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D Pozo

Publications and source records attributed to D Pozo.

At least 19 recordsLinked to original sources
PubMed2000

Functional and molecular characterization of VIP receptor--effector system in rat developing immunocompetent cells: G protein involvement.

Changes in the functional characteristics for vasoactive intestinal peptide (VIP) receptor-effector system were evaluated in rat developing immunocompetent cells (from 1-week-old animals up to 12-week-old animals). These characteristics include [125I]VIP binding studies, cell cyclic AMP (cAMP) generation, analysis of [125I]VIP-receptor complexes by cross-linking experiments, as well as developed-associated G proteins assayed by cholera and pertussis toxin-catalyzed ADP-ribosylation and Western blot. The Scatchard analysis of binding data was consistent with the existence of two classes of VIP binding sites with K(d) values unaltered and B(max) increased during postnatal development. The efficiency of VIP stimulation of cAMP generation increased from 1-week-old rats to adult conditions. The VIP-receptor complex apparent molecular mass (52-55 kDa) remains unaltered, but it was significantly lower in 2-week-old than in 8-week-old rats. ADP-ribosylated material by cholera toxin (CTx) was higher from 8-week-old than from 2-week-old animals, while ADP-ribosylation by pertussis toxin (PTx) was quantitatively higher in 8-week-old rats. Results were confirmed when immunoblots for different G protein subunits were performed.

Adenosine Diphosphate Ribose↗
PubMed2000

Correlation between nuclear melatonin receptor expression and enhanced cytokine production in human lymphocytic and monocytic cell lines.

The report shows that melatonin enhances IL-2 and IL-6 production by two human lymphocytic (Jurkat) and monocytic (U937) cell lines via a nuclear receptor-mediated mechanism. Jurkat cells express nuclear (RZRalpha, RORalpha1 and RORalpha2) and membrane (mt1) melatonin receptors, and melatonin binds to Jurkat nuclei and membranes with the same affinity described for human peripheral blood mononuclear cells (PBMCs). Melatonin enhances IL-2 production by Jurkat cells activated by either phytohemagglutinin (PHA) or phorbol myristate acetate (PMA). PHA activation of Jurkat cells does not change the profile of melatonin receptor expression; on the contrary, PMA activation negatively regulates the mtl receptor. In the absence of the membrane receptor, melatonin still activates IL-2 production. U937 cells express only the mtl receptor. Although melatonin binds to both U937 nuclei and membranes, CGP 52608, a ligand of the nuclear receptor for melatonin, does not inhibit melatonin binding to U937 nuclei, suggesting that a protein other than the RZR/RORalpha receptor was involved in the process. In U937 cells, melatonin did not modify basal production of IL-6 or when activated by PMA plus LPS (lipopolysaccharide), a treatment that downregulates the expression of the mtl receptor. However, in U937 cells activated with IFN-gamma, which induces the expression of the RORgamma1 and RORalpha2 nuclear receptors and represses the expression of the mt1 receptor, melatonin can activate IL-6 production. These results show that the expression of nuclear melatonin receptor is sufficient for melatonin to activate cytokine production in human lymphocytic and monocytic cell lines.

Acetamides↗
PubMed2000

Identification of G-protein coupled receptor subunits in normal human dental pulp.

To respond appropriately to their environment, dental pulp cells must integrate informational input from multiple ligands, such as neuropeptides, growth factors, and vasoactive amines. These ligands act through multiple intracellular signaling pathways. G-protein coupled receptor subunits play a major role in this process, providing a mechanism for coordinated regulation of both messengers and effectors. Increasing number of neuropeptides have been found in pulpal tissue. However, there is no data about molecular identification of G-protein subunits in human dental pulp. To identify the postreceptor mechanism involved in dental pulp cell signal transduction, we performed a Western blot analysis of different G-protein subunits. Biopsy specimens of human dental pulp were prepared and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by analysis with appropriate antibodies. We detected G alpha q/alpha 11, short and long forms of G alpha s, beta common, Gio-3, and Gil-2 antigens with a molecular weight approximately 42 kDa, 42 and 45 kDa, 36 kDa, 40 kDa, and 40 kDa, respectively. These results indicate that human pulp cells possess the cellular machinery to respond to sensory neuropeptides when they are released from the peptidergic nerve fibers. On this basis, the relationships of postdevelopmental, age-dependent, and pathophysiological disorders of G-proteins subunits in dental pulp could be studied.

Dental Pulp↗
PubMed2000

Involvement of nuclear receptors in the enhanced IL-2 production by melatonin in Jurkat cells.

This report shows that melatonin enhances IL-2 production by Jurkat cells via a nuclear receptor-mediated mechanism. Jurkat cells express nuclear (RZR alpha, ROR alpha 1, and ROR alpha 2) and membrane (mt1) melatonin receptors, and melatonin binds to Jurkat nuclei and membranes with the same affinity described for human peripheral blood mononuclear cells (PBMCs). Melatonin enhances IL-2 production by Jurkat cells activated by either phytohemagglutinin (PHA) or phorbol myristate acetate (PMA). PHA activation of Jurkat cells does not change the profile of melatonin receptor expression; on the contrary, PMA activation negatively regulates the mt1 receptor. In the absence of the membrane receptor, melatonin still activates the IL-2 production. These results show that the expression of the nuclear melatonin receptor is sufficient for melatonin to activate IL-2 production by Jurkat cells.

Humans↗
PubMed1999

Characterization of membrane melatonin receptor in mouse peritoneal macrophages: inhibition of adenylyl cyclase by a pertussis toxin-sensitive G protein.

Melatonin binding sites were characterized in mouse peritoneal macrophages. Binding of 2-[125I]melatonin by macrophages fulfills all criteria for binding to a receptor site. Thus, binding was dependent on time, temperature and cell concentration, stable, reversible, saturable and specific. Stoichiometric studies showed a high-affinity binding site with a Kd of 0.58-0.71 nM. These data are in close agreement with data obtained from kinetic studies (Kd = 0.29 nM). The affinity of these binding sites suggests that they may recognize the physiological concentrations of melatonin in serum. Moreover, binding experiments using macrophage crude membranes showed that melatonin bound specifically to the membranes. Additionally, in competition studies we observed a low-affinity binding site (Kd = 2.02 microM). Melatonin inhibited significantly forskolin-stimulated cyclic AMP accumulation in a dose-dependent manner. This effect was blocked by luzindole, an antagonist of the melatonin membrane receptor. Pretreatment of macrophages with pertussis toxin blocked the inhibitory effect of melatonin. Pertussis toxin ADP-rybosilation and Western blot experiments demonstrated both alpha(i1/2) and alpha(i3/o) G protein subunits expression in mouse peritoneal macrophages membranes. Our results demonstrate the existence of melatonin receptors in mouse peritoneal macrophages, and a pertussis toxin-sensitive melatonin signal transduction pathway that involves the inhibition of adenylyl cyclase.

Adenosine Diphosphate Ribose↗
PubMed1999

Vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide inhibit endotoxin-induced TNF-alpha production by macrophages: in vitro and in vivo studies.

Vasoactive intestinal peptide (VIP) is a neuropeptide synthesized by immune cells that can modulate several immune aspects, including the function of cells involved in the inflammatory response, such as macrophages and monocytes. The production and release of cytokines by activated phagocytes are important events in the pathogenesis of ischemia-reperfusion injury. There is abundant evidence that the proinflammatory cytokine TNF-alpha is an important mediator of shock and organ failure complicating Gram-negative sepsis. VIP has been shown to attenuate the deleterious consequences of this pathologic phenomenon. In this study we have investigated the effects of VIP and the structurally related neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP38) on the production of TNF-alpha by endotoxin-activated murine peritoneal macrophages. Both neuropeptides rapidly and specifically inhibit the LPS-stimulated production of TNF-alpha, exerting their action through the binding to VPAC1 receptor and the subsequent activation of the adenylate cyclase system. VIP and PACAP regulate the production of TNF-alpha at a transcriptional level. In vitro results were correlated with an inhibition of both TNF-alpha expression and release in endotoxemic mice in vivo. The immunomodulatory role of VIP in vivo is supported by the up-regulation of VIP release in serum and peritoneal fluid by LPS and proinflammatory cytokines such as TNF-alpha, IL-1beta, and IL-6. These findings support the idea that under toxicity conditions associated with high LPS doses, VIP and PACAP could act as protective mediators that regulate the excessive release of TNF-alpha to reduce inflammation or shock.

Animals↗
PubMed1999

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activation polypeptide (PACAP) protect mice from lethal endotoxemia through the inhibition of TNF-alpha and IL-6.

The neuropeptides vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) down-regulate cytokine production. Because human septic shock involves excessive cytokine production, the effect of VIP/PACAP was investigated in a high endotoxemia murine model. Both peptides protect against endotoxin-induced lethality and prevent septic shock-associated histopathological alterations. VIP/PACAP reduce serum and peritoneal TNF-alpha and IL-6, suggesting that the protective effect is exerted by inhibiting the production of endogenous TNF-alpha/IL-6. Consistent with this mechanism, VIP does not protect against septic shock induced by exogenous TNF-alpha. The immunomodulatory role of VIP in vivo is supported by the appearance of high levels of VIP in serum and peritoneal fluid following LPS administration. Thus, the neuropeptides VIP/PACAP protect from the lethal effect of high endotoxemia, presumably by down-regulating TNF-alpha and IL-6 production, and may offer an alternative in the treatment of human septic shock syndrome.

Animals↗
PubMed1999

Melatonin activates Th1 lymphocytes by increasing IL-12 production.

Melatonin could act on immune system by regulating cytokine production of immunocompetent cells. The hormone enhances IL-2, IFN-gamma and IL-6 production by cultured human mononuclear cells. As enhancement of IL-6 production is related to monocyte activation by melatonin, the hormone acts on human lymphoid cells causing a Th1-type response. This paper shows that melatonin seems to promote a Th1-response by increasing IL-12 production. The hormone enhances IL-12 production by cultured monocytes under suboptimal stimulation in a dose-dependent way. The effect of the hormone increases when PBMCs are incubated with melatonin before monocyte isolation. Enhanced IL-12 production by melatonin can also be shown in cultured human mononuclear cells.

Cells, Cultured↗
PubMed1999

Melatonin inhibits expression of the inducible NO synthase II in liver and lung and prevents endotoxemia in lipopolysaccharide-induced multiple organ dysfunction syndrome in rats.

We evaluated the role of melatonin in endotoxemia caused by lipopolysaccharide (LPS) in unanesthetized rats. The expression of inducible isoform of nitric oxide synthase (iNOS) and the increase in the oxidative stress seem to be responsible for the failure of lungs, liver, and kidneys in endotoxemia. Bacterial LPS (10 mg/kg b. w) was i.v. injected 6 h before rats were killed and melatonin (10-60 mg/kg b.w.) was i.p. injected before and/or after LPS. Endotoxemia was associated with a significant rise in the serum levels of aspartate and alanine aminotransferases, gamma-glutamyl-transferase, alkaline phosphatase, creatinine, urea, and uric acid, and hence liver and renal dysfunction. LPS also increased serum levels of cholesterol and triglycerides and reduced glucose levels. Melatonin administration counteracted these organ and metabolic alterations at doses ranging between 20 and 60 mg/kg b. w. Melatonin significantly decreased lung lipid peroxidation and counteracted the LPS-induced NO levels in lungs and liver. Our results also show an inhibition of iNOS activity in rat lungs by melatonin in a dose-dependent manner. Expression of iNOS mRNA in lungs and liver was significantly decreased by melatonin (60 mg/kg b. w., 58-65%). We conclude that melatonin inhibits NO production mainly by inhibition of iNOS expression. The inhibition of NO levels may account for the protection of the indoleamine against LPS-induced endotoxemia in rats.

Animals↗
PubMed1998

VIP and PACAP enhance IL-6 release and mRNA levels in resting peritoneal macrophages: in vitro and in vivo studies.

Vasoactive intestinal peptide (VIP), a neuropeptide produced by lymphocytes has been previously reported to modulate cytokine expression in T lymphocytes. In this study, we investigated the effects of VIP and of the structurally related neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP38) on the production of IL-6 in unstimulated murine peritoneal macrophages. Both neuropeptides stimulate rapidly, specifically, and similarly the production of IL-6, exerting their action through two different receptor/signal transduction systems, i.e., primarily through the binding to VIP1/PACAP receptor followed by adenylate cyclase activation, and partially through the activation of protein kinase C following binding to PACAP-R. VIP and PACAP38 regulate the production of IL-6 at a transcriptional level, affecting the de novo synthesis of this cytokine. The stimulatory in vitro effect correlates with the stimulation of IL-6 expression and release in vivo. These studies suggest that VIP/PACAP play a role in immune system homeostasis, participating in the intricate cytokine network and controlling local immune responses. In addition, the understanding of the factors that regulate the expression and release of IL-6 by macrophages is important for the elucidation of the role of IL-6 in health and disease.

Animals↗
PubMed1998

Vasoactive intestinal peptide and pituitary adenylate cyclase-activating polypeptide modulate endotoxin-induced IL-6 production by murine peritoneal macrophages.

Vasoactive intestinal peptide (VIP) is a neuropeptide synthesized by immune cells that can modulate several immune aspects, including the function of cells involved in the inflammatory response, such as macrophages and monocytes. Production and release of cytokines by activated mononuclear phagocytes is an important event in the pathogenesis of ischemia-reperfusion injury. VIP has been shown to attenuate the deleterious consequences of this pathologic phenomenon. We have investigated the effects of VIP and PACAP38 on the production of interleukin-6 (IL-6), a proinflammatory cytokine, by endotoxin-activated murine macrophages. Both neuropeptides exhibit a dual effect on the IL-6 production by peritoneal macrophages. Whereas VIP and PACAP inhibit with similar dose-response curves the release of IL-6 from macrophages stimulated with a LPS dose range from 100 pg/mL to 10 microg/mL, both neuropeptides enhance IL-6 secretion in unstimulated macrophages and in macrophages stimulated with very low LPS concentrations (1-10 pg/mL). The inhibition on LPS-induced IL-6 production is specific, presumably mediated through a subtype of the PACAP-R. VIP and PACAP regulate the production of IL-6 at a transcriptional level. These results were correlated with an inhibition on both IL-6 expression and release in endotoxemic mice in vivo. These findings support the idea that in the absence of stimulation or in the presence of low doses of LPS, VIP and PACAP could play a role in immune system homeostasis. However, under toxicity conditions associated with high LPS doses, VIP and PACAP could act as protective mediators that regulate the excessive release of IL-6 in order to reduce inflammation or shock.

Adenylyl Cyclases↗
PubMed1997

Functional characterization and mRNA expression of pituitary adenylate cyclase activating polypeptide (PACAP) type I receptors in rat peritoneal macrophages.

The present work characterizes the mRNA expression of PACAP type I receptors in rat peritoneal macrophages but not in peritoneal lymphocytes by both retrotranscriptase and polymerase chain reaction (RT-PCR) and homologous Southern hybridization and the stimulation by PACAP27, PACAP38 and vasoactive intestinal peptide (VIP) of sn-1,2-diacylglycerol production in rat peritoneal macrophage membranes. The binding of [125I]PACAP27 was time and cell concentration dependent. Scatchard analysis of displacement of the bound tracer by unlabeled PACAP27 indicates the existence of two classes of binding sites. The dissociation constant (Kd) was 0.64 +/- 0.08 nM and the maximal binding capacity (Bmax) was 8.85 +/- 1.45 fmol/10(6) cells for the high affinity binding site. The low affinity binding site had a Kd of 0.10 +/- 0.06 microM with a Bmax of 300 +/- 21.9 fmol/10(6) cells. Scatchard analysis of VIP displacement data indicated the presence of two classes of binding sites with a Kd and Bmax different to those of PACAP27. These results suggest that PACAP binds to two binding sites, PACAP type I receptors and PACAP type II receptors. The PACAP27-stimulated diacylglycerol production was not affected by treatment with pertussis toxin. However, the presence of GTP partially inhibited this PACAP27 stimulation of 1,2-diacylglycerol in a dose dependent manner, although GTP alone stimulates diacylglycerol accumulation. In conclusion, for the first time we demonstrate by biochemical and molecular biology criteria the existence of PACAP type I receptors on rat peritoneal macrophages and the evidence for coupling with a pertussis toxin-insensitive G regulatory protein.

Adenylate Cyclase Toxin↗
PubMed1997

Signal transduction for melatonin in human lymphocytes: involvement of a pertussis toxin-sensitive G protein.

We analyzed the melatonin signal transduction in human blood lymphocytes. High affinity melatonin receptors were identified by specific binding of 2-[125I]melatonin ([125I]MEL) to human lymphocyte membranes. Scatchard analysis of [125I]MEL binding revealed high affinity receptors, with a dissociation constant (Kd) of 0.45 nM and a binding capacity (Bmax) of 7.8 fmol/mg protein. Specific [125I]MEL binding was reduced markedly by GTP and its nonhydrolyzable analogues guanosine 5'-beta, gamma-imidotriphosphate (Gpp(NH)p) and guanosine 5'-O-(3-triphosphate) (GTP-gamma-S). Gpp(NH)p inhibited in a dose-dependent manner the [125I]MEL specifically bound to human lymphocyte membranes with a half-maximal inhibition (IC50) of 3.5 +/- 0.6 microM Gpp(NH)p. Treatment of human lymphocyte membranes with Gpp(NH)p increased the Kd value (Kd = 1.20 nM). Melatonin inhibited significantly and in a dose-dependent manner forskolin-stimulated cAMP production in intact human lymphocytes. Melatonin was able to stimulate diacylglycerol production in a dose-dependent manner in human lymphocyte membranes. Pertussis toxin treatment inhibited the specific [125I]MEL binding and blocked the ability of melatonin to both inhibit forskolin-stimulated cAMP production and stimulate diacylglycerol production. Pertussis toxin ADP-ribosylation and Western blot experiments demonstrated the protein expression of alpha i1/2, alpha i3/0, and beta gamma complexes of G proteins in human lymphocyte membranes. The results strongly suggest a pertussis toxin-sensitive melatonin signal transduction pathway in human lymphocytes that involves the inhibition of adenylyl cyclase and the stimulation of phospholipase C.

Adenylate Cyclase Toxin↗
PubMed1997

Inhibition of cerebellar nitric oxide synthase and cyclic GMP production by melatonin via complex formation with calmodulin.

Constitutive rat cerebellar nitric oxide synthase (NOS) activity is shown to be inhibited by physiological concentrations of the pineal hormone melatonin. The inhibition was dose-dependent and was coupled to an inhibition of the cyclic GMP production activated by L-arginine. Results also show that calmodulin appears to be involved in this process because its presence in the incubation medium was able to prevent the effect of melatonin on both NOS activity and cyclic GMP production. Moreover, polyacrylamide gel electrophoresis studies suggest that melatonin can interact with calmodulin modifying the binding of the peptide to the synthetic NOS peptide encompassing the calmodulin-binding domain of constitutive NOS from rat cerebellum, the natural mechanism by which calmodulin activates cerebellar NOS.

Animals↗
PubMed1997

Characterization of VIP receptor-effector system antagonists in rat and mouse peritoneal macrophages.

In the present study we show that the synthetic peptides [4-Cl-D-Phe6,Leu17]VIP and the growth hormone releasing factor (GRF) analog [Ac-Tyr1,D-Phe2]GRF-(1-29)-NH2 inhibit in a competitive manner the specific [125I]VIP binding to both rat and mouse peritoneal macrophages. In rat peritoneal macrophages, the order of potency of the different peptides, as expressed by the IC50 values was: VIP (IC50 = 1.90 +/- 0.16 nM) > [4-Cl-D-Phe6,Leu17]VIP (IC50 = 125.8 +/- 13.2 nM) > [Ac-Tyr1,D-Phe2]GRF-(1-29)-NH2 (IC50 = 354.8 +/- 21.2 nM). In mouse peritoneal macrophages a similar pattern of potency was observed: VIP (IC50 = 1.58 +/- 0.12 nM) > [4-Cl-D-Phe6,Leu17]VIP (IC50 = 110.8 +/- 10.7 nM) > [Ac-Tyr1,D-Phe2]GRF-(1-29)-NH2 (IC50 = 251 +/- 19.2 nM). The behavior as VIP receptor antagonists of both [4-Cl-D-Phe6,Leu17]VIP and [Ac-Tyr1,D-Phe2]GRF-(1-29)-NH2 in rat and mouse peritoneal macrophages was confirmed by: (a) the shift to the right of VIP dose-stimulated cyclic AMP production curves in the presence of the two antagonists; (b) the agreement between the order of efficacy of the two peptides in competition experiments with the corresponding inhibition of cyclic AMP production; (c) the inefficiency of the two antagonists on the stimulation of cyclic AMP production by the beta-adrenoceptor agonist isoproterenol, which indicates the specificity of the interaction; (d) the synergic effect of VIP on isoproterenol-stimulated cyclic AMP production was completely abolished by [4-Cl-D-Phe6,Leu17]VIP or [Ac-Tyr1,D-Phe2]GRF-(1-29)-NH2, suggesting that both antagonists acted via specific VIP receptors. Moreover, propranolol, a beta-adrenoceptor antagonist, did not affect the VIP-stimulated cyclic AMP production and the antagonist role of [4-Cl-D-Phe6,Leu17]VIP or [Ac-Tyr1,D-Phe2]GRF-(1-29)-NH2; (e) in cross-linking experiments, the intensity of the labeling of the [125I]VIP/receptor complexes was significantly lower with the antagonists than in the control experimental situation in both mouse and rat peritoneal macrophage membranes.

Adrenergic beta-Agonists↗