Rosa Maria Gaion

Synthesis and cardiotonic activity of esters of 2-substituted 5-cyano-1,6-dihydro-6-oxo-3-pyridinecar☐ylic acids. Crystal structure of 2-methyl, 2-t-butyl and 2-phenyl esters

Abstract The synthesis of ethyl and methyl esters of 2-substituted 5-cyano-1,6-dihydro-6-oxo-3-pyridinecar☐ylic acids by reaction of ethyl or methyl 2-dimethylaminomethylene-3-oxoalkanoates with sodium cyanoacetamide is described. These esters gave by alkaline hydrolysis the corresponding car☐ylic acids, which were decar☐ylated to 6-substituted 1,2-dihydro-2-oxo-3-pyridinecarbonitriles. As milrinone analogues, nearly all the above compounds were tested on contractile activity and frequency rate of spontaneously beating atria and electrically driven left atria from guinea pigs. Among the esters, ethyl 5-cyano-1,6-dihydro-2-methyl-6-oxo-3-pyridinecar☐ylate induced positive inotropic and chronotropic effects superior to those caused by milrinone. By increasing or branching the 2-substitutent, the activity decreased until faded or even reversed. Car☐ylic acids and nitriles were less active than milrinone. Some aspects of the structure-activity relationship of these compounds are discussed on the basis of X-ray structural analyses of 2-methyl, 2- t -butyl and 2-phenyl esters.

Low- and medium-dose diltiazem in chronic atrial fibrillation: Comparison with digoxin and correlation with drug plasma levels

The safety and efficacy of diltiazem were compared with digoxin maintenance therapy for control of ventricular response in 19 patients with chronic atrial fibrillation. The relationship between drug plasma levels and cardiovascular effects was also investigated. After 7 days of combined therapy with diltiazem (60 mg three times a day in 10 patients and four times a day in nine patients) and digoxin (0.125 mg/day in two patients and 0.250 mg/day in 17 patients), the 24-hour mean heart rate derived from ambulatory ECG recording was reduced by 16.3% in comparison with digoxin therapy alone; the serum digoxin level was not significantly changed (1.06 +/- 0.43 vs 1.05 +/- 0.61 ng/ml). After a standardized bicycle exercise test (50 watts for 3 minutes), maximal heart rate was reduced by 19.9%, diastolic blood pressure was decreased by 8.9%, and systolic pressure-rate product was decreased by 12.5%. Diltiazem plasma levels (mean 120.9 +/- 63.3 ng/ml) were linearly correlated with percentage variations in maximal heart rate, diastolic blood pressure, systolic blood pressure, and pressure-rate product during exercise. Eighteen patients in succession discontinued digoxin therapy; after 14 days of diltiazem alone, the 24-hour mean heart rate returned to control values of digoxin therapy, whereas maximal heart rate and pressure-rate product during exercise were significantly reduced (-17.2% and -14.1%, respectively), with no changes in blood pressure. Diltiazem plasma levels (135.0 +/- 83.2 ng/ml) showed a linear correlation with the percentage of reduction in maximal heart rate.(ABSTRACT TRUNCATED AT 250 WORDS)

Critical role of COX-1 in prostacyclin production by human endothelial cells under modification of hydroperoxide tone

We aimed at evaluating the relative contribution of cyclooxygenase (COX) ‐1 and COX‐2 to the synthesis of prostacyclin in endothelial cells under static conditions in the presence or absence of exogenous arachidonic acid and/or altered intracellular redox balance. Selective inhibitors of either COX‐1 (SC560 and FR122047) or COX‐2 (SC236) concentration dependently (1–300 nM) reduced basal and interleukin (IL) ‐1β‐induced prostacyclin production in human umbilical vein endothelial cells by 70% or more; compound selectivity was confirmed using a whole‐blood assay (IC50 COX‐1/COX‐2: 13 nM/930 nM for SC‐560; 9 μM/457 nM for SC‐236). The observed concomitant formation of isoprostane appeared to be associated with COX enzyme activity, while formation of COX‐1/COX‐2 heterodimers was detected by immunoprecipitation. In the presence of arachidonic acid and 12‐hydroperoxy‐eicosatetraenoic acid, either exogenous or provided by platelet activation, or after glutathione depletion, COX‐1 inhibition but not COX‐2 inhibition concentration dependently decreased prostacyclin production. Both isoforms appear to contribute to basal prostacyclin production by endothelial cells, with COX‐2 providing the hydroperoxide tone required for COX‐1 activity. Conversely, in the case of intracellular glutathione depletion or enhanced availability of arachidonic acid and hydroperoxides, selective COX‐2 inhibition did not significantly affect the production of endothelial prostacyclin. These findings contribute to a better understanding of the effects of cyclooxygenase inhibitors on prostacyclin production.—Bolego, C, Buccellati, C, Prada, A., Gaion, R. M., Folco, G., Sala, A. Critical role of COX‐1 in prostacyclin production by human endothelial cells under modification of hydroperoxide tone. FASEB J. 23, 605–612 (2009)

Dual effect of (-)-N6-phenylisopropyl adenosine on guinea-pig trachea.

1 The effect of (−)‐N6‐phenylisopropyl adenosine (PIA), a metabolically stable P1‐receptor agonist, was investigated on guinea‐pig isolated trachea. 2 PIA showed two opposite effects: contraction, evident at low concentrations (10−7 to 2–5 × 10−6m), and relaxation at higher doses. 3 Relaxation by PIA was antagonized in an apparently competitive manner by two antagonists of extracellular (P1) adenosine receptors: theophylline (Theo) and 8‐phenyltheophylline (PT). 4 Contraction by PIA was not inhibited by methylxanthines and was not mediated by stimulation of cholinergic or histaminergic systems. 5 Inhibitors of arachidonic acid cascade acting at different levels, i.e. indomethacin, nordihyd‐roguaiaretic acid (NDGA) and BW755C, all inhibited the contraction by PIA, while they potentiated the relaxation in a concentration‐dependent manner. Mepacrine, an inhibitor of phospholipase A2, inhibited the contraction by PIA, but did not affect the relaxation. 6 These results indicate that the contractile effect induced by PIA is supported by an indirect mechanism involving the release of arachidonic acid derivatives (via P2‐purinoceptor?). Thus the balance between the two opposite effects of adenosine on tracheal tone is possibly modulated by the prostaglandin turnover.

Vasoprotective activity of standardized Achillea millefolium extract.

We investigated the effects of Achillea millefolium extract in vitro on the growth of primary rat vascular smooth muscle cells (VSMCs) as well as the potential involvement of estrogen receptors (ERs) in this process. In addition, the ability of A. millefolium extract to modulate the NF-κB pathway was tested in human umbilical vein endothelial cells (HUVECs). The fingerprinting of the extract was carried out by HPLC-DAD and LC-MS(n) and main constituents were flavonoids (10%) and dicaffeolylquinic acid derivatives (12%). The extract enhanced VSMC growth at least in part by acting through ERs and impaired NF-κB signaling in HUVECs. The various compounds may act with different mode of actions thus contributing to the final effect of the extract. Our findings support some of the traditional uses of A. millefolium, and suggest potential modes of action as related to its effects on vascular inflammation. Therefore, A. millefolium may induce novel potential actions in the cardiovascular system.

Control of enteric neuromuscular functions by purinergic A3 receptors in normal rat distal colon and experimental bowel inflammation

BACKGROUND AND PURPOSE Adenosine A3 receptors mediate beneficial effects in experimental colitis, but their involvement in enteric neuromuscular functions during bowel inflammation is undetermined. This study investigated the regulatory role of A3 receptors on colonic motility in the presence of experimental colitis.

Cyclooxygenase-1 and Prostacyclin Production by Endothelial Cells in the Presence of Mild Oxidative Stress

This study aimed at evaluating the relative contribution of endothelial cyclooxygenase-1 and -2 (COX-1 and COX-2) to prostacyclin (PGI2) production in the presence of mild oxidative stress resulting from autooxidation of polyphenols such as (-)-epigallocatechin 3-gallate (EGCG), using both endothelial cells in culture and isolated blood vessels. EGCG treatment resulted in an increase in hydrogen peroxide formation in human umbilical vein endothelial cells. In the presence of exogenous arachidonic acid and EGCG, PGI2 production was preferentially inhibited by a selective COX-1 inhibitor. This effect of selective inhibition was also substantially reversed by catalase. In addition, EGCG caused vasorelaxation of rat aortic ring only partially abolished by a nitric oxide synthase inhibitor. Concomitant treatment with a selective COX-1 inhibitor completely prevented the vasorelaxation as well as the increase in PGI2 accumulation in the perfusate observed in EGCG-treated aortic rings, while a selective COX-2 inhibitor was completely uneffective. Our data strongly support the notions that H2O2 generation affects endothelial PGI2 production, making COX-1, and not COX-2, the main source of endothelial PGI2 under altered oxidative tone conditions. These results might be relevant to the reappraisal of the impact of COX inhibitors on vascular PGI2 production in patients undergoing significant oxidative stress.

Selective estrogen receptor-α agonist provides widespread heart and vascular protection with enhanced endothelial progenitor cell mobilization in the absence of uterotrophic action

The beneficial effects of estrogens on the cardiovascular system are associated with adverse effects on reproductive tissues. On the basis of previous work indicating a major role for estrogen receptor (ER)‐α in maintaining cardiovascular health, we evaluated the tissue selectivity of the ERα‐selective agonist propyl pyrazole triol (PPT) compared with 17ß‐estradiol (E2) invivo. Four weeks postovariectomy, equimolar doses of PPT and E2 were administered to rats in subcutaneous implants for 5 d. Both treatments restored rapid vasorelaxation of aortic tissue to estrogenic agents and prevented coronary hyperresponsiveness to angiotensin II in isolated heart preparations. Accordingly, multiple endpoints of myocardial ischemia‐reperfusion injury exacerbated by ovariectomy returned to baseline following treatment. These protective effects were linked to increased in vivo levels of endothelial progenitor cells (EPCs). Human EPC function was enhanced in vitro after PPT treatment. In sharp contrast to E2, PPT treatment had no effect on uterine weight and histomorphology except for vessel density, and failed to up‐regulate classic estrogen target genes. Dissection of the effects on vascular reactivity and uterine morphology was also observed following increased exposure to PPT at a higher dose for longer time. These data provide the first in vivo evidence for tissue‐specific ERa activation. By conferring cardiovascular protection dissected from unwanted uterotrophic effects, ERa‐selective agonists may represent a potential safer alternative to natural hormones.—Bolego, C, Rossoni, G., Fadini, G. P., Vegeto, E., Pinna, C., Albiero, M., Boscaro, E., Agostini, C., Avogaro, A., Gaion, R M., Cignarella, A Selective estrogen receptor‐a agonist provides widespread heart and vascular protection with enhanced endothelial progenitor cell mobilization in the absence of uterotrophic action. FASEBJ. 24, 2262–2272 (2010). www.fasebj.org

Distinct roles of estrogen receptor-alpha and beta in the modulation of vascular inducible nitric-oxide synthase in diabetes.

Estrogen is known to affect vascular function and diabetes development, but the relative contribution of estrogen receptor (ER) isoforms is unclear. The aim of this study was to determine how individual ER isoforms modulate inflammatory enzymes in the vascular wall of control and streptozotocin (STZ)-injected rodents. Primary cultures of rat aortic smooth muscle cells (SMCs) were stimulated with inflammatory agents in the presence or absence of increasing concentrations of the ERα and ERβ-selective agonists 4,4′,4′′-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT) and diarylpropionitrile (DPN), respectively. The production of inducible nitric-oxide synthase (iNOS), a classical indicator of vascular inflammation, was significantly reduced by PPT in control but not diabetic SMCs, whereas it was further enhanced by DPN treatment in both groups. This distinct action profile was not related to changes in ER transcriptional activity. However, extracellular signal-regulated kinase 1/2 signaling was activated by DPN but not by PPT in cytokine-treated SMCs. In cultured aortic rings from both normoglycemic and STZ-diabetic mice, pharmacological activation of ERα attenuated cytokine-driven iNOS induction by 30 to 50%. Vascular iNOS levels were decreased consistently when adding 1 nM 17β-estradiol to aortic tissues from ERβ- but not ERα-knockout mice. These findings suggest a possible role for ERα-selective ligands in reducing vascular inflammatory responses under normo- and hyperglycemic conditions.

Herpes Simplex Virus Type 1 Infection of the Rat Enteric Nervous System Evokes Small-Bowel Neuromuscular Abnormalities

BACKGROUND & AIMS Infectious agents, such as neurotropic viruses, are proposed to disrupt the enteric neuromuscular system, leading to dysmotility, although the mechanisms are unknown. Our purpose was to assess whether herpes simplex virus type-1 (HSV-1) establishes an enteric-neuronal infection and induces gut dysmotility. METHODS Rats were inoculated with HSV-1 intranasally and after 4 weeks intragastrically. After 1-10 weeks, infection was determined by molecular analysis whereas neuromuscular function was evaluated by pharmacologic/electrical stimulation of longitudinal ileal segments and by gastrointestinal transit and by [(3)H]acetylcholine release measurements. Inflammation in the neuromuscular layer was assessed by myeloperoxidase and cytokine levels and by anti-CD3(+) immunohistochemistry. RESULTS After 1-10 weeks of intragastric inoculation, HSV-1 latency-associated messenger RNA transcripts were detected in the brain and in ileal neurons with no signs of illness or histologic gut abnormalities. By using a recombinant HSV-1 carrying the lacZ gene, HSV-1 virions were localized in myenteric ganglia by in situ X-gal staining. Interleukin-2 and IFN-gamma levels were increased significantly 1 and 6 weeks after inoculation. CD3(+) cells were found around the myenteric ganglia 6 weeks after inoculation. Smooth muscle responses to carbachol, CaCl(2), and gut transit were increased significantly after 1 and 6 weeks, whereas KCl- and electrical field stimulation-mediated contractions were modified significantly only 1-2 weeks after HSV-1 administration. The release of [(3)H]acetylcholine was reduced significantly in ileum segments after 1 and 6 weeks. CONCLUSIONS After intragastric inoculation, HSV-1 establishes a latent infection in the rat myenteric ganglia, which leads to gut dysmotility.