Gabriel Nava

In vivo release and gene upregulation of brain prolactin in response to physiological stimuli

Although prolactin (PRL) actions and expression in the brain have been shown, dynamic changes in its intracerebral release and gene expression have still not been demonstrated. Using push‐pull perfusion, the in vivo release of PRL was monitored within the paraventricular nucleus (PVN) and medial preoptic area (MPOA) of virgin female, lactating and male rats in response to various stimuli. Perfusion with a depolarizing medium (56 mm K+) increased local release of PRL within both the PVN (P < 0.05) and MPOA (P < 0.05) of urethane‐anaesthetized rats, indicating release from excitable neuronal structures. The PRL in perfusates was verified by radioimmunoassay, Nb2 cell bioassays and western blot. Systemic osmotic stimulation (3 m NaCl i.p., 8 mL/kg b.w.) raised PRL concentration in plasma (P < 0.01) but not within the PVN, suggesting independent release from the pituitary and in distinct brain regions. Immobilization for 30 min increased PRL release within the PVN (P < 0.05) and the MPOA (P < 0.01) of virgin female and male (P < 0.05 each) rats and increased hypothalamic PRL mRNA expression (P = 0.008) after 30 and 90 min as revealed by real‐time polymerase chain reaction. This indicates a stress‐induced activation of both PRL release from and synthesis in hypothalamic neurons. Additionally, PRL was significantly released within, but not outside, the PVN (P < 0.01) and the MPOA (P < 0.05) of lactating rats during suckling and this was accompanied by a significant increase of PRL mRNA (P < 0.05) in the hypothalamus 60 min after suckling. This is the first demonstration of stimulus‐induced, locally restricted release and gene upregulation of PRL within the brain, emphasizing the involvement of this ‘novel’ neuropeptide in various brain functions.

Matrix metalloproteases from chondrocytes generate an antiangiogenic 16 kDa prolactin

The 16 kDa N-terminal fragment of prolactin (16K-prolactin) is a potent antiangiogenic factor. Here, we demonstrate that matrix metalloproteases (MMPs) produced and secreted by chondrocytes generate biologically functional 16K-prolactin from full-length prolactin. When incubated with human prolactin at neutral pH, chondrocyte extracts and conditioned medium, as well as chondrocytes in culture, cleaved the Ser155-Leu156 peptide bond in prolactin, yielding - upon reduction of intramolecular disulfide bonds - a 16 kDa N-terminal fragment. This 16K-prolactin inhibited basic fibroblast growth factor (FGF)-induced endothelial cell proliferation in vitro. The Ser155-Leu156 site is highly conserved, and both human and rat prolactin were cleaved at this site by chondrocytes from either species. Conversion of prolactin to 16K-prolactin by chondrocyte lysates was completely abolished by the MMP inhibitors EDTA, GM6001 or 1,10-phenanthroline. Purified MMP-1, MMP-2, MMP-3, MMP-8, MMP-9 and MMP-13 cleaved human prolactin at Gln157, one residue downstream from the chondrocyte protease cleavage site, with the following relative potency: MMP-8>MMP-13 >MMP-3>MMP-1=MMP-2>MMP-9. Finally, chondrocytes expressed prolactin mRNA (as revealed by RT-PCR) and they contained and released antiangiogenic N-terminal 16 kDa prolactin (detected by western blot and endothelial cell proliferation). These results suggest that several matrix metalloproteases in cartilage generate antiangiogenic 16K-prolactin from systemically derived or locally produced prolactin.

17-Beta-Estradiol Directly Regulates the Expression of Adrenergic Receptors and Kisspeptin/GPR54 System in GT1-7 GnRH Neurons

Estradiol plays a critical role in the feedback regulation of reproduction, in part by modulating the neurosecretory activity of gonadotropin-releasing hormone (GnRH) neurons. While indirect effects of estradiol on GnRH neurons have been clearly demonstrated, direct actions are still controversial. In the current study, we examined direct effects of 17β-estradiol upon the expression of receptors for afferent signals at the level of the GnRH neuron, using immortalized GT1-7 cells. Using RT-PCR, we confirmed the expression of mRNA for the adrenergic receptors (AR) α1A-, α1B-, α1D-, α2A-, α2C-, and β1-AR, and showed for the first time that mRNAs for α2B-, β2- and β3-AR, for kisspeptin and its receptor GPR54 and for the novel estrogenic receptor GPR30 are expressed in GT1-7 cells. After treatment with 10 nM 17β-estradiol, α1B-AR mRNA was significantly increased (14-fold) after 6 h as determined by real-time PCR, while α1B- and α1D-AR mRNA were significantly increased (19- and 23-fold, respectively) after 24 h. The expression of KiSS-1 and GPR54 mRNAs were also significantly increased (8- and 6-fold, respectively) after 24 h treatment of GT1-7 cells with estradiol. GPR30 mRNA expression was not affected by estradiol. Our data also showed that kisspeptin-10 (1–10 nM) can significantly stimulate GnRH release and GnRH mRNA expression in GT1-7 cells. These results suggest that the complex physiologic effects of estradiol on the function of the reproductive axis could be mediated partly through direct modulation of the expression of receptors for afferent signals in GnRH neurons.

Prolactin stimulates integrin-mediated adhesion of circulating mononuclear cells to endothelial cells

Attachment of leukocytes to endothelial cells is an essential step for the extravasation and recruitment of cells at sites of inflammation. The pituitary hormone prolactin (PRL) is involved in the inflammatory process. Here, we show that treatment with PRL of human peripheral blood mononuclear cells (PBMC) stimulates their adhesion to human umbilical vein endothelial cells (HUVEC) activated by interleukin-1β. Stimulation of adhesion by PRL is mediated via integrins leukocyte functional antigen-1 (LFA-1) and very late antigen-4 (VLA-4), because immunoneutralization of both integrins prevents PRL action. Also, PRL promotes the adhesion of PBMC to immobilized intercellular adhesion molecule-1 and fibronectin, ligands for LFA-1 and VLA-4, respectively. Stimulation of integrin-mediated cell adhesion by PRL may involve the activation of chemokine receptors, because PRL upregulates the expression of the G-protein-coupled chemokine receptor CXCR3 in PBMC, and pertussis toxin, a specific G-protein inhibitor, blocks PRL stimulation of PBMC adhesion to HUVEC. In addition, PRL stimulates tyrosine phosphorylation pathways leading to leukocyte adhesion. PRL triggered the tyrosine phosphorylation of Janus kinase-2, of signal transducer and activator of transcription-3 and 5, and of the focal adhesion protein paxillin. Furthermore, genistein, a tyrosine kinase inhibitor, blocked PRL-stimulated adhesion of PBMC and Jurkat T-cells to HUVEC. These results suggest that PRL promotes integrin-mediated leukocyte adhesion to endothelial cells via chemokine receptors and tyrosine phosphorylation signaling pathways.

Cytokine induction of prolactin receptors mediates prolactin inhibition of nitric oxide synthesis in pulmonary fibroblasts.

Prolactin (PRL) has been implicated as a modulator of immune function, and some of its actions may be linked to NO synthesis. Because NO acts as a mediator of inflammation, we speculated that an inflammatory milieu could unmask pathways by which PRL could affect NO synthesis. Here, we show that pro‐inflammatory cytokines induce the expression of PRL receptors in pulmonary fibroblasts, allowing PRL to inhibit cytokine‐induced NO production and the expression of the inducible nitric oxide synthase (iNOS). Inhibition of iNOS expression by PRL correlates with the phosphorylation of STAT‐5b (signal transducer and activator of transcription 5b) and the suppression of expression of IRF‐1 (interferon regulatory factor 1), a transcription factor for iNOS. These results reveal previously unrecognized mechanisms by which PRL and PRL receptors may play significant modulatory roles during immune–inflammatory processes.

GABA Inhibition of Cyclic AMP Production in Immortalized GnRH Neurons Is Mediated by Calcineurin-Dependent Dephosphorylation of Adenylyl Cyclase 9

The neurotransmitter γ-aminobutyric acid (GABA) is an important modulator of gonadotropin-releasing hormone (GnRH), and consequently of reproduction. GABA, acting via ionotropic GABAA receptors, exerts a biphasic effect on GnRH secretion in immortalized GnRH cells. The initial increase in GnRH secretion is triggered by a sharp rise in [Ca2+]i, while the progressive decline of GnRH levels that follows is paralleled by reduced levels of intracellular cAMP. The experiments described here were designed to explore the potential signaling pathways involved in this novel GABAA ionotropic inhibition of cAMP synthesis in GT1–7 cells. Using RT-PCR and real-time PCR, we found that GT1–7 cells express 8 of 9 known membrane adenylyl cyclase (AC) isoforms, including a large proportion of AC3 and AC9, as well as AC5 and AC6, all of which are negatively regulated by increases in [Ca2+]i. In contrast, isoforms of AC that are positively regulated by [Ca2+]i were barely detectable (AC1) or undetectable (AC8). Pharmacological activation of L-type voltage-operated calcium channels with BayK 8644 produced a decrease in [cAMP]i similar to that induced by GABA, while blocking these calcium channels with verapamil reversed the effect of GABA on cAMP synthesis. Furthermore, blocking calcineurin with deltamethrin, FK-506 or cyclosporin A blocked the inhibitory effect of GABA on [cAMP]i, supporting the involvement of AC9 in this effect. In addition, blocking Ca2+/calmodulin-dependent protein kinase II (CamKII) with KN-62 partially reversed the action of GABA, suggesting that AC3 may also be involved in this effect. Finally, GABA increased phosphatase activity in a calcium-dependent manner, an effect blocked by calcineurin inhibitors. Collectively, our results show that the ionotropic action of GABA via the activation of GABAA receptors can decrease AC activity in immortalized GnRH neurons, and that the effect of GABA appears to be mediated by a transient increase in [Ca2+]i followed by activation of calcineurin and CamKII, leading to dephosphorylation of AC9 and phosphorylation of AC3, respectively, and subsequently reducing the synthesis of cAMP.

Small-size ribosomal RNA species in Trypanosoma cruzi.

Trypanosoma cruzi ribosomal RNA was analyzed electrophoresis. On agarose gels, where both large- and small-size species are grossly fractionable, it revealed two bands in the small-size region. These were similar in size to the mammalian 5.8 S and 5 S species. Increased resolution, however, showed these two bands to be composite. The pseudo 5.8 S band contained three, and the pseudo 5 S two, discretely sized molecules. The ribosomal binding of four of these five novel species is apparently dependent on large ribosomal subunit proteins. One species is hydrogen bonded to the beta species of 24 S ribosomal RNA. The five species were estimated to be 261, 217, 197, 141 and 110 nucleotides long.

Vasoinhibins Prevent Bradykinin-Stimulated Endothelial Cell Proliferation by Inactivating eNOS via Reduction of both Intracellular Ca2+ Levels and eNOS Phosphorylation at Ser1179

Vasoinhibins, a family of antiangiogenic peptides derived from prolactin proteolysis, inhibit the vascular effects of several proangiogenic factors, including bradykinin (BK). Here, we report that vasoinhibins block the BK-induced proliferation of bovine umbilical vein endothelial cells. This effect is mediated by the inactivation of endothelial nitric oxide synthase (eNOS), as the NO donor DETA-NONOate reverted vasoinhibin action. It is an experimentally proven fact that the elevation of intracellular Ca2+ levels ([Ca2+]i) upon BK stimulation activates eNOS, and vasoinhibins blocked the BK-mediated activation of phospholipase C and the formation of inositol 1,4,5-triphosphate leading to a reduced release of Ca2+ from intracellular stores. The [Ca2+]i rise evoked by BK also involves the influx of extracellular Ca2+ via canonical transient receptor potential (TRPC) channels. Vasoinhibins likely interfere with TRPC-mediated Ca2+ entry since La3+, which is an enhancer of TRPC4 and TRPC5 channel activity, prevented vasoinhibins from blocking the stimulation by BK of endothelial cell NO production and proliferation, and vasoinhibins reduced the BK-induced increase of TRPC5 mRNA expression. Finally, vasoinhibins prevented the BK-induced phosphorylation of eNOS at Ser1179, a post-translational modification that facilitates Ca2+-calmodulin activation of eNOS. Together, our data show that vasoinhibins, by lowering NO production through the inhibition of both [Ca2+]i mobilization and eNOS phosphorylation, prevent the BK-induced stimulation of endothelial cell proliferation. Thus, vasoinhibins help to regulate BK effects on angiogenesis and vascular homeostasis.

Modelo de gestión logística para pequeñas y medianas empresas en México

La apertura de los mercados y la globalizacion de las cadenasde suministro demandan cambios estructurales en los que lalogistica juega un papel estrategico. Actualmente, los clientesevaluan la calidad del producto, el valor agregado del mismoy su disponibilidad en tiempo y forma, de ahi la necesidad dehacer eficientes los procesos. Diversos expertos han propuestomodelos de gestion logistica para elevar la competitividad enel mercado; algunos de ellos son ambiciosos para las pequenasy medianas empresas (Pyme) debido a la estructura informal ycarencia de conocimientos tecnicos de las mismas; otros hacenreferencia indirecta a los flujos de informacion interna, implicandouna desintegracion total del sistema por la debil interrelacionentre areas. La Pyme en Mexico representa el 4.2% delas empresas, genera el 31.5% del empleo y aporta el 37% delProducto Interno Bruto; de ahi surge la importancia de fortalecersu posicion competitiva en el mercado. Esta investigacionpresenta el diseno de un modelo conceptual de gestion logisticapara Pyme que podria dar solucion integral a traves delcontrol de las variables involucradas en los procesos logisticos;para verificar que las variables consideradas en cada dimensionidentificada son las correctas se utilizo el analisis factorial.