Stress Hormones Norepinephrine Research Articles

Cytokine production profiles of immune cells of stressed beta2-adrenergic receptor knockout mouse model during Chlamydia muridarumgenital infection

Abstract Chlamydia genital infection by Chlamydia trachomatisis the most common bacterial sexually transmitted disease worldwide. The relationships between stress and chlamydia genital infection remain unknown. Beta2-adrenergic receptor(β2-AR), the major receptor of the stress hormone norepinephrine (NE), is known to impair the function of immune cells. Stress was induced by immersing mice in cold water for five minutes daily for 21 days before infection. This study determined profiles of immune cells of stressed β2-AR KO and stressed WT C57BL/6J after 48 h of Chlamydia muridarumgenital infection. Splenic T cells and differentiated bone marrow-derived macrophages and dendric cells (DCs) were purified by negative selection and stimulated by seeding T cells on anti-CD3/CD28 antibodies coated wells and by treating macrophages and DCs with LPS. After 48 h proliferation, cytokine levels in culture supernatants of the cells were determined using ELISA. Results show increased IFN-γ and IL-1β secretions but deceased IL-4 secretion by CD4+ T cells of β2-AR KO compared to WT (p<0.05). The secretion of IL-13, IL-5, and IL-10 but not IL-23 in CD4 T cells of stressed β2-AR KO mice was two-fold lower than that of stressed WT. TNF-α and IL-1β secretions in macrophages and DCs or IL-12 secretion in DCs was high in stressed β2-AR KO compared to WT (p<0.05). Overall, this study provides insights into the β2-AR signaling pathway’s role in suppressing the secretion of protective cytokines suggesting that inhibiting β2-AR signaling pathway may restore the function of immune cells during chlamydia genital infection. However, further study is warranted. This work was funded by NIH grant # 1R15AI124156-01 awarded to Bluefield State University

Characterization of immune cells of stressed beta2-adrenergic receptor homozygous knockout mouse model during Chlamydia muridarum genital infection

Abstract The relationship of stress in chlamydia genital disease pathogenesis and its influence on the host immune response remains unknown. Beta2-adrenergic receptor(β2-AR), the major receptor of the stress hormone norepinephrine (NE), is known to impair the function of immune cells. This study was undertaken to determine whether cold-induced stress (CIS) results in differential viability percent of immune cells β2-AR KO and WT C57BL/6J treated with NE, β2-AR agonist(Fenoterol), or antagonist(ICI 118,551), and patterns of cytokine production. CIS was induced by immersing mice in cold water for five minutes daily for 21 days. Total CD4+, CD8+, differentiated bone marrow-derived monocytes were purified by negative selection and treated with chemicals for one hour before proliferation for 48 h proliferation. The vi-cell viability analyzer showed that NE results in a 30 and 40 % decrease in viability of WT C57BL/6J CD4+ T and CD8+ cells, respectively, compared to KO (p<0.05). Similarly, NE and Fenoterol resulted in a 20 and 27% decrease in viability of bone marrow-derived monocytes compared to the β2-AR KO mice. Cells treated with ICI 118, 551 showed the slightest reduction in viability (12%) compared to those treated with NE. ELISA results show decreased IL-4 and increased IFN-g secretion by CD4+ T cells of β2-AR KO compared to WT. The secretion of TNF-a by CD8+ cells and bone marrow cells treated with NE and β2-AR agonist or antagonist was high in all treatment groups. This study provides insights for the first time to better understand the NE-β2-AR pathway during chlamydia genital infection. Supported by WV-INBRE of NIH Grant P20GM103434 and NIH grant # 1R15AI124156-01 given to BSC.

Production of CD4+ T Cell Subsets in a Beta2-Adrenergic Receptor Knockout Stress Mouse Model during Chlamydia muridarum Genital Infection

Kaitlyn Cook*, Tesfaye Belay, Ph.D*., *Department of Applied Science and Mathematics, Bluefield State College, Bluefield WV 24701 Production of CD4+ T Cell Subsets in a Beta2-Adrenergic Receptor Knockout Stress Mouse Model during Chlamydia muridarum Genital Infection
 Chlamydia is the most widely spread sexually transmitted disease, with more than one hundred million new cases occurring every year worldwide. Studies show the effect of stress promoting infection, but little is known about Chlamydia and the impact of stress. Cold-induced stress was used to simulate stress in mice to understand the relationship between the stress hormone norepinephrine (NE) and the immune system. This study aims to determine the role of beta2-adrenergic receptor (β2-AR) under stressful conditions during infection. We hypothesize the β2-AR suppresses T helper cell 1 (Th1) and promotes the production of Th2 cytokines. Following stressing, infection, sacrificing, and dissection of mice, the spleen, lymph node, and genital tract cells were proliferated for 72 hours. ELISA data for IL-13 concentration shows stressed knockout (KO) mice had 273.6pg/mL compared to stressed wildtype (WT) mice with 120.6pg/mL. Various treatment of CD4+ T cells resulted in production of TNF- α, ranging from 595pg/mL to 398.7pg/mL. Viability of CD4+ T cells treated with β2-AR agonist Fenoterol, and antagonist ICI 118,551 were analyzed. The viability of CD4+ T cells treated with NE was 70.7% in KO, compared to 69.1% and 70.2% for the agonist and antagonist, respectively. NE, Fenoterol, and ICI 118,551 treatment resulted in 72.2%, 83.6% and 84.4% respectively for WT. No significant difference is found between the agonist and antagonist. Chlamydia muridarum isolation from the genital tract of treatment groups is underway. The ELISA data implies that β2-AR mice restored increased production of protective cytokines more than their WT counterparts.

Stress hormone norepinephrine incites resistance of oral cancer cells to chemotherapy.

This study investigated whether norepinephrine (NE) and epinephrine (E) interfere in the response of head and neck squamous cell carcinoma (SCC) cell lines to cisplatin and explored the mechanisms of chemoresistance. Head and neck SCC-derived cell lines SCC-9, Cal27, SCC-25, and FaDu were stimulated with NE or E and treated with the inhibitory concentration of cisplatin for 24 h. As for adrenergic receptors (ADRB) inhibition, cells were treated with propranolol. The results showed that, when combined with NE, cisplatin effectiveness against SCC-9 and Cal27 but not SCC-25 and FaDu cells were notably reduced. E did not affect the response of the cells to cisplatin. Further experiments were performed with the responsive SCC-9 and SCC-25 cell lines and the hormone NE. The time course assay showed that stimulation of oral SCC cells with NE decreased the cleavage of caspase-3 and expression of multidrug resistance protein 1 (MDR-1) but only transiently affected ATP-binding cassette (ABC) subfamily G, isoform 2 protein (ABCG2) expression. The expression of cleaved caspase-3 and Bcl-2 were, respectively, decreased and increased by the combination of NE and cisplatin in SCC-9 and Cal27 cells. NE-induced resistance was reverted by previous treatment with propranolol. Expressions of ABCG2, and p-Akt but not of MDR-1, were enhanced by NE plus cisplatin when compared to cisplatin only in both cell lines. Migratory activity of oral SCC cells challenged with cisplatin was not affected by NE. These findings reveal for the first time that stress hormone NE induces resistance of oral cancer cells to cisplatin in vitro through the ADRB/Akt/ABCG2 pathway, pumping the drug out of the cell and inhibiting apoptosis.

Integrative transcriptomic and proteomic analyses of pathogenic Aeromonas hydrophila in response to stress hormone norepinephrine

Stress is commonly considered as a significant contributing factor in fish disease, and stress hormones could directly influence the pathogenicity of bacteria. To identify the mechanism of Aeromonas hydrophila that responds to norepinephrine (NE), we integrated transcriptomic and proteomic analyses of A. hydrophila grown in serum-SAPI medium with or without NE using RNA Sequencing (RNA-seq) and isobaric tags for relative and absolute quantification (iTRAQ) analyses. There were 406 differentially expression genes (296 upregulated and 110 downregulated) and 62 differentially expression proteins (31 upregulated and 31 downregulated). The integrative analyses showed that 14 genes displayed consistent expression at both transcript and protein levels, while 7 genes exhibited contrasting expression. Validation with real-time quantitative PCR showed that the expression of four selected DEGs was consistent with the pattern of transcriptome profile. Further analyses revealed that the expression of virulence factor transcripts was significantly increased, including haemolysin and structural toxin protein RtxA. Furthermore, the transcripts and proteins involved in iron metabolism were also changed. The expression of bacterioferritin and iron-containing proteins was enhanced at transcript level; conversely, the genes involved in amonabactin synthesis and transport were repressed at both transcript and protein levels. Notably, novel observations revealed that the gene expression of type 6 secretion system (T6SS) and pilus-related loci was inhibited in A. hydrophila after exposure to NE. These results corroborate previous findings that NE can not only improve the growth and virulence of A. hydrophila, but also provide a new suggestion about the T6SS role in bacterial cell–cell interaction for further research.

Stress hormones promote DNA damage in human oral keratinocytes

Chronic stress increases the systemic levels of stress hormones norepinephrine and cortisol. As well as tobacco-specific carcinogen NNK (4-(methylnitrosamine)-1-(3-pyridyl)-1-butanone), they can induce expressive DNA damage contributing to the cancer development. However, it is unknown whether stress hormones have genotoxic effects in oral keratinocytes. This study investigated the effects of stress hormones on DNA damage in a human oral keratinocyte cell line (NOK-SI). NOK-SI cells stimulated with norepinephrine or cortisol showed higher DNA damage compared to untreated cells. Norepinephrine-induced DNA damage was reversed by pre-treatment with beta-adrenergic blocker propranolol. Cells treated with NNK combined to norepinephrine displayed reduced levels of caspases 3 and 7. Cortisol also reduced the activity of pro-apoptotic enzymes. NNK or norepinephrine promoted single-strand breaks and alkali-label side breaks in the DNA of NOK-SI cells. Pre-treatment of cells with propranolol abolished these effects. Carcinogen NNK in the presence or absence of cortisol also induced DNA damage of these cells. The genotoxic effects of cortisol alone and hormone combined with NNK were blocked partially and totally, respectively, by the glucocorticoid receptor antagonist RU486. DNA damage promoted by NNK or cortisol and carcinogen combined to the hormone led to intracellular γH2AX accumulation. The effects caused by NNK and cortisol were reversed by propranolol and glucocorticoid receptor antagonist RU486, respectively. Propranolol inhibited the oxidation of basis induced by NNK in the presence of DNA-formamidopyrimidine glycosylase. DNA breaks induced by norepinephrine in the presence or absence of NNK resulted in higher 8OHdG cellular levels. This effect was also induced through beta-adrenergic receptors. Together, these findings indicate that stress hormones induce DNA damage of oral keratinocytes and could contribute to oral carcinogenesis.

Workplace bullying increases the risk of anxiety through a stress-induced \u03b22-adrenergic receptor mechanism: a multisource study employing an animal model, cell culture experiments and human data

ObjectivesSeveral studies show that severe social stressors, e.g., in the form of exposure to workplace bullying in humans, is associated with negative mental health effects such as depression and anxiety among those targeted. However, the understanding of the underlying biological mechanisms that may explain the relationship between exposure to bullying and such negative health outcomes is scarce. The analyses presented here focus on understanding the role of the β2-adrenergic receptors (ADRB2) on this association.MethodsFirst, a resident-intruder paradigm was used to investigate changes in circulating norepinephrine (NE) in rat serum induced by repeated social defeat and its relationship with subsequent social behavior. Second, the direct effects of the stress-hormones NE and cortisol, i.e., synthetic dexamethasone (DEX), on the ADRB2 expression (qPCR) and monocyte chemoattractant protein-1 (MCP-1) release (immunoassay) was examined in cultured EL-1 cells. Third, in a probability sample of 1052 Norwegian employees, the 9-item short version of the Negative Acts Questionnaire—Revised (S-NAQ) inventory, Hopkins Symptom Checklist and genotyping (SNP TaqMan assay) were used to examine the association between social stress in the form of workplace bullying and anxiety moderated by the ADRB2 genotype (rs1042714) in humans.ResultsThe present study showed a clear association between reduced social interaction and increased level of circulating NE in rats previously exposed to repeated social defeat. Parallel cell culture work, which was performed to examine the direct effects of NE and DEX on ADRB2, demonstrated ADRB2 downregulation and MCP-1 upregulation in cultured EL-1 cells. Genotyping with regard to the ADRB2 genotype; rs1042714 CC vs CG/GG, on human saliva samples, showed that individuals with CC reported more anxiety following exposure to bullying behaviors as compared to the G carriers.ConclusionWe conclude that workplace bullying promotes anxiety and threaten well-being through an ADRB2 associated mechanism.

Effects of norepinephrine on colonic tight junction protein expression during heat stress.

Stress induced by changes in the internal or external environment in humans and animals leads to intestinal epithelial damage, in a manner that is associated with impaired intestinal barrier function. However, the role of the stress hormone norepinephrine (NE) in impairments in barrier function remains poorly understood. In the present study, a rat heat-exposed model was used to observe changes in the tight junction proteins Occludin and zonula occludens-1 (ZO-1), in addition to those in protease-activated receptor 2 (PAR-2) and transient receptor potential ankyrin 1 channel (TRPA1) in colon. The levels of plasma NE were detected using an ELISA kit. Different concentrations of NE were used to culture the human colon cell line Caco-2 for 6 and 24 h to investigate the cell viability using Cell Counting Kit-8 assay, whilst the expression levels of Occludin, ZO-1, PAR-2 and TRPA1 were examined using western blotting and immunofluorescence in Caco-2 cells and immunohistrochemistry in rat colon tissues. Although there was no clear histological damage to the rat colonic mucosa, there were decreased expression levels of tight junction proteins Occludin and ZO-1 after heat exposure. In addition, PAR-2 expression was increased by heat exposure. It was found that TRPA1 expression was concentrated to the luminal surface of the colon in the heat exposed group compared with that in the control group. After the administration of increasing concentrations of NE for 6 h, treatment did not affect cell viability. Furthermore, after application of NE for 24 h, cell viability gradually increased as the NE concentration was elevated from 10 to 100 µM. However, no significant increase in viability was observed when the cells were treated with 120 and 160 µM NE. Occludin expression was decreased when 10 µM NE was applied for 6 or 24 h. By contrast, 60 µM NE significantly downregulated Occludin expression in the 6 h group, but caused an insignificant decrease in the 24 h group. It was found that ZO-1 expression was upregulated after treatment with 10 µM NE for 6 h, whilst downregulation was observed after treatment with 10 µM NE for 24 h. PAR-2 protein expression was increased after application of NE for both 6 and 24 h, but not after treatment with 60 µM NE. In addition, TRPA1 expression was not affected by the treatment of NE, but increased positive staining was observed on the luminal side of the mucosa, which appeared to be concentrated in the cells of the luminal side in the rat colon after heat exposure. Collectively, the present results suggested that expression of tight junction proteins Occludin and ZO-1, in addition to that of PAR-2, can be regulated by NE, which may contribute to impairments in barrier function observed during heat stress.

Enhanced Virulence of Aeromonas hydrophila Is Induced by Stress and Serial Passaging in Mice.

Simple SummaryAeromonas hydrophila, which is an opportunistic zoonotic bacterium, has the ability to infect animals with injuries involving the condition of the aquatic environments. Factors including poor sanitation and water quality, stress, overcrowding, and rough handling can make animals more sensitive to infections and trigger outbreaks of A. hydrophila. A. hydrophila was previously isolated from an African black-footed penguin that died while in captivity at a zoo, following clinical signs of depression and anorexia, and in this study, we investigated the effect of stress and serial passaging in mice on A. hydrophila virulence. Serial passaging in mice enhanced the virulence of A. hydrophila, and A. hydrophila infection combined with administration of stress hormones or fasting increased mortality.Aeromonas hydrophila was isolated from an African black-footed penguin (Spheniscus demersus) that died while in zoo captivity. At necropsy, the virulence of A. hydrophila appeared to be enhanced by stress, so was assessed in the presence of in vitro and in vivo stressors and serial passaging in mice. Virulence genes from the isolate were amplified by PCR. In vitro assays were conducted to test the hemolytic activity, cytotoxicity, and effect of stress hormones on A. hydrophila virulence. In vivo assays were conducted to test the stress effect on mortality of A. hydrophila-infected mice and virulence in mice. Two virulence genes coding for hemolysin (ahh1) and aerolysin (aerA) were detected, and the cytotoxic potential of the isolate was demonstrated in baby hamster kidney and Vero cells. Some or all mice inoculated with A. hydrophila and exposed to stress hormones (epinephrine and norepinephrine) or low temperature died, while mice inoculated with A. hydrophila and exposed to fasting or agitation stressors or no stressors survived. We concluded that stress can be fatal in mice experimentally infected with A. hydrophila and that serial passaging in mice dramatically enhances the virulence of A. hydrophila.

Extended Contact Lens Wear Promotes Corneal Norepinephrine Secretion and Pseudomonas aeruginosa Infection in Mice.

PurposeExtended contact lens (CL) wear predisposes the wearer to Pseudomonas aeruginosa infection of the cornea, but the mechanism involved remains incompletely understood. The purpose of this study was to investigate the role of the stress hormone norepinephrine (NE) in the pathogenesis of CL-induced P. aeruginosa keratitis.MethodsA total 195 adult C57BL/6 mice were used in this study. Corneal NE content was measured after 48 hours of sterile CL wear in mice. The effect of NE on P. aeruginosa adhesion and biofilm formation on the CL surface was examined in vitro. Moreover, mouse eyes were covered with P. aeruginosa-contaminated CLs, and either 500-µM NE was topically applied or the eyes were subconjunctivally injected with 100 µg of N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) to deplete local NE. Clinical scores, neutrophil infiltration, proinflammatory cytokine levels, and bacterial load on the corneas and CLs were evaluated.ResultsCorneal NE content was elevated with extended CL wear in mice. In vitro, NE promoted the adhesion and biofilm formation of P. aeruginosa on the CL surface. In mice, topical application of NE aggravated P. aeruginosa infection, accompanied with increased clinical scores, neutrophil infiltration, proinflammatory cytokine expression, and bacterial burden on the corneas and CLs. However, pre-depletion of local NE with DSP-4 significantly alleviated the severity of P. aeruginosa keratitis.ConclusionsExtended CL wear elevates corneal NE content, which promotes the pathogenesis of CL-induced P. aeruginosa keratitis in mice. Targeting NE may provide a potential strategy for the treatment of CL-related corneal infection caused by P. aeruginosa.

Norepinephrine-stimulated HSCs secrete sFRP1 to promote HCC progression following chronic stress via augmentation of a Wnt16B/\u03b2-catenin positive feedback loop

BackgroundSustained adrenergic signaling secondary to chronic stress promotes cancer progression; however, the underlying mechanisms for this phenomenon remain unclear. Hepatocellular carcinoma (HCC) frequently develops within fibrotic livers rich in activated hepatic stellate cells (HSCs). Here, we examined whether the stress hormone norepinephrine (NE) could accelerate HCC progression by modulating HSCs activities.MethodsHCC cells were exposed to conditioned medium (CM) from NE-stimulated HSCs. The changes in cell migration and invasion, epithelial-mesenchymal transition, parameters of cell proliferation, and levels of cancer stem cell markers were analyzed. Moreover, the in vivo tumor progression of HCC cells inoculated with HSCs was studied in nude mice subjected to chronic restraint stress.ResultsCM from NE-treated HSCs significantly promoted cell migration and invasion, epithelial-mesenchymal transition (EMT), and expression of cell proliferation-related genes and cancer stem cell markers in HCC cells. These pro-tumoral effects were markedly reduced by depleting secreted frizzled related protein 1 (sFRP1) in CM. The pro-tumoral functions of sFRP1 were dependent on β-catenin activation, and sFRP1 augmented the binding of Wnt16B to its receptor FZD7, resulting in enhanced β-catenin activity. Additionally, sFRP1 enhanced Wnt16B expression, reinforcing an autocrine feedback loop of Wnt16B/β-catenin signaling. The expression of sFRP1 in HSCs promoted HCC progression in an in vivo model under chronic restraint stress, which was largely attenuated by sFRP1 knockdown.ConclusionsWe identify a new mechanism by which chronic stress promotes HCC progression. In this model, NE activates HSCs to secrete sFRP1, which cooperates with a Wnt16B/β-catenin positive feedback loop. Our findings have therapeutic implications for the treatment of chronic stress-promoted HCC progression.

The stress hormone norepinephrine promotes tumor progression through β2-adrenoreceptors in oral cancer

ObjectiveChronic stress hormone norepinephrine (NE) has been previously reported to play a role in the development of cancer, but the correlation between NE and oral squamous cell carcinoma (OSCC) progression is not well understood. MethodTo address this, the expression of adrenergic receptors (ARs) in human OSCC cell lines and clinic OSCC samples was detected, and the role of NEin vivo and in vitro was further investigated. ResultsIt was found that β2-AR was the main AR of NE in OSCC. Stimulation of OSCC cells with NE significantly increased the OSCC proliferation and invasion, which was, however, blocked by β2-AR inhibitor. NE could induce the phosphorylation of extracellular regulated protein kinases (ERK) and cAMP-response element binding protein (CREB). Inhibition of ERK and CREB pathway abrogated NE-induced OSCC invasion and proliferation. NE could enhance cancer stem cells (CSCs)-like phenotype and up-regulate the expression of stemness marker. In tumor-bearing nude mice, it was found that consecutive administration of NE significantly promoted the tumor growth, while daily injection of β2-AR inhibitor blocked this phenomenon. ConclusionsThose findings indicated a critical role of the chronic stress hormone NE in OSCC progression. Inhibition of β2-AR may serve as a potential therapeutic strategy for protecting OSCC patients from chronic stress related deleterious effect.

Salivary alpha-amylase enzyme is a non-invasive biomarker of acute stress in Japanese macaques (Macaca fuscata).

Salivary alpha-amylase (sAA) enzyme functions as a digestive enzyme in many species that consume starch in their diet. Human studies have also revealed that sAA enzyme activity levels are positively correlated with the release of the stress hormone norepinephrine, allowing sAA to act as a biomarker for sympathetic nervous system activity. Recent non-human primate studies have incorporated sAA as a physiological stress marker. However, no published reports have investigated the time course of sAA from a stressful event to return to baseline levels in non-human primates. Furthermore, no validation of sAA as a stress biomarker has been reported for Japanese macaques (Macaca fuscata). This study had two primary aims: (1) to develop a systematic method for non-invasive saliva collection and, (2) to investigate sAA as a biomarker of acute stress in M. fuscata in order to better understand its acute stress-related characteristics. We developed a non-invasive method for cooperative saliva collection using positive reinforcement training (PRT) and tracked individual progress over 595 trials in ten individually housed Japanese macaques. We detected sAA enzyme in M. fuscata via kinetic reaction assay, then performed 22 acute stress tests. Four tests met conditions for interpreting sAA in response to an acute stressor and these results show that on average sAA activity rapidly increased post-stressor (mean ± SD = 4.2 ± 0.9min) and returned to baseline shortly thereafter (10.4 ± 0.6min). Our report reveals for the first time the temporal dynamics of sAA when applying acute stress to Japanese macaques and could be a useful tool for assessing animal welfare.

Cx32 mediates norepinephrine-promoted EGFR-TKI resistance in a gap junction-independent manner in non-small-cell lung cancer.

The second-generation EGFR-TKI Afatinib is an irreversible ErbB family blocker used to treat patients with non-small-cell lung cancer (NSCLC). Unfortunately, resistance to this drug develops over time, and patients are always under great psychological pressure. A previous study showed that chronic stress hormones participate in EGFR-TKI resistance via β2 -AR signaling via an IL-6 dependent mechanism. Our study further explores a novel potential underlying mechanism. In the present study, we show that the stress hormone norepinephrine (NE) promotes Afatinib resistance by upregulating Cx32 expression. Furthermore, we, for the first time, find that Cx32 is a target gene for transcription factor CREB and NE enhances Cx32 mRNA expression by activation of CREB. We also demonstrate that Cx32 promotes Afatinib resistance by decreasing the degradation of EGFR-TKI resistance-associated proteins (MET, IGF-1R) and by increasing their transcription levels. Together, these results reveal that the stress hormone NE accelerates Afatinib resistance by increasing the expression of Cx32, which augments MET and IGF-1R levels in cancer cells and provides a promising therapeutic strategy against EGFR-TKI Afatinib resistance in NSCLC.

Adrenergic modulation of AMPK‑dependent autophagy by chronic stress enhances cell proliferation and survival in gastric cancer.

Epidemiological data show that chronic stress has adverse effects on the incidence and progression of cancer. As a critical target organ for stress hormones, the stomach is frequently subjected to stress-related injury. However, few reports regarding the association between stress and gastric cancer (GC) have been published. The present study aimed to investigate the effect of chronic stress on the growth and survival of GC, and the role of the autophagy process. A restraint-stress procedure over 21 days was used to establish a chronic stress mouse model. Subcutaneous xenografts and gastric orthotopic xenografts were established in BALB/c nude mice. Alzet osmotic minipumps containing either PBS or propranolol hydrochloride was inserted on the nape of the neck 7 days prior to the initiation of restraint stress. The presence of autophagosomes and autolysosomes were examined by electron microscopy. The stress hormone norepinephrine significantly enhanced the proliferation of GC cells. By inhibiting adrenoreceptor expression, it was demonstrated that β2-adrenergic receptor (ADRB2) was the specific β-adrenergic receptor subtype responsible for catecholamine release. In addition, it was demonstrated that the induction of autophagy was a novel consequence of β2-adrenergic activation in GC cells. This was demonstrated by the appearance of double-membrane vesicles, punctuate GFP-RFP-microtubule-associated protein 1 light chain 3 distribution in the cytoplasm and a corresponding increase in autophagic flux. Notably, norepinephrine-induced autophagy was shown to have a tumor-promoting role under conditions of chronic stress in vitro and in vivo. It was further demonstrated that, upon activation of cAMP-response element binding protein, chronic stress promoted autophagic flux through the adenosine 5′-monophosphate-activated protein kinase-unc-51 like autophagy activating kinase 1 (AMPK-ULK1) pathway. Tissue microarray analysis revealed a negative correlation between the expression of ADRB2 and autophagic marker p62/sequestosome-1 in GC tumor samples. Additionally, high protein levels of ADRB2 correlated positively with tumor, node, metastasis stage and poor prognosis in patients with GC. These results establish a novel pathway that chronic stress activates tumor-promoting autophagy to accelerate the progression of GC. The present study is the first, to the best of our knowledge, providing preclinical evidence that chronic stress serves a role in the progression of GC.

The Role of Norepinephrine and α-Adrenergic Receptors in Acute Stress-Induced Changes in Granulocytes and Monocytes.

Acute stress induces redistribution of circulating leucocytes in humans. Although effects on lymphocytes as adaptive immune cells are well understood, the mechanisms underlying stress effects on granulocytes and monocytes as innate immune blood cells are still elusive. We investigated whether the stress hormone norepinephrine (NE) and α-adrenergic receptors (α-ADRs) may play a mediating role. In a stress study, we cross-sectionally tested 44 healthy men for associations between stress-induced NE increases and simultaneous granulocyte and monocyte cell count increases, as measured immediately before and several times after the Trier Social Stress Test. In a subsequent infusion study, 21 healthy men participated in three different experimental trials with sequential infusions of 1- and 15-minute duration with varying substances (saline as placebo, the nonspecific α-ADR blocker phentolamine [2.5 mg/min], and NE [5 μg/min]): trial 1 = saline+saline, trial 2 = saline+NE, trial 3 = phentolamine+NE. Granulocyte and monocyte cell numbers were assessed before, immediately after, 10 minutes, and 30 minutes after infusion procedures. In the stress study, higher NE related to higher neutrophil stress changes (β = .31, p = .045, R change = .09), but not epinephrine stress changes. In the infusion study, saline+NE induced significant increases in neutrophil (F(3/60) = 43.50, p < .001, η = .69) and monocyte (F(3/60) = 18.56, p < .001, η = .48) numbers compared with saline+saline. With phentolamine+NE, neutrophil (F(3/60) = 14.41, p < .001, η = .42) and monocyte counts (F(2.23/44.6) = 4.32, p = .016, η = .18) remained increased compared with saline+saline but were lower compared with saline+NE (neutrophils: F(3/60) = 19.55, p < .001, η = .494, monocytes: F(3/60) = 2.54, p = .065, η = .11) indicating partial mediation by α-ADRs. Trials did not differ in eosinophil and basophil count reactivity. Our findings suggest that NE-induced immediate increases in neutrophil and monocyte numbers resemble psychosocial stress effects and can be reduced by blockade of α-ADRs.

The stress hormone norepinephrine increases the growth and virulence of Aeromonas hydrophila.

Stress is an important contributing factor in the outbreak of infectious fish diseases. To comprehensively understand the impact of catecholamine stress hormone norepinephrine (NE) on the pathogenicity of Aeromonas hydrophila, we assessed variations in bacterial growth, virulence‐related genes expression and virulence factors activity after NE addition in serum‐SAPI medium. Further, we assessed the effects of NE on A. hydrophila virulence in vivo by challenging fish with pathogenic strain AH196 and following with or without NE injection. The NE‐associated stimulation of A. hydrophila strain growth was not linear‐dose‐dependent, and only 100 μM, or higher concentrations, could stimulate growth. Real‐time PCR analyses revealed that NE notably changed 13 out of the 16 virulence‐associated genes (e.g. ompW, ahp, aha, ela, ahyR, ompA, and fur) expression, which were all significantly upregulated in A. hydrophila AH196 (p < 0.01). NE could enhance the protease activity, but not affect the lipase activity, hemolysis, and motility. Further, the mortality of crucian carp challenged with A. hydrophila AH196 was significantly higher in the group treated with NE (p < 0.01). Collectively, our results showed that NE enhanced the growth and virulence of pathogenic bacterium A. hydrophila.

In vitro reduction of antibacterial activity of tigecycline against multidrug-resistant Acinetobacter baumannii with host stress hormone norepinephrine

The host stress hormone norepinephrine (NE), also called noradrenaline, is reported to augment bacterial growth and pathogenicity, but few studies have focused on the effect of NE on the activity of antimicrobials. The aim of this study was to clarify whether NE affects antimicrobial activity against multidrug-resistant Acinetobacter baumannii (MDR-AB). Time–kill studies of tigecycline (TIG) and colistin (COL) against MDR-AB as well as assays for factors contributing to antibiotic resistance were performed using MDR-AB clinical strains both in the presence and absence of 10 µM NE. In addition, expression of three efflux pump genes (adeB, adeJ and adeG) in the presence and absence of NE was analysed by quantitative reverse transcription PCR. Viable bacterial cell counts in TIG-supplemented medium containing NE were significantly increased compared with those in medium without NE. In contrast, NE had little influence on viable bacterial cell counts in the presence of COL. NE-supplemented medium resulted in an ca. 2 log increase in growth and in bacterial cell numbers adhering on polyurethane, silicone and polyvinylchloride surfaces. Amounts of biofilm in the presence of NE were ca. 3-fold higher than without NE. Expression of the adeG gene was upregulated 4–6-fold in the presence of NE. In conclusion, NE augmented factors contributing to antibiotic resistance and markedly reduced the in vitro antibacterial activity of TIG against MDR-AB. These findings suggest that NE treatment may contribute to the failure of TIG therapy in patients with MDR-AB infections.

Resveratrol attenuates norepinephrine-induced ovarian cancer invasiveness through downregulating hTERT expression.

Stress hormone norepinephrine (NE) has been associated with acquisition of cancer progression, and naturally occurring phytoalexin resveratrol (REV) has been known to suppress cancer growth and progression. In the present study, we determine the effect of REV on NE-induced ovarian cancer invasiveness. Pretreatment of REV significantly inhibited NE-induced ovarian cancer cell epithelial-to-mesenchymal transition with concomitant recovery of E-cadherin expression. In addition, our data showed that REV downregulates NE-induced human telomerase reverse transcriptase (hTERT) expression through inhibiting Src phosphorylation and HIF-1α expression. Further, REV reduced NE-induced Slug expression and subsequent ovarian cancer invasion. More importantly, combined treatment of REV with a pharmacological inhibitor of beta adrenergic receptor significantly attenuated NE-induced ovarian cancer invasion compared to single treatment. Therefore, we demonstrate interference of a Src and HIF-1α/hTERT/Slug signaling cascade by REV, providing potential therapeutic targets and inhibition of ovarian cancer.

The stress hormone norepinephrine increases migration of prostate cancer cells in vitro and in vivo.

The metastatic process is the most serious cause of cancer death. Norepinephrine, secreted in chronic stress conditions, stimulates the motility of breast and colon cells through β-adrenergic receptor. On these bases, we examined its possible role in metastasis formation and development invitro and in vivo. Treatments with norepinephrine (β2-adrenoreceptor agonist) in mice xenografted with human DU145 prostate cancer cells increased the metastatic potential of these cells. Specifically, we showed that treatment of mice with norepinephrine induced a significant increase of the migratory activity of cancer cells in a concentration-dependent manner and that this process was blocked by propanolol (β-adrenergic antagonist). Mice treated with norepinephrine, displayed an increased number of metastatic foci of DU145 cells in inguinal lymph nodes and also showed an increased expression of MMP2 and MMP9 in tumor samples compared to controls. Moreover, we demonstrated that propanolol induced in norepinephrine treated DU145 cells a E-cadherin finger-like membrane protrusions driven by vimentin remodeling. Altogether these data suggest that β2-AR plays an important role in prostate cancer metastasis formation and that the treatment with antagonist propanolol, could represents an interesting tool to control this process in cells overexpressing β2AR.