Relaxin family peptide receptors Rxfp1 and Rxfp2: mapping of the mRNA and protein distribution in the reproductive tract of the male rat.

BACKGROUND: Relaxin is the endogenous ligand of the G-protein coupled receptor RXFP1, previously known as LGR7. In humans relaxin can also activate, but with lower affinity, the closely related receptor for the insulin-like peptide from Leydig cells, RXFP2, previously known as LGR8. The lack of relaxin impairs male fertility but the precise distribution and the function of relaxin receptors in the male reproductive tract is not known. We investigated the distribution of Rxfp1 and Rxfp2 in the reproductive tract of the male rat and the function of relaxin in the vas deferens, a tissue with high expression of both receptors. METHODS: The presence of mRNA for Rxfp1 and Rxfp2 was investigated in testes, cultured Sertoli cells, epididymis, vas deferens, seminal vesicle, prostate, and spermatozoa by RT-PCR and Southern blot. Protein expression in the testis, vas deferens, primary culture of Sertoli cells, and spermatozoa was assessed by immunohistochemistry and immunofluorescence. The role of relaxin in the vas deferens was evaluated by contractility studies and radioimmunoassay of cAMP production. The effect of relaxin on mRNA levels for metalloproteinase-7 was measured by Northern blot. RESULTS: Transcripts for Rxfp1 and Rxfp2 were present in almost all parts of the male reproductive tract, with high levels in testis and vas deferens. Both receptors were immunolocalized in late stage germ cells but not in mature spermatozoa, although mRNAs for both receptors were also present in mature spermatozoa. Rxfp1 but not Rxfp2 was detected in cultured Sertoli cells. Strong immunostaining for Rxfp1 and Rxfp2 was seen in muscular and epithelial layers of the vas deferens and in arteriolar walls. Relaxin did not affect contractility and cyclic AMP production of the vas deferens, but increased the levels of mRNA for metalloproteinase-7. CONCLUSION: Rxfp1 and Rxfp2 are widely and similarly distributed throughout the male reproductive tract. Our results suggest that Rxfp1 on spermatids and Sertoli cells may be important in spermatogenesis. Relaxin in the vas deferens does not affect contractility, but may affect vascular compliance and collagen and matrix remodeling.

Cells positive for microtubule-associated protein 1B (MAP 1B) are present along rat and human efferent ductules and epididymis.

Microtubule-associated protein 1B (MAP 1B) is a neuronal cytoskeleton marker with predominant expression in the developing nervous system. The present study provides evidence for the expression of this cytoskeleton protein in non-neuronal and neuronal cells along rat and human efferent ductules and epididymis (initial segment, caput, and cauda). Reverse transcription/polymerase chain reaction and Western blot analysis were used to confirm the presence of MAP 1B (mRNA and protein) in rat tissues. Immunohistochemical studies revealed MAP-1B-positive staining in columnar ciliated cells present in efferent ductules and in narrow cells located in the initial segment, in both rat and human. MAP-1B-positive basal cells, located underneath the columnar cells, were only identified in the initial segment and caput epididymidis of the rat. Qualitative analysis of tissues from 40-day-old and 120-day-old rats indicated that the number of MAP-1B-positive ciliated, narrow, and basal cells per tubule increased with sexual maturation. These immunoreactive cells did not stain for dopamine beta-hydroxylase or acetylcholinesterase, indicating that they were not adrenergic or cholinergic in nature. Immunohistochemical studies also revealed the presence of MAP-1B-positive staining in interstitial nerve fibers in caput and cauda epididymidis from both rat and human. Thus, the expression of MAP 1B is not confined to a specific cell type in rat and human efferent ductules and epididymis. The functional significance of this cytoskeleton protein in tissues from the male reproductive tract requires further investigation.

Molecular structure and transcriptional regulation by nuclear factor-kappaB of the mouse kinin B1 receptor gene.

Kinins are important mediators in cardiovascular homeostasis, inflammation, and nociception. Two kinin receptors have been described, B 1 and B 2 . The B 1 receptor is normally absent in healthy tissues, but is highly induced under pathological conditions. To understand the molecular mechanism of B 1 receptor up-regulation, we determined the mouse B 1 receptor gene structure, isolated and characterized the promoter region and studied its transcriptional regulation. The mouse B 1 receptor gene contains two exons (with the entire coding region located in the second exon) and a TATA-less promoter with multiple transcription start sites. A 7.7-kbp portion of the 5'-flanking region was examined for promoter activity in vascular smooth muscle cells (VSMCs). A minimal 92-bp fragment, located immediately upstream of the transcription start region, exerted basal and lipopolysaccharide (LPS)-inducible transcription activity in the sense and antisense orientation, and was thereby identified as an enhancer element. Nuclear extracts from VSMCs showed basal and LPS-inducible binding activity of nuclear factor (NF)-kappaB at this sequence. B 1 receptor transcription activation in response to LPS was abolished by cotransfection with IkappaBalphaDeltaN, an NF-kappaB repressor. In summary, our results reveal the structure of the mouse B 1 receptor gene and the involvement of NF-kappaB in the inducible mouse kinin B 1 receptor expression under pathological conditions.

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

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 microm) for 48 hr in the presence or absence of the glucocorticoid receptor (GR) antagonist, mifepristone (1.0 microm), the proteasome inhibitor, N-acetyl-leucinyl-leucinyl-norleucinal-H (ALLN, 12.5 microm) or the antagonist of the nuclear factor kappa B (NFkappaB), pyrrolidinedithiocarbamate (PDTC, 12.5 microm). 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 microm 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 NFkappaB nuclear translocation was demonstrated by electromobility shift assay. Therefore, corticosterone potentiates noradrenaline-induced melatonin and NAS production through GR inhibition of NFkappaB 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.

Bradykinin B1 receptor expression induced by tissue damage in the rat portal vein: a critical role for mitogen-activated protein kinase and nuclear factor-kappaB 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 IkappaBalpha and translocation of nuclear transcription factor-kappaB (NF-kappaB) to the nucleus. The blockade of p38 MAPK, JNK or NF-kappaB, 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.

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 B(1) 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-kappaB activation, and neutrophil influx for the functional and molecular up-regulation of the bradykinin B(1) receptor. Treatment with LPS resulted in a rapid and sustained functional up-regulation of B(1) receptors in the rat paw that correlated with the increase in B(1) receptor mRNA levels. B(1) receptor up-regulation is preceded by the rapid activation of the transcription factor NF-kappaB and the production of proinflammatory cytokines, including TNF-alpha and IL-1beta. More importantly, blockade of NF-kappaB translocation, TNF-alpha, or IL-1beta prevented the functional and molecular up-regulation of B(1) 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-kappaB and functional B(1) receptor up-regulation. Blockade of neutrophil influx with platelet-activating factor receptor antagonists or cell adhesion molecule blockers prevented B(1) receptor up-regulation. Thus, by acting in cooperation and in a coordinated, timely manner, TNF-alpha, IL-1beta, neutrophils, and the transcription factor NF-kappaB are major and essential players in the ability of LPS to induce B(1) receptor expression in vivo.

Transcriptional regulation of the rat bradykinin B2 receptor gene: identification of a silencer element.

Kinins are involved in a variety of physiological and pathophysiological processes related to cardiovascular homeostasis, inflammation, blood flow, and nociception. Under physiological conditions, the bradykinin B2 (BKB2) receptor is constitutively expressed and mediates most of kinins' actions. However, the mechanisms regulating BKB2 receptor gene expression are still poorly understood. In this study, 4.6 kilobases of the 5'-flanking region from the rat BKB2 receptor gene were sequenced, and computer analysis revealed several sites for transcriptional factors. Nine promoter mutants were cloned in luciferase reporter gene vectors and transfected in NG108-15 cells and rat aorta vascular smooth muscle cells (VSMCs), showing several positive and negative regulatory elements. A classical silencer with 56 base pairs (bp) caused a decrease in reporter gene activity in NG108-15 cells and VSMCs and was able to inhibit the thymidine kinase promoter. Using electrophoretic mobility shift assay and surface plasmon resonance assay, protein-DNA interactions in the silencer region were determined and specific sets of protein-silencer complexes were detected in both cell types. More intense complexes were observed in the central 21 bp of the silencer and mutation in a putative SRE-1 site strongly impaired the protein-DNA binding. Down-regulation of the BKB2 receptor population in NG108-15 cells promoted by N(6), 2'-O-dibutyryladenosine 3':5'-cyclic monophosphate was paralleled by an increase in the amount of nuclear proteins bound to the silencer sequence showing an inverse relationship between protein-silencer complexes and the transcription of the BKB2 receptor gene. In summary, these data highlight the cell-specific regulation of the BKB2 receptor and the importance of a silencer element present in the regulatory region of the gene.

Age-related changes in the neuromodulatory effect of opioid peptides on sympathetic nerve terminals

A alteraçäo, dependente da idade, do efeito neuromodulador da ß-endorfina nos terminais nervosos simpáticos foi estudada na porçäo prostática de vasa diferentia de ratos de 4 e 20 meses. O efeito da ß-endorfina e naloxona sobre a contraçäo induzida pelo estímulo transmural dos terminais nervosos (0.2 Hz, 0.5 ms, metade da voltagem supramáxima) foi analisado. Na ausência da droga näo ocorreram alteraçöes dependentes da idade na contraçäo induzida pelo estímulo nervoso. ß-Endorfina causava inibiçäo dose-dependente da contraçäo muscular. A sensibilidade da inibiçäo pela ß-endorfina duplicava entre 4 e 20 meses. Naloxona potenciava de maneira dose-dependente as contraçöes campo-estimuladas induzidas. A potência da naloxona era seis vezes maior em animais de 20 do que de 4 meses. Os dados deste trabalho säo comparados com as alteraçöes relacionadas à idade na reatividade pós-juncional, e é proposto o desenvolvimento de mecanismos compensatórios da idade no nível pré-juncional