxation in ASM via an OPN receptor-mediated pathway.

Sympathetic Nerve Hyperactivity in the Spleen: Causal for Nonpathogenic-Driven Chronic Immune-Mediated Inflammatory Diseases (IMIDs)?

Immune-Mediated Inflammatory Diseases (IMIDs) is a descriptive term coined for an eclectic group of diseases or conditions that share common inflammatory pathways, and for which there is no definitive etiology. IMIDs affect the elderly most severely, with many older individuals having two or more IMIDs. These diseases include, but are not limited to, type-1 diabetes, obesity, hypertension, chronic pulmonary disease, coronary heart disease, inflammatory bowel disease, and autoimmunity, such as rheumatoid arthritis (RA), Sjogren's syndrome, systemic lupus erythematosus, psoriasis, psoriatic arthritis, and multiple sclerosis. These diseases are ostensibly unrelated mechanistically, but increase in frequency with age and share chronic systemic inflammation, implicating major roles for the spleen. Chronic systemic and regional inflammation underlies the disease manifestations of IMIDs. Regional inflammation and immune dysfunction promotes targeted end organ tissue damage, whereas systemic inflammation increases morbidity and mortality by affecting multiple organ systems. Chronic inflammation and skewed dysregulated cell-mediated immune responses drive many of these age-related medical disorders. IMIDs are commonly autoimmune-mediated or suspected to be autoimmune diseases. Another shared feature is dysregulation of the autonomic nervous system and hypothalamic pituitary adrenal (HPA) axis. Here, we focus on dysautonomia. In many IMIDs, dysautonomia manifests as an imbalance in activity/reactivity of the sympathetic and parasympathetic divisions of the autonomic nervous system (ANS). These major autonomic pathways are essential for allostasis of the immune system, and regulating inflammatory processes and innate and adaptive immunity. Pathology in ANS is a hallmark and causal feature of all IMIDs. Chronic systemic inflammation comorbid with stress pathway dysregulation implicate neural-immune cross-talk in the etiology and pathophysiology of IMIDs. Using a rodent model of inflammatory arthritis as an IMID model, we report disease-specific maladaptive changes in ß2-adrenergic receptor (AR) signaling from protein kinase A (PKA) to mitogen activated protein kinase (MAPK) pathways in the spleen. Beta2-AR signal "shutdown" in the spleen and switching from PKA to G-coupled protein receptor kinase (GRK) pathways in lymph node cells drives inflammation and disease advancement. Based on these findings and the existing literature in other IMIDs, we present and discuss relevant literature that support the hypothesis that unresolvable immune stimulation from chronic inflammation leads to a maladaptive disease-inducing and perpetuating sympathetic response in an attempt to maintain allostasis. Since the role of sympathetic dysfunction in IMIDs is best studied in RA and rodent models of RA, this IMID is the primary one used to evaluate data relevant to our hypothesis. Here, we review the relevant literature and discuss sympathetic dysfunction as a significant contributor to the pathophysiology of IMIDs, and then discuss a novel target for treatment. Based on our findings in inflammatory arthritis and our understanding of common inflammatory process that are used by the immune system across all IMIDs, novel strategies to restore SNS homeostasis are expected to provide safe, cost-effective approaches to treat IMIDs, lower comorbidities, and increase longevity.

Cardiac hyporesponsiveness in severe sepsis is associated with nitric oxide-dependent activation of G protein receptor kinase.

G protein-coupled receptor kinase isoform 2 (GRK2) has a critical role in physiological and pharmacological responses to endogenous and exogenous substances. Sepsis causes an important cardiovascular dysfunction in which nitric oxide (NO) has a relevant role. The present study aimed to assess the putative effect of inducible NO synthase (NOS2)-derived NO on the activity of GRK2 in the context of septic cardiac dysfunction. C57BL/6 mice were submitted to severe septic injury by cecal ligation and puncture (CLP). Heart function was assessed by isolated and perfused heart, echocardiography, and ß-adrenergic receptor binding. GRK2 was determined by immunofluorescence and Western blot analysis in the heart and isolated cardiac myocytes. Sepsis increased NOS2 expression in the heart, increased plasma nitrite + nitrate levels, and reduced isoproterenol-induced isolated ventricle contraction, whole heart tension development, and ß-adrenergic receptor density. Treatment with 1400W or with GRK2 inhibitor prevented CLP-induced cardiac hyporesponsiveness 12 and 24 h after CLP. Increased labeling of total and phosphorylated GRK2 was detected in hearts after CLP. With treatment of 1400W or in hearts taken from septic NOS2 knockout mice, the activation of GRK2 was reduced. 1400W or GRK2 inhibitor reduced mortality, improved echocardiographic cardiac parameters, and prevented organ damage. Therefore, during sepsis, NOS2-derived NO increases GRK2, which leads to a reduction in ß-adrenergic receptor density, contributing to the heart dysfunction. Isolated cardiac myocyte data indicate that NO acts through the soluble guanylyl cyclase/cGMP/PKG pathway. GRK2 inhibition may be a potential therapeutic target in sepsis-induced cardiac dysfunction.NEW & NOTEWORTHY The main novelty presented here is to show that septic shock induces cardiac hyporesponsiveness to isoproterenol by a mechanism dependent on nitric oxide and mediated by G protein-coupled receptor kinase isoform 2. Therefore, G protein-coupled receptor kinase isoform 2 inhibition may be a potential therapeutic target in sepsis-induced cardiac dysfunction.

The right ventricle in pulmonary arterial hypertension: disorders of metabolism, angiogenesis and adrenergic signaling in right ventricular failure.

The right ventricle (RV) is the major determinant of functional state and prognosis in pulmonary arterial hypertension. RV hypertrophy (RVH) triggered by pressure overload is initially compensatory but often leads to RV failure. Despite similar RV afterload and mass some patients develop adaptive RVH (concentric with retained RV function), while others develop maladaptive RVH, characterized by dilatation, fibrosis, and RV failure. The differentiation of adaptive versus maladaptive RVH is imprecise, but adaptive RVH is associated with better functional capacity and survival. At the molecular level, maladaptive RVH displays greater impairment of angiogenesis, adrenergic signaling, and metabolism than adaptive RVH, and these derangements often involve the left ventricle. Clinically, maladaptive RVH is characterized by increased N-terminal pro-brain natriuretic peptide levels, troponin release, elevated catecholamine levels, RV dilatation, and late gadolinium enhancement on MRI, increased (18)fluorodeoxyglucose uptake on positron emission tomography, and QTc prolongation on the ECG. In maladaptive RVH there is reduced inotrope responsiveness because of G-protein receptor kinase-mediated downregulation, desensitization, and uncoupling of ß-adrenoreceptors. RV ischemia may result from capillary rarefaction or decreased right coronary artery perfusion pressure. Maladaptive RVH shares metabolic abnormalities with cancer including aerobic glycolysis (resulting from a forkhead box protein O1-mediated transcriptional upregulation of pyruvate dehydrogenase kinase), and glutaminolysis (reflecting ischemia-induced cMyc activation). Augmentation of glucose oxidation is beneficial in experimental RVH and can be achieved by inhibition of pyruvate dehydrogenase kinase, fatty acid oxidation, or glutaminolysis. Therapeutic targets in RV failure include chamber-specific abnormalities of metabolism, angiogenesis, adrenergic signaling, and phosphodiesterase-5 expression. The ability to restore RV function in experimental models challenges the dogma that RV failure is irreversible without regression of pulmonary vascular disease.

IL-13 desensitizes ß2-adrenergic receptors in human airway epithelial cells through a 15-lipoxygenase/G protein receptor kinase 2 mechanism.

BACKGROUND: ß2-Adrenergic receptor (ß2AR) agonists are critical treatments for asthma. However, receptor desensitization can lead to loss of therapeutic effects. Although desensitization to repeated use of ß2-agonists is well studied, type 2 inflammation could also affect ß2AR function. OBJECTIVE: We sought to evaluate the effect of the type 2 cytokine IL-13 on ß2AR desensitization in human airway epithelial cells (HAECs) and determine whether 15-lipoxygenase-1 (15LO1) binding with phosphatidylethanolamine-binding protein 1 (PEBP1) contributes to desensitization through release of G protein receptor kinase 2 (GRK2). METHODS: HAECs in air-liquid interface culture with or without IL-13 (48 hours) or isoproterenol hydrochloride (ISO; 30 minutes) pretreatment were stimulated with ISO (10 minutes). Cyclic adenosine 3, 5-monophosphate (cAMP) levels were measured using ELISA, and ß2AR and GRK2 phosphorylation was measured using Western blotting. Short interfering RNA was used for 15LO1 knockdown. Interactions of GRK2, PEBP1, and 15LO1 were detected by means of immunoprecipitation/Western blotting and immunofluorescence. HAECs and airway tissue from control subjects and asthmatic patients were evaluated for I5LO1, PEBP1, and GRK2. RESULTS: Pretreatment with ISO or IL-13 decreased ISO-induced cAMP generation compared with ISO for 10 minutes alone paralleled by increases in ß2AR and GRK2 phosphorylation. GRK2 associated with PEBP1 after 10 minutes of ISO in association with low phosphorylated GRK2 (pGRK2) levels. In contrast, in the presence of IL-13 plus ISO (10 minutes), binding of GRK2 to PEBP1 decreased, whereas 15LO1 binding and pGRK2 levels increased. 15LO1 knockdown restored ISO-induced cAMP generation. These findings were recapitulated in freshly brushed HAECs from cells and tissue of asthmatic patients. CONCLUSION: IL-13 treatment of HAECs leads to ß2AR desensitization, which involves 15LO1/PEBP1 interactions to free GRK2, and allows it to phosphorylate (and desensitize) ß2ARs, suggesting that the beneficial effects of ß2-agonists could be blunted in patients with type 2 associated asthma.

Pharmacogenetics: implications of race and ethnicity on defining genetic profiles for personalized medicine.

Pharmacogenetics is being used to develop personalized therapies specific to subjects from different ethnic or racial groups. To date, pharmacogenetic studies have been primarily performed in trial cohorts consisting of non-Hispanic white subjects of European descent. A "bottleneck" or collapse of genetic diversity associated with the first human colonization of Europe during the Upper Paleolithic period, followed by the recent mixing of African, European, and Native American ancestries, has resulted in different ethnic groups with varying degrees of genetic diversity. Differences in genetic ancestry might introduce genetic variation, which has the potential to alter the therapeutic efficacy of commonly used asthma therapies, such as ß2-adrenergic receptor agonists (ß-agonists). Pharmacogenetic studies of admixed ethnic groups have been limited to small candidate gene association studies, of which the best example is the gene coding for the receptor target of ß-agonist therapy, the ß2-adrenergic receptor (ADRB2). Large consortium-based sequencing studies are using next-generation whole-genome sequencing to provide a diverse genome map of different admixed populations, which can be used for future pharmacogenetic studies. These studies will include candidate gene studies, genome-wide association studies, and whole-genome admixture-based approaches that account for ancestral genetic structure, complex haplotypes, gene-gene interactions, and rare variants to detect and replicate novel pharmacogenetic loci.

The role of G protein-coupled receptor kinases in GLP-1R ß-arrestin recruitment and internalisation.

The glucagon-like peptide 1 receptor (GLP-1R) is a validated clinical target for the treatment of type 2 diabetes and obesity. Unlike most G protein-coupled receptors (GPCRs), the GLP-1R undergoes an atypical mode of internalisation that does not require ß-arrestins. While differences in GLP-1R trafficking and ß-arrestin recruitment have been observed between clinically used GLP-1R agonists, the role of G protein-coupled receptor kinases (GRKs) in affecting these pathways has not been comprehensively assessed. In this study, we quantified the contribution of GRKs to agonist-mediated GLP-1R internalisation and ß-arrestin recruitment profiles using cells where endogenous ß-arrestins, or non-visual GRKs were knocked out using CRISPR/Cas9 genome editing. Our results confirm the previously established atypical ß-arrestin-independent mode of GLP-1R internalisation and revealed that GLP-1R internalisation is dependent on the expression of GRKs. Interestingly, agonist-mediated GLP-1R ß-arrestin 1 and ß-arrestin 2 recruitment were differentially affected by endogenous GRK knockout with ß-arrestin 1 recruitment more sensitive to GRK knockout than ß-arrestin 2 recruitment. Moreover, individual overexpression of GRK2, GRK3, GRK5 or GRK6 in a newly generated GRK2/3/4/5/6 HEK293 cells, rescued agonist-mediated ß-arrestin 1 recruitment and internalisation profiles to similar levels, suggesting that there is no specific GRK isoform that drives these pathways. This study advances mechanistic understanding of agonist-mediated GLP-1R internalisation and provides novel insights into how GRKs may fine-tune GLP-1R signalling.

Characterizing Adrenergic Regulation of Glucose Transporter 4-Mediated Glucose Uptake and Metabolism in the Heart.

Whereas adrenergic stimulation promotes cardiac function that demands more fuel and energy, how this receptor controls cardiac glucose metabolism is not defined. This study shows that the cardiac ß2 adrenoreceptor (ß2AR) is required to increase glucose transporter 4 (GLUT4)-mediated glucose uptake in myocytes and glucose oxidation in working hearts via activating the cardiac ß2AR and promotes the G inhibitory-phosphoinositide 3-kinase-protein kinase B cascade to increase phosphorylation of TBC1D4 (aka AS160), a Rab guanosine triphosphatase-activating protein, which is a key enzyme to mobilize GLUT4. Furthermore, deleting G-protein receptor kinase phosphorylation sites of ß2AR blocked adrenergic stimulation of GLUT4-mediated glucose uptake in myocytes and hearts. This study defines a molecular pathway that controls cardiac GLUT4-mediated glucose uptake and metabolism under adrenergic stimulation.

Ketamine Improves Desensitization of µ-Opioid Receptors Induced by Repeated Treatment with Fentanyl but Not with Morphine.

The issue of tolerance to continuous or repeated administration of opioids should be addressed. The ability of ketamine to improve opioid tolerance has been reported in clinical studies, and its mechanism of tolerance may involve improved desensitization of µ-opioid receptors (MORs). We measured changes in MOR activity and intracellular signaling induced by repeated fentanyl and morphine administration and investigated the effects of ketamine on these changes with human embryonic kidney 293 cells expressing MOR using the CellKey™, cADDis cyclic adenosine monophosphate, and PathHunter® ß-arrestin recruitment assays. Repeated administration of fentanyl or morphine suppressed the second MOR responses. Administration of ketamine before a second application of opioids within clinical concentrations improved acute desensitization and enhanced ß-arrestin recruitment elicited by fentanyl but not by morphine. The effects of ketamine on fentanyl were suppressed by co-treatment with an inhibitor of G-protein-coupled receptor kinase (GRK). Ketamine may potentially reduce fentanyl tolerance but not that of morphine through modulation of GRK-mediated pathways, possibly changing the conformational changes of ß-arrestin to MOR.

Determining the Effects of Differential Expression of GRKs and ß-arrestins on CLR-RAMP Agonist Bias.

Signalling of the calcitonin-like receptor (CLR) is multifaceted, due to its interaction with receptor activity modifying proteins (RAMPs), and three endogenous peptide agonists. Previous studies have focused on the bias of G protein signalling mediated by the receptor and receptor internalisation of the CLR-RAMP complex has been assumed to follow the same pattern as other Class B1 G Protein-Coupled Receptors (GPCRs). Here we sought to measure desensitisation of the three CLR-RAMP complexes in response to the three peptide agonists, through the measurement of ß-arrestin recruitment and internalisation. We then delved further into the mechanism of desensitisation through modulation of ß-arrestin activity and the expression of GPCR kinases (GRKs), a key component of homologous GPCR desensitisation. First, we have shown that CLR-RAMP1 is capable of potently recruiting ß-arrestin1 and 2, subsequently undergoing rapid endocytosis, and that CLR-RAMP2 and -RAMP3 also utilise these pathways, although to a lesser extent. Following this we have shown that agonist-dependent internalisation of CLR is ß-arrestin dependent, but not required for full agonism. Overexpression of GRK2-6 was then found to decrease receptor signalling, due to an agonist-independent reduction in surface expression of the CLR-RAMP complex. These results represent the first systematic analysis of the importance of ß-arrestins and GRKs in CLR-RAMP signal transduction and pave the way for further investigation regarding other Class B1 GPCRs.

A novel GPCR target in correlation with androgen deprivation therapy for prostate cancer drug discovery.

Most prostate carcinomas require androgen stimulation to grow, and for nearly 70 years, androgen ablation therapy has been one of the central therapeutic strategies against advanced prostate cancer. Although most tumours initially respond to this therapy, some will be acquired resistant and progress to metastatic castration-resistant (mCRPC) disease which clinically tends to progress more rapidly than earlier disease manifestations. The underlying molecular biology of mCRPC is highly complex, and numerous mechanisms have been proposed that promote and retain androgen independence. In various clinical and preclinical data explored, the nature of intracellular signalling pathways mediating mitogenic acquired resistant effects of GPCRs in prostate cancer is poorly defined. G-protein-coupled receptor kinase 2 (GRK2) contributes to the modulation of basic cellular functions-such as cell proliferation, survival or motility-and is involved in metabolic homeostasis, inflammation or angiogenic processes. Moreover, altered GRK2 levels are starting to be reported in different tumoural contexts and shown to promote breast tumourigenesis or to trigger the tumoural angiogenic switch. Thus, we are exploring recent findings that present unexpected opportunities to interfere with major tumourigenic signals by manipulating GPCR-mediated pathways.

Development of "Plug and Play" Fiducial Marks for Structural Studies of GPCR Signaling Complexes by Single-Particle Cryo-EM.

"Universal" synthetic antibody (sAB)-based fiducial marks have been generated by customized phage display selections to facilitate the rapid structure determination of G protein-coupled receptor (GPCR) signaling complexes by single-particle cryo-electron microscopy (SP cryo-EM). sABs were generated to the two major G protein subclasses: trimeric Gi and Gs, as well as mini-Gs, and were tested to ensure binding in the context of their cognate GPCRs. Epitope binning revealed that multiple distinct epitopes exist for each G(αßγ) protein. Several Gßγ-specific sABs, cross-reactive between trimeric Gi and Gs, were identified suggesting they could be used across all subclasses in a "plug and play" fashion. sABs were also generated to a representative of another class of GPCR signaling partner, G protein receptor kinase 1 (GRK1) and evaluated further, supporting the generalizability of the approach. EM data suggested that the subclass-specific sABs provide effective single and dual fiducials for multiple GPCR signaling complexes.

Evolution of the shut-off steps of vertebrate phototransduction.

Different isoforms of the genes involved in phototransduction are expressed in vertebrate rod and cone photoreceptors, providing a unique example of parallel evolution via gene duplication. In this study, we determine the molecular phylogeny of the proteins underlying the shut-off steps of phototransduction in the agnathan and jawed vertebrate lineages. For the G-protein receptor kinases (GRKs), the GRK1 and GRK7 divisions arose prior to the divergence of tunicates, with further expansion during the two rounds of whole-genome duplication (2R); subsequently, jawed and agnathan vertebrates retained different subsets of three isoforms of GRK. For the arrestins, gene expansion occurred during 2R. Importantly, both for GRKs and arrestins, the respective rod isoforms did not emerge until the second round of 2R, just prior to the separation of jawed and agnathan vertebrates. For the triplet of proteins mediating shut-off of the G-protein transducin, RGS9 diverged from RGS11, probably at the second round of 2R, whereas Gß5 and R9AP appear not to have undergone 2R expansion. Overall, our analysis provides a description of the duplications and losses of phototransduction shut-off genes that occurred during the transition from a chordate with only cone-like photoreceptors to an ancestral vertebrate with both cone- and rod-like photoreceptors.

Matrix Signaling Subsequent to a Myocardial Infarction: A Proteomic Profile of Tissue Factor Microparticles.

This study investigated the release and proteomic profile of tissue factor microparticles (TFMPs) prospectively (up to 6 months) following a myocardial infarction (MI) in a chronic porcine model to establish their utility in tracking cellular level activities that predict physiologic outcomes. Our animal groups (n = 6 to 8 each) consisted of control, noninfarcted (negative control); infarcted only (positive control); and infarcted animals treated with cardiac resynchronization therapy (CRT) and a ß-blocker (BB) (metoprolol succinate). The authors found different protein profiles in TFMPs between the control, infarcted only group, and the CRT + BB treated group with predictive impact on the outward phenotype of pathological remodeling after an MI within and between groups. This novel approach of monitoring cellular level activities by profiling the content of TFMPs has the potential of addressing a shortfall of the current crop of cardiac biomarkers, which is the inability to capture composite molecular changes associated with chronic maladaptive signaling in a spatial and temporal manner.