Elisa Cairrão

Cyclic nucleotide-dependent relaxation pathways in vascular smooth muscle.

Vascular smooth muscle tone is controlled by a balance between the cellular signaling pathways that mediate the generation of force (vasoconstriction) and release of force (vasodilation). The initiation of force is associated with increases in intracellular calcium concentrations, activation of myosin light-chain kinase, increases in the phosphorylation of the regulatory myosin light chains, and actin-myosin crossbridge cycling. There are, however, several signaling pathways modulating Ca2+ mobilization and Ca2+ sensitivity of the contractile machinery that secondarily regulate the contractile response of vascular smooth muscle to receptor agonists. Among these regulatory mechanisms involved in the physiological regulation of vascular tone are the cyclic nucleotides (cAMP and cGMP), which are considered the main messengers that mediate vasodilation under physiological conditions. At least four distinct mechanisms are currently thought to be involved in the vasodilator effect of cyclic nucleotides and their dependent protein kinases: (1) the decrease in cytosolic calcium concentration ([Ca2+]c), (2) the hyperpolarization of the smooth muscle cell membrane potential, (3) the reduction in the sensitivity of the contractile machinery by decreasing the [Ca2+]c sensitivity of myosin light-chain phosphorylation, and (4) the reduction in the sensitivity of the contractile machinery by uncoupling contraction from myosin light-chain phosphorylation. This review focuses on each of these mechanisms involved in cyclic nucleotide-dependent relaxation of vascular smooth muscle under physiological conditions.

PDE4 and PDE5 regulate cyclic nucleotides relaxing effects in human umbilical arteries

Cyclic 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.

The effects of phthalates in the cardiovascular and reproductive systems: A review.

Every year millions of tons of plastic are produced around the world and humans are increasingly exposed to them. This constant exposure to plastics has raised some concerns against human health, particularly when it comes to phthalates. These compounds have endocrine-disrupting properties, as they have the ability to bind molecular targets in the body and interfere with hormonal function and quantity. The main use of phthalates is to give flexibility to polyvinyl chloride (PVC) polymers. Phthalates are found in a variety of industrial and consumer products, and as they are not covalently bound to the plastic, phthalates contaminate the environment from which human exposure occurs. Studies in human and animal populations suggest a correlation between phthalate exposure and adverse health outcomes, particularly at the reproductive and cardiovascular systems, however there is much less information about the phthalate toxicity of the later. Thus, the main purpose of this review is to present the studies relating the effects already stated of phthalates on the cardiovascular and reproductive systems, and also present the link between these two systems.

PKG is involved in testosterone-induced vasorelaxation of human umbilical artery.

The cyclic nucleotides involvement in the vasorelaxation induced by testosterone in human umbilical artery was investigated. The effect of this hormone on denuded human umbilical arteries contracted by serotonin (5-HT), histamine or KCl was analysed. Testosterone effect on potassium current (IK) was also studied in human umbilical artery vascular smooth muscle cells (HUASMC). In general, the relaxant effects of testosterone, sodium nitroprusside (SNP) and atrial natriuretic peptide (ANP) are similar. The testosterone relaxant effect is not different to the induced by the conjoint application of ANP and testosterone. However, the effects of SNP and testosterone seem additive. The inhibition of protein kinase A (PKA) did not modify the testosterone relaxant effect, but protein kinase G (PKG) inhibition significantly reduced the testosterone effect independently of the contractile stimuli. In HUASMC, the IK is mainly constituted by potassium exit through voltage sensitive (KV) and large-conductance Ca2+ activated (BKCa) potassium channels. Testosterone significantly activates the basal IK. SNP does not induce a significant modification in basal or testosterone stimulated IK. In contrast, ANP stimulates the basal IK, but does not increase the testosterone stimulation on IK. The IK increases induced by testosterone or by ANP are not significantly affected by the PKA inhibition, but are completely inhibited by the PKG inhibition. Our results show that testosterone and ANP stimulate the activity of BKCa and KV channels due to PKG activation and suggest that this hormone relaxes by activating particulate guanylate cyclase which increases the cGMP intracellular level.

Isolation and culture of human umbilical artery smooth muscle cells expressing functional calcium channels

The 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.

Testosterone and cholesterol vasodilation of rat aorta involves L-type calcium channel inhibition.

Testosterone has rapid nongenomic vasodilator effects which could be involved in protective cardiovascular actions. Several authors suggested specific mechanisms to explain this effect, but this matter was not clarified yet. We studied the actions of testosterone and cholesterol on endothelium-denuded rat aorta and their effects on the L-type Ca2+ current (ICa,L) and potassium current (IK). Testosterone (1–100 μM) totally relaxed, in a rapid and concentration-dependent way, the aortic rings contracted by KCl or by (−)-Bay K8644 (BAY). Cholesterol also fully relaxed the contractions induced by KCl. None of the potassium channel antagonists tested (glibenclamide, tetraethylammonium and 4-aminopyridine) modified significantly the relaxant effect of testosterone. The antagonist of classic testosterone receptors, flutamide, did not modify the vasorelaxant effect of testosterone. Furthermore, testosterone and cholesterol inhibited either basal and BAY-stimulated ICa,L in A7r5 cells and they have no effects on IK. In summary, our results demonstrate that cholesterol and testosterone relax rat aorta by inhibiting LTCC. This effect of testosterone is not mediated by the classic hormone receptor or by potassium channel activation. These results suggest that the vasodilator mechanism of cholesterol and testosterone is the same.

Regulation of Human Umbilical Artery Contractility By Different Serotonin and Histamine Receptors

We 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-HT 2A and 5HT1B/D agonists contracts HUA. The 5-HT-induced contractions were fully inhibited by ketanserin (5-HT2A antagonist). The 5-HT 7-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 (H 2 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 H3-activation lead to vasorelaxation.

Targeting of mitochondria-endoplasmic reticulum by fluorescent macrocyclic compounds.

Background Useful probes of the intracellular environment that target a specific organelle in order to allow direct observation of the changes in these regions is of high current interest. Macrocyclic ligands have already revealed themselves as important selective hosts in some biological applications, forming stable and specific complexes. Therefore, in this paper, several macrocyclic ligands are evaluated as potential molecular probes. Methodology Four polyammonium macrocycles and one macrotricyclic bearing pyridine and phenanthroline chromophores have been synthesised and evaluated as molecular probes. The cytotoxicity of the compounds has been analyzed using human breast cancer cells (MCF-7), non-cancerous human dermal fibroblasts (NHDF) and human adult dermal skin fibroblasts from a breast cancer patient (P14). All the compounds showed low toxicity at concentrations ranging from 10 nM to 10 µM, except for [32]phen2N4 which proved to be highly cytotoxic for MCF-7 cells. Flow cytometry studies evidenced that the percentage of apoptotic and necrotic MCF-7 and NHDF cells induced by the compounds is considerably low. Also, flow cytometry analysis showed that some compounds seem to modify the mitochondrial membrane potential (MMP) of the cells. Fluorescence microscopy evidenced that compounds easily cross the plasma membrane (5 min) and accumulated into the mitochondria, as confirmed by co-localization with MitoTracker Green™. The fluorescence images also evidenced an intact mitochondria structure after 48 h. Moreover, reticular staining suggestive of endoplasmic reticulum (ER) localization, in addition to the mitochondrial one, has been found by confocal microscopy. Conclusion Our study reveals that compounds Me2[28]py2N6, cryptphen, [16]phenN2, [30]phen2N6, have low toxicity and localize in mitochondria and ER. The ability of these compounds for translocating the cellular membrane (5 min) without special conditioning of the cells or derivatization of the probe, the time-dependent localization (48 h) and the cellular viability provide a proof-of-concept towards their use as promising probes towards biomedical studies.

Cyclic guanosine monophosphate compartmentation in human vascular smooth muscle cells

AIMS The role of different vascular subtypes of phosphodiesterases (PDE) in cGMP compartmentalization was evaluated in human smooth muscle cells. METHODS AND RESULTS To understand how the cGMP conveys different information we infected smooth muscle cells with adenovirus containing mutants of the rat olfactory cyclic nucleotide-gated (CNG) channel-subunit and we recorded the associated cGMP-gated current (ICNG). The whole cell configuration of patch clamp technique was used to measure the ICNG and also the potassium current (IK) in human umbilical artery smooth muscle cells (HUASMC). ANP (0.1μM) induced a clear activation of basal ICNG, whereas SNP (100 μM) had a slight effect. The nonselective PDE inhibitor (IBMX; 100 μM), the PDE5 inhibitor (T0-156; 1 μM) and the PDE3 inhibitor (cilostamide; 10 μM), all had a tiny effects on the basal ICNG current. Concerning potassium channels, we observed that ANP and testosterone induced activation of IK and this activation is bigger than that elicited by SNP, cilostamide and T0-156. Cilostamide and T0-156 decreased the CNG stimulation induced by ANP and testosterone, suggesting that pGC pool is controlled by PDE3 and 5. Thus, the effects of SNP show the existence of two separated pools, one localized next to the plasma membrane and controlled by the PDE5 and PDE3, and a second pool localized in the cytosol of the cells that is regulated mainly by PDE3. CONCLUSIONS Our results show the existence of cGMP compartmentalization in human vascular smooth muscle cells and this phenomenon can open new perspectives concerning the examination of PDE families as therapeutic targets.

Testosterone and atrial natriuretic peptide share the same pathway to induce vasorelaxation of human umbilical artery.

Abstract: We recently observed in human umbilical artery smooth muscle cells that testosterone activates protein kinase G and stimulates large-conductance Ca2+ activated (BKCa) and voltage sensitive (KV) potassium channels. In the same work, we also show that atrial natriuretic peptide (ANP), an activator of particulate guanylate cyclase (pGC), stimulates the activity of BKCa and KV channels because of protein kinase G activation. The aim of this work was to prove that the relaxant effects of testosterone are also because of the increase of cGMP because of activation of the pGC. Subsarcolemmal cGMP signals were monitored in single cells by recording the cGMP-gated current (ICNG) in human umbilical artery smooth muscle cells expressing the wild-type rat olfactory cyclic nucleotide-gated (CNG) channel. Sodium nitroprusside (10 and 100 &mgr;M), ANP (0.1 and 1 &mgr;M), or testosterone (0.1, 1, and 10 &mgr;M) induced activation of ICNG. This activation induced by testosterone and ANP is bigger than that elicited by sodium nitroprusside. In summary, our study reveals that testosterone and ANP activate the pGC and induce vasorelaxation of human umbilical artery.