RGS2 Knockout Mice Research Articles

RGS4 controls airway hyperresponsiveness through GAP-independent mechanisms

Regulators of G protein signaling (RGS proteins) constrain G protein-coupled receptor (GPCR)-mediated and other responses throughout the body primarily, but not exclusively, through their GTPase activating (GAP) activity. Asthma is a highly prevalent condition characterized by airway hyper-responsiveness (AHR) to environmental stimuli resulting in part from amplified GPCR-mediated airway smooth muscle (ASM) contraction. Rgs2 or Rgs5 gene deletion in mice enhances AHR and ASM contraction whereas RGS4 knockout (KO) mice unexpectedly have decreased AHR due to increased production of the bronchodilator prostaglandin E2 (PGE2) by lung epithelial cells. Here we found that knockin mice harboring Rgs4 alleles encoding a point mutation (N128A) that sharply curtails RGS4 GAP activity had increased AHR, reduced airway PGE2 levels, and augmented GPCR-induced bronchoconstriction compared to either RGS4 KO mice or WT controls. RGS4 interacted with the p85α subunit of PI3 kinase (PI3K) and inhibited PI3K-dependent PGE2 secretion elicited by TGFβ in airway epithelial cells. Together these findings suggest that RGS4 affects asthma severity in part by regulating the airway inflammatory milieu in a G protein-independent manner.

Tianeptine promotes lasting antiallodynic effects in a mouse model of neuropathic pain.

Tricyclic antidepressants (TCAs), such as desipramine (DMI), are effective at managing neuropathic pain symptoms but often take several weeks to become effective and also lead to considerable side effects. Tianeptine (TIAN) is an atypical antidepressant that activates the mu-opioid receptor but does not produce analgesic tolerance or withdrawal in mice, nor euphoria in humans, at clinically-relevant doses. Here, we evaluate the efficacy of TIAN at persistently alleviating mechanical allodynia in the spared nerve injury (SNI) model of neuropathic pain, even well after drug clearance. After finding an accelerated onset of antiallodynic action compared to DMI, we used genetically modified mice to gain insight into RGS protein-associated pathways that modulate the efficacy of TIAN relative to DMI in models of neuropathic pain. Because we observed similar behavioral responses to both TIAN and DMI treatment in RGS4, RGSz1, and RGS9 knockout mice, we performed RNA sequencing on the NAc of TIAN- and DMI-treated mice after prolonged SNI to further clarify potential mechanisms underlying TIANs faster therapeutic actions. Our bioinformatic analysis revealed distinct transcriptomic signatures between the two drugs, with TIAN more directly reversing SNI-induced differentially expressed genes, and further predicted several upstream regulators that may be implicated in onset of action. This new understanding of the molecular pathways underlying TIAN action may enable the development of novel and more efficacious pharmacological approaches for the management of neuropathic pain.

Abstract P209: Loss Of Rgs2 Specifically In CD4+ T Cells Decreases The Hypertensive Response To Vasopressin

Regulator of G protein Signaling (RGS) family members can modulate multiple cardiovascular hormones and are associated with hypertension and preeclampsia, a hypertensive disorder in pregnancy. We previously observed a 9-fold increase in RGS2 in CD4+ T cells isolated from women with preeclampsia compared to normotensive women. Further, in non-pregnant mice, we showed that loss of RGS2 in CD4+ T cells prevented angiotensin II-induced hypertension (measured via radiotelemetry) and resulted in increased levels of the anti-inflammatory cytokines interleukin 4 and transforming growth factor beta. We hypothesize that modulating RGS2 in CD4+ T cells may be a therapeutic strategy for hypertension. The objective of this study was to determine if loss of RGS2 specifically in CD4+ T cells protects against the development of arginine vasopressin-induced hypertension in mixed background CD4+ RGS2 knockout mice (CD4 RGS2 KO ). To generate mice wherein RGS2 is specifically knocked out in CD4+ T cells, CD4-Cre+ mice (C57BL/6J) were crossed with RGS2 flox/flox mice (B6SJLF1/J). Female 8-12 week old CD4 RGS2 KO or littermate control mice (n=5 per group) from this mixed strain were administered 24 ng/hr vasopressin for 21 days via mini-osmotic pump. Systolic blood pressure (SBP), diastolic blood pressure (DBP), mean blood pressure (MP), and heart rate (HR) were assessed using a high throughput non-invasive blood pressure system. Prior to vasopressin infusion, KO and CTL mice showed no differences in SBP, DBP, MP, or HR. At 9-13 days of vasopressin-infusion, KO mice had a significantly lower 24-hr SBP [KO 138.7 ±4.7 vs CTL 155.9 ±3.4 mmHg, p<0.05], DBP [KO 106.9 ±4.1 vs CTL 122.8 ±2.9 mmHg, p<0.05], MP [KO 117.2 ±4.3 vs CTL 133.5 ±3.0 mmHg, p<0.05], and HR [KO 378 ±18.7 vs CTL 445 x±12.8 BPM, p<0.05] compared to CTL mice. Here, we demonstrate that in a mixed strain CD4+ RGS2 KO mouse, loss of RGS2 specifically in CD4+ T cells prevented vasopressin-induced hypertension. Therefore, RGS2 expression in CD4+ T cells may play an expanded role in the modulation of hypertension.

RGS4 controls Gαi3-mediated regulation of Bcl-2 phosphorylation on TGN38-containing intracellular membranes.

Intracellular pools of the heterotrimeric G-protein α-subunit Gαi3 (encoded by GNAI3) have been shown to promote growth factor signaling, while at the same time inhibiting the activation of JNK and autophagic signaling following nutrient starvation. The precise molecular mechanisms linking Gαi3 to both stress and growth factor signaling remain poorly understood. Importantly, JNK-mediated phosphorylation of Bcl-2 was previously found to activate autophagic signaling following nutrient deprivation. Our data shows that activated Gαi3 decreases Bcl-2 phosphorylation, whereas inhibitors of Gαi3, such as RGS4 and AGS3 (also known as GPSM1), markedly increase the levels of phosphorylated Bcl-2. Manipulation of the palmitoylation status and intracellular localization of RGS4 suggests that Gαi3 modulates phosphorylated Bcl-2 levels and autophagic signaling from discreet TGN38 (also known as TGOLN2)-labeled vesicle pools. Consistent with an important role for these molecules in normal tissue responses to nutrient deprivation, increased Gαi signaling within nutrient-starved adrenal glands from RGS4-knockout mice resulted in a dramatic abrogation of autophagic flux, compared to wild-type tissues. Together, these data suggest that the activity of Gαi3 and RGS4 from discreet TGN38-labeled vesicle pools are critical regulators of autophagic signaling that act via their ability to modulate phosphorylation of Bcl-2.

Abstract 842: Protein kinase G - Regulator of G protein Signaling 2 Axis of Cardiac Myocytes Critically Determines Cardiac Performance in Early Cardiac Remodeling

Introduction: Regulator of G protein signaling 2 (RGS2) is a negative regulator of G protein-coupled signaling, and is potentiated by cGMP-PKG. Our prior work using a global deletion model demonstrated the protective role for RGS2 against mal-adaptive cardiac remodeling, which is essential to the cGMP-PDE5 inhibitor efficacy; however, cell type-specific contribution remains to be determined, given the expression of RGS2 in myocytes, fibroblasts and vascular smooth muscle cells. We hypothesized that myocyte RGS2 might serve as a primary contributor to the cardio-protection and mediates cGMP-PKG benefits. Method: We generated cardiomyocyte-specific RGS2 knockout mice (cKO) on a C57BL/6 background. Pressure overload was produced by transverse aortic constriction (TAC) in male RGS2-cKO mice (RGS2cKO) and their littermate controls (WT). To investigate cGMP-PKG enhancing effects, a soluble guanylyl cyclase stimulator (riociguat) was orally administrated daily (3mg/kg/day). Invasive hemodynamics, anatomy, histology and molecular signaling were assessed at 1wk after TAC. Results: RGS2-cKO revealed exacerbated dilative cardiac remodeling (LVDD: cKO 3.57±0.36 vs WT 2.90±0.16) and severely impaired cardiac function (dPdt/IP: cKO 166±3.49 vs WT 195±7.40), while their basal phenotype was undistinguishable from WT. cKO-TAC hearts showed larger cardiomyocyte size and more fibrosis than WT-TAC hearts, associated with aberrant gene expression induction including Myh6, Myh7, Col1a2, and TGFβ . Intriguingly, concomitant riociguat treatment reduced LV chamber size and fibrosis but failed to improve cardiac performance in cKO-TAC hearts, while riociguat ameliorated all these in WT-TAC hearts. Conlusion: These results suggest that RGS2-PKG axis in cardiac myocytes critically contributes to cardiac functional performance and that cGMP-PKG exerts anti-fibrotic effects independently of myocyte conditions.

Regulator of G protein signaling 2 differentially regulates nicotine-induced anxiolytic- and antidepressant-like effects in mice.

This study assessed the role of regulator of G protein signaling 2 (RGS2) in nicotine-induced anxiolytic- and antidepressant-like effects using RGS2 wildtype (WT) and RGS2 knockout (KO) mice. RGS2 negatively regulates monoaminergic neurotransmission, which is implicated in the pathology of anxiety and depression. We hypothesized that deletion of RGS2 would enhance nicotine-induced anxiolytic- and antidepressant-like effects, which were assessed using the elevated plus maze and tail suspension tests, respectively. Anxiolytic-like effects were observed in both RGS2 WT and KO mice after administration of low dose of nicotine (0.05mg/kg, base) compared to respective saline controls. Additionally, administration of nicotine (0.1mg/kg, base) compared to saline resulted in anxiolytic-like effects in RGS2 KO mice, but not RGS2 WT mice, suggesting genetic deletion of RGS2 facilitated anxiolytic-like effects of nicotine. Administration of nicotine (0.5 and 1mg/kg, base) compared to saline resulted in antidepressant-like effects in RGS2 WT mice. Antidepressant-like effects were observed in RGS2 KO mice only at the highest tested dose of nicotine (1mg/kg, base) compared to saline controls, suggesting that genetic deletion of RGS2 decreased sensitivity to antidepressant-like effects of nicotine. Together, the data suggest that RGS2 differentially regulated nicotine-induced affective behavioral responses. These data suggest that individuals with RGS2 polymorphisms may experience differential affective responses to tobacco smoking, which may make them vulnerable to developing nicotine addiction.

A novel regulatory role of RGS4 in STAT5B activation, neurite outgrowth and neuronal differentiation

The Regulator of G protein Signalling 4 (RGS4) is a multitask protein that interacts with and negatively modulates opioid receptor signalling. Previously, we showed that the δ-opioid receptor (δ-OR) forms a multiprotein signalling complex consisting of Gi/Go proteins and the Signal Transducer and Activator of Transcription 5B (STAT5B) that leads to neuronal differentiation and neurite outgrowth upon δ-ΟR activation. Here, we investigated whether RGS4 could participate in signalling pathways to regulate neurotropic events. We demonstrate that RGS4 interacts directly with STAT5B independently of δ-ΟR presence both in vitro and in living cells. This interaction involves the N-terminal portion of RGS4 and the DNA-binding SH3 domain of STAT5B. Expression of RGS4 in HEK293 cells expressing δ-OR and/or erythropoietin receptor results in inhibition of [D-Ser2, Leu5, Thr6]-enkephalin (DSLET)-and erythropoietin-dependent STAT5B phosphorylation and subsequent transcriptional activation. DSLET-dependent neurite outgrowth of neuroblastoma cells is also blocked by RGS4 expression, whereas primary cortical cultures of RGS4 knockout mice (RGS4-/-) exhibit enhanced neuronal sprouting after δ-OR activation. Additional studies in adult brain extracts from RGS4-/- mice revealed increased levels of p-STAT5B. Finally, neuronal progenitor cultures from RGS4-/- mice exhibit enhanced proliferation with concomitant increases in the mRNA levels of the anti-apoptotic STAT5B target genes bcl2 and bcl-xl. These observations suggest that RGS4 is implicated in opioid dependent neuronal differentiation and neurite outgrowth via a “non-canonical” signaling pathway regulating STAT5B-directed responses.

The role of regulator of G protein signaling 4 in delta-opioid receptor-mediated behaviors.

Regulator of G protein signaling (RGS) proteins act as negative modulators of G protein signaling. RGS4 has been shown to negatively modulate G protein signaling mediated by the delta opioid receptor (DOPr) in vitro. However, the role of RGS4 in modulating DOPr-mediated behaviors in vivo has not been elucidated. The aim of this study was to compare the ability of the DOPr agonist SNC80 to induce DOPr-mediated antinociception, antihyperalgesia, antidepressant-like effects, and convulsions in wild-type and RGS4 knockout mice. Antinociception was assessed in the acetic acid stretch assay. Antihyperalgesia was measured in a nitroglycerin-induced thermal hyperalgesia assay. Antidepressant-like effects were evaluated in the forced swim and tail suspension tests. Mice were also observed for convulsive activity post-SNC80 treatment. SNC80-induced phosphorylation of MAP kinase in striatal tissue from RGS4 wild-type and knockout mice was quantified by Western blot. DOPr number from forebrain tissue was measured using [3H]DPDPE saturation binding. Elimination of RGS4 potentiated SNC80-induced antinociception and antihyperalgesia. SNC80-induced antidepressant-like effects were potentiated in RGS4 knockout mice in the forced swim test but not in the tail suspension test. Additionally, RGS4 knockout did not alter SNC80-induced convulsions. SNC80-induced phosphorylation of MAP kinase was potentiated in striatum from RGS4 knockout mice. Loss of RGS4 did not affect total DOPr number. Overall, these findings demonstrate that reduction of RGS4 functionally may increase the therapeutic index of SNC80. These results provide the first evidence of differential regulation of DOPr-mediated behaviors by RGS proteins and G protein signaling pathways.

The Effects of RGS4 on Delta Opioid Receptor‐mediated Behaviors in Mice

The delta opioid receptor (DOPr) is member of the opioid receptor family of G protein‐coupled receptors. Activation of DOPr induces antinociception and antidepressant‐like effects in animal models without the constipation, respiratory depression, and abuse liability associated with mu opioid receptor agonists such as morphine. In addition, some DOPr agonists cause convulsions, hindering their development as therapeutics in humans. Regulator of G protein signaling 4 (RGS4) proteins have been shown to act as negative modulators of DOPr‐mediated G protein signaling in vitro. However, the role of RGS4 in modulating DOPr‐mediated behaviors in vivo has yet to be determined. To this end, we compared the ability of the DOPr agonist SNC80 to induce antinociception, antidepressant‐like effects, and convulsions in wildtype and RGS4 knockout mice. Loss of RGS4 potentiated SNC80‐induced peripherally‐mediated antinociception in the acetic acid stretch assay and centrally‐mediated antihyperalgesia in a nitroglycerin‐induced thermal hyperalgesia assay in a DOPr‐dependent manner. There was no change in the dose of SNC80 needed to induce a convulsion. Furthermore, in the tail suspension test, RGS4 knockout mice showed no significant difference in SNC80‐induced antidepressant‐like effects. Taken together, these data suggest that RGS4 negatively modulates some, but not all DOPr‐mediated behaviors. Because RGS4 modulates DOPr‐mediated G protein signaling, these data are consistent with G protein‐independent signaling mechanisms generating DOPr‐mediated antidepressant‐like effects and convulsions. Future studies will focus on determining the mechanisms underlying differential modulation of DOPr‐mediated behaviors by RGS4. Research support was provided by start‐up funds to EMJ.

Intrathecal RGS4 inhibitor, CCG50014, reduces nociceptive responses and enhances opioid-mediated analgesic effects in the mouse formalin test.

The regulator of G-protein signaling protein type 4 (RGS4) accelerates the guanosine triphosphatase activity of G(αi) and G(αo), resulting in the inactivation of G-protein-coupled receptor signaling. An opioid receptor (OR), a G(αi)-coupled receptor, plays an important role in pain modulation in the central nervous system. In this study, we examined whether (1) spinal RGS4 affected nociceptive responses in the formalin pain test, (2) this RGS4-mediated effect was involved in OR activation, and (3) the µ-OR agonist-induced antinociceptive effect was modified by RGS4 modulation. Formalin (1%, 20 µL) was injected subcutaneously into the right hindpaws of male 129S4/SvJae×C57BL/6J (RGS4(+/+) or RGS4(-/-)) mice, and the licking responses were counted for 40 minutes. The time periods (seconds) spent licking the injected paw during 0 to 10 minutes (early phase) and 10 to 40 minutes (late phase) were measured as indicators of acute nociception and inflammatory pain response, respectively. An RGS4 inhibitor, CCG50014, and/or a µ-OR agonist, [D-Ala², N-MePhe⁴, Gly-ol]-enkephalin (DAMGO), were intrathecally injected 5 minutes before the formalin injection. A nonselective OR antagonist, naloxone, was intraperitoneally injected 30 minutes before the CCG50014 injection. Mice that received the formalin injection exhibited typical biphasic nociceptive behaviors. The nociceptive responses in RGS4-knockout mice were significantly decreased during the late phase but not during the early phase. Similarly, intrathecally administered CCG50014 (10, 30, or 100 nmol) attenuated the nociceptive responses during the late phase in a dose-dependent manner. The antinociceptive effect of the RGS4 inhibitor was totally blocked by naloxone (5 mg/kg). In contrast, intrathecal injection of DAMGO achieved a dose-dependent reduction of the nociceptive responses at the early and late phases. This analgesic effect of DAMGO was significantly enhanced by the genetic depletion of RGS4 or by coadministration of CCG50014 (10 nmol). These findings demonstrated that spinal RGS4 inhibited the endogenous or exogenous OR-mediated antinociceptive effect in the formalin pain test. Thus, the inhibition of RGS4 activity can enhance OR agonist-induced analgesia. The enhancement of OR agonist-induced analgesia by coadministration of the RGS4 inhibitor suggests a new therapeutic strategy for the management of inflammatory pain.

Regulator of G-protein signaling 2 repression exacerbates airway hyper-responsiveness and remodeling in asthma.

G protein-coupled receptors (GPCRs) are important regulators of cell functions in asthma. We recently reported that regulator of G-protein signaling (RGS) 2, a selective modulator of Gq-coupled GPCRs, is a key regulator of airway hyper-responsiveness (AHR), the pathophysiologic hallmark of asthma. Because RGS2 protein levels in airway cells were significantly lower in patients with asthma compared with patients without asthma, we further investigated the potential pathological importance of RGS2 repression in asthma. The human RGS2 gene maps to chromosome 1q31. We first screened patients with asthma for RGS2 gene promoter single-nucleotide polymorphisms (SNPs) and found significant differences in the distribution of two RGS2 SNPs (A638G, rs2746071 and C395G, rs2746072) between patients with asthma and nonasthmatic subjects. These two SNPs are always associated with each other and have the same higher prevalence in patients with asthma (65%) as compared with nonasthmatic subjects (35%). Point mutations corresponding to these SNPs decrease RGS2 promoter activity by 44%. The importance of RGS2 down-regulation was then determined in an acute IL-13 mouse model of asthma. Intranasal administration of IL-13 in mice also decreased RGS2 expression in lungs by ∼50% and caused AHR. Although naive RGS2 knockout (KO) mice exhibit spontaneous AHR, acute IL-13 exposure further increased AHR in RGS2 KO mice. Loss of RGS2 also significantly enhanced IL-13-induced mouse airway remodeling, including peribronchial smooth muscle thickening and fibrosis, without effects on goblet cell hyperplasia or airway inflammation in mice. Thus, genetic variations and increased inflammatory cytokines can lead to RGS2 repression, which exacerbates AHR and airway remodeling in asthma.

Effects of gender on locomotor sensitivity to amphetamine, body weight, and fat mass in regulator of G protein signaling 9 (RGS9) knockout mice

Regulator of G-protein signaling (RGS) protein 9-2 is enriched in the striatum where it modulates dopamine and opioid receptor-mediated signaling. RGS9 knockout (KO) mice show increased psychostimulant-induced behavioral sensitization, as well as exhibit higher body weights and greater fat accumulation compared to wild-type (WT) littermates. In the present study, we found gender influences on each of these phenotypic characteristics. Female RGS9 KO mice exhibited greater locomotor sensitization to amphetamine (1.0mg/kg) treatment as compared to male RGS9 KO mice. Male RGS9 KO mice showed increased body weights as compared to male WT littermates, while no such differences were detected in female mice. Quantitative magnetic resonance showed that male RGS9 KO mice accumulated greater fat mass vs. WT littermates at 5months of age. Such observations could not be explained by increased caloric consumption since male and female RGS9 KO mice demonstrated equivalent daily food intake as compared to their respective WT littermates. Although indirect calorimetry methods found decreased oxygen consumption and carbon dioxide production during the 12-hour dark phase in male RGS9 KO vs. WT mice which are indicative of less energy expenditure, male RGS9 KO mice exhibited lower levels of locomotor activity during this period. Genotype had no effect on metabolic activities when KO and WT groups were compared under fasting vs. feeding treatments. In summary, these results highlight the importance of factoring gender into the experimental design since many studies conducted in RGS9 KO mice utilize locomotor activity as a measured outcome.

Regulator of G protein signaling 2 (RGS2) deficiency accelerates the progression of kidney fibrosis

The regulator of G protein signaling 2 (RGS2) is a potent negative regulator of Gq protein signals including the angiotensin II (AngII)/AngII receptor signal, which plays a critical role in the progression of fibrosis. However, the role of RGS2 on the progression of kidney fibrosis has not been assessed. Here, we investigated the role of RGS2 in kidney fibrosis induced by unilateral ureteral obstruction (UUO) in mice. UUO resulted in increased expression of RGS2 mRNA and protein in the kidney along with increases of AngII and its type 1 receptor (AT1R) signaling and fibrosis. Furthermore, UUO increased the levels of F4/80, Ly6G, myeloperoxidase, and CXCR4 in the kidneys. RGS2 deficiency significantly enhanced these changes in the kidney. RGS2 deletion in the bone marrow-derived cells by transplanting the bone marrow of RGS2 knock-out mice into wild type mice enhanced UUO-induced kidney fibrosis. Overexpression of RGS2 in HEK293 cells, a human embryonic kidney cell line, and RAW264.7 cells, a monocyte/macrophage line, inhibited the AngII-induced activation of ERK and increase of CXCR4 expression. These findings provide the first evidence that RGS2 negatively regulates the progression of kidney fibrosis following UUO, likely by suppressing fibrogenic and inflammatory responses through the inhibition of AngII/AT1R signaling.

Nucleus Accumbens-Specific Interventions in RGS9-2 Activity Modulate Responses to Morphine

Regulator of G protein signalling 9-2 (Rgs9-2) modulates the actions of a wide range of CNS-acting drugs by controlling signal transduction of several GPCRs in the striatum. RGS9-2 acts via a complex mechanism that involves interactions with Gα subunits, the Gβ5 protein, and the adaptor protein R7BP. Our recent work identified Rgs9-2 complexes in the striatum associated with acute or chronic exposures to mu opioid receptor (MOR) agonists. In this study we use several new genetic tools that allow manipulations of Rgs9-2 activity in particular brain regions of adult mice in order to better understand the mechanism via which this protein modulates opiate addiction and analgesia. We used adeno-associated viruses (AAVs) to express forms of Rgs9-2 in the dorsal and ventral striatum (nucleus accumbens, NAc) in order to examine the influence of this protein in morphine actions. Consistent with earlier behavioural findings from constitutive Rgs9 knockout mice, we show that Rgs9-2 actions in the NAc modulate morphine reward and dependence. Notably, Rgs9-2 in the NAc affects the analgesic actions of morphine as well as the development of analgesic tolerance. Using optogenetics we demonstrate that activation of Channelrhodopsin2 in Rgs9-2-expressing neurons, or in D1 dopamine receptor (Drd1)-enriched medium spiny neurons, accelerates the development of morphine tolerance, whereas activation of D2 dopamine receptor (Drd2)-enriched neurons does not significantly affect the development of tolerance. Together, these data provide new information on the signal transduction mechanisms underlying opiate actions in the NAc.

Atrial Fibrillation Promotion by Endurance Exercise: Demonstration and Mechanistic Exploration in an Animal Model

The goal of this study was to assess mechanisms underlying atrial fibrillation (AF) promotion by exercise training inan animal model. High-level exercise training promotes AF, but the underlying mechanisms are unclear. AF susceptibility was assessed by programmed stimulation in rats after 8 (Ex8) and 16 (Ex16) weeks of daily 1-h treadmill training, along with 4 and 8 weeks after exercise cessation and time-matched sedentary (Sed) controls. Structural remodeling was evaluated by using serial echocardiography and histopathology, autonomic nervous system with pharmacological tools, acetylcholine-regulated potassium current (IKACh) with patch clamp recording, messenger ribonucleic acid expression with quantitative polymerase chain reaction, and regulators of G protein-signaling (RGS) 4 function in knockout mice. AF inducibility increased after 16 weeks of training (e.g., AF >30 s in 64% of Ex16 rats vs 15% of Sed rats; p< 0.01) and rapidly returned to baseline levels with detraining. Atropine restored sinus rhythm in 5 of 5 Ex rats with AF sustained >15 min. Atrial dilation and fibrosis developed after 16 weeks of training and failed to fully recover with exercise cessation. Parasympathetic tone was increased in Ex16 rats and normalized within 4 weeks of detraining. Baroreflex heart rate responses to phenylephrine-induced blood pressure elevation and IKACh sensitivity to carbachol were enhanced in Ex16 rats, implicating both central and end-organ mechanisms in vagal enhancement. Ex rats showed unchanged cardiac adrenergic and cholinergic receptor and IKACh-subunit gene expression, but significant messenger ribonucleic acid downregulation of IKACh-inhibiting RGS proteins was present at 16 weeks. RGS4 knockout mice showed significantly enhanced sensitivity to AF induction in the presence of carbachol. Chronic endurance exercise increased AF susceptibility in rats, with autonomic changes, atrial dilation, and fibrosis identified as potential mechanistic contributors. Vagal promotion is particularly important and occurs via augmented baroreflex responsiveness and increased cardiomyocyte sensitivity to cholinergic stimulation, possibly due to RGSprotein downregulation.

RGS2 repression increases susceptibility of mice to interleukin‐ 13‐induced airway hyperresponsiveness

G‐protein coupled receptors (GPCRs) are important regulators of cells involved in asthma. We recently reported that RGS2, a selective modulator of Gq‐coupled GPCRs, is a key regulator of airway hyperresponsiveness (AHR), the pathophysiologic hallmark of asthma. Since immunohistochemistry staining showed 50% repression of RGS2 protein in bronchial epithelium and smooth muscle of asthma patients as compared to non‐asthmatics, we further investigated the pathological importance of RGS2 repression in asthma. Th2 cytokine interleukin‐13 (IL‐13) is necessary and sufficient to induce AHR in mice. We found that intranasal administration of IL‐13 decreased RGS2 expression in airways and caused AHR in mice, assessed by Western blot and invasive tracheostomy. Although both hetero‐ and homozygous naïve RGS2 knockout (KO) mice exhibit spontaneous AHR, IL‐13 treatment further augmented AHR in RGS2 KO mice. Interestingly, deletion of RGS2 gene significantly enhanced IL‐13‐ induced airway remodeling, including increased peribronchial fibrosis and smooth muscle in mice. Thus, we speculate that RGS2 is an important gene regulating AHR, and RGS2 repression increases susceptibility of mice to IL‐13‐induced AHR in part through the promotion of airway remodeling. American Asthma Foundation (to Y.T.), Nebraska LB595 and 692 (to Y.T. and T.B.C.), NIH R01 HL097796 and P30 ES013508 (to R.A.P.).

Defective expression of the regulator of G-protein signaling (RGS) 4 increases MUC5AC overproduction in the epithelia of nasal polyps

Nasal polyps are believed that they may be caused by allergy and cystic fibrosis. Despite elimination by surgery, nasal polyps are observed to recur about 70%. Most importantly, the pathogenesis of nasal polyps has been unknown. Therefore, the molecular mechanisms that increase mucous hypersecretion in nasal polyps should be identified. The understanding of these mechanisms is important for developing new therapeutic strategies. The aim of this study was to identify the effect of the regulator of G protein signaling 4 protein (RGS4) on nasal polyps and to investigate the role of the ATP-sensitive potassium (KATP) channel on lipopolysaccharide (LPS)-induced mucin-5AC protein (MUC5AC) expression in vivo. MUC5AC and RGS4 expressions were studied by immunoblotting in human nasal polyp and in lungs from Rgs4 knock-out mice. Glybenclamide and apyrase were challenged i.p. injection for 2 hr in prior to LPS instillation using tracheostomy. We found that the expression of RGS4 was dramatically suppressed to about 70% in the polyp specimens compared to that in the normal nasal mucosa. This finding suggests that defective RGS4 expression may have a role in the increased MUC5AC expression in the epithelia of human nasal polyps. Inhibition of the KATP channel leads to diminished MUC5AC overexpression in vivo. Since the KATP channel can be mediated in LPS-induced MUC5AC expression in vivo, the KATP channel may be a good target for regulating mucous hypersecretion in respiratory diseases.

Regulator of G protein signaling 2 is a key modulator of airway hyperresponsiveness

Drugs targeting individual G protein-coupled receptors are used as asthma therapies, but this strategy is limited because of G protein-coupled receptor signal redundancy. Regulator of G protein signaling 2 (RGS2), an intracellular selective inhibitor of multiple bronchoconstrictor receptors, may play a central role in the pathophysiology and treatment of asthma. We defined functions and mechanisms of RGS2 in regulating airway hyperresponsiveness (AHR), the pathophysiologic hallmark of asthma. Real-time PCR and Western blot were used to determine changes in RGS2 expression in ovalbumin-sensitized/-challenged mice. We also used immunohistochemistry and real-time PCR to compare RGS2 expression between human asthmatic and control subjects. The AHR of RGS2 knockout mice was assessed by using invasive tracheostomy and unrestrained plethysmography. Effects of loss of RGS2 on mouse airway smooth muscle (ASM) remodeling, contraction, intracellular Ca(2+), and mitogenic signaling were determined in vivo and in vitro. RGS2 was highly expressed in human and murine bronchial epithelium and ASM and was markedly downregulated in lungs of ovalbumin-sensitized/-challenged mice. Lung tissues and blood monocytes from asthma patients expressed significantly lower RGS2 protein (lung) and mRNA (monocytes) than from nonasthma subjects. The extent of reduction of RGS2 on human monocytes correlated with increased AHR. RGS2 knockout caused spontaneous AHR in mice. Loss of RGS2 augmented Ca(2+) mobilization and contraction of ASM cells. Loss of RGS2 also increased ASM mass and stimulated ASM cell growth via extracellular signal-regulated kinase and phosphatidylinositol 3-kinase pathways. We identified RGS2 as a potent modulator of AHR and a potential novel therapeutic target for asthma.

Molecular Mechanism of Macrophage Activation by Red Ginseng Acidic Polysaccharide from Korean Red Ginseng

Red ginseng acidic polysaccharide (RGAP), isolated from Korean red ginseng, displays immunostimulatory and antitumor activities. Even though numerous studies have been reported, the mechanism as to how RGAP is able to stimulate the immune response is not clear. In this study, we aimed to explore the mechanism of molecular activation of RGAP in macrophages. RGAP treatment strongly induced NO production in RAW264.7 cells without altering morphological changes, although the activity was not strong compared to LPS-induced dendritic-like morphology in RAW264.7 cells. RGAP-induced NO production was accompanied with enhanced mRNA levels of iNOS and increases in nuclear transcription factors such as NF-κB, AP-1, STAT-1, ATF-2, and CREB. According to pharmacological evaluation with specific enzyme inhibitors, Western blot analysis of intracellular signaling proteins and inhibitory pattern using blocking antibodies, ERK, and JNK were found to be the most important signaling enzymes compared to LPS signaling cascade. Further, TLR2 seems to be a target surface receptor of RGAP. Lastly, macrophages isolated from RGS2 knockout mice or wortmannin exposure strongly upregulated RGAP-treated NO production. Therefore, our results suggest that RGAP can activate macrophage function through activation of transcription factors such as NF-κB and AP-1 and their upstream signaling enzymes such as ERK and JNK.

Controlling Parasympathetic Regulation of Heart Rate: A Gatekeeper Role for RGS Proteins in the Sinoatrial Node

Neurotransmitters released from sympathetic and parasympathetic nerve terminals in the sinoatrial node (SAN) exert their effects via G-protein-coupled receptors. Integration of these different G-protein signals within pacemaker cells of the SAN is critical for proper regulation of heart rate and function. For example, excessive parasympathetic signaling can be associated with sinus node dysfunction (SND) and supraventricular arrhythmias. Our previous work has shown that one member of the regulator of G-protein signaling (RGS) protein family, RGS4, is highly and selectively expressed in pacemaker cells of the SAN. Consistent with its role as an inhibitor of parasympathetic signaling, RGS4-knockout mice have reduced basal heart rates and enhanced negative chronotropic responses to parasympathetic agonists. Moreover, RGS4 appears to be an important part of SA nodal myocyte signaling pathways that mediate G-protein-coupled inwardly rectifying potassium channel (GIRK) channel activation/deactivation and desensitization. Since RGS4 acts immediately downstream of M2 muscarinic receptors, it is tempting to speculate that RGS4 functions as a master regulator of parasympathetic signaling upstream of GIRKs, HCNs, and L-type Ca2+ channels in the SAN. Thus, loss of RGS4 function may lead to increased susceptibility to conditions associated with increased parasympathetic signaling, including bradyarrhythmia, SND, and atrial fibrillation.