Francesca Uberti

Intracoronary Genistein Acutely Increases Coronary Blood Flow in Anesthetized Pigs through β-Adrenergic Mediated Nitric Oxide Release and Estrogenic Receptors

Various studies have suggested that the phytoestrogen genistein has beneficial cardioprotective and vascular effects. However, there has been scarce information regarding the primary effect of genistein on coronary blood flow and its mechanisms including estrogen receptors, autonomic nervous system, and nitric oxide (NO). The present study was planned to determine the primary effect of genistein on coronary blood flow and the mechanisms involved. In anesthetized pigs, changes in left anterior descending coronary artery caused by intracoronary infusion of genistein at constant heart rate and arterial pressure were assessed using ultrasound flowmeters. In 25 pigs, genistein infused at 0.075 mg/min increased coronary blood flow by about 16.3%. This response was graded in a further five pigs by increasing the infused dose of the genistein between 0.007 and 0.147 mg/min. In the 25 pigs, blockade of cholinergic receptors (iv atropine; five pigs) and alpha-adrenergic receptors (iv phentolamine; five pigs) did not abolish the coronary response to genistein, whose effects were prevented by blockade of beta(2)-adrenergic receptors (iv butoxamine; five pigs), nitric oxide synthase (intracoronary N(omega)-nitro-L-arginine methyl ester; five pigs) and estrogenic receptors (ERs; ERalpha/ERbeta; intracoronary fulvestrant; five pigs). In porcine aortic endothelial cells, genistein induced the phosphorylation of endothelial nitric oxide synthase and NO production through ERK 1/2, Akt, and p38 MAPK pathways, which was prevented by the concomitant treatment by butoxamine and fulvestrant. In conclusion, genistein primarily caused coronary vasodilation the mechanism of which involved ERalpha/ERbeta and the release of NO through vasodilatory beta(2)-adrenoreceptor effects.

Intracoronary intermedin 1-47 augments cardiac perfusion and function in anesthetized pigs: role of calcitonin receptors and β-adrenoreceptor-mediated nitric oxide release

Systemic intermedin (IMD)1-47 administration has been reported to result in vasodilation and marked hypotension through calcitonin-related receptor complexes. However, its effects on the coronary circulation and the heart have not been examined in vivo. The present study was therefore planned to determine the primary in vivo effect of IMD1-47 on coronary blood flow and cardiac function and the involvement of the autonomic nervous system and nitric oxide (NO). In 35 anesthetized pigs, IMD1-47, infused into the left anterior descending coronary artery at doses of 87.2 pmol/min, at constant heart rate and arterial blood pressure, augmented coronary blood flow and cardiac function. These responses were graded in a further five pigs by increasing the infused dose of IMD1-47 between 0.81 and 204.1 pmol/min. In the 35 pigs, the blockade of cholinergic receptors (intravenous atropine, 5 pigs), alpha-adrenoceptors (intravenous phentolamine, 5 pigs), and beta1-adrenoceptors (intravenous atenolol, 5 pigs) did not abolish the cardiac response to IMD1-47, the effects of which were prevented by blockade of beta2-adrenoceptors (intravenous butoxamine, 5 pigs), NO synthase (intracoronary N(omega)-nitro-l-arginine methyl ester, 5 pigs), and calcitonin-related receptors (intracoronary CGRP8-37/AM22-52, 10 pigs). In porcine coronary endothelial cells, IMD1-47 induced the phosphorylation of endothelial NO synthase and NO production through cAMP signaling leading to ERK, Akt, and p38 activation, which was prevented by the inhibition of beta2-adrenoceptors, calcitonin-related receptor complexes, and K+ channels. In conclusion, IMD1-47 primarily augmented coronary blood flow and cardiac function through the involvement of calcitonin-related receptor complexes and beta2-adrenoreceptor-mediated NO release. The intracellular signaling involved cAMP-dependent activation of kinases and the opening of K+ channels.

Levosimendan modulates programmed forms of cell death through K(ATP) channels and nitric oxide

Levosimendan exerts cardioprotection through mitochondrial KATP (mitoKATP) channels opening. In addition, intracoronary levosimendan was found to modulate programmed forms of cell death by nitric oxide (NO) involvement. The aim of this study was to examine the role of mitoKATP channels and NO in the effects of levosimendan on apoptosis/autophagy. In H9c2 cells treated with hydrogen peroxide apoptosis/autophagy, survival signaling, cell viability, mitochondrial membrane potential, and permeability transition pore opening were analyzed through Western blot and colorimetric and fluorescence assays. Pretreatment of H9c2 cells with levosimendan was able to counteract the oxidative injuries caused by hydrogen peroxide. The effects of levosimendan were potentiated by diazoxide and were similar to those elicited by the autophagic activator rapamycin. The autophagic inhibitor 3-methyladenine reduced the effects of levosimendan, whereas after the pan-caspases inhibitor N-Acetyl-Asp-Glu-Val-Asp-al (Z-VAD.FMK), cell survival and autophagy in response to levosimendan increased. Both the mitoKATP channels inhibition and the NO synthase blocking attenuated the cardioprotection elicited by levosimendan. The results have shown that levosimendan protects H9c2 cells against oxidative injuries through the modulation of the interplay between autophagy and apoptosis and the activation of survival signaling. The mitoKATP channels and NO may be involved in such cardioprotection through interference with mitochondrial functioning.

Intracoronary melatonin increases coronary blood flow and cardiac function through β-adrenoreceptors, MT1/MT2 receptors, and nitric oxide in anesthetized pigs.

Abstract:  Melatonin is involved in the regulation of the cardiovascular system through the modulation of sympathetic function and the nitric oxide (NO)‐related pathway and interaction with MT1/MT2 receptors. However, information regarding its direct actions on coronary blood flow and cardiac function is scarce. This study therefore determined the primary in vivo effect of melatonin on cardiac function and perfusion and the involvement of the autonomic nervous system, MT1/MT2 receptors, and NO. In 35 pigs, melatonin infused into the coronary artery at 70 pg for each mL/min of coronary blood flow while preventing changes in heart rate and arterial pressure increased coronary blood flow, dP/dtmax, segmental shortening, and cardiac output by about 12%, 14%, 8%, and 23% of control values (P < 0.05), respectively. These effects were accompanied by an increase in coronary NO release of about 46% (P < 0.05) of control values. The aforementioned responses were graded in a further five pigs. Moreover, the blockade of muscarinic cholinoreceptors (n = 5) and α‐adrenoreceptors (n = 5) did not abolish the observed responses to melatonin. After β1‐adrenoreceptors blocking (n = 5), melatonin failed to affect cardiac function, whereas β2‐adrenoreceptors (n = 5) and NO synthase inhibition (n = 5) prevented the coronary response and the effect of melatonin on NO release. Finally, all effects were prevented by MT1/MT2 receptor inhibitors (n = 10). In conclusion, melatonin primarily increased coronary blood flow and cardiac function through the involvement of MT1/MT2 receptors, β‐adrenoreceptors, and NO release. These findings add new information about the mechanisms through which melatonin physiologically modulates cardiovascular function and exerts cardioprotective effects.

Urocortin II induces nitric oxide production through cAMP and Ca2+ related pathways in endothelial cells.

Background: Urocortin II has previously been shown in anesthetized pigs to increase coronary blood flow through activation of the endothelial nitric oxide synthase (eNOS) pathway and involvement of the subtype 2 of corticotropin releasing factor receptors (CRFR2). However, little information has been available regarding the intracellular signalling through these receptors and leading to the release of nitric oxide (NO). Aim: The present study was therefore planned to determine the mechanism involved in such signalling. Methods: In porcine aortic endothelial cells (PAE) the effects of urocortin II on NO production and ERK, Akt, p38 and eNOS phosphorylation were examined in absence or presence of the adenylyl cyclase agonist forskolin and antagonist 2’5’ dideoxyadenosine, the Ca2+ ionophore A23187, the Ca2+-calmodulin-kinase inhibitor KN93, the CRFR2 blocker astressin 2B and of the protein kinases specific inhibitors UO126, wortmannin and SB203580. Results: Urocortin II caused a significant increase of NO production, which was amplified by forskolin and A23187 (P <0.05). All effects of urocortin II were abolished by l-NAME, 2’5’ dideoxyadenosine, KN93, astressin 2B and by pre-treatment of cells with UO126, wortmannin and SB203580. Western Blot analysis confirmed the involvement of ERK, Akt and p38 in the eNOS activation. Conclusion: In PAE urocortin II interaction with CRFR2 caused a cAMP-dependent and Ca2+-related phoshorylation of ERK, Akt and p38 leading to eNOS activation.

Biochemical and immunochemical characterization of different varieties of amaranth (Amaranthus L. ssp.) as a safe ingredient for gluten-free products.

Celiac disease is a food intolerance triggered by the ingestion of gluten-containing cereals; the only therapy is a strict gluten-free diet for life. In recent years, amaranth flour has received considerable attention as an interesting source for the formulation of gluten-free products due to its high nutritional value and low content of prolamins, the toxic proteins for celiacs. The aim of this study was to characterize 40 amaranth varieties using both SDS-PAGE/immunoblotting and ELISA to assess their possible tolerance by celiac subjects. All of the amaranth samples studied showed similar binding affinities for both specific anti-gliadin antibodies and human IgAs. In most amaranth grains, the content of gluten-like proteins measured by ELISA was <20 ppm. The molecular characterization of amaranth proteins suggests that amaranth is safe for celiacs to consume. It is recommended that the most suitable amaranth varieties are those having the lowest content of proteins cross-reacting with anti-gliadin antibodies.

Diversity of oat varieties in eliciting the early inflammatory events in celiac disease

AbstractPurpose Celiac disease (CD) is an autoimmune enteropathy, triggered by dietary gluten. The only treatment is a strict gluten-free diet. Oats are included in the list of gluten-free ingredients by European Regulation, but the safety of oats in CD is still a matter of debate. The present study examined the capability of different oat cultivars of activating the gliadin-induced transglutaminase-2 (TG2)-dependent events in some in vitro models of CD. In addition, we compared this capability with the electrophoresis pattern of peptic–tryptic digests of the proteins of the oat cultivars.MethodsK562(S) cells agglutination, transepithelial electrical resistance of T84-cell monolayers, intracellular levels of TG2 and phosphorylated form of protein 42–44 in T84 cells were the early gliadin-dependent events studied.ResultsThe results showed that the Nave oat cultivar elicited these events, whereas Irina and Potenza varieties did not. The ability of a cultivar to activate the above-described events was associated with the electrophoretic pattern of oat proteins and their reactivity to anti-gliadin antibodies.ConclusionWe found significant differences among oat cultivars in eliciting the TG2-mediated events of CD inflammation. Therefore, the safety of an oat cultivar in CD might be screened in vitro by means of biochemical and biological assays, before starting a clinical trial to definitely assess its safety.

Extracorporeal life support in a severe Taxus baccata poisoning

Introduction. Yew (Taxus baccata) is a conifer known to be toxic since ancient times. Taxine A and taxine B, the toxic alkaloids of Taxus, block cardiac sodium and calcium channels causing nausea, vomiting, abdominal pain, cardiac arrhythmias, respiratory distress, coma, seizures, and death in yew poisoning. Case report. A 44-year-old male farmer was admitted to the hospital because of a suspected myocardial infarction. First bradycardia and then ventricular tachycardia were present and a severe right ventricular dilatation with biventricular dysfunction was observed but with normal coronary arteriography. He was resistant to conventional therapy and, 6 h after hospital admission, extracorporeal support with membrane oxygenation was applied. The patient recovered. Nine days later, a large number of yew leaves were unexpectedly observed in his feces. Botanical and laboratory analysis confirmed the poisoning. Blood (651 ng/mL) and urinary (5.6 mcg/mL) levels of 3,5-dimethoxyphenol (metabolite of taxicatine) were greater than previously reported in lethal cases. The patient was transferred to a psychiatric unit 17 days after admission. Conclusions. Intensive treatment of severe cardiovascular symptoms with antiarrhythmic drugs, temporary pacemaker, intra-aortic balloon pump, extracorporeal membrane oxygenation, and extracorporeal life support can be life-saving even after a potentially lethal ingestion of T. baccata leaves.

Could 1,3 dimethylamylamine (DMAA) in food supplements have a natural origin?

1,3 dimethylamylamine or methylexaneamine (DMAA) is a synthetic pharmaceutical patented in the 1940s as a nasal decongestant which can be used as a recreational stimulant. Alleged to occur in nature, DMAA has become a widely used ingredient in sports food supplements, despite its status as a doping agent and concerns over its safety. There is now some doubt as to whether it can be sourced naturally or whether it actually occurs naturally at all. The presence of DMAA was investigated by high performance liquid chromatography (HPLC) in extracts of the leaves and stems of four geranium species and of three well-known cultivars. The amounts of DMAA in commercial geranium (Pelargonium graveolens) oil and the leading sports supplement which uses the ingredient were also measured. DMAA was not found in any of the leaves or stems or in the commercial geranium oil included in this study. Approximately 30 mg per daily dose was found in the food supplement. Therefore, the amount of DMAA found in the supplement is most unlikely to have been sourced in nature, and it must be concluded that synthetic DMAA, known to be capable of causing severe adverse physiological effects, has been added.

Biochemical and immunochemical evidences supporting the inclusion of quinoa (Chenopodium quinoa Willd.) as a gluten-free ingredient.

To date, the only acceptable therapeutic approach for celiac disease (CD) is a strict elimination from the diet of gluten-containing foods, but this diet does not always guarantee an adequate nutritional intake. Pseudocereals are receiving considerable attention as interesting alternatives for the formulation of gluten-free products, and quinoa grains arise as nutritive substitutes of conventional cereals. The aim of this study was the characterization of different quinoa samples corresponding to 11 quinoa varieties, using polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) and immunoblotting techniques to assess their suitability for celiac subjects. Some of these varieties were grown in Italy to assess if the reproduction in a new habitat can guarantee the retention of the “safe” protein pattern. None of the quinoa varieties studied presented protein bands with electrophoretic mobility comparable with those of wheat gliadins, the toxic protein for celiac subjects. All the quinoa samples showed a low binding affinity for both specific anti-gliadin antibodies and IgAs from celiac subjects, confirming that quinoa can be considered as a safe ingredient for celiac patients. However, reliable varieties should be previously selected since the immuno cross-reactivity with anti-gliadin antibodies can vary significantly.