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- Role of the endothelial system in Bay-K-8644 enantiomer and nifedipine vasomodulator action in rat aortaPublication . Verde, Ignacio; Gil-Longo, José; Orallo, Francisco; Campos, Manuel; Calleja, José MariaThe potential importance of the endothelial system in regulating the effects of (−)-Bay K 8644 (0.1 μM), (+)-Bay K 8644 (0.1 μM) and nifedipine (10 nM) on resting tension, on contractile responses to noradrcnaline (NA) and Ca2+ (in a Ca2+-free high-K+ solution), and on basal, NA-induced and K+-induced 45Ca2+ uptake, was investigated in rat aorta rings. Mechanical removal of endothclium considerably potentiated the contractile response induced by NA in standard medium and by Ca2+ in Ca2+-free high-K' (15 mM) medium, but did not modify the response induced by Ca2+ in Ca2+-frcc high-K+ (55 mM) medium or by NA in Ca2+-free medium. Furthermore, the basal 45Ca2+ uptake and that induced by NA (10 μM) or KCl (15 and 55 mM) were similar in endothelium-rubbed and intact rings. (−)-Bay K 8644 (0.1 μM) shifted the NA and Ca2+ concentration-response curves to the left with potentialion of the maximal contraction. However, (+)-Bay K 8644 (0.1 μM) and nifedipine (10 nM) caused a shift to the right, with depression of the maximal contraction. The NA concentration-response curves, and those of Ca2+ in Ca2+-free high-K+ (55 mM) medium, were affected by the drugs to similar extents, and were not modified by the presence or absence of endothelial cells. The drugs tested did not affect resting tension. Basal 45Ca2+ uptake was not modified by either nifedipine or the Bay K 8644 enantiomers. On the other hand, (−)-Bay K 8644 increased with equal effectives both NA- and KCl-induccd 45Ca2+ uptake, whilst (+)-Qay K 8644 and nifedipine inhibited both uptakes. The presence or absence of endothelium did not modify these effects. These results suggest that, in rat aorta, the endothelial system does not modulate either the agonist effect of (−)-Bay K 8644 or the antagonistic effects of (+)-Bay K S644 and nifedipine. Furthermore, our data indicate that the effects of Bay K 8644 enantiomers and nifedipine on the contractile responses and 45Ca2+ uptake elicited by NA and high-K+ (55 mM) solutions are similar.
- Characterization of the cyclic nucleotide phosphodiesterase subtypes involved in the regulation of the L-type Ca2+ current in rat ventricular myocytesPublication . Verde, Ignacio; Vandecasteele, Grégoire; Lezoualc'h, Frank; Fischmeister, RodolphePhosphorylation of cardiac L-type Ca2+ channels by cyclic AMP-dependent protein kinase (PKA) plays a determinant role in the hormonal regulation of myocardial contraction. PKA increases the mean open probability of individual Ca2+ channels which results in an increase in the macroscopic L-type calcium current (ICa) (McDonald et al., 1994). Activation of PKA usually results from an increased production of cyclic AMP by activation of membrane receptors positively coupled to adenylyl cyclase via stimulatory G proteins (Gs). The best documented of such a regulation is the positive inotropic effect of sympathomimetic amines, such as isoprenaline (Hartzell et al., 1991; Hove-Madsen et al., 1996). However, cardiac myocytes, as most other cell types, also possess a negative feedback mechanism to adenylyl cyclase activation which is constituted of the cyclic nucleotide phosphodiesterases (PDEs), a family of enzymes that break down cyclic AMP into 5'-AMP (Beavo, 1995). Cyclic nucleotide PDE activity, at any given location within the cell, will counterbalance the synthesis of cyclic AMP and determine the extent of PKA activation and, hence, of protein phosphorylation. In particular, at the sarcolemmal membrane, this balance between adenylyl cyclase and PDE activities will control the degree of ICa stimulation upon hormonal activation (Fischmeister & Hartzell, 1991; Hove-Madsen et al., 1996). Other factors are involved, such as cyclic AMP compartmentation (Jurevicius & Fischmeister, 1996), PKA tethering to the membrane (Gao et al., 1997), or phosphatase activity (Wiechen et al., 1995). [...]
- A study of glaucine-induced relaxation of rat aortaPublication . Verde, Ignacio; Orallo, Francisco; Loza, Isabel; Gil-Longo, José; Calleja, José Maria; Cadavid, IsabelThe vasorelaxant effect of glaucine, the major alkaloid of Platycapnos spicata (L.) Bernh., was studied. At concentrations of 10 microM-0.3 mM in normal Krebs solution it was almost equieffective in relaxing K(+)-induced and noradrenaline-induced tension in rat aortic rings without endothelium, with IC50 values of 160 +/- 16 microM and 90 +/- 14 microM respectively. In experiments in a calcium-free medium, 10 microM glaucine strongly inhibited noradrenaline-induced contractions. Glaucine (0.3 mM) did not affect basal uptake of 45Ca, but induced uptake was reduced to 100% (K+) and 97.7% (noradrenaline) of the basal value. These results suggest that glaucine has an intracellular effect and also acts on the cell membrane by blocking voltage-dependent and receptor-operated calcium channels.
- Potassium channels are involved in testosterone-induced vasorelaxation of human umbilical arteryPublication . Verde, Ignacio; Cairrão, Elisa; Álvarez, Ezequiel; Silva, António José SantosRecent studies have shown that testosterone induces relaxation of different arteries, although the mechanism of this action is still under debate. We investigated the involvement of potassium channels in this mechanism. Using standard organ bath techniques, rings of human umbilical arteries (HUA) without endothelium were contracted by serotonin (5-HT, 1 μM), histamine (10 μM) and potassium chloride (KCl, 30 and 60 mM), and the vasorelaxant effect of testosterone was analysed. Testosterone (100 μM) relaxed human umbilical arteries contracted with 5-HT (30.1±3.2%), histamine (55.1±2.6%), KCl 30 mM (52.9±8.3%) and KCl 60 mM (54.8±6.3%). Flutamide (10 μM), an inhibitor of classical intracellular testosterone receptor, and glibenclamide, an ATP-sensitive potassium-channels (KATP) inhibitor, did not influence the testosterone relaxant effect. 4-aminopyridine, a voltagesensitive potassium-channels (Kv) inhibitor, decreased the effect of testosterone on histamine- and 5-HT-contracted arteries. Tetraethylammonium (TEA), which inhibits Kv channels and large-conductance Ca2+-activated potassium channels (BKCa), decreased the effect of testosterone on KCl (60 mM)-contracted and 5-HT-contracted HUA. In conclusion, testosterone induces relaxation of HUA, and this effect does not appear to be mediated via a classic intracellular testosterone receptor-dependent mechanism. Our results suggest that this relaxation is partially mediated by activation of BKCa and KV channels. The involvement of these two channels in testosterone-relaxant mechanism is dependent on the pathways activated by the contractile agent used.
- 17 Beta-Estradiol and progesterone inhibit L-type Ca2+ current of rat aorta smooth muscle cellsPublication . Verde, Ignacio; Cairrão, Elisa; Carvas, João; Silva, António José Santos; Alvarez, EzequielSex hormones like 17ß-estradiol (ßES) and progesterone have shown rapid non-genomic vasodilator effects, which could be involved in the protection of cardiovascular system. However, the precise mechanism by which this effect occurs has not been elucidated yet, even if Ca2+ influx inhibition seems to be implicated. The aim of this study was to study the influence of ßES and progesterone on the L-type Ca2+ current measured by whole cell voltage-clamp in A7r5 cells. Voltage-operated Ca2+ currents were elicited by square-step voltage pulses and pharmacologically characterized as L-type currents by (-)-Bay K8644 (BAY) and nifedipine. Both ßES and progesterone (1-100 µM), rapidly and reversibly inhibited, in a concentration dependent manner, either non-stimulated or BAY-stimulated Ca2+ currents registered in A7r5 cells. These results suggest that ßES and progesterone inhibit L-type voltage-operated Ca2+ channels through a non-genomic pathway. Consequently, these hormones inhibit the Ca2+ entry into smooth muscle cells from rat aorta, an effect that can contribute for the protection of the cardiovascular system.
- Cyclic GMP regulation of the L-type Ca2+ channel current in human atrial myocytesPublication . Verde, Ignacio; Vandecasteele, Grégoire; Rucker-Martin, C.; Donzeau-Gouge, P.; Fischmeister, RodolpheThe cardiac L-type Ca2+ channel current (ICa) is an important determinant of myocardial contractility. Its regulation by neurotransmitters, hormones, and paracrine factors contributes to the control of cardiac output to meet the demands of the body. A large number of these extracellular first messengers, acting on specific membrane receptors in cardiac myocytes, regulate the activity of adenylyl cyclase which in turn controls the intracellular concentration of cAMP, the activity of the cAMPdependent protein kinase (PKA), and the degree of phosphorylation and stimulation of L-type Ca2+ channels (Hartzell, 1988; McDonald et al. 1994; Hove-Madsen et al. 1996; Striessnig, 1999). A typical example of such regulation is the control of heart function by the sympathetic and parasympathetic nervous systems, which act via adrenoceptors and muscarinic receptors (Brodde & Michel, 1999). In addition to the cAMP cascade, other factors regulate heart function by acting primarily on the cGMP cascade; these include atrial and brain natriuretic peptides (de Bold et al.1996) and nitric oxide (NO) (Paulus & Shah, 1999; Shah & MacCarthy, 2000).
- Pharmacological study of several effects of hydralazine in the bisected rat vas deferensPublication . Verde, Ignacio; Campos-Toimil, Manuel; Orallo, Francisco; Gil-Longo, José; Loza, Isabel; Fernandez-Alzueta, AlejandroWe have studied several effects of hydralazine in the bisected rat vas deferens. Hydralazine produced a shift to the left of the concentration-response curve for noradrenaline, with potentiation of the maximal response in both portions of the vas deferens. In contrast it caused a shift to the right of the concentration-response curve for noradrenaline in preparations pretreated with cocaine (inhibitor of catecholamine neuronal uptake), and of the curve for methoxamine and for CaCl2 (in depolarizing medium with K+ 55 mM), in all cases with depression of the maximal response. Hydralazine enhanced the contractions induced by noradrenaline in Ca2+-free medium, except in the presence of cocaine. It had no effect on [3H]noradrenaline neuronal uptake into noradrenergic neurons of the vas deferens, nor did it affect basal or K+-induced 45Ca2+ uptake. These results suggest that hydralazine potentiates the contractions elicited by noradrenergic by a mechanism other than blockade of the neuronal uptake of this catecholamine. Our results also suggest that the inhibition by hydralazine of the contraction elicited by Ca2+ (in Ca2-free depolarizing high-K+ 55 mM solution) and by methoxamine is not due to an action on voltage-dependent Ca2+ channels, but may reflect an intracellular site of action.
- Fosfolipasa A2 asociada a lipoproteínas y patología vascular cerebralPublication . Verde, Ignacio; Perez, FranciscoLa fosfolipasa A2 asociada a lipoproteínas (FLA2-Lp) es una enzima perteneciente a la superfamilia de las fosfolipasas A2. El 80% circula unido al colesterol ligado a lipoproteínas de baja densidad (colesterol-LDL), con cuyos niveles plasmáticos se correlaciona positivamente. Su función principal es hidrolizar los fosfolípidos oxidados del colesterol- LDL, formando derivados proinflamatorios en la placa del ateroma. Múltiples estudios han relacionado el aumento de FLA2-Lp y el riesgo vascular. Objetivo. Revisar los datos disponibles sobre la relación entre FLA2-Lp y la patología vascular, específicamente el ictus. Desarrollo. Diferentes estudios de cohortes y de casos y controles han demostrado una asociación entre los niveles superiores de FLA2-Lp y el riesgo de sufrir un primer evento cardiovascular (cardiopatía isquémica, ictus isquémico) o su recurrencia, la progresión de la lesión coronaria evaluada mediante tomografía computarizada cardíaca y la muerte de causa cardiovascular. Las medidas de riesgo publicadas son inferiores a 2, y se ha demostrado que los niveles elevados de la enzima son un factor de riesgo independiente de los factores de riesgo cardiovascular clásicos y de los niveles de colesterol no ligado a lipoproteínas de alta densidad y de proteína C reactiva. Los datos clínicos y el conocimiento de su papel en la desestabilización de la placa han justificado el desarrollo de fármacos inhibidores. Conclusiones. La evidencia existente permite considerar a los pacientes en el rango superior de FLA2-Lp como sujetos con mayor riesgo de padecer un episodio vascular. La aplicación al ictus isquémico precisa definir mejor su papel fisiopatológico y conocer los resultados de ensayos clínicos de fase II actualmente en curso en cardiopatía isquémica.
- Efectos del haloperidol y nuevas butirofenonas de sintesis sobre las contracciones inducidas por dopamina en conducto deferente de rataPublication . Verde, Ignacio; Loza, M.; Orallo, Francisco; Cadavid, Isabel; Gato, A.; Calleja, José MariaEI objetivo inicial de este trabajo fué el de estudiar los efectos de dos potenciales agentes neurolépticos (el p-clorobenzoato de pseudotropanol (I) y el p-F-fenil 2-4-o metoxifenilpiperazinometilciclopenitilcetona (II) frente a las contracciones inducidas por dopamina en conducto deferente aislado de rata. Pero, aunque está clara la presencia de dopamina (DA) y noradrenalina (NA) (1), al revisar la bibiografía encontramos datos contradictorios sobre la existencia de receptores postsinápticos específicos para la dopamina en esta preparación. Así, por una parte algunos autores (2) proponen que la DA activa una población de receptores postsinápticos que se diferencian de los activados por la NA, basándose en el hecho de que encontraron valores de pA2, distintos para la NA y para la DA utilizando varias antagonistas. Por otro lado, otros autores (3, 4) proponen que tanto la NA como la DA activan una población homogénea de receptores en vaso deferente de rata. Para demostrarlo utilizan una solución de Krebs especial a la que añaden inhibidores de la recaptaci6n, bloqueantes de receptores beta-adrenérgicos, y además modifican el tiempo de preincubación del antagonista. En nuestro trabajo estudiamos el efecto del haloperidol frente a las contracciones inducidas por NA y DA en ausencia y en presencia de inhibidores de la recaptaci6n neuronal (cocaina), extraneuronal (estradiol), y de un bloqueante beta adrenérgico (propranolol); y el de los dos potenciales neurolépticos (compuestos I y II) frente a las contracciones inducidas por dopamina en condiciones normales.
- Effects of trans- and cis-resveratrol on Ca2+ handling in A7r5 vascular myocytesPublication . Verde, Ignacio; Campos-Toimil, Manuel; Elies, Jacobo; Alvarez, Ezequiel; Orallo, FranciscoAlthough the natural polyphenol resveratrol posses a direct vasorelaxant effect, its effects on cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) in vascular cells remain still unclear. Here, we have investigated the effects of the isomers trans- and cis-resveratrol on agonist- and high-K(+)-induced [Ca(2+)](i) increases and on voltage-activated transmembrane Ca(2+) fluxes using imaging and patch-clamp techniques in vascular A7r5 myocytes. Arginine vasopressin (AVP) or angiotensin II caused a biphasic increase in [Ca(2+)](i) that was reduced by preincubation with trans-resveratrol and cis-resveratrol. Both isomers also reduced the agonist-induced increase in [Ca(2+)](i) in absence of extracellular Ca(2+). In high-K(+) Ca(2+)-free solution, reintroduction of Ca(2+) caused a sustained rise in [Ca(2+)](i) that was reduced by preincubation with trans-resveratrol or cis-resveratrol. When the isomers were applied during the plateau phase of the agonist- or the high-K(+)-induced response, a biphasic change in [Ca(2+)](i) was observed: a transient reduction of the plateau (<5 min) followed by an increase (>10 min). Finally, trans-resveratrol and cis-resveratrol inhibited voltage-dependent L-type Ca(2+) currents (I(Ca(L))). In conclusion, resveratrol isomers exert a dual effect on [Ca(2+)](i) handling in A7r5 myocytes: 1) a blockade of I(Ca(L)) and 2) an increase in [Ca(2+)](i) by depletion of intracellular Ca(2+) stores (which interferes with the agonist-induced release of intracellular Ca(2+)) and influx of Ca(2+), mainly due to activation of capacitative Ca(2+) entry, although other Ca(2+)-permeable channels are also involved. Taken together, these effects may explain, in part, the endothelium-independent vasorelaxant effects of resveratrol in rat aorta.
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