EXPERIMENTAL PHARMACOLOGY

Author: SEGURA AGULLO MIREIA.
Year: 2003.
University: POMPEU FABRA [www.upf.edu].
Place of defense: DEPARTAMENTO DE CIENCIAS EXPERIMENTALES Y DE LA SALUD.
Place of preparation: DEPARTAMENTO DE CIENCIAS EXPERIMENTALES Y DE LA SALUD.

Summary: The 3,4-metilenedioximetamfetamina (MDMA, Ecstasy) is a synthetic drug that is commonly consumed by young people in a recreational, this thesis has deepened in the metabolism of MDMA in humans. Specifically, the metabolic pathway that leads, through mostly CYP2D6, the metabolite catechol 3,4-dihidroximetamfetamina (HHMA). This thesis has also been targeted to study the contribution of CYP2D6 in cleansing overall MDMA. It has conducted a clinical trial in healthy volunteers which has been administered three doses of paroxetine, an antidepressant drug that is both substrate and inhibitor of CYP2D6, before the administration of the MDGs, in order to inhibit the activity this enzyme. To assess this inhibition and related drug interaction between MDMA and paroxetine have been developed and validated analytical techniques for the determination of the different compounds in biological fluids at the same time that has been chemically synthesized paroxetine, their metabolites and HHMA. The analysis of plasma samples and originates has shown that the metabolic HHMA is relevant from a quantitative metabolic cleansing of the MDAM resulting in plasma concentrations (Cmax 154ug / L) and urinary tract (18% of dose) quantitatively similar to MDMA itself. The assessment of the pharmacokinetic parameters and urinary recovery of the MDAM and its main metabólitos indicate that the MDAM affected their metabolic cleansing when administered in conjunction with the proxetina. The inhibition of oxidative metabolism in vivo of a MDMA translates into an increase in the plasma concentrations of MDAM of 30% that would be the contribution of CYP2D6 in vivo in O-demetilenación of MDMA. Thus, other isoenzymes could contribute significantly to the overall metabolic cleansing MDMA.

Author: LAFUENTE LOPEZ NURIA.
Year: 2004.
University: AUTÓNOMA DE MADRID [www.uam.es].
Place of defense: FACULTAD DE MEDICINA.
Place of preparation: FACULTAD DE MEDICINA.

Summary: The vascular complications involving the leading cause of morbidity and mortality associated with long-term diabetes mellitus. In recent years, the arterial diabetes has been associated with a chronic inflammatory process of the vascular wall, associated with increased circulating levels of inflammatory cytokines and other markers of inflammation. The inducible nitric oxide synthase (iNOS) is one of the most important enzymes proinflammatory the vascular wall, as it once induced by several factors, generates nitric oxide (NO) in large quantities and so poorly regulated. In addition to his involvement in other inflammatory statements of the vascular wall as atherosclerosis, some studies have described an increase in iNOS activity in the cardiovascular system in relacíón with diabetes mellitus. Moreover, it is now fully accepted that hyperglycemia is at the base of cellular processes, including inflammation, leading to the development and progression of vascular damage associated with diabetes. In this paper, it wanted to analyze the role of the cytokine interleuquina-1beta (IL-1beta) and glucose, either alone or in combination, on the expression and activity of the enzyme iNOS, using cultured human aorta smooth muscle. First, we explored the effect of IL-1 beta (0.1, 0.5, 1, 5 and 10 ng / ml) added to a culture medium containing 5.5 mM O-glucosa, with a increased levels of the enzyme from the concentration of 5 ng / ml. The maximum increase in the levels of iNOS observed with 10 ng / ml IL-1beta was accompanied by unRumento the liberation of nitrites used as markers for the synthesis of NO. In contrast to the effect of IL-1beta, O-glucosa to increasing concentrations (5,5,8,11 and 22 mM) was unable to índucir synthesis of iNOS in CMLAH.Sin embargo, cuando se íncubó concentration higher O-glucosa (22 mM) with IL-1 beta (10 ng / ml), there was a clear aumento-de synthesis of iNOS, in relation to the levels attained in presencía of IL-1 beta in conjunction with 5.5 mM O-glucosa, from 10 hours of incubation. This leverage effect on the levels of iNOS was directly proportional to the concentration of O-glucosa used, and was accompanied by a parallel increase in the production of nitrites. It ruled out a possible effect of hiperosmolaridad using L-glucosa, not reproduced the results obtained with O-glucosa. Furthermore, it was noted that this increase in protein levels  WE correlacionaba was an increase of messenger RNA for the enzyme. Moreover, it was observed that this effect enhancer of O-glucosa, was related to an increase in the activity of transcription factor-related phenomena inflammation NF-kappaB. Although it has been suggested that much of the effects of O-glucosa at high concentrations is mediated by the generation of stress oxidatívo, in the present work pharmacological interference with different antioxidants have failed to reverse the leverage effect of O-glucosa on the actions of IL-1 beta. By contrast, the effect potencíador of O-glucosa was inhibited in the presence of an inhibitor of the activity of iNOS (1400W), suggesting an involvement of itself nitrico oxide generated by the iNOS. Moreover, it was observed by quantifying waste nitrotirosina, that the combination of 22 mM glucose and IL-1beta increased generation of peroxynitrite in comparison with the IL-1beta together with 5.5 mM glucose. In conclusion, these results suggest that elevated levels of glucose power phenomena in the smooth muscle inflammation induced by cytokines and in particular, IL-1 beta. The possible mechanism involved represents an aum 8 nt of 1ee activity NF-kappaB, which could be mediated by an increase of NO and peroxynitrite.

Author: MATESANZ PARELLADA NURIA.
Year: 2004.
University: AUTÓNOMA DE MADRID [www.uam.es].
Place of defense: FACULTAD MEDICINA.
Place of preparation: FACULTAD DE MEDICINA.

Summary: Cardiovascular diseases pose one of the leading causes of morbidity and mortality worldwide. Thus, in situations such as hypertension, atherosclerosis, or arterial diabetic, among others, appear impairment of vascular function that is often accompanied by changes in the structure of the glass as the disease progresses. These structural changes, generally irreversible, known as vascular remodeling, it is associated with a worse prognosis of vascular disease and are primarily determined by changes in the rate of migration, growth and / or death of vascular smooth muscle. Oxidative stress plays an important role in vascular disease. Among the sources of oxidative stress vascular, is the enzyme xanthine oxidase, xanthine that transforms into uric acid anions through reactions that generate superoxide (02 .-). The XO seems participate in the endothelial dysfunction, but little is known about their possible involvement in vascular remodeling. Therefore, in this paper we wanted to analyze the possible role of XO on the growth of cultured smooth muscle cells from human aorta. First, we checked the capacity laXO (25-500 UU / ml) for generating O2 .- both in a sistema-acelular as in the presence of cells cultivated object of this study. It was also found, by far the levels of intracellular antioxidant glutathione, which in the concentrations used, XO had no toxic effects for this cell type. As for a possible influence of XO on the growth of CMLAH, the enzyme did not induce cell proliferation, but a significant increase in cell size accompanied by an increase in protein synthesis, indicating XO's ability to produce hypertrophy cell. This process resulted in the generation extracellular O2 .- since it was abolished in the presence of the enzyme impervious to the plasma membrane superoxide dismutase (SOD: 200 U / ml). To identify potential intracellular signaling pathways sensitive to oxidative stress involved in the hypertrophic effect, initially discussed the role of the transcription factor involved in cell growth AP-1. The XO (50 Â ¡. Â ¡U / ml) was able to increase levels cell c-Jun, one of the main components of AP-1, and the transcriptional activity of this factor. In addition, XO was able to activate different protein kinases activated by mitogenic (MAPK). The XO did not affect the kinases ERK1 / 2 yAkt / PKB, but stimulated the activity of JNK kinases associated with stress and p38. While JNK activation introduced a peak of 10 minutes, followed by a rapid return to baseline levels, stimulation of p38 occurred gradually, with a peak of activity in the 30 minutes which was maintained at 60 minutes. Both the activation of AP-1 and JNK and p38 was abolished in the presence of 800. Using 8P600125 (10 UM) AND 8B203580 (5 UM), specific inhibitors of JNK and p38, respectively, confirmed the involvement of both kinases in enhancing the activity of AP-1 induced by XO, if only p38 was involved in the increased levels of c-Jun and French horn in hypertrophic effect induced by XO. In conclusion, XO produced hypertrophy in vascular smooth muscle cells of humans through a mechanism of oxidative stress dependent. A better understanding of the signaling pathways involved in this effect, could allow ideritificar new therapeutic targets to prevent remodeling associated with vascular disease.

Author: PERNAS SUEIRAS OCTAVIO.
Year: 2005.
University: SANTIAGO DE COMPOSTELA [www.usc.es].
Place of defense: FACULTAD DE VETERINARIA.
Place of preparation: FACULTAD DE VETERINARIA.

Summary: The mast cells are crucial in inflammatory and immune to the type of allergies, hives, as well as autoimmune diseases. For his study, have been routinely using rat mast cells, mast cells obtained by the cultivation of hematopoietic precursor cells, as well as various cell lines. Among the latter, the line HMC-1 is the best known and most widely used, with its man-a great advantage in terms of being able to extrapolate data obtained from the study to knowledge of human mast cells. The objective of this thesis is to characterize the process of activation of the line HMC-1, which is still little known. For this you use the parameter release of histamine. In addition, it wants to study the possible influence on the process of two major metabolic pathways, which are involved in regulating many cellular processes: * The calcium signal, a second messenger universally recognized. * The intracellular pH, a parameter physicochemistry that previous studies suggests a possible role as a new signal transduction. The conclusions of this study are: 1-A alkalization intracellular is by itself a signal sufficient to trigger exocytosis in cells HMC-1. 2, cell-HMC-1, an increase of cytosolic calcium is not a sufficient signal to induce release of histamine. 3-cells HMC-1 presents reservoirs intracellular calcium adjustable for various drugs, which emptied induces an influx of calcium through channels SOC. 4, cell-HMC-1, an active alkalization intracellular calcium exit through the calcium ATPase of the plasma membrane. 5, - stimulation of PKC increases intracellular alkalization and release of histamine-induced ammonium chloride cells HMC-1. 6, In-cell HMC-1, the increase in the levels of intracellular cAMP alkalization power and the release of histamine-induced ammonium chloride.

Author: SOLÍS GARRIDO LUISA MARÍA.
Year: 2005.
University: AUTÓNOMA DE MADRID [www.uam.es].
Place of defense: FACULTAD DE MEDICINA.
Place of preparation: FACULTAD DE MEDICINA.

Summary: In 1991, Shirvan and colleagues identified a new population "atypical" cholinergic receptors coupled to the secretion of catecholamines in cells cromafines cattle. These receptors, which they termed "muscatínicos" possessed the extraordinary capacity to file a pharmacology halfway between for the nicotinic and muscarinic receptor. However, after 15 years of its initial identification, still remains unresolved the molecular nature of this receptor "atypical". The neuronal nicotinic receptors for acetylcholine (nAChRs) are complex protein pentaméricos built by the combination of several subunits of which, so far, have been identified and cloned twelve (alfa2-alfa10, beta2-beta4). Curiously, the nAChR consists of subunits alfa9 and alfa10, the last two being identified and cloned in olfactory epithelium and rat cochlea, has pharmacological properties that resemble those described for the receiver muscatínico the cell cromafín. Therefore, the central objective of this thesis is to explore whether this receptor was also expressed by atypical cells cromafines rat and if it corresponds to nAChR subtype alfa9alfa10. In this study we have combined a number of experimental approaches including molecular techniques, western blot, immunocytochemistry and confocal microscopy, pharmacological and electrophysiological. The analysis of our data in single cell RT-PCR indicates that the 54% and 78% of the cells cromafines discussed express the mRNAS for subunits nicotínicas alfa3 and alfa7 respectively; meanwhile, almost all of them expressed the transcribed alfa9 and alfa10. Moreover, using techniques inmunocitoquímicas shows that the latter transcripts are translated into the corresponding protein. Both subunits appear to be combined to form a native functional receptor in the cell croamfín, as is apparent from the records electrophysiological of patch-clamp in whole cell configuration showing how oxotremorina-M (Oxo-M) generates a current which nicotine is reversibly blocked by alfa-bungarotoxina (alfa-Bgtx). After obtaining PCR by the cDNAs corresponding to the subunits alfa9 and alfa10 the cell cromafín rat, these are secuenciaron and cloned in pGEMHE. We identified two variants alfa10 cell cromafín, identical to suhomónima of cochlea, along with a second isoform alfa10 "short" which lacks domain amino-terminal. For its part, the subunit alfa9 of cromafín only differed in the two residues cloned from olfactory epithelium (V157M and A458T). Injection of mRNA in vitro transcribed from cDNA of variant alfa10 "long" in the Xenopus oocytes produced no functional receptors but potentiate about 100 times the flow alfa9 activated by ACh in oocytes that coexpresan both subunits. The pharmacological and biophysical characteristics of nAChR alfa9alfa10 cell cromafín expressed in oocytes reproduced to a large extent, those described for the receiver cochlea. Taken together, our data suggest that cells cromafines express a cholinergic receptor unusual it is for the nAChR subtype alfa9 alfa10 previously identified in cochlea.

Author: LÓPEZ PEÑA RAFAEL.
Year: 2006.
University: VALENCIA [www.uv.es].
Place of defense: FACULTAD DE MEDICINA Y ODONTOLOGÍA.
Place of preparation: DEPARTAMENTO DE FARMACOLOGÍA - SECCIÓN DEPARTAMENTAL DE MEDICINA - UNIVERSIDAD DE VALENCIA.

Summary: INTRODUCTION TO THE PROBLEM OF STUDY SUBJECT: The smooth muscle cells in the walls of many bodies, are vital to many functions and tampering contributes to a host of diseases. The expulsion of the baby from the womb, breathlessness and asthma spasm of the coronary arteries are the result of the contraction of smooth muscle cells that form the main part of the walls of the uterus, blood vessels, airways, stomach, urinary bladder and intestines (Somlyo & Somlyo, 1994). The contraction of smooth muscle, although based on a filament-oscillating mechanism similar to that of striated muscle, its function is regulated by both mechanisms electromechanical coupling mechanisms for coupling farmacomecánicos. The contraction of smooth muscle is regulated primarily by changes in the intracellular concentration of Ca2 +-free. Increases in the concentration of Ca2 + intracellular cause contraction while its decrease cause smooth muscle relaxation. The complex Ca2 + -calmomodulina stimulates the kinase of myosin light chains and the subsequent phosphorylation of the regulatory light chains of myosin caused by the contraction of smooth muscle (1994, Kamm et al., 1989, Somlyo, AP & Somlyo , AV 1994). It has been discovered that the protein tyrosine kinase have a prominent role in the mobilization of Ca2 + intracellular and the contraction of smooth muscle, has been observed in different cell types, several growth factors mitogénicos such as epidermal growth factor (EGF) and growth factor plaquetar (PDGF) increase the concentration of Ca2 +-free intracellular and have contractile effects on different varieties of smooth muscles (Berk et al., 1985, Hollemberg, 1994). The use of drugs that inhibit protein tyrosine kinases attenuated smooth muscle contraction induced by growth factors and by vasoactive peptides which invokes a share in the phosphorylation of the protein tyrosine kinases in the mechanisms of transmission signal contraction smooth muscle (Di Salvo et al., 1993, Savineau et al., 1996). It has been shown that some agonists G-protein coupled receptors can stimulate, through a mechanism transactivation, receptor tyrosine kinase and route ERK / MAPK (via extracellular signal regulated kinase / protein kinase activated by mitógeno) and the transmission, forward in this way mitogenic, activated by a G-protein coupled receptor specifically not working in isolation and that the different signal paths are connected in networks of intracellular proteins, in which there are multiple crossings of roads between individual tracks (Liebmann, 2001 , Reinhard Wetzker and Frank-D Bomer, 2003). The problem under study is to investigate the role played by phosphorylation by protein kinase in the mechanism of contraction in human pulmonary arteries and veins. The establishment of this causal relationship between tyrosine phosphorylation of the activated receptor and the subsequent increases in the concentration of Ca2 + receiver activated pathophysiological implications can be significant (Di Salvo et al., 1997): 1 .- Changes to the rules governing counterpoint to one or more sites in the paths signal evoked by the activation of the receptor may produce undesirable increases in the concentration of Ca2 + intracellular, which can cause vascular spasm which may culminate in Myocardial Myocardium or Cerebral Hemorrhage. It may also be a contributing factor to some forms of progressive hypertension. 2 .- Changes in the regulation anterógrad 8 to the 1ff8 pathways of signal can contribute to failure in the peripheral vasoconstriction favoring tabling Shock or limit regulation neurohumoral in blood pressure. 3 .- Arterioesclerosis or peripheral vascular disease associated with diseases such as diabetes mellitus (Schwartz et al., 1990, Ross, 1995), because the tyrosine phosphorylation of the protein is an important mechanism for regulating cell growth and certain agents neurohumorales vasoactive as adrenalin, serotonin, angiotensin II, endothelin 1 and atrial vasoactive peptide can modulate the growth of vascular smooth muscle cells. 4 .- Remodeling and vascular restenosis after balloon angioplasty (Pyles et al, 1997) by changes in the regulation of MAPK. Therefore, the identification of changes in the signal paths can be a valuable aid for the development of new drugs aimed at correcting these changes, for example, because of their impact on transactivation, agonists or antagonists of G-protein coupled receptors, too used in the clinic, may present a carcinogenic risk or on the contrary, have beneficial effects in the treatment of cancer (Asuka et al., 2002, Gschwind et al., 2002). OBJETIVOSLa movement of the lower body features that differentiate it from the general circulation, such as the pulmonary vessels react to certain situations like hypoxia so opposite from those systemic vessels. Another feature no less striking is the movement that supports lower pressures much lower. This is a translation morphological in pulmonary arteries, which have a much thinner wall, with a muscular layer well below the arteries from the general circulation to the point that, once isolated from the body, it may be difficult to distinguish an artery a human pulmonary vein. Undoubtedly, the most important pathology at the smaller circulation is pulmonary hypertension. We do not know whether it was the cause or a consequence, but in cases of pulmonary hypertension, pulmonary arteries suffer some drastic changes, increase muscle size and the wall becomes thickened to the point of acquiring a morphology similar to that of any systemic artery. The question is whether all these changes might result from excessive and prolonged stimulation of the pulmonary vascular contraction. Perhaps the intracellular effects of the adrenergic stimulation of a receiver, not limited merely to contraction, but they are put in place in parallel phenomena of vascular remodeling, processes cell hyperplasia or hypertrophy. Classically has been associated phenomena of cell proliferation with the tyrosine kinase activity and the stimulation of receptor tyrosine kinase activity. On the other hand, some writers have described that stimulation of the family of G protein coupled receptors can lead to stimulation of tyrosine kinase activity. But also, has been reported that stimulation of certain receptors with tyrosine kinase activity may lead to the contraction of blood vessels. All this suggests an interesting overlap of the two intracellular transduction pathways, which raises several questions: Â What happens in humans?, Â Would a physiological stimulus as norepinephrine is capable of launching the road tyrosine kinase?, Â Does the tyrosine kinase activity is limited to participate in cell proliferation as it has been classically associated or involved with the contraction?, Â What happens to the mitógeno activated kinase (MAPK)?. These questions and many others that we could probably make, are still unknowns, which is why we are exploring the involvement of tyrosine kinases and the MAPK in the stimulation caused by noradrenaline in vessels. However, all approaches that could acquire work: cell proliferation, modification of apoptosis, neovascularization, etc., given the extent that it could acquire the work, we decided to limit ourselves to a study farmacomécanico, focusing on study participation these kinases in contraction. All this in pulmonary arteries and veins, in order to see if there were differences in the contraction and the mechanisms responsible for it between the two types Vascular. Therefore, we set out the following objectives: 1. Studying the effects of cryopreservation of human pulmonary arteries and veins and study the feasibility of conservation of valid samples for further studies farmacomecánicos. 2. Study on the involvement of protein tyrosine kinases in the contraction caused by noradrenaline using drugs as a nonspecific inhibitor genistein and tirfostina. 3. Study on the involvement of protein tyrosine phosphatases in the contraction caused by noradrenaline using drugs as inhibitors oxide fenilarsina, sodium vanadate and pervanadato. 4. Study of the role of calcium in the contraction of noradrenaline in pulmonary arteries and veins dilucidando whether there are differences between arteries and veins, and in that way the protein tyrosine kinase modulates the cellular calcium homeostasis in these vessels. 5. Using specific inhibitors of different drugs protein tyrosine kinase and MAPK who might be involved in the contraction by adrenergic stimulation of human pulmonary arteries and veins. CONCLUSIONS: 1. The cryopreservation of human pulmonary arteries and veins does not alter in a significant way the contractile responses to norepinephrine and potassium chloride, so that this procedure is feasible in the conservation of valid samples for further studies farmacomecánicos. 2. In human pulmonary arteries and veins, there was no significant correlation between the initial contractile responses evoked by potassium chloride and subsequently obtained a noradrenaline. On the contrary, there is a significant correlation between the contractile responses obtained in two curves concentración-respuesta to norepinephrine constructed consecutively in the same vascular prepared. Consequently, the normalization of the response to norepinephrine as a percentage of the initial contraction potassium chloride is not justified, which is why our results have been presented with noradrenaline in the form of gramos-fuerza or as a percentage of the first turn control this agonist. 3. Tyrosine kinase inhibitors, genistein and tirfostina A23, inhibit, so concentración-dependiente, the contractile responses to norepinephrine obtained in human pulmonary artery and vein, lacking the appropriate analogues inactive, daidzeína and tirfostina A1, biological activity in these preparations. The tyrosine phosphatase inhibitors studied oxide fenilarsina, vanadate and pervanadato not altered significantly curves concentración-respuesta to noradrenaline in human pulmonary artery and vein. 4. The pharmacological blockade of the entry of calcium channel operated by L-type voltage by verapamil, significantly reduced contractile response to norepinephrine in human pulmonary artery, but not in the vein. The pharmacological blockade of intracellular calcium deposits using drugs with different mechanisms of action as dantrolene, tapsigargina and SKF96365, did not alter in a significant way the contractile response to norepinephrine in human pulmonary artery, but fell sharply produced by the contraction in this agonist human pulmonary vein. The entry of calcium capacitativo does not induce a contractile response in human pulmonary artery while, on the other hand, leads to a robust contractile response in the pulmonary vein is removed by SKF96365. Taken together, these results allow us to state that the handling of calcium activator is completely different in the pulmonary vessels human of the 8 s vertie b5e nten arterial and venous. 5. The inhibition of tyrosine kinase by genistein not result in potentiation of the effects of drugs modulating signal calcium used in our study on the contractile response to norepinephrine in human pulmonary artery and vein. However, genistein inhibits the contraction caused by calcium capacitativo in pulmonary vein. Taken together, these results suggest that tyrosine kinases may have some role on the generation of activator calcium in these preparations. 6. Drugs specific inhibitors of Src kinases (PP2), fosfatidil-inositol 3-quinasa (LY294002) and the NADPH oxidase (apocianina) do not produce significant changes on the curve concentración-respuesta to noradrenaline in human pulmonary artery and vein. The specific inhibition of ERK1 / 2 by PD98059 and U0126, and the p38-MAPK by SB202190 produces a tendency to increase contraction of noradrenaline in pulmonary vein and artery which suggests involvement of this route of MAPK in the mechanisms involved in the contractile response to norepinephrine in human pulmonary vessels. Finally, the depression of the contraction caused by noradrenaline in human pulmonary artery and vein after incubation with AG1478, a specific inhibitor of the activity of tyrosine kinase receptor for epidermal growth factor, and GM6001, an inhibitor of metaloproteasas that impedes the release of an endogenous ligand of this receptor show that the transactivation receptor for epidermal growth factor is involved in the mechanisms of the contractile response to norepinephrine. 7. In short, all the results provided in this work presents experimental evidence of the involvement generic tyrosine kinases, as well as some specific kinases, the response evoked after activation adrenoceptor-a by the neurotransmitter norepinephrine in pulmonary vessels adrenergic human. These mechanisms may have potential interest not only in the pulmonary vasoconstriction but also for understanding the mechanisms underlying angiogenesis and remodeling of the pulmonary vessels that occur in human pathological circumstances.

Author: BOVAIRA GARCÍA MARÍA JOSÉ DEL ROSARIO.
Year: 2006.
University: MURCIA [www.um.es].
Place of defense: FACULTAD DE VETERINARIA.
Place of preparation: FACULTAD DE VETERINARIA.