Non-genomic vasorelaxant effects of 17ß-estradiol and progesterone in rat aorta are mediated by L-type Ca2+ current inhibition.

AIM: The sex hormones 17ß-estradiol (ßES) and progesterone (PRG) induce rapid non-genomic vasodilator effects which could be protective for the cardiovascular system. The purpose of this study was to analyze the mechanisms underlying their vasodilator effect in rat aortic smooth muscle preparations. METHODS: Endothelium-denuded aorta artery rings were prepared from male Wistar rats and incubated in an organ bath. The contractions of the preparation were recorded through isometric transducers. The effects of the hormones on K(+) current and L-type Ca(2+) current (LTCC) were analyzed by using the whole cell voltage-clamp technique in A7r5 cells. RESULTS: Both ßES and PRG (1-100 µmol/L) concentration-dependently relaxed the endothelium-denuded aortic rings contracted by (-)-Bay K8644 (0.1 µmol/L) or by KCl (60 mmol/L). The IC(50) values of the two hormones were not statistically different. The K(V) channel blocker 4-aminopyridine (2 mmol/L), BK(Ca) channel blocker tetraethylammonium (1 mmol/L) and K(ATP) channel blocker glibenclamide (10 µmol/L) did not significantly modify the relaxant effect of the hormones. On the other hand, the blockage of the intracellular ßES and PRG receptors with estradiol receptor antagonists ICI 182,780 (1 µmol/L) and PRG receptor antagonist mifepristone (30 µmol/L), respectively, did not significantly modify the relaxant action of the hormones. In A7r5 cells, both the hormones (1-100 µmol/L) rapidly and reversibly inhibited the basal and BAY-stimulated LTCC. However, these hormones had no effect on the basal K(+) current. CONCLUSION: The vasorelaxant effects of ßES and PRG are due to the inhibition of LTCC. The K(+) channels are not involved in the effects.

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 Ca(2+) mobilization and Ca(2+) 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 ([Ca(2+)]c), (2) the hyperpolarization of the smooth muscle cell membrane potential, (3) the reduction in the sensitivity of the contractile machinery by decreasing the [Ca(2+)]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.

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.

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 5HT(1B/D) agonists contracts HUA. The 5-HT-induced contractions were fully inhibited by ketanserin (5-HT(2A) antagonist). The 5-HT( 7)-activation also relaxes and increases intracellular cyclic adenosine monophosphate (cAMP). Among the histamine receptor agonists, only betahistine (H(1) agonist) induced significant contractile effect. Histamine-induced contraction was partially relaxed by pyrilamine (H(1) antagonist). Betahistine-induced contraction was partially blocked by dimaprit (H( 2) agonist) and by the H(3) agonist when a low concentration of forskolin is present. Both, H(2) and H(3) agonists increased the cAMP intracellular levels in HUA smooth muscle. These findings show that in HUA, 5-HT(2A)- and 5-HT(1B/1D)-activation lead to vasoconstriction and 5-HT(7)-activation induces vasorelaxation. Concerning histamine receptors, H(1)-activation induces contraction and H(2)- and H(3)-activation lead to vasorelaxation.

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.

Potassium channels are involved in testosterone-induced vasorelaxation of human umbilical artery.

Recent 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 microM), histamine (10 microM) and potassium chloride (KCl, 30 and 60 mM), and the vasorelaxant effect of testosterone was analysed. Testosterone (100 microM) 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 microM), an inhibitor of classical intracellular testosterone receptor, and glibenclamide, an ATP-sensitive potassium-channels (K(ATP)) inhibitor, did not influence the testosterone relaxant effect. 4-aminopyridine, a voltage-sensitive 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 Ca(2+)-activated potassium channels (BK(Ca)), 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 BK(Ca) and K(V) channels. The involvement of these two channels in testosterone-relaxant mechanism is dependent on the pathways activated by the contractile agent used.