beta-AR Stimulation Research Articles

β-Arrestin Mediates β1-Adrenergic Receptor-Epidermal Growth Factor Receptor Interaction and Downstream Signaling

beta1-Adrenergic receptor (beta1AR) stimulation confers cardioprotection via beta-arrestin-de pend ent transactivation of epidermal growth factor receptors (EGFRs), however, the precise mechanism for this salutary process is unknown. We tested the hypothesis that the beta1AR and EGFR form a complex that differentially directs intracellular signaling pathways. beta1AR stimulation and EGF ligand can each induce equivalent EGFR phosphorylation, internalization, and downstream activation of ERK1/2, but only EGF ligand causes translocation of activated ERK to the nucleus, whereas beta1AR-stimulated/EGFR-transactivated ERK is restricted to the cytoplasm. beta1AR and EGFR are shown to interact as a receptor complex both in cell culture and endogenously in human heart, an interaction that is selective and undergoes dynamic regulation by ligand stimulation. Although catecholamine stimulation mediates the retention of beta1AR-EGFR interaction throughout receptor internalization, direct EGF ligand stimulation initiates the internalization of EGFR alone. Continued interaction of beta1AR with EGFR following activation is dependent upon C-terminal tail GRK phosphorylation sites of the beta1AR and recruitment of beta-arrestin. These data reveal a new signaling paradigm in which beta-arrestin is required for the maintenance of a beta1AR-EGFR interaction that can direct cytosolic targeting of ERK in response to catecholamine stimulation.

Regulation of gap-junction protein connexin 43 by β-adrenergic receptor stimulation in rat cardiomyocytes

beta-adrenergic receptor (beta-AR) agonists are among the most potent factors regulating cardiac electrophysiological properties. Connexin 43 (Cx43), the predominant gap-junction protein in the heart, has an indispensable role in modulating cardiac electric activities by affecting gap-junction function. The present study investigates the effects of short-term stimulation of beta-AR subtypes on Cx43 expression and gap junction intercellular communication (GJIC) function. The level of Cx43 expression in neonatal rat cardiomyocytes (NRCM) was detected by a Western blotting assay. The GJIC function was evaluated by scrape loading/dye transfer assay. Stimulation of beta-AR by the agonist isoproterenol for 5 min induces the up-regulation of nonphosphorylated Cx43 protein level, but not total Cx43. Selective beta(2)-AR inhibitor ICI 118551, but not beta(1)-AR inhibitor CGP20712, could fully abolish the effect. Moreover, pretreatment with both protein kinase A inhibitor H89 and G(i) protein inhibitor pertussis toxin also inhibited the isoproterenol-induced increase of nonphosphorylated Cx43 expression. Isoproterenol-induced up-regulation of nonphosphorylated Cx43 is accompanied with enhanced GJIC function. Taken together, beta(2)-AR stimulation increases the expression of nonphosphorylated Cx43, thereby enhancing the gating function of gap junctions in cardiac myocytes in both a protein kinase A- and G(i)-dependent manner.Acta Pharmacologica Sinica (2009) 30: 928-934; doi: 10.1038/aps.2009.92.

Antioxidants but not Doxycycline Treatments Restore Depressed Beta-Adrenergic Responses of the Heart in Diabetic Rats

Reactive oxygen species (ROS) play important roles in the development of diabetic cardiomyopathy. Matrix metalloproteinases (MMPs) can get activated by ROS and contribute to loss of myocardial contractile function in oxidative stress injury. Previously we have shown that either a MMP-2 inhibitor doxycycline or an antioxidant selenium treatment in vivo prevented diabetes-induced cardiac dysfunction significantly. In addition, there is an evidence for impaired cardiac responsiveness to beta-adrenoceptor (beta AR) stimulation in experimental animals with diabetes. The exact nature of linkage between the functional depression in cardiac responses to catecholamines and the variations in uncoupling of beta AR in diabetes has not been clearly defined. Therefore, we aimed to evaluate the effect of in vivo administration of doxycycline on beta AR responses of isolated hearts from diabetic rats and compare these data with two well-known antioxidants; sodium selenate and (n-3) fatty acid-treated diabetic rats. We examined the changes in the basal cardiac function in response to the beta AR stimulation, adenylate cyclase activity, and beta AR affinity to its agonist, isoproterenol. These results showed that antioxidant treatment of diabetic rats could protect the hearts against diabetes-induced depression in beta AR responses, significantly while doxycycline did not have any significant beneficial action on these parameters. As a summary, present data, in part, demonstrate that antioxidants and MMP inhibitors could both regulate MMP function but may also utilize different mechanisms of action in cardiomyocytes, particularly related with beta AR signaling pathway.

Differential roles of NHERF1, NHERF2, and PDZK1 in regulating CFTR-mediated intestinal anion secretion in mice

The epithelial anion channel CFTR interacts with multiple PDZ domain-containing proteins. Heterologous expression studies have demonstrated that the Na+/H+ exchanger regulatory factors, NHERF1, NHERF2, and PDZK1 (NHERF3), modulate CFTR membrane retention, conductivity, and interactions with other transporters. To study their biological roles in vivo, we investigated CFTR-dependent duodenal HCO3- secretion in mouse models of Nherf1, Nherf2, and Pdzk1 loss of function. We found that Nherf1 ablation strongly reduced basal as well as forskolin-stimulated (FSK-stimulated) HCO3- secretory rates and blocked beta2-adrenergic receptor (beta2-AR) stimulation. Conversely, Nherf2-/- mice displayed augmented FSK-stimulated HCO3- secretion. Furthermore, although lysophosphatidic acid (LPA) inhibited FSK-stimulated HCO3- secretion in WT mice, this effect was lost in Nherf2-/- mice. Pdzk1 ablation reduced basal, but not FSK-stimulated, HCO3- secretion. In addition, laser microdissection and quantitative PCR revealed that the beta2-AR and the type 2 LPA receptor were expressed together with CFTR in duodenal crypts and that colocalization of the beta2-AR and CFTR was reduced in the Nherf1-/- mice. These data suggest that the NHERF proteins differentially modulate duodenal HCO3- secretion: while NHERF1 is an obligatory linker for beta2-AR stimulation of CFTR, NHERF2 confers inhibitory signals by coupling the LPA receptor to CFTR.

Modulation Of Cardiac Contractility By Antagonism Of Pleckstrin-homology Domain And Akt-1 Silencing

The pleckstrin-homology (PH) domain is involved in PI3-Kinase-mediated membrane recruitment, and subsequent activation, of signaling pathways, including Akt. PI3-Kinase pathway may modulate beta-adrenergic inotropic effect and Akt disregulation has a central role in diabetic cardiomyopathy. Recent data suggest that Akt may directly modulate sarcoplasmic reticulum (SR) function. Aims: to investigate modulation of cardiac excitation-contraction (EC) coupling by 1) two chemically unrelated compounds with PH-domain affinity (compounds A and B); 2) selective Akt-1 isoform silencing by small RNA-interference (sRNAi). Methods: rat ventricular myocytes were studied at 36.5 °C. Twitch amplitude was measured during field stimulation (2 Hz). Intracellular Ca2+ transients (FLUO 4-AM) was recorded in V-clamped myocytes; SR Ca2+ uptake function was estimated from Ca2+−transient features under inhibition of the Na+/Ca2+ exchanger (by Na+−free conditions). Akt-1 silencing was performed by myocyte transfection with Akt1-specific ds-iRNA oligos, labelled with the fluorescent probe Cy3. Akt phosphorylation levels and activity were tested by western-blot and ELISA. The effect of all interventions was tested in basal conditions and under weak adrenergic activation (isoproterenol 10 nM). Results: Akt-1 phosphorylation and activity were decreased by compounds A and B. Both compounds increased (p<0.05) twitch amplitude in basal condition; these effects was enhanced during weak beta-AR stimulation, also the compounds effects are significantly reduced during beta-AR blockade. Akt-1 silencing increased twitch amplitude, enhanced its response to beta-AR stimulation and completely occluded the effect of compounds. The compounds increased SR Ca reuptake rate and EC-coupling gain. Conclusions: 1) chemical antagonism of PH-domain increased contractility by stimulating SR Ca2+ uptake; 2) this effect is likely to result from inhibition of the Akt-1 pathway and involve interaction of the latter with beta-AR-mediated signaling; 3) PH-domain is a novel putative target for inotropical support through enhancement of SR function.

Phosphodiesterase 4 and Phosphatase 2A Differentially Regulate cAMP/Protein Kinase A Signaling for Cardiac Myocyte Contraction under Stimulation of β1 Adrenergic Receptor

Activation of the beta adrenergic receptor (betaAR) induces a tightly controlled cAMP/protein kinase A (PKA) activity to ensure an agonist dose-dependent and saturable contraction response in animal heart. We have found that stimulation of beta(1)AR by isoproterenol induces maximal contraction responses at the dose of 1 microM in cardiac myocytes; however, cAMP accumulation continues to increase with higher agonist concentrations. Dose-dependent cAMP accumulation is tightly controlled by negative regulator phosphodiesterase 4 (PDE4) that hydrolyzes cAMP. At 1 nM isoproterenol, cAMP accumulation is minimal because of the hydrolysis of cAMP by PDE4, which leads to a small increase in PKA phosphorylation of phospholamban and troponin I (TnI), and contraction responses. Inhibition of PDE4 activity with rolipram enhances cAMP accumulation, yields maximal PKA phosphorylation of phospholamban and TnI, and myocyte contraction responses. In contrast, at 10 microM isoproterenol, despite the negative effect of PDE4, cAMP accumulation is sufficient for maximal PKA phosphorylation of phospholamban and TnI. Inhibition of PDE4 with rolipram enhances cAMP accumulation, but not PKA phosphorylation and contraction responses. It is interesting that activities of both PKA and protein phosphatase 2A (PP2A) are enhanced under beta(1)AR activation with 10 microM isoproterenol, and PP2A is recruited to PKA/A kinase-anchoring protein complex. Inhibition of PP2A with okadaic acid further enhances the phosphorylation of phospholamban and TnI as well as contraction responses induced by 10 microM isoproterenol. Therefore, PP2A plays a key role in limiting PKA phosphorylation of phospholamban and TnI for myocyte contraction responses under beta(1)AR stimulation.

β3‐Adrenoceptors modulate left ventricular relaxation in the rat heart via the NO‐cGMP‐PKG pathway

Using a model of isolated and Langendorff-perfused rat heart we analysed whether activation of beta3-adrenergic receptors (beta3-ARs) influences ventricular lusitropic performance. We also focused on the NOS/NO/cGMP/PKG cascade as the signal transduction mechanism. Hearts were treated with increasing concentrations (from 10(-12) to 10(-6) m) of BRL(37344), a selective beta3-AR agonist, and cardiac performance was evaluated by analysing both lusitropic parameters and coronary motility. Cardiac preparations were also perfused with BRL(37344) in the presence of either isoproterenol (ISO) or nadolol, or pertussis toxin (PTx), or selective inhibitors of the NOS/NO/cGMP/PKG pathway. BRL(37344) caused a significant concentration-dependent reduction in (LVdP/dt)(min), a decrease in half time relaxation significant starting from 10(-12) m, and an increase in (LVdP/dt)(max)/(LVdP/dt)(min) ratio (T/-t). BRL(37344) abolished the ISO-mediated positive lusitropism. beta3-AR-dependent effects on relaxation were insensitive to beta(1)/beta2-AR inhibition by nadolol (100 nm), and were abolished by G(i/o) protein inhibition by PTx (0.01 nm). NO scavenging by haemoglobin (10 microm), and nitric oxide synthase (NOS) inhibition by NG-monomethyl-l-arginine (10 microm) revealed the involvement of NO signalling in BRL(37344) response. Pre-treatment with inhibitors of either soluble guanylate cyclase (ODQ; 10 microm) or PKG (KT(5823); 100 nm) abolished beta3-AR-dependent negative lusitropism. In contrast, anantin (10 nm), an inhibitor of particulate guanylate cyclase, did not modify the effect of BRL(37344) on relaxation. Taken together, our findings provide functional evidence for beta3-AR modulation of ventricular relaxation in the rat heart which involves PTx-sensitive inhibitory Gi protein and occurs via an NO-cGMP-PKG cascade. Whether the effects of beta3-AR stimulation on lusitropism are beneficial or detrimental remains to be established.

Tonic β-Adrenergic Drive Provokes Proinflammatory and Proapoptotic Changes in Aging Mouse Heart

Tonic activation of adrenergic drive has been found to be associated with aging, and its further activation is also seen in aging patients with major surgery or congestive heart failure. Nevertheless, its potential effect on the aging heart remains enigmatic. In the present study, at baseline, significant inflammatory and apoptotic changes were found in the aging mouse (20 months old), as evidenced by increases in inducible nitric oxide synthase (iNOS) expression, myocardial apoptosis in the heart, and C-reactive protein (CRP) release in the circulation. These phenotypic changes in aging animals can be induced in young animals (3 months old) by chronic beta-adrenergic receptor (AR) stimulation with isoproterenol (ISO), and they can be markedly reduced in aging animals by chronic beta-blockade with propranolol. Compared with young animals, chronic beta-AR stimulation with ISO in aging animals induced larger increases in iNOS expression, nitrotyrosine formation in the heart, and nitric oxide (NO) production and CRP release in the circulation; it also accelerated myocardial apoptosis and resulted in an enlarged infarct size when animals were subjected to myocardial ischemia and reperfusion (MI/R). However, the pretreatment of 1400W (N-(3-(aminomethyl) benzyl)acetamidine)-a specific iNOS inhibitor-significantly reduced iNOS-mediated nitrative stress associated with a marked decrease in myocardial apoptosis and infarct size in aging mice. These results demonstrate that tonic activation of the beta-adrenergic system associated with aging induces proinflammatory and proapoptotic changes in the heart and that additional beta-AR stimulation results in an exaggerated nitrative stress, mediated by iNOS, that is associated with more severe myocardial injury in aging mice.

Adrenergic regulation of a key cardiac potassium channel can contribute to atrial fibrillation: evidence from an IKs transgenic mouse

Inherited gain-of-function mutations of genes coding for subunits of the heart slow potassium (I Ks) channel can cause familial atrial fibrillation (AF). Here we consider a potentially more prevalent mechanism and hypothesize that beta-adrenergic receptor (beta-AR)-mediated regulation of the I Ks channel, a natural gain-of-function pathway, can also lead to AF. Using a transgenic I Ks channel mouse model, we studied the role of the channel and its regulation by beta-AR stimulation on atrial arrhythmias. In vivo administration of isoprenaline (isoproterenol) predisposes I Ks channel transgenic mice but not wild-type (WT) littermates that lack I Ks to prolonged atrial arrhythmias. Patch-clamp analysis demonstrated expression and isoprenaline-mediated regulation of I Ks in atrial myocytes from transgenic but not WT littermates. Furthermore, computational modelling revealed that beta-AR stimulation-dependent accumulation of open I Ks channels accounts for the pro-arrhythmic substrate. Our results provide evidence that beta-AR-regulated I Ks channels can play a role in AF and imply that specific I Ks deregulation, perhaps through disruption of the I Ks macromolecular complex necessary for beta-AR-mediated I Ks channel regulation, may be a novel therapeutic strategy for treating this most common arrhythmia.

Insulin inhibits β-adrenergic action in ischemic/reperfused heart: a novel mechanism of insulin in cardioprotection

Sympathetic overactivity is closely connected with cell injury and contractile dysfunction during myocardial ischemia/reperfusion (MI/R). Insulin exerts protection for the I/R heart and the underlying mechanisms remain unclear. This study aimed to investigate the ability of insulin to modulate beta-adrenergic actions on myocardial contraction and post-ischemic injury in acute MI/R and the underlying mechanism. Isolated hearts from adult SD rats were subjected to MI/R (30 min/2 h) and treated with isoproterenol (ISO) or/and insulin. Myocardial contraction, cardiomyocyte apoptosis, myocardial injury and infarction were assessed. In a separate study, isolated ventricular myocytes were subjected to simulated I/R (15/30 min) and myocyte shortening and intracellular Ca2+ transient in response to ISO during reperfusion were assessed with presence or absence of insulin. In isolated I/R hearts, insulin largely reversed the ISO-associated contractile functional impairment at 2 h after MI/R, inhibiting ISO-induced declines in heart rate and left ventricular systolic pressure by 34.0% and 23.0% and preventing ISO-induced elevation in left ventricular end-diastolic pressure by 28.7% respectively (all P < 0.05). In addition, ISO alone resulted in enlarged infarct size, elevated CK and LDH activity and increased apoptotic index in I/R hearts compared with vehicle, which were inhibited by treatment of insulin (all P < 0.05). Interestingly, in SI/R cardiomyocytes, insulin alone at 10(-7 )mol/l increased cell contraction whereas attenuated the positive inotropic response to ISO (10(-9 )mol/l) during R as evidenced by a 18.7% reduction in peak twitch amplitude and a 23.9% reduction in calcium transient amplitude (both P < 0.05). Moreover, insulin blunted ISO-mediated increase in PKA activity, enhanced the PKA-dependent phosphorylation of phospholamban (PLB), resulting in increased sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) activity. Insulin attenuated the contractile response to beta-AR stimulation and suppressed ISO-elicited cardiac dysfunction and cell injury in MI/R. The inhibitory effect of insulin on the beta-adrenergic action involved the inhibition of PKA-mediated Ca2+ transient and promotion of post-ischemic Ca2+ handling.

Role of the acidic N′ region of cardiac troponin I in regulating myocardial function

Cardiac troponin I (cTnI) phosphorylation modulates myocardial contractility and relaxation during beta-adrenergic stimulation. cTnI differs from the skeletal isoform in that it has a cardiac specific N' extension of 32 residues (N' extension). The role of the acidic N' region in modulating cardiac contractility has not been fully defined. To test the hypothesis that the acidic N' region of cTnI helps regulate myocardial function, we generated cardiac-specific transgenic mice in which residues 2-11 (cTnI(Delta2-11)) were deleted. The hearts displayed significantly decreased contraction and relaxation under basal and beta-adrenergic stress compared to nontransgenic hearts, with a reduction in maximal Ca(2+)-dependent force and maximal Ca(2+)-activated Mg(2+)-ATPase activity. However, Ca(2+) sensitivity of force development and cTnI-Ser(23/24) phosphorylation were not affected. Chemical shift mapping shows that both cTnI and cTnI(Delta2-11) interact with the N lobe of cardiac troponin C (cTnC) and that phosphorylation at Ser(23/24) weakens these interactions. These observations suggest that residues 2-11 of cTnI, comprising the acidic N' region, do not play a direct role in the calcium-induced transition in the cardiac regulatory or N lobe of cTnC. We hypothesized that phosphorylation at Ser(23/24) induces a large conformational change positioning the conserved acidic N region to compete with actin for the inhibitory region of cTnI. Consistent with this hypothesis, deletion of the conserved acidic N' region results in a decrease in myocardial contractility in the cTnI(Delta2-11) mice demonstrating the importance of acidic N' region in regulating myocardial contractility and mediating the response of the heart to beta-AR stimulation.

Antiarrhythmic effects of beta3-adrenergic receptor stimulation in a canine model of ventricular tachycardia

Beta3-adrenergic receptor (beta3-AR) stimulation inhibits cardiac contractility. This study sought to test the hypothesis that beta3-AR stimulation is antiarrhythmic. We implanted a radio transmitter for continuous electrocardiogram monitoring in 18 dogs with a tendency for high incidence of spontaneous ventricular tachycardia (VT). Ten of 18 had subcutaneous continuous BRL37344 (beta3-AR agonist) infusion (experimental group) for 1 month. The other dogs were controls. Western blotting studies were performed on tissues sampled from the noninfarcted left ventricular free wall of all dogs that survived the 60-day follow-up period. Phase 2 VT appeared significantly later in the experimental group than in the control group (P <.05). The number of VT episodes in the experimental group was significantly lower than in the control group during both the first month (0.5 +/- 0.95 episodes/day vs. 2.6 +/- 2.3 episodes/day) and the second month (0.2 +/- 0.2 episode/day vs. 1.2 +/- 1.1 episodes/day, P <.05 for both). The experimental group had shorter QTc than control (P <.002). The experimental group had decreased protein levels for sodium calcium exchanger and dihydropyridine receptor, increased beta3-AR expression, without changes in beta1-AR, beta2-AR. The average heart weight and the left ventricular free wall thickness in the experimental group (226 +/- 17 g and 15.1 +/- 1.2 mm, respectively) was significantly lower than in the control group (265 +/- 21 g and 17.4 +/- 2.5 mm, respectively, P <.05 for both). There was no difference in the incidences of sudden cardiac death in these 2 groups of dogs. Beta3-AR stimulation significantly reduces the occurrence of ventricular tachycardia.

Sex Differences in Lipolysis‐Regulating Mechanisms in Overweight Subjects: Effect of Exercise Intensity

To explore sex differences in the regulation of lipolysis during exercise, the lipid-mobilizing mechanisms in the subcutaneous adipose tissue (SCAT) of overweight men and women were studied using microdialysis. Subjects matched for age, BMI, and physical fitness performed two 30-minute exercise bouts in a randomized fashion: the first test at 30% and 50% of their individual maximal oxygen uptake (Vo(2max)) and the second test at 30% and 70% of their Vo(2max). In both groups, an exercise-dependent increment in extracellular glycerol concentration (EGC) was observed. Whatever the intensity, phentolamine [alpha-adrenergic receptor (AR) antagonist] added to a dialysis probe potentiated exercise-induced lipolysis only in men. In a probe containing phentolamine plus propranolol (beta-AR antagonist), no changes in EGC occurred when compared with the control probe when exercise was performed at 30% and 50% Vo(2max). A significant reduction of EGC (when compared with the control probe) was observed in women at 70% Vo(2max). At each exercise power, the plasma non-esterified fatty acid and glycerol concentrations were higher in women. Exercise-induced increase in plasma catecholamine levels was lower in women compared with men. Plasma insulin decreased and atrial natriuretic peptide increased similarly in both groups. Overweight women mobilize more lipids (assessed by glycerol) than men during exercise. alpha(2)-Anti-lipolytic effect was functional in SCAT of men only. The major finding is that during low-to-moderate exercise periods (30% and 50% Vo(2max)), lipid mobilization in SCAT relies less on catecholamine-dependent stimulation of beta-ARs than on an increase in plasma atrial natriuretic peptide concentrations and the decrease in plasma insulin.

Increased thermogenic responsiveness to intravenous β‐adrenergic stimulation in habitually exercising humans is not related to skeletal muscle β2‐adrenergic receptor density

Habitually exercising adults demonstrate greater thermogenic responsiveness to beta-adrenergic receptor (beta-AR) stimulation compared with their sedentary peers, but the molecular mechanisms involved are unknown. To determine the possible role of increased beta-AR density, we studied 32 healthy adults: 17 habitual aerobic exercisers (age 45 +/- 5 years, 11 males) and 15 sedentary (49 +/- 5 years, 7 males). Maximal oxygen uptake (43.7 +/- 2.5 versus 31.6 +/- 2.9 ml kg(-1) min(-1), P = 0.002, mean +/- S.E.M.) and vastus lateralis muscle maximal citrate synthase activity (1.70 +/- 0.36 versus 0.58 +/- 0.11 micromol min(-1) g(-1), P = 0.008) were higher in the habitually exercising subjects. Resting energy expenditure (EE) adjusted for fat-free mass (FFM) was similar in the habitually exercising (5903 +/- 280 kJ day(-1)) and sedentary adults (6054 +/- 289 kJ day(-1), P = 0.43). The percentage increase in EE (DeltaEE%; indirect calorimetry, ventilated hood) above resting EE in response to beta-AR stimulation (intravenous isoproterenol at 6, 12 and 24 ng (kg FFM)(-1) min(-1)) was greater (7.1 +/- 1.2, 13.7 +/- 1.0, 20.7 +/- 1.3 versus 5.9 +/- 0.9, 9.9 +/- 1.4, 15.9 +/- 1.70%, respectively, P = 0.04), and the dose of isoproterenol required to increase EE by 10% above resting EE was lower (8.2 +/- 1.5 versus 17.1 +/- 4.1 ng (kg FFM)(-1) min(-1), P = 0.03) in the habitually exercising adults. In contrast, vastus lateralis muscle beta(2)-AR density was similar in the habitually exercising and sedentary subjects (7.46 +/- 0.29 versus 7.44 +/- 0.60 fmol (mg dry weight muscle)(-1), P = 0.98), and was not related to DeltaEE% (r = 0.02, P = 0.94) or to the isoproterenol dose required to increase EE by 10% above resting EE (r = -0.06, P = 0.76). These findings indicate that increased beta(2)-AR density is not a mechanism contributing to the greater thermogenic responsiveness to beta-AR stimulation in adult humans who regularly perform aerobic exercise.

A Major Functional Role for Phosphodiesterase 4D5 in Human Airway Smooth Muscle Cells

Relaxation of airway smooth muscle is dependent predominantly upon elevation of cell cAMP content. Although the processes involved in elevation of cell cAMP content are reasonably well established, the mechanisms governing subsequent control of cAMP turnover are less clear. Breakdown of cAMP is solely regulated by phosphodiesterase (PDE) isoenzymes. We have previously reported that PDE4 family members are likely to be important in this process, and that expression of PDE4D variants is actively regulated at the transcriptional level. Here, we demonstrate a key role for PDE4D5 in the control of beta(2)-adrenoceptor (beta(2)AR)-stimulated cAMP activity in human airway smooth muscle cells using splice variant-specific small interfering RNA knockdown. Furthermore, we show, using an Epac (exchange protein directly activated by cAMP)-based, cAMP-sensitive fluorescent probe, that these intracellular cAMP gradients are controlled both temporally and dynamically by PDE4D5. Elevation of cAMP within the cytoplasm after beta(2)AR stimulation is rapid and shows no distinct spatial compartmentalization in these cells. These data suggest that PDE4D5, despite being a minor component of the tissue PDE pool, is the key physiological regulator of beta(2)AR-induced cAMP turnover within human airway smooth muscle.

Downregulation of CuZn-superoxide dismutase contributes to β-adrenergic receptor-mediated oxidative stress in the heart

Sustained beta-adrenergic receptor (beta-AR) activation augments oxidative stress in the heart; whether alterations in antioxidant enzymes contribute to this effect is unknown. Adult male Wistar rats were implanted with osmotic minipumps to infuse either l-isoproterenol (ISO, 25 microg/kg/h) or saline (SAL). After 7-days, ISO-treated hearts exhibited significant (p<0.005): 1) concentric hypertrophy and augmentation of systolic function, 2) reductions of end-systolic wall stress, and 3) augmentation of oxidative stress, with a approximately 3-fold increase in 4-hydroxy-2-nonenal-and malondialdehyde-protein adducts. ISO-treated hearts also exhibited significant (p<0.01) reductions of CuZn-superoxide dismutase (SOD) enzyme activity (30%), protein (40%), and mRNA (60%), without changes in Mn-SOD, catalase, or glutathione peroxidase. Elk-1 and YinYang1 (YY1) are transcription factors that positively and negatively regulate CuZn-SOD expression, respectively. ISO-treated hearts exhibited a 3-fold increase in YY1 and a 2-fold reduction in Elk-1 DNA binding activity, strongly favoring CuZn-SOD gene repression. In isolated cardiomyocytes, sustained (24 h) ISO stimulation significantly (p<0.01) increased reactive oxygen species (ROS), an effect blocked by CGP20712A, a beta1-AR antagonist, but not by ICI118,551, a beta2-AR antagonist. CuZn-SOD downregulation paralleled the increase in ROS, and were similarly blocked by beta1- but not beta2-AR blockade. There were no changes in CuZn-SOD mRNA stability or myocyte size with ISO treatment. However, nuclear run-on revealed a 40% reduction in CuZn-SOD mRNA expression (p<0.01), consistent with transcriptional repression. ISO also depressed total cellular antioxidant capacity, reduced glutathione (GSH) levels, and the GSH:GSSG ratio. Moreover, CuZn-SOD siRNA transfection of H9c2 cardiomyocytes to suppress CuZn-SOD protein by approximately 40-50% (analogous to the in vivo changes) induced cellular apoptosis. Sustained beta-AR stimulation transcriptionally downregulates CuZn-SOD in myocardium via the beta1-AR, thereby contributing to beta-AR-mediated oxidative stress.

A chemical biology approach identifies a beta-2 adrenergic receptor agonist that causes human tumor regression by blocking the Raf-1/Mek-1/Erk1/2 pathway

A chemical biology approach identifies a beta 2 adrenergic receptor (beta2AR) agonist ARA-211 (Pirbuterol), which causes apoptosis and human tumor regression in animal models. beta2AR stimulation of cAMP formation and protein kinase A (PKA) activation leads to Raf-1 (but not B-Raf) kinase inactivation, inhibition of Mek-1 kinase and decreased phospho-extracellular signal-regulated kinase (Erk)1/2 levels. ARA-211 inhibition of the Raf/Mek/Erk1/2 pathway is mediated by PKA and not exchange protein activated by cAMP (EPAC). ARA-211 is selective and suppresses P-Erk1/2 but not P-JNK, P-p38, P-Akt or P-STAT3 levels. beta2AR stimulation results in inhibition of anchorage-dependent and -independent growth, induction of apoptosis in vitro and tumor regression in vivo. beta2AR antagonists and constitutively active Mek-1 rescue from the effects of ARA-211, demonstrating that beta2AR stimulation and Mek kinase inhibition are required for ARA-211 antitumor activity. Furthermore, suppression of growth occurs only in human tumors where ARA-211 induces cAMP formation and decreases P-Erk1/2 levels. Thus, beta2AR stimulation results in significant suppression of malignant transformation in cancers where it blocks the Raf-1/Mek-1/Erk1/2 pathway by a cAMP-dependent activation of PKA but not EPAC.

Advanced‐glycation end products (AGEs) derived from glycated albumin suppress early β1‐adrenergic preconditioning

Ischemic heart disease in diabetic patients might be linked to the accumulation of advanced-glycation end products (AGEs). In ischemic rat hearts, expression of receptor for AGEs and its ligands is significantly enhanced and involved in cardiac ischemia/reperfusion (I/R) injury even in the absence of diabetes. It has recently been reported that diabetic human myocardium cannot be protected by preconditioning. In this context, our hypothesis was that beta1-adrenergic preconditioning might be altered in the presence of AGEs. Using an isolated non-working rat heart model, this study investigated the effect of AGEs on cardioprotection induced by transient beta1-adrenoceptor (beta1-AR) stimulation with xamoterol (Xa). After 6-hydroxydopamine (6-OHDA) pre-treatment and a 20-min stabilization period, hearts were perfused at constant pressure for 20 min, then subjected to 40 min of global ischemia and 30 min of reperfusion (I/R, Ctrl); and exposed to 0.01 microm Xa for 5 min framed with or without 15.2 microm albumin (Alb) or glycated albumin (Gly Alb). The main endpoints were the mean coronary flow (MCF), the left ventricular end-diastolic pressure (LVEDP), rate-pressure product (RPP) and creatine kinase (CK) release and necrosis area. XA induced an increase in the MCF after I/R (t = 85 min), a protective effect on the LVEDP, an improvement in RPP, a decrease of CK release during reperfusion and a reduction of necrotic area. The beneficial effects induced by Xa during reperfusion were suppressed by the administration of Gly Alb during Xa infusion, whereas Alb did not hamper Xa-induced protection. These results suggest that AGEs suppress the cardioprotection resulting from the activation of beta1-ARs and thus might contribute to cardiovascular damages seen in diabetic patients.

Chronic Direct Stimulation of Adenylyl Cyclase Induces Cardiac Desensitization to Catecholamine and Beta-Adrenergic Receptor Downregulation in Rabbits

Chronic stimulation of beta-adrenergic receptors (betaARs) induces betaAR downregulation. However, it is not known whether continuous activation of adenylyl cyclase without direct stimulation of betaARs leads to receptor downregulation. This study investigated the effects of chronic stimulation of adenylyl cyclase with colforsin, on hemodynamic variables, and on myocardial betaAR density. In all, 55 rabbits received intravenous colforsin (1.6 microg/kg/min, n = 20), isoproterenol (ISO; 0.4 microg/kg/min, n = 16), or saline (n = 19) for two weeks. After chronic drug administration, responses of systolic (Delta% peak LV +dP/dt) and diastolic function (Delta% peak LV -dP/dt), and heart rate (Delta% heart rate), to acute administration of ISO (0.05 to 0.2 microg/kg/min) or colforsin (5 to 20 nmol/kg/min) were decreased compared to those before chronic administration. betaAR density in the colforsin group (69.8 +/- 13.8 fmol/ml protein) was less than that in the saline group (79.8 +/- 15.0 fmol/ml protein, P < 0.05), but was greater than that in the ISO group (56.3 +/- 8.4 fmol/ml protein, P < 0.05). Thus, chronic direct stimulation of adenylyl cyclase elicited systolic and diastolic functional desensitization to betaAR stimulation or adenylyl cyclase stimulation, and myocardial betaAR downregulation.

Mechanisms underlying β2‐adrenoceptor‐mediated nitric oxide generation by human umbilical vein endothelial cells

Endothelial beta(2)-adrenoceptor (beta(2)AR) stimulation increases nitric oxide (NO) generation, but the underlying cellular mechanisms are unclear. We examined the role of l-arginine transport and of phosphorylation of NO synthase 3 (NOS-3) in beta(2)AR-mediated NO biosynthesis by human umbilical vein endothelial cells (HUVEC). To this end, we assessed l-arginine uptake, NOS activity (from l-arginine to l-citrulline conversion), membrane potential (using [(3)H]tetraphenylphosphonium), as well as serine phosphorylation of NOS-3 (by Western blotting and mass spectrometry), in HUVEC treated with betaAR agonists or cyclic AMP-elevating agents. beta(2)AR stimulation increased l-arginine transport, as did cyclic AMP elevation with either forskolin or dibutyryl cyclic AMP, and this increase was inhibitable by N-ethylmaleimide. Blockade of l-arginine uptake by l-lysine inhibited NOS activity and, conversely, blockade of NOS using N(omega)-nitro-l-arginine methyl ester (l-NAME) inhibited l-arginine transport. beta(2)AR stimulation also caused a membrane hyperpolarization inhibitable by l-NAME, suggesting that the increase in l-arginine uptake occurred in response to NO-mediated hyperpolarization. beta(2)AR activation also increased NOS activity and phosphorylation of NOS-3 on serine-1177, and these increases were attenuated by inhibition of protein kinase A (PKA), phosphatidylinositol 3-kinase (PI3K) or Akt, and abolished by coinhibition of PKA and Akt. These findings suggest that beta(2)AR-mediated NOS-3 activation in HUVEC is mediated through phosphorylation of NOS-3 on serine-1177 through both the PKA and the PI3K/Akt systems, and is sustained by an increase in l-arginine uptake resulting from NO-mediated membrane hyperpolarization.