Distinct binding conformations of epinephrine with α- and ß-adrenergic receptors.

Agonists targeting α2-adrenergic receptors (ARs) are used to treat diverse conditions, including hypertension, attention-deficit/hyperactivity disorder, pain, panic disorders, opioid and alcohol withdrawal symptoms, and cigarette cravings. These receptors transduce signals through heterotrimeric Gi proteins. Here, we elucidated cryo-EM structures that depict α2A-AR in complex with Gi proteins, along with the endogenous agonist epinephrine or the synthetic agonist dexmedetomidine. Molecular dynamics simulations and functional studies reinforce the results of the structural revelations. Our investigation revealed that epinephrine exhibits different conformations when engaging with α-ARs and ß-ARs. Furthermore, α2A-AR and ß1-AR (primarily coupled to Gs, with secondary associations to Gi) were compared and found to exhibit different interactions with Gi proteins. Notably, the stability of the epinephrine-α2A-AR-Gi complex is greater than that of the dexmedetomidine-α2A-AR-Gi complex. These findings substantiate and improve our knowledge on the intricate signaling mechanisms orchestrated by ARs and concurrently shed light on the regulation of α-ARs and ß-ARs by epinephrine.

Metabolomic profiling of pheochromocytomas in dogs: Catecholamine phenotype and tricarboxylic acid cycle metabolites.

BACKGROUND: In humans with pheochromocytomas (PCCs), targeted metabolomics is used to determine the catecholamine phenotype or to uncover underlying pathogenic variants in tricarboxylic acid (TCA) cycle genes such as succinate dehydrogenase subunits (SDHx). HYPOTHESIS/OBJECTIVES: To analyze catecholamine contents and TCA cycle metabolites of PCCs and normal adrenals (NAs). ANIMALS: Ten healthy dogs, 21 dogs with PCC. METHODS: Prospective observational study. Dogs diagnosed with PCC based on histopathological and immunohistochemical confirmation were included. Tissue catecholamine contents and TCA metabolites in PCCs and NAs were measured by liquid chromatography with mass spectrometry or electrochemical detection. RESULTS: Compared to NAs, PCCs had significantly higher tissue proportion of norepinephrine (88% [median: range, 38%-98%] vs 14% [11%-26%]; P < .001), and significantly lower tissue proportion of epinephrine (12% [1%-62%] vs 86% [74%-89%]; P < .001). Pheochromocytomas exhibited significantly lower fumarate (0.4-fold; P < .001), and malate (0.5-fold; P = .008) contents than NAs. Citrate was significantly higher in PCCs than in NAs (1.6-fold; P = .015). One dog in the PCC group had an aberrant succinate : fumarate ratio that was 25-fold higher than in the other PCCs, suggesting an SDHx mutation. CONCLUSIONS AND CLINICAL IMPORTANCE: This study reveals a distinct catecholamine content and TCA cycle metabolite profile in PCCs. Metabolite profiling might be used to uncover underlying pathogenic variants in TCA cycle genes in dogs.

Epinephrine promotes breast cancer metastasis through a ubiquitin-specific peptidase 22-mediated lipolysis circuit.

Chronic stress-induced epinephrine (EPI) accelerates breast cancer progression and metastasis, but the molecular mechanisms remain unclear. Herein, we found a strong positive correlation between circulating EPI levels and the tumoral expression of ubiquitin-specific peptidase 22 (USP22) in patients with breast cancer. USP22 facilitated EPI-induced breast cancer progression and metastasis by enhancing adipose triglyceride lipase (ATGL)-mediated lipolysis. Targeted USP22 deletion decreased ATGL expression and lipolysis, subsequently inhibiting EPI-mediated breast cancer lung metastasis. USP22 acts as a bona fide deubiquitinase for the Atgl gene transcription factor FOXO1, and EPI architects a lipolysis signaling pathway to stabilize USP22 through AKT-mediated phosphorylation. Notably, USP22 phosphorylation levels are positively associated with EPI and with downstream pathways involving both FOXO1 and ATGL in breast cancers. Pharmacological USP22 inhibition synergized with ß-blockers in treating preclinical xenograft breast cancer models. This study reveals a molecular pathway behind EPI's tumor-promoting effects and provides a strong rationale for combining USP22 inhibition with ß-blockers to treat aggressive breast cancer.

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.

Adrenergic microenvironment driven by cancer-associated Schwann cells contributes to chemoresistance in patients with lung cancer.

Doublecortin (DCX)-positive neural progenitor-like cells are purported components of the cancer microenvironment. The number of DCX-positive cells in tissues reportedly correlates with cancer progression; however, little is known about the mechanism by which these cells affect cancer progression. Here we demonstrated that DCX-positive cells, which are found in all major histological subtypes of lung cancer, are cancer-associated Schwann cells (CAS) and contribute to the chemoresistance of lung cancer cells by establishing an adrenergic microenvironment. Mechanistically, the activation of the Hippo transducer YAP/TAZ was involved in the acquisition of new traits of CAS and DCX positivity. We further revealed that CAS express catecholamine-synthesizing enzymes and synthesize adrenaline, which potentiates the chemoresistance of lung cancer cells through the activation of YAP/TAZ. Our findings shed light on CAS, which drive the formation of an adrenergic microenvironment by the reciprocal regulation of YAP/TAZ in lung cancer tissues.

Stress-induced epinephrine promotes hepatocellular carcinoma progression via the USP10-PLAGL2 signaling loop.

Hepatocellular carcinoma (HCC) is associated with a poor prognosis. Our previous study demonstrated that Pleomorphic adenoma gene like-2 (PLAGL2) was a potential therapeutic target in HCC. However, the mechanisms that lead to the upregulation of PLAGL2 in HCC remain unclear. The present study revealed that stress-induced epinephrine increased the expression of PLAGL2, thereby promoting the progression of HCC. Furthermore, PLAGL2 knockdown inhibited epinephrine-induced HCC development. Mechanistically, epinephrine upregulated ubiquitin-specific protease 10 (USP10) to stabilize PLAGL2 via the adrenergic ß-receptor-2-c-Myc (ADRB2-c-Myc) axis. Furthermore, PLAGL2 acted as a transcriptional regulator of USP10, forming a signaling loop. Taken together, these results reveal that stress-induced epinephrine activates the PLAGL2-USP10 signaling loop to enhance HCC progression. Furthermore, PLAGL2 plays a crucial role in psychological stress-mediated promotion of HCC progression.

Study on the Mechanism of the Adrenaline-Evoked Procoagulant Response in Human Platelets.

Adrenaline has recently been found to trigger phosphatidylserine (PS) exposure on blood platelets, resulting in amplification of the coagulation process, but the mechanism is only fragmentarily established. Using a panel of platelet receptors' antagonists and modulators of signaling pathways, we evaluated the importance of these in adrenaline-evoked PS exposure by flow cytometry. Calcium and sodium ion influx into platelet cytosol, after adrenaline treatment, was examined by fluorimetric measurements. We found a strong reduction in PS exposure after blocking of sodium and calcium ion influx via Na+/H+ exchanger (NHE) and Na+/Ca2+ exchanger (NCX), respectively. ADP receptor antagonists produced a moderate inhibitory effect. Substantial limitation of PS exposure was observed in the presence of GPIIb/IIIa antagonist, phosphoinositide-3 kinase (PI3-K) inhibitors, or prostaglandin E1, a cyclic adenosine monophosphate (cAMP)-elevating agent. We demonstrated that adrenaline may develop a procoagulant response in human platelets with the substantial role of ion exchangers (NHE and NCX), secreted ADP, GPIIb/IIIa-dependent outside-in signaling, and PI3-K. Inhibition of the above mechanisms and increasing cytosolic cAMP seem to be the most efficient procedures to control adrenaline-evoked PS exposure in human platelets.

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.

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.

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.

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.

Sympathetic activation leads to Schlemm's canal expansion via increasing vasoactive intestinal polypeptide secretion from trabecular meshwork.

We previously demonstrated vasoactive intestinal polypeptide (VIP) eyedrops reduce intraocular pressure (IOP) and stabilize cytoskeleton of the Schlemm's canal (SC) endothelium in a chronic ocular hypertension rat model. Here we determine if the trabecular meshwork (TM) releases endogenous VIP and affect SC in paracrine manner, and whether this cellular interaction via VIP is strengthened under stimulated sympathetic activity. A rat model of moderate-intensity exercise was established to stimulate sympathetic activation. IOP post exercise was measured by a rebound tonometer. Sympathetic nerve activity at the TM was immunofluorescence-stained with DßH and PGP9.5. Morphological changes of TM and SC were quantitatively measured by hematoxylin-eosin (HE) staining. Further, epinephrine was applied to mimic sympathetic excitation on primary rat TM cells, and ELISA to measure VIP levels in the medium. The cytoskeleton protective effect of VIP in the epinephrine-stimulated conditioned medium (Epi-CM) was evaluated in oxidative stressed human umbilical vein endothelial cells (HUVECs). Elevated sympathetic nerve activity was found at TM post exercise. Changes accompanying the sympathetic excitation included thinned TM, expanded SC and decreased IOP, which were consistent with epinephrine treatment. Epinephrine decreased TM cell size, enhanced VIP expression and release in the medium in vitro. Epi-CM restored linear F-actin and cell junction integrity in H2O2 treated HUVECs. Blockage of VIP receptor by PG99-465 attenuated the protective capability of Epi-CM. VIP expression was upregulated at TM and the inner wall of SC post exercise in vivo. PG99-465 significantly attenuated exercise-induced SC expansion and IOP reduction. Thus, the sympathetic activation promoted VIP release from TM cells and subsequently expanded SC via stabilizing cytoskeleton, which resulted in IOP reduction.

Catecholamine-dependent hyperpolarization of the junctional membrane via ß2- adrenoreceptor/Gi-protein/α2-Na-K-ATPase pathway.

We investigated the effects of catecholamines, adrenaline and noradrenaline, as well as ß-adrenoceptor (AR) modulators on a resting membrane potential at the junctional and extrajunctional regions of mouse fast-twitch Levator auris longus muscle. The aim of the study was to find which AR subtypes, signaling molecules and Na,K-ATPase isoforms are involved in the hyperpolarizing action of catecholamines and whether this action could be accompanied by changes in the pump abundance on the sarcolemma. Adrenaline, noradrenaline and specific ß2-AR agonist induced hyperpolarization of both junctional and extrajunctional membrane, but the underlying mechanisms were different. In the junctional membrane the hyperpolarization depended on α2 isoform of the Na,K-ATPase and Gi-protein, whereas in the extrajunctional regions the hyperpolarization mainly relied on α1 isoform of Na,K-ATPase and adenylyl cyclase activities. In both junctional and extrajunctional regions, AR activation caused an increase in Na,K-ATPase abundance in the plasmalemma in a protein kinase A-dependent manner. Thus, the compartment-specific mechanisms are responsible for catecholamine-mediated hyperpolarization in the skeletal muscle.

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.

The Pharmacological Effects of Silver Nanoparticles Functionalized with Eptifibatide on Platelets and Endothelial Cells.

Purpose: In the search for new drug delivery platforms for cardiovascular diseases and coating of medical devices, we synthesized eptifibatide-functionalized silver nanoparticles (AgNPs-EPI) and examined the pharmacological activity of AgNPs-EPI on platelets and endothelial cells in vitro and ex vivo. Methods: Spherical AgNPs linked to eptifibatide were synthesized and characterized. Cytotoxicity was measured in microvascular endothelial cells (HMEC-1), platelets and red blood cells. Platelet mitochondrial respiration was measured using the Oxygraph-2k, a high-resolution modular respirometry system. The effect of AgNPs-EPI on the aggregation of washed platelets was measured by light aggregometry and the ex vivo occlusion time was determined using a reference laboratory method. The surface amount of platelet receptors such as P-selectin and GPIIb/IIIa was measured. The influence of AgNPS-EPI on blood coagulation science was assessed. Finally, the effect of AgNPs-EPI on endothelial cells was measured by the levels of 6-keto-PGF1alpha, tPa, cGMP and vWF. Results: We describe the synthesis of AgNPs using eptifibatide as the stabilizing ligand. The molecules of this drug are directly bonded to the surface of the nanoparticles. The synthesized AgNPs-EPI did not affect the viability of platelets, endothelial cells and erythrocytes. Preincubation of platelets with AgNPs-EPI protected by mitochondrial oxidative phosphorylation capacity. AgNPs-EPI inhibited aggregation-induced P-selectin expression and GPIIb/IIIa conformational changes in platelets. AgNPs-EPI caused prolongation of the occlusion time in the presence of collagen/ADP and collagen/adrenaline. AgNPs-EPI regulated levels of 6-keto-PGF1alpha, tPa, vWf and cGMP produced in thrombin stimulated HMEC-1 cells. Conclusion: AgNPs-EPI show anti-aggregatory activity at concentrations lower than those required by the free drug acting via regulation of platelet aggregation, blood coagulation, and endothelial cell activity. Our results provide proof-of-principle evidence that AgNPs may be used as an effective delivery platform for antiplatelet drugs.

N-Stearoylethanolamine Inhibits Integrin-Mediated Activation, Aggregation, and Adhesion of Human Platelets.

N-stearoylethanolamine (NSE), a lipid mediator that belongs to the N-acylethanolamine (NAE) family, has anti-inflammatory, antioxidant, and membranoprotective actions. In contrast to other NAEs, NSE does not interact with cannabinoid receptors. The exact mechanism of its action remains unclear. The aim of this study is to evaluate the action of NSE on activation, aggregation, and adhesion of platelets that were chosen as a model of cellular response. Aggregation of platelets was measured to analyze the action of NSE (10-6-10-10 M) on platelet reactivity. Changes in granularity and shape of resting platelets and platelets stimulated with ADP in the presence of NSE were monitored by flow cytometry, and platelet deganulation was monitored by spectrofluorimetry. In vivo studies were performed using obese insulin-resistant rats. Binding of fibrinogen to the GPIIb/IIIa receptor was estimated using indirect ELISA and a scanning electron microscopy (SEM). It was found that NSE inhibits the activation and aggregation of human platelets. Our results suggest that NSE may decrease the activation and subsequent aggregation of platelets induced by ristocetin, epinephrine, and low doses of ADP. NSE also reduced the binding of fibrinogen to GPIIb/IIIa on activated platelets. These effects could be explained by the inhibition of platelet activation mediated by integrin receptors: the GPIb-IX-V complex for ristocetin-induced activation and GPIIb/IIIa when epinephrine and low doses of ADP were applied. The anti-platelet effect of NSE complements its anti-inflammatory effect and allows us to prioritize studies of NSE as a potent anti-thrombotic agent. SIGNIFICANCE STATEMENT: N-stearoylethanolamine (NSE) was shown to possess inhibitory action on platelet activation, adhesion, and aggregation. The mechanism of inhibition possibly involves integrin receptors. This finding complements the known anti-inflammatory effects of NSE.

Adrenergic Receptors in the Mechanism of Regulation of Mitochondrial and Cytoplasmic Enzymes of Cardiomyocytes by Catecholamines.

We studied the role of adrenoceptors in the regulation of activity of mitochondrial and cytoplasmic enzymes in cardiomyocytes by catecholamines and their metabolites. Different types of adrenergic receptors (AR) agonists acting either on both α- and ß-AR or selectively on α- or ß-AR, as well as quinoid metabolites of catecholamines were used. It was found that the activating effect of ß-AR agonist isadrin (isoproterenol) on succinate dehydrogenase of the mitochondria in the heart is prevented by ß-adrenergic blockade. The activating effect of dopamine, epinephrine, and isoproterenol on cytochrome C-oxidase and the inhibitory effect of dopamine, norepinephrine, epinephrine, and isoproterenol on Mg-activated ATPase was not mediated by adrenoreceptors. Hormones of the sympathoadrenal system epinephrine, dopamine, norepinephrine, isoproterenol, and catecholamine metabolites (adrenochrome and adrenoxyl) modulating activity of the respiratory chain enzymes of mitochondria in the heart regulate the processes of tissue respiration by transferring mitochondria into a state of "loose" phosphorylation and respiration coupling. Epinephrine as a ß-AR agonist increased activity of cytosolic enzymes catalyzing metabolism of purine nucleotides (adenosine deaminase and AMP deaminase), enzymes of antioxidant defense (glutathione peroxidase and catalase), and the level of malondialdehyde and diene conjugates. ß-AR blockade with metoprolol abolished the activating effect of epinephrine on glutathione reductase, glutathione peroxidase, and catalase and reduced the level of malondialdehyde and diene conjugates.

Contribution of adrenergic mechanisms for the stress-induced breast cancer carcinogenesis.

Breast cancer is the most common and deadliest type of cancer in women. Stress exposure has been associated with carcinogenesis and the stress released neurotransmitters, noradrenaline and adrenaline, and their cognate receptors, can participate in the carcinogenesis process, either by regulating tumor microenvironment or by promoting systemic changes. This work intends to provide an overview of the research done in this area and try to unravel the role of adrenergic ligands in the context of breast carcinogenesis. In the initiation phase, adrenergic signaling may favor neoplastic transformation of breast epithelial cells whereas, during cancer progression, may favor the metastatic potential of breast cancer cells. Additionally, adrenergic signaling can alter the function and activity of other cells present in the tumor microenvironment towards a protumor phenotype, namely macrophages, fibroblasts, and by altering adipocyte's function. Adrenergic signaling also promotes angiogenesis and lymphangiogenesis and, systemically, may induce the formation of preneoplastic niches, cancer-associated cachexia and alterations in the immune system which contribute for the loss of quality of life of breast cancer patients and their capacity to fight cancer. Most studies points to a major contribution of ß2 -adrenoceptor activated pathways on these effects. The current knowledge of the mechanistic pathways activated by ß2 -adrenoceptors in physiology and pathophysiology, the availability of selective drugs approved for clinical use and a deeper knowledge of the basic cellular and molecular pathways by which adrenergic stimulation may influence cancer initiation and progression, opens the possibility to use new therapeutic alternatives to improve efficacy of breast cancer treatments.

Changes in esophagus interstitial cells of Cajal in response to acute stress.

BACKGROUND: Thoracic trauma is common, and traffic accident-related traumatic injury can cause acute stress leading to esophageal motility disorders. Interstitial cells of Cajal (ICCs) are regarded as gastrointestinal pacemaker cells. AIM: This study explored the mechanism underlying changes in lower esophagus ICCs under acute stress conditions. METHODS: Fifty adult rabbits, randomly divided into one healthy control and four study groups, were subjected to right chest puncture using a Hopkinson bar. Thereafter, one group was immediately subjected to lower esophagectomy, whereas the other three groups were maintained for 24, 48 and 72 h after puncture and subjected to lower esophagectomy. Immunohistochemistry was used to detect ICC distribution, morphology and density, and TUNEL assays were used to determine ICC apoptosis. Enzyme-linked immunosorbent assays (ELISAs) were used to measure cortisol, epinephrine, dopamine, IL-9, cholecystokinin (CCK) and vasoactive intestinal peptide (VIP). Western blotting and RT-PCR were performed to detect changes in SCF/c-kit and nNOS pathways. RESULTS: After puncture, lung tissue was hemorrhaged, alveoli in puncture areas were destroyed, esophageal pH was decreased, and serum cortisol, epinephrine and dopamine levels increased. ICC numbers increased and apoptotic ICCs decreased in all stress groups after puncture (all p < .01). IL-9, CCK and VIP levels in lower esophagus tissue were increased after puncture (all p < .01). Moreover, SCF/c-kit and nNOS pathways were upregulated in response to stress (all p < .01). CONCLUSIONS: Acute stress promotes increases in lower esophageal ICCs that might affect esophagus ICC functions and esophageal motility.