ß-Adrenergic Stimulation-Induced PVAT Dysfunction in Male Sex: A Role for 11ß-Hydroxysteroid Dehydrogenase-1.

Long-term ß-adrenoceptor (ß-AR) stimulation is a pathological mechanism associated with cardiovascular diseases resulting in endothelial and perivascular adipose tissue (PVAT) dysfunction. In this study, we aimed to identify whether ß-adrenergic signaling has a direct effect on PVAT. Thoracic aorta PVAT was obtained from male Wistar rats and cultured ex vivo with the ß-AR agonist isoproterenol (Iso; 1 µM) or vehicle for 24 hours. Conditioned culture medium (CCM) from Iso-treated PVAT induced a marked increase in aorta contractile response, induced oxidative stress, and reduced nitric oxide production in PVAT compared to vehicle. In addition, Iso-treated PVAT and PVAT-derived differentiated adipocytes exhibited higher corticosterone release and protein expression of 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1), an enzyme responsible for de novo synthesis of corticosterone. Macrophages exposed to Iso also exhibited increased corticosterone release in response to ß-AR stimulation. Incubation of Iso-treated PVAT and PVAT-derived differentiated adipocytes with ß3-AR antagonist restored aorta contractile function modulated by Iso-CCM and normalized 11ß-HSD1 protein expression. These results show that ß3-AR signaling leads to upregulation of 11ß-HSD1 in PVAT, thus increasing corticosterone release and contributing to impair the anticontractile function of this tissue.

Mechano-sensitivity of ß2-adrenoceptors enhances constitutive activation of cAMP generation that is inhibited by inverse agonists.

The concept of agonist-independent signalling that can be attenuated by inverse agonists is a fundamental element of the cubic ternary complex model of G protein-coupled receptor (GPCR) activation. This model shows how a GPCR can exist in two conformational states in the absence of ligands; an inactive R state and an active R* state that differ in their affinities for agonists, inverse agonists, and G-protein alpha subunits. The proportion of R* receptors that exist in the absence of agonists determines the level of constitutive receptor activity. In this study we demonstrate that mechanical stimulation can induce ß2-adrenoceptor agonist-independent Gs-mediated cAMP signalling that is sensitive to inhibition by inverse agonists such as ICI-118551 and propranolol. The size of the mechano-sensitive response is dependent on the cell surface receptor expression level in HEK293G cells, is still observed in a ligand-binding deficient D113A mutant ß2-adrenoceptor and can be attenuated by site-directed mutagenesis of the extracellular N-glycosylation sites on the N-terminus and second extracellular loop of the ß2-adrenoceptor. Similar mechano-sensitive agonist-independent responses are observed in HEK293G cells overexpressing the A2A-adenosine receptor. These data provide new insights into how agonist-independent constitutive receptor activity can be enhanced by mechanical stimulation and regulated by inverse agonists.

ß-Adrenergic signaling drives structural and functional maturation of mouse cardiomyocytes.

Cardiac maturation represents the last phase of heart development and is characterized by morphofunctional alterations that optimize the heart for efficient pumping. Its understanding provides important insights into cardiac regeneration therapies. Recent evidence implies that adrenergic signals are involved in the regulation of cardiac maturation, but the mechanistic underpinnings involved in this process are poorly understood. Herein, we explored the role of ß-adrenergic receptor (ß-AR) activation in determining structural and functional components of cardiomyocyte maturation. Temporal characterization of tyrosine hydroxylase and norepinephrine levels in the mouse heart revealed that sympathetic innervation develops during the first 3 wk of life, concurrent with the rise in ß-AR expression. To assess the impact of adrenergic inhibition on maturation, we treated mice with propranolol, isolated cardiomyocytes, and evaluated morphofunctional parameters. Propranolol treatment reduced heart weight, cardiomyocyte size, and cellular shortening, while it increased the pool of mononucleated myocytes, resulting in impaired maturation. No changes in t-tubules were observed in cells from propranolol mice. To establish a causal link between ß-AR signaling and cardiomyocyte maturation, mice were subjected to sympathectomy, followed or not by restoration with isoproterenol treatment. Cardiomyocytes from sympathectomyzed mice recapitulated the salient immaturity features of propranolol-treated mice, with the additional loss of t-tubules. Isoproterenol rescued the maturation deficits induced by sympathectomy, except for the t-tubule alterations. Our study identifies the ß-AR stimuli as a maturation promoting signal and implies that this pathway can be modulated to improve cardiac regeneration therapies.NEW & NOTEWORTHY Maturation involves a series of morphofunctional alterations vital to heart development. Its regulatory mechanisms are only now being unveiled. Evidence implies that adrenergic signaling regulates cardiac maturation, but the mechanisms are poorly understood. To address this point, we blocked ß-ARs or performed sympathectomy followed by rescue experiments with isoproterenol in neonatal mice. Our study identifies the ß-AR stimuli as a maturation signal for cardiomyocytes and highlights the importance of this pathway in cardiac regeneration therapies.

Targeting S100A9 Prevents ß-Adrenergic Activation-Induced Cardiac Injury.

Altered cardiac innate immunity is highly associated with the progression of cardiac disease states and heart failure. S100A8/A9 is an important component of damage-associated molecular patterns (DAMPs) that is critically involved in the pathogenesis of heart failure, thus considered a promising target for pharmacological intervention. In the current study, initially, we validated the role of S100A8/A9 in contributing to cardiac injury and heart failure via the overactivation of the ß-adrenergic pathway and tested the potential use of paquinimod as a pharmacological intervention of S100A8/A9 activation in preventing cardiac dysfunction, collagen deposition, inflammation, and immune cell infiltration in ß-adrenergic overactivation-mediated heart failure. This finding was further confirmed by the cardiomyocyte-specific silencing of S100A9 via the use of the adeno-associated virus (AAV) 9-mediated short hairpin RNA (shRNA) gene silencing system. Most importantly, in the assessment of the underlying cellular mechanism by which activated S100A8/A9 cause aggravated progression of cardiac fibrosis and heart failure, we discovered that the activated S100A8/A9 can promote fibroblast-macrophage interaction, independent of inflammation, which is likely a key mechanism leading to the enhanced collagen production. Our results revealed that targeting S100A9 provides dual beneficial effects, which is not only a strategy to counteract cardiac inflammation but also preclude cardiac fibroblast-macrophage interactions. The findings of this study also indicate that targeting S100A9 could be a promising strategy for addressing cardiac fibrosis, potentially leading to future drug development.

The renal vasodilatation from ß-adrenergic activation in vivo in rats is not driven by KV7 and BKCa channels.

The mechanisms behind renal vasodilatation elicited by stimulation of ß-adrenergic receptors are not clarified. As several classes of K channels are potentially activated, we tested the hypothesis that KV7 and BKCa channels contribute to the decreased renal vascular tone in vivo and in vitro. Changes in renal blood flow (RBF) during ß-adrenergic stimulation were measured in anaesthetized rats using an ultrasonic flow probe. The isometric tension of segmental arteries from normo- and hypertensive rats and segmental arteries from wild-type mice and mice lacking functional KV7.1 channels was examined in a wire-myograph. The ß-adrenergic agonist isoprenaline increased RBF significantly in vivo. Neither activation nor inhibition of KV7 and BKCa channels affected the ß-adrenergic RBF response. In segmental arteries from normo- and hypertensive rats, inhibition of KV7 channels significantly decreased the ß-adrenergic vasorelaxation. However, inhibiting BKCa channels was equally effective in reducing the ß-adrenergic vasorelaxation. The ß-adrenergic vasorelaxation was not different between segmental arteries from wild-type mice and mice lacking KV7.1 channels. As opposed to rats, inhibition of KV7 channels did not affect the murine ß-adrenergic vasorelaxation. Although inhibition and activation of KV7 channels or BKCa channels significantly changed baseline RBF in vivo, none of the treatments affected ß-adrenergic vasodilatation. In isolated segmental arteries, however, inhibition of KV7 and BKCa channels significantly reduced the ß-adrenergic vasorelaxation, indicating that the regulation of RBF in vivo is driven by several actors in order to maintain an adequate RBF. Our data illustrates the challenge in extrapolating results from in vitro to in vivo conditions.

Effects of protein concentration and beta-adrenergic agonists on ruminal bacterial communities in finishing beef heifers.

To improve animal performance and modify growth by increasing lean tissue accretion, beef cattle production has relied on use of growth promoting technologies such as beta-adrenergic agonists. These synthetic catecholamines, combined with the variable inclusion of rumen degradable (RDP) and undegradable protein (RUP), improve feed efficiency and rate of gain in finishing beef cattle. However, research regarding the impact of beta-adrenergic agonists, protein level, and source on the ruminal microbiome is limited. The objective of this study was to determine the effect of different protein concentrations and beta-adrenergic agonist (ractopamine hydrochloride; RAC) on ruminal bacterial communities in finishing beef heifers. Heifers (n = 140) were ranked according to body weight and assigned to pens in a generalized complete block design with a 3 × 2 factorial arrangement of treatments of 6 different treatment combinations, containing 3 protein treatments (Control: 13.9% CP, 8.9% RDP, and 5.0% RUP; High RDP: 20.9% CP, 14.4% RDP, 6.5% RUP; or High RUP: 20.9% CP, 9.7% RDP, 11.2% RUP) and 2 RAC treatments (0 and 400 mg/day). Rumen samples were collected via orogastric tubing 7 days before harvest. DNA from rumen samples were sequenced to identify bacteria based on the V1-V3 hypervariable regions of the 16S rRNA gene. Reads from treatments were analyzed using the packages 'phyloseq' and 'dada2' within the R environment. Beta diversity was analyzed based on Bray-Curtis distances and was significantly different among protein and RAC treatments (P < 0.05). Alpha diversity metrics, such as Chao1 and Shannon diversity indices, were not significantly different (P > 0.05). Bacterial differences among treatments after analyses using PROC MIXED in SAS 9 were identified for the main effects of protein concentration (P < 0.05), rather than their interaction. These results suggest possible effects on microbial communities with different concentrations of protein but limited impact with RAC. However, both may potentially act synergistically to improve performance in finishing beef cattle.

The mechanism of 25-hydroxycholesterol-mediated suppression of atrial ß1-adrenergic responses.

25-Hydroxycholesterol (25HC) is a biologically active oxysterol, whose production greatly increases during inflammation by macrophages and dendritic cells. The inflammatory reactions are frequently accompanied by changes in heart regulation, such as blunting of the cardiac ß-adrenergic receptor (AR) signaling. Here, the mechanism of 25HC-dependent modulation of responses to ß-AR activation was studied in the atria of mice. 25HC at the submicromolar levels decreased the ß-AR-mediated positive inotropic effect and enhancement of the Ca2+ transient amplitude, without changing NO production. Positive inotropic responses to ß1-AR (but not ß2-AR) activation were markedly attenuated by 25HC. The depressant action of 25HC on the ß1-AR-mediated responses was prevented by selective ß3-AR antagonists as well as inhibitors of Gi protein, Gßγ, G protein-coupled receptor kinase 2/3, or ß-arrestin. Simultaneously, blockers of protein kinase D and C as well as a phosphodiesterase inhibitor did not preclude the negative action of 25HC on the inotropic response to ß-AR activation. Thus, 25HC can suppress the ß1-AR-dependent effects via engaging ß3-AR, Gi protein, Gßγ, G protein-coupled receptor kinase, and ß-arrestin. This 25HC-dependent mechanism can contribute to the inflammatory-related alterations in the atrial ß-adrenergic signaling.

Control of lipolysis by a population of oxytocinergic sympathetic neurons.

Oxytocin (OXT), a nine-amino-acid peptide produced in the hypothalamus and released by the posterior pituitary, has well-known actions in parturition, lactation and social behaviour1, and has become an intriguing therapeutic target for conditions such as autism and schizophrenia2. Exogenous OXT has also been shown to have effects on body weight, lipid levels and glucose homeostasis1,3, suggesting that it may also have therapeutic potential for metabolic disease1,4. It is unclear, however, whether endogenous OXT participates in metabolic homeostasis. Here we show that OXT is a critical regulator of adipose tissue lipolysis in both mice and humans. In addition, OXT serves to facilitate the ability of ß-adrenergic agonists to fully promote lipolysis. Most surprisingly, the relevant source of OXT in these metabolic actions is a previously unidentified subpopulation of tyrosine hydroxylase-positive sympathetic neurons. Our data reveal that OXT from the peripheral nervous system is an endogenous regulator of adipose and systemic metabolism.

In silico identification of a biarylamine acting as agonist at human ß3 adrenoceptors and exerting BRL37344-like effects on mouse metabolism.

Human ß3-adrenoceptor (ß3AR) agonists were considered potential agents for the treatment of metabolic disorders. However, compounds tested as ß3AR ligands have shown marked differences in pharmacological profile in rodent and human species, although these compounds remain attractive as they were successfully repurposed for the therapy of urinary incontinence. In this work, some biarylamine compounds were designed and tested in silico as potential ß3AR agonists on 3-D models of mouse or human ß3ARs. Based on the theoretical results, we identified, synthesized and tested a biarylamine compound (polibegron). In CHO-K1 cells expressing the human ß3AR, polibegron and the ß3AR agonist BRL 37344 were partial agonists for stimulating cAMP accumulation (50 and 57% of the response to isoproterenol, respectively). The potency of polibegron was 1.71- and 4.5-fold higher than that of isoproterenol and BRL37344, respectively. These results indicate that polibegron acts as a potent, but partial, agonist at human ß3ARs. In C57BL/6N mice with obesity induced by a high-fat diet, similar effects of the equimolar intraperitoneal administration of polibegron and BRL37344 were observed on weight, visceral fat and plasma levels of glucose, cholesterol and triglycerides. Similarities and differences between species related to ligand-receptor interactions can be useful for drug designing.

The ß-Blocker Carvedilol and Related Aryloxypropanolamines Promote ERK1/2 Phosphorylation in HEK293 Cells with K A Values Distinct From Their Equilibrium Dissociation Constants as ß 2-Adrenoceptor Antagonists: Evidence for Functional Affinity.

The determination of affinity by using functional assays is important in drug discovery because it provides a more relevant estimate of the strength of interaction of a ligand to its cognate receptor than radioligand binding. However, empirical evidence for so-called, "functional affinity" is limited. Herein, we determined whether the affinity of carvedilol, a ß-adrenoceptor antagonist used to treat heart failure that also promotes extracellular signal-regulated kinases 1 and 2 (ERK1/2) phosphorylation, differed between these two pharmacological activities. Four structurally related ß-adrenoceptor antagonists (alprenolol, carazolol, pindolol, propranolol) that also activated ERK1/2 were included as comparators to enhance our understanding of how these drugs work in the clinical setting. In HEK293 cells stably expressing the human ß 2-adrenoceptor carvedilol and related aryloxypropanolamines were partial agonists of ERK1/2 phosphorylation with potencies ([A]50s) that were lower than their equilibrium dissociation constants (K Bs) as ß 2-adrenoceptor antagonists. As the [A]50 of a partial agonist is a good approximation of its K B, then these data indicated that the affinities of carvedilol and related ligands for these two activities were distinct. Moreover, there was a significant negative rank order correlation between the [A]50 of each ligand to activate ERK1/2 and their intrinsic activities (i.e., as intrinsic activity for ERK1/2 phosphorylation increased, so did affinity). Genome editing revealed that the transducer that coupled the ß 2-adrenoceptor to ERK1/2 phosphorylation in response to carvedilol and other ß 2-adrenoceptor antagonists was Gαs. Collectively, these data support the concept of "functional affinity" and indicate that the ability of the ß 2-adrenoceptor to recruit Gαs may influence the affinity of the activating ligand. SIGNIFICANCE STATEMENT: In HEK293 cells overexpressing the human ß2-adrenoceptor carvedilol and four related aryloxypropanolamines behaved as ß2-adrenoceptor antagonists and partial agonists of ERK1/2 phosphorylation with rank orders of affinity that were distinct. These data imply that carvedilol and other ß-blockers can stabilize the ß2-adrenoceptor in different affinity conformations that are revealed when functionally distinct responses are measured. This is the basis for the pharmacological concept of "functional affinity."

Treatment with ß-Adrenoceptor Agonist Isoproterenol Reduces Non-parenchymal Cell Responses in LPS/D-GalN-Induced Liver Injury.

There is an increasing evidence indicating the involvement of the sympathetic nervous system (SNS) in liver disease development. To achieve an extensive comprehension of the obscure process by which the SNS alleviates inflammatory damage in non-parenchymal liver cells (NPCs) during acute liver failure (ALF), we employ isoproterenol (ISO), a beta-adrenoceptor agonist, to mimic SNS signaling. ISO was administered to C57BL/6J mice to establish an acute liver failure (ALF) model using LPS/D-GalN, which was defined as ISO + ALF. Non-parenchymal cells (NPCs) were isolated from liver tissues and digested for tandem mass tag (TMT) labeled proteomics to identify differentially expressed proteins (DEPs). The administration of ISO resulted in a decreased serum levels of pro-inflammatory cytokines, e.g., TNF-α, IL-1ß, and IL-6 in ALF mice, which alleviated liver damage. By using TMT analysis, it was possible to identify 1587 differentially expressed proteins (DEPs) in isolated NPCs. Notably, over 60% of the DEPs in the ISO + ALF vs. ALF comparison were shared in the Con vs. ALF comparison. According to enrichment analysis, the DEPs influenced by ISO in ALF mice were linked to biological functions of heme and fatty acid metabolism, interferon gamma response, TNFA signaling pathway, and mitochondrial oxidation function. Protein-protein interaction network analysis indicated Mapk14 and Caspase3 may serve as potentially valuable indicators of ISO intervention. In addition, the markers on activated macrophages, such as Mapk14, Casp1, Casp8, and Mrc1, were identified downregulated after ISO initiation. ISO treatment increased the abundance of anti-inflammatory markers in mouse macrophages, as evidenced by the immunohistochemistry (IHC) slides showing an increase in Arg + staining and a reduction in iNOS + staining. Furthermore, pretreatment with ISO also resulted in a reduction of LPS-stimulated inflammation signaling markers, Mapk14 and NF-κB, in human THP-1 cells. Prior treatment with ISO may have the potential to modify the biological functions of NPCs and could serve as an innovative pharmacotherapy for delaying the pathogenesis and progression of ALF.

Development of novel ß2-adrenergic receptor agonists for the stimulation of glucose uptake - The importance of chirality and ring size of cyclic amines.

ß2-Adrenergic receptor (ß2AR) agonists have been reported to stimulate glucose uptake (GU) by skeletal muscle cells and are therefore highly interesting as a possible treatment for type 2 diabetes (T2D). The chirality of compounds often has a great impact on the activity of ß2AR agonists, although this has thus far not been investigated for GU. Here we report the GU for a selection of synthesized acyclic and cyclic ß-hydroxy-3-fluorophenethylamines. For the N-butyl and the N-(2-pentyl) compounds, the (R) and (R,R) (3d and 7e) stereoisomers induced the highest GU. When the compounds contained a saturated nitrogen containing 4- to 7-membered heterocycle, the (R,R,R) enantiomer of the azetidine (8a) and the pyrrolidine (9a) had the highest activity. Altogether, these results provide pivotal information for designing novel ß2AR agonist for the treatment of T2D.

Impaired vascular ß-adrenergic relaxation in spontaneously hypertensive rats: The differences between conduit and resistance arteries.

It was suggested that impaired ß-adrenergic relaxation in spontaneously hypertensive rats (SHR) might contribute to their high blood pressure (BP). Our study was focused on isoprenaline-induced dilatation of conduit femoral or resistance mesenteric arteries and on isoprenaline-induced BP reduction in SHR and Wistar-Kyoto rats (WKY). We confirmed decreased ß-adrenergic relaxation of SHR femoral arteries due to the absence of its endothelium-independent component, whereas endothelium-dependent component of ß-adrenergic smooth muscle relaxation was similar in both strains. Conversely, isoprenaline-induced relaxation of resistance mesenteric arteries was similar in both strains and this was true for endothelium-dependent and endothelium-independent components. We observed moderately reduced sensitivity of SHR mesenteric arteries to salmeterol (ß2-adrenergic agonist) and this strain difference disappeared after endothelium removal. However, there was no difference in mesenteric arteries relaxation by dobutamine (ß1-adrenergic agonist) which was independent of endothelium. The increasing isoprenaline doses elicited similar BP decrease in both rat strains, although BP sensitivity to isoprenaline was slightly decreased in SHR. The blockade of cyclooxygenase (indomethacin) and NO synthase (L-NAME) further reduced BP sensitivity to isoprenaline in SHR. On the other hand, salmeterol elicited similar BP decrease in both strains and the blockade of cyclooxygenase and NO synthase increased BP sensitivity to salmeterol in SHR as compared to WKY. In conclusion, attenuated ß-adrenergic vasodilatation of conduit arteries of SHR but similar ß-adrenergic relaxation of resistance mesenteric arteries from WKY and SHR and their similar BP response to ß-adrenergic agonists do not support major role of altered ß-adrenergic vasodilatation for high BP in genetic hypertension.

Dapagliflozin attenuates cardiac remodeling and dysfunction in rats with ß-adrenergic receptor overactivation through restoring calcium handling and suppressing cardiomyocyte apoptosis.

Background: Long-term ß-adrenergic receptor (ß-AR) activation can impair myocardial structure and function. Dapagliflozin (DAPA) has been reported to improve clinical prognosis in heart failure patients, whereas the exact mechanism remains unclear. Here, we investigated the effects of DAPA against ß-AR overactivation toxicity and explored the underlying mechanism.Methods and Results: Rats were randomized to receive saline + placebo, isoproterenol (ISO, 5 mg/kg/day, intraperitoneally) + placebo, or ISO + DAPA (1 mg/kg/day, intragastrically) for 2-week. DAPA treatment improved cardiac function, alleviated myocardial fibrosis, prevented cardiomyocytes (CMs) apoptosis, and decreased the expression of ER stress-mediated apoptosis markers in ISO-treated hearts. In isolated CMs, 2-week ISO stimulation resulted in deteriorated kinetics of cellular contraction and relaxation, increased diastolic intracellular Ca2+ level and decay time constant of Ca2+ transient (CaT) but decreased CaT amplitude and sarcoplasmic reticulum (SR) Ca2+ level. However, DAPA treatment prevented abnormal Ca2+ handling and contractile dysfunction in CMs from ISO-treated hearts. Consistently, DAPA treatment upregulated the expression of SR Ca2+-ATPase protein and ryanodine receptor 2 (RyR2) but reduced the expression of phosphorylated-RyR2, Ca2+/calmodulin-dependent protein kinase II (CaMKII), and phosphorylated-CaMKII in ventricles from ISO-treated rats.Conclusion: DAPA prevented myocardial remodeling and cardiac dysfunction in rats with ß-AR overactivation via restoring calcium handling and suppressing ER stress-related CMs apoptosis.

Cardiac Protein Kinase D1 ablation alters the myocytes ß-adrenergic response.

ß-adrenergic (ß-AR) signaling is essential for the adaptation of the heart to exercise and stress. Chronic stress leads to the activation of Ca2+/calmodulin-dependent kinase II (CaMKII) and protein kinase D (PKD). Unlike CaMKII, the effects of PKD on excitation-contraction coupling (ECC) remain unclear. To elucidate the mechanisms of PKD-dependent ECC regulation, we used hearts from cardiac-specific PKD1 knockout (PKD1 cKO) mice and wild-type (WT) littermates. We measured calcium transients (CaT), Ca2+ sparks, contraction and L-type Ca2+ current in paced cardiomyocytes under acute ß-AR stimulation with isoproterenol (ISO; 100 nM). Sarcoplasmic reticulum (SR) Ca2+ load was assessed by rapid caffeine (10 mM) induced Ca2+ release. Expression and phosphorylation of ECC proteins phospholambam (PLB), troponin I (TnI), ryanodine receptor (RyR), sarcoendoplasmic reticulum Ca2+ ATPase (SERCA) were evaluated by western blotting. At baseline, CaT amplitude and decay tau, Ca2+ spark frequency, SR Ca2+ load, L-type Ca2+ current, contractility, and expression and phosphorylation of ECC protein were all similar in PKD1 cKO vs. WT. However, PKD1 cKO cardiomyocytes presented a diminished ISO response vs. WT with less increase in CaT amplitude, slower [Ca2+]i decline, lower Ca2+ spark rate and lower RyR phosphorylation, but with similar SR Ca2+ load, L-type Ca2+ current, contraction and phosphorylation of PLB and TnI. We infer that the presence of PKD1 allows full cardiomyocyte ß-adrenergic responsiveness by allowing optimal enhancement in SR Ca2+ uptake and RyR sensitivity, but not altering L-type Ca2+ current, TnI phosphorylation or contractile response. Further studies are necessary to elucidate the specific mechanisms by which PKD1 is regulating RyR sensitivity. We conclude that the presence of basal PKD1 activity in cardiac ventricular myocytes contributes to normal ß-adrenergic responses in Ca2+ handling.

The secretory ability of newly formed secretory granules is regulated by pro-cathepsin B and amylase in parotid glands.

Parotid glands are exocrine glands that release saliva into the oral cavity. Acinar cells of parotid glands produce many secretory granules (SGs) that contain the digestion enzyme amylase. After the generation of SGs in the Golgi apparatus, they mature by enlarging and membrane remodeling. VAMP2, which is involved in exocytosis, accumulates in the membrane of mature SGs. The remodeling of SG membranes is regarded as a preparation process for exocytosis but its detailed mechanism remains unknown. To address that subject, we investigated the secretory ability of newly formed SGs. Although amylase is a useful indicator of secretion, the cell leakage of amylase might affect the measurement of secretion. Thus, in this study, we focused on cathepsin B (CTSB), a lysosomal protease, as an indicator of secretion. It has been reported that some procathepsin B (pro-CTSB), which is a precursor of CTSB, is initially sorted to SGs after which it is transported to lysosomes by clathrin-coated vesicles. Because pro-CTSB is processed to mature CTSB after its arrival in lysosomes, we can distinguish between the secretion of SGs and cell leakage by measuring the secretion of pro-CTSB and mature CTSB, respectively. When acinar cells isolated from parotid glands were stimulated with isoproterenol (Iso), a ß-adrenergic agonist, the secretion of pro-CTSB was increased. In contrast, mature CTSB was not detected in the medium although it was abundant in the cell lysates. To prepare parotid glands rich in newly formed SGs, the depletion of per-existing SGs was induced by an intraperitoneal injection of Iso into rats. At 5 h after that injection, newly formed SGs were observed in parotid acinar cells and the secretion of pro-CTSB was also detected. We confirmed that the purified newly formed SGs contained pro-CTSB, but not mature CTSB. At 2 h after Iso injection, few SGs were observed in the parotid glands and the secretion of pro-CTSB was not detected, which proved that the Iso injection depleted pre-existing SGs and the SGs observed at 5 h were newly formed after the Iso injection. These results suggest that newly formed SGs have a secretory ability prior to membrane remodeling.

ß-adrenergic receptor agonist promotes ductular expansion during 3,5-diethoxycarbonyl-1,4-dihydrocollidine-induced chronic liver injury.

Intrahepatic nerves are involved in the regulation of metabolic reactions and hepatocyte-based regeneration after surgical resection, although their contribution to chronic liver injury remains unknown. Given that intrahepatic nerves are abundant in the periportal tissue, they may be correlated also with cholangiocyte-based regeneration. Here we demonstrate that isoproterenol (ISO), a ß-adrenergic receptor agonist, promoted ductular expansion induced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) in vivo. Immunofluorescence analysis shows that nerve fibers positive for tyrosine hydroxylase form synaptophysin-positive nerve endings on epithelial cell adhesion molecule-positive (EpCAM+) cholangiocytes as well as on Thy1+ periportal mesenchymal cells (PMCs) that surround bile ducts, suggesting that the intrahepatic biliary tissue are targeted by sympathetic nerves. In vitro analyses indicate that ISO directly increases cAMP levels in cholangiocytes and PMCs. Mechanistically, ISO expands the lumen of cholangiocyte organoids, resulting in promotion of cholangiocyte proliferation, whereas it increases expression of fibroblast growth factor 7, a growth factor for cholangiocytes, in PMCs. Taken together, the results indicate that intrahepatic sympathetic nerves regulate remodeling of bile ducts during DDC-injury by the activation of ß-adrenergic receptors on cholangiocytes and PMCs.

Repurposing of carvedilol to alleviate bleomycin-induced lung fibrosis in rats: Repressing of TGF-ß1/α-SMA/Smad2/3 and STAT3 gene expressions.

Idiopathic pulmonary fibrosis (IPF) is the most widely studied interstitial lung disease. IPF eventually leads to respiratory insufficiency, lung cancer, and death. Carvedilol (CAR) is a third-generation ß-adrenergic receptor antagonist with an α1-blocking effect. CAR demonstrates antifibrotic activities in various experimental models of organ fibrosis. AIMS: This work is designed to explore the possible alleviating effects of CAR on bleomycin (BLM)-induced lung fibrosis in rats. MAIN METHODS: The BLM rat model of lung fibrosis was achieved by intratracheal delivery of a single dose of 5 mg/kg of BLM. Seven days following BLM injection, either prednisolone or CAR was orally administered at doses of 10 mg/kg once daily for 21 days to the rats. The actions of CAR were evaluated by lung oxidant/antioxidant parameters, protein concentration and total leucocyte count (TLC) in bronchoalveolar lavage fluid (BALF), fibrosis regulator-related genes along with the coexistent lung histological changes. KEY FINDINGS: CAR effectively decreased lung malondialdehyde level, increased superoxide dismutase activity, declined both protein concentration and TLC in BALF, downregulated TGF-ß1/α-SMA/Smad2/3 and STAT3 gene expressions, and repaired the damaged lung tissues. SIGNIFICANCE: CAR conferred therapeutic potential against BLM-induced lung fibrosis in rats, at least in part, to its antioxidant, anti-inflammatory, and antifibrotic activities. CAR could be utilized as a prospective therapeutic option in patients with lung fibrosis in clinical practice.

Effect of stress on the chronotropic and inotropic responses to ß-adrenergic agonists in isolated atria of KOß2 mice.

Altered sensitivity to the chronotropic and inotropic effects of catecholamines and reduction in ß1/ß2-adrenoceptor (ß1/ß2-AR) ratio were reported in failing and in senescent human heart, as well as in isolated atria and ventricle of rats submitted to stress. This was due to downregulation of ß1-AR with or without up-regulation of ß2-AR. AIMS: To investigate the stress-induced behavior of ß1-AR in the heart of mice expressing a non-functional ß2-AR subtype. The guiding hypothesis is that the absence of ß2-AR signaling will not affect the behavior of ß1-AR during stress and that those are independent processes. MATERIALS AND METHODS: The chronotropic and inotropic responses to ß-AR agonists in isolated atria of stressed mice expressing a non-functional ß2-AR were analyzed. The mRNA and protein expressions of ß1- and ß2-AR were also determined. KEY FINDINGS: No deaths were observed in mice under stress protocol. Atria of stressed mice displayed reduced sensitivity to isoprenaline compared to the controls, an effect that was abolished by the ß2- and ß1-AR antagonists 50 nM ICI118,551 and 300 nM CGP20712A, respectively. Sensitivity and maximum response to the ß-agonists dobutamine and salbutamol were not altered by stress or ICI118,551. The responses to dobutamine and salbutamol were prevented by CGP20712A. The expression of ß1-AR was reduced at protein levels. SIGNIFICANCE: Collectively, our data provide evidence that the cardiac ß2-AR is not essential for survival in a stressful situation and that the stress-induced reduction of ß1-AR expression was independent of the ß2-AR presence.

A comprehensive pharmacological analysis of fenoterol and its derivatives to unravel the role of ß2-adrenergic receptor in zebrafish.

ß-adrenergic receptors (ßARs) belong to a key molecular targets that regulate the most important processes occurring in the human organism. Although over the last decades a zebrafish model has been developed as a model complementary to rodents in biomedical research, the role of ß2AR in regulation of pathological and toxicological effects remains to elucidate. Therefore, the study aimed to clarify the role of ß2AR with a particular emphasis on the distinct role of subtypes A and B of zebrafish ß2AR. As model compounds selective ß2AR agonists - (R,R)-fenoterol ((R,R)-Fen) and its new derivatives: (R,R)-4'-methoxyfenoterol ((R,R)-MFen) and (R,R)-4'-methoxy-1-naphtylfenoterol ((R,R)-MNFen) - were tested. We described dose-dependent changes observed after fenoterols exposure in terms of general toxicity, cardiotoxicity and neurobehavioural responses. Subsequently, to better characterise the role of ß2-adrenergic stimulation in zebrafish, we have performed a series of molecular docking simulations. Our results indicate that (R,R)-Fen displays the highest affinity for subtype A of zebrafish ß2AR and ß2AAR might be involved in pigment depletion. (R,R)-MFen shows the lowest affinity for zebrafish ß2ARs out of the tested fenoterols and this might be associated with its cardiotoxic and anxiogenic effects. (R,R)-MNFen displays the highest affinity for subtype B of zebrafish ß2AR and modulation of this receptor might be associated with the development of malformations, increases locomotor activity and induces a negative chronotropic effect. Taken together, the presented data offer insights into the functional responses of the zebrafish ß2ARs confirming their intraspecies conservation, and support the translation of the zebrafish model in pharmacological and toxicological research.