Maria Christina W. Avellar

Chronic Unpredictable Stress Exacerbates Lipopolysaccharide-Induced Activation of Nuclear Factor-κB in the Frontal Cortex and Hippocampus via Glucocorticoid Secretion

Although the anti-inflammatory actions of glucocorticoids (GCs) are well established in the periphery, these stress hormones can increase inflammation under some circumstances in the brain. The transcription factor nuclear factor-κB (NF-κB), which is inhibited by GCs, regulates numerous genes central to inflammation. In this study, the effects of stress, GCs, and NMDA receptors on lipopolysaccharide (LPS)-induced activation of NF-κB in the brain were investigated. One day after chronic unpredictable stress (CUS), nonstressed and CUS rats were treated with saline or LPS and killed 2 h later. CUS potentiated the increase in LPS-induced activation of NF-κB in frontal cortex and hippocampus but not in the hypothalamus. This stress effect was blocked by pretreatment of rats with RU-486, an antagonist of the GC receptor. MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d] cyclohepten-5,10-imine maleate], an NMDA receptor antagonist, also reduced the effect of LPS in all three brain regions. However, the combined antagonism of both GC and NMDA receptors produced no further reduction in NF-κB activation when compared with the effect of each treatment alone. Our results indicate that stress, via GC secretion, can increase LPS-induced NF-κB activation in the frontal cortex and hippocampus, agreeing with a growing literature demonstrating proinflammatory effects of GCs.

Kinin B1 Receptor Up-Regulation after Lipopolysaccharide Administration: Role of Proinflammatory Cytokines and Neutrophil Influx

Several studies have now clearly established the ability of LPS to induce bradykinin B1 receptor up-regulation in vivo and the functional relevance of this up-regulation for the pathophysiological effects of LPS. Using an in vivo system in which LPS is injected locally into the rat paw, we have examined the potential contribution of proinflammatory cytokines, NF-κB activation, and neutrophil influx for the functional and molecular up-regulation of the bradykinin B1 receptor. Treatment with LPS resulted in a rapid and sustained functional up-regulation of B1 receptors in the rat paw that correlated with the increase in B1 receptor mRNA levels. B1 receptor up-regulation is preceded by the rapid activation of the transcription factor NF-κB and the production of proinflammatory cytokines, including TNF-α and IL-1β. More importantly, blockade of NF-κB translocation, TNF-α, or IL-1β prevented the functional and molecular up-regulation of B1 receptors. Injection of LPS also induced the influx of neutrophils that followed the peak of cytokine production and associated with the persistent activation of NF-κB and functional B1 receptor up-regulation. Blockade of neutrophil influx with platelet-activating factor receptor antagonists or cell adhesion molecule blockers prevented B1 receptor up-regulation. Thus, by acting in cooperation and in a coordinated, timely manner, TNF-α, IL-1β, neutrophils, and the transcription factor NF-κB are major and essential players in the ability of LPS to induce B1 receptor expression in vivo.

Corticosterone modulates noradrenaline-induced melatonin synthesis through inhibition of nuclear factor kappa B

Abstract:  In chronically inflamed animals, adrenal hormones exert a positive control on the secretion of melatonin by the pineal gland. In this paper, the mechanism of corticosterone as a modulator of melatonin and N‐acetylserotonin (NAS) was determined. Rat pineal glands in culture, stimulated for 5 hr with noradrenaline (10 nm), were previously incubated with corticosterone (1.0 nm–1.0 μm) for 48 hr in the presence or absence of the glucocorticoid receptor (GR) antagonist, mifepristone (1.0 μm), the proteasome inhibitor, N‐acetyl‐leucinyl‐leucinyl‐norleucinal‐H (ALLN, 12.5 μm) or the antagonist of the nuclear factor kappa B (NFκB), pyrrolidinedithiocarbamate (PDTC, 12.5 μm). Corticosterone potentiated noradrenaline‐induced melatonin and NAS production in a bell‐shaped manner. The increase in NAS (12.9 ± 2.7, n = 6 versus 34.3 ± 8.3 ng per pineal) and melatonin (16.3 ± 2.0, n = 6 versus 44.3 ± 12.9 ng per pineal) content induced by 1 μm corticosterone was blocked by mifepristone, and mimicked by ALLN and PDTC. The presence of GRs was shown by [3H]‐dexamethasone binding (0.30 ± 0.09 pmol/mg protein) and corticosterone inhibition of NFκB nuclear translocation was demonstrated by electromobility shift assay. Therefore, corticosterone potentiates noradrenaline‐induced melatonin and NAS production through GR inhibition of NFκB nuclear translocation. To the best of our knowledge, this is the first time that this relevant pathway for passive and acquired immune response is shown to modulate melatonin production in pineal gland.

MK-801 and 7-Ni attenuate the activation of brain NF-κB induced by LPS

The activation of nuclear factor-kappaB (NF-kappaB) leads to an increase in the expression of genes involved in important events in the central nervous system (CNS), such as development, plasticity and inflammation. It has been shown that inflammatory stimulus in the brain increases excitatory glutamatergic transmission, especially at N-methyl-D-aspartate (NMDA) receptor. These receptors have an important role in glutamate neurotoxicity and are in general coupled with the generation of nitric oxide (NO) through the activation of neuronal nitric oxide synthase (NOS). We have investigated the involvement of NMDA-NO pathway in LPS induction of NF-kappaB in CNS. Our results demonstrate that systemic LPS activates NF-kappaB in several regions of the CNS, which was partially reduced by the NMDA receptor antagonist dizolcipine (MK-801) and by the selective brain NOS inhibitor 7-Nitroindazol (7-Ni). 7-Ni effects were not synergic to MK-801 effects, suggesting that these compounds act through the same pathway. Dexamethasone caused a stronger reduction in LPS induction of NF-kappaB in CNS, demonstrating that MK-801 and 7-Ni act on a pathway that is responsible only by a fraction of the overall NF-kappaB activation. These results suggest that a considerable part of NF-kappaB activation by LPS is linked to the NMDA/NO pathway in CNS.

Bradykinin B1 Receptor Expression Induced by Tissue Damage in the Rat Portal Vein A Critical Role for Mitogen-Activated Protein Kinase and Nuclear Factor-κB Signaling Pathways

The bradykinin B1 receptor (B1R) is normally absent under physiological conditions, but is highly inducible during inflammatory conditions or following tissue damage. The present study attempted to determine some of the mechanisms underlying B1R upregulation following tissue injury in rat portal vein. Damage induced by tissue isolation and in vitro incubation caused a significant and time-dependent increase in des-Arg9–bradykinin (des-Arg9–BK) responsiveness that paralleled the B1R mRNA expression, as confirmed by real-time quantitative PCR. In vitro incubation of rat portal vein also induced the activation of some members of the mitogen activated protein kinase (MAPK) family, namely, extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and p38 MAPK, an effect accompanied by degradation of the inhibitory protein IκB&agr; and translocation of nuclear transcription factor-κB (NF-κB) to the nucleus. The blockade of p38 MAPK, JNK or NF-κB, but not ERK pathways with selective inhibitors, resulted in a significant reduction of the upregulated contractile response caused by the selective B1R agonist des-Arg9–BK, and largely prevented the induction of B1R mRNA expression in the rat portal vein. Together, these results demonstrate that in vitro tissue damage induces activation of several intracellular signaling pathways that have a key role in the control of B1R expression. B1R could exert a pivotal role in the development of the cardiovascular response associated with vascular damage.

Activation of Toll-Like Receptor 4 (TLR4) by In Vivo and In Vitro Exposure of Rat Epididymis to Lipopolysaccharide from Escherichia Coli

Abstract This study provides the first evidence that rat epididymis is fully capable of initiating an inflammatory response to lipopolysaccharide (LPS) from Escherichia coli through activation of Toll-like receptor 4 (TLR4). TLR4 functionality was demonstrated by in vivo LPS challenge, which induced a time- and dose-dependent activation of the transcription factor nuclear factor kappa B (NFKB) in caput and cauda epididymides. NFKB activation by LPS in caput epididymidis was abrogated when rats were pretreated with the NFKB inhibitor PDTC, confirming the specificity of this response. Within 2 h of LPS treatment (0.01 and 1 mg/kg, i.v.), NFKB activation in caput and cauda was accompanied by upregulation of Il1b, Nfkbia, and Cd14, but not Tlr4, mRNA. These effects, however, were not sustained after 24 h of LPS treatment. Lipopolysaccharide systemic effects were not restricted to epididymides, since Il1b, Nfkbia, and Cd14 mRNAs were also upregulated in other male reproductive tissues from LPS-treated rats (1 mg/kg, i.v., 2 h). Constitutive TLR4 was immunolocalized in some, but not all, epididymal epithelial cells and in interstitial cells, some of them identified as resident ED2-positive macrophages. No change in TLR4 immunostaining pattern was observed when epididymides from control and LPS-treated rats were compared (1 mg/kg, i.v., 2 h and 24 h). Significant NFKB activation was also achieved within 1 min of in vitro incubation of caput epididymidis with LPS (0.01–5 μg/ml), confirming that components for TLR4 signaling cascade activation are fully active in this tissue. This study contributes to a better understanding of the innate immune response in the epididymis and other tissues from the male reproductive tract.

Androgens and the male reproductive tract: an overview of classical roles and current perspectives

Androgens are steroid hormones that play key roles in the development and maintenance of male phenotype and reproductive function. These hormones also affect the function of several non-reproductive organs, such as bone and skeletal muscle. Endogenous androgens exert most of their effects by genomic mechanisms, which involve hormone binding to the androgen receptor (AR), a ligand-activated transcription factor, resulting in the modulation of gene expression. AR-induced non-genomic mechanisms have also been reported. A large number of steroidal and non-steroidal AR-ligands have been developed for therapeutic use, including the treatment of male hypogonadism (AR agonists) and prostate diseases (AR antagonists), among other pathological conditions. Here, the AR gene and protein structure, mechanism of action and AR gene homologous regulation were reviewed. The AR expression pattern, its in vivo regulation and physiological relevance in the developing and adult testis and epididymis, which are sites of sperm production and maturation, respectively, were also presented.

Differential Expression and Antibacterial Activity of Epididymis Protein 2 Isoforms in the Male Reproductive Tract of Human and Rhesus Monkey (Macaca mulatta)

Abstract The epididymis protein 2 (EP2) gene, the fusion of two ancestral β-defensin genes, is highly expressed in the epididymis and subject to species-specific regulation at the levels of promoter selection, transcription, and mRNA splicing. EP2 mRNA expression is also androgen dependent, and at least two of the secreted proteins bind spermatozoa. Alternative splicing produces more than 17 different EP2 mRNA variants. In this article, the expression of EP2 variants was profiled in different tissues from the human and rhesus monkey (Macaca mulatta) male reproductive tract using reverse transcriptase-polymerase chain reaction. Different EP2 mRNA variants were identified not only in human and rhesus testis and epididymis but also in the novel sites, seminal vesicle and prostate. Immunolocalization of EP2 protein in epithelial cells from rhesus and human seminal vesicle demonstrated that EP2 transcripts are translated in these tissues. In addition, two novel splicing variants, named EP2R and EP2S, were discovered. EP2C was the only splice variant expressed in all tissues tested from rhesus monkey. However, expression was not detected in human testis or seminal vesicle. For the first time, bactericidal function was demonstrated for EP2C, EP2K, and EP2L. Taken together, the results indicate that EP2 expression is more widespread in the male reproductive tract than realized previously. Whereas the activity of every EP2 variant tested thus far is antibacterial, further investigation may reveal additional physiological roles for EP2 peptides in the primate male reproductive tract.

Expression and pharmacological characterization of α1-adrenoceptors in rat seminal vesicle

In the present study, we investigated the alpha1-adrenoceptor subtypes mediating adrenaline-induced contractions of the rat seminal vesicle by using functional studies. The reverse transcription combined with polymerase chain reaction (RT-PCR) was also used to identify of alpha1-adrenoceptor mRNA subtypes. The rank order of potency of alpha1-adrenoceptor antagonists in blocking the contractile effects of adrenaline was: prazosin = WB 4101 >>> BMY 7378 > chloroethylclonidine, indicating the presence of alpha1A-adrenoceptors in the rat seminal vesicle. In the presence of nifedipine, there was a 76% reduction in the adrenaline-induced contractions. The nifedipine-insensitive component (24%) of the contractile response to adrenaline was unaffected by chloroethylclonidine. A small pool of spare alpha1-adrenoceptors for adrenaline (0.10%) was also detected. All three alpha1-adrenoceptor subtypes were amplified when RT-PCR was performed on total RNA isolated from rat seminal vesicle. In conclusion, these data indicate the presence of three alpha1-adrenoceptor mRNA subtypes, but only alpha1A-adrenoceptors are involved in the rat seminal vesicle contraction.

Role of transient receptor potential vanilloid 4 in rat joint inflammation.

OBJECTIVE To determine whether activation of transient receptor potential vanilloid 4 (TRPV-4) induces inflammation in the rat temporomandibular joint (TMJ), and to assess the effects of TRPV-4 agonists and proinflammatory mediators, such as a protease-activated receptor 2 (PAR-2) agonist, on TRPV-4 responses. METHODS Four hours after intraarticular injection of carrageenan into the rat joints, expression of TRPV-4 and PAR-2 in trigeminal ganglion (TG) neurons and in the TMJs were evaluated by real-time reverse transcription-polymerase chain reaction and immunofluorescence, followed by confocal microscopy. The functionality of TRPV-4 and its sensitization by a PAR-2-activating peptide (PAR-2-AP) were analyzed by measuring the intracellular Ca(2+) concentration in TMJ fibroblast-like synovial cells or TG neurons. Plasma extravasation, myeloperoxidase activity, and the head-withdrawal threshold (index of mechanical allodynia) were evaluated after intraarticular injection of selective TRPV-4 agonists, either injected alone or coinjected with PAR-2-AP. RESULTS In the rat TMJs, TRPV-4 and PAR-2 expression levels were up-regulated after the induction of inflammation. Two TRPV-4 agonists specifically activated calcium influx in TMJ fibroblast-like synovial cells or TG neurons. In vivo, the agonists triggered dose-dependent increases in plasma extravasation, myeloperoxidase activity, and mechanical allodynia. In synovial cells or TG neurons, pretreatment with PAR-2-AP potentiated a TRPV-4 agonist-induced increase in [Ca(2+) ](i) . In addition, TRPV-4 agonist-induced inflammation was potentiated by PAR-2-AP in vivo. CONCLUSION In this rat model, TRPV-4 is expressed and functional in TG neurons and synovial cells, and activation of TRPV-4 in vivo causes inflammation in the TMJ. Proinflammatory mediators, such as PAR-2 agonists, sensitize the activity of TRPV-4. These results identify TRPV-4 as an important signal of inflammation in the joint.