Erythrocytes membrane fluidity changes induced by adenylyl cyclase cascade activation: study using fluorescence recovery after photobleaching.

In this study, fluorescence recovery after photobleaching (FRAP) experiments were performed on RBC labeled by lipophilic fluorescent dye CM-DiI to evaluate the role of adenylyl cyclase cascade activation in changes of lateral diffusion of erythrocytes membrane lipids. Stimulation of adrenergic receptors with epinephrine (adrenaline) or metaproterenol led to the significant acceleration of the FRAP recovery, thus indicating an elevated membrane fluidity. The effect of the stimulation of protein kinase A with membrane-permeable analog of cAMP followed the same trend but was less significant. The observed effects are assumed to be driven by increased mobility of phospholipids resulting from the weakened interaction between the intermembrane proteins and RBC cytoskeleton due to activation of adenylyl cyclase signaling cascade.

"Photo-Adrenalines": Photoswitchable ß2 -Adrenergic Receptor Agonists as Molecular Probes for the Study of Spatiotemporal Adrenergic Signaling.

ß2 -adrenergic receptor (ß2 -AR) agonists are used for the treatment of asthma and chronic obstructive pulmonary disease, but also play a role in other complex disorders including cancer, diabetes and heart diseases. As the cellular and molecular mechanisms in various cells and tissues of the ß2 -AR remain vastly elusive, we developed tools for this investigation with high temporal and spatial resolution. Several photoswitchable ß2 -AR agonists with nanomolar activity were synthesized. The most potent agonist for ß2 -AR with reasonable switching is a one-digit nanomolar active, trans-on arylazopyrazole-based adrenaline derivative and comprises valuable photopharmacological properties for further biological studies with high structural accordance to the native ligand adrenaline.

Ligustilide enhances pregnancy outcomes via improvement of endometrial receptivity and promotion of endometrial angiogenesis in rats.

Ligustilide (LIG) is the main active ingredient of Angelica sinensis (Oliv.) Diels, which could promote focal angiogenesis to exert neuroprotection. However, there was no report that verified the exact effects of LIG on endometrial angiogenesis and the pregnancy outcomes. To explore the effects of LIG on low endometrial receptivity (LER) and angiogenesis, pregnancy rats were assigned into Control (saline treatment), LER (hydroxyurea-adrenaline treatment), LIG 20 mg/kg and LIG 40 mg/kg groups. Hematoxylin and eosin (H&E) staining was performed to evaluate endometrial morphology. Quantitative real-time PCR, immunofluorescence staining, western blot and immunohistochemistry staining were employed to assess the expression of endometrial receptivity factors and angiogenesis-related gene/protein, respectively. RNA sequencing was used to analyze the effects of LIG on LER caused by Kidney deficiency and blood stasis. We found that endometrial thickness and the implanted embryo number were substantially reduced in the hydroxyurea-adrenaline-treated pregnancy rats. At the same time, the gene and protein expressions of ERα, LIF, VEGFA and CD31 in the endometrium were markedly reduced, while the expressions of MUC1, E-cadherin were increased in the LER group. Administration of LIG raised the endometrial thickness and implanted embryos, as well as reversed the expressions of these factors. Collectively, our findings revealed that LIG could facilitate embryo implantation via recovery of the endometrium receptivity and promotion of endometrial angiogenesis.

Epinephrine inhibits PI3Kα via the Hippo kinases.

The phosphoinositide 3-kinase p110α is an essential mediator of insulin signaling and glucose homeostasis. We interrogated the human serine, threonine, and tyrosine kinome to search for novel regulators of p110α and found that the Hippo kinases phosphorylate p110α at T1061, which inhibits its activity. This inhibitory state corresponds to a conformational change of a membrane-binding domain on p110α, which impairs its ability to engage membranes. In human primary hepatocytes, cancer cell lines, and rodent tissues, activation of the Hippo kinases MST1/2 using forskolin or epinephrine is associated with phosphorylation of T1061 and inhibition of p110α, impairment of downstream insulin signaling, and suppression of glycolysis and glycogen synthesis. These changes are abrogated when MST1/2 are genetically deleted or inhibited with small molecules or if the T1061 is mutated to alanine. Our study defines an inhibitory pathway of PI3K signaling and a link between epinephrine and insulin signaling.

CP-25 inhibits the hyperactivation of rheumatic synoviocytes by suppressing the switch in Gαs-Gαi coupling to the ß2-adrenergic receptor.

In essence, the ß2 adrenergic receptor (ß2AR) plays an antiproliferative role by increasing the intracellular cyclic 3',5'-adenosine monophosphate (cAMP) concentration through Gαs coupling, but interestingly, ß2AR antagonists are able to effectively inhibit fibroblast-like synoviocytes (FLSs) proliferation, thus ameliorating experimental RA, indicating that the ß2AR signalling pathway is impaired in RA FLSs via unknown mechanisms. The local epinephrine (Epi) level was found to be much higher in inflammatory joints than in normal joints, and high-level stimulation with Epi or isoproterenol (ISO) directly promoted FLSs proliferation and migration due to impaired ß2AR signalling and cAMP production. By applying inhibitor of receptor internalization, and small interfering RNA (siRNA) of Gαs and Gαi, and by using fluorescence resonance energy transfer and coimmunoprecipitation assays, a switch in Gαs-Gαi coupling to ß2AR was observed in inflammatory FLSs as well as in FLSs with chronic ISO stimulation. This Gαi coupling was then revealed to be initiated by G protein coupled receptor kinase 2 (GRK2) but not ß-arrestin2 or protein kinase A-mediated phosphorylation of ß2AR. Inhibiting the activity of GRK2 with the novel GRK2 inhibitor paeoniflorin-6'-O-benzene sulfonate (CP-25), a derivative of paeoniflorin, or the accepted GRK2 inhibitor paroxetine effectively reversed the switch in Gαs-Gαi coupling to ß2AR during inflammation and restored the intracellular cAMP level in ISO-stimulated FLSs. As expected, CP-25 significantly inhibited the hyperplasia of FLSs in a collagen-induced arthritis (CIA) model (CIA FLSs) and normal FLSs stimulated with ISO and finally ameliorated CIA in rats. Together, our findings revealed the pathological changes in ß2AR signalling in CIA FLSs, determined the underlying mechanisms and identified the pharmacological target of the GRK2 inhibitor CP-25 in treating CIA. Video Abstract.

Effects of epinephrine, lidocaine, and prilocaine on viability and differentiation capacity of human adipose stem cells.

INTRODUCTION: Local anesthetics (LAs) are routinely administered in plastic and reconstructive surgery, e.g., as tumescent anesthesia adjunct in liposuction. Historically, these substances were assumed to act cytotoxically. Thus, the application of LA was avoided when handling adipose stem cells (ASCs). We recently determined that most LAs are not cytotoxic when ASCs are exposed to concentrations used for tumescent liposuction. However, there is limited information when combining LA with epinephrine and about the effects of prilocaine on ASCs. METHODS: We analyzed the effects of prilocaine or lidocaine in co-exposure with epinephrine on the viability of primary human ASCs, i.e., proliferation, metabolic activity, and cytotoxicity, using crystal violet-staining, PrestoBlue®-, and WST-1 assay. We quantified the impact of short-term incubation of lidocaine and epinephrine on the differentiation of ASCs into the adipogenic, chondrogenic, and osteogenic lineage. RESULTS: After 2 h, prilocaine (10 mM) significantly reduced metabolic activity and cell numbers, whereas lidocaine only inhibited metabolic activity. After 6 h, prilocaine (10 mM) and lidocaine significantly decreased metabolic activity as well as cell numbers. The application of high concentrations of epinephrine did not affect cell numbers but diminished metabolic activity. Combining lidocaine with epinephrine had no additional cytotoxic effect. Differentiation into the chondrogenic lineage was significantly inhibited by epinephrine. CONCLUSIONS: Deducing from our data, neither lidocaine combined with epinephrine nor prilocaine has a cytotoxic impact on ASCs in vitro at concentrations equivalent to those in tumescent anesthesia and has no long-lasting effect on the differentiation capacity of ASCs into the osteogenic and adipogenic lineage.

Changes in vital signs during adrenaline administration for hemostasis in intracordal injection: an observational study with a hypothetical design of endotracheal adrenaline administration in cardiopulmonary arrest.

BACKGROUND: The background is that intravenous adrenaline administration is recommended for advanced cardiovascular life support in adults and endotracheal administration is given low priority. The reason is that the optimal dose of adrenaline in endotracheal administration is unknown, and it is ethically difficult to design studies of endotracheal adrenaline administration with non-cardiopulmonary arrest. We otolaryngologists think so because we administered adrenaline to the vocal folds for hemostasis after intracordal injection under local anesthesia, but have had few cases of vital changes. We hypothesized that examining vital signs before and after adrenaline administration for hemostasis would help determine the optimal dose of endotracheal adrenaline. METHODS: We retrospectively examined the medical records of 79 patients who visited our hospital from January 2018 to December 2020 and received adrenaline in the vocal folds and trachea for hemostasis by intracordal injection under local anesthesia to investigate changes in heart rate and systolic blood pressure before and after the injection. RESULTS: The mean heart rates before and after injection were 83.96 ± 18.51 (standard deviation) beats per minute (bpm) and 81.50 ± 15.38 (standard deviation) bpm, respectively. The mean systolic blood pressure before and after the injection were 138.13 ± 25.33 (standard deviation) mmHg and 135.72 ± 22.19 (standard deviation) mmHg, respectively. Heart rate and systolic blood pressure had P-values of 0.136, and 0.450, respectively, indicating no significant differences. CONCLUSIONS: Although this study was an observational, changes in vital signs were investigated assuming endotracheal adrenaline administration. The current recommended dose of adrenaline in endotracheal administration with cardiopulmonary arrest may not be effective. In some cases of cardiopulmonary arrest, intravenous and intraosseous routes of adrenaline administration may be difficult and the opportunity for resuscitation may be missed. Therefore, it is desirable to have many options for adrenaline administration. Therefore, if the optimal dose and efficacy of endotracheal adrenaline administration can be clarified, early adrenaline administration will be possible, which will improve return of spontaneous circulation (ROSC) and survival discharge rates.

Regulatory effects of baicalin, a flavonoid compound, on adipocyte metabolism.

Baicalin is a plant-derived, biologically active compound exerting numerous advantageous effects. Adipocytes store and release energy in the process of lipogenesis and lipolysis. Rodent studies have shown that baicalin treatment positively affects fat tissue, however, data on the direct influence of this compound on adipocyte metabolism is lacking. In the present research, the short-term effects of 25, 50, and 100 µM baicalin on glucose transport, conversion to lipids, and oxidation, and also on lipolysis in primary rat adipocytes were explored. Lipolysis was measured as glycerol release from adipocytes. It was shown that 100 µM baicalin reduced glucose oxidation but at any concentration did not affect glucose transport and lipogenesis. Baicalin significantly increased the adipocyte response to physiological and pharmacological lipolytic stimuli (such as epinephrine - adrenergic agonist, DPCPX - adenosine A1 receptor antagonist, and amrinone - cAMP phosphodiesterase inhibitor). The stimulatory effects of baicalin on epinephrine-induced lipolysis were markedly diminished by insulin (activator of cAMP phosphodiesterases) and H-89 (PKA inhibitor). It was also demonstrated that baicalin evoked a similar rise in epinephrine-induced lipolysis in the presence of glucose and alanine. Our results provided evidence that baicalin may reduce glucose oxidation and is capable of enhancing lipolysis in primary rat adipocytes. The action on lipolysis is glucose-independent and covers both the adrenergic and adenosine A1 receptor pathways. The rise in cAMP content is proposed to be responsible for the observed potentiation of the lipolytic process.

Adrenaline blocks key cell cycle genes and exhibits antifibrotic and vasoconstrictor effects in glaucoma surgery.

Adrenaline is a sympathomimetic drug used to maintain pupil dilation and to decrease the risk of bleeding. The aim of this study was to demonstrate if adrenaline could exert antifibrotic effects in glaucoma surgery. Adrenaline was tested in fibroblast-populated collagen contraction assays and there was a dose-response decrease in fibroblast contractility: matrices decreased to 47.4% (P = 0.0002) and 86.6% (P = 0.0036) with adrenaline 0.0005% and 0.01%, respectively. There was no significant decrease in cell viability even at high concentrations. Human Tenon's fibroblasts were also treated with adrenaline (0%, 0.0005%, 0.01%) for 24 h and RNA-Sequencing was performed on the Illumina NextSeq 2000. We carried out detailed gene ontology, pathway, disease and drug enrichment analyses. Adrenaline 0.01% upregulated 26 G1/S and 11 S-phase genes, and downregulated 23 G2 and 17 M-phase genes (P < 0.05). Adrenaline demonstrated similar pathway enrichment to mitosis and spindle checkpoint regulation. Adrenaline 0.05% was also injected subconjunctivally during trabeculectomy, PreserFlo Microshunt and Baerveldt 350 tube surgeries, and patients did not experience any adverse effects. Adrenaline is a safe and cheap antifibrotic drug that significantly blocks key cell cycle genes when used at high concentrations. Unless contraindicated, we recommend subconjunctival injections of adrenaline (0.05%) in all glaucoma bleb-forming surgeries.

Saikosaponin D reverses epinephrine- and norepinephrine-induced gemcitabine resistance in intrahepatic cholangiocarcinoma by downregulating ADRB2/glycolysis signaling.

Intrahepatic cholangiocarcinoma (iCCA) is a highly fatal malignancy with rapidly increasing incidence and mortality worldwide. Currently, gemcitabine-based systemic chemotherapy is the main clinical therapeutic regimen; however, its efficacy is poor, and its mechanism has not been elucidated. In this study, we use a Seahorse Extracellular Flux analyser to measure glycolysis capacity (extracellular acidification rate, ECAR) and oxygen consumption rate (OCR). The glucose uptake or lactic acid content is detected, and the effects of saikosaponin D, an active compound derived from Bupleuri Radix (a traditional Chinese medicine for soothing the liver and relieving depression), on gemcitabine cytotoxicity in norepinephrine-stimulated iCCA cells are analysed. We find that adrenergic signaling plays a fundamental role in chronic stress-induced therapeutic resistance in iCCA. Norepinephrine (NE) and epinephrine (E) enhance the proliferation of iCCA cells and interfere with the response to gemcitabine through activation of the ß2-adrenergic receptor (ADRB2). Furthermore, we find that NE upregulates the expressions of several drug efflux-related genes (such as ABCG2 and MDR1) and promotes glycolysis in iCCA cells. In addition, saikosaponin D reverses the poor response of iCCA cells to gemcitabine by downregulating ADRB2 level. Furthermore, saikosaponin D inhibits drug efflux and glycolysis in iCCA cells by regulating the expressions of MDR1, ABCG2, HK2, and GLUT1. Collectively, saikosaponin D enhances the antitumor effect of gemcitabine by controlling glucose metabolism and drug efflux by inhibiting the ADRB2 signaling. Therefore, the combination of saikosaponin D and gemcitabine may be a potential therapeutic strategy for the treatment of iCCA.

Multicenter evaluation of light transmission platelet aggregation reagents: communication from the ISTH SSC Subcommittee on Platelet Physiology.

BACKGROUND: Light transmission aggregation (LTA) is used widely by the clinical and research communities. Although it is a gold standard, there is a lack of interlaboratory harmonization. OBJECTIVES: The primary objective was to assess whether sources of activators (mainly adenosine diphosphate [ADP], collagen, arachidonic acid, epinephrine, and thrombin receptor activating peptide6) and ristocetin contribute to poor LTA reproducibility. The secondary objective was to evaluate interindividual variability of results to appreciate the distribution of normal values and consequently better interpret pathologic results. METHODS: An international multicenter study involving 28 laboratories in which we compared LTA results obtained with center-specific activators and a comparator that we supplied. RESULTS: We report variability in the potency (P) of activators in comparison with the comparator. Thrombin receptor activating peptide 6 (P, 1.32-2.68), arachidonic acid (P, 0.87-1.43), and epinephrine (P, 0.97-1.34) showed the greatest variability. ADP (P, 1.04-1.20) and ristocetin (P, 0.98-1.07) were the most consistent. The data highlighted clear interindividual variability, notably for ADP and epinephrine. Four profiles of responses were observed with ADP from high-responders, intermediate-responders, and low-responders. A fifth profile corresponding to nonresponders (5% of the individuals) was observed with epinephrine. CONCLUSION: Based on these data, the establishment and adoption of simple standardization principles should mitigate variability due to activator sources. The observation of huge interindividual variability for certain concentrations of activators should lead to a cautious interpretation before reporting a result as abnormal. Confidence can be taken from the fact that difference between sources is not exacerbated in patients treated with antiplatelet agents.

The Secretome of Parental and Bone Metastatic Breast Cancer Elicits Distinct Effects in Human Osteoclast Activity after Activation of ß2 Adrenergic Signaling.

The sympathetic nervous system (SNS), particularly through the ß2 adrenergic receptor (ß2-AR), has been linked with breast cancer (BC) and the development of metastatic BC, specifically in the bone. Nevertheless, the potential clinical benefits of exploiting ß2-AR antagonists as a treatment for BC and bone loss-associated symptoms remain controversial. In this work, we show that, when compared to control individuals, the epinephrine levels in a cohort of BC patients are augmented in both earlier and late stages of the disease. Furthermore, through a combination of proteomic profiling and functional in vitro studies with human osteoclasts and osteoblasts, we demonstrate that paracrine signaling from parental BC under ß2-AR activation causes a robust decrease in human osteoclast differentiation and resorption activity, which is rescued in the presence of human osteoblasts. Conversely, metastatic bone tropic BC does not display this anti-osteoclastogenic effect. In conclusion, the observed changes in the proteomic profile of BC cells under ß-AR activation that take place after metastatic dissemination, together with clinical data on epinephrine levels in BC patients, provided new insights on the sympathetic control of breast cancer and its implications on osteoclastic bone resorption.

Stress biomarkers in individuals with fibromyalgia syndrome: a systematic review with meta-analysis.

ABSTRACT: Evidence suggests an involvement of hypothalamic-pituitary-adrenal (HPA) axis dysregulation in the development and maintenance of fibromyalgia syndrome (FMS). However, studies on the stress response via the HPA-axis in individuals with FMS show conflicting results. To better understand the relationship between FMS and HPA-axis dysregulation, we (1) systematically summarized the current level of evidence on HPA biomarkers in individuals with FMS compared with individuals without and (2) evaluated whether FMS is associated with a specific pattern of HPA dysregulation. The main outcome measures were cortisol, adrenocorticotropic hormone (ACTH), corticotropin-releasing hormone (CRH), epinephrine, and norepinephrine. A systematic search of MEDLINE, EMBASE, and PsychMed yielded 47 studies eligible for meta-analysis, including 1465 individuals with FMS and 1192 FMS-free controls. No main effect of FMS was found on altered levels of blood cortisol, ACTH, CRH, and epinephrine. Compared with controls, salivary and urinary cortisol levels were decreased in individuals with FMS, whereas blood levels of norepinephrine were increased. However, heterogeneity of data was high with significant evidence for publication bias. Overall, the data are compatible with association of FMS with adrenocortical hypofunction in the presence of increased sympathetic tone. However, the data are partially contradictory, so it must be assumed that the data are highly dependent on the respective study designs, patient samples, and analytical methods and do not necessarily demonstrate an abnormal HPA-axis function in FMS.

ß-adrenoreceptor-triggered PKA activation negatively regulates the innate antiviral response.

Upon viral infection, cytoplasmic pattern recognition receptors detect viral nucleic acids and activate the adaptor protein VISA/MAVS- or MITA/STING-mediated innate antiviral response. Whether and how the innate antiviral response is regulated by neuronal endocrine functions is unclear. Here, we show that viral infection reduced the serum levels of the ß-adrenergic hormones epinephrine and norepinephrine as well as the cellular levels of their receptors ADRB1 and ADRB2. We further show that an increase in epinephrine/norepinephrine level inhibited the innate antiviral response in an ADRB1-/2-dependent manner. Mechanistically, epinephrine/norepinephrine stimulation activated the downstream kinase PKA, which catalyzed the phosphorylation of MITA at S241, S243 and T263, inhibiting MITA activation and suppressing the innate immune response to DNA virus. In addition, phosphorylation of VISA at T54 by PKA antagonized the innate immune response to RNA virus. These findings reveal the regulatory mechanisms of innate antiviral responses by epinephrine/norepinephrine and provide a possible explanation for increased host susceptibility to viral infection in stressful and anxiety-promoting situations.

Investigation on the positive chronotropic action of 6-nitrodopamine in the rat isolated atria.

6-Nitrodopamine (6-ND) is released from rat isolated atria being 100 times more potent than noradrenaline and adrenaline, and 10,000 times more potent than dopamine as a positive chronotropic agent. The present study aimed to investigate the interactions of 6-ND with the classical catecholamines, phosphodiesterase (PDE)-3 and PDE4, and the protein kinase A in rat isolated atria. Atrial incubation with 1 pM of dopamine, noradrenaline, or adrenaline had no effect on atrial frequency. Similar results were observed when the atria were incubated with 0.01 pM of 6-ND. However, co-incubation of 6-ND (0.01 pM) with dopamine, noradrenaline, or adrenaline (1 pM each) resulted in significant increases in atrial rate, which persisted over 30 min after washout of the agonists. The increased atrial frequency induced by co-incubation of 6-ND with the catecholamines was significantly reduced by the voltage-gated sodium channel blocker tetrodotoxin (1 µM, 30 min), indicating that the positive chronotropic effect of 6-ND is due in part to activation of nerve terminals. Pre-treatment of the animals with reserpine had no effect on the positive chronotropic effect induced by dopamine, noradrenaline, or adrenaline; however, reserpine markedly reduced the 6-ND (1 pM)-induced positive chronotropic effect. Incubation of the rat isolated atria with the protein kinase A inhibitor H-89 (1 µM, 30 min) abolished the increased atrial frequency induced by dopamine, noradrenaline, and adrenaline, but only attenuated the increases induced by 6-ND. 6-ND induces catecholamine release from adrenergic terminals and increases atrial frequency independently of PKA activation.

Inhibition of signaling downstream of beta-2 adrenoceptor by propranolol in prostate cancer cells.

BACKGROUND: There is accumulating evidence that propranolol, an antagonist of beta-1 and beta-2 adrenoreceptors, extends survival of patients with prostate cancer; yet it is not known whether propranolol inhibits beta-adrenergic signaling in prostate cancer cells, or systemic effects of propranolol play the leading role in slowing down cancer progression. Recently initiated clinical studies offer a possibility to test whether administration of propranolol inhibits signaling pathways in prostate tumors, however, there is limited information on the dynamics of signaling pathways activated downstream of beta-2 adrenoreceptors in prostate cancer cells and on the inactivation of these pathways upon propranolol administration. METHODS: Western blot analysis was used to test the effects of epinephrine and propranolol on activation of protein kinase (PKA) signaling in mouse prostates and PKA, extracellular signal-regulated kinase (ERK), and protein kinase B/AKT (AKT) signaling in prostate cancer cell lines. RESULTS: In prostate cancer cell lines epinephrine induced robust phosphorylation of PKA substrates pS133CREB and pS157VASP that was evident 2 min after treatments and lasted for 3-6 h. Epinephrine induced phosphorylation of AKT in PTEN-positive 22Rv1 cells, whereas changes of constitutive AKT phosphorylation were minimal in PTEN-negative PC3, C42, and LNCaP cells. A modest short-term increase of pERK in response to epinephrine was observed in all tested cell lines. Incubation of prostate cancer cells with 10-fold molar excess of propranolol for 30 min inhibited all downstream pathways activated by epinephrine. Subjecting mice to immobilization stress induced phosphorylation of S133CREB, whereas injection of propranolol at 1.5 mg/kg prevented the stress-induced phosphorylation. CONCLUSIONS: The analysis of pS133CREB and pS157VASP allows measuring activation of PKA signaling downstream of beta-2 adrenoreceptors. Presented results on the ratio of propranolol/epinephrine and the time needed to inhibit signaling downstream of beta-2 adrenoreceptors will help to design clinical studies that examine the effects of propranolol on prostate tumors.

Effects of Intracameral Drugs and Dyes on Corneal Endothelial Cell Apoptosis in a Rat Model: An In Vivo and In Vitro Analysis.

Objectives: To evaluate the effects of intracameral drugs and dyes on rat corneal endothelial apoptosis and cell morphology. Materials and Methods: The right eyes of 72 rats were injected intracamerally with 1% lidocaine, 0.01% adrenaline, triamcinolone acetonide (TA) 4 mg/mL, 1% trypan blue (TB), 0.5% indocyanine green (ICG), and fortified balanced salt solution as control. Corneal samples were taken 1 day and 1 week post-injection. Corneal endothelial apoptosis was assessed by the TUNEL technique, and the ratio of apoptotic cells in each group was compared with the control. Corneal endothelial cell morphology was evaluated in each specimen by transmission electron microscopy. Results: The mean apoptotic endothelial cell ratio was significantly higher at 1 day and 1 week after intracameral adrenaline injection when compared to controls (p=0.03 and 0.021, respectively). TB caused a significantly higher apoptotic cell ratio when compared to controls at 1 week after injection (p=0.043). Lidocaine caused a higher apoptotic cell ratio compared to TA and ICG at 1 week, although not statistically significant (p=0.058, 0.09, 0.69, respectively). In all experimental specimens, transmission electron microscopy showed morphological changes associated with apoptosis. Conclusion: This study showed that intracameral adrenaline, TB, and lidocaine injections may have toxic effects on corneal tissue, as indicated by ultrastructural and histopathological alterations. Therefore, these agents should be used with caution in intraocular surgery.

Effects of adrenergic-stimulated lipolysis and cytokine production on in vitro mouse adipose tissue-islet interactions.

Inflammatory cytokines and non-esterified fatty acids (NEFAs) are obesity-linked factors that disturb insulin secretion. The aim of this study was to investigate whether pancreatic adipose tissue (pWAT) is able to generate a NEFA/cytokine overload within the pancreatic environment and as consequence to impact on insulin secretion. Pancreatic fat is a minor fat depot, therefore we used high-fat diet (HFD) feeding to induce pancreatic steatosis in mice. Relative Adipoq and Lep mRNA levels were higher in pWAT of HFD compared to chow diet mice. Regardless of HFD, Adipoq and Lep mRNA levels of pWAT were at least 10-times lower than those of epididymal fat (eWAT). Lipolysis stimulating receptors Adrb3 and Npr1 were expressed in pWAT and eWAT, and HFD reduced their expression in eWAT only. In accordance, HFD impaired lipolysis in eWAT but not in pWAT. Despite expression of Npr mRNA, lipolysis was stimulated solely by the adrenergic agonists, isoproterenol and adrenaline. Short term co-incubation of islets with CD/HFD pWAT did not alter insulin secretion. In the presence of CD/HFD eWAT, glucose stimulated insulin secretion only upon isoproterenol-induced lipolysis, i.e. in the presence of elevated NEFA. Isoproterenol augmented Il1b and Il6 mRNA levels both in pWAT and eWAT. These results suggest that an increased sympathetic activity enhances NEFA and cytokine load of the adipose microenvironment, including that of pancreatic fat, and by doing so it may alter beta-cell function.

Effects of diacylglycerol O-acyltransferase 1 (DGAT1) on endoplasmic reticulum stress and inflammatory responses in adipose tissue of ketotic dairy cows.

Adipose tissue of ketotic dairy cows exhibits greater lipolytic rate and signs of inflammation, which further aggravate the metabolic disorder. In nonruminants, the endoplasmic reticulum (ER) is a key organelle coordinating metabolic adaptations and cellular functions; thus, disturbances known as ER stress lead to inflammation and contribute to metabolic disorders. Enhanced activity of diacylglycerol O-acyltransferase 1 (DGAT1) in murine adipocytes undergoing lipolysis alleviated ER stress and inflammation. The aim of the present study was to investigate the potential role of DGAT1 on ER stress and inflammatory response of bovine adipose tissue in vivo and in vitro. Adipose tissue and blood samples were collected from cows diagnosed as clinically ketotic (n = 15) or healthy (n = 15) following a veterinary evaluation based on clinical symptoms and serum concentrations of ß-hydroxybutyrate, which were 4.05 (interquartile range = 0.46) and 0.52 mM (interquartile range = 0.14), respectively. Protein abundance of DGAT1 was greater in adipose tissue of ketotic cows. Among ER stress proteins measured, ratios of phosphorylated PKR-like ER kinase (p-PERK) to PERK and phosphorylated inositol-requiring enzyme 1 (p-IRE1) to IRE1, and protein abundance of cleaved ATF6 protein were greater in adipose tissue of ketotic cows. Furthermore, ratios of phosphorylated RELA subunit of NF-κB (p-RELA) to RELA and phosphorylated c-jun N-terminal kinase (p-JNK) to JNK were greater, whereas protein abundance of NF-κB inhibitor α (NFKBIA) was lower in adipose tissue of ketotic cows. In addition, mRNA abundance of proinflammatory cytokines including TNF and IL-6 was greater in adipose tissue of ketotic cows. To better address mechanistic aspects of these responses, primary bovine adipocytes isolated from the harvested adipose tissue of healthy cows were subjected to lipolysis-stimulating conditions via incubation with 1 µM epinephrine (EPI) for 2 h. In another experiment, adipocytes were cultured with DGAT1 overexpression adenovirus and DGAT1 small interfering RNA for 48 h, respectively, followed by EPI (1 µM) exposure for 2 h. Treatment with EPI led to greater ratios of p-PERK to PERK, p-IRE1 to IRE1, p-RELA to RELA, p-JNK to JNK, and cleaved ATF6 protein, whereas EPI stimulation inhibited protein abundance of NFKBIA. Furthermore, treatment with EPI upregulated the secretion of proinflammatory cytokines into culture medium, including TNF-α and IL-6. Overexpression of DGAT1 in EPI-treated adipocytes attenuated ER stress, the activation of NF-κB and JNK signaling pathways, and the secretion of inflammatory cytokines. In contrast, silencing DGAT1 further aggravated EPI-induced ER stress and inflammatory responses. Overall, these data indicated that activation of DGAT1 may act as an adaptive mechanism to dampen metabolic dysregulation in adipose tissue. As such, it contributes to relief from ER stress and inflammatory responses.

ß2-Adrenergic Receptor Signaling Pathway Stimulates the Migration and Invasion of Cancer Cells via Src Activation.

Chronic stress has been reported to stimulate the release of catecholamines, including norepinephrine (NE) and epinephrine (E), which promote cancer progression by activating the adrenergic receptor (AR). Although previous studies showed that ß2-AR mediated chronic stress-induced tumor growth and metastasis, the underlying mechanism has not been fully explored. In this study, we aimed to investigate the molecular mechanism by which ß2-AR exerts a pro-metastatic function in hepatocarcinoma (HCC) cells and breast cancer (BC) cells. Our results showed that Hep3B human HCC cells and MDA-MB-231 human BC cells exhibited the highest ADRB2 expression among diverse HCC and BC cell lines. NE, E, and isoprenaline (ISO), adrenergic agonists commonly increased the migration and invasion of Hep3B cells and MDA-MB-231 cells. The phosphorylation level of Src was significantly increased by E/NE. Dasatinib, a Src kinase inhibitor, blocked E/NE-induced migration and invasion, indicating that AR agonists enhanced the mobility of cancer cells by activating Src. ADRB2 knockdown attenuated E/NE-induced Src phosphorylation, as well as the metastatic ability of cancer cells, suggesting the essential role of ß2-AR. Taken together, our results demonstrate that chronic stress-released catecholamines promoted the migration and invasion of HCC cells and BC cells via ß2-AR-mediated Src activation.