Tyrosyl-tRNA synthetase: A potential kyotorphin synthetase in mammals.

Kyotorphin (KTP; L-tyrosyl-l-arginine), an opioid-like analgesic discovered in the bovine brain, is potentially a neuromodulator because of its localization in synaptosomes, the existence of a specific KTP receptor, and the presence of its biosynthetic enzyme in the brain. KTP is formed in the brain from its constituent amino acids, L-tyrosine and L-arginine, by an enzyme termed KTP synthetase. However, the latter has never been identified. We aimed to test the hypothesis that tyrosyl-tRNA synthetase (TyrRS) is also KTP synthetase. We found that recombinant hTyrRS synthesizes KTP from tyrosine, arginine, and ATP, with Km = 1400 µM and 200 µM for arginine and tyrosine, respectively. TyrRS knockdown of PC12 cells with a small interfering RNA (siRNA) in the presence of 1.6 mM tyrosine, arginine, proline, or tryptophan significantly reduced the level of KTP, but not those of tyrosine-tyrosine, tyrosine-proline, or tyrosine-tryptophan. siRNA treatment did not affect cell survival or proliferation. In mice, TyrRS levels were found to be greater in the midbrain and medulla oblongata than in other brain regions. When arginine was administered 2 h prior to brain dissection, the KTP levels in these regions plus olfactory bulb significantly increased, although basal brain KTP levels remained relatively even. Our conclusion is further supported by a positive correlation across brain regions between TyrRS expression and arginine-accelerated KTP production.

Involvement of spinal release of α-neo-endorphin on the antinociceptive effect of TAPA.

The antinociceptive effect of i.t.-administered Tyr-d-Arg-Phe-ß-Ala (TAPA), an N-terminal tetrapeptide analog of dermorphin, was characterized in ddY mice. In the mouse tail-flick test, TAPA administered i.t. produced a potent antinociception. The antinociception induced by TAPA was significantly attenuated by i.t. pretreatment with the κ-opioid receptor antagonist nor-binaltorphimine, as well as by the µ-opioid receptor antagonist ß-funaltrexamine and the µ1-opioid receptor antagonist naloxonazine. TAPA-induced antinociception was also significantly suppressed by co-administration of the µ1-opioid receptor antagonist Tyr-d-Pro-Phe-Phe-NH2 (d-Pro(2)-endomorphin-2) but not by co-administration of the µ2-opioid receptor antagonists Tyr-d-Pro-Trp-Phe-NH2 (d-Pro(2)-endomorphin-1) and Tyr-d-Pro-Trp-Gly-NH2 (d-Pro(2)-Tyr-W-MIF-1). In CXBK mice whose µ1-opioid receptors were naturally reduced, the antinociceptive effect of TAPA was markedly suppressed compared to the parental strain C57BL/6ByJ mice. Moreover, the antinociception induced by TAPA was significantly attenuated by i.t. pretreatment with antiserum against the endogenous κ-opioid peptide α-neo-endorphin but not antisera against other endogenous opioid peptides. In prodynorphin-deficient mice, the antinociceptive effect of TAPA was significantly reduced compared to wild-type mice. These results suggest that the spinal antinociception induced by TAPA is mediated in part through the release of α-neo-endorphin in the spinal cord via activation of spinal µ1-opioid receptors.

Peritoneal administration of Met-RANTES attenuates inflammatory and nociceptive responses in a murine neuropathic pain model.

UNLABELLED: The C-C motif chemokine ligand 5 (CCL5; also known as regulated on activation, normal T expressed and secreted, or RANTES) is a member of the CC family of chemokines that specifically attract and activate leukocytes to sites of inflammation. Although CCL5 has been implicated in the processing of pain, its detailed mechanisms of action are still unknown. In this study, we investigated the potential of the Met-RANTES, a selective CCL5 receptor antagonist, via peritoneal administration to modulate the recruitment of inflammatory cells in injured sites and attenuate nociceptive responses in a neuropathic pain model in mice. Nociceptive sensitization, immune cell infiltration, multiple cytokine secretion, and opioid peptide expression in damaged nerves were studied. Our results indicated that Met-RANTES-treated mice had less behavioral hypersensitivity after partial sciatic nerve ligation. Macrophage infiltration, pro-inflammatory cytokine (TNFα, IL-1ß, IL-6, and IFNγ) protein secretion, and enkephalin, ß-endorphin, and dynorphin mRNA expression in damaged nerves following partial sciatic nerve ligation were significantly decreased, and anti-inflammatory cytokine (IL-10) protein was significantly increased in Met-RANTES-treated mice. These results suggest that CCL5 is capable of regulating the microenvironment that controls behavioral hypersensitivity at the level of the peripheral injured site in a murine chronic neuropathic pain model. PERSPECTIVE: The present study identifies the potent pro-inflammatory potential of CCL5 and verifies the possible role of selective CCL5 receptor inhibitor in a murine neuropathic pain model.

Premenstrual dysphoric disorder: neuroendocrine interferences.

Premenstrual dysphoric disorder (PMDD) consists in severe cognitive and mood changes, more aggressive as seen in premenstrual syndrome (PMS). These two syndromes are situated at the border between gynecology and psychiatry but the link between the two domains remains the neuroendocrine underlying mechanisms. In present, there are some molecular systems certainly proved as being involved, like estrogens. The hormonal pattern consists not in different levels of the hormones but different response to normal hormonal levels. The cyclical biochemical triggers are related to neurotransmitters as serotonin, endorphin and gamma-amino butyric acid (GABA). The heritability of the syndrome is sustained by genetic polymorphism in ESR1 gene. Thus, the PMDD is the result of multiple disturbances regarding neuroendocrine systems.

In vitro effect of hormones on the hormone content of rat peritoneal and thymic cells. Is there an endocrine network inside the immune system?

OBJECTIVE: The cells of the immune system contain hormones and receptors for hormones. However, the hormonal interactions between the cells are not elucidated. In the present experiments the effect of four hormones (insulin, oxytocin, gonadotropin and epidermal growth factor) on the production of three hormones (ACTH, endorphin, triiodothyronine) in immune cells was studied. METHODS: Hormone content of peritoneal immune cells (lymphocytes, monocyte-macrophage-granulocyte group [mo-gran], mast cells) as well as thymic cells of 100 g male rats, in vitro-treated or not treated with hormones mentioned above, were determined by flow cytometry, using specific antibodies. RESULTS: All of the hormones studied influenced the hormone concentration of immune cells significantly. The width of the effects was different in the following order: oxytocin > gonadotropin > EGF > insulin. In the case of oxytocin, 8/12 of the hormone/cell group were affected, while treatment with insulin affected only 4/12. The reaction measured by the hormone production was also different: ACTH production was reduced everywhere except in the insulin-thymocyte setup. Endorphin and T3 production was always elevated. Cells of the mo-gran group were more sensitive to hormonal regulation than peritoneal lymphocytes. CONCLUSION: The results mean that immune cells can be regulated by the neuroendocrine system and could also be able to play a role in endocrine regulation. In addition, taking into account the localization of these cells in the immune organs, there is the possibility of autocrine and paracrine regulation inside the immune system. Considering that the hormones, contained or sensed by the cells of the immune system are influencing each other, the concept of a hormonal network within the immune system can be proposed.

Dysregulation of dynorphins in Alzheimer disease.

The opioid peptides dynorphins may be involved in pathogenesis of Alzheimer disease (AD) by inducing neurodegeneration or cognitive impairment. To test this hypothesis, the dynorphin system was analyzed in postmortem samples from AD and control subjects, and subjects with Parkinson or cerebro-vascular diseases for comparison. Dynorphin A, dynorphin B and related neuropeptide nociceptin were determined in the Brodmann area 7 by radioimmunoassay. The precursor protein prodynorphin, processing convertase PC2 and the neuroendocrine pro7B2 and 7B2 proteins required for PC2 maturation were analyzed by Western blot. AD subjects displayed robustly elevated levels of dynorphin A and no differences in dynorphin B and nociceptin compared to controls. Subjects with Parkinson or cerebro-vascular diseases did not differ from controls with respect to any of the three peptides. PC2 levels were also increased, whereas, those of prodynorphin and pro7B2/7B2 were not changed in AD. Dynorphin A levels correlated with the neuritic plaque density. These results along with the known non-opioid ability of dynorphin A to induce neurodegeneration suggest a role for this neuropeptide in AD neuropathology.

Synthesis of morphiceptin (Tyr-Pro-Phe-Pro-NH(2)) by dipeptidyl aminopeptidase IV derived from Aspergillus oryzae.

Morphiceptin (Tyr-Pro-Phe-Pro-NH(2)), tetrapeptide, was synthesized using dipeptidyl aminopeptidase IV (DP IV, EC 3.4.14.5) derived from Aspergillus oryzae RIB 915 as a catalyst. Tyr-Pro-OEt was incubated with Phe-Pro-NH(2) in the presence of DP IV under various conditions of temperature, concentrations of ethylene glycol, pH, reaction time, and others. Morphiceptin was obtained at 40% yield under the optimal reaction conditions: substrate, 4 mM Tyr-Pro-OEt.HCl and 20 mM Phe-Pro-NH(2).HCl; enzyme, DP IV, 0.275 nkat; solvent, 60% ethylene glycol containing 20 mM phosphate buffer at pH 7.0; amine, 4.2 mM diisopropylamine at 4 degrees C for 24 h. Amino group protection was unnecessary for synthesis of morphiceptin by DP IV.

Time course of opioid and cannabinoid gene transcription alterations induced by repeated administration with fluoxetine in the rat brain.

This study examined the time course effects (8, 16 and 31 days) of fluoxetine administration (1 mg/kg, p.o./day) on serotonin transporter (5-HTT), opioid, tyrosine hydroxylase (TH) and cannabinoid CB1 receptor gene expressions in selected regions of the rat brain. Treatment with fluoxetine progressively decreased (35-55%) 5-HTT gene expression in dorsal raphe nucleus at 8, 16 and 31 days. The results revealed that fluoxetine administration decreased (30%) proenkephalin gene expression in nucleus accumbens shell (AcbS) and caudate-putamen (CPu) (31 days) but was without effect in nucleus accumbens core AcbC. A pronounced and time related decrease (25-65%) in prodynorphin gene expression was detected in AcbC, AcbS, CPu, hypothalamic supraoptic and paraventricular nuclei at all time points as well as in proopiomelanocortin gene expression (20-30%) in the arcuate nucleus (ARC) of the hypothalamus. On days 16 and 31, tyrosine hydroxylase gene expression in ventral tegmental area and substantia nigra and cannabinoid CB1 receptor gene expression in the CPu decreased (approximately 45-50% from vehicle). In conclusion, fluoxetine by inhibiting the reuptake of serotonin produced pronounced and time related alterations in genes involved in the regulation of emotional behaviour, suggesting that these neuroplastic changes may be involved, at least in part, in the clinical efficacy of this drug in neuropsychiatric disorders.

Different mechanisms of intrinsic pain inhibition in early and late inflammation.

Neuroimmune interactions control pain through activation of opioid receptors on sensory nerves by immune-derived opioid peptides. Here we evaluate mechanisms of intrinsic pain inhibition at different stages of Freund's adjuvant-induced inflammation of the rat paw. We use immunohistochemistry and paw pressure testing. Our data show that in early (6 h) inflammation leukocyte-derived beta-endorphin, met-enkephalin and dynorphin A activate peripheral mu-, delta- and kappa-receptors to inhibit nociception. In addition, central opioid mechanisms seem to contribute significantly to this effect. At later stages (4 days), antinociception is exclusively produced by leukocyte-derived beta-endorphin acting at peripheral mu and delta receptors. Corticotropin-releasing hormone (CRH) is an endogenous trigger of these effects at both stages. These findings indicate that peripheral opioid mechanisms of pain inhibition gain functional relevance with the chronicity of inflammation.

Close apposition of dynorphin-positive nerve fibres to lymphocytes in the liver suggests opioidergic neuroimmunomodulation.

The liver is innervated by sympathetic efferent, spinal afferent, vagal afferent and probably also vagal efferent fibres. To assess potential functional roles of the various neuronal subsets, data on transmitter systems are of crucial importance. This study was aimed at elucidating a possible opioidergic system in the mouse and rat liver. In particular relationships of opioidergic neurons to immune cells were emphasised. Material from perfusion-fixed mice ( n=29) of different strains (BALB/c, NMRI, C57Bl6, SV 129 inbred) and Wistar rats ( n=7) was cryosectioned at 12-14 microm and incubated for single or double immunofluorescence. Antibodies directed against dynorphin A, met-enkephalin, endomorphin 1 and 2, mu, kappa- and delta-opioid receptors (MOR, KOR, DOR), tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH), CD4, CD8 and macrophages were used. Binding sites were detected using Cy3-, FITC-, DTAF-, Cy2-, Alexa 555- and Texas red-tagged secondary antibodies. Specimens were analysed using confocal laser scanning microscopy (CLSM). Numerous nerve fibres staining for dynorphin were found in periportal areas of both mouse and rat livers. Neither met-enkephalin nor endomorphin could be detected in axons. No immunopositive neuronal cell bodies or other cellular elements were seen. All dynorphin positive fibres costained for TH while not every TH-positive fibre costained for dynorphin. Thus, most if not all dynorphin-positive nerve fibres may be of sympathetic origin. KOR immunostaining could be localised to round mononuclear cells which often costained for CD4, less frequently for CD8 and rarely for the pan-macrophage marker BM8. Altogether, about 45% of KOR-positive cells were identified as T-lymphocytes. In some instances, close appositions of dynorphin-positive axons to KOR-positive cells were revealed by CLSM. No KOR immunoreactivity was detected in nerve fibres. Hence, sympathetic neurons innervating the liver may interfere with inflammatory processes, in addition to their well-established beta(2)-adrenergic effect, via an opioidergic action on immune cells.

Protein kinase C signaling transduces endorphin-primed cardiogenesis in GTR1 embryonic stem cells.

The prodynorphin gene and its product, dynorphin B, have been found to promote cardiogenesis in embryonic cells by inducing the expression of GATA-4 and Nkx-2.5, two transcription factor-encoding genes essential for cardiogenesis. The molecular mechanism(s) underlying endorphin-induced cardiogenesis remain unknown. In the present study, we found that GTR1 embryonic stem (ES) cells expressed cell surface kappa opioid receptors, as well as protein kinase C (PKC)-alpha, -beta1, -beta2, -delta, -epsilon, and -zeta. Cardiac differentiation was associated with a marked increase in the Bmax value for a selective opioid receptor ligand and complex subcellular redistribution of selected PKC isozymes. PKC-alpha, -beta1, -beta2, -delta, and -epsilon all increased in the nucleus of ES-derived cardiac myocytes, compared with nuclei from undifferentiated cells. In both groups of cells, PKC-delta and -epsilon were mainly expressed at the nuclear level. The nuclear increase of PKC-alpha, -beta1, and -beta2 was due to a translocation from the cytosolic compartment. In contrast, the increase of both PKC-delta and PKC-epsilon in the nucleus of ES-derived cardiomyocytes occurred independently of enzyme translocation, suggesting changes in isozyme turnover and/or gene expression during cardiogenesis. No change in PKC-zeta expression was observed during cardiac differentiation. Opioid receptor antagonists prevented the nuclear increase of PKC-alpha, PKC-beta1, and PKC-beta2 and reduced cardiomyocyte yield but failed to affect the nuclear increase in PKC-delta and -epsilon. PKC inhibitors prevented the expression of cardiogenic genes and dynorphin B in ES cells and abolished their development into beating cardiomyocytes.

Orexin/hypocretin neurons: chemical phenotype and possible interactions with melanin-concentrating hormone neurons.

We showed earlier that a specific neuron population of the rat lateral hypothalamus, differing from the codistributed melanin-concentrating hormone (MCH) neurons, express both dynorphin (DYN) and secretogranin II (SgII) genes. We demonstrated later that this population corresponds in fact to the newly identified orexin/hypocretin (OX/Hcrt) neurons. In the present study, by revisiting the chemical phenotype of these neurons, we confirm that all of them contain DYN B- and SgII-immunoreactive materials. The roles played by these peptide/protein in OX/Hcrt neurons are still unclear. Double immunocytochemical stainings highlight putative somasomatic, axosomatic and axodendritic contacts between OX/Hcrt and MCH neurons. Adding OX/Hcrt to the culture medium of hypothalamic slices from 8-day-old rats results either in a significant increase of MCH mRNA after 24 h survival or a strong fall after 10 days culture. These results taken together suggest that OX/Hcrt can directly and/or indirectly affect MCH expression, and that both OX/Hcrt and MCH neuron populations interact to respond in a coordinated manner to central and peripheral signals.

Opioid peptide gene expression primes cardiogenesis in embryonal pluripotent stem cells.

Zinc finger-containing transcription factor GATA-4 and homeodomain Nkx-2.5 govern crucial developmental fates and have been found to promote cardiogenesis in embryonic cells exposed to the differentiating agent DMSO. Nevertheless, intracellular activators of these transcription factors are largely unknown. In this study, pluripotent P19 cells expressed the prodynorphin gene, an opioid gene encoding for the dynorphin family of opioid peptides. P19 cells were also able to synthesize and secrete dynorphin B, a biologically active end product of the prodynorphin gene. DMSO-primed GATA-4 and Nkx-2.5 gene expression was preceded by a marked increase in prodynorphin gene expression and dynorphin B synthesis and secretion. The DMSO effect occurred at the transcriptional level. In the absence of DMSO, dynorphin B triggered GATA-4 and Nkx-2.5 gene expression and led to the appearance of both alpha-myosin heavy chain and myosin light chain-2V transcripts, two markers of cardiac differentiation. Moreover, dynorphin B-exposed cells were positively stained in the presence of MF 20, a mouse monoclonal antibody raised against the alpha-myosin heavy chain. Opioid receptor antagonism and inhibition of opioid gene expression by a prodynorphin antisense phosphorothioate oligonucleotide blocked DMSO-induced cardiogenesis, suggesting an autocrine role of an opioid gene in developmental decisions.

Role of thymic peptides as transmitters between the neuroendocrine and immune systems.

Thymic peptides, a heterogenous family of polypeptidic hormones synthesized within the thymus, not only exert important regulatory effects within both the immune and neuroendocrine systems but are also themselves subject to control by hormones derived from the hypothalamic-pituitary-adrenal axis (HPA) and other endocrine glands. Regarding thymic hormonal function, thymulin production is up-regulated by several hormones, including prolactin, growth hormone and thyroid hormones. Other aspects of the physiology of thymic epithelial cells can also be modulated by hormones and neuropeptides, particularly cytokeratin expression, cell growth and production of extracellular matrix proteins, thus characterizing the pleiotrophic action of these molecules on the thymic epithelium. Conversely, thymic-derived peptides also regulate hormone release from the HPA axis and may act directly on target endocrine glands of this axis, modulating gonadal tissues. In addition, it has recently been shown that thymulin can modulate some peripheral nervous sensory functions, including those related to sensitivity to pain. According to the dose given, thymulin induces or reduces hyperalgesia related to both mechanical and thermal nociceptors and thus represents an important interface between the immune, endocrine and nervous systems.

El tacto, un sentido muy especial / Tacto, a very special felt

El autor destaca la importancia de la experiencia táctil como estímulo del desarrollo y la adaptación social del ser humano, a partir de la vida intrauterina. Subraya la influencia del contacto con otro ser humano y diferencia sus afectos positivos o negativos en función de la sensibilidad del niño en etapas muy tempranas

El tacto, un sentido muy especial / Tacto, a very special felt

El autor destaca la importancia de la experiencia táctil como estímulo del desarrollo y la adaptación social del ser humano, a partir de la vida intrauterina. Subraya la influencia del contacto con otro ser humano y diferencia sus afectos positivos o negativos en función de la sensibilidad del niño en etapas muy tempranas (AU)

Effects of repeated psychostimulant administration on the prodynorphin system activity and kappa opioid receptor density in the rat brain.

The prodynorphin system is implicated in the neurochemical mechanism of psychostimulants. To elucidate the activity of the endogenous prodynorphin system upon treatment with psychostimulants, we investigated the effect of single and repeated cocaine and amphetamine on the prodynorphin messenger RNA level, the prodynorphin-derived peptide alpha-neoendorphin tissue level, and its in vitro release in the nucleus accumbens and striatum of rats. The density of kappa opioid receptors in those brain regions was also assessed. Rats were injected with cocaine following a "binge" administration pattern, 20 mg/kg i.p. every hour for 3 h, one (single treatment) or five days (chronic treatment). Amphetamine, 2.5 mg/kg i.p. was administered once (single treatment) or twice a day for five days (chronic treatment). As shown by an in situ hybridization study, the prodynorphin messenger RNA levels in the nucleus accumbens and striatum were raised following single (at 3 h) and chronic (at 3 and 24 h) cocaine administration. The prodynorphin messenger RNA level in the nucleus accumbens was markedly elevated after single or repeated amphetamine administration. A similar tendency was observed in the striatum. Acute cocaine and amphetamine administration had no effect on the alpha-neoendorphin tissue level, whereas chronic administration of those drugs elevated the alpha-neoendorphin level in the nucleus accumbens and striatum at the late time-points studied. Acute and repeated cocaine administration had no effect on alpha-neoendorphin release in both the nucleus accumbens and striatum at 3 and 48 h after drug injection. In contrast, single and chronic (at 24 and 48 h) amphetamine administration profoundly elevated the release of alpha-neoendorphin in both these structures. Addition of cocaine or amphetamine to the incubation medium (10(-5)-10(-6) M) decreased the basal release of alpha-neoendorphin in the nucleus accumbens slices of naive rats, but it did not change the stimulated release (K+, 57 mM). On the other hand, in the striatum slices, addition of cocaine to the incubation medium depressed basal and stimulated release of the peptide; no significant changes were observed after addition of amphetamine. Cocaine and amphetamine evoked profound and long-term down-regulation of the kappa opioid receptors in both structures. The above data indicate that the amphetamine-induced changes were more abundant than those caused by cocaine; only treatment with amphetamine markedly enhanced the release of prodynorphin-derived peptide. Furthermore, the psychostimulant-induced enhancement of biosynthetic activity of prodynorphin neurons was correlated with a marked and persistent decrease in the kappa opioid receptor density at a late withdrawal time.

Neokyotorphin formation and quantitative evolution following human hemoglobin hydrolysis with cathepsin D.

In vitro human hemoglobin hydrolysis by cathepsin D was investigated. The quantitative evolution of neokyotorphin following the hydrolysis was determined by high-performance liquid chromatography coupled with a photodiode array detector. Spectral comparisons allowed us to identify neokyotorphin in the hydrolysates all along the hydrolysis. Second order derivative spectrometry was used in order to verify the presence of tyrosine in the peptide. This provided informations about the mechanism of cathepsin D activity towards hemoglobin. Moreover it confirmed that hemoglobin could appear as a precursor of some bioactive peptides following proteolytic degradation.