Epinephrine Stimulation Research Articles

Ex vivo evaluation of the whole heart function allowing selective investigation of the right and left heart

Objectives. The aim was to demonstrate a reliable ex vivo method to test the function of the whole heart. Design. Pigs of varying sizes (44–80 kg) were exposed to dose response of adrenaline. Blood pressures and cardiac output were measured. The explanted hearts were tested in a novel ex vivo system to see if we could replicate the in vivo values at maximal adrenaline stimulation. The perfusion solution was STEEN Solution™ with erythrocytes and continuous infusion of essential drugs. In contrast to normal body circulation which is sequential, the heart evaluation system is divided into left and right heart circuits which are operating in parallel, making it possible to test the right and left heart individually or as a whole. The system provides coronary flow measurements. The nonlinear dynamic resistances are constructed to stabilize systolic and diastolic pressures in a broad range and independently from cardiac output. It is important for the functional evaluation to avoid pumping help for the heart; therefore, atrial vortexes are constructed to minimize pump flow directionality and energy from entering atria. Results. Ex vivo evaluation was able to match the maximal in vivo effect of adrenaline on cardiac output and blood pressures. After 2 h of evaluation, the blood gases and lactate were normal and free haemoglobin was zero. Autopsy of the hearts showed no macroscopic pathology. Conclusions. The system is able to give a reliable functional evaluation of the heart ex vivo.

Chronic stimulation desensitizes β2-adrenergic receptor responses in natural killer cells.

Adrenergic receptors (ARs) are preferentially expressed by innate lymphocytes such as natural killer (NK) cells. Here, we study the effect of epinephrine-mediated stimulation of the β2-adrenergic receptor (β2AR) on the function of human NK cells. Epinephrine stimulation inhibited early NK cell signaling events and blocked the function of the integrin LFA-1. This reduced the adhesion of NK cells to ICAM-1, explaining how NK cells are mobilized into the peripheral blood upon epinephrine release during acute stress or exercise. Additionally, epinephrine stimulation transiently reduced NK cell degranulation, serial killing, and cytokine production and affected metabolic changes upon NK cell activation via the cAMP-protein kinase A (PKA) pathway. Repeated exposure to β2AR agonists resulted in the desensitization of the β2AR via a PKA feedback loop-initiated G-protein switch. Therefore, acute epinephrine stimulation of chronically β2AR stimulated NK cells no longer resulted in inhibited signaling and reduced LFA-1 activity. Sustained stimulation by long-acting β2-agonists (LABA) not only inhibited NK cell functions but also resulted in desensitization of the β2AR. However, peripheral NK cells from LABA-treated asthma patients still reacted unchanged to epinephrine stimulation, demonstrating that local LABA administration does not result in detectable systemic effects on NK cells.

The fully activated open state of KCNQ1 controls the cardiac "fight-or-flight" response.

The increase of I Ks potassium currents with adrenalin stimulation is important for "fight-or-flight" responses. Mutations of the IKs channel reducing adrenalin responses are associated with more lethal form of the type-1 long-QT syndrome (LQT). The alpha subunit of the IKs channel, KCNQ1 opens in two distinct open states, the intermediate-open (IO) and activated-open (AO) states, following a two-step voltage sensing domain (VSD) activation process. We found that the AO state, but not the IO state, is responsible for the adrenalin response. Modulators that specifically enhance the AO state occupancy can enhance adrenalin responses of the WT and LQT-associated mutant channels. These results reveal a mechanism of state dependent modulation of ion channels and provide an anti-arrhythmic strategy.

Chronic Epinephrine-Induced Endoplasmic Reticulum and Oxidative Stress Impairs Pancreatic β-Cells Function and Fate.

Epinephrine influences the function of pancreatic β-cells, primarily through the α2A-adrenergic receptor (α2A-AR) on their plasma membrane. Previous studies indicate that epinephrine transiently suppresses insulin secretion, whereas prolonged exposure induces its compensatory secretion. Nonetheless, the impact of epinephrine-induced α2A-AR signaling on the survival and function of pancreatic β-cells, particularly the impact of reprogramming after their removal from sustained epinephrine stimulation, remains elusive. In the present study, we applied MIN6, a murine insulinoma cell line, with 3 days of high concentration epinephrine incubation and 2 days of standard incubation, explored cell function and activity, and analyzed relevant regulatory pathways. The results showed that chronic epinephrine incubation led to the desensitization of α2A-AR and enhanced insulin secretion. An increased number of docked insulin granules and impaired Syntaxin-2 was found after chronic epinephrine exposure. Growth curve and cell cycle analyses showed the inhibition of cell proliferation. Transcriptome analysis showed the occurrence of endoplasmic reticulum stress (ER stress) and oxidative stress, such as the presence of BiP, CHOP, IRE1, ATF4, and XBP, affecting cellular endoplasmic reticulum function and survival, along with UCP2, OPA1, PINK, and PRKN, associated with mitochondrial dysfunction. Consequently, we conclude that chronic exposure to epinephrine induces α2A-AR desensitization and leads to ER and oxidative stress, impairing protein processing and mitochondrial function, leading to modified pancreatic β-cell secretory function and cell fate.

Decreased platelet activation predicts hepatic decompensation and mortality in patients with cirrhosis.

Patients with cirrhosis show alterations in primary hemostasis, yet prognostic implications of changes in platelet activation remain controversial, and assay validity is often limited by thrombocytopenia. We aimed to study the prognostic role of platelet activation in cirrhosis, focusing on bleeding/thromboembolic events, decompensation, and mortality. We prospectively included 107 patients with cirrhosis undergoing a same-day hepatic venous pressure gradient (HVPG) and platelet activation measurement. Platelet activation was assessed using flow cytometry after protease-activated receptor (PAR)-1, PAR-4, or epinephrine stimulation. Over a follow-up of 25.3 (IQR: 15.7-31.2) months, first/further decompensation occurred in 29 patients and 17 died. More pronounced platelet activation was associated with an improved prognosis, even after adjusting for systemic inflammation, HVPG, and disease severity. Specifically, higher PAR-4-inducible platelet activation was independently linked to a lower decompensation risk [adjusted HR per 100 MFI (median fluorescence intensity): 0.95 (95% CI: 0.90-0.99); p =0.036] and higher PAR-1-inducible platelet activation was independently linked to longer survival [adjusted HR per 100 MFI: 0.93 (95% CI: 0.87-0.99); p =0.040]. Thromboembolic events occurred in eight patients (75% nontumoral portal vein thrombosis [PVT]). Higher epinephrine-inducible platelet activation was associated with an increased risk of thrombosis [HR per 10 MFI: 1.07 (95% CI: 1.02-1.12); p =0.007] and PVT [HR per 10 MFI: 1.08 (95% CI: 1.02-1.14); p =0.004]. In contrast, of the 11 major bleedings that occurred, 9 were portal hypertension related, and HVPG thus emerged as the primary risk factor. Preserved PAR-1- and PAR-4-inducible platelet activation was linked to a lower risk of decompensation and death. In contrast, higher epinephrine-inducible platelet activation was a risk factor for thromboembolism and PVT.

Glaesserella parasuis QseBC two-component system senses epinephrine and regulates capD expression.

Glaesserella parasuis, a common inhabitant of the upper respiratory tract of pigs, is the etiological agent of Glässer's disease, which is characterized by fibrinous polyserositis, polyarthritis, and meningitis. Decreased production of capsular polysaccharide (CPS) promotes bacterial adhesion and uptake, whereas maximal expression of CPS is essential to evade the host immune response. For bacterial survival under highly variable host conditions, a coordinated regulation of CPS expression is necessary. In the present study, we investigated how epinephrine (Epi) affects the CPS production, biofilm formation, and adhesion ability of G. parasuis, as well as the specific mechanism of G. parasuis changes in response to Epi. The results demonstrated that Epi stimulation dramatically inhibited CPS expression, and both the action of Epi and the deletion of CPS biosynthesis gene capD had detrimental impacts on biofilm formation, and had the enhanced ability for cell adhesion. QseBC two-component system is a typical adrenaline-sensing system in bacteria. In the present study, by quantitative real-time PCR, Western blot, and electrophoretic mobility shift assay, we discovered that in the presence of Epi, G. parasuis negatively regulated capD expression via the interaction of QseB with the promoter of capD, thereby affecting the synthesis of CPS. In conclusion, we explored the impact of Epi on CPS synthesis and defined its regulatory mechanism in G. parasuis for the first time, which provides an important theoretical basis for understanding the pathogenic mechanism of G. parasuis. IMPORTANCE The key bacterial pathogen Glaesserella parasuis, which can cause Glässer's disease, has caused significant financial losses to the swine industry worldwide. Capsular polysaccharide (CPS) is an important virulence factor for bacteria, providing the ability to avoid recognition and killing by the host immune system. Exploring the alteration of CPS synthesis in G. parasuis in response to epinephrine stimulation can lay the groundwork for revealing the pathogenic mechanism of G. parasuis as well as providing ideas for Glässer's disease control.

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 β-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.

Recurrent hypoglycemia increases hepatic gluconeogenesis without affecting glycogen metabolism or systemic lipolysis in rat

IntroductionRecurrent hypoglycemia (RH) impairs secretion of counterregulatory hormones. Whether and how RH affects responses within metabolically important peripheral organs to counterregulatory hormones are poorly understood. ObjectiveTo study the effects of RH on metabolic pathways associated with glucose counterregulation within liver, white adipose tissue and skeletal muscle. MethodsUsing a widely adopted rodent model of 3-day recurrent hypoglycemia, we first checked expression of counterregulatory hormone G-protein coupled receptors (GPCRs), their inhibitory regulators and downstream enzymes catalyzing glycogen metabolism, gluconeogenesis and lipolysis by qPCR and western blot. Then, we examined epinephrine-induced phosphorylation of PKA substrates to validate adrenergic sensitivity in each organ. Next, we measured hepatic and skeletal glycogen content, degree of breakdown by epinephrine and abundance of phosphorylated glycogen phosphorylase under hypoglycemia and that of phosphorylated glycogen synthase during recovery to evaluate glycogen turnover. Further, we performed pyruvate and lactate tolerance tests to assess gluconeogenesis. Additionally, we measured circulating FFA and glycerol to check lipolysis. The abovementioned studies were repeated in streptozotocin-induced diabetic rat model. Finally, we conducted epinephrine tolerance test to investigate systemic glycemic excursions to counterregulatory hormones. Saline-injected rats served as controls. ResultsRH increased counterregulatory hormone GPCR signaling in liver and epidydimal white adipose tissue (eWAT), but not in skeletal muscle. For glycogen metabolism, RH did not affect total content or epinephrine-stimulated breakdown in liver and skeletal muscle. Although RH decreased expression of phosphorylated glycogen synthase 2, it did not affect hepatic glycogen biosynthesis during recovery from hypoglycemia or after fasting-refeeding. For gluconeogenesis, RH upregulated fructose 1,6-bisphosphatase 1 and monocarboxylic acid transporter 1 that imports lactate as precursor, resulting in a lower blood lactate profile during hypoglycemia. In agreement, RH elevated fasting blood glucose and caused higher glycemic excursions during pyruvate tolerance test. For lipolysis, RH did not affect circulating levels of FFA and glycerol after overnight fasting or upon epinephrine stimulation. Interestingly, RH upregulated the trophic fatty acid transporter FATP1 and glucose transporter GLUT4 to increase lipogenesis in eWAT. These aforementioned changes of gluconeogenesis, lipolysis and lipogenesis were validated in streptozotocin-diabetic rats. Finally, RH increased insulin sensitivity to accelerate glucose disposal, which was attributable to upregulated visceral adipose GLUT4. ConclusionsRH caused metabolic adaptations related to counterregulation within peripheral organs. Specifically, adrenergic signaling was enhanced in liver and visceral fat, but not in skeletal muscle. Glycogen metabolism remained unchanged. Hepatic gluconeogenesis was augmented. Systemic lipolysis was unaffected, but visceral lipogenesis was enhanced. Insulin sensitivity was increased. These findings provided insights into mechanisms underlying clinical problems associated with intensive insulin therapy, such as high gluconeogenic flux and body weight gain.

Abstract 2543: Chronic stress promotes tumor-associated inflammation in high-grade serous ovarian cancer

Abstract A cancer diagnosis increases stress hormones levels and leads to altered psychological states. In the context of ovarian cancer (OC), chronic stress promotes tumor growth, chemoresistance and modulates immune cell populations in the tumor microenvironment (TME). Moreover, previous work from our team suggests that chronic stress promotes an increased inflammatory response in OC. Our data show an altered CD4+/CD8+ T-cell ratio and a heterogeneous expression of exhaustion markers in patients with high-grade serous ovarian cancer (HGSOC). Therefore, we hypothesized that chronic stress results in chronic inflammation and an immunosuppressed TME. To address this, we obtained ascites from 66 patients with HGSOC and measured cytokine levels using a comprehensive Cytokine/Chemokine immunoassay. Cortisol, corticosterone, and stress hormone metabolites (metanephrine and normetanephrine) levels from ascites were measured by ELISA. CD8+ T-cells isolated from OC patient ascites were stimulated with epinephrine, and flow cytometry was used to measure co-expression of CD38 activation marker and Granzyme B, an essential mediator of CD8+ T-cell killing capacity. To further establish the impact of chronic stress on tumor progression, we subjected IG10 or ID8 tumor-bearing C57/BL6 female mice to daily restraint stress. Mice were sacrificed 8-12 weeks after inoculation, data collected, and ascites stored. Results showed an increase in inflammatory cytokines (Eotaxin, IL-6, and IL-7) in recurrent tumors of HGSOC patients. IP-10 and IFN-γ were negatively associated with cortisol levels. Moreover, normetanephrine levels positively correlated with inflammatory cytokines: IL-6, MCP-1, MCP-3, VEGF, GRO, and IL-7. Metanephrine was also positively correlated with inflammatory cytokines: SCD40L, FGF-2, and MIP1α. Ascites-derived CD8+ T-cells treated with epinephrine showed decreased co-expression CD38 and Granzyme B. Additionally, data show that daily restraint stress led to increased tumor growth in ID8 and IG10 syngeneic mouse models of OC. Ascites derived from our animal models suggest a positive correlation of inflammatory cytokines VEGF, TNFα and Eotaxin, and corticosterone levels in ID8 tumor-bearing mice. These results suggest a role for stress hormones in inflammation and immunosuppression. In conclusion, inflammatory cytokines are upregulated in recurrent HGSOC ascites samples. Cytokines that regulate T-cell function were negatively associated with cortisol levels, while stress hormone metabolites correlated with higher inflammatory cytokines. Moreover, epinephrine stimulation decreased ascites-derived CD8+ T-cell function. Inflammatory cytokines correlated with corticosterone levels in ID8 tumor-bearing mice. Overall, these data suggest a role for chronic stress in inflammation and immunosuppression, impacting the efficacy of therapies that aim to restore T-cell function. Citation Format: Alexandra N. Aquino-Acevedo, Hope Knochenhauer, Melanie Ortiz-León, Yadiel A. Rivera-López, Margarita Bonilla-Claudio, John S. Yi, Rebecca A. Previs, Guillermo N. Armaiz-Pena. Chronic stress promotes tumor-associated inflammation in high-grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2543.

The diagnostic performance of platelet function analyzer-100 (PFA-100) in Thai children with mucocutaneous bleeding disorder

BackgroundClosure time (CT), a parameter measured by platelet function analyzer (PFA-100), is used to assess primary hemostasis. However, some factors (e.g., ethnicity and age) may affect its cut-off value or reference range. Moreover, the diagnostic performance of CT in the Thai and Southeast Asian populations has not been reported in any study. Therefore, this study aims to evaluate the diagnostic performance of CT in patients with mucocutaneous bleeding disorder using the established cut-off value from Thai children. MethodsCT from both collagen/epinephrine (COL/EPI) and collagen/adenosine 5- diphosphate (COL/ADP) cartridges and complete blood count were performed in both healthy volunteers and known cases of primary hemostatic disorders. The cut-off value and diagnostic performance of CT was determined using receiver operating characteristic curve analysis. ResultsThe cut-off value of both COL/EPI and COL/ADP in this study was somewhat prolonged than the other studies because of the higher prevalence of non-responsiveness of platelets to epinephrine stimulation in the Asian than the Western. Nonetheless, the diagnostic performance was comparable to the previous studies. In addition, the diagnostic performance could be enhanced when the CT from both cartridges was interpreted together. ConclusionThe cut-off value in this study was more prolonged than in other studies due to the variation of ethnicity. Consequently, each laboratory should establish and/or verify its own CT reference range prior to use in clinical practice. Furthermore, CT interpretation of both cartridges together resulted in a better diagnostic performance than interpreting each cartridge separately.

Clinical Update on Vasopressors and Titration Strategies.

Clinical Update on Vasopressors and Titration Strategies.

Abstract 11420: Platelet Activity Mediates Increased Cardiovascular Risk in Patients with End-Stage Kidney Disease and Peripheral Artery Disease

Background: End-stage kidney disease (ESKD) and peripheral artery disease (PAD) are frequently co-prevalent and each associated with increased cardiovascular (CV) risk. Platelets drive PAD pathogenesis and mediate atherothrombosis. Their function and influence on CV risk in ESKD remain unclear. We hypothesized that ESKD is associated with heightened platelet activity and incident cardiac and limb events in patients with PAD. Methods: The Platelet Activity and Cardiovascular Events study enrolled 289 patients with PAD undergoing lower extremity revascularization (LER). We measured platelet activity prior to LER via light transmission aggregometry in response to submaximal ADP, collagen, serotonin, epinephrine, and arachidonic acid. We followed patients for a median of 18 months for a primary endpoint of major adverse CV event (MACE; MI, stroke, death) and a secondary composite of major adverse CV or limb event (MACLE; MACE or acute limb ischemia). Results: Among enrolled subjects, 25 (9%) had ESKD. They were younger and more likely to have a history of coronary artery disease (CAD), heart failure, and critical limb ischemia (CLI) than those without ESKD (P<0.05 for each). Aggregation in response to epinephrine 2.0 uM after 5 minutes and at maximum was greater in those with compared to without ESKD (61% vs. 48%; P=0.004 and 77% vs. 60%; P=0.001, respectively). After multivariable adjustment, patients with vs. without ESKD were at greater risk for CV events ( Figure ). Platelet aggregation in response to submaximal epinephrine stimulation had a 32% and 45% mediating effect on the excess risk for MACE and MACLE, respectively, associated with ESKD ( Figure ). Conclusions: In patients with PAD, ESKD was associated with increased platelet aggregation in response to submaximal epinephrine that partially mediated increased risk for incident cardiac and limb events. Platelet activity appears to be an important mechanism underlying CV risk in ESKD.

Elevated MAOA impairs intracellular β 1 ‐adrenegic signaling and myocardial contractility in heart failure

Rationale: We recently identified a pool of β1ARs at the sarcoplasmic reticulum (SR) senses cytosolic catecholamines and promotes cardiac contractility. Monoamine oxidase A (MAOA), the major enzyme metabolizing myocardial catecholamines is upregulated in heart failure (HF) together with blunted β1AR signaling. However, the regulation of intracellular β1ARs signaling in HF remains unknown. Objective To uncover the role of MAOA in the spatiotemporal regulation of intracellular β1AR signaling and cardiac contractility. Methods and Results Echocardiogram analysis showed that cardiac-specific deletion (cKO) or pharmacological inhibition of MAOA did not affect the baseline cardiac output but significantly amplified the inotropy of catecholamine (epinephrine) in vivo. Enzyme immunoassay revealed elevated catecholamine concentrations and cAMP/PKA activities in MAOA-cKO mice hearts compared with their flox/flox (FF) littermates. We measured the subcellular-localized β1AR in living cardiomyocytes using PKA biosensors targeted at SR or plasma membrane (PM). Compared with FF cardiomyocytes, MAOA-cKO exhibited an increased PKA activity at SR, but not PM upon epinephrine stimulation. Consistently, phosphorylation of phospholamban (PLB), a major target of SR-localized β1AR signals, was enhanced by MAOA-cKO. More importantly, deleting MAOA increased the response of calcium transients and contractility to epinephrine. Blocking the entry of catecholamines by organic cation transporter 3 (OCT3) inhibitor abolished the differential effects in MAOA-cKO vs FF. Similarly, the effect of MAOA-cKO on intracellular β1AR/PKA/PLB signaling and cardiac excitation-contraction coupling (EC-coupling) was recapitulated by pharmacological inhibition of MAOA. In contrast, overexpression of MAOA in cardiomyocytes suppressed epinephrine-induced PKA activation at the SR, PLB phosphorylation and contractility. Furthermore, the increased expression of MAOA in failing cardiomyocytes suppressed activation of intracellular β1AR and attenuated PKA phosphorylation of PLB and inotropic responses. Inhibition of MAOA restored intracellular β1AR signaling and EC coupling in failing cardiomyocytes, and rescued the inotropy response in HF mice. Conclusion MAOA modulates catecholamine-promoted EC coupling via tuning the intracellular β1AR signaling and PKA phosphorylation of PLB. Inhibition of MAOA restores β1AR/PKA signaling at the SR and cardiac contractility in HF.

Adrenergic signaling promotes the expansion of cancer stem-like cells of malignant peripheral nerve sheath tumors

Malignant peripheral nerve sheath tumor (MPNST), a highly malignant tumor that arises in peripheral nerve tissues, is known to be highly resistant to radiation and chemotherapy. Although there are several reports on genetic mutations and epigenetic changes that define the pathogenesis of MPNST, there is insufficient information regarding the microenvironment that contributes to the malignancy of MPNST. In the present study, we demonstrate that adrenaline increases the cancer stem cell population in MPNST. This effect is mediated by adrenaline stimulation of beta-2 adrenergic receptor (ADRB2), which activates the Hippo transducer, YAP/TAZ. Inhibition and RNAi experiments revealed that inhibition of ADRB2 attenuated the adrenaline-triggered activity of YAP/TAZ and subsequently attenuated MPNST cells stemness. Furthermore, ADRB2-YAP/TAZ axis was confirmed in the MPNST patients’ specimens. The prognosis of patients with high levels of ADRB2 was found to be significantly worse. These data show that adrenaline exacerbates MPNST prognosis and may aid the development of new treatment strategies for MPNST.

POS-087 ACUTE METABOLIC ACIDOSIS IN ALCOHOL ABUSERS IS HIGHLY ASSOCIATED WITH LACTIC ACIDOSIS

It has long been known that alcohol abuse combined with dehydration and starvation causes acute metabolic acidosis. Recently, it has attracted attention because of its pathogenic similarity to SGLT2i-induced euglycemic ketoacidosis. In these conditions, the accumulation of acids, specifically lactic acid, other than ketones is considered rare except in special circumstances, such as sepsis and vitamin deficiencies. However, its incidence in clinical practice seems to be more frequent. Therefore, we reviewed the present situation at our hospital to determine the etiology of lactic acid accumulation. From January 2013 to June 2020, we extracted data of patients with blood gas conditions of pH <7.30 and HCO3 <18 mEq/l in our hospital. Then, we defined cases with documented illnesses suggestive of alcohol abuse and a clear indication of prior heavy drinking as the alcohol-related acidosis (AIA) group. To understand the direct effect of alcohol, cases with proven infection were excluded. As a control, diabetic ketoacidosis (DKA) patients >18 years of age with the same blood gas condition were included. In 7.5 years, 28,697 blood gas analyses were performed, of which 1404 met the defined blood gas conditions. Among them, 25 AIA and 20 DKA patients met the inclusion criteria. Urinary ketones were 90% and 67% positive in DKA and AIA patients, respectively. As expected, lactate was significantly higher in AIA than in DKA (16.4 ± 8.4 vs. 4.6 ± 2.7 mmol/l p<0.001). There was no difference in oxygenation or renal function between the two groups at the time of admission. However, blood pressure and blood glucose levels were lower and liver deviation enzymes were higher in the AIA group than in the DKA group. Although in AIA, lactic acid is thought to be a contributing factor for circulatory failure due to dehydration, there was no significant difference in the amount of fluid infused up to 6 h from the start of treatment between the two groups (2.6 L in AIA vs. 3.0 L in DKA, p=0.4). Additionally, there was no difference in the time from the start of treatment to improvement in acidosis (pH>7.3. 6.7 h in AIA and 7.5 h in DKA, p=0.5). Moreover, there was no correlation between the amount of fluid and time to improve acidosis in the 22 AIA patients who were treated with fluid alone (r = -0.004). Among study participants divided into two groups according to median fluid intake in the AIA group (mean 1.3. vs. 3.9 L / 6 hours, respectively; p<0.001), there was no difference in time to improvement in acidosis (7.0 vs. 6.2 hours, p=0.6) Acute metabolic acidosis that occurs in alcohol abusers was accompanied by a high frequency of lactic acid accumulation and ketones. Furthermore, the standard therapy of dehydration correction was not an effective treatment. Recently, in the area of sepsis, it is believed that the cause of elevated lactate is not circulating insufficiency but overproduction due to adrenaline stimulation or decreased excretion due to hepatic and renal damage; therefore, excessive fluid infusion should be avoided. These results suggest that alcohol-induced lactic acid accumulation may be due to similar reasons.

Influence of rs1042713 and rs1042714 polymorphisms of β2-adrenergic receptor gene with erythrocyte cAMP in sickle cell disease patients from Odisha State, India.

The vaso-occlusive crisis (VOCs) in sickle cell disease (SCD) is often associated with stress. Epinephrine released during stress acts via beta 2-adrenergic receptors (β2-AR or ADRB2) to stimulate the synthesis of cyclic adenosine monophosphate (cAMP) in the red blood cells (RBCs). Higher cAMP levels promote adhesion of sickled RBCs to vascular endothelium, a major contributor for VOCs. Several single-nucleotide polymorphisms (SNPs) of the β2-AR gene have been reported; two of them at codon 16 (rs1042713) and codon 27 (rs1042714) have been extensively studied for their clinical relevance. Therefore, we assessed the influence of polymorphism at these two sites of the β2-AR gene on the RBC cAMP concentrations with and without epinephrine stimulation in SCD subjects. We determined the frequency distribution of different genotypes of codon 16 and codon 27 of the β2-AR gene using the Sanger sequencing method in the SCD subjects. We measured the RBC-cAMP levels at baseline and after stimulation with epinephrine, to ascertain the influence of different genotypes in determining cAMP levels. There was no difference in the socio-demographic and hematological indicators in different genotypes of both codon 16 and 27. In the sham-treated erythrocytes, the cAMP levels were significantly different with three genotypes of codon 16 (F = 3.39, P = 0.036; one way ANOVA) but not with different genotypes of codon 27. A significant increase in cAMP levels was noticed with epinephrine treatment in all genotypes of codons 16 and 27 (P = 0.001; Wilcoxon signed-rank test). However, the extent of increase in the epinephrine-treated cAMP values from the sham-treated (baseline) cAMP values was significantly different between the three genotypes of codon 16 (H = 8.74; P = 0.012; Kruskal-Wallis test) but not in codon 27 genotypes. Polymorphism in codon 16 (rs1042713) of the β2-AR gene influences cAMP concentrations in the RBC both before and after epinephrine treatment. Higher cAMP levels may lead to increased adhesion of sickle cell RBCs to vascular endothelium and may increase the frequency of VOCs.

STATE OF THE PLATELET HEMOSTASIS IN THE MIDDLE-AGED AND THE ELDERLY HYPERTENSIVE PATIENTS

It has long been known that complications of essential hypertension are associated mostly with disorders of blood coagulation system and degree of absolute risk of cardiovascular events is growing with increasing of age. That is why much attention is paid to more detailed study of mechanisms of thrombosis as the component of complications of essential hypertension through lens of hemostatic changes at present. Therefore, regulatory mechanisms of initiation of thrombus formation and participation of platelet hemostasis are studied in hypertensive patients insufficiently today.&#x0D; The aim of the study was to identify the baseline levels of the platelet hemostasis indices in the middle-aged and the elderly hypertensive patients.&#x0D; Material and methods. We performed the comparative assessment of status of the platelet hemostasis in 63 middle-aged (group I) and 53 elderly patients (group II) respectively to clarify this issue in our work. Patients of the surveyed groups were comparable in age, sex and related pathology. The control group consisted of 15 patients for every of the surveyed groups (group III the middle-aged and group IV the elderly respectively) matched with basic by age and gender.&#x0D; Conclusions. Therefore, there is the increase in platelet activity depending on age in the middle-aged and the elderly hypertensive patients that is manifested by adrenaline stimulation most clearly. In addition, the most significant changes are determined in the spontaneous and the adrenaline-induced aggregation namely in amplitude and slope of aggregation in the spontaneous and time of maximal aggregation in the adrenaline-induced aggregation in the patients of older age group compared with the middle-aged patients. The substantial deceleration of time of maximal aggregation during the adrenaline stimulated aggregation in 1,6 times (p&lt;0,001) in the elderly patients confirms the expressed thrombogenicity and sensitiveness to neurohumoral influences too. Thus, the blood plasma thrombogenicity revealed in the study of the platelet hemostasis in the elderly indicates a high probability of thrombotic complications developing in this category of the patients and this is consistent with literature.

Spatiotemporal Control of ERK Activity by b2‐adrenergic Signaling

G‐protein coupled receptors (GPCRs) play vital roles in regulating physiological functions, such as sympathetic nervous regulation, and senses of vision and olfaction. Signaling of the β2‐adrenergic receptor (β2‐AR), which is a prototypical class A GPCR, has been proposed to control activation of ERK, subsequently regulating different processes including gene expression. However, the molecular mechanism of ERK activation downstream of β2AR signaling is not clearly understood. Here we investigate the molecular mechanism of β2‐AR mediated ERK activation using improved version of FRET‐based ERK biosensors targeted to various subcellular regions which enable us to visualize spatial and temporal regulation of ERK activity in living cells. Epinephrine stimulation leads to increases of ERK activity at the endosome and in the nucleus but not at the plasma membrane. An Nb37 nanobody that stables the active Gαsconformation enhances and prolongs the endosomal and nuclear ERK activity. We propose a new model that an endosome‐localized active β2AR recruits critical signaling components to facilitate ERK activation on the endosome. These findings provide a molecular basis for location selective activation of ERK by β2‐AR signaling.Support or Funding InformationThis work was supported by the National Institutes of Health (R01 DK073368 to J.Z.)

β-Adrenergic stimuli and rotating suspension culture enhance conversion of human adipogenic mesenchymal stem cells into highly conductive cardiac progenitors.

Clinical trials using human adipogenic mesenchymal stem cells (hAdMSCs) for the treatment of cardiac diseases have shown improvement in cardiac function and were proven safe. However, hAdMSCs do not convert efficiently into cardiomyocytes (CMs) or vasculature. Thus, reprogramming hAdMSCs into myocyte progenitors may fare better in future investigations. To reprogramme hAdMSCs into electrically conductive cardiac progenitor cells, we pioneered a three-step reprogramming strategy that uses proven MESP1/ETS2 transcription factors, β-adrenergic and hypoxic signalling induced in three-dimensional (3D) cardiospheres. In Stage 1, ETS2 and MESP1 activated NNKX2.5, TBX5, MEF2C, dHAND, and GATA4 during the conversion of hAdMSCs into cardiac progenitor cells. Next, in Stage 2, β2AR activation repositioned cardiac progenitors into de novo immature conductive cardiac cells, along with the appearance of RYR2, CAV2.1, CAV3.1, NAV1.5, SERCA2, and CX45 gene transcripts and displayed action potentials. In Stage 3, electrical conduction that was fostered by 3D cardiospheres formed in a Synthecon®, Inc. rotating bioreactor induced the appearance of hypoxic genes: HIF-1α/β, PCG 1α/β, and NOS2, which coincided with the robust activation of adult contractile genes including MLC2v, TNNT2, and TNNI3, ion channel genes, and the appearance of hyperpolarization-activated and cyclic nucleotide-gated channels (HCN1-4). Conduction velocities doubled to ~200 mm/s after hypoxia and doubled yet again after dissociation of the 3D cell clusters to ~400 mm/s. By comparison, normal conduction velocities within working ventricular myocytes in the whole heart range from 0.5 to 1 m/s. Epinephrine stimulation of stage 3 cardiac cells in patches resulted in an increase in amplitude of the electrical wave, indicative of conductive cardiac cells. Our efficient protocol that converted hAdMSCs into highly conductive cardiac progenitors demonstrated the potential utilization of stage 3 cells for tissue engineering applications for cardiac repair.

Left Ventricular Function During Epinephrine Stimulation and Hypothermia: Effects at Spontaneous and Paced Heart Rates in a Porcine Model.

Postcardiac arrest patients treated with hypothermia, frequently require vasopressors and inotropic medication. The aim of this experimental study was to investigate the effect of epinephrine on left ventricular (LV) function during hypothermia. In an open-chest porcine model, seven animals were equipped with LV micromanometer and epicardial ultrasound transducers to provide LV pressure, Tau, and wall thickness and thickening velocities in systole (S') and early diastole (e'). Arterial, central venous, and pulmonal artery pressures were recorded. Cardiac output (CO) was measured by transit-time flow probe on the ascending aorta. Hypothermia was induced using a cooling catheter through the femoral vein. Pacemaker leads were attached to the right atrium for pacing. LV volumes were obtained by two-dimensional echocardiography. Measurements were made at normothermia (38°C) and hypothermia (33°C), without and with epinephrine infusion (0.03 μg/kg/min), at spontaneous and paced heart rates (HRs) 120 and 140 beats/min. Hypothermia reduced LV stroke volume (SV). Epinephrine during hypothermia increased the SV with reduced end-systolic volumes. LV dP/dtmax and wall-thickening velocity increased. During normothermia, epinephrine increased CO mainly due to accelerated HR, but during hypothermia, the increased CO resulted from augmented SV and, to a lesser degree, elevated HR. The incomplete relaxation and shortened diastolic filling time and the following reduction in SV seen in hypothermic animals, was repealed by epinephrine. The CO remained elevated also due to a shortened systolic duration, which gave time for complete relaxation during higher HRs. Epinephrine infusion improved systolic and diastolic function during hypothermia, and thereby reversed the effects induced by hypothermia considerably. Epinephrine augmented CO at hypothermia through increases in both SV and HR, in contrast to a mainly HR-dependent effect during normothermia. Systolic duration was shortened, which gave sufficient diastolic duration for complete relaxation. This allowed diastolic filling and maintained CO at elevated HRs.