Roles of ß-adrenoceptor Subtypes and Therapeutics in Human Cardiovascular Disease: Heart Failure, Tachyarrhythmias and Other Cardiovascular Disorders.

ß-Adrenoceptors (ß-ARs) provide an important therapeutic target for the treatment of cardiovascular disease. Three ß-ARs, ß1-AR, ß2-AR, ß3-AR are localized to the human heart. Activation of ß1-AR and ß2-ARs increases heart rate, force of contraction (inotropy) and consequently cardiac output to meet physiological demand. However, in disease, chronic over-activation of ß1-AR is responsible for the progression of disease (e.g. heart failure) mediated by pathological hypertrophy, adverse remodelling and premature cell death. Furthermore, activation of ß1-AR is critical in the pathogenesis of cardiac arrhythmias while activation of ß2-AR directly influences blood pressure haemostasis. There is an increasing awareness of the contribution of ß2-AR in cardiovascular disease, particularly arrhythmia generation. All ß-blockers used therapeutically to treat cardiovascular disease block ß1-AR with variable blockade of ß2-AR depending on relative affinity for ß1-AR vs ß2-AR. Since the introduction of ß-blockers into clinical practice in 1965, ß-blockers with different properties have been trialled, used and evaluated, leading to better understanding of their therapeutic effects and tolerability in various cardiovascular conditions. ß-Blockers with the property of intrinsic sympathomimetic activity (ISA), i.e. ß-blockers that also activate the receptor, were used in the past for post-treatment of myocardial infarction and had limited use in heart failure. The ß-blocker carvedilol continues to intrigue due to numerous properties that differentiate it from other ß-blockers and is used successfully in the treatment of heart failure. The discovery of ß3-AR in human heart created interest in the role of ß3-AR in heart failure but has not resulted in therapeutics at this stage.

The associations of cytokines and gens polymorphisms of ß-adrenoceptors in patients with heart failure and some thyroid pathology (literature review and own observations).

OBJECTIVE: Aim: To analyze the role of cytokines in the progression of heart failure (HF) in patients with concomitant pathology of the thyroid gland. PATIENTS AND METHODS: Materials and Methods: The systematization of literature data on the role of cytokines in the progression of HF in patients with concomitant thyroid pathology (TP) was carried out. The results of our own research were presented. CONCLUSION: Conclusions: The final chapter in the history of the role of cytokines in the progression of HF has not yet been written. Further studies, including genetic ones, are necessary. The patients with HF have higher levels of TNFß and IL-6, and a lower concentration of IL-4, compared to the control group. Patients with a fatal outcome of the disease, in contrast to those who survived for two years, have an increased level of TNFß. In patients with concomitant TP, who had repeated hospitalization, a lower level was registered, compared to that under conditions of a more favorable course of heart failure. Concentrations of cytokines in the blood of patients with HF are associated with gene polymorphisms of the ß-adrenoreceptor system: the C-allele of the Gly389A polymorphism of the ß1-adrenoceptor gene leads to a decrease in the risk of increasing TNFα; IL-1α increases in the presence of the A-allele of the Ser49Gly polymorphism of this gene. In patients with HF and concomitant thyroid pathology, the risk of IL-6 growth increases in homozygous (C) patients for the Ser275 polymorphism of the ß3 subunit of the G-protein.

ß-Adrenoceptor Blockade Moderates Neuroinflammation in Male and Female EAE Rats and Abrogates Sexual Dimorphisms in the Major Neuroinflammatory Pathways by Being More Efficient in Males.

Our previous studies showed more severe experimental autoimmune encephalomyelitis (EAE) in male compared with female adult rats, and moderating effect of propranolol-induced ß-adrenoceptor blockade on EAE in females, the effect associated with transcriptional stimulation of Nrf2/HO-1 axis in spinal cord microglia. This study examined putative sexual dimorphism in propranolol action on EAE severity. Propranolol treatment beginning from the onset of clinical EAE mitigated EAE severity in rats of both sexes, but to a greater extent in males exhibiting higher noradrenaline levels and myeloid cell ß2-adrenoceptor expression in spinal cord. This correlated with more prominent stimulatory effects of propranolol not only on CX3CL1/CX3CR1/Nrf2/HO-1 cascade, but also on Stat3/Socs3 signaling axis in spinal cord microglia/myeloid cells (mirrored in the decreased Stat3 and the increased Socs3 expression) from male rats compared with their female counterparts. Propranolol diminished the frequency of activated cells among microglia, increased their phagocyting/endocyting capacity, and shifted cytokine secretory profile of microglia/blood-borne myeloid cells towards an anti-inflammatory/neuroprotective phenotype. Additionally, it downregulated the expression of chemokines (CCL2, CCL19/21) driving T-cell/monocyte trafficking into spinal cord. Consequently, in propranolol-treated rats fewer activated CD4+ T cells and IL-17+ T cells, including CD4+IL17+ cells coexpressing IFN-γ/GM-CSF, were recovered from spinal cord of propranolol-treated rats compared with sex-matched saline-injected controls. All the effects of propranolol were more prominent in males. The study as a whole disclosed that sexual dimorphism in multiple molecular mechanisms implicated in EAE development may be responsible for greater severity of EAE in male rats and sexually dimorphic action of substances affecting them. Propranolol moderated EAE severity more effectively in male rats, exhibiting greater spinal cord noradrenaline (NA) levels and myeloid cell ß2-adrenoceptor (ß2-AR) expression than females. Propranolol affected CX3CR1/Nrf2/HO-1 and Stat3/Socs3 signaling axes in myeloid cells, favored their anti-inflammatory/neuroprotective phenotype and, consequently, reduced Th cell reactivation and differentiation into highly pathogenic IL-17/IFN-γ/GM-CSF-producing cells.

Activation of ß-Adrenoceptors Promotes Lipid Droplet Accumulation in MCF-7 Breast Cancer Cells via cAMP/PKA/EPAC Pathways.

Physiologically, ß-adrenoceptors are major regulators of lipid metabolism, which may be reflected in alterations in lipid droplet dynamics. ß-adrenoceptors have also been shown to participate in breast cancer carcinogenesis. Since lipid droplets may be seen as a hallmark of cancer, the present study aimed to investigate the role of ß-adrenoceptors in the regulation of lipid droplet dynamics in MCF-7 breast cancer cells. Cells were treated for up to 72 h with adrenaline (an endogenous adrenoceptor agonist), isoprenaline (a non-selective ß-adrenoceptor agonist) and salbutamol (a selective ß2-selective agonist), and their effects on lipid droplets were evaluated using Nile Red staining. Adrenaline or isoprenaline, but not salbutamol, caused a lipid-accumulating phenotype in the MCF-7 cells. These effects were significantly reduced by selective ß1- and ß3-antagonists (10 nM atenolol and 100 nM L-748,337, respectively), indicating a dependence on both ß1- and ß3-adrenoceptors. These effects were dependent on the cAMP signalling pathway, involving both protein kinase A (PKA) and cAMP-dependent guanine-nucleotide-exchange (EPAC) proteins: treatment with cAMP-elevating agents (forskolin or 8-Br-cAMP) induced lipid droplet accumulation, whereas either 1 µM H-89 or 1 µM ESI-09 (PKA or EPAC inhibitors, respectively) abrogated this effect. Taken together, the present results demonstrate the existence of a ß-adrenoceptor-mediated regulation of lipid droplet dynamics in breast cancer cells, likely involving ß1- and ß3-adrenoceptors, revealing a new mechanism by which adrenergic stimulation may influence cancer cell metabolism.

Chronic isoprenaline/phenylephrine vs. exclusive isoprenaline stimulation in mice: critical contribution of alpha1-adrenoceptors to early cardiac stress responses.

Hyperactivity of the sympathetic nervous system is a major driver of cardiac remodeling, exerting its effects through both α-, and ß-adrenoceptors (α-, ß-ARs). As the relative contribution of subtype α1-AR to cardiac stress responses remains poorly investigated, we subjected mice to either subcutaneous perfusion with the ß-AR agonist isoprenaline (ISO, 30 mg/kg × day) or to a combination of ISO and the stable α1-AR agonist phenylephrine (ISO/PE, 30 mg/kg × day each). Telemetry analysis revealed similar hemodynamic responses under both ISO and ISO/PE treatment i.e., permanently increased heart rates and only transient decreases in mean blood pressure during the first 24 h. Echocardiography and single cell analysis after 1 week of exposure showed that ISO/PE-, but not ISO-treated animals established α1-AR-mediated inotropic responsiveness to acute adrenergic stimulation. Morphologically, additional PE perfusion limited concentric cardiomyocyte growth and enhanced cardiac collagen deposition during 7 days of treatment. Time-course analysis demonstrated a diverging development in transcriptional patterns at day 4 of treatment i.e., increased expression of selected marker genes Xirp2, Nppa, Tgfb1, Col1a1, Postn under chronic ISO/PE treatment which was either less pronounced or absent in the ISO group. Transcriptome analyses at day 4 via RNA sequencing demonstrated that additional PE treatment caused a marked upregulation of genes allocated to extracellular matrix and fiber organization along with a more pronounced downregulation of genes involved in metabolic processes, muscle adaptation and cardiac electrophysiology. Consistently, transcriptome changes under ISO/PE challenge more effectively recapitulated early transcriptional alterations in pressure overload-induced experimental heart failure and in human hypertrophic cardiomyopathy.

Comparative characterization of ß-adrenoceptors in the bladder, heart, and lungs of rats: Alterations in spontaneously hypertensive rats.

The present study aimed to characterize and compare ß-adrenoceptors in the rat bladder with those in the heart and lungs of SD rats (8-10 weeks old) using subtype-selective agonists and antagonists in a radioligand binding assay with (-)-[125I]cyanopindolol ([125I]CYP), and also to clarify alterations in ß-adrenoceptors in the bladder of spontaneously hypertensive rats (SHR) at 14 weeks old, from those of Wistar-Kyoto rats (WKY) and Wistar rats at the same age. A radioligand binding assay with [125I]CYP was used to measure ß-adrenoceptor binding activity in rat tissues. Metoprolol exhibited the highest affinity to specific binding sites of [125I]CYP in the rat heart, indicating the dominance of ß1-adrenoceptors. ß3-selective agonists (BRL37344 and CL316243) and antagonist (SR59230A) exhibited higher affinity to specific binding sites of [125I]CYP in the bladder than in the heart and lungs. Furthermore, the binding affinity of the ß2-selective antagonist, ICI118551 was the highest in the bladder. The Bmax of specific [125]CYP binding in the bladder was significantly lower in WKY and SHR than in Wistar rats. The present study provides further evidence for the coexistence of ß2-and ß3-adrenoceptors in the rat bladder, and indicates that ß-adrenoceptor density is lower in the bladders of WKY and SHR.

ß-Adrenoceptor blockade prevents carotid body hyperactivity and elevated vascular sympathetic nerve density induced by chronic intermittent hypoxia.

Carotid body (CB) hyperactivity promotes hypertension in response to chronic intermittent hypoxia (CIH). The plasma concentration of adrenaline is reported to be elevated in CIH and our previous work suggests that adrenaline directly activates the CB. However, a role for chronic adrenergic stimulation in mediating CB hyperactivity is currently unknown. This study evaluated whether beta-blocker treatment with propranolol (Prop) prevented the development of CB hyperactivity, vascular sympathetic nerve growth and hypertension caused by CIH. Adult male Wistar rats were assigned into 1 of 4 groups: Control (N), N + Prop, CIH and CIH + Prop. The CIH paradigm consisted of 8 cycles h-1, 8 h day-1, for 3 weeks. Propranolol was administered via drinking water to achieve a dose of 40 mg kg-1 day-1. Immunohistochemistry revealed the presence of both ß1 and ß2-adrenoceptor subtypes on the CB type I cell. CIH caused a 2-3-fold elevation in basal CB single-fibre chemoafferent activity and this was prevented by chronic propranolol treatment. Chemoafferent responses to hypoxia and mitochondrial inhibitors were attenuated by propranolol, an effect that was greater in CIH animals. Propranolol decreased respiratory frequency in normoxia and hypoxia in N and CIH. Propranolol also abolished the CIH mediated increase in vascular sympathetic nerve density. Arterial blood pressure was reduced in propranolol groups during hypoxia. Propranolol exaggerated the fall in blood pressure in most (6/7) CIH animals during hypoxia, suggestive of reduced sympathetic tone. These findings therefore identify new roles for ß-adrenergic stimulation in evoking CB hyperactivity, sympathetic vascular hyperinnervation and altered blood pressure control in response to CIH.

Effects of sitagliptin on ß-adrenoceptor mediated relaxation in streptozotocin-diabetic rat aorta

Background/aim: Dipeptidyl peptidase-4 (DPP4) inhibitors, a class of oral antidiabetic drugs, have been shown to be protective on the vascular system because of their antiinflammatory, antiatherosclerotic, and vasodilatory effects. ß2-adrenoceptors (ß2-ARs) mediate the vasorelaxation in the aorta. However, ß3-adrenoceptor-mediated relaxation has not been studied in diabetic aorta yet. Thus, we aimed to study the effect of sitagliptin treatment on ß2- and ß3-adrenoceptor-mediated relaxations in the diabetic rat aorta. Materials and methods: Eight-week old Sprague Dawley rats were divided into three groups: control, diabetic, sitagliptin treated diabetic. Diabetes was induced by injection of streptozotocin (35 or 40 mg/kg, intraperitoneally). After 10 weeks of diabetes, some of the diabetic rats were treated with sitagliptin (orally, 10mg/kg/day). ß2- and ß3-AR-mediated relaxation responses were evaluated by using isoprenaline and CL 316,243, respectively. ß3-AR-mediated relaxation experiments were repeated in presence of L-NAME. Western blotting and immunohistochemistry were performed to determine the abundance of ß3-adrenoceptor and endothelial nitric oxide synthase (eNOS). Results: The isoprenaline-mediated relaxation response was impaired in the diabetic group and sitagliptin treatment did not improve it. There was no significant change in CL316,243 mediated-relaxation or protein expression of ß3-ARs among the groups. However, the ratio of phosphorylated eNOS/NOS protein was increased markedly in the sitagliptin treated group, which points the stimulating effect of this drug towards the eNOS pathway. Conclusion: Our results indicate that sitagliptin treatment does not alter ß-AR-mediated relaxation in streptozotocin-diabetic rat aorta; however, it significantly stimulates the eNOS pathway. Future studies are needed to clarify the relationship between the eNOS pathway and DPP-4 inhibition.

ß-Adrenoceptor Activation in Breast MCF-10A Cells Induces a Pattern of Catecholamine Production Similar to that of Tumorigenic MCF-7 Cells.

Adrenaline, which participates in the neuroendocrine response that occurs during stress and perimenopause, may be tumorigenic. This exploratory study aimed at investigating whether non-tumorigenic and tumorigenic human breast epithelial cell lines are able to synthesize adrenaline. The study was carried out in non-tumorigenic (MCF-10A) and tumorigenic (MCF-7) human breast cell lines. Expression of enzymes involved in adrenaline synthesis was characterized by RT-qPCR, immunocytochemistry and western blot. Catecholamines and analogue compounds were quantified by HPLC-ECD. Functional assessment of the impact of drugs on cells' tumorigenic potential was assessed by determination of cell viability and clonogenic ability. Both MCF-10A and MCF-7 cells produce catecholamines, but the capacity to produce adrenaline is lower in MCF-10A cells. ß-adrenoceptor activation increases the capacity of MCF-10A cells to produce adrenaline and favor both cell viability and colony formation. It is concluded that exposure of human breast epithelial cells to ß-adrenoceptor agonists increases cell proliferation and the capacity to produce adrenaline, creating an autocrine potential to spread these adrenergic effects in a feed-forward loop. It is conceivable that these effects are related to tumorigenesis, bringing a new perspective to understand the claimed anticancer effects of propranolol and the increase in breast cancer incidence caused by stress or during perimenopause.

The N-termini of GRK2 and GRK3 simulate the stimulating effects of RKIP on ß-adrenoceptors.

The Raf kinase inhibitor protein (RKIP) activates ß-adrenoceptors (ß-AR) and thereby induces a well-tolerated cardiac contractility and prevents heart failure in mice. Different to RKIP-mediated ß-AR activation, chronic activation of ß-AR by catecholamines was shown to be detrimental for the heart. RKIP is an endogenous inhibitor of G protein coupled receptor kinase 2 (GRK2); it binds GRK2 and thereby inhibits GRK2 mediated ß-AR phosphorylation and desensitization. Here, we evaluate RKIP-mediated effects on ß-AR to explore new strategies for ß-AR modulation. Co-immunoprecipitation assays and pull-down assays revealed subtype specificity of RKIP for the cardiac GRK isoforms GRK2 and GRK3 - not GRK5 - as well as several RKIP binding sites within their N-termini (GRK21-185 and GRK31-185). Overexpression of these N-termini prevented ß2-AR phosphorylation and internalization, subsequently increased receptor signaling in HEK293 cells and cardiomyocyte contractility. Co-immunoprecipitation assays of ß2-AR with these N-terminal GRK fragments revealed a direct interaction suggesting a steric interference of the fragments with the functional GRK-receptor interaction. Altogether, N-termini of GRK2 and GRK3 efficiently simulate RKIP effects on ß-AR signaling in HEK293 cells and in cardiomyocytes by their binding to ß2-AR and, thus, provide important insights for the development of new strategies to modulate ß2-AR signaling.

ß2 -Adrenoceptors indirectly support impaired ß1 -adrenoceptor responsiveness in the isolated type 2 diabetic rat heart.

NEW FINDINGS: What is the central question of this study? Are there specific contributions of ß1 - and ß2 -adrenoceptor subtypes to the impaired ß-adrenoceptor responsiveness of the type 2 diabetic heart? What is the main finding and its importance? In hearts isolated from the Zucker diabetic fatty rat model of type 2 diabetes, we showed that the ß1 -adrenoceptors are the main subtype to regulate heart rate, contraction and relaxation. Notably, the ß2 -adrenoceptor subtype actions seem to support function in the diabetic heart indirectly. ABSTRACT: Impaired ß-adrenoceptor (ß-AR) responsiveness causes cardiac vulnerability in patients with type 2 diabetes, but the independent contributions of ß1 - and ß2 -AR subtypes to ß-AR-associated cardiac dysfunction in diabetes are unknown. Our aim was to determine the specific ß1 - and ß2 -AR responsiveness of heart rate (HR), contraction and relaxation in the diabetic heart. Isolated Langendorff-perfused hearts of Zucker type 2 diabetic fatty (ZDF) rats were stimulated with the ß-AR agonist isoprenaline (1 × 10-11 to 3 × 10-8  mol l-1 ) with or without the selective ß1 -AR antagonist CGP20712A (3 × 10-8  mol l-1 ) or the ß2 -AR antagonist ICI-118,551 (5 × 10-8  mol l-1 ), and HR, contraction and relaxation were measured. Diabetic hearts showed lower basal HR (non-diabetic 216 ± 17 beats min-1 versus diabetic 151 ± 23 beats min-1 , P < 0.05). However, the ß-AR-induced increase in HR was similar and was completely blocked by the ß1 -AR antagonist, but not by the ß2 -AR antagonist. The ß-AR-induced increase in contraction and acceleration of relaxation was impaired in diabetic hearts, completely blocked by the ß1 -AR antagonist and partly impaired by the ß2 -AR antagonist. Western blots revealed 41% higher phosphorylation levels of AMP kinase (AMPK), a key regulator of cardiac energy metabolism, in diabetic hearts (non-diabetic 1.62 ± 0.19 a.u. versus diabetic 2.30 ± 0.25 a.u., P < 0.05). In conclusion, the ß1 -AR is the main subtype regulating chronotropic, inotropic and lusitropic ß-AR responses in the healthy heart and the type 2 diabetic heart. The ß2 -AR subtype indirectly supports the ß1 -AR functional response in the diabetic heart. This suggests that ß2 -ARs could be an indirect target to improve the function of the heart in type 2 diabetes.

Autonomic dysregulation at multiple sites is implicated in age-associated underactive bladder in female mice.

AIMS: To evaluate the functional and molecular alterations of contractile and relaxant machinery in the bladder and urethra that lead to the underactive bladder (UAB) in old female mice. METHODS: Female young (3-months) and old (18-months) C57BL/6 mice were used. Urodynamic was assessed in awake and anaesthetized mice. Electrical-field stimulation (EFS) and concentration-response curves to contractile and relaxing agents in isolated bladders and urethras were performed. Messenger RNA (mRNA) expressions of muscarinic, adrenergic, and transient receptor potential vanilloid-4 (TRPV4), and of the enzymes tyrosine hydroxylase and neuronal nitric oxide synthase (nNOS) were determined. Bladder cyclic adenosine monophosphate (cAMP) levels were measured. RESULTS: Cystometry in old mice showed incapacity to produce bladder emptying. On filter paper, old mice showed reduced urinary spots. Compared to the young group, bladder contractions induced by EFS and carbachol were lower in old mice. The ß3 -adrenoceptor agonist mirabegron promoted higher bladder relaxation and elevation of cAMP levels in old mice. In old mice urethras, the α1a -adrenoceptor agonist phenylephrine produced higher contractions, but no differences were found for the NO donor sodium nitroprusside-induced relaxations. In old mice, increased mRNA expressions of ß3 - and α1a -adrenoceptors in bladder and urethra were found, respectively, whereas the muscarinic M2 and M3 receptors and ß2 -adrenoceptors did not change between groups. Reduced mRNA expressions of tyrosine hydroxylase and nNOS were found in old mouse urethras. Additionally, TRPV4 expression was reduced in bladder urothelium from old mice. CONCLUSION: Age-associated mouse UAB is the result of autonomic dysfunction at multiple levels leading to the less sensitive and overrelaxed bladder, along with urethral hypercontractility.

ß-1 adrenoceptors and AT1 receptors may not be involved in catecholamine-induced lethal arrhythmias 1.

An excessive amount of catecholamines produce arrhythmias, but the exact mechanisms of this action are not fully understood. For this purpose, Sprague-Dawley rats were treated with or without atenolol, a ß1-adrenoceptor blocker (20 mg/kg per day), for 15 days followed by injections of epinephrine for cumulative doses of 4 to 128 µg/kg. Another group of animals were pretreated with losartan, an angiotensin receptor (AT1) blocker (20 mg/kg per day), for comparison. Control animals received saline. Varying degrees of ventricular arrhythmias were seen upon increasing the dose of epinephrine, but the incidence and duration of the rhythm abnormalities as well as the number of episodes and severity of arrhythmias were not affected by treating the animals with atenolol or losartan. The levels of both epinephrine and norepinephrine were increased in the atenolol-treated rats but were unchanged in the losartan-treated animals after the last injection of epinephrine; the severity of arrhythmias did not correlate with the circulating catecholamine levels. These results indicate that both ß1-adrenoceptors and AT1 receptors may not be involved in the pathogenesis of catecholamine-induced arrhythmias and support the view that other mechanisms, such as the oxidation products of catecholamines, may play a crucial role in the occurrence of lethal arrhythmias.

A New Class of ß-Pyrrolidino-1,2,3-Triazole Derivatives as ß-Adrenergic Receptor Inhibitors: Synthesis, Pharmacological, and Docking Studies.

New 1,4-disubstituted ß-pyrrolidino-1,2,3-triazoles were synthesized using a reusable copper-iodide-doped neutral alumina catalyst. Synthesis of diversely substituted triazoles and recyclability of CuI catalyst explains the broad scope of this protocol. The synthesized compounds were screened for their antimicrobial and anticancer properties. Most of the compounds showed significant antimicrobial activities against all the tested microorganisms compared to standard drugs. Furthermore, compounds 5a, 5e, 5g, 5h, 5i, and 5j showed moderate to potent activities against A549 and HepG-2 cells. In addition, compounds 5g and 5h displayed potential cytotoxicity activity against A549 cells with IC50 values of 72 ± 3.21 and 58 ± 2.31 µM, respectively. The molecular docking study revealed that some of the synthesized compounds exhibited comparable binding as co-crystalized ligands with the DNA topoisomerase IV and anaplastic lymphoma kinase receptors.

Melatonin modulate the expression of α1- and ß2-adrenoceptors in the hippocampus of rats subjected to unpredictable chronic mild stress.

OBJECTIVE: This study investigated the effects of chronic melatonin treatment on gene expression of α1-, α2-, ß1- and ß2-adrenoceptors in the hippocampus of rats subjected to chronic unpredictable mild stress (CUMS). BACKGROUND: Preclinical studies have also shown that melatonin prevented short- and long-term memory impairments and exhibited antidepressant-like actions. METHODS: For this study, we used 24 animals, which were divided into four groups, and the experiment lasted 4 weeks. We quantified the changes in mRNA and protein levels of α1-, α2-, ß1- and ß2-adrenoceptors in the hippocampus after melatonin treatment. RESULTS: Our results demonstrated a decreased gene expression of α1-, α2- and ß2-adrenoceptors in the hippocampus of rats subjected to unpredictable chronic mild stress, while there was no change in gene expression of ß1-adrenoceptors. Melatonin treatment in the CUMS rats prevented the stress-induced decrease in mRNA and protein levels of α1-and ß2-adrenoceptors, whereas did not affect either on mRNA or protein level of ß1-and α2-adrenoceptors. CONCLUSION: Our data suggest that melatonin, by increasing reduced levels of α1- and ß2-adrenoceptors mRNA and protein in the hippocampus of chronic stressed rats, may be beneficial in conditions such as chronic stress and provides an experimental opportunity to probe into further molecular mechanisms underlying the regulation of these receptor subtype (Fig. 2, Ref. 28).

Cardiac sympathetic innervation, from a different point of (re)view.

The audience of basic and clinical scientists is familiar with the notion that the sympathetic nervous system controls heart function during stresses. However, evidence indicates that the neurogenic control of the heart spans from the maintenance of housekeeping functions in resting conditions to the recruitment of maximal performance, in the fight-or-flight responses, across a whole range of intermediate states. To perform such sophisticated functions, sympathetic ganglia integrate both peripheral and central inputs, and transmit information to the heart via 'motor' neurons, directly interacting with target cardiomyocytes. To date, the dynamics and mode of communication between these two cell types, which determine how neuronal information is adequately translated into the wide spectrum of cardiac responses, are still blurry. By combining the anatomical and structural information brought to light by recent imaging technologies and the functional evidence in cellular systems, we focus on the interface between neurons and cardiomyocytes, and advocate the existence of a specific 'neuro-cardiac junction', where sympathetic neurotransmission occurs in a 'quasi-synaptic' way. The properties of such junctional-type communication fit well with those of the physiological responses elicited by the cardiac sympathetic nervous system, and explain its ability to tune heart function with precision, specificity and elevated temporal resolution.

ß1 -Adrenoceptor, but not ß2 -adrenoceptor, subtype regulates heart rate in type 2 diabetic rats in vivo.

NEW FINDINGS: What is the central question of the study? The sympathetic system regulates heart rate via ß-adrenoceptors; this is impaired during diabetes. However, the specific ß-adrenoceptor subtype contributions in heart rate regulation in diabetes in vivo are unknown. What is the main finding and its importance? Telemetric recordings in conscious non-diabetic and type 2 diabetic rats demonstrated that the ß1 -adrenoceptor subtype, and not the ß2 -adrenoceptor, regulated the lower resting heart rate and increased ß-adrenoceptor responsiveness in diabetes in vivo. This provides new physiological insight into the dysregulation of heart rate in type 2 diabetes, which is important for improving therapeutic strategies targeting the diabetic chronotropic incompetence. ß-Adrenoceptor blockers are widely used to reduce heart rate, the strongest predictor of mortality in cardiac patients, but are less effective in diabetic patients. This study aimed to determine the specific contributions of ß1 - and ß2 -adrenoceptor subtypes to chronotropic responses in type 2 diabetes in vivo, which are currently unknown. Type 2 diabetic and non-diabetic rats were implanted with radiotelemeters to measure arterial blood pressure and derive heart rate in conscious conditions. Vascular access ports were implanted to inject isoprenaline (ß1 - and ß2 -adrenoceptor agonist, 0.1-300 µg kg-1 ) in the presence of atenolol (ß1 -adrenoceptor antagonist, 2000 µg kg-1 ) or nadolol (ß1 - and ß2 -adrenoceptor agonist, 4000 µg kg-1 ) to determine the chronotropic contributions of the ß-adrenoceptor subtypes. Resting heart rate was reduced in diabetic rats (388 ± 62 versus 290 ± 37 beats min-1 non-diabetic versus diabetic, P < 0.05, mean ± SD), which remained after atenolol or nadolol administration. Overall ß-adrenoceptor chronotropic responsiveness was increased in diabetic rats (change in heart rate at highest dose of isoprenaline: 135 ± 66 versus 205 ± 28 beats min-1 , non-diabetic versus diabetic, P < 0.05), a difference that diminished after ß1 -adrenoceptor blockade with atenolol (change in heart rate at highest dose of isoprenaline: 205 ± 37 versus 195 ± 22 beats min-1 , non-diabetic versus diabetic, P < 0.05). In conclusion, the ß1 -adrenoceptor is the main subtype to modulate chronotropic ß-adrenoceptor responses in healthy and diabetic rats. This study provides new insights into the pathological basis of dysregulation of heart rate in type 2 diabetes, which could be important for improving the current therapeutic strategies targeting diabetic chronotropic incompetence.

Implication of Rho-kinase and soluble guanylyl cyclase enzymes in prostate smooth muscle dysfunction in middle-aged rats.

AIMS: Aging is highly associated with benign prostate hyperplasia (BPH). We investigated here the alterations of the contractile and relaxant machinery in prostates of middle-aged rats, focusing on the Rho-kinase, nitric oxide (NO)-soluble guanylyl cyclase (sGC), α1- and ß-adrenoceptor pathways. METHODS: Male Wistar young (3.5-month old) and middle-aged rats (10-month old) were used. Quantitative image analysis of prostates and functional assays evaluating the prostate contractions and relaxations were employed. Measurement of [3 H]-noradrenaline efflux, western blotting for α1 and ß1 sGC subunits, and cyclic nucleotide levels were carried out. RESULTS: Prostates of middle-aged rats showed significant increases in lumen and smooth muscle cells, but no alterations in the relative prostate weight were observed. In vivo, noradrenaline (10-7 -10-4 g/kg) produced greater prostatic contractions in middle-aged compared with control rats. Likewise, the in vitro contractions to phenylephrine (1 nM-100 µM) and α,ß-methylene ATP (1-10 µM) were greater in middle-aged rats. Electrical-field stimulation (EFS, 1-32 Hz) promoted higher [3 H]-noradrenaline efflux and prostate contractions in middle-aged rats. Reduced expressions of α1 and ß1 sGC subunits and diminished NO-mediated prostate relaxations in middle-age were observed. Isoproterenol-induced relaxations and cAMP levels were reduced in prostates of middle-aged rats. The Rho-kinase inhibitor fasudil (50 mg/kg, 2 weeks) normalized the prostate hypercontractility in middle-age rats. CONCLUSIONS: Prostate hypercontractility in middle-aging is associated with increased release of noradrenaline and Rho-kinase pathway, as well as with impairments of NO-sGC and ß-adrenoceptor pathways. Middle-aged rats are suitable to explore the enhanced prostatic tone in the absence of prostate overgrowth. Neurourol. Urodynam. 36:589-596, 2017. © 2016 Wiley Periodicals, Inc.

Classical and novel pharmacological insights offered by the simple chick cardiomyocyte cell culture model: a valuable teaching aid and a primer for "real" research.

The chick embryo cardiomyocyte model of cell culture is a staple technique in many physiology and pharmacology laboratories. Despite the relative simplicity, robustness, and reproducibility inherent in this model, it can be used in a variety of ways to yield important new insights that help facilitate student understanding of underlying physiological and pharmacological concepts as well as, more generally, the scientific method. Using this model, this paper will show real data obtained by undergraduate students in the authors' laboratories. It will first demonstrate classical pharmacological concepts such as full and partial agonism, inverse agonism, and competitive reversible antagonism and then move on to more complex pharmacology involving the characterization of novel receptors in these cells.

Agonist-induced ß2-adrenoceptor desensitization and downregulation enhance pro-inflammatory cytokine release in human bronchial epithelial cells.

It is not clear whether increased asthma severity associated with long-term use of ß2-adrenoceptor (ß2-AR) agonists can be attributed to receptor degradation and increased inflammation. We investigated the cross-talk between ß-AR agonist-mediated effects on ß2-AR function and expression and cytokine release in human bronchial epithelial cells. In 16HBE14o(-) cells grown in the presence and absence of ß-AR agonists and/or antagonists, the ß2-AR density was assessed by radioligand binding; the receptor protein and mRNA was determined using laser scanning cytometer and RT-PCR; cAMP generation, the cytokines IL-6 and IL-8 release were determined using AlphaScreen Assay and ELISA, respectively. Isoprenaline (ISO) and salbutamol (Salbu) induced a concentration- and time-dependent significant decrease in ß2-AR density. Both Salbu and ISO reduced cAMP generation in a concentration-dependent manner while in same cell culture the IL-6 and IL-8 release was significantly enhanced. These effects were antagonized to a greater extent by ICI 118.551 than by propranolol, but CGP 20712A had no effect. Reduction of the ß2-AR protein and mRNA could be seen when cells were treated with ISO for 24 h. Our findings indicate a direct link between cytokine release and altered ß2-AR expression and function in airway epithelial cells. ß2-AR desensitization and downregulation induced by long-term treatment with ß2-AR agonists during asthma may account for adverse reactions also due to enhanced release of pro-inflammatory mediators and should, thus, be considered in asthma therapy.