Biomedical subjects

D Pozo

Publications and source records attributed to D Pozo.

34 records · Page 2Linked to original sources

PubMed1997

Expression of the Mel1a-melatonin receptor mRNA in T and B subsets of lymphocytes from rat thymus and spleen.

In the present work we analyze by reverse transcription, polymerase chain reaction, cDNA cloning, and sequence analysis the expression of membrane melatonin receptors in rat thymus and spleen. Results show, for the first time, that the melatonin receptor mRNA is expressed in both the thymus and spleen. Moreover, the melatonin receptor mRNA was expressed in all the lymphocyte subpopulations (CD4+,CD8+, double positive, double negative, and B cells) studied from the rat thymus. The Southern blot analysis with the melatonin receptor probe and sequence data also showed the identity of the DNA fragments in thymus, spleen, and the lymphocyte subpopulations studied. The melatonin receptor fragments amplified from rat brain, thymus, and spleen share identical nucleotide sequences with the rat Mel1a-melatonin receptor subtype. No signal was obtained with primers used to amplify the rat Mel1b-melatonin receptor subtype in both thymus and spleen. Finally, the melatonin receptor mRNA transcript distribution throughout the rat thymus was examined. Using digoxigenin-labeled cRNA probe to the specific melatonin receptor mRNA, examination of the whole thymus revealed a clear hybridization signal in both cortex and medulla. Melatonin receptor gene expression in the thymus and spleen supports the notion of the immunomodulatory role of melatonin.

Animals↗

PubMed1997

Differential adrenergic regulation of rat pineal cyclic AMP production and N-acetyltransferase activity during postnatal development: involvement of G alpha s and G alpha i1-2 proteins.

We have studied why rat pineal N-acetyltransferase (NAT) activity is relatively insensitive to isoproterenol in young rats when compared with adult rats. We report that activation by isoproterenol of pineal cyclic AMP production and NAT activity is higher in adult than in 2-week-old rats. However, the effect of dibutyryl cyclic AMP, which enters the pinealocyte and duplicates the effect of cyclic AMP, on NAT activity was similar at both ages. Moreover, we found that both alpha- and beta-adrenergic receptors are highly specific at both ages, since the binding of the specific radioligands used to their receptors could be displaced only by their corresponding agonists and antagonists. However, we observed differences between pineals from young and adult rats when several families of the alpha subunit of G-proteins were studied in cell membranes. ADP-ribosylation and immunoblot studies have shown clear differences in both 42 and 45 kDa forms of the Gs alpha Both forms exhibit low values in pineals from 2-week-old animals when compared with 6-week-old. We also show that the later appearance of both Gs alpha forms is roughly similar to the potent activation of cyclic AMP production and NAT activity in adult rats when compared with young rats. In conclusion, the results presented suggest that the relative lack of sensitivity of rat pineal gland to beta-adrenergic receptor agonists early in the postnatal development may be explained by the low levels of membrane Gs alpha, rather than postreceptor-mediated mechanisms or changes in the characteristics of the beta-adrenergic receptors on the pinealocyte membrane.

Adrenergic alpha-Agonists↗

PubMed1996

Characterization of adenylyl cyclase stimulated by VIP in rat and mouse peritoneal macrophage membranes.

Vasoactive intestinal peptide (VIP) stimulated adenylyl cyclase activity in rat and mouse peritoneal macrophage membranes. GTP potentiated the stimulatory effect of VIP so that it was routinely included at 10 microM GTP. Other agents like GTP, Gpp(NH)p, GTP-gamma-S, sodium fluoride, and forskolin, at a concentration of 0.1 mM, increased the basal activity of enzyme by 3.1, 5.7, 4.7, 3.6, and 7.8-fold, respectively. The stimulation of adenylyl cyclase by VIP was time, temperature, and membrane concentration dependent. Half-maximal enzyme activation (ED50) was very similar in rat and mouse peritoneal macrophage membranes (1.5 +/- 0.1 nM and 1.0 +/- 0.1 nM, respectively). However, VIP showed more efficacy in mouse macrophages membranes (about 3.1-fold basal values) than that in rat macrophage membranes (about 2.5-fold basal values). The relative potency of several peptides upon stimulation of adenylyl cyclase activity showed the following potency in both species: VIP = PACAP38 = PACAP27 > helodermin > PHI > secretin. On the other hand, a M(r)-45 kDa alpha s subunit of Gs protein was demonstrated by both ADP-ribosylation and immunoblot in mouse and rat peritoneal macrophage membranes. The present results, together other previous, strongly suggest that VIP play an important role in the regulation of macrophage function.

Adenosine Diphosphate Ribose↗

PubMed1996

Characterization of binding sites for beta-adrenergic agonists and vasoactive intestinal peptide in the rat harderian gland.

Vasoactive intestinal peptide (VIP) receptors and beta-adrenergic receptors were investigated in rat Harderian gland membranes using 125I-VIP and 125I-cyanopindolol (125I-CYP), respectively, as ligands. The receptor bindings were rapid, reversible, saturable, specific, and dependent on time, temperature, and membrane concentration. The stoichiometric data suggested the presence of two classes of VIP receptors with Kd values of 0.36 and 65.37 nM and binding capacities of 323 and 39,537 fmol VIP/mg protein, respectively. The interaction showed a high degree of specificity, as suggested by competitive displacement experiments with several peptides structurally or not structurally related to VIP as follows: VIP > helodermin > rGRF > PHI > > secretin. Glucagon, somatostatin, insulin, and pancreastatin were ineffective at concentrations up to 1 microM. However, the stoichiometric data suggest the presence of one class of binding sites for 125I-CYP. The Kd for the single site was 290 pM with a binding capacity of 32 pmol/L. The pharmacological characterization of 125I-CYP binding to membranes showed that only isoproterenol, a beta-adrenergic agonist, and norepinephrine, an alpha beta-adrenergic agonist, was as effective as propranolol in inhibiting 125I-CYP binding to Harderian gland membranes. However, alpha 1- and alpha 2-adrenergic agonists and blockers such as methoxamine, prazosin, clonidine, and yohimbine were shown to be ineffective. These results demonstrate the presence of specific VIP and beta-adrenergic receptors in the Harderian gland and suggest a role for VIP and beta-adrenergic agonists in the physiology of this gland.

Animals↗

PubMed1996

Characterization of gene expression of VIP and VIP1-receptor in rat peritoneal lymphocytes and macrophages.

In the present report we show the gene expression pattern of VIP and VIP1 receptor in two peritoneal cell populations, macrophages and lymphocytes by reverse transcription (RT) and polymerase chain reaction (PCR). Only in the lymphoid cells we have obtained a specific VIP cDNA product of 458 bp identical in size to the one obtained from cerebral cortex. On the other hand, we have obtained in both peritoneal populations lymphocytes and macrophages, a specific VIP1 receptor cDNA product of 311 bp identical in size to that obtained from lung. These results have been confirmed by Southern blot hybridization. Our findings suggest an autocrine/paracrine action of VIP in peritoneal microenvironment, supporting an immunoregulatory role for this neuropeptide.

Animals↗

PubMed1996

Thymosin alpha 1 interacts with the VIP receptor-effector system in rat and mouse immunocompetent cells.

Thymic peptide thymosin alpha 1 (10(-11) to 10(-6) M) is shown to interact with the VIP receptor-effector system in rat and mouse peritoneal macrophages, and both rat peripheral blood lymphocytes and spleen lymphocytes. In all models, thymosin alpha 1 inhibits 125I-VIP binding with a potency that is in a range 1000-1700 times lower than that of the native VIP. Interaction of thymosin alpha 1 with VIP receptors is compared with that of some structurally VIP-related peptides such as helodermin, PHI, secretin, and glucagon. The order of potency in inhibiting 125I-VIP binding was VIP > helodermin > PHI > secretin > thymosin alpha 1. Thymosin alpha 1 (10(-10) to 10(-6) M) was weak in stimulating adenylyl cyclase activity. Its efficacy is in a range 900-1800 times lower than that of native VIP in all cell types studied. The analysis of the sequence of both complete and N-terminal portion of thymosin alpha 1 reveals close structural and physicochemical similarities with the members of the so-called VIP family of polypeptides. Taken together, experimental data support that thymosin alpha 1 must be included like the lowest partial agonist of the VIP family of polypeptides and it is a VIP receptor antagonist with weak intrinsic activity.

Animals↗

PubMed1996

Expression of vasoactive intestinal peptide binding sites in rat peritoneal macrophages is stimulated by inflammatory stimulus.

Vasoactive intestinal peptide (VIP) binding to resident and stimulated-rat peritoneal macrophages was studied. No specific VIP binding was obtained with resident rat peritoneal macrophages. In contrast, VIP bound specifically to casein-elicited macrophages. The Scatchard analysis of binding data was consistent with the presence of two classes of VIP binding sites, but may represent a receptor site and internalized VIP. Both specific VIP binding and number of specific high affinity binding sites for VIP augmented progressively after sodium caseinate injection, reaching maximum at days 4-5. Macrophages obtained 1 day after injection showed a minimal specific VIP binding (0.3 +/- 0.1% of total), but cells obtained 4 days after injection showed a maximal binding to the peptide (3.1+/-0.2% of total). The number of high affinity binding sites per cell raised also progressively after sodium caseinate injection: 2650+/-301 at day 2, 4939 +/-723 at day 3, 6684+/-903 at day 4 and 9636+/-1626 at day 5 (P = 0.0035). The number of low affinity binding sites per cell exhibited the same changes. In contrast, the Kd values of both high and low affinity VIP binding sites did not vary significantly (P>0.05). These results demonstrate that VIP binding sites are only displayed by stimulated macrophages, suggesting that VIP binding sites could be considered to be a pre-activation marker in macrophages and could be used to recognize inflammatory or stimulated macrophages.

Animals↗

PubMed1996

Functional and molecular characterization of VIP receptors and signal transduction in human and rodent immune systems.

In the last few decades, as a result of the interaction between different areas of research, the new interdisciplinary and exciting field of neuroimmunology has emerged. In this context, it has been demonstrated that small peptides may function in a communication network that links nervous, endocrine, and immune systems. Thus, each peptide may function as a neurotransmitter, peptide hormone, or cytokine, depending on its site of release and the target cell with which it interacts. Among these peptides, vasoactive intestinal peptide (VIP) has been shown to play a very important role in the regulation of immune function. The first stage in the action of VIP with immunocompetent cells is the binding to specific plasma membrane receptors and the generation of an intracellular signal. In this review, we focus and present data about the signal transduction pathway of VIP in both human and rodent immunocompetent cells.

Animals↗

PubMed1996

Melatonin reduces nitric oxide synthase activity in rat hypothalamus.

In this report, rat hypothalamic nitric oxide synthase (NOS) activity is shown to be partially inhibited by physiological concentrations of the pineal hormone melatonin. In vitro studies demonstrate that 1 nM melatonin, which approximates the physiological concentration of the hormone at night, significantly inhibited NOS activity. In vivo studies show that administering melatonin or collecting the hypothalamus from animals at night, when endogenous melatonin levels are elevated, results in a significant decrease of NOS activity. Results also show that calmodulin may be involved in this process since its presence in the incubation medium prevents the inhibitory effect of melatonin on NOS activity.

Animals↗

PubMed1995

Specific binding of 2-[125I]iodomelatonin by rat splenocytes: characterization and its role on regulation of cyclic AMP production.

In the present paper we show that pineal hormone melatonin interacts with rat splenocytes through high-affinity binding sites. Binding of 2-[125I]iodomelatonin ([125I]MEL) by splenocytes fulfills all criteria for binding to a receptor site. Binding exhibited properties such as dependence on time and temperature as well as reversibility, saturability, high affinity, specificity, and increased under constant light exposure. Results suggest binding to a single class of binding sites without cooperative interactions. The dissociation constant (Kd) for the single site was 0.34 nM with a binding capacity of 2.25 fmol/10(7) cells. These data are in close agreement with data obtained from kinetic studies, in which the kinetically derived value of the dissociation constant was 0.20 nM. The affinity of these binding sites suggests that they may recognize the physiological concentrations of melatonin in serum. Moreover, pharmacological doses of melatonin also inhibited cyclic AMP production stimulated by forskolin, a potent activator of adenylate cyclase system. The demonstration of [125I]MEL binding sites in the spleen, in addition to those described in blood mononuclear cells and thymus, provides evidence to support a direct mechanism of action of melatonin on immune system.

Animals↗

PubMed1995

Homologous regulation of vasoactive intestinal peptide (VIP) receptors on rat peritoneal macrophages.

In the present study, we examined the effect of pretreatment with VIP and various peptides structurally related to VIP such us PHI, helodermin, and secretin on VIP receptor number and affinity, as well as VIP-stimulated cyclic AMP production in rat peritoneal macrophages. Short-term (5-30 min) exposures of rat peritoneal macrophages to 0.1 microM VIP induced a rapid reduction in specific binding. Pretreatment for 15 and 30 min caused 26% (SEM = 6) and 48% (SEM = 4) reduction in specific binding, respectively. The maximal effect was observed at 120 min, causing a decrease of 67% (SEM = 6) in specific binding. Pretreatment with 0.1 microM VIP for 15, 30, and 120 min caused 23% (SEM = 9), 52% (SEM = 4), and 76% (SEM = 4) reduction in cyclic AMP production, respectively. Only VIP concentrations at the nanomolar level and higher were shown to be effective. The potency of VIP and related peptides to desensitize was similar to their potency to occupy receptors and to activate cyclic AMP production. The internalization of radioiodinated VIP was also studied. It was shown that receptor-bound ligand is internalized during the downregulation process. However, the diminution in VIP binding to macrophages was not completely explained by internalization.

Animals↗

PubMed1995

VIP receptor-effector system in rat harderian gland and its coupling to activation of type II thyroxine 5'-deiodinase.

Vasoactive intestinal peptide (VIP) receptors were investigated in rat Harderian gland membranes using [125I]VIP as ligand. The receptor binding was rapid, reversible, saturable, specific, and dependent on time, temperature, and membrane concentration. At 30 degrees C, the stoichiometric data suggested the presence of two classes of VIP receptors with Kd values of 0.36 +/- 0.06 and 65.37 +/- 8.08 nM and binding capacities of 323 +/- 54 and 39,537 +/- 3100 fmol VIP/mg protein, respectively. The interaction showed a high degree of specificity, as suggested by competitive displacement experiments with several peptides structurally or not structurally related to VIP. The binding of [125I]VIP to membranes was sensitive to guanine nucleotides in a dose-dependent manner. The molecular characterization of VIP receptors was realized by chemical cross-linking; sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the solubilized membrane proteins revealed the presence of two specific [125I]VIP-protein complexes of M(r) 57 and 35 kDa as estimated in denaturing conditions. VIP stimulated adenylate cyclase activity in rat Harderian gland membranes in a dose-dependent manner. Finally, VIP stimulated in vivo the type II thyroxine 5'-deiodinase activity. These results demonstrate the presence of specific and functional VIP receptors in Harderian gland and suggest a role for VIP in the physiology of this gland.

Adenylyl Cyclases↗

PubMed1995

Immunomodulatory role of melatonin: specific binding sites in human and rodent lymphoid cells.

This paper reviews the evidence that supports the hypothesis of the existence of specific binding sites for melatonin on immune cells. These binding sites have been described in human blood lymphocytes and granulocytes, and thymus, spleen, and bursa of Fabricius from different rodents and birds. The dissociation constant values of these binding sites are in the 0.1-1 nM range, suggesting that melatonin may play a physiological role in lymphocyte regulation. Moreover, melatonin binding sites appear to be modulated by guanine nucleotides. Therefore, in addition to other mechanisms described for the regulation of immune function by melatonin, a direct mechanism of regulation can be involved via binding of melatonin by immunocompetent cells.

Animals↗

PubMed1994

Peptide T from human immunodeficiency virus does not interact with VIP receptor-effector system in immunocompetent cells of rat and mouse.

Human immunodeficiency virus (HIV) infection is initiated by attachment of the virus to specific target cells. An octapeptide sequence contained within the envelope of HIV, peptide T, mediates the viral binding. Since there is an appreciable structural similarity between peptide T and an eight amino acid sequence of VIP, it is interesting to investigate the interaction of peptide T with the VIP receptor-effector system of immunocompetent cells from both rat and mouse. In this paper, we show the lack of interaction between peptide T and VIP receptor-effector system in peripheral blood lymphocytes, spleen lymphocytes and macrophages of rat and in macrophages of mouse. These results do not support the hypothesis that HIV through peptide T may interact with the VIP receptor-effector system present in immunocompetent cells.

Animals↗

PubMed1994

Physiological concentrations of melatonin inhibit nitric oxide synthase in rat cerebellum.

In the present paper we show the inhibitory effect of melatonin on rat cerebellar nitric oxide synthase (NOS) activity. NO production was monitored by the stoichiometric conversion of L-arginine to L-citrulline. The inhibitory effect of melatonin was dose-dependent, with an IC50 value of about 0.1 mM. However, a significant inhibition of enzyme activity (> 22%) was observed at 1 nM melatonin which is in the range of the physiological serum concentration of the hormone at night. The inhibitory effect of melatonin was observed exclusively in the presence of Ca++. Results suggest a new and important role of the pineal hormone melatonin on central nervous system processes, i.e., by modulating NO production.

Amino Acid Oxidoreductases↗

PubMedDate not supplied

Nuclear receptors are involved in the enhanced IL-6 production by melatonin in U937 cells.

This report shows that melatonin enhances IL-6 production by U937 cells via a nuclear receptor-mediated mechanism. Resting U937 cells only express membrane (mt1) melatonin receptors. In these cells, melatonin did not modify basal production of IL-6 or when activated by PMA plus lipopolysaccharide, a treatment that downregulates the expression of mt1 receptor. However, in U937 cells activated with IFN-gamma, which induces the expression of the ROR alpha 1 and ROR alpha 2 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 but not membrane receptors is sufficient for melatonin to activate cytokine production in human lymphocytic and monocytic cell lines.

Down-Regulation↗