Loading...
8 results
Search Results
Now showing 1 - 8 of 8
- 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.
- Polyazamacrocycles as potential antitumor agents for human prostate cancer cellsPublication . Cruz, Carla; Cairrão, Elisa; Lourenço, Olga; Almeida, Paulo; Verde, Ignacio; Queiroz, JoãoPolyazamacrocycles are currently being studied and used in a variety of applications beyond their traditional place in supramolecular and co-ordination chemistry. This study suggests additional applications of these compounds with particular emphasis on their use as antiproliferative agents that could be potentially used to treat cancer. Four polyazamacrocycles were tested in human prostate cancer LNCaP and prostate epithelial PNTA1 cells to analyze changes in cell proliferation and cell death capabilities. Their intracellular localization was also evaluated by confocal microscopy. The results show a decrease in proliferation rate and cell viability of LNCaP and PNTA1, after treatment with these compounds. The decrease in the number of viable cells is similar for the majority of the compounds studied, and at higher concentration, the proliferation efficiency decreased significantly in the cell lines studied. Also, our results suggest that L and L2 induce early apoptosis in PNTA1 cells and late apoptosis/necrosis in LNCaP cells. The compounds did not induce a significant increase in necrosis of both cell types. Although the compounds did not localize in a unique organelle, all of them have as main target the Golgi apparatus and other localization profiles differed depending on the cell line.
- 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.
- 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.
- Long- and short-term effects of androgens in human umbilical artery smooth musclePublication . Saldanha, Paulo; Cairrão, Elisa; Baptista, Cláudio; Verde, IgnacioThe aim of the present study was to determine the effects of androgens in the regulation of human umbilical artery (HUA) contractility. The short-term effects of testosterone on the tone of the HUA were investigated, as were the long-term effects of dihydrotestosterone (DHT) on the expression of some proteins involved in the contractile process. Endothelium-denuded HUA were treated for 24 h with DHT (2 μmol/L) or the vehicle control (ethanol) to analyse the genomic effects of androgens. Twenty-four hour treatment of HUA with DHT increased the mRNA expression of the β(1)-subunit of the large-conductance Ca(2+)-activated (BK(Ca)) channel and decreased expression of the α-subunit of L-type calcium channels. In organ bath studies, testosterone (1-100 μmol/L) produced similar relaxant responses in DHT- and vehicle-treated HUA rings precontracted with 5-HT, histamine and KCl. However, the relaxation response obtained by the combined application of testosterone (100 μmol/L) and nifedipine (10 μmol/L) was significantly greater in DHT- compared with vehicle-treated HUA. The results indicate that the rapid vasorelaxant effects of testosterone that are dependent on both BK(Ca) and voltage-sensitive potassium (K(V)) channel activity in control arteries become dependent solely on K(V) channel activity in DHT-treated HUA. Thus, the present study reveals the importance of the investigation of both the short- and long-term effects of androgens in human arteries.
- 5α-Dihydrotestosterone regulates the expression of L-type calcium channels and calcium-binding protein regucalcin in human breast cancer cells with suppression of cell growthPublication . Marques, Ricardo; Peres, Carina; Vaz, Cátia; Gomes, Inês; Figueira, Marília I; Cairrão, Elisa; Verde, Ignacio; Baptista, Cláudio; Socorro, SílviaAndrogens have been associated with the development of normal breast, and their role in mammary gland carcinogenesis has also been described. Several studies reported that androgens inhibit breast cancer cell growth, whereas others linked their action with the modulation of calcium (Ca(2+)) pumps, Ca(2+) channels and Ca(2+)-binding proteins. Also, it is known that deregulated Ca(2+) homeostasis has been implicated in the pathophysiology of breast. The L-type Ca(2+) channels (LTCCs) were found to be up-regulated in colon, colorectal and prostate cancer, but their presence in breast tissues remains uncharacterized. On the other hand, regucalcin (RGN) is a Ca(2+)-binding protein involved in the control of mammary gland cell proliferation, which has been identified as an androgen target gene in distinct tissues except breast. This study aimed to confirm the expression and activity of LTCCs in human breast cancer cells and investigate the effect of androgens in regulating the expression of α1C subunit (Cav1.2) of LTCCs and Ca(2+)-binding protein RGN. PCR, Western blot, immunofluorescence and electrophysiological experiments demonstrated the expression and activity of Cav1.2 subunit in MCF-7 cells. The MCF-7 cells were treated with 1, 10 or 100 nM of 5α-dihydrotestosterone (DHT) for 24-72 h. The obtained results showed that 1 nM DHT up-regulated the expression of Cav1.2 subunit while diminishing RGN protein levels, which was underpinned by reduced cell viability. These findings first confirmed the presence of LTCCs in breast cancer cells and opened new perspectives for the development of therapeutic approaches targeting Ca(2+) signaling.