CGP 20712A Research Articles

Mechanistic insights into the therapeutic potential of Angiotensin type 2 receptors (AT2R) in shaping behavior of astrocytes and microglia during hypertensive state

AbstractBackgroundAstrocytes are the major glial cell type in the central nervous system (CNS) and play a critical role in the pathogenesis of neuroinflammatory and neurodegenerative diseases. In particular, astrocytic activation during neuroinflammatory conditions perpetuates the inflammatory response and brain damage and neurodegeneration. Conversely, Angiotensin type 2 receptor (AT2R) activation is known to promote neuroprotection.MethodTherefore, the present study evaluated the role of AT2R in astrocytic activation in vitro in primary astrocytes and in vivo in hypertensive rats.ResultHere, we demonstrated that Ang II‐induced astrogliosis in primary astrocytes as apparent from increased expression of GFAP and iNOS, ROS generation, inflammatory imbalance and mitochondrial dysfunction in the primary astrocytes. On the contrary, AT2R activation by CGP42112A (CGP) abrogated Ang II induced astrocytic activation by inhibition of ROS production, mitochondrial dysfunction and inflammatory imbalance. However, this anti‐inflammatory action of AT2R activation by CGP was reversed by AT2R antagonist, PD123319 in both in vitro and in vivo conditions. Mechanistically, we found that AT2R via protein phosphatase‐2A (PP2A) activation abrogated the Ang II induced NFкB activation, ROS generation and consequent pro‐inflammatory activation of astrocytes. Importantly, PP2A antagonist, okadaic acid, reversed the anti‐inflammatory action of CGP to the comparable level as that by AT2R antagonist PD123319.ConclusionThus, the present study suggests that AT2R by the activation of PP2A abrogates the NFкB inflammatory signalling and inflammatory activation of astrocytes. Therefore, the therapeutic strategies targeting AT2R might be advantageous for neuroinflammatory or degenerative diseases perpetuated by astrocytic activation.

Involvement of β-adrenoceptors in the cardiovascular responses induced by selective adenosine A2A and A2B receptor agonists.

A2A and A2B adenosine receptors produce regionally selective regulation of vascular tone and elicit differing effects on mean arterial pressure (MAP), whilst inducing tachycardia. The tachycardia induced by the stimulation of A2A or A2B receptors has been suggested to be mediated by a reflex increase in sympathetic activity. Here, we have investigated the role of β1‐ and β2‐adrenoceptors in mediating the different cardiovascular responses to selective A2A and A2B receptor stimulation. Hemodynamic variables were measured in conscious male Sprague‐Dawley rats (350–450 g) via pulsed Doppler flowmetry. The effect of intravenous infusion (3 min per dose) of the A2A‐selective agonist CGS 21680 (0.1, 0.3, 1.0 µg.kg−1.min−1) or the A2B‐selective agonist BAY 60–6583 (4.0, 13.3, 40.0 µg.kg−1.min−1) in the absence or following pre‐treatment with the non‐selective β‐antagonist propranolol (1.0 mg.kg−1), the selective β1‐antagonist CGP 20712A (200 µg.kg−1), or the selective β2‐antagonist ICI 118,551 (2.0 mg.kg−1) was investigated (maintenance doses also administered). CGP 20712A and propranolol significantly reduced the tachycardic response to CGS 21680, with no change in the effect on MAP. ICI 118,551 increased BAY 60–6583‐mediated renal and mesenteric flows, but did not affect the heart rate response. CGP 20712A attenuated the BAY 60–6583‐induced tachycardia. These data imply a direct stimulation of the sympathetic activity via cardiac β1‐adrenoceptors as a mechanism for the A2A‐ and A2B‐induced tachycardia. However, the regionally selective effects of A2B agonists on vascular conductance were independent of sympathetic activity and may be exploitable for the treatment of acute kidney injury and mesenteric ischemia.

The Protective Effects of AT2R Agonist, CGP42112A, Against Angiotensin II-Induced Oxidative Stress and Inflammatory Response in Astrocytes: Role of AT2R/PP2A/NFκB/ROS Signaling.

Angiotensin II receptor type 2 (AT2R) agonists have been known to promote neuroprotection by limiting ischemic insult, neuronal proliferation, and differentiation. Further, AT2R agonists have also been associated with the suppression of neuroinflammation and neurodegeneration. Of note, brain astrocytes play a critical role in these neuroinflammatory and neurodegenerative processes. However, the role of AT2R in astrocytic activation remains elusive. Therefore, this study evaluated the role and molecular mechanism of AT2R agonist CGP42112A (CGP) against Angiotensin II (Ang II)-induced astrocytic activation in primary astrocytes, and in a rat model of hypertension. Here, we demonstrated that AT2R activation by CGP abrogated Ang II-induced astrocytic activation, by mitigating the ROS production, mitochondrial dysfunction, IκB-α degradation, NFκB nuclear translocation, and release of TNF-α in astrocytes. However, AT2R-mediated anti-inflammatory effects were reversed by AT2R antagonist, PD123319 (PD), in both in vitro and in vivo conditions. Mechanistically, AT2R via protein phosphatase-2A (PP2A) abrogated the Ang II-induced NFκB activation, ROS generation, and subsequent astrocytic activation. Importantly, PP2A antagonist, okadaic acid, reversed the anti-inflammatory effects of AT2R in Ang II-stimulated primary astrocytes and in the cortex of hypertensive rats. Thus, the present study suggests that AT2R by activating PP2A inhibits oxidative stress and NFκB activation, thereby preventing the astrocytic pro-inflammatory activation. Therefore, AT2R might be advantageous therapeutic target for neuroinflammatory/neurodegenerative diseases perpetuated by astrocytic activation.

The Rho kinase (ROCK) inhibitor Y-27632 reduces the β2-adrenoceptor density but enhance cAMP formation in primary equine bronchial epithelial cells

The present study addresses the effect of the Rho-kinase (ROCK) inhibitor Y-27632 on the β2-adrenoceptor density and β-agonist-stimulated intracellular second messenger cAMP formation in primary equine bronchial epithelial cells (EBEC). Y-27632 significantly decreased the β2-adrenoceptor number (Bmax) without markedly affecting the receptor affinity (dissociation constant, KD) to the radioligand [125I]-iodocyanopindolol (ICYP). In contrast, Y-27632 augmented the β-agonist-stimulated intracellular cAMP production. Herein, Y-27632 markedly increased the maximal cAMP responses (Emax) (isoproterenol > epinephrine > norepinephrine) but did not shift the β-agonist concentration-effect curves to the left. The β2-selective antagonist ICI 118.551 and the β1/β2-antagonsit propranolol but not the β1-selctive antagonist CGP 20712A reversed the isoproterenol-induced cAMP formation equally in Y-27632-treated and control EBEC, suggesting the effect was merely related to the β2-subtype. These results show that Y-27632 differentially regulates the receptor density and function. Thus, these findings provide the first evidence that the functional interaction of the β2-adrenoceptor and Rho-kinase (ROCK) signaling pathways decreases the receptor expression but enhances receptor downstream cAMP formation. This differential regulation of the receptor density and function by Y-27632 should be further reconsidered with regard to the beneficial effect of the drug in asthma therapy.

No impact of sex and age on beta-adrenoceptor-mediated inotropy in human right atrial trabeculae.

There is an increasing awareness of the impact of age and sex on cardiovascular diseases (CVDs). Differences in physiology are suspected. Beta-adrenoceptors (beta-ARs) are an important drug target in CVD and potential differences might have significant impact on the treatment of many patients. To investigate whether age and sex affects beta-AR function, we analysed a large data set on beta-AR-induced inotropy in human atrial trabeculae. We performed multivariable analysis of individual atrial contractility data from trabeculae obtained during heart surgery of patients in sinus rhythm (535 trabeculae from 165 patients). Noradrenaline or adrenaline were used in the presence of the beta2 -selective antagonist (ICI 118551, 50nmol/L) or the beta1 -selective antagonist (CGP 20712A, 300nmol/L) to stimulate beta1 -AR or beta2 -AR respectively. Agonist concentration required to achieve half-maximum inotropic effects (EC50 ) was taken as a measure of beta-AR sensitivity. Impact of clinical variables was modelled using multivariable mixed model regression. As previously reported, chronic treatment with beta-blockers sensitized beta-AR. However, there was no significant interaction between basal force, maximum force and beta-AR sensitivity when age and sex were modelled continuously. In addition, there was no statistically significant effect of body mass index or diabetes on atrial contractility. Our large, multivariable analysis shows that neither age nor sex affects beta-AR-mediated inotropy or catecholamine sensitivity in human atrial trabeculae. These findings may have important clinical implications because beta-ARs, as a common drug target in CVD and heart failure, do not behave differently in women and men across age decades.

Adrenoceptor Responses in Human Embryonic Stem Cell-Derived Cardiomyocytes: a Special Focus on Electrophysiological Property.

Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) have become a promising cell source for cardiovascular research. The electrophysiological characteristic of hESC-CMs has been generally studied, but little is known about electrophysiological response to adrenergic receptor (AR) activation. This study aims to characterize electrophysiological response of hESC-CMs to adrenergic stimulation in terms of the conduction velocity (CV) and action potential (AP) shape. The H9 hESC-CMs were acquired by a classic differentiation protocol and cultured to achieve confluent cell monolayers. The AP shape and CV among the monolayers were recorded using optical mapping during electrophysiological and pharmacological stimulation experiments. Quantitative real-time polymerase chain reaction and Western blot were adopted to determine the expression levels of Connexin and ion channel gene and protein. Chronic β-AR stimulation by isoproterenol for 24 hours in hESC-CM monolayers increased CV by approximately 50%, whereas α-AR or acute β-AR stimulation had no significant effect; chronic β-AR stimulation resulted in a significant Connexin (Cx) 43 and Nav1.5 upregulation at both protein and mRNA level. Isoproterenol-induced CV accelerating and Cx43 and Nav1.5 upregulation in hESC-CMs, which was attenuated by selective β1-adrenoceptor antagonist CGP 20712A but not selective β2-antagonist ICI 118551. Moreover, pretreatment with protein kinase A (PKA) inhibitor H89, mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (MEK) inhibitor SB203580, and MAPK inhibitor PD98059 suppressed the isoproterenol-induced CV accelerating and Cx43 upregulation, whereas it had no significant effect on Nav1.5 upregulation. The AP shape in hESC-CM monolayers was less susceptible by either β-AR or α-AR stimulation. It was β1-AR not β2-AR contributing to the modification of conduction velocity among hESC-CM monolayers. Chronic β1-AR stimulation accelerates CV by upregulating Cx43 via PKA/MEK/MAPK pathway. SIGNIFICANCE STATEMENT: These data provide new insight into the electrophysiological characteristics of human embryonic stem cell-derived cardiomyocytes (hESC-CMs) and depict a concise signaling pathway in the adrenergic receptor (AR) regulation of action potential shape and electrical propagation across hESC-CM monolayer. It is β1-AR not β2-AR contributing to the modification of conduction velocity in hESC-CMs and accelerating conduction velocity by upregulating Connexin 43 via protein kinase A/ mitogen-activated protein kinase (MAPK)-extracellular signal-regulated kinase/MAPK pathway.

Presence and function of β-adrenergic receptors in primary equine bronchial epithelia cells

The β-adrenergic receptor (β-AR) plays an important role in regulating a variety of cell and organ functions in different animal species and is an important target in asthma pathogenesis and therapy. The β-AR expression and function in equine bronchial epithelial cells (EBEC) were not known but innervation and significant decrease in receptor level were reported in the equine bronchial tissues from asthmatic horses. 125I-iodocyanopindolol (ICYP) binding studies were undertaken in primary freshly isolated and cultured EBEC to identify the presence of the β-ARs. The receptor distribution was assessed using subtype-selective β-AR antagonists (ICI 118 551 (β2) and CGP 20712A (β1). The β-AR function was confirmed by measuring the agonist-induced intracellular cAMP accumulation in freshly isolated and cultured EBEC. In both freshly isolated and cultured EBEC, the specific ICYP binding was saturable and of high affinity. The maximal receptor density (Bmax) was 9763 ± 140 binding sites/cell (mean ± SEM, n = 7) and 10575 ± 194 binding sites/cell (mean ± SEM, n = 5) in freshly isolated and cultured EBEC, respectively. The receptor affinity to the ligand (KD) was also not different between the two cell conditions. ICI 118.551 displaced ICYP with 25 000-fold higher affinity than CGP 20712A. Moreover, in both fresh isolated and cultured EBEC, cAMP-accumulation was stimulated with a rank-order of potency of isoproterenol > adrenaline > noradrenaline. These results highlight the β2-AR to be a key subtype in both freshly isolated and cultured primary EBEC.

AT2R Activation Prevents Microglia Pro-inflammatory Activation in a NOX-Dependent Manner: Inhibition of PKC Activation and p47phox Phosphorylation by PP2A.

Microglia-induced reactive oxygen species (ROS) production and inflammation play an imperative role in neurodegenerative diseases like Alzheimer's disease (AD) and Parkinson's disease (PD). It has been established that angiotensin II type-2 receptor (AT2R) activation is neuroprotective in central nervous system diseases like stroke and AD. However, the involvement of AT2R in NADPH oxidase (NOX)-mediated microglia activation is still elusive. Therefore, the present study investigated the role of AT2R in angiotensin II (Ang II) or Phorbol 12-myristate 13-acetate (PMA)-induced microglia activation in BV2 cells, primary microglia, p47phox knockout (p47KO) microglia, and in vivo. Treatment of microglia with Ang II or PMA induced a significant ROS generation and promoted pro-inflammatory microglia in a NOX-dependent manner. In contrast, AT2R activation by CGP42112A (CGP) inhibited NOX activation, ROS production, and pro-inflammatory microglia activation, while promoting the immunoregulatory microglia. This inhibitory effect of AT2R on NOX and pro-inflammatory activation was attenuated by AT2R antagonist, PD123319. Essentially, NOX inhibition (by DPI) or scavenging cellular ROS (by NAC) or p47KO microglia were immune to Ang II- or PMA-induced pro-inflammatory microglia activation. Mechanistically, AT2R, via activation of protein phosphatase-2A (PP2A), prevented the Ang II- or PMA-induced protein kinase C (PKC) activation and phosphorylation of p47phox, an effect that was reversed by the addition of PP2A inhibitor, Okadaic acid (OA). Importantly, PKC inhibitor, Rottlerin, inhibited the Ang II- or PMA-induced p47phox phosphorylation and ROS generation to the similar extent as AT2R activation. In addition, AT2R activation or p47KO prevented ROS production, pro-inflammatory microglial activation, and sickness behavior in mice model of neuroinflammation. Therefore, the present findings suggested that AT2R, via PP2A-mediated inhibition of PKC, prevents the NOX activation, ROS generation, and subsequent pro-inflammatory activation of microglia.

A Non-imaging High Throughput Approach to Chemical Library Screening at the Unmodified Adenosine-A3 Receptor in Living Cells.

Recent advances in fluorescent ligand technology have enabled the study of G protein-coupled receptors in their native environment without the need for genetic modification such as addition of N-terminal fluorescent or bioluminescent tags. Here, we have used a non-imaging plate reader (PHERAstar FS) to monitor the binding of fluorescent ligands to the human adenosine-A3 receptor (A3AR; CA200645 and AV039), stably expressed in CHO-K1 cells. To verify that this method was suitable for the study of other GPCRs, assays at the human adenosine-A1 receptor, and β1 and β2 adrenoceptors (β1AR and β2AR; BODIPY-TMR-CGP-12177) were also carried out. Affinity values determined for the binding of the fluorescent ligands CA200645 and AV039 to A3AR for a range of classical adenosine receptor antagonists were consistent with A3AR pharmacology and correlated well (R2 = 0.94) with equivalent data obtained using a confocal imaging plate reader (ImageXpress Ultra). The binding of BODIPY-TMR-CGP-12177 to the β1AR was potently inhibited by low concentrations of the β1-selective antagonist CGP 20712A (pKi 9.68) but not by the β2-selective antagonist ICI 118551(pKi 7.40). Furthermore, in experiments conducted in CHO K1 cells expressing the β2AR this affinity order was reversed with ICI 118551 showing the highest affinity (pKi 8.73) and CGP20712A (pKi 5.68) the lowest affinity. To determine whether the faster data acquisition of the non-imaging plate reader (~3 min per 96-well plate) was suitable for high throughput screening (HTS), we screened the LOPAC library for inhibitors of the binding of CA200645 to the A3AR. From the initial 1,263 compounds evaluated, 67 hits (defined as those that inhibited the total binding of 25 nM CA200645 by ≥40%) were identified. All compounds within the library that had medium to high affinity for the A3AR (pKi ≥6) were successfully identified. We found three novel compounds in the library that displayed unexpected sub-micromolar affinity for the A3AR. These were K114 (pKi 6.43), retinoic acid p-hydroxyanilide (pKi 6.13) and SU 6556 (pKi 6.17). Molecular docking of these latter three LOPAC library members provided a plausible set of binding poses within the vicinity of the established orthosteric A3AR binding pocket. A plate reader based library screening using an untagged receptor is therefore possible using fluorescent ligand opening the possibility of its use in compound screening at natively expressed receptors.

The β3 -adrenoceptor agonist mirabegron increases human atrial force through β1 -adrenoceptors: an indirect mechanism?

Mirabegron has been classified as a β3 -adrenoceptor agonist approved for overactive bladder syndrome. We investigated possible cardiac effects of mirabegron in the absence or presence of β-adrenoceptor subtype antagonists. In view of its phenylethanolamine structure, we investigated whether mirabegron has indirect sympathomimetic activity by using neuronal uptake blockers. Right atrial trabeculae, from non-failing hearts, were paced and contractile force measured at 37°C. Single concentrations of mirabegron were added in the absence or presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX), β3 (L-748,337), β1 (CGP 20712A), β2 (ICI 118,551) -adrenoceptor antagonists, neuronal uptake inhibitors desipramine or phenoxybenzamine. Mirabegron significantly increased contractile force in human right atrium (1μM, 7.6±2.6%, n=7; 10μM, 10.2±1.5%, n=22 compared with (-)-isoprenaline P<0.05). In the presence of IBMX, mirabegron (10μM) caused a greater contraction. L-748,337 (100nM) had no effect on the increase in contractile force caused by mirabegron (10μM). In contrast, mirabegron (10μM) reduced contractile force in the presence of CGP 20712A, which was not affected by L-748,337 (100nM) or ICI 118,551 (50nM). Mirabegron (10μM) also reduced contractile force in the presence of desipramine or phenoxybenzamine. Mirabegron increases human atrial force through β1 - but not β3 -adrenoceptors. Desipramine and phenoxybenzamine block neuronal uptake and conceivably prevent mirabegron from releasing noradrenaline. A non-specific cardiodepressant effect is not mediated through β3 (or β2 )-adrenoceptors, consistent with lack of β3 -adrenoceptor function on human atrial contractility.

AkrinorTM, a Cafedrine/ Theodrenaline Mixture (20:1), Increases Force of Contraction of Human Atrial Myocardium But Does Not Constrict Internal Mammary Artery In Vitro.

Background: Intraoperative hypotension is a common problem and direct or indirect sympathomimetic drugs are frequently needed to stabilize blood pressure. AkrinorTM consists of the direct and the indirect sympathomimetic noradrenaline and norephedrine. Both substances are covalently bound to the phosphodiesterase (PDE) inhibitor theophylline, yielding theodrenaline and cafedrine, respectively. We investigated pharmacodynamic effects of AkrinorTM and its constituents on contractile force and tension in human atrial trabeculae and internal A. mammaria rings.Methods: Isometric contractions were measured in human atrial trabeculae at 1 Hz and 37°C. CGP 20712A and ICI 118,551 were used to elaborate β1- and β2-adrenoceptor (AR) subtypes involved and phenoxybenzamine to estimate indirect sympathomimetic action. PDE-inhibition was measured as a potentiation of force increase upon direct activation of adenylyl cyclase by forskolin. Human A. mammaria preparations were used to estimate intrinsic vasoconstriction and impact on the noradrenaline-induced vasoconstriction.Results: Clinically relevant concentrations of AkrinorTM (4.2–420 mg/l) robustly increased force in human atrial trabeculae (EC50 41 ± 3 mg/l). This direct sympathomimetic action was mediated via β1-AR and the effect size was as large as with high concentrations of calcium. Only the highest and clinically irrelevant concentration of AkrinorTM increased the potency of forskolin to a minor extent. Norephedrine has lost its indirect sympathomimetic effect when bound to theophylline. Increasing concentrations of AkrinorTM (4.2–168 mg/l) alone did not affect the tension of human A. mammaria interna rings, but shifted the noradrenaline curve rightward from -logEC50 6.18 ± 0.08 to 5.23 ± 0.05 M.Conclusion: AkrinorTM increased cardiac contractile force by direct sympathomimetic actions and PDE inhibition, did not constrict A. mammaria preparations, but shifted the concentration-response curve to the right, compatible with an α-AR antagonistic effect or PDE inhibition. The pharmacodynamic profile and potency of AkrinorTM differs from noradrenaline and norephedrine in vitro. We anticipate metabolism of theodrenaline and cafedrine resulting in a different pharmacodynamic profile of AkrinorTM in vivo.

Influence of cell confluence on the cAMP signalling pathway in vascular smooth muscle cells

The influence of cell confluence on the β-adrenoceptor (β-AR)/cAMP/phosphodiesterase (PDE) pathway was investigated in cultured rat aortic smooth muscle cells (RASMCs).Cells were plated either at low density (LD: 3·103cells/cm2) or high density (HD: 3·104cells/cm2) corresponding to non-confluent or confluent cells, respectively, on the day of experiment.β-AR-stimulated cAMP was monitored in real-time using the fluorescence resonance energy transfer (FRET)-based cAMP sensor, Epac2-camps. A brief application (15s) of the β-AR agonist isoprenaline (Iso) induced a typical transient FRET signal, reflecting cAMP production followed by its rapid degradation. The amplitude of this response, which increased with the concentration of Iso (10 or 100nM), was higher in HD than in LD cells, whatever the Iso concentration used. However, activation of adenylyl cyclase by L-858051 (100μM) induced a similar saturating response in both LD and HD cells. A β1-AR antagonist (CGP 20712A, 100nM) reduced the Iso (100nM) response in HD but not LD cells, whereas a β2-AR antagonist (ICI 118,551, 5nM) reduced this response in HD cells and almost abolished it in LD cells. Competitive [125I]-ICYP binding experiments with betaxolol, a β-AR ligand, identified two binding sites in HD cells, corresponding to β1- and β2-ARs with a proportion of 11% and 89%, respectively, but only one binding site in LD cells, corresponding to β2-ARs. Total cAMP-PDE activity (assessed by a radioenzymatic assay) was increased in HD cells compared to LD cells. This increase was associated with a rise in mRNA expression of five cAMP-PDEs subtypes (PDE1A, 3A, 4A, 4B and 7B) in HD cells, and a decrease in basal [cAMP]i (assessed by an EIA assay). PDE4 inhibition with Ro-20-1724 (10μM) strongly prolonged the Iso response in LD and HD cells, whereas PDE3 inhibition with cilostamide (1μM) slightly prolonged Iso response only in LD cells. Interestingly, inhibition of PDE4 unmasked an effect of PDE3 in HD cells.Our results show that in cultured RASMCs, the β-AR/cAMP/PDE signalling pathway is substantially modulated by the cell density. In HD cells, Iso response involves both β1- and β2-AR stimulation and is mainly controlled by PDE4, PDE3 being recruited only after PDE4 inhibition. In LD cells, Iso response involves only β2-AR stimulation and is controlled by PDE4 and to a lower degree by PDE3. This low density state is associated with an absence of membrane expression of the β1-AR, a lower cAMP-PDE activity and a higher basal [cAMP]i. This study highlights the critical role of the cellular environment in controlling the vascular β-AR signalling.

Involvement of NF2 Signaling Pathway in β‐Adrenergic Receptor‐Stimulated Cardiac Myocyte Apoptosis

β‐adrenergic receptor (β‐AR) stimulation induces cardiac myocyte apoptosis in vitro and in vivo. Tumor suppressor protein NF2 (neurofibromin 2, also known as merlin) is a member of the ezrin/radixin/moesin (ERM) family of proteins. Post‐translational modifications such as phosphorylation and sumoylation affect NF2 activity, subcellular localization and function. Role of NF2 in inhibition of cell growth and apoptosis is well established in a variety of cell types. However, the role of NF2 in β‐AR‐stimulated cardiac myocyte apoptosis remains to be investigated. Here we provide evidence that treatment of adult rat ventricular myocytes (ARVMs) with β‐AR agonist (isoproterenol, ISO, 10 μM) increases phosphorylation (serine 518) and sumoylation of NF2. Activation of adenylyl cyclase using forskolin mimicked the effects of ISO. ISO‐mediated increase in phosphorylation of NF2 was inhibited by protein kinase A inhibitor (H89) and β1‐AR receptor antagonist (CGP 20712A). Knockdown of NF2 using siRNA decreased ISO‐mediated increase in the number of apoptotic ARVMs (CTL, 8.3±0.6; ISO, 23.2±1.0*; negSiRNA+ISO, 21.3±1.2*; NF2‐SiRNA+ISO, 12.0±1.5$; *p&lt;0.05 vs CTL; $p&lt;0.05 vs ISO and negSiRNA+ISO, n=3–4). Adenoviral‐mediated expression of WT‐NF2 led to increased phosphorylation and sumoylation of NF2, and apoptosis. It also activated JNKs, a pro‐apoptotic kinase. Treatment with ISO and forskolin, or adenoviral‐mediated expression of NF2 increased phosphorylation (inactivation) of YAP, a downstream target of NF2. In H9C2 cardiomyocytes, knockdown of NF2 using siRNA decreased YAP phosphorylation. These data suggest that β‐AR stimulation affects post‐translational modification of NF2 via the involvement of PKA pathway, and NF2 plays a pro‐apoptotic role in β‐AR‐stimulated myocyte apoptosis.Support or Funding InformationSupported by Department of Veterans Affairs (BX002332), NIH R15HL129140 (NHLBI) and Institutional Research and Improvement account.

β-Adrenoceptor-mediated Relaxation of Urinary Bladder Muscle in β2-Adrenoceptor Knockout Mice.

Background and Objective: In order to characterize the β-adrenoceptor (AR) subtypes involved in agonist-stimulated relaxation of murine urinary bladder we studied the effects of (-)-isoprenaline and CL 316,243 on tonic contraction and spontaneous contractions in detrusor strips of wild-type (WT) and β2-AR knockout (β2-AR KO) mice.Materials and Methods: Urinary bladders were isolated from male WT and β2-AR KO mice. β-AR subtype expression was determined with quantitative real-time PCR. Intact muscle strips pre-contracted with KCl (40 mM) were exposed to cumulatively increasing concentrations of (-)-isoprenaline or β3-AR agonist CL 316,243 in the presence and absence of the subtype-selective β-AR blockers CGP 20712A (β1-ARs), ICI 118,551 (β2-ARs), and L748,337 (β3-ARs).Results: Quantitative real-time PCR confirmed lack of β2-AR expression in bladder tissue from β2-AR KO mice. In isolated detrusor strips, pre-contraction with KCl increased basal tone and enhanced spontaneous activity significantly more in β2-AR KO than in WT. (-)-Isoprenaline relaxed tonic tension and attenuated spontaneous activity with similar potency, but the concentrations required were two orders of magnitude higher in β2-AR KO than WT. The concentration-response curves (CRCs) for relaxation were not affected by CGP 20712A (300 nM), but were shifted to the right by ICI 118,551 (50 nM) and L748,337 (10 μM). The -logEC50 values for (-)-isoprenaline in WT and β2-AR KO tissue were 7.98 and 6.00, respectively, suggesting a large receptor reserve of β2-AR. (-)-CL 316,243 relaxed detrusor and attenuated spontaneous contractions from WT and β2-AR KO mice with a potency corresponding to the drug’s affinity for β3-AR. L743,337 shifted the CRCs to the right.Conclusion: Our findings in β2-AR KO mice suggest that there is a large receptor reserve for β2-AR in WT mice so that this β-AR subtype will mediate relaxation of tone and attenuation of spontaneous activity under physiological conditions. Nevertheless, upon removal of this reserve, β3-AR can also mediate murine detrusor relaxation.

Abstract P072: Adipocyte AT 2 Receptor Suppresses UCP1 Transcription and Thereby Resting Metabolic Rate

The renin-angiotensin system (RAS) contributes to energy balance through opposing actions in the brain and periphery. We hypothesize that tissue- and receptor-specific RAS modulation may represent a novel therapeutic approach to obesity. Transgenic “sRA” mice exhibit brain-specific RAS hyperactivity through expression of human renin in neurons (synapsin promoter) and human angiotensinogen via its own promoter. Previously we documented that sRA mice exhibit a suppressed circulating RAS, and an elevated resting metabolic rate (RMR) that is sensitive to replacement of circulating angiotensin II or the AT 2 receptor (AT 2 R) agonist, CGP-42112a (CGP, 100 ng/kg/min, s.c.). sRA mice exhibit a robust and specific elevation in uncoupling protein-1 (UCP1) expression and glucose uptake in inguinal white adipose tissue (iWAT). Chronic infusion of the AT 2 R agonist, CGP-42112a (CGP) into sRA mice increased weight gain and normalized RMR and inguinal fat expression of UCP1, but had no effect on food intake or digestive efficiency. RMR suppression by CGP was not mediated through attenuation of adipose sympathetic nervous activity (SNA, measured via multifiber nerve recordings), leading to the hypothesis that AT 2 R action on the adipocyte suppresses adipose sensitivity to SNA. Cultured primary mouse adipocytes were isolated from the iWAT, and norepinephrine (NE, 1 nM) treatment induced UCP1 mRNA (597 -fold of vehicle, P&lt;0.05), however this UCP1 induction was attenuated with CGP co-treatment (10 nM, 0.2 -fold of NE alone, P&lt;0.05). In human adipocytes, activation of AT 2 R also prevented the NE-induced increase in UCP1 mRNA. NE additionally significantly increased lipolysis by adipocytes (measured via free glycerol in the media; P&lt;0.05), but AT 2 R activation had no effect (vehicle= 15.9 ug/mL; NE= 70.8 ug/mL; NE + CGP=64.7 ug/mL). These data support a suppressive action of AT 2 R upon RMR that is mediated specifically through the suppression of UCP1 expression, but not its activation by lipolysis, within subcutaneous adipocytes. Ongoing studies are examining the transcriptional regulation of UCP1 by adipose AT 2 R activation.

β1 Adrenoceptor antagonistic effects of the supposedly selective β2 adrenoceptor antagonist ICI 118,551 on the positive inotropic effect of adrenaline in murine hearts

Studies on the relative contribution of β1- and β2-adrenoceptors (AR) generally employ selective β1- and β2-AR antagonists such as CGP 20712A and ICI 118,551, respectively, and assume that antagonism by one of these compounds indicates mediation by the respective AR subtype. Here, we evaluated the β2-AR-selectivity of ICI 118,551 in ventricular muscle strips of transgenic mice lacking β1-AR (β1-KO), β2-AR (β2-KO), or both (β1/β2-KO). Strips were electrically driven and force development was measured. In wild type (WT), ICI 118,551 (100 nmol/L) shifted the concentration–response curve (CRC) for adrenaline by about 0.5 log units to the right, corresponding to the known affinity of ICI 118,551 to β1-AR but not to β2-AR. Conversely, the phosphodiesterase inhibitor rolipram (10 μmol/L) shifted the CRC to the left, but did not enlarge the ICI 118,551 shift, indicating exclusive β1-AR mediation even when PDE4 is inactive. In line with this, rolipram and ICI 118,551 had similar effects in β2-KO than in WT. In contrast, β1-KO did not show any inotropic reaction to adrenaline (+/− rolipram). In WT, the β1-AR selective antagonist CGP 20712A (100 nmol/L) shifted the CRC for isoprenaline by 2.1 log units, corresponding to the affinity of CGP 20712A to β1-AR. Rolipram increased the sensitivity to adrenaline independently of the presence of CGP 20712A. We conclude that effects sensitive to the β2-AR antagonist ICI 118,551 are not necessarily β2-AR-mediated and CGP 20712A-resistant effects cannot be simply interpreted as β2-AR-mediated. Catecholamine effects in murine ventricles strictly depend on β1-AR, even if PDE 4 is blocked.

Characterization of β-adrenergic receptors in the heart chambers of adult turkeys

The presence, distribution and characteristics of chamber-specific β-adrenergic receptors in adult turkey hearts were investigated by radioligand binding studies using (−)-[125I]-iodocyanopindolol (ICYP). The β1-selective (CGP 20712A) and β2-selective (ICI 118.551) antagonists as well as the nonselective β-agonists isoproterenol, epinephrine and norepinephrine were used in displacement studies.In all cardiac chambers, ICI 118.551 and CGP 20712A displacement curves were monophasic and steep, with the affinity of CGP 20712A higher than that of ICI 118.551, indicating the exclusive presence of the β1-adrenergic receptor subtype. The agonist rank order of potency was isoproterenol > norepinephrine ≥ epinephrine, typical for the β1-receptor subtype. In all chambers, the density of β-adrenergic receptors was ~40 fmol/mg protein and the KD was ~30 pM. The study revealed similar β-adrenergic receptor density mainly of the β1-subtype in all cardiac chambers, indicating that this receptor subtype could contribute equally to regulate cardiac physiological function and pathophysiology.

β-Adrenoceptor-Mediated Relaxation of Carbachol-Pre-Contracted Mouse Detrusor

Aims: To study the β-adrenoceptor subtypes involved in the relaxation responses to (-)-isoprenaline in carbachol-pre-contracted (CCh) mouse detrusor muscle with intact and denuded mucosa. Methods: Isolated muscle strips from the urinary bladder of male C57BL6 mice or β₂-adrenoceptor knockout mice were pre-contracted with CCh, 1 µ<smlcap>M</smlcap> and relaxed with increasing concentrations of the β-adrenoceptor (β-AR) agonist (-)-isoprenaline and forskolin. For estimating the β-AR subtypes involved, subtype-selective receptor blockers were used, that is, CGP 20712A (β₁-ARs), ICI 118,551 (β₂-ARs), and L748,337 (β₃-ARs). Results: Unlike in KCl-pre-contracted muscle, the mucosa did not affect the sensitivity of the relaxation response to (-)-isoprenaline in CCh-pre-contracted murine detrusor strips. Increasing concentrations of (-)-isoprenaline produced a biphasic concentration-relaxation response without any difference both during the presence and absence of mucosa. The relaxation fraction produced by low (-)-isoprenaline concentrations was mediated by β₂-AR as evidenced by a shift of the concentration-response curve to higher concentrations with ICI 118,551, but not with CGP 20712A and L748,337, and by the absence of this fraction in β₂-AR-KO mice. The relaxation response with low sensitivity to (-)-isoprenaline was not affected by any of the β-AR subtype-selective blockers and was the only response detected in detrusor strips from β₂-AR-KO mice. Conclusions: In CCh-pre-contracted mouse detrusor, β₂-ARs are responsible for the relaxation component with high sensitivity to (-)-isoprenaline as indicated by the conversion of a biphasic into a monophasic CRC with ICI 118,551 or by its absence in β₂-AR KO mice. The mucosa does not impair relaxation under these conditions.

Concomitant inhibition of primary equine bronchial fibroblast proliferation and differentiation by selective β2-adrenoceptor agonists and dexamethasone

Altered airway cell proliferation plays an important role in the pathogenesis of human bronchial asthma and chronic obstructive pulmonary disease (COPD) as well as the equine recurrent airway obstruction (RAO) with consistent changes, i.e. narrowing the airway wall, explained by proliferation and differentiation of fibroblasts. In permanent cell lines, it has been suggested that β2-adrenoceptor agonists and glucocorticoids regulate cell proliferation via the β2-adrenoceptor pathway; indeed, no study was carried out in fresh isolated primary equine bronchial fibroblasts (EBF). We characterized the β-adrenoceptors in EBF, and compared effects of long-acting (clenbuterol) and short-acting (salbutamol and isoproterenol) β2-agonists and dexamethasone on proliferation, differentiation and collagen synthesis. High density (Bmax; 5037±494 sites/cell) of β2-adrenoceptor subtype was expressed in EBF. β2-agonists inhibited concentration-dependently EBF proliferation with potency of clenbuterol>salbutamol »isoproterenol which was inhibited by ICI 118.551 and propranolol but not by CGP 20712A. In contrast, dexamethasone alone inhibited less EBF proliferation, but the effect was high when dexamethasone was combined with β2-agonists. Transforming growth factor-β1 (TGF-β1) increased transformation of fibroblasts into myofibroblasts, which was inhibited by clenbuterol and dexamethasone alone and drug combination resulted in high inhibition rate. Collagen synthesis in EBF was rather hampered by dexamethasone than by β-agonists. Collectively, the expression of β2-adrenoceptor subtype in EBF and the anti-proliferative effect of clenbuterol suggest that β2-adrenoceptors are growth inhibitory and anti-fibrotic in EBF. These β2-agonist effects in EBF were synergistically enhanced by dexamethasone, providing the additive effects of glucocorticoids to counteract airway remodelling and morbidity of asthma and RAO.

P343Effect of T3 on human induced pluripotent stem cell-derived cardiomyocyte maturation

Purpose: Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) hold great potential for regenerative medicine and in vitro screening. However, the phenotype is immature and more closely resembles foetal/neonatal cardiomyocytes. The aim of this study is to drive hiPSC-CM to a more mature state using thyroid hormone, triiodothyronine (T3), which has a role in cardiomyocyte maturation during development. Methods: hiPSC-CM (Cellular Dynamics) were treated with 30nM T3 for up to 4 weeks. Expression of β1 adrenergic receptor (β1AR) was assessed by qPCR (Taqman). Beating rates were determined using a Nikon Eclipse TE2000E microscope with Digital sight camera and NIS Elements3.2 software video tracking. Ca2+ dynamics were studied using Fluo-4AM loaded cells imaged with a Nikon Eclipse T6200 and Redshirt NeuroCMOS camera at 0.5KHz with 1Hz, 15V, 5ms field stimulation. Comparative proteomic analysis (4 week T3) was performed by mass spectrometry using isobaric tags. Data expressed as mean ±SEM; statistical significance determined by student's t-test (n=3). Results: 4 week T3 treatment resulted in a 1.5-fold increase in basal beating rate. T3 increased β1AR gene expression and remained stable for up to 2 weeks (p<0.05). CGP 20712A (10μM), a β1AR inverse agonist, decreased the beating rate of T3-treated cells (39.3±9.7bpm) to basal levels in untreated cells (25.7±9bpm). Ca2+ transient analysis revealed alterations in Ca2+ handling. 4 week T3 treatment resulted in decreased time to transient peak from 244±4.0 ms to 90.7±15 (p<0.0006). Time to 50% decay also decreased from 309.2±6.6ms to 287.3±2.3ms (p<0.05) and transient duration (start to 75% decay) decreased from 695.4±6.4ms to 537.9±21.3ms (p<0.002). Proteomic analysis revealed increases in proteins involved in regulating sarcoplasmic reticulum (SR) function including; phospholamban (17.2%), SERCA (27.6%) and calsequestrin2 (303.8%). In addition increases in mitochondrial enzymes, including ATP5B (32.7%), and α/βMHC ratio were detected along with decreases in slow skeletal troponin I (-43.7%) and C (-36.2%) isoforms. Conclusions: T3 treatment induced alterations in expression of proteins involved in SR function, mitochondrial metabolism and cytoskeletal composition. In addition, T3 treatment improved Ca2+ handling and increased β1AR function, with increases in beating rate possibly driven by constitutive β1AR activity. Collectively these data provide evidence for a shift towards a more mature phenotype and indicate that this may be an effective strategy to drive maturation of hiPSC-CM.