Browsing by Author "Alvarez, Ezequiel"
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- 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.
- 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.
- Isolation and culture of human umbilical artery smooth muscle cells expressing functional calcium channelsPublication . Cairrão, Elisa; Santos-Silva, António; Alvarez, Ezequiel; Correia, Ilídio Joaquim Sobreira; Verde, IgnacioThe human umbilical cord is a biological sample that can be easily obtained just after birth. A methodology was developed to perform cultures of human umbilical artery smooth muscle cells (HUASMC) expressing contractile proteins and functional ionic channels. To avoid fibroblast and endothelial cell contamination, we mechanically separated the tunica media, which only contains HUASMC and matrix proteins. To isolate the cells, collagenase V and elastase were used as hydrolyzing enzymes. The isolated cells were plated in collagen-coated dishes to obtain cultures of HUASMC. The cells obtained after different passages (1 to 6) exhibit the characteristic vascular smooth cell morphology and express smooth muscle alpha-2 actin, myosin heavy chain SM1, and alpha subunits of L- and T-type calcium channels (Cav 1.2, Cav 1.2, and Cav 3.2). Electrophysiology recordings for L- and T-type calcium channels were made, indicating that these channels are functional in the cultured cells. In conclusion, the procedure developed allows obtaining cultures of HUASMC expressing contractile proteins and also functional ionic channels. These cells could be used to study cellular and molecular aspects about the regulation of the vascular function.