Adrenergic Receptor Knockout Mice Research Articles

The β 1 -Adrenergic Receptor Contributes to Sepsis-Induced Immunosuppression Through Modulation of Regulatory T-Cell Inhibitory Function.

Although cardiovascular benefits of β 1 -adrenergic receptor blockade have been described in sepsis, little is known about its impact on the adaptive immune response, specifically CD4 T cells. Herein, we study the effects of β 1 -adrenergic receptor modulation on CD4 T-cell function in a murine model of sepsis. Experimental study. University laboratory. C57BL/6 mice. High-grade sepsis was induced by cecal ligation and puncture in wild-type mice (β 1+/+ ) with or without esmolol (a selective β 1 -adrenergic receptor blocker) or in β 1 -adrenergic receptor knockout mice (β 1-/- ). At 18 hours after surgery, echocardiography was performed with blood and spleen collected to analyze lymphocyte function. At 18 hours, β 1+/+ cecal ligation and puncture mice exhibited characteristics of high-grade sepsis and three surrogate markers of immunosuppression, namely decreased splenic CD4 T cells, reduced CD4 T-cell proliferation, and increased regulatory T lymphocyte cell proportions. Pharmacologic and genetic β 1 -adrenergic receptor blockade reversed the impact of sepsis on CD4 T and regulatory T lymphocyte proportions and maintained CD4 T-cell proliferative capacity. β 1 -adrenergic receptor blocked cecal ligation and puncture mice also exhibited a global decrease in both pro- and anti-inflammatory mediators and improved in vivo cardiovascular efficiency with maintained cardiac power index despite the expected decrease in heart rate. β 1 -adrenergic receptor activation enhances regulatory T lymphocyte inhibitory function and thus contributes to sepsis-induced immunosuppression. This can be attenuated by β 1 -adrenergic receptor blockade, suggesting a potential immunoregulatory role for this therapy in the management of sepsis.

The Role of Beta‐2 Adrenergic Receptors in Cardiac Bioenergetics Following Severe Burns

Severe burn injury is characterized by increased energy expenditure, and altered mitochondrial function is thought to be central to the hypermetabolic response to burns. As catecholamines are an important driver of the hypermetabolic stress response to burn, we set out to determine the role of adrenergic signaling through Beta‐2 adrenergic receptors in cardiac mitochondrial function following burn. We hypothesized that a loss of adrenergic signaling would attenuate cardiac mitochondrial dysfunction in response to burns.We used beta ‐2 adrenergic receptor knockout mice (ADRB2‐KO) and wild type controls (WT). Mice received a 30% of total body surface area scald burn or a sham burn and were sacrificed one week following injury. Hearts were excised and mitochondrial function was measured in saponin‐permeabilized myofiber bundles by high‐resolution respirometry. Data were analyzed using two‐way ANOVA and Bonferroni post‐test to compare all groups.Mitochondrial respiratory capacity was significantly reduced in both WT (P<0.01) and KO (P<0.01) mice following burn, although there was no difference between WT and KO mice. In WT animals, respiration coupled to ATP production was significantly lower in the burn animals compared to shams (p< 0.05). In contrast, respiration coupled to ATP production was not different in sham and burn treated ADRB2‐KO mice. Mitochondrial flux control ratios (leak and couple respiration normalized to maximal uncoupled respiration) were significantly higher in burned WT mice compared to sham (P<0.05). However, mitochondrial flux control was not significantly altered by burn in KO mice.Collectively, these data underscore the role of catecholamines and adrenergic stress in the metabolic response to burns. Specifically, ablation of beta 2 adrenergic signaling in the heart prevents cardiac mitochondrial dysfunction in response to severe burn trauma.Support or Funding InformationThis study was supported by grants from the National Institute of Health (P50‐GM60338, R01‐GM56687, and GM 112936‐01), Shriners Hospitals for Children (80100, 87300, 71008, 70900, 85116, 71000), and SSF grant for Craig Porter. This study was also conducted with the support of the Institute for Translational Sciences at the University of Texas Medical Branch, supported in part by a Clinical and Translational Science Award (UL1TR000071) from the National Center for Advanc‐ing Translational Sciences, National Institutes of Health.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A stress response pathway in mice upregulates somatostatin level and transcription in pancreatic delta cells through Gs and β-arrestin 1.

Somatostatin secretion from islet delta cells plays an important role in regulating islet function and is tightly controlled by environmental changes. Activation of the adrenergic system promoted somatostatin secretion from islet delta cells; however, the role of the adrenergic system in regulating somatostatin content and transcription has not been defined. An imbalance between the somatostatin content and its secretion may cause dysfunctions in the islet delta cells. We have investigated the role of the adrenergic system in the modulation of somatostatin content and transcription in pancreatic delta cells and the detailed underlying mechanisms of this regulation. The stress hormone adrenaline (epinephrine), specific adrenergic agonists or specific adrenergic antagonists were applied to islets from either wild-type or specific adrenergic receptor knockout mice and pancreatic delta cell lines to investigate their effects on somatostatin content and transcription. The GloSensor assay, quantitative real-time PCR, western blots and the dual luciferase assay were used to monitor the cAMP level, somatostatin expression, activations of kinases and transcriptional factors. Arrb1 knockout mice, specific Creb or Pax6 mutations and specific kinase inhibitors were used to dissect the signalling pathway. Adrenaline and isoprenaline increased somatostatin content and transcription through the activation of β1-/β2-adrenergic receptors (β1-/β2ARs). The somatostatin content in β1AR(-/-) /β2AR(-/-) (Adrb1/Adrb2 knockout) mice was 50% lower than in β1AR(+/+)/β2AR (+/+) mice. Two parallel signalling pathways, Gs-cAMP-protein kinase A (PKA)-cAMP response element binding protein (CREB) and β-arrestin 1-extracellular signal-related kinase (ERK)-paired box protein 6 (PAX6), cooperatively regulated isoprenaline-induced somatostatin transcription. A stress pathway increased somatostatin content and transcription through β-adrenergic agonism. β-Arrestin1, ERK and PAX6 are important pancreatic delta cell regulators in addition to cAMP, PKA and CREB. Dysfunction of β-adrenergic agonism may impair pancreatic delta cell function.

Integrative Effect of Carvedilol and Aerobic Exercise Training Therapies on Improving Cardiac Contractility and Remodeling in Heart Failure Mice

The use of β-blockers is mandatory for counteracting heart failure (HF)-induced chronic sympathetic hyperactivity, cardiac dysfunction and remodeling. Importantly, aerobic exercise training, an efficient nonpharmacological therapy to HF, also counteracts sympathetic hyperactivity in HF and improves exercise tolerance and cardiac contractility; the latter associated with changes in cardiac Ca2+ handling. This study was undertaken to test whether combined β–blocker and aerobic exercise training would integrate the beneficial effects of isolated therapies on cardiac structure, contractility and cardiomyocyte Ca2+ handling in a genetic model of sympathetic hyperactivity-induced HF (α2A/α2C- adrenergic receptor knockout mice, KO). We used a cohort of 5–7 mo male wild-type (WT) and congenic mice (KO) with C57Bl6/J genetic background randomly assigned into 5 groups: control (WT), saline-treated KO (KOS), exercise trained KO (KOT), carvedilol-treated KO (KOC) and, combined carvedilol-treated and exercise-trained KO (KOCT). Isolated and combined therapies reduced mortality compared with KOS mice. Both KOT and KOCT groups had increased exercise tolerance, while groups receiving carvedilol had increased left ventricular fractional shortening and reduced cardiac collagen volume fraction compared with KOS group. Cellular data confirmed that cardiomyocytes from KOS mice displayed abnormal Ca2+ handling. KOT group had increased intracellular peak of Ca2+ transient and reduced diastolic Ca2+ decay compared with KOS group, while KOC had increased Ca2+ decay compared with KOS group. Notably, combined therapies re-established cardiomyocyte Ca2+ transient paralleled by increased SERCA2 expression and SERCA2:PLN ratio toward WT levels. Aerobic exercise trained increased the phosphorylation of PLN at Ser16 and Thr17 residues in both KOT and KOCT groups, but carvedilol treatment reduced lipid peroxidation in KOC and KOCT groups compared with KOS group. The present findings provide evidence that the combination of carvedilol and aerobic exercise training therapies lead to a better integrative outcome than carvedilol or exercise training used in isolation.

β1-Adrenergic receptors increase UCP1 in human MADS brown adipocytes and rescue cold-acclimated β3-adrenergic receptor-knockout mice via nonshivering thermogenesis

With the finding that brown adipose tissue is present and negatively correlated to obesity in adult man, finding the mechanism(s) of how to activate brown adipose tissue in humans could be important in combating obesity, type 2 diabetes, and their complications. In mice, the main regulator of nonshivering thermogenesis in brown adipose tissue is norepinephrine acting predominantly via β(3)-adrenergic receptors. However, vast majorities of β(3)-adrenergic agonists have so far not been able to stimulate human β(3)-adrenergic receptors or brown adipose tissue activity, and it was postulated that human brown adipose tissue could be regulated instead by β(1)-adrenergic receptors. Therefore, we have investigated the signaling pathways, specifically pathways to nonshivering thermogenesis, in mice lacking β(3)-adrenergic receptors. Wild-type and β(3)-knockout mice were either exposed to acute cold (up to 12 h) or acclimated for 7 wk to cold, and parameters related to metabolism and brown adipose tissue function were investigated. β(3)-knockout mice were able to survive both acute and prolonged cold exposure due to activation of β(1)-adrenergic receptors. Thus, in the absence of β(3)-adrenergic receptors, β(1)-adrenergic receptors are effectively able to signal via cAMP to elicit cAMP-mediated responses and to recruit and activate brown adipose tissue. In addition, we found that in human multipotent adipose-derived stem cells differentiated into functional brown adipocytes, activation of either β(1)-adrenergic receptors or β(3)-adrenergic receptors was able to increase UCP1 mRNA and protein levels. Thus, in humans, β(1)-adrenergic receptors could play an important role in regulating nonshivering thermogenesis.

Gene expression profiling: Classification of mice with left ventricle systolic dysfunction using microarray analysis*

We tested the hypothesis that a set of differentially expressed genes could be used to classify mice according to cardiovascular phenotype after prolonged catecholamine stress. Prospective, randomized study. University-based research laboratory. One hundred seventy-three male mice were studied: wild-type (WT) C57, WT FVB, WT B6129SF2/J, and beta2 adrenergic receptor knockout. Mice of each genotype were randomly assigned to 14-day infusions of isoproterenol (120 microg/g/day) or no treatment. Approximately half of the animals underwent left ventricle pressure volume loop analysis. The remaining animals were killed for extraction of messenger RNA from whole heart preparations for microarray analysis. We observed that WT FVB and beta2 adrenergic receptor knockout mice developed systolic dysfunction in response to continuous catecholamine infusion, whereas WT C57 mice developed diastolic dysfunction. Using these mice as the derivation cohort, we identified a set of 83 genes whose differential expression correlated with left ventricle systolic dysfunction. The gene set was then used to accurately classify mice from a separate group (WT B6129SF2/J) into the cohort that developed left ventricle systolic dysfunction after catecholamine stress. The differential expression pattern of 83 genes can be used to accurately classify mice according to physiological phenotype after catecholamine stress.

Reduced Wheel Running Activity in β1/β2 Adrenergic Receptor Knockout Mice is not Caused by Anxiety

Mice lacking β1 and β2 adrenergic receptors (β1/β2ADR ‐/‐) exhibit reduced locomotor activity as measured by telemetry transmitter. In the present experiments we have examined whether the reduced locomotor activity resulted of augmented anxiety levels. Wild type (WT) and β1/β2ADR ‐/‐ mice were housed in running wheel (RW) cages for a 2 week period at a 12h light:12h dark (LD) cycle followed by a 2 week period at 12h dark: 12h dark (DD) conditions. The 24 hour average of RW revolutions/min was significantly lower in β1/β2ADR‐/‐ mice (LD: 3 ¡± 1; DD: 4 ± 1; n=7) compared to WT mice (LD: 18 ± 3; DD: 17 ± 4; n=8; p < 0.05). The activity showed a circadian pattern in both genotypes, but the periodic strength was significantly greater in WT (LD: 1117 ± 152; DD: 842 ± 135; n=8) than in β1/β2ADR‐/‐ (LD: 561 ± 114; DD: 414 ± 105; n=7; p < 0.05). To study anxiety levels behavior was analyzed in an open field test by videotracking for 5 min. 30‐35 min after placement into the open field, total distance traveled was 1382 ± 283 cm in WT (n=4) and 823 ± 150 cm (n=4; p<0.05) consistent with reduced locomotor activity. However, β1/β2ADR‐/‐ mice were found to spend 73 ± 8 % of total time in the open inner field while WT mice only spent 25 ± 9 % in the open field preferring proximity of walls for 75% of the time (n=4; p<0.05). In conclusion, absence of β1/β2ADR causes reduced voluntary locomotor activity and strength of circadian rhythmicity despite reduced levels of anxiety.

Clinical implications from studies of α1 adrenergic receptor knockout mice

α1-Adrenergic receptors (α1-ARs) modulate a large number of physiological functions in cardiovascular and noncardiovascular tissues. Because individual members of the α1-AR family (α1A-, α1B-, and α1D-ARs) have overlapping expression profiles in most tissues, elucidation of the precise physiological roles of individual α1-AR subtypes remains a challenging task. To alleviate this constraint, a gene targeting approach has been employed to generate mutant mice lacking one or two α1-AR genes. Recent studies on these mutant mouse strains are discussed in this article, with an emphasis on the role of α1-AR in the central nervous system and lower urinary tracts. These are two major tissues of particular interest for the development of new therapeutic strategies targeted to the α1-ARs. By combining gene targeting techniques with pharmacological tools, the specific roles of α1-AR subtypes could be delineated.

Enhanced proliferation of astrocytes from β2‐adrenergic receptor knockout mice is influenced by the IGF system

In the present study, we investigated the IGF system in neonatal astrocytes derived from mice with a targeted disruption of the beta-2 adrenergic receptor (beta(2)AR). beta(2)AR knockout astrocytes demonstrated higher proliferation rates and increased expression of the astrogliotic marker GFAP, as compared with wild-type cells. beta(2)AR deletion also regulated molecules of the IGF system. Although IGF-1 levels remained unaltered, IGF-2 and type 1 IGF receptor expression was increased in beta(2)AR knockout cells. Furthermore, conditioned medium from knockout astrocytes contained lower levels of IGF binding protein-2 and -4. Our data suggest a deficit of beta(2)AR on astrocytes, as previously reported in multiple sclerosis, may have implications on proliferative status of astrocytes, a feature that might be attributed to regulation of IGF mitogenic actions.

Alpha-1B adrenergic receptor knockout mice are protected against methamphetamine toxicity.

The psychostimulant methamphetamine (MA) is toxic to nigro-striatal dopaminergic terminals in both experimental animals and humans. In mice, three consecutive injections of MA (5 mg/kg, i.p. with 2 h of interval) induced a massive degeneration of the nigro-striatal pathway, as reflected by a 50% reduction in the striatal levels of dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC), by a substantial reduction in striatal tyrosine hydroxylase and high-affinity DA transporter immunostaining, and by the development of reactive gliosis. MA-induced nigro-striatal degeneration was largely attenuated in mice lacking alpha1b-adrenergic receptors (ARs). MA-stimulated striatal DA release (measured by microdialysis in freely moving animals) and locomotor activity were also reduced in alpha1b-AR knockout mice. Pharmacological blockade of alpha-adrenergic receptors with prazosin also protected wild-type mice against MA toxicity. These results suggests that alpha1b-ARs may play a role in the toxicity of MA on nigro-striatal DA neurons.

Alpha(2C)-Adrenergic receptors mediate spinal analgesia and adrenergic-opioid synergy.

The alpha(2A)-adrenergic receptor (AR) subtype mediates antinociception induced by the alpha(2)AR agonists clonidine, dexmedetomidine, norepinephrine, and 5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine (UK-14,304) as well as antinociceptive synergy of UK-14,304 with opioid agonists [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin and deltorphin II. Differential localization of alpha(2)-adrenergic (alpha(2A)-, alpha(2B)-(,) alpha(2C)-) and opioid (mu-, delta-, kappa-) subtypes suggests differential involvement of subtype pairs in opioid-adrenergic analgesic synergy. The present study applies a novel imidazoline(1)/alpha(2)-adrenergic receptor analgesic, moxonidine, to test for involvement of alpha(2B)- and alpha(2C)ARs in antinociception and antinociceptive synergy, because spinal antinociceptive activity of moxonidine shows minimal dependence on alpha(2A)AR. Intrathecal administration of moxonidine produced similar (2-3-fold) decreases in both mutant mice with a functional knockout of alpha(2A)AR (D79N-alpha(2A)AR) and alpha(2C)AR knockout (KO) mice. The potency of moxonidine was not altered in alpha(2B)KO mice, indicating that this subtype does not participate in moxonidine-induced spinal antinociception. Moxonidine-mediated antinociception was dose dependently inhibited by the selective alpha(2)-receptor antagonist SK&F 86466 in both D79N-alpha(2A) mice and alpha(2C)KO mice, indicating that alpha(2)AR activation is required in the absence of either alpha(2A)- or alpha(2C)AR. Spinal administration of antisense oligodeoxynucleotides directed against the alpha(2C)AR decreased both alpha(2C)AR immunoreactivity and the antinociceptive potency of moxonidine. Isobolographic analysis demonstrates that moxonidine-deltorphin antinociceptive synergy is present in the D79N-alpha(2A) mice but not in the alpha(2C)AR-KO mice. These results confirm that the alpha(2C)AR subtype contributes to spinal antinociception and synergy with opioids.

Activation and Deactivation Kinetics of α2A- and α2C-Adrenergic Receptor-activated G Protein-activated Inwardly Rectifying K+ Channel Currents

Although G protein-coupled receptor-mediated signaling is one of the best studied biological events, little is known about the kinetics of these processes in intact cells. Experiments with neurons from alpha(2A)-adrenergic receptor knockout mice suggested that the alpha(2A)-receptor subtype inhibits neurotransmitter release with higher speed and at higher action potential frequencies than the alpha(2C)-adrenergic receptor. Here we investigated whether these functional differences between presynaptic alpha(2)-adrenergic receptor subtypes are the result of distinct signal transduction kinetics of these two receptors and their coupling to G proteins. alpha(2A)- and alpha(2C)-receptors were stably expressed in HEK293 cells at moderate ( approximately 2 pmol/mg) or high (17-24 pmol/mg) levels. Activation of G protein-activated inwardly rectifying K(+) (GIRK) channels was similar in extent and kinetics for alpha(2A)- and alpha(2C)-receptors at both expression levels. However, the two receptors differed significantly in their deactivation kinetics after removal of the agonist norepinephrine. alpha(2C)-Receptor-activated GIRK currents returned much more slowly to base line than did alpha(2A)-stimulated currents. This observation correlated with a higher affinity of norepinephrine at the murine alpha(2C)- than at the alpha(2A)-receptor subtype and may explain why alpha(2C)-adrenergic receptors are especially suited to control sympathetic neurotransmission at low action potential frequencies in contrast to the alpha(2A)-receptor subtype.

A Behavioral Study of Alpha-1b Adrenergic Receptor Knockout Mice: Increased Reaction to Novelty and Selectively Reduced Learning Capacities

Knockout mice lacking the alpha-1b adrenergic receptor were tested in behavioral experiments. Reaction to novelty was first assessed in a simple test in which the time taken by the knockout mice and their littermate controls to enter a second compartment was compared. Then the mice were tested in an open field to which unknown objects were subsequently added. Special novelty was introduced by moving one of the familiar objects to another location in the open field. Spatial behavior and memory were further studied in a homing board test, and in the water maze. The alpha-1b knockout mice showed an enhanced reactivity to new situations. They were faster to enter the new environment, covered longer paths in the open field, and spent more time exploring the new objects. They reacted like controls to modification inducing spatial novelty. In the homing board test, both the knockout mice and the control mice seemed to use a combination of distant visual and proximal olfactory cues, showing place preference only if the two types of cues were redundant. In the water maze the alpha-1b knockout mice were unable to learn the task, which was confirmed in a probe trial without platform. They were perfectly able, however, to escape in a visible platform procedure. These results confirm previous findings showing that the noradrenergic pathway is important for the modulation of behaviors such as reaction to novelty and exploration, and suggest that this is mediated, at least partly, through the alpha-1b adrenergic receptors. The lack of alpha-1b adrenergic receptors in spatial orientation does not seem important in cue-rich tasks but may interfere with orientation in situations providing distant cues only.