Lucia Trevisi

Isolation of callipeltins A–C and of two new open-chain derivatives of callipeltin A from the marine sponge Latrunculia sp. A revision of the stereostructure of callipeltins

Two new callipeltin-related acyclic peptides (2 and 3) have been isolated, together with callipeltins A–C from the marine sponge Latrunculia sp. collected at the Vanuatu Islands. The gross structures of new compounds were elucidated by spectroscopic data. The application of the Marfeys analysis on the new derivatives and on callipeltin A (1), allowed us to revise the configuration of two amino acid units in callipeltin A.

Endothelin-1-induced arachidonic acid release by cytosolic phospholipase A2 activation in rat vascular smooth muscle via extracellular signal-regulated kinases pathway.

The present study investigates whether endothelin-1 (ET-1), like noradrenaline (NA), stimulates the release of arachidonic acid (AA) via cytosolic phospholipase A2 (cPLA2) in rat tail artery. In tail artery segments labelled with [3H]AA, ET-1-induced AA release in a concentration-dependent manner with an EC50 of 1.3 nM. The effect of ET-1 was inhibited by bosentan and was insensitive to BQ788, suggesting the involvement of ETA receptor. The stimulation of AA release induced by ET-1 was prevented by arachydonyl trifluoromethyl ketone (AACOCF3), a selective inhibitor of cPLA2 and not by RHC80267, a diacylglycerol lipase inhibitor. Furthermore, PD98059, inhibitor of mitogen-activated protein kinase kinase (MEK) cascade and calphostin C, a protein kinase C (PKC) inhibitor, prevented the stimulation of AA release induced by ET-1 and NA. Immunoblotting of the cytosolic fraction of rat tail arteries stimulated with ET-1 or NA showed an increase in extracellular signal-regulated kinases (ERKs) phosphorylation and this effect was abolished by calphostin C treatment. These findings show that in rat tail artery ET-1 and NA induce a sequential activation of protein kinase C and extracellular signal-regulated kinases that results in stimulation of AA release via cPLA2 activation. This may represent a general pathway by which G-proteins coupled receptors stimulate AA release and its metabolites in vascular smooth muscle.

Cardiac glycoside ouabain induces autophagic cell death in non-small cell lung cancer cells via a JNK-dependent decrease of Bcl-2.

Cardiac glycosides are Na/K-ATPase inhibitors, clinically used for congestive heart failure and cardiac arrhythmias. Epidemiological studies have reported that patients on cardiac glycosides treatment are protected from some types of cancers. This evidence together with the demonstration that cardiac glycosides show selective cytotoxicity against cancer cells has raised new interest on the anticancer properties of these drugs. This study examines the mechanism involved in the anticancer effect of ouabain in non-small cell lung cancer cells lines (A549 and H1975). Ouabain inhibited cell proliferation and induced cell death in a concentration-dependent manner. Cell death was caspase-independent and showed classical patterns of autophagic cell death: conversion of LC3-I to LC3-II, increase of LC3 puncta and increase of autophagic flux. Moreover, cell death was completely blocked by the class III phosphatidylinositol-3 kinase inhibitor 3-methyladenine. Here we show that ouabain caused the reduction of Bcl-2 protein levels, with no change in the expression of the autophagic protein Beclin 1. Early signalling events of ouabain exposure were ERK1/2 and JNK activation, however only JNK inhibition with SP600125 or JNK knockdown by shRNA were able to prevent Bcl-2 decrease, conversion of LC3-I to LC3-II and cell death. We propose that JNK activation by ouabain leads to a decrease of Bcl-2 levels, resulting in disruption of the inhibitory interaction of Bcl-2 with Beclin 1, that promotes autophagy. These findings indicate that pharmacological modulation of autophagy by cardiac glycosides could be exploited for anticancer therapy.

Vasorelaxant properties of norbormide, a selective vasoconstrictor agent for the rat microvasculature.

1 The effects of norbormide on the contractility of endothelium‐deprived rat, guinea‐pig, mouse, and human artery rings, and of freshly isolated smooth muscle cells of rat caudal artery were investigated. In addition, the effect of norbormide on intracellular calcium levels of A7r5 cells was evaluated. 2 In resting rat mesenteric, renal, and caudal arteries, norbormide (0.5–50 μm) induced a concentration‐dependent contractile effect. In rat caudal artery, the contraction was very slowly reversible on washing, completely abolished in the absence of extracellular calcium, and antagonized by high concentrations (10–800 μm) of verapamil. The norbormide effect persisted upon removal of either extracellular Na+ or K+. The contractile effect of norbormide was observed also in single, freshly isolated smooth muscle cells from rat caudal artery. 3 In resting rat and guinea‐pig aortae, guinea‐pig mesenteric artery, mouse caudal artery, and human subcutaneous resistance arteries, norbormide did not induce contraction. When these vessels were contracted by 80 mM KCl, norbormide (10–100 μm) caused relaxation. Norbormide inhibited the response to Ca2+ of rat aorta incubated in 80 mM KCl/Ca2+‐free medium. Norbormide (up to 100 μm) was ineffective in phenylephrine‐contracted guinea‐pig and rat aorta. 4 In A7r5 cells, a cell line from rat aorta, norbormide prevented high K+‐but not 5‐hydro‐xytryptamine‐induced intracellular calcium transients. 5 These findings indicate that in vitro, norbormide induces a myogenic contraction, selective for the rat small vessels, by promoting calcium entry in smooth muscle cells, presumably through calcium channels. In rat aorta and arteries from other mammals, norbormide behaves like a calcium channel entry blocker.

Autocrine-paracrine role of endothelin-1 in the regulation of aldosterone synthase expression and intracellular Ca2+ in human adrenocortical carcinoma NCI-H295 cells.

The role played by endothelin (ET-1) and its receptor subtypes A and B (ETA and ETB) in the functional regulation of human NCI-H295 adrenocortical carcinoma cells has been investigated. Reverse transcription-PCR with primers specific for prepro-ET-1, human ET-1 converting enzyme-1, ETA, and ETB complementary DNAs consistently demonstrated the expression of all genes in NCI-H295 cells. The presence of mature ET-1 and both its receptor subtypes was confirmed by immunocytochemistry and autoradiography, respectively. Aldosterone synthase (AS) messenger RNA was also detected in NCI-H295 cells, and AS gene expression was enhanced by both ET-1 and the specific ETB agonist IRL-1620; this effect was not inhibited by either the ETA antagonist BQ-123 or the ETB antagonist BQ-788. A clear-cut increase in the intracellular Ca2+ concentration in NCI-H295 cells in response to ETB, but not ETA, activation was observed. In light of these findings, the following conclusions can be drawn: 1) NCI-H295 cells possess an active...

Calcium‐antagonist effects of norbormide on isolated perfused heart and cardiac myocytes of guinea‐pig: a comparison with verapamil

Cardiac effects of norbormide and verapamil were compared in single ventricular myocytes, right atria, and Langendorff perfused hearts isolated from guinea‐pigs. In ventricular myocytes, norbormide 50 μm inhibited the peak calcium current (ICa) by 49.6±3.9% without altering the shape of the current‐voltage relationship; verapamil 1 μm inhibited ICa by 83.2±3.3%. Neither norbormide nor verapamil affected ICa at the first beat after a 3 min quiescence period; during repeated depolarizations, both drugs cumulatively blocked ICa (use‐dependence), with time constants of 23.0±7.0 s for norbormide and 91.3±8.4 s for verapamil. In constant‐flow perfused hearts electrically driven at 2.5 Hz or 3.3 Hz, both norbormide and verapamil concentration‐dependently decreased ventricular contractility (dP/dtmax), atrio‐ventricular (AV) conduction velocity and coronary pressure. Intraventricular conduction velocity was slightly decreased by norbormide but not by verapamil. At an equivalent change in AV conduction, norbormide depressed heart contractility less than verapamil. The effects of norbormide on AV conduction, intraventricular conduction, and contractility were frequency‐dependent. Furthermore, the curves correlating the mechanical and electrical effects of norbormide at the two frequencies used were apparently coincident, while those of verapamil were clearly separated. In spontaneously beating right atria, norbormide and verapamil decreased the frequency of sinus node (SA) in a concentration‐dependent way. At an equivalent effect on the AV conduction, norbormide exerted a greater effect on sinus frequency than verapamil. These results indicate that in guinea‐pig heart norbormide has the pharmacological profile of a Ca‐antagonist with strong electrophysiological properties. In comparison with verapamil, norbormide is more selective on SA and AV node tissues and exerts a weaker negative inotropic effect on ventricles. In principle, this pattern of effects may be an advantage in treating supraventricular tachyarrhythmias in patients with heart failure. The effect of norbormide on intraventricular conduction may represent an additional antiarrhythmic mechanism.

Inhibition of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) endocytosis by ouabain in human endothelial cells

3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) uptake and reduction is widely used to evaluate cell proliferation and viability. MTT is taken up by the cells through endocytosis. We find that ouabain (1–200 nM) inhibits MTT reduction in human umbilical vein endothelial cells (HUVEC) without affecting cell viability. Ouabain does not inhibit MTT reduction when cell lysates substituted for the intact cells. Disruption of caveolae by cholesterol depletion, completely prevents the effect of ouabain. Treatment of HUVEC with Src inhibitor 4‐amino‐5‐(4‐chlorophenyl)‐7‐(t‐butyl)pyrazolo[3,4‐d]pyrimidine partially abrogates the inhibitory effect of ouabain. The data suggest that ouabain interaction with caveolar Na/K‐ATPase inhibits MTT endocytosis through the activation of signaling proteins such as Src kinase.

Antiapoptotic and Proliferative Effects of Low Concentrations of 7β-Hydroxycholesterol in Human Endothelial Cells via ERK Activation

The atherogenic potential of oxidized low-density lipoproteins (oxLDL) has been correlated to their 7β-hydroxycholesterol (7βOHC) content; oxLDLs have a dual effect on endothelial cell viability, inducing apoptosis or proliferation depending on the concentration. Considering that 7βOHC is apoptotic for endothelial cells at concentrations ≧20 µg/ml, a study on the effect of lower concentrations of 7βOHC on human umbilical vein endothelial cells (HUVECs) was undertaken. 7βOHC (1–10 µg/ml) increased 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction of growth-factor-deprived HUVECs. This effect was due to the increased cell proliferation, determined by [3H]thymidine incorporation, and the reduction of apoptosis, revealed by the decreased caspase-3 activation and annexin V staining. 7βOHC also protected against staurosporine apoptosis. 7βOHC induced an increase in intracellular ROS antagonized by N-acetylcysteine; however, HUVECs treatment with the antioxidant did not inhibit the effects of 7βOHC. 7βOHC produced an increase in extracellular signal-regulated kinase (ERK) phosphorylation that was blocked by inhibitors of store-operated calcium entry 2-aminoethoxydiphenyl borate and gadolinium. MEK inhibition with PD98059 or U0126 as well as store-operated calcium entry inhibition antagonized the effect of 7βOHC. The results suggest that 7βOHC promotes HUVECs survival and proliferation by a mechanism independent of ROS production and involving calcium-dependent activation of ERK.

Effect of long-term ouabain treatment on contractile responses of rat aortae.

Male Sprague-Dawley rats were infused with 50 microg/kg/day of ouabain for 4 weeks to address the question whether prolonged exposure to the drug affects blood pressure, the in vitro contractile responses to agonists and high K+ of their aortae, and the influence of endothelium on these responses. Systolic blood pressure was not affected by ouabain treatment. The responsiveness of endothelium-intact aortae from ouabain-treated rats to endothelin-1 increased, that to phenylephrine decreased, and that to high K+ was unchanged, as compared with control. The responses of endothelium-free aortae to endothelin-1, phenylephrine, and high K+ were lower in ouabain-treated than in control rats. The removal of endothelium increased the response to phenylephrine and decreased that to high K+ in either control or ouabain-treated rat aortae, whereas it did not affect the response to endothelin-1 in control rat aortae and decreased it in ouabain-treated rat aortae. The response to caffeine was unaffected by either ouabain treatment or endothelium removal. Thus rat ouabain long-term treatment induces opposing effects on the responsiveness of their intact aortae to an alpha-adrenergic agonist and endothelin-1. If these effects observed in the ex vivo experiments occur also in vivo on rat microvasculature, they could balance out and contribute to the lack of effect on systolic blood pressure of prolonged ouabain treatment.

Effects of canrenone on aorta and right ventricle of the rat.

Canrenone is a major active metabolite of spironolactone and, in addition to the antimineralocorticoid effect, shares with the parent compound the action as a partial agonist with respect to ouabain on the Na+-K+ ATPase. We have investigated whether canrenone, through its action on Na+-K+ ATPase, reverses rat aorta contractions induced by ouabain and has vasorelaxant properties unrelated to its interaction with ouabain. Contractile responses of endothelium-deprived aorta to 1 m M ouabain, 0.1 &mgr;M phenylephrine, 10 &mgr;M serotonin, and 60 m M K+ were relaxed by canrenone (50–250 &mgr;M), with maximum inhibitions of 85.3%, 55.3%, 56.7%, and 64.2%, respectively. Canrenone shifted to the right the concentration-response curve for Ca2+ in depolarized aorta and did not affect the response to 10 m M caffeine. In rat right ventricular strips driven at 0.1 Hz, canrenone exerted negative inotropic effect. The relaxation of ouabain-induced contraction may be due, at least in part, to an interaction between canrenone and ouabain on the Na+-K+ ATPase. Inhibition of calcium entry through calcium channels either in aorta or ventricles is the most parsimonious hypothesis of mechanism underlying the effect of canrenone on contractile responses of rat aorta to agonists and high K+ and the negative inotropic effect on ventricular strips.