Adrenergic Receptor Activation Research Articles

Young rats exhibit an age- and sex-dependent bladder response to alpha-antagonists but not beta-agonists

Previous studies have suggested that the onset of alpha- (α) and beta- (β) adrenergic receptor activity is delayed in young animals. The use of alpha1- (α1-) antagonists for dysfunctional voiding, and beta3- (β3-) agonists for overactive bladder in younger children may not be indicated if maturation is required before bladder and urethral adrenergic receptors are active. To determine the sex- and age-dependent responses of the bladder and external urethral sphincter (EUS) to α- and β-adrenergic agents in neonatal and young adult rats. A total of 72 naïve Sprague-Dawley rats (36 female, 36 male) and 15 bladder-reduced (BR) female Sprague-Dawley rats underwent cystometry and EUS electromyography at 3, 6, and 9 weeks of life. Following administration of WAY 100,635 (0.3 mg/kg, serotonergic receptor antagonist), the non-selective α-agonist phenylephrine (0.3 mg/kg), α-antagonist phentolamine (1-3 mg/kg), β-agonist isoprenaline (3 mg/kg) and β-antagonist propranolol (3 mg/kg) were delivered intravenously. The maximum intravesical pressure (IVP), pressure threshold (PT), intermicturition interval (IMI), contraction duration (CD), burst amplitude and burst frequency were compared after each drug. The α-antagonist phentolamine lowered the IVP in 9-week-old males without lowering the PT. In contrast, the β-agonist isoprenaline lowered the IVP in both males and females of all ages, also without affecting the PT. Isoprenaline was also effective at shortening the CD in females, suggesting more effective bladder emptying. The α-agonist phenylephrine increased the IVP in 3-week-old and 6-week-old females and 3-week-old males, but this effect was blocked by pretreatment with phentolamine. The β-antagonist propranolol increased the PT in both males and females, and shortened the IMI in females, which was consistent with retention. Phenylephrine increased the burst duration in 9-week-old naïve females, while isoprenaline increased the burst amplitude and duration in 9-week-old BR females. In the neonatal rat, both α- and β-adrenergic receptors actively regulate bladder function by 3 weeks of life, but the desired effect of decreasing IVP by α-antagonists was delayed until 9 weeks in male rats. In contrast, β-agonists were effective at decreasing IVP in both male and female rats of all ages, which suggests that they are better agents for enhancing bladder emptying in female and young male rats.

Cell membrane chromatography coupled with UHPLC-ESI-MS/MS method to screen target components from Peucedanum praeruptorum Dunn acting on α1A adrenergic receptor.

Peucedanum praeruptorum Dunn (BaiHuaQianHu in Chinese) is a traditional Chinese medicine that has a long history of use in China. In this study, HEK 293 α1A adrenergic cell membrane chromatography was coupled with UHPLC-ESI-MS/MS and successfully used to identify active components from Peucedanum praeruptorum Dunn. Paeruptorin A, paeruptorin B, and paeruptorin C were identified with α1A adrenergic receptor activity. Pharmacological assays showed that tamsulosin hydrochloride, paeruptorin A, paeruptorin B, and paeruptorin C in concentrations of 1×10(-8) to 1×10(-4)mol/mL could relax prostate strips pre-contracted with adrenalin in a concentration dependent manner. Therefore, the HEK293 α1A cell membrane chromatography coupled UHPLC-ESI-MS/MS system may be a potentially useful drug discovery method for screening for medicinal herbal components with α1A adrenergic receptor inhibitory activity.

Beta 2-adrenergic receptor activation enhances neurogenesis in Alzheimer's disease mice.

Impaired hippocampal neurogenesis is one of the early pathological features of Alzheimer's disease. Enhancing adult hippocampal neurogenesis has been pursued as a potential therapeutic strategy for Alzheimer's disease. Recent studies have demonstrated that environmental novelty activates β2-adrenergic signaling and prevents the memory impairment induced by amyloid-β oligomers. Here, we hypothesized that β2-adrenoceptor activation would enhance neurogenesis and ameliorate memory deficits in Alzheimer's disease. To test this hypothesis, we investigated the effects and mechanisms of action of β2-adrenoceptor activation on neurogenesis and memory in amyloid precursor protein/presenilin 1 (APP/PS1) mice using the agonist clenbuterol (intraperitoneal injection, 2 mg/kg). We found that β2-adrenoceptor activation enhanced hippocampal neurogenesis, ameliorated memory deficits, and increased dendritic branching and the density of dendritic spines. These effects were associated with the upregulation of postsynaptic density 95, synapsin 1 and synaptophysin in APP/PS1 mice. Furthermore, β2-adrenoceptor activation decreased cerebral amyloid plaques by decreasing APP phosphorylation at Thr668. These findings suggest that β2-adrenoceptor activation enhances neurogenesis and ameliorates memory deficits in APP/PS1 mice.

Isoproterenol Enhances Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand-Induced Apoptosis in Human Embryonic Kidney Cells through Death Receptor 5 up-Regulation.

Background and ObjectivesChronic impairment of β-adrenergic receptor signaling increases cardiac apoptosis, hypertrophy and fibrosis. The aim of this study was to investigate whether isoproterenol (ISO), an agonist of the adrenergic receptor, can enhance tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in human embryonic kidney (HEK) 293 cells.Materials and MethodsHEK 293 cells were treated with ISO and/or TRAIL for 24 hours. Cell viability was evaluated by microscopy and an established viability assay, and apoptotic cell death was analyzed by staining with fluorescein isothiocynate-annexin-V/propidium iodide (PI) and caspase activation. To confirm the mechanism of cell death induced by co-treatment with ISO and TRAIL, expression of TRAIL receptor 2 (death receptor 5, DR5) was evaluated by immunoblotting.ResultsAlthough ISO or TRAIL treatment decreased HEK 293 cell viability by 13% and 17%, respectively, co-treatment with ISO and TRAIL resulted in a markedly higher death rate of 35% after 24 hours. Increases were evident in early apoptotic cells (i.e., annexin-V positive/PI negative; 19.4%), late apoptotic cells (i.e., annexin-V positive/PI positive; 6.3%) and dead cells (i.e., annexin-V negative/PI positive; 1.1%) when cells were co-treated with ISO and TRAIL, compared to cells treated with either ISO or TRAIL. In addition, marked increases of cleaved cas-3, cleaved poly (adenosine diphosphate-ribose) polymerase and DR5 were observed in HEK 293 cells co-treated with ISO and TRAIL.ConclusionTreatments combining ISO with TRAIL may be responsible for death of HEK 293 cells through DR5 up-regulation. Activation of adrenergic receptors is responsible for the synergistic cell death observed with TRAIL.

Role of sympathetic nervous system in rat model of chronic visceral pain.

Changes in central pain modulation have been implicated in generalized pain syndromes such as irritable bowel syndrome (IBS). We have previously demonstrated that reduced descending inhibition unveils a role of sympathoneuronal outflow in decreasing peripheral sensory thresholds, resulting in stress-induced hyperalgesia. We investigated whether sympathetic nervous system (SNS) exacerbation of pain sensation when central pain inhibition is reduced is relevant to chronic pain disorders using a rat colon irritation (CI) model of chronic visceral hypersensitivity with hallmarks of IBS. Rats were treated to a series of colorectal balloon distensions (CRD) as neonates resulting in visceral and somatic hypersensitivity and altered stool function that persists into adulthood. The visceral sensitivity was assessed by recording electromyographic (EMG) responses to CRD. Somatic sensitivity was assessed by paw withdrawal thresholds to radiant heat. The effects on the hypersensitivity of (i) inhibiting sympathoneuronal outflow with pharmacological and surgical interventions and (ii) enhancing the outflow with water avoidance stress (WAS) were tested. The alpha2-adrenergic agonist, clonidine, and the alpha1-adrenergic antagonist, prazosin, reduced the visceral hypersensitivity and WAS enhanced the pain. Chemical sympathectomy with guanethidine and surgical sympathectomy resulted in a loss of the chronic visceral hypersensitivity. The results support a role of the SNS in driving the chronic visceral and somatic hypersensitivity seen in CI rats. The findings further suggest that treatments that decrease sympathetic outflow or block activation of adrenergic receptors on sensory nerves could be beneficial in the treatment of generalized pain syndromes.

Abstract 11879: Control of Fibroblast Function by Adrenergic/EPAC System and Potential Role in Atrial Remodeling

Introduction: Heart failure (HF) produces left atrial (LA)-selective fibrosis and atrial fibrillation. HF also causes adrenergic activation, which contributes to remodeling via a variety of pathways mediated by altered signaling molecules, including the exchange protein activated by cAMP (Epac). Hypothesis: To evaluate the effects of adrenergic receptor (AR) and Epac1 signaling on LA fibroblast (FB) function and its potential role in HF-induced atrial remodeling. Methods: HF was induced in dogs by ventricular tachypacing (VTP, 240 bpm) for various periods. mRNA and protein expression were measured in freshly isolated LA FBs by qPCR and immunoblot, respectively. Results: Epac1 expression decreased in LA FBs within 12 hrs (-3.9 fold) of VTP onset (Fig. A). The selective Epac activator, 8-pCPT (33 μM) reduced, whereas the Epac blocker ESI-09 (1 μM) enhanced, LA FB collagen expression (Fig. B). Norepinephrine (NE, 1 μM) decreased Epac1 expression, an effect blocked by prazosin, and increased FB collagen protein production (Fig. C). Isoproterenol (ISO) increased Epac1 expression (Fig. D), an effect antagonized by ICI (β2-AR antagonist, 2 μM), but not CGP (β1-AR antagonist, 2 μM). β2-AR activation with ISO decreased collagen expression (Fig. E), an effect mimicked by salbutamol (10 μM) and blocked by ICI but not CGP. Transforming growth factor (TGF) β1 (10 ng/ml), known to be activated in HF, suppressed Epac1 expression, an effect blocked by the Smad3 inhibitor SIS3 (Fig. F). Conclusions: AR activation has complex effects on FBs, with NE/α-AR-activation suppressing Epac1 and increasing collagen expression, and β2-ARs having opposite effects. HF reduces LA FB Epac1 expression, likely via increased NE release from adrenergic nerve endings, along with TGF β1 activation. Epac1 signaling reduces FB collagen expression, so Epac1 downregulation in HF contributes to the atrial profibrotic milieu and may be a novel target for prevention of profibrillatory atrial remodeling.

Disruption of Spinal Noradrenergic Activation Delays Recovery of Acute Incision-Induced Hypersensitivity and Increases Spinal Glial Activation in the Rat

Results of clinical studies suggest that descending inhibitory controls from the brainstem are important for speeding recovery from pain after surgery. We examined the effects of destroying spinally projecting noradrenergic neurons via intrathecally administered antibody to dopamine β-hydroxylase conjugated to saporin (DβH-saporin) on recovery in an acute incisional pain model. Mechanical and thermal paw withdrawal thresholds and nonevoked spontaneous guarding scores were tested for several weeks postoperatively and analyzed using mixed effects growth curve modeling. DβH-saporin treatment resulted in a significant prolongation in the duration of mechanical and to a lesser degree thermal hypersensitivity in the ipsilateral paw of incised rats but did not increase the duration of spontaneous guarding. DβH-saporin treatment was also associated with increased microglial and astrocyte activation in the ipsilateral spinal cord 21 days after incision compared with immunoglobulin G-saporin treated controls. Chronic intrathecal administration of the α2 adrenergic receptor antagonist atipamezole (50–200 μg/d) produced similar effects. These data suggest that spinally projecting noradrenergic pathways and spinal α2 adrenergic receptor activation are important for speeding recovery from hypersensitivity after surgical incision possibly by reducing spinal glial activation. Interventions that augment the noradrenergic system might be important to speed recovery from pain after surgery. PerspectiveEndogenous descending spinal noradrenergic activation promotes resolution of incision-induced hypersensitivity and inhibits spinal microglial and astrocyte activation in part through α2 adrenergic receptors.

Prescriptions séquentielles : arguments en faveur d’une modification des schémas thérapeutiques dans les dépressions résistantes

Among the therapeutic strategies in treatment of resistant depression, the use of sequential prescriptions is discussed here. A number of observations, initially quite isolated and few controlled studies, some large-scale, have been reported, which showed a definite therapeutic effect of certain requirements in sequential treatment of depression. The Sequenced Treatment Alternatives to Relieve Depression Study (STAR*D) is up to now the largest clinical trial exploring treatment strategies in non psychotic resistant depression in real-life conditions with an algorithm of sequential decision. The main conclusions of this study are the following: after two unsuccessful attempts, the chance of remission decreases considerably. A 12-months follow-up showed that the higher the use of the processing steps were high, the more common the relapses were during this period. The pharmacological differences between psychotropic did not cause clinically significant difference. The positive effect of lithium in combination with antidepressants has been known since the work of De Montigny. Antidepressants allow readjustment of physiological sequence involving different monoaminergic systems together. Studies with tricyclic antidepressant-thyroid hormone T3: in depression, decreased norepinephrine at the synaptic receptors believed to cause hypersensitivity of these receptors. Thyroid hormones modulate the activity of adrenergic receptors. There would be a balance of activity between alpha and beta-adrenergic receptors, depending on the bioavailability of thyroid hormones. ECT may in some cases promote pharmacological response in case of previous resistance, or be effective in preventing relapse. Cognitive therapy and antidepressant medications likely have an effect on different types of depression. We can consider the interest of cognitive therapy in a sequential pattern after effective treatment with an antidepressant effect for treatment of residual symptoms, preventing relapses and recurrences, in antidepressant maintenance. These data support the interest of therapeutic strategies based on evolutionary criteria. Sequential models inspired by statistical methods may incorporate the effects of a future treatment by measuring the current one.

Abstract P204: Food Restriction Increases the Cardiovascular Response Evoked by Angiotensin-[1-10] and Angiotensin-[1-7] in Female Fischer Rats

Anorexia is associated with cardiovascular dysfunction including hypotension, bradycardia and cardiac arrhythmias. In a rat model of food restriction (FR)-induced hypotension and bradycardia, we previously demonstrated that the activity of endothelial α1 adrenergic receptors and brain type 1 angiotensin receptors (AT1R) were increased. The aim of this study was to determine the role of the vasodilator, angiotensin (Ang)-[1-7] and the precursor Ang-[I-10] in the periphery and brain. Methods: Female Fischer rats (220g, 4 mo) were maintained for 14 days on a FR diet, that was 40% of the control group (C) food intake. Cannulas were implanted in the lateral ventricle (LV) on day 7. On day 14, the animals were catheterized with a polyethylene tube in the femoral artery for recording of mean arterial pressure (MAP) and heart rate (HR). Results: After 14 days, body weight (g) [C: 202±2, n=13 vs. FR: 176±2, n=14; p<0.05], MAP (mm Hg) [C: 105±1, n=35 vs. FR: 102±2, n=36; p<0.05] and HR (bpm) [C: 386±5, n=35 vs. FR: 353±8, n=36, p<0.001) were reduced in the FR compared to control rats. Injection of Ang-[1-7] caused a greater increase in MAP (Δ mm Hg) in the FR compared to C group when delivered either into the LV (25pmol) (C: Δ -2±1, n=8 vs. FR: Δ 7±1, n=10; p<0.05) or the femoral vein (1910pmol) [C: Δ 1±1, n=9 vs. FR: Δ 3±1, n=6; p<0.05]. Moreover, there was a greater increase in HR (Δ bpm) in the FR compared to the C group after LV injection (C: Δ 9±3, n=8 vs FR: Δ 29±9, n=10; p<0.05). Injection of Ang-[I-10] caused a greater increase in MAP (Δ mm Hg) in the FR compared to C group when delivered into the LV (25pmol) (C: 5±1, n=7 vs. FR: 13±2, n=7; p<0.005) or i.v (1910pmol) (C: Δ 1±1, n=7 vs. FR: Δ 15±2, n=8; p<0.05); however, there was no effect on HR. This Ang-[l-10]-induced increase in MAP was attenuated by administering captopril into the LV, in both the FR and C groups, suggesting this increase in MAP was mediated by angiotensin converting enzyme. Conclusion: These results suggest that increased Ang-[1-7] and Ang-[I-10] activity both in the periphery and brain play a role in the increased sympathetic activity observed in anorexia.

From one generation to the next: a comprehensive account of sympathetic receptor control in branching arteriolar trees.

The effect of the sympathetic nervous system on blood flow distribution within skeletal muscle microvasculature is conditional upon regional activation of receptors for sympathetic neurotransmitters. Previous studies have shown that proximal arterioles are largely governed by adrenergic activation, whereas it is speculated that distal branches are controlled by peptidergic and purinergic activation. However, no study has systematically evaluated the activation of adrenergic, peptidergic and purinergic receptors in continuously branching arteriolar trees of an individual skeletal muscle model. Therefore, in the present study, sympathetic agonists were used to evaluate the constriction responses along first to fifth order arterioles in continuously branching arteriolar trees of a in vivo rat gluteus maximus muscle preparation with respect to specific activation of receptors for sympathetic neurotransmitters (α1R, α2R, NPY1R and P2X1R). Constriction responses were incorporated into a mathematical blood flow model to estimate the total flow, resistance and red blood cell flow heterogeneity within a computationally reconstructed gluteus maximus arteriolar network. For the first time, the effects of activating receptors for sympathetic neurotransmitters on vasoconstrictor responses and the ensuing haemodynamics in continuously branching arteriolar trees of skeletal muscle were characterized, where proximal arterioles responded most to α1R and α2R adrenergic activation, whereas distal arterioles responded most to Y1R and P2X1R activation. Total flow and resistance changed with activation of all receptors, whereas red blood cell flow heterogeneity was largely affected by peptidergic and purinergic activation in distal arterioles. The reported data highlight the functional consequences of topologically-dependent sympathetic control and may serve as novel input parameters in computational modelling of network flow.

Adrenergic Control of the Cutaneous Circulation during Cryotherapy

Cryotherapy application is a widely clinically used therapeutic technique using ice or cold water applied to the skin surface to reduce local blood flow. While beneficial, cryotherapy treatment does have the potential to induce some detrimental physiological side effects. For example, cryotherapy treatment induces pronounced tissue ischemia that is sustained for hours even during a post-treatment rewarming period. PURPOSE: To investigate the role of adrenergic receptor activation on cutaneous blood flow during 30 min of cryotherapy as well as during the subsequent 1 hr of passive rewarming. We hypothesized that adrenergic-mediated vasoconstriction is involved in pronounced and sustained cutaneous vasoconstriction that occurs during cooling and passive rewarming. METHODS: To date 4 complete data sets have been collected with 2 partial, incomplete datasets. A commercially available cryotherapy unit with a water perfused bladder was used. The bladder was placed on the lateral portion of the calf. Four microdialysis membranes were threaded through the dermis of the skin directly underneath the water bladder. One site was perfused with lactated ringers solution (CON), one with bretylium tosylate (BRE), one with propranolol+yohimbine+BIBP (PBY), and one with propranolol+BIBP (PB). Skin temperature (SkT) and skin vascular conductance (SkVC) was measured at each site. Subjects had 0 °C water perfused through the cryotherapy bladder for 30 min, followed by passive rewarming (i.e. water shut off) for 1 hr, followed by direct heating with 46 °C for 10 min. RESULTS: SkT fell from 33°C to 17.7°C during 30 min of cold water application across all sites. SkVC at CON was reduced ∼80% after 30 min of cooling and was not significantly different than PB (p>0.05). Vasoconstriction was attenuated at the BRE and PBY sites during cooling compared the control (SkVC: BRE = 58% reduction, PBY = 37% reduction, CON 80% reduction (p<0.05 for each comparison). Following 10 min of active warming the BRE site was not different than CON (SkBF: 46% reduction vs. 28% reduction (P>0.05). CONCLUSION: Our preliminary finding was that adrenergic function mediates the vasoconstriction that occurs with cryotherapy application. This is in line with previous literature investigating the adrenergic mechanisms of vasoconstriction with skin cooling.

Inhibition of type 5 phosphodiesterase counteracts β2-adrenergic signalling in beating cardiomyocytes.

Compartmentalization of cAMP and PKA activity in cardiac muscle cells plays a key role in maintaining basal and enhanced contractility stimulated by sympathetic nerve activity. In cardiomyocytes, activation of adrenergic receptor increases cAMP production, which is countered by the hydrolytic activity of selective phosphodiesterases (PDEs). The intracellular regional dynamics of cAMP production and hydrolysis modulate downstream signals resulting in different biological responses. The interplay between beta receptors (βARs) signalling and phosphodiesterase 5 (PDE5) activity remains to be addressed. Using combined strategies with pharmacological inhibitors and genetic deletion of PDEs and βAR isoforms, we revealed a specific pool of cAMP that is under dual regulation by PDE2 and, indirectly, PDE5 activity. Inhibition of PDE5 with sildenafil produces a cGMP-dependent activation of PDE2 that attenuates cAMP generation induced by βAR agonists, with concomitant modulation of stimulated contraction rate and calcium transients. PDE2 haploinsufficiency abolished the effects of sildenafil. The negative chronotropic effect of PDE5 inhibition through PDE2 activation was also observed in sinoatrial node tissue from adult mice. PDE5 inhibition selectively lowered contraction rate stimulated by β2AR, but not β1AR activation, supporting a compartmentalization of the cGMP-modulated pool of cAMP. These data identify a new effect of PDE5 inhibitors on the modulation of cardiomyocyte response to adrenergic stimulation via PDE5-PDE2-mediated cross-talk.

β3‐ Adrenergic Receptor Activation and Endoplasmic Reticulum Stress via Modulation of Intracellular Free Zn2+in Hyperglycemic Cardiomyocytes

Beta‐adrenergic receptor (β‐AR)‐signaling plays important role in normal cardiac function while β3‐AR activation depresses cardiac contractile force. Since intracellular free Zn2+releases are originated either from metallothionin‐nitrolisation or endoplasmic reticulum (ER) and disturbing excitation‐contraction cyclying in cardiomyocytes, we examined role of β3‐AR activation on modulation of intracellular free Zn2+and ER stress (ERS) in hyperglycemic cardiomyocytes. Protein and mRNA levels of β3‐AR were increased in diabetic cardiomyocytes being associated with depressed left ventricular developed pressure. β3‐AR stimulation with BRL37344 induced significant increase in intracellular free Zn2+in diabetic cardiomyocytes which was diminished either with NOS inhibitor L‐NAME or β3‐AR antagonist SR59230 while it was further increased with non‐selective β‐blocker nadolol. Additionally, either BRL37344 or NO‐donor SNP induced significant increases in intracellular free Zn2+in normal cardiomyocytes while they were diminished either with L‐NAME or ODQ. These data are strongly demonstrated an association between increased intracellular free Zn2+ and β3‐AR activation in diabetic cardiomyocytes. BRL37344 stimulation of only diabetic cardiomyocytes presented also marked increases in ERS‐associated proteins which are very similar to those of diabetics. Taken together, our data demonstrated that β3‐AR activation in cardiomyocytes under hyperglycemia induces marked increase in intracellular free Zn2+which seems to be closely related with ERS, thereby both actions can underlie the depressed cardiac function in diabetics.(Supported by TUBITAK‐SBAG‐113S466)

Evidence for ETB receptor mediated pressor effects mediated by alpha‐adrenergic receptors

The hypertensive response to exogenous ET-1 is largely mediated by ETA receptors located on vascular smooth muscle. However, there is an ETB receptor contribution, but the origin of this response is not clear. We hypothesized that stimulation of ETB receptors on neuronal tissues leads to a pressor through simulation of alpha-adrenergic receptors. To test this hypothesis, male rats that express ETB receptors only on sympathetic nerves were anesthetized and implanted with a venous catheter for infusion of the ETB agonist sarafotoxin 6c (S6c). An arterial catheter was placed in the carotid artery for mean arterial pressure (MAP) recording and all experiments were carried out in the presence of a ganglion blocker (chlorasondamine, 5 mg/kg) to inhibit central input. Bolus i.v. infusion of S6c (0.1, 0.3, and 1.0 nmol/kg) significantly increased MAP in a dose dependent manner (ΔMAP; 7±2, 15±8, and 27±11 mmHg, respectively). In the presence of the alpha-1 adrenergic blocker, prazosin (1mg/kg), acute infusion of S6c caused a reduction in blood pressure (ΔMAP; -10±2, -19±3, and -15±3 mmHg, respectively). Interestingly, beta-adrenergic blockade (propranolol, 5mg/kg) exacerbated the pressor response to S6c (ΔMAP; 52±14, 94±5, and 82±1 mmHg, respectively). These data suggest that activation of neuronal ETB receptors causes constriction primarily through the activation of alpha-1 adrenergic receptors while beta-adrenergic activity opposes this response to limit ETB mediated increases in blood pressure.

Phosphatidylethanolamine-binding protein is not involved in μ-opioid receptor-mediated regulation of extracellular signal-regulated kinase

Stimulation of the μ-opioid receptor activates extracellular signal-regulated kinase (ERK), however, the mechanism by which this occurs remains to be elucidated. Phosphatidylethanolamine-binding protein (PEBP) has been reported to act as a negative regulator of the ERK cascade (Raf-MEK-ERK) by binding to Raf-1 kinase. In the present study, the role of PEBP in μ-opioid receptor-mediated ERK activation was investigated in Chinese hamster ovary/μ cells and SH-SY5Y cells, as well as in human embryonic kidney 293 cells expressing other types of G protein-coupled receptors. The acute activation of μ-opioid receptors by morphine or (D-Ala2, MePhe4, Gly5-ol) enkephalin induced a rapid activation of ERK. Prolonged morphine treatment did not affect the phosphorylation level of ERK compared with control cells, but the phosphorylation level of ERK decreased markedly when cells were precipitated with naloxone following chronic morphine treatment. For the phosphorylation of PEBP, no change was identified under the designated drug treatment and exposure duration. A total of two other types of G protein-coupled receptors, including Gs-coupled dopamine D1 receptors and Gq-coupled adrenergic α1A receptors were also investigated and only the activation of adrenergic α1A receptors induced an upregulated phosphorylation of PEBP, which was protein kinase C activity dependent. Thus, PEBP did not have a significant role in μ-opioid receptor-mediated regulation of ERK.

β2 adrenergic receptor activation governs cardiac repolarization and arrhythmogenesis in a guinea pig model of heart failure.

β2-AR activation increases the risk of sudden cardiac death (SCD) in heart failure (HF) patients. Non-selective β-AR blockers have greater benefits on survival than selective β1-AR blockers in chronic HF patients, indicating that β2-AR activation contributes to SCD in HF. This study investigated the role of β2-AR activation on repolarization and ventricular arrhythmia (VA) in the experimental HF model. The guinea pig HF was induced by descending aortic banding. The effective refractoriness period (ERP), corrected QT (QTc) and the incidence of VA were examined using Langendorff and programmed electrical stimulation. Ikr and APD were recorded by the whole cell patch clamp. Selective β2-AR agonist salbutamol significantly increased the incidence of VA, prolonged QTc and shortened ERP. These effects could be prevented by the selective β2-AR antagonist, ICI118551. Salbutamol prolonged APD90 and reduced Ikr in guinea pig HF myocytes. The antagonists of cAMP (Rp-cAMP) and PKA (KT5720) attenuated Ikr inhibition and APD prolongation induced by salbutamol. However, the antagonists of Gi protein (PTX) and PDE III (amrinone) showed opposite effects. This study indicates that β2-AR activation increases the incidence of VA in the experimental HF model via activation of Gs/cAMP/PKA and/or inhibition of Gi/PDE pathways.

The Inhibitory Mechanism of Gentamicin on Electrical Field Stimulation Response in Rat Bladder Smooth Muscle.

To see the inhibitory mechanism of gentamicin in response to electrical field stimulation (EFS) using the rat bladder smooth muscle, atropine or guanethidine was treated but had no effect. Methylsergide, a non-selective 5-HT1, 5-HT2 receptor antagonist was also treated but had on effect. Kinase inhibitors, such as chelerythrine (PKC inhibitor), ML-9 (MLCK inhibitor), or Y27632 (rho kinase inhibitor) were pretreated before gentamicin treatment, but did not have effect. For U73122, a phospholipase C (PLC) inhibitor however, the inhibitory effect to gentamicin was significantly attenuated in all frequencies given by the EFS. Therefore gentamicin induced inhibitory effect on EFS response in rat bladder smooth muscle was not mediated by the activation of adrenergic, cholinergic, or serotonergic receptor. The inhibition of gentamicin might be mediated through the PLC dependent pathway, but not through the PKC, MLCK or rho kinase dependent pathway.

Adrenal G protein-coupled receptor kinase-2 in regulation of sympathetic nervous system activity in heart failure.

Heart failure (HF), the number one cause of death in the western world, is caused by the insufficient performance of the heart leading to tissue underperfusion in response to an injury or insult. It comprises complex interactions between important neurohormonal mechanisms that try but ultimately fail to sustain cardiac output. The most prominent such mechanism is the sympathetic (adrenergic) nervous system (SNS), whose activity and outflow are greatly elevated in HF. SNS hyperactivity confers significant toxicity to the failing heart and markedly increases HF morbidity and mortality via excessive activation of adrenergic receptors, which are G protein-coupled receptors. Thus, ligand binding induces their coupling to heterotrimeric G proteins that transduce intracellular signals. G protein signaling is turned-off by the agonist-bound receptor phosphorylation courtesy of G protein-coupled receptor kinases (GRKs), followed by βarrestin binding, which prevents the GRK-phosphorylated receptor from further interaction with the G proteins and simultaneously leads it inside the cell (receptor sequestration). Recent evidence indicates that adrenal GRK2 and βarrestins can regulate adrenal catecholamine secretion, thereby modulating SNS activity in HF. The present review gives an account of all these studies on adrenal GRKs and βarrestins in HF and discusses the exciting new therapeutic possibilities for chronic HF offered by targeting these proteins pharmacologically.

Adrenergic and imidazoline mechanisms of arterial baroreceptor reflex in the short and long-term regulation of blood pressure

Objective. The purpose of this work was to study how selective activation of сentral adrenergic and imidazoline receptors influence functional state of the blood baroreceptor reflex (BR) in the short and long-term regulation of blood pressure (BP) at rest and under emotional stress. Design and methods. Experiments were conducted on awake Wistar rats. Clonidine (1 and 10 mg/kg) was used for adrenergic receptors activation, moxonidine (10 and 100 mg/kg) was used for imidazoline receptor activation. Emotional stress was caused by sound signal (a ring). Results. Studies demonstrated that the activation of central imidazoline receptors does not change BP level, while alpha 2‑adrenergic systems play a major role in the central regulation of vascular tone and cardiac function. Both imidazoline and alpha 2‑adrenergic systems participate in functioning of baroreceptor reflex. Conclusions. Emotional stress-associated imidazoline receptor activation restores initial BR and reduces BP elevation in rats. Alpha 2‑adrenergic systems do not have a significant impact on cardiovascular manifestations of emotional tension.

Insights into the role of Asp79(2.50) in β2 adrenergic receptor activation from molecular dynamics simulations.

Achieving a molecular-level understanding of G-protein-coupled receptor (GPCR) activation has been a long-standing goal in biology and could be important for the development of novel drugs. Recent breakthroughs in structural biology have led to the determination of high-resolution crystal structures for the β2 adrenergic receptor (β2AR) in inactive and active states, which provided an unprecedented opportunity to understand receptor signaling at the atomic level. We used molecular dynamics (MD) simulations to explore the potential roles of ionizable residues in β2AR activation. One such residue is the strongly conserved Asp79(2.50), which is buried in a transmembrane cavity and becomes dehydrated upon β2AR activation. MD free energy calculations based on β2AR crystal structures suggested an increase in the population of the protonated state of Asp79(2.50) upon activation, which may contribute to the experimentally observed pH-dependent activation of this receptor. Analysis of MD simulations (in total > 100 μs) with two different protonation states further supported the conclusion that the protonated Asp79(2.50) shifts the conformation of the β2AR toward more active-like states. On the basis of our calculations and analysis of other GPCR crystal structures, we suggest that the protonation state of Asp(2.50) may act as a functionally important microswitch in the activation of the β2AR and other class A receptors.