Maria Cristina Breschi

Vasorelaxing effects of flavonoids: investigation on the possible involvement of potassium channels

A flavonoid-rich diet has been associated with a lower incidence of cardiovascular diseases, probably because of the antioxidant and vasoactive properties of flavonoids. Indeed, many flavonoids show vasorelaxing properties, due to different and often not yet completely clarified mechanisms of action. Among them, the activation of vascular potassium channels has been indicated as a possible pathway, accounting, at least in part, for the vasodilatory action of some flavonoid derivatives, such as apigenin and dioclein. Therefore, this work aims at evaluating, on in vitro isolated rat aortic rings, the endothelium-independent vasorelaxing effects of a number of flavonoid derivatives, to identify a possible activation of calcium-activated and/or ATP-sensitive potassium channels and to indicate some possible structure–activity relationships. Among the several flavonoids submitted to the pharmacological assay, only baicalein and quercetagetin were almost completely ineffective, while quercetin, hesperidin, quercitrin and rhoifolin exhibited only a partial vasorelaxing effect. On the contrary, acacetin, apigenin, chrysin, hesperetin, luteolin, pinocembrin, 4′-hydroxyflavanone, 5-hydroxyflavone, 5-methoxyflavone, 6-hydroxyflavanone and 7-hydroxyflavone, belonging to the chemical classes of flavones and flavanones, showed full vasorelaxing effects. The vasodilatory activity of hesperetin, luteolin, 5-hydroxyflavone and 7-hydroxyflavone were antagonised by tetraethylammonium chloride, indicating the possible involvement of calcium-activated potassium channels. Moreover, iberiotoxin clearly antagonised the effects of 5-hydroxyflavone, indicating the probable importance of a structural requirement (the hydroxy group in position 5) for a possible interaction with large-conductance, calcium-activated potassium channels. Finally, glibenclamide inhibited the vasorelaxing action of luteolin and 5-hydroxyflavone, suggesting that ATP-sensitive potassium channels may also be involved in their mechanism of action.

Hydrogen sulphide: novel opportunity for drug discovery

Hydrogen sulphide (H2S) is emerging as an important endogenous modulator, which exhibits the beneficial effects of nitric oxide (NO) on the cardiovascular (CV) system, without producing toxic metabolites. H2S is biosynthesized in mammalian tissues by cystathionine‐β‐synthase and cystathionine‐γ‐lyase. H2S exhibits the antioxidant properties of inorganic and organic sulphites, behaving as a scavenger of reactive oxygen species. There is also clear evidence that H2S triggers other important effects, mainly mediated by the activation of ATP‐sensitive potassium channels (KATP). This mechanism accounts for the vasorelaxing and cardioprotective effects of H2S. Furthermore, H2S inhibits smooth muscle proliferation and platelet aggregation. In non‐CV systems, H2S regulates the functions of the central nervous system, as well as respiratory, gastroenteric, and endocrine systems. Conversely, H2S deficiency contributes to the pathogenesis of hypertension. Likewise, impairment of H2S biosynthesis is involved in CV complications associated with diabetes mellitus. There is also evidence of a cross‐talk between the H2S and the endothelial NO pathways. In particular, recent observations indicate a possible pathogenic link between deficiencies of H2S activity and the progress of endothelial dysfunction. These biological aspects of endogenous H2S have led several authors to look at this mediator as “the new NO” that has given attractive opportunities to develop innovative classes of drugs. In this review, the main biological actions of H2S are discussed. Moreover, some examples of H2S‐donors are shown, as well as some hybrids, in which H2S‐releasing moieties are added to well‐known drugs, for improving their pharmacodynamic profile or reducing the potential for adverse effects, are reported.

The role of nitric oxide in anthracycline toxicity and prospects for pharmacologic prevention of cardiac damage

Anthracycline antibiotics are potent antitumor agents whose activity is severely limited by a cumulative dose‐dependent chronic cardiotoxicity that results from the summation of multiple biochemical pathways of cellular damage, which ultimately yields to disruption of myocardiocyte integrity and loss of cardiac function. Nitric oxide (NO) is a key molecule involved in the pathophysiology of heart; dysregulation of activity of NO synthases (NOSs) and of NO metabolism seems to be a common feature in various cardiac diseases. The contribution of NO to anthracycline cardiac damage is suggested by evidence demonstrating anthracycline‐mediated induction of NOS expression and NO release in heart and the ability of NOSs to promote anthracycline redox cycling to produce reactive oxygen species (ROS), including O2− and H2O2. Overproduction of ROS and NO yields to reactive nitrogen species, particularly the powerful oxidant molecule peroxynitrite (ONOO−), which may produce the marked reduction of cardiac contractility. This review focuses on the anthracycline‐mediated deregulation of NO network and presents an unifying viewpoint of the main molecular mechanisms involved in the pathogenesis of anthracycline cardiotoxicity, including iron, free radicals, and novel mechanistic notions on cardiac ceramide signaling and apoptosis. The data presented in the literature encourage the development of strategies of pharmacological manipulation of NO metabolism to be used as a novel approach to the prevention of cardiotoxicity induced by anthracyclines.—Fogli, S., Nieri, P., Breschi, M. C. The role of nitric oxide in anthracycline toxicity and prospects for pharmacologic prevention of cardiac damage. FASEB J. 18, 664–675 (2004)

Altered prejunctional modulation of intestinal cholinergic and noradrenergic pathways by α2-adrenoceptors in the presence of experimental colitis

This study investigates the influence of intestinal inflammation on: (1) the control of intestinal neurotransmission and motility by prejunctional α2‐adrenoceptors and (2) the expression of intestinal α2‐adrenoceptors. Experimental colitis was induced by intrarectal administration of 2,4‐dinitrobenzenesulphonic acid (DNBS) to rats. UK‐14,304 inhibited atropine‐sensitive electrically evoked contractions of ileal and colonic longitudinal muscle preparations. UK‐14,304 acted with similar potency, but higher efficacy, on tissues from DNBS‐treated animals; its effects were antagonized with greater potency by phentolamine than rauwolscine. Electrically induced [3H]noradrenaline release from ileal preparations was reduced in the presence of colitis. Tritium outflow was decreased by UK‐14,304 and stimulated by rauwolscine or phentolamine: these effects were enhanced in preparations from animals with colitis. Reverse transcription–polymerase chain reaction and Western blot assay demonstrated the protein expression of α2A‐adrenoceptors in mucosal and muscular tissues isolated from ileum and colon. The induction of colitis increased α2A‐adrenoceptor expression in both ileal and colonic muscular layers, without concomitant changes in mucosal tissues. Induction of colitis reduced gastrointestinal propulsion of a charcoal suspension in vivo. In this setting, the gastrointestinal transit was inhibited by intraperitoneal (i.p.) UK‐14,304 and stimulated by i.p. rauwolscine. After pretreatment with guanethidine, the stimulant action of rauwolscine no longer occurred, and UK‐14,304 exerted a more prominent inhibitory effect that was antagonized by rauwolscine. The present results indicate that, in the presence of intestinal inflammation, prejunctional α2‐adrenoceptors contribute to an enhanced inhibitory control of cholinergic and noradrenergic transmission both at inflamed and noninflamed distant sites. Evidence was obtained that such modulatory actions depend on an increased expression of α2A‐adrenoceptors within the enteric nervous system.

Gastrin promotes human colon cancer cell growth via CCK‐2 receptor‐mediated cyclooxygenase‐2 induction and prostaglandin E2 production

1 The present study investigates the effects of gastrin‐17 on human colon cancer HT‐29 cells to examine whether gastrin receptor (CCK‐2), cyclooxygenase (COX‐1, COX‐2) isoforms and prostaglandin receptor pathways interact to control cell growth. 2 Reverse transcription (RT)–polymerase chain reaction (PCR) analysis demonstrated that HT‐29 cells are endowed with the naïve expression of CCK‐2 receptor (short splice variant), COX‐1, COX‐2 and prostaglandin EP4 receptor, but not gastrin. 3 Gastrin‐17 significantly promoted cell growth and DNA synthesis. Both these stimulating effects were abolished by L‐365,260 or GV150013 (CCK‐2 receptor antagonists), but were unaffected by SC‐560 (COX‐1 inhibitor). L‐745,337 (COX‐2 inhibitor) or AH‐23848B (EP4 receptor antagonist) partly reversed gastrin‐17‐induced cell growth, while they fully antagonized the enhancing action on DNA synthesis. 4 HT‐29 cells responded to gastrin‐17 with a significant increase in prostaglandin E2 release. This enhancing effect was completely counteracted by L‐365,260, GV150013 or L‐745,337, while it was insensitive to cell incubation with SC‐560. 5 Exposure of HT‐29 cells to gastrin‐17 was followed by an increased phosphorylation of both extracellular regulated kinases (ERK‐1/ERK‐2) and Akt. Moreover, gastrin‐17 enhanced the transcriptional activity of COX‐2 gene promoter and stimulated COX‐2 expression. These latter effects were antagonized by L‐365,260 or GV150013, and could be blocked also by PD98059 (inhibitor of ERK‐1/ERK‐2 phosphorylation) or wortmannin (inhibitor of phosphatidylinositol 3‐kinase). Analogously, gastrin‐17‐induced prostaglandin E2 release was prevented by PD98059 or wortmannin. 6 The present results suggest that (a) in human colon cancer cells endowed with CCK‐2 receptors, gastrin‐17 is able to enhance the transcriptional activity of COX‐2 gene through the activation of ERK‐1/ERK‐2‐ and phosphatidylinositol 3‐kinase/Akt‐dependent pathways; (b) these stimulant actions lead to downstream increments of COX‐2 expression, followed by prostaglandin E2 production and EP4 receptor activation; (c) the recruitment of COX‐2/prostaglandin pathways contributes to the growth‐promoting actions exerted by gastrin‐17.

Arylthioamides as H2S Donors: l-Cysteine-Activated Releasing Properties and Vascular Effects in Vitro and in Vivo

A small library of arylthioamides 1-12 was easily synthesized, and their H2S-releasing properties were evaluated both in the absence or in the presence of an organic thiol such as l-cysteine. A number of arylthioamides (1-3 and 7) showed a slow and l-cysteine-dependent H2S-releasing mechanism, similar to that exhibited by the reference slow H2S-releasing agents, such as diallyl disulfide (DADS) and the phosphinodithioate derivative GYY 4137. Compound 1 strongly abolished the noradrenaline-induced vasoconstriction in isolated rat aortic rings and hyperpolarized the membranes of human vascular smooth muscle cells in a concentration-dependent fashion. Finally, a significant reduction of the systolic blood pressure of anesthetized normotensive rats was observed after its oral administration. Altogether these results highlighted the potential of arylthioamides 1-3 and 7 as H2S-donors for basic studies, and for the rational design/development of promising pharmacotherapeutic agents to treat cardiovascular diseases.

Cardioprotective effects of different flavonoids against myocardial ischaemia/reperfusion injury in Langendorff-perfused rat hearts

Flavonoids are important components of ‘functional foods’, with beneficial effects on the cardiovascular function, mainly due to their antioxidant activity. Many flavonoids exert antihypertensive, anti‐atherosclerotic and antiplatelet activity and positive effects against endothelial dysfunction. Recent evidence indicates that they exert cardioprotective effects against myocardial ischaemia/reperfusion (I/R) injury. The aim of this work was to investigate these properties for flavonoids with different structural characteristics.

Cannabinoid derivatives induce cell death in pancreatic MIA PaCa-2 cells via a receptor-independent mechanism

Cannabinoids (CBs) are implicated in the control of cell survival in different types of tumors, but little is known about the role of CB system in pancreatic cancer. Herein, we investigated the in vitro antitumor activity of CBs and the potential role of their receptors in human pancreatic cancer cells MIA PaCa‐2. Characterization tools used for this study included growth inhibition/cell viability analyses, caspase 3/7 induction, DNA fragmentation, microarray analysis and combination index‐isobologram method. Our results demonstrate that CBs produce a significant cytotoxic effect via a receptor‐independent mechanism. The CB1 antagonist N‐(piperidin‐1‐1yl)‐5‐(4‐iodophenyl)‐1‐(2,4‐dichlorophenyl)‐4‐methyl‐1H‐pyrazole‐3‐carboxamide (AM251) was the most active compound with an IC50 of 8.6 ± 1.3 μM after 72 h. AM251 induces apoptosis, causes transcriptional changes of genes in janus kinase/signal transducers and activators of transcription signaling network and synergistically interacts with the pyrimidine analogue, 5‐fluorouracil. These findings exclude the involvement of CB receptors in the regulation of MIA PaCa‐2 cell growth and put AM251 forward as a candidate for the development of novel compounds worthy to be tested in this type of neoplasia.

Pharmacological characterization of the vascular effects of aryl isothiocyanates: Is hydrogen sulfide the real player?

Hydrogen sulfide (H₂S) is an endogenous gasotransmitter, which mediates important physiological effects in the cardiovascular system. Accordingly, an impaired production of endogenous H₂S contributes to the pathogenesis of important cardiovascular disorders, such as hypertension. Therefore, exogenous compounds, acting as H₂S-releasing agents, are viewed as promising pharmacotherapeutic agents for cardiovascular diseases. Thus, this paper aimed at evaluating the H₂S-releasing properties of some aryl isothiocyanate derivatives and their vascular effects. The release of H₂S was determined by amperometry, spectrophotometry and gas/mass chromatography. Moreover, the vascular activity of selected isothiocyanates were tested in rat conductance (aorta) and coronary arteries. Since H₂S has been recently reported to act as an activator of vascular Kv7 potassium channels, the possible membrane hyperpolarizing effects of isothiocyanates were tested on human vascular smooth muscle (VSM) cells by spectrofluorescent dyes. Among the tested compounds, phenyl isothiocyanate (PhNCS) and 4-carboxyphenyl isothiocyanate (PhNCS-COOH) exhibited slow-H₂S-release, triggered by organic thiols such as L-cysteine. These compounds were endowed with vasorelaxing effects on conductance and coronary arteries. Moreover, these two isothiocyanates caused membrane hyperpolarization of VSM cells. The vascular effects of isothiocyanates were strongly abolished by the selective Kv7-blocker XE991. In conclusion, the isothiocyanate function can be viewed as a suitable slow H₂S-releasing moiety, endowed with vasorelaxing and hypotensive effects, typical of this gasotransmitter. Thus, such a chemical moiety can be employed for the development of novel chemical tools for basic studies and promising cardiovascular drugs.

New chitosan derivatives for the preparation of rokitamycin loaded microspheres designed for ocular or nasal administration.

Acanthamoeba spp. are the causative agents of granulomatous amoebic encephalitis (GAE) and amoebic keratitis. Recent studies performed by Rassu et al. showed that, compared with the free drug, the loading of rokitamycin in chitosan microspheres improves and prolongs the in vitro antiamoebic activity of rokitamycin. This could be useful in transporting the drug for either ocular application to treat amoebic keratitis or nasal administration as an alternative route for the administration of the drug to the brain in GAE therapy. Starting from the previous study, our goal was to optimize the technological parameters in order to obtain chitosan microparticles loaded with rokitamycin and to evaluate the use of new quaternary ammonium chitosan derivatives in the preparation of spray dried microspheres containing the macrolide; these derivatives showed better characteristics (solubility, penetration enhancement) compared with chitosan itself. Toxicity studies on new polymers were performed. Spray dried loaded microspheres based on chitosan or chitosan derivatives were obtained by using appropriate preparative parameters. Microparticles containing chitosan derivatives showed similar or often better properties than formulations made of chitosan with respect to size, in vitro release behaviour and mucoadhesiveness thus making them more suitable for ocular or nasal administration. New polymers did not demonstrate cytotoxicity.