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- 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.
- Regulation of human umbilical artery contractility by different serotonin and histamine receptorsPublication . Verde, Ignacio; Silva, António José Santos; Cairrão, Elisa; Marques, BrunoWe studied the role of several serotonin (5-HT) and histamine receptors in the regulation of human umbilical artery (HUA) contractility. Among the 5-HT agonists used, only the 5-HT2A and 5HT1B/D agonists contracts HUA. The 5-HT-induced contractions were fully inhibited by ketanserin (5-HT2A antagonist). The 5-HT7-activation also relaxes and increases intracellular cyclic adenosine monophosphate (cAMP). Among the histamine receptor agonists, only betahistine (H1 agonist) induced significant contractile effect. Histamine-induced contraction was partially relaxed by pyrilamine (H1 antagonist). Betahistine-induced contraction was partially blocked by dimaprit (H2 agonist) and by the H3 agonist when a low concentration of forskolin is present. Both, H2 and H3 agonists increased the cAMP intracellular levels in HUA smooth muscle. These findings show that in HUA, 5-HT2A- and 5-HT1B/1D-activation lead to vasoconstriction and 5-HT7-activation induces vasorelaxation. Concerning histamine receptors, H1-activation induces contraction and H2- and H 3-activation lead to vasorelaxation.
- PDE4 and PDE5 regulate cyclic nucleotides relaxing effects in human umbilical arteriesPublication . Verde, Ignacio; Silva, António José Santos; Cairrão, Elisa; Morgado, Manuel; Álvarez, EzequielCyclic nucleotides (cAMP and cGMP) are the main second messengers linked to vasodilatation. They are synthesized by cyclases and degraded by different types of phosphodiesterases (PDE). The effect of PDE inhibition and cyclases stimulation on 5-hydroxytryptamine (5-HT; 1 microM) and histamine (10 microM) contracted arteries was analysed. Stimulation of guanylate cyclase or adenylate cyclase relaxed the histamine- and 5-HT-induced contractions indicating that intracellular increase of cyclic nucleotides leads to vasodilatation of the human umbilical artery. We investigated the role of different PDE families in the regulation of this effect. The presence of the different PDE types in human umbilical artery smooth muscle was analysed by RT-PCR and the expression of PDE1B, PDE3A, PDE3B, PDE4C, PDE4D and PDE5A was detected. The unspecific PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX; 50 microM) relaxed histamine-contracted human umbilical artery on 47.4+/-7.2%. This effect seems to be due to PDE4 and PDE5 inhibition because among the selective PDE inhibitors used only the PDE4 inhibitor (rolipram; 1 microM) and the PDE5 inhibitors (dipyridamole and T0156; 3 microM and 1 microM respectively) induced significant relaxation (39.0+/-8.7, 30.4+/-6.0 and 36.3+/-2.8 respectively). IBMX, dipyridamole and T0156 produced similar relaxation on 5-HT-induced contraction. After forskolin, the addition of IBMX or rolipram increased the effect of the adenylate cyclase stimulator and almost completely relaxed the human umbilical artery contracted by histamine (92.5+/-4.9 and 90.9+/-4.7 respectively), suggesting a main role of PDE4. The data obtained with 5-HT contracted arteries confirmed this, because only rolipram and IBMX significantly increased the forskolin vasodilator effect. The administration of dipyridamole and T0156 after sodium nitroprusside (SNP) induced a significant increase of the SNP relaxant effect on histamine-contracted arteries, but PDE1 and PDE3 inhibition did not increase the effect of the guanylate cyclase stimulator. Similar effects were obtained in 5-HT contracted arteries, the SNP induced relaxation was increased by the PDE5 inhibition, but not by PDE1 or PDE3 inhibition. In summary, our results demonstrate that: 1) the increase of cAMP and/or cGMP levels induces relaxation of the human umbilical vascular smooth muscle; 2) four families of PDE are expressed in this smooth muscle: PDE1, PDE3, PDE4 and PDE5; 3) between these families, PDE4 and PDE5 are the key enzymes involved in the regulation of the relaxation associated to cAMP and cGMP, respectively.