Elaboración de videos didácticos como alternativa a los ensayos in vivo en las prácticas de farmacología / Didactic video as an alternative to in vivo assays in practical lessons of pharmacology

La Farmacología es una ciencia eminentemente práctica, en la que tiene gran relevancia la investigación "in vivo" con animales de experimentación. Los conocimientos impartidos en las clases teóricas, seminarios y tutorías, se completan con la enseñanza en las sesiones prácticas. Sin embargo, algunos aspectos importantes de esta enseñanza práctica, que incluyen la realización de ensayos “in vivo”, resulta problemático impartirlos correctamente con la normativa actual sobre la utilización de animales de laboratorio. Por ello, nos planteamos la realización de unos vídeos demostrativos de las técnicas experimentales utilizadas en algunas de las sesiones prácticas de las asignaturasde Farmacologia I y II del Grado en Farmacia. En cada sesión el profesor realiza una breve introducción del modelo experimental, indicando los objetivos que se plantea el investigador asi como las posibilidades de dicha técnica. A continuación en los vídeos, los estudiantes ven el desarrollo completo del experimento, los materiales necesarios y las condiciones experimentales adecuadas para su realización, asi como los diferentes parámetros y variables que se pueden medir. Al finalizar la proyección del vídeo se plantean dos tipos de tareas a los estudiantes:- diseño de un protocolo de evaluación de un fármaco con la metodología descrita- análisis, presentación y discusión de resultados, tras proporcionarles ejemplos de datos obtenidos en el ensayo.El procedimiento seguido para la elaboración de los vídeos es: 1) Diseño del ensayo de laboratorio. 2) Preparación del material necesario y las condiciones para una correcta grabación. 3) Grabación. Montaje de las imágenes (incluye fotografías, esquemas) y del sonido. 4) Edición del material filmado (una versión para Video- DVD y otra para incluirla en el Servidor Multimedia de la plataforma de la Universitat de València)(AU)

Pharmacology is a primarily practical science, in which “in vivo” research using experimental animal models plays a relevant role. The topics covered in the theoretical classes, seminars and tutorials are complemented with learning in practical sessions. However, certain important aspects of the practical learning, which include performing “in vivo” assays, represents a challenge given the actual regulations regarding laboratory animal utilization. Therefore, we proposed to produce didactic videos for various practical sessions. The teacher gives a brief introduction of the selected experimental animal model, indicating the intended objectives to be achieved. The students can see in the video the complete experiment progression, necessary materials and the proper experimental conditions toperform the assay, as well as the different parameters and variables to be measured.- a protocol design to evaluate a drug with the described methodology- analysis, result presentation and discussion of given example data obtained with the assay.The procedure used for the video elaboration was: 1) Design of the laboratory assay. 2) Preparation of the necessary materials and conditions for a correct recording. 3) Recording. Image (including photographs, schemes, figures) and sound download. 4) Editing of the filmed material (one version for Video- DVD and another one to be included in the virtual platform Multimedia Server of the University of Valencia)(AU)

Adenosine A2A receptors in diffuse dermal fibrosis: pathogenic role in human dermal fibroblasts and in a murine model of scleroderma.

OBJECTIVE: Adenosine regulates inflammation and tissue repair, and adenosine A2A receptors promote wound healing by stimulating collagen matrix production. We therefore examined whether adenosine A2A receptors contribute to the pathogenesis of dermal fibrosis. METHODS: Collagen production by primary human dermal fibroblasts was analyzed by real-time polymerase chain reaction, 14C-proline incorporation, and Sircol assay. Intracellular signaling for dermal collagen production was investigated using inhibitors of MEK-1 and by demonstration of ERK phosphorylation. In vivo effects were studied in a bleomycin-induced dermal fibrosis model using adenosine A2A receptor-deficient wild-type littermate mice, C57BL/6 mice, and mice treated with adenosine A2A receptor antagonist. Morphometric features and levels of hydroxyproline were determined as measures of dermal fibrosis. RESULTS: Adenosine A2A receptor occupancy promoted collagen production by primary human dermal fibroblasts, which was blocked by adenosine A2A, but not A1 or A2B, receptor antagonism. Adenosine A2A receptor ligation stimulated ERK phosphorylation, and A2A receptor-mediated collagen production by dermal fibroblasts was blocked by MEK-1 inhibitors. Adenosine A2A receptor-deficient and A2A receptor antagonist-treated mice were protected from developing bleomycin-induced dermal fibrosis. CONCLUSION: These results demonstrate that adenosine A2A receptors play an active role in the pathogenesis of dermal fibrosis and suggest a novel therapeutic target in the treatment and prevention of dermal fibrosis in diseases such as scleroderma.

Immune complexes and IFN-gamma decrease cholesterol 27-hydroxylase in human arterial endothelium and macrophages.

The enzyme cholesterol 27-hydroxylase, expressed by arterial endothelium and monocytes/macrophages, is one of the first lines of defense against the development of atherosclerosis. By catalyzing the hydroxylation of cholesterol to 27-hydroxycholesterol, which is more soluble in aqueous medium, the enzyme promotes the removal of cholesterol from the arterial wall. Prior studies have suggested that immune reactants play a role in the pathogenesis of atherosclerosis; we report here that immune reactants, IFN-gamma and immune complexes bound to C1q, but not interleukin-1 and tumor necrosis factor, diminish the expression of cholesterol 27-hydroxylase in human aortic endothelial cells, peripheral blood mononuclear cells, monocyte-derived macrophages, and the human monocytoid cell line THP-1. In addition, our studies demonstrate that immune complexes down-regulate cholesterol 27-hydroxylase only after complement fixation via interaction with the 126-kD C1qRp protein on endothelial cells and THP-1 cells. These results are consistent with the prior demonstration that IFN-gamma contributes to the pathogenesis of atherosclerosis and suggest a role for C1q receptors in the atherogenic process. Moreover, these observations suggest that one mechanism by which immune reactants contribute to the development of atherosclerosis is by down-regulating the expression of the enzymes required to maintain cholesterol homeostasis in the arterial wall.

Inflammatory cytokines regulate function and expression of adenosine A(2A) receptors in human monocytic THP-1 cells.

Adenosine, acting at its receptors, particularly A(2A) receptors, is a potent endogenous anti-inflammatory agent that modulates the functions and differentiation of inflammatory and immune cells. Because the inflammatory milieu abounds in proinflammatory cytokines, we investigated the effects of Th1-inflammatory cytokines on function and expression of adenosine A(2A) receptors in the human monocytic cell line THP-1. We found that, consistent with previous reports, adenosine and 2-[p-(2-carnonylethyl)phenylethylamino]-5'-N-ethylcarboxamidoadenosine (CGS-21680), a selective A(2A) receptor agonist, suppress IL-12 production but increase IL-10 production in LPS-activated THP-1 cells. These effects were blocked by the A(2A) receptor antagonist 4-(2-[7-amino-2-(2-furyl)[1,2,4-triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM-241385). More importantly, the suppressive effect of adenosine and CGS-21680 on IL-12 production was significantly enhanced in cells pretreated with either IL-1 (10 U/ml) or TNF-alpha (100 U/ml) but markedly attenuated in cells pretreated with IFN-gamma (100 U/ml). Similarly, IL-1 and TNF-alpha treatment potentiated the stimulatory effect of adenosine and CGS-21680 on IL-10 production, whereas IFN-gamma treatment almost completely abolished this effect. CGS-21680 stimulated an increase in intracellular cAMP in a time- and dose-dependent manner in IL-1- and TNF-alpha-treated cells but not in control or IFN-gamma-treated cells. Both IL-1 and TNF-alpha increased A(2A) receptor mRNA and protein. In parallel with its effect on A(2A) receptor function, IFN-gamma down-regulated A(2A) receptor message and protein. Because adenosine mediates many of the antiinflammatory effects of drugs such as methotrexate, these observations suggest that local changes in the cytokine milieu may influence the therapeutic response to those drugs by altering the expression and function of adenosine receptors on inflammatory cells.

Reversal of the antiinflammatory effects of methotrexate by the nonselective adenosine receptor antagonists theophylline and caffeine: evidence that the antiinflammatory effects of methotrexate are mediated via multiple adenosine receptors in rat adjuvant arthritis.

OBJECTIVE: Weekly low-dose methotrexate (MTX) remains the mainstay of second-line therapy for rheumatoid arthritis (RA). We have previously reported that adenosine, acting at specific receptors on inflammatory cells, mediates the antiinflammatory effects of MTX in both in vitro and in vivo models of acute inflammation, but the mechanism by which MTX suppresses the chronic inflammation of arthritis remains controversial. The present study was undertaken to further investigate the means by which adenosine mediates the antiinflammatory effects of MTX. METHODS: The effects of 2 nonselective adenosine receptor antagonists, theophylline and caffeine, were examined, using the rat adjuvant arthritis model of RA. These agents were given alone and in conjunction with MTX, and arthritis severity was assessed clinically, radiologically, and histologically. Since rodent adenosine A3 receptors are not blocked by theophylline, selective A1, A2A, and A2B receptor antagonists were tested as well. RESULTS: Control animals developed severe arthritis, which was markedly attenuated by weekly treatment with MTX (0.75 mg/kg/week). Neither theophylline alone nor caffeine alone (each at 10 mg/kg/day) significantly affected the severity of the arthritis, but both agents markedly reversed the effect of MTX as measured by a severity index, hindpaw swelling, and hindpaw ankylosis. Radiographic and histologic analyses confirmed these observations. Neither A1, A2A, nor A2B receptor antagonists affected the capacity of MTX to ameliorate inflammation in adjuvant arthritis. CONCLUSION: These results provide strong evidence that adenosine mediates the antiinflammatory effects of MTX in this model of RA. Moreover, the findings suggest that abstinence from caffeine, a ubiquitous food additive and medication, may enhance the therapeutic effects of MTX in RA.

Sites of action for future therapy: an adenosine-dependent mechanism by which aspirin retains its antiinflammatory activity in cyclooxygenase-2 and NFkappaB knockout mice.

The antiinflammatory action of aspirin is generally attributed to inhibition of cyclooxygenases 1 and 2, but additional mechanisms are at work. These include inhibition of NFkappaB translocation to the nucleus and the capacity of aspirin to promote accumulation of adenosine, a potent antiinflammatory autocoid. We tested these hypotheses in the murine air pouch model of acute inflammation in wild type mice and in cyclooxygenase 2 or NFkappaB knockouts. The antiinflammatory effects of aspirin, sodium salicylate and indomethacin did not correlate with inhibition of cyclooxygenase in either group. Indeed, aspirin retained its antiinflammatory properties even in COX-2 knockouts. Similarly, aspirin was no less antiinflammatory in mice rendered deficient for NFkappaB (p105) than in wild type controls. In contrast, dexamethasone lost its antiinflammatory capacity in NFkappaB knockouts. Aspirin and sodium salicylate dramatically increased concentrations of adenosine in exudates, a property shared with methotrexate and sulfasalazine. Removal of adenosine by adenosine deaminase or specific antagonism of adenosine at A(2)receptors completely reversed the antiinflammatory effects of aspirin and sodium salicylate, but not those of dexamethasone. This adenosine-dependent, antiinflammatory effect of aspirin points to another target of drug development.

Salicylates and sulfasalazine, but not glucocorticoids, inhibit leukocyte accumulation by an adenosine-dependent mechanism that is independent of inhibition of prostaglandin synthesis and p105 of NFkappaB.

The antiinflammatory action of aspirin generally has been attributed to direct inhibition of cyclooxygenases (COX-1 and COX-2), but additional mechanisms are likely at work. These include aspirin's inhibition of NFkappaB translocation to the nucleus as well as the capacity of salicylates to uncouple oxidative phosphorylation (i.e., deplete ATP). At clinically relevant doses, salicylates cause cells to release micromolar concentrations of adenosine, which serves as an endogenous ligand for at least four different types of well-characterized receptors. Previously, we have shown that adenosine mediates the antiinflammatory effects of other potent and widely used antiinflammatory agents, methotrexate and sulfasalazine, both in vitro and in vivo. To determine in vivo whether clinically relevant levels of salicylate act via adenosine, via NFkappaB, or via the "inflammatory" cyclooxygenase COX-2, we studied acute inflammation in the generic murine air-pouch model by using wild-type mice and mice rendered deficient in either COX-2 or p105, the precursor of p50, one of the components of the multimeric transcription factor NFkappaB. Here, we show that the antiinflammatory effects of aspirin and sodium salicylate, but not glucocorticoids, are largely mediated by the antiinflammatory autacoid adenosine independently of inhibition of prostaglandin synthesis by COX-1 or COX-2 or of the presence of p105. Indeed, both inflammation and the antiinflammatory effects of aspirin and sodium salicylate were independent of the levels of prostaglandins at the inflammatory site. These experiments also provide in vivo confirmation that the antiinflammatory effects of glucocorticoids depend, in part, on the p105 component of NFkappaB.

Methotrexate and sulfasalazine promote adenosine release by a mechanism that requires ecto-5'-nucleotidase-mediated conversion of adenine nucleotides.

We and others have shown that an increased extracellular concentration of adenosine mediates the antiinflammatory effects of methotrexate and sulfasalazine both in vitro and in vivo, but the mechanism by which these drugs increase extracellular adenosine remains unclear. The results of the experiments reported here provide three distinct lines of evidence that adenosine results from the ecto-5'-nucleotidase- mediated conversion of adenine nucleotides to adenosine. First, pretreatment of a human microvascular endothelial cell line (HMEC-1) with methotrexate increases extracellular adenosine after exposure of the pretreated cells to activated neutrophils; the ecto-5'-nucleotidase inhibitor alpha, beta-methylene adenosine-5'-diphosphate (APCP) abrogates completely the increase in extracellular adenosine. Second, there is no methotrexate-mediated increase in extracellular adenosine concentration in the supernate of cells deficient in ecto-5'-nucleotidase, but there is a marked increase in extracellular adenosine concentration in the supernates of these cells after transfection and surface expression of the enzyme. Finally, as we have shown previously, adenosine mediates the antiinflammatory effects of methotrexate and sulfasalazine in the murine air pouch model of inflammation, and injection of APCP, the ecto-5'-nucleotidase inhibitor, abrogates completely the increase in adenosine and the decrement in inflammation in this in vivo model. These results not only show that ecto-5'-nucleotidase activity is a critical mediator of methotrexate- and sulfasalazine-induced antiinflammatory activity in vitro and in vivo but also indicate that adenine nucleotides, released from cells, are the source of extracellular adenosine.

Wound healing is accelerated by agonists of adenosine A2 (G alpha s-linked) receptors.

The complete healing of wounds is the final step in a highly regulated response to injury. Although many of the molecular mediators and cellular events of healing are known, their manipulation for the enhancement and acceleration of wound closure has not proven practical as yet. We and others have established that adenosine is a potent regulator of the inflammatory response, which is a component of wound healing. We now report that ligation of the G alpha s-linked adenosine receptors on the cells of an artificial wound dramatically alters the kinetics of wound closure. Excisional wound closure in normal, healthy mice was significantly accelerated by topical application of the specific A2A receptor agonist CGS-21680 (50% closure by day 2 in A2 receptor antagonists. In rats rendered diabetic (streptozotocin-induced diabetes mellitus) wound healing was impaired as compared to nondiabetic rats; CGS-21680 significantly increased the rate of wound healing in both nondiabetic and diabetic rats. Indeed, the rate of wound healing in the CGS-21680-treated diabetic rats was greater than or equal to that observed in untreated normal rats. These results appear to constitute the first evidence that a small molecule, such as an adenosine receptor agonist, accelerates wound healing in both normal animals and in animals with impaired wound healing.

Adenosine A2 receptor occupancy regulates stimulated neutrophil function via activation of a serine/threonine protein phosphatase.

Adenosine modulates generation of superoxide anion by neutrophils via occupancy of specific adenosine A2A receptors. However, the intracellular signal transduction pathways by which occupancy of neutrophil adenosine A2A receptors inhibits superoxide anion generation (O2.-) are not well understood. We, therefore, tested the hypothesis that signaling at polymorphonuclear leukocyte (PMN) adenosine receptors proceeds via activation of a serine/threonine protein phosphatase (pp). Both the specific pp1 inhibitor calyculin A (10 nM) and the pp2A inhibitor okadaic acid (10 microM) enhanced O2.- generation (185 +/- 24 and 189 +/- 35% of control, respectively, p < 0.0001 for both, n = 8), as reported previously. Calyculin A, but not okadaic acid, completely reversed inhibition of stimulated O2.- generation by the adenosine A2 receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA; IC50 = 30 nM; p < 0.0001, analysis of variance). Calyculin A also reversed the adenosine receptor-mediated desensitization of bound chemoattractant receptors in neutrophils. Treatment of PMNs with NECA increased the pp1 activity of crude membrane preparations in a time- and dose-dependent fashion (EC50 = 40 nM; p < 0.001, analysis of variance, n = 5). NECA inhibited cytosolic protein phosphatase activity by 78 +/- 12% (p < 0.003, n = 6) but did not shift pp1 catalytic subunit from cytosol to plasma membrane. Similar changes were observed in neutrophil cytoplasts depleted of organelles and nucleus. Moreover, the selective protein kinase A inhibitor KT5720 (10 microM) reversed the capacity of dibutyryl cAMP but not NECA to increase pp1 activity (p < 0.01, n = 5) in keeping with its effects on O2.- generation. Western blot analysis of PMN subcellular fractions demonstrated the presence of pp1alpha and pp1gamma1 but not pp1gamma2 isotypes in both cytosol and plasma membrane but not in azurophil or specific granules. We conclude from these studies that signal transduction by adenosine in PMN proceeds via a novel pathway: cAMP-independent activation of a serine/threonine protein phosphatase in the plasma membrane.

The anti-inflammatory mechanism of sulfasalazine is related to adenosine release at inflamed sites.

The anti-inflammatory mechanism of sulfasalazine is not well understood. It has recently been shown that sulfasalazine inhibits 5-aminoimidazole-4-carboxamidoribonucleotide (AICAR) transformylase, an enzyme involved in de novo purine biosynthesis. We recently demonstrated that methotrexate promotes intracellular AICAR accumulation, thereby increasing adenosine release and diminishing inflammation, so we tested the hypothesis that sulfasalazine similarly promotes intracellular AICAR accumulation. We studied adenosine release and the state of inflammation in in vitro and in vivo models of the inflammatory process. The adhesion of stimulated neutrophils (FMLP) to endothelial cells preincubated with sulfasalazine was inhibited in a dose-dependent manner. Elimination of extracellular adenosine by addition of adenosine deaminase or inhibition of adenosine by the adenosine A2 receptor antagonist 3,7-dimethyl-1-propargylxanthine (DMPX) completely reversed the anti-inflammatory effect of sulfasalazine (at concentrations <1 microM in this in vitro model. To determine whether this phenomenon was relevant to inhibition of inflammation in vivo, we studied the effect of sulfasalazine (100 mg/kg/day by gastric gavage for 3 days) on leukocyte accumulation in the murine air pouch model of inflammation. Treatment with sulfasalazine markedly decreased the number of leukocytes that accumulated in the inflamed (carrageenan, 2 mg/ml) air pouch. Injection of either adenosine deaminase or DMPX, but not the A1 receptor antagonist 8-cyclopentyl-dipropylxanthine, significantly reversed the anti-inflammatory effects of sulfasalazine treatment. Sulfasalazine increased the exudate adenosine concentration from 127 +/- 64 nM to 869 +/- 47 nM. Moreover, sulfasalazine treatment promoted a marked increase in splenocyte AICAR concentration from 35 +/- 6 to 96 +/- 3 pmols/10(6) splenocytes, which is consistent with the in vitro observation that sulfasalazine inhibits AICAR transformylase. These results indicate that sulfasalazine, like methotrexate, enhances adenosine release at an inflamed site and that adenosine diminishes inflammation via occupancy of A2 receptors on inflammatory cells. Our studies provide evidence that sulfasalazine and methotrexate may be described as a newly recognized family of anti-inflammatory agents that share the property of using adenosine as an antagonist of inflammation.

Antioxidant profile of mono- and dihydroxylated flavone derivatives in free radical generating systems.

A number of free radical generating systems were used to investigate the antioxidant properties and structure-activity relationships of a series of monohydroxylated and dihydroxylated flavones. Ortho-dihydroxylated flavones showed the highest inhibitory activity on enzymic and non-enzymic microsomal lipid peroxidation as well as on peroxyl radical scavenging. Most flavones were weak scavengers of hydroxyl radical, while ortho-dihydroxylated flavones interacted with superoxide anion generated by an enzymic system or by human neutrophils. This series of compounds did not exert cytotoxic effects on these cells. Scavenging of superoxide and peroxyl radicals may determine the antioxidant properties of these active flavones.