Caracterización farmacológica del efecto vasodilatador de extractos de bayas de calafate y de sus tres principales antocianinas glicosiladas

Calafate (Berberis microphylla) es una planta nativa de la Patagonia de Chile y Argentina, sus bayas son de color púrpura oscuro y posee la mayor capacidad antioxidante entre frutas comerciales y endémicas de Chile. Las bayas tienen alto contenido de compuestos fenólicos que incluyen varias antocianinas, flavonoles y ácidos fenólicos. Dado que la etnomedicina une las bayas chilenas con un efecto clínico benéfico, propusimos que los extractos de bayas de calafate tienen propiedades vasodilatadoras relacionadas a la variada naturaleza de sus compuestos. Con este propósito, extractos hidroalcohólicos de calafate (0.1-300 μg/mL) y 3 principales antocianinas glicosiladas (1nM-10 μM), fueron evaluadas en la red vascular mesentérica de rata previa contracción con noradrenalina, se determinó la dependencia del endotelio y producción de óxido nítrico. Extractos de calafate inducen una respuesta dilatadora concentración-dependiente (EC50 3.7 μg/mL); la respuesta máxima de vasodilatación (75-80%) se alcanzó con 10-30 μg/mL. La vasodilatación fue significativamente reducida por la remoción del endotelio (saponina 0.1%) (70 a 30%) e inhibición de eNOS con 150 μM de L-NNA (70 a 10%). El perfil químico del extracto (HPLC-ESI-MS/MS) identificó delfinidina (D3G), petunidina y malvidina como 3-glucósidos, las principales antocianinas del extracto. Las 3 antocianinas provocaron una relajación vascular endotelio-dependiente con diferente potencia y similar eficacia (40-50%). La principal antocianina D3G es más potente pero menos efectiva que los extractos de calafate, lo cual explica parcialmente que otros compuestos no antocianos del extracto enmascaran la respuesta inducida por D3G pero incrementan la eficacia del extractoFil: Calfío, C.. Universidad de Santiago de Chile.Fil: Huidobro-Toro, JP. Universidad de Santiago de Chile

Nanomolar clodronate induces adenosine accumulation in the perfused rat mesenteric bed and mesentery-derived endothelial cells

The vesicular nucleotide transporter (VNUT) is critical for sympathetic co-transmission and purinergic transmission maintenance. To examine this proposal, we assessed whether the bisphosphonate clodronate, claimed as a potent in vitro VNUT blocker, modified spontaneous and/or the electrically evoked overflow of ATP/metabolites and NA from mesentery sympathetic perivascular nerve terminals. Additionally, in primary endothelial cell cultures derived from this tissue, we also evaluated whether clodronate interfered with ATP/metabolite cell outflow and metabolism of N6-etheno adenosine 5′-triphosphate (eATP), N6-etheno adenosine (eADO), and adenosine deaminase enzyme activity. Rat mesenteries were perfused in the absence or presence of .01–1,000 nM clodronate, 1–1,000 nM Evans blue (EB), and 1–10 µM DIDS; tissue perfusates were collected to determine ATP/metabolites and NA before, during, and after perivascular electrical nerve terminal depolarization. An amount of 1–1,000 nM clodronate did not modify the time course of ATP or NA overflow elicited by nerve terminal depolarization, and only 10 nM clodronate significantly augmented perfusate adenosine. Electrical nerve terminal stimulation increased tissue perfusion pressure that was significantly reduced only by 10 nM clodronate [90.0 ± 18.6 (n = 8) to 35.0 ± 10.4 (n = 7), p = .0277]. As controls, EB, DIDS, or reserpine treatment reduced the overflow of ATP/metabolites and NA in a concentration-dependent manner elicited by nerve terminal depolarization. Moreover, mechanical stimulation of primary endothelial cell cultures from the rat mesentery added with 10 or 100 nM clodronate increased adenosine in the cell media. eATP was metabolized by endothelial cells to the same extent with and without 1–1,000 nM clodronate, suggesting the bisphosphonate did not interfere with nucleotide ectoenzyme metabolism. In contrast, extracellular eADO remained intact, indicating that this nucleoside is neither metabolized nor transported intracellularly. Furthermore, only 10 nM clodronate inhibited (15.5%) adenosine metabolism to inosine in endothelial cells as well as in a commercial crude adenosine deaminase enzyme preparation (12.7%), and both effects proved the significance (p < .05). Altogether, present data allow inferring that clodronate inhibits adenosine deaminase activity in isolated endothelial cells as in a crude extract preparation, a finding that may account for adenosine accumulation following clodronate mesentery perfusion

The Cellular Prion Protein Prevents Copper-Induced Inhibition of P2X4 Receptors

Although the physiological function of the cellular prion protein (PrPC) remains unknown, several evidences support the notion of its role in copper homeostasis. PrPC binds Cu2+ through a domain composed by four to five repeats of eight amino acids. Previously, we have shown that the perfusion of this domain prevents and reverses the inhibition by Cu2+ of the adenosine triphosphate (ATP)-evoked currents in the P2X4 receptor subtype, highlighting a modulatory role for PrPC in synaptic transmission through regulation of Cu2+ levels. Here, we study the effect of full-length PrPC in Cu2+ inhibition of P2X4 receptor when both are coexpressed. PrPC expression does not significantly change the ATP concentration-response curve in oocytes expressing P2X4 receptors. However, the presence of PrPC reduces the inhibition by Cu2+ of the ATP-elicited currents in these oocytes, confirming our previous observations with the Cu2+ binding domain. Thus, our observations suggest a role for PrPC in modulating synaptic activity through binding of extracellular Cu2+

Nitric oxide synthase-independent release of nitric oxide induced by KCl in the perfused mesenteric bed of the rat

The aim of the present study was to test whether the contractile responses elicited by KCl in the rat mesenteric bed are coupled to the release of nitric oxide (NO). Contractions induced by 70 mM KCl were coincident with the release of NO to the perfusate. The in vitro exposure to the nitric oxide synthase (NOS) inhibitor L-N(ω)-nitro-L-arginine methyl ester, L-NAME (1-100 μM) potentiated the vascular responses to 70 mM KCl and, unexpectedly, increased the KCl-stimulated release of NO. Moreover, even after the chronic treatment with L-NAME (70 mg/kg/day during 4 weeks), the KCl-induced release of NO was not reduced, whereas the potentiation of contractile responses was indeed achieved. The possibility that NOS had not been completely inhibited under our experimental conditions can be precluded because NOS activity was significantly inhibited after both L-NAME treatments. After the in vitro treatment with 1 to 100 μM L-NAME, the inhibition of NOS was concentration-dependent (from 50% to 90%). With regard to the basal release of NO, the inhibition caused by L-NAME was not concentration-dependent and reached a maximum of 40%, suggesting that basal NO outflow is only partially dependent on NOS activity. An eventual enhancement of NOS activity caused by KCl was disregarded because the activity of this enzyme measured in homogenates from mesenteric beds perfused with 70 mM KCl was significantly reduced. On the other hand, endothelium removal, employed as a negative control, almost abolished NOS activity, whereas the incubation with the Ca2+ ionophore A23187, employed as a positive control, induced an increase in NOS activity. It is concluded that in the mesenteric arterial bed of the rat, the contractile responses elicited by depolarization through KCl are coincident with a NOS-independent release of NO. This observation, which differs from the results obtained with noradrenaline, do not support the use of KCl as an alternative contractile agent whenever the participation of NO is under study. (C) 2000 Elsevier Science B.V.Fil: Mendizabal, Victoria Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Farmacológicas. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Investigaciones Farmacológicas; ArgentinaFil: Poblete, I.. Pontificia Universidad Católica de Chile; ChileFil: Lomniczi, A.. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Besuhli, Valeria. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Huidobro Toro, J. P.. Pontificia Universidad Católica de Chile; ChileFil: Adler, Edda. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Farmacológicas. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Investigaciones Farmacológicas; Argentin

Peptidergic receptors

The term neuropeptides refers to a relatively large number of biologically active molecules localized to discrete cell populations of nervous system, including autonomic ganglion neurons and their effector cells. Different combinations of transmitters and peptides are found in ganglionic cells of the autonomic system which comprise a functional “chemical code” for neurons subserving specific actions. Generally, peptidergic receptors are G-protein coupled receptors acting through multiple transduction pathways which reflect the pleiotropic actions of peptides. This section represents a current view of the most important actions of peptides and their receptors in the autonomic nervous system and in peripheral organs.L'articolo è disponibile sul sito dell'editore http://www.sciencedirect.com

Functional and Structural Analysis of the Internal Ribosome Entry Site Present in the mRNA of Natural Variants of the HIV-1

The 5′untranslated regions (UTR) of the full length mRNA of the HIV-1 proviral clones pNL4.3 and pLAI, harbor an internal ribosomal entry site (IRES). In this study we extend this finding by demonstrating that the mRNA 5′UTRs of natural variants of HIV-1 also exhibit IRES-activity. Cap-independent translational activity was demonstrated using bicistronic mRNAs in HeLa cells and in Xenopus laevis oocytes. The possibility that expression of the downstream cistron in these constructs was due to alternative splicing or to cryptic promoter activity was ruled out. The HIV-1 variants exhibited significant 5′UTR nucleotide diversity with respect to the control sequence recovered from pNL4.3. Interestingly, translational activity from the 5′UTR of some of the HIV-1 variants was enhanced relative to that observed for the 5′UTR of pNL4.3. In an attempt to explain these findings we probed the secondary structure of the variant HIV-1 5′UTRs using enzymatic and chemical approaches. Yet subsequent structural analyses did not reveal significant variations when compared to the pNL4.3-5′UTR. Thus, the increased IRES-activity observed for some of the HIV-1 variants cannot be ascribed to a specific structural modification. A model to explain these findings is proposed

Nucleotide P2Y1 receptor regulates EGF receptor mitogenic signaling and expression in epithelial cells

Epidermal growth factor receptor (EGFR) function is transregulated by a variety of stimuli, including agonists of certain G-protein-coupled receptors (GPCRs). One of the most ubiquitous GPCRs is the P2

Recurrent Cellulitis Associated with Long-Term Intrathecal Opioid Infusion Therapy: A Case Report and Review of the Literature

Lower-limb edema is recognized as an untoward side effect of intrathecal opioid therapy. Cellulitis, an acute, spreading pyogenic inflammation of the dermis and subcutaneous tissue, predisposed by persistent leg edema, can become problematic in patients on intraspinal opioid infusion therapy.To present a case of recurrent cellulitis in an elderly lady with persistent leg edema associated with intrathecal morphine/hydromorphone infusion therapy.Sixty-one-year-old woman with intractable chronic low back pain and bilateral leg pain treated with an intrathecal infusion of morphine up to 5 mg/day over 3 months with satisfactory pain control developed progressive lower extremity edema, complicated by recurrent cellulitis, requiring repeated hospitalization and intravenous antibiotic treatment. Switching to intrathecal hydromorphone helped minimally. Intrathecal baclofen and clonidine infusion resulted in complete resolution of leg edema and pain relief over the following 12 months.Intrathecal Baclofen and Clonidine may be used as alternatives to provide spinally mediated antinociception when intraspinal opioid fails due to pharmacological side effects such as persistent edema.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/79174/1/j.1526-4637.2010.00854.x.pd

Lipopolysaccharide Inhibits the Channel Activity of the P2X7 Receptor

The purinergic P2X7 receptor (P2X7R) plays an important role during the immune response, participating in several events such as cytokine release, apoptosis, and necrosis. The bacterial endotoxin lipopolysaccharide (LPS) is one of the strongest stimuli of the immune response, and it has been shown that P2X7R activation can modulate LPS-induced responses. Moreover, a C-terminal binding site for LPS has been proposed. In order to evaluate if LPS can directly modulate the activity of the P2X7R, we tested several signaling pathways associated with P2X7R activation in HEK293 cells that do not express the TLR-4 receptor. We found that LPS alone was unable to induce any P2X7R-related activity, suggesting that the P2X7R is not directly activated by the endotoxin. On the other hand, preapplication of LPS inhibited ATP-induced currents, intracellular calcium increase, and ethidium bromide uptake and had no effect on ERK activation in HEK293 cells. In splenocytes-derived T-regulatory cells, in which ATP-induced apoptosis is driven by the P2X7R, LPS inhibited ATP-induced apoptosis. Altogether, these results demonstrate that LPS modulates the activity of the P2X7R and suggest that this effect could be of physiological relevance