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This observational retrospective study analysed the association of adherence to statins with the achievement of a target total cholesterol level (CL, <200mg/dl), and any association of adherence with the time to first hospital admission for coronary event in hypercholesterolemic patients treated with statins, in one Italian Local Health Authority between 1998 and 2003. The study population consisted of 3516 patients who were prescribed statins and for whom full cholesterol results were available. After three months of treatment, there were significant reductions in CL (p<0.001) in the three treatment groups stratified by adherence (good adherents -24%, poor adherents -22%, and nonadherents -14%). Patients more likely to achieve the target CL were older, male and more adherent to the statins. The risk of first hospitalization was associated positively with increased age and male gender. Patients with co-treatments were more likely to be hospitalized. Surprisingly, better adherence to statin treatment increased the risk of hospitalization.

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We examined the interaction between the selective serotonin reuptake inhibitor, fluoxetine, and group-II metabotropic glutamate (mGlu) receptors using progenitor cells isolated from cultured cerebellar granule cells, considered as an in vitro model of antidepressant-drug induced neurogenesis. These cells expressed mGlu3 receptors negatively coupled to adenylyl cyclase. A 72-h treatment with either fluoxetine or low concentrations of mGlu2/3 receptor agonists (LY379268 or 2R,4R-APDC) enhanced cell proliferation. The action of fluoxetine was mediated by the activation of 5-HT(1A) receptors. We found a strong synergism between fluoxetine and LY379268 in enhancing cell proliferation and inhibiting cAMP formation. The increased cell proliferation induced by fluoxetine+LY379268 was abrogated by the cAMP analogue, 8-Br-cAMP, as well as by drugs that inhibit the mitogen-activated protein kinase and phosphatidyilinositol-3-kinase pathways. Interestingly, fluoxetine and LY379268 also acted synergistically in promoting neuronal differentiation when progenitor cells were incubated in the presence of serum. These data support the hypothesis that a combination between classical antidepressants and mGlu2/3 receptor agonists may be helpful in the experimental treatment of depression.

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Both preclinical and clinical evidence suggested that antidepressant drugs upregulate hippocampal cell proliferation and neurogenesis. In addition, direct evidence was recently published that hippocampal de novo cell proliferation is necessary for antidepressant action. Within this frame, we used primary cultures of rat cerebellar granule cells (CGC) as an in vitro model of central nervous system (CNS) to investigate whether a neurogenic response could be elicited also in the cerebellum, upon chronic treatment with selective serotonin reuptake inhibitors (SSRIs). Furthermore, we assayed the presence of neural precursor cells in CGC, possibly responsive to proliferation and differentiation stimuli. We found that 1 microM fluoxetine increased cell proliferation, as assayed by [3H]-thymidine incorporation. CGC immunocytochemical analysis with neural cell-specific markers revealed the presence of granule neurons, glial cells, and a cell component that we named "round cells." Because only round cells displayed proliferation ability, as revealed by 5-bromo-2'-deoxyuridine (BrdU) labeling, they were further characterized. For this purpose, round cells were isolated and expanded by culturing in a serum-free medium, containing basic fibroblast growth factor (bFGF), before immunocytochemical analysis. We found that round cells were not immunoreactive for glial, neuronal, and oligodendrocyte markers, whereas they were immunoreactive for several immature neuronal markers. Accordingly, round cells could be induced to differentiate into astrocytes, neurons, and oligodendrocytes, either by withdrawing the mitogen bFGF or by exposing them to fluoxetine. These findings suggest that round cells in CGC possess the features and potentials of neural precursors, able to differentiate in mature neural cells upon a pharmacological simulum.

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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 mM ouabain, 0.1 microM phenylephrine, 10 microM serotonin, and 60 mM K+ were relaxed by canrenone (50-250 microM), 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 mM 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.

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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.

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In the present study, we investigated whether phospholipase A2 (PLA2)/lysophospholipase activity producing glycerophosphoinositols from phosphoinositides was operating in rat heart and could be stimulated by alpha1-adrenergic agonists. PLA2/lysophospholipase activity was found in homogenates from rat right ventricles. The stimulation of PLA2/lysophospholipase activity by noradrenaline (NA) was prevented either by the alpha1-adrenergic antagonist prazosin or arachidonyl trifluoromethyl ketone, a selective inhibitor of the 85-110 kDa, sn-2-arachidonyl-specific cytosolic PLA2. The selective alpha1-adrenergic agonist phenylephrine induced a concentration- and time-dependent increase in glycerophosphoinositol (GroPIns) and glycerophosphoinositol 4-phosphate (GroPIns4P) in rat right ventricle slices prelabelled with D-myo-[3H]inositol. In electrically driven strips of rat right ventricles, prelabelled with D-myo-[3H]inositol, the positive inotropic effect induced by 20 microM NA in the presence of propranolol was accompanied by the formation of GroPIns and GroPIns4P. The concentration of the formed GroPIns4P (1.33+/-0.12 microM, N = 6) was similar to that previously reported to inhibit the Na+/Ca2+ exchanger in cardiac sarcolemmal vesicles (Luciani S, Antolini M, Bova S, Cargnelli G, Cusinato F, Debetto P, Trevisi L and Varotto R, Biochem Biophys Res Commun 206: 674-680, 1995). These findings show that the stimulation of alpha1-adrenoceptors in rat heart is followed by an increase in the formation of GroPIns4P, which may contribute to the positive inotropic effect of alpha1-adrenergic agonists by inhibition of the Na+/Ca2+ exchanger.

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We studied the contractile responses to endothelin-1 (ET-1) of aortic strips from female transgenic rats, TGR(mRen2)27, heterozygous for the Ren-2 mouse gene, during the phases of developing (blood pressure in rats aged 5 weeks; 156 +/- 8 mmHg), steady (blood pressure in rats aged 11 weeks: 206 +/- 27 mmHg), and reversed (blood pressure in rats aged 35 weeks: 151 +/- 17 mmHg) hypertension. These responses were compared with those of aortae from sex- and age-matched, genetically homogeneous, normotensive Sprague-Dawley (SD) rats. Aortic strips from both transgenic and SD rats were deprived of endothelium before isometrically recording developed tension to cumulatively added ET-1. Aortic strips from 5- and 11-week-old female transgenic TGR(mRen2)27 (hfTG) rats responded to ET-1 with higher Emax values and lower EC50 values than those of age-matched SD rats. Conversely, aortic strips from 35-week-old hfTG rats exhibited lower Emax and higher EC50 values than aortic strips from SD rats. Within the hfTG rats, aortic strips from 11-week-old rats showed increased Emax and decreased EC50 of ET-1 as compared with either 5- or 35-week-old hfTG rats. These data are in keeping with the hypothesis that ET-1 contributes to the hypertension of hfTG rats and suggest that an altered vascular responsiveness to the peptide may be implicated in the changes of their systolic blood pressure occurring with ageing in this animal model.

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In this study, three rapid assay techniques for the determination of glutathione, one enzymatic, one fluorometric and one newly patented colorimetric method, were compared by measuring reduced (GSH) and oxidized (GSSG) glutathione in guinea-pig heart and liver. The HPLC technique was used as a standard, since it is considered the most reliable assay method. In heart, all methods measured the same levels of GSH (about 1 mumole/g wet tissue), whereas in liver the fluorometric assay gave GSH levels about half as high as those measured by the other methods (about 3 vs. 7 mumoles/g wet tissue). Conversely, the fluorometric assay grossly overestimated GSSG concentration (by 5 to 8 times) in both heart and liver. These results confirm previous doubts about the use of the fluorometric technique for GSSG determination in mammalian tissues and also raise some questions about its use for the measurement of GSH in liver. In this tissue, the GSH concentration determined by the fluorometric method was shown to be inversely correlated with the size of the sample, suggesting the presence of some quenching material.

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1. Cardiac effects on norbormide and verapamil were compared in single ventricular myocytes, right atria, and Langendorff perfused hearts isolated from guinea-pigs. 2. In ventricular myocytes, norbormide 50 microM inhibited the peak calcium current (ICa) by 49.6 +/- 3.9% without altering the shape of the current-voltage relationship; verapamil 1 microM 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. 3. 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. 4. 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. 5. 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.

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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 microM) 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 microM) 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 microM) caused relaxation. Norbormide inhibited the response to Ca2+ of rat aorta incubated in 80 mM KCl/Ca2(+)-free medium. Norbormide (up to 100 microM) 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-hydroxytryptamine-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.

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The enrichment in phosphatidylinositol 4-phosphate and phosphatidylinositol 4-5-bisphosphate of cardiac sarcolemmal vesicles stimulate Na+/Ca2+ exchange activity. On the contrary the deacylation products of polyphosphoinositides are powerful inhibitors of Na+/Ca2+ exchange: half maximal inhibition of 1.6 and 2.1 microM have been observed for glycerophosphoinositol 4-phosphate and glycerophosphoinositol 4-5-bisphosphate, respectively. The data indicate a bidirectional regulation of Na+/Ca2+ exchanger and suggest that phosphorylated glycerophosphoinositols can be regarded as novel signal transducers of intracellular Ca2+ regulation in heart cell.

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The effect of amiloride on the positive inotropic and toxic effects of ouabain in guinea-pig left atria has been studied. In atria driven at 1 Hz, amiloride (0.3 and 0.5 mM) decreased the EC50 but did not affect the maximal tension developed by ouabain. At 0.1 Hz, amiloride did not change either the EC50 or the maximal tension developed by ouabain. Ouabain toxicity (onset of arrhythmias) was not changed by amiloride at either frequency of stimulation. Therefore, amiloride did not antagonize either the positive inotropic or the toxic effect of ouabain. The positive inotropic effect of amiloride has been ascribed to the inhibition of the Na+/Ca2+ exchanger. Since amiloride inhibits also the Na+/H+ exchanger, 5-(N-ethyl-N-isopropyl)amiloride (EIPA), an amiloride derivative which selectively inhibits the Na+/H+ exchange, has been tested to evaluate the role of the Na+/H+ exchange in the amiloride-ouabain interaction. EIPA increased the EC50 values of ouabain and decreased the maximal developed tension by the glycoside in atria driven at 0.1 and 1 Hz, but did not antagonize the toxic response (arrhythmias) of atria to ouabain. It is suggested that the inhibition of Ca2+ exit through the Na+/Ca2+ exchange by amiloride and ouabain may explain the observation that the positive inotropic effects of amiloride and ouabain are additive.

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1. The effect of ouabain at a concentration (0.8 microM) that does not induce contractile response in guinea-pig aortic strips has been studied on endothelium-denuded strips repeatedly stimulated with 1 microM noradrenaline or 60 mM K+ applied for 5 min every 30 min. 2. The resting tone (i.e. the tone between one noradrenaline stimulation and the following) of the aortic strips exposed to ouabain increased progressively, whereas the control strips (no ouabain) completely relaxed on washout of the agonist. In the aortic strips stimulated by 60 mM K+, the resting tone did not increase. 3. The calcium antagonist, verapamil, did not affect the increase in tone, that was nevertheless strictly dependent on external calcium, since the contracted strips completely relaxed on calcium removal and promptly contracted again on calcium readdition. This finding indicates a mechanism independent of voltage-gated calcium channels. 4. Caffeine-induced contractions, taken as a measure of sarcoplasmic reticulum calcium content, were amplified by the presence of ouabain in aortic strips either stimulated by noradrenaline or unstimulated, with a larger increase in the former. 5. These results suggest that the repeated stimulation of guinea-pig aortic strips by noradrenaline in the presence of ouabain, by raising both intracellular Na+ and Ca2+, decreases the ouabain threshold concentration required for contraction, thus increasing the responsiveness of vascular smooth muscle to the glycoside.

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The effect of amiloride on Na+/Ca2+ exchange activity and adenylate energy charge in isolated rat cardiac myocytes has been studied. Amiloride inhibits Na+/Ca2+ exchange activity in rat cardiac myocytes with an IC50 of 1.76 mM when added to the external side of the cell membrane, whereas it is a weaker inhibitor when present at the cytoplasmic side. The adenylate energy charge in rat cardiac myocytes is unaffected by amiloride.

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Inhibition of cell growth was compared in V79 and HeLa cell cultures treated for 60 hr with nine of the first 10 MEIC chemicals; FeSO(4) could not be tested because it produced artefacts. Whereas the IC(50) of digoxin was at least three orders of magnitude lower in HeLa cells, all the other chemicals were almost equally toxic in the two cell lines. The IC(50) values showed good correlation with the in vivo human toxic concentrations, but the correlation was better with HeLa cells, which allowed the species-related sensitivity to digoxin to be detected. The effects of acute exposures to the same compounds on the cytosolic free Ca(2+) of PC12 cells, a neurosecretory cell line derived from a rat phaeochromocytoma, were measured fluorometrically by the fura-2 method. Amitriptyline, methanol, ethanol and isopropanol increased resting [Ca(2+)](i), both in the presence of extracellular Ca(2+) and, to a lesser extent, in Ca(2+)-free medium. Diazepam, digoxin and ethylene glycol were effective exclusively in the former condition. The changes of resting [Ca(2+)](i) appear to be sensitive indicators of early cytotoxicity induced by different toxic chemicals.

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Phosphatidylserine (PS) vesicles incorporated into rat brain synaptosomes increased total Ca2+ uptake. Total Ca2+ uptake was resolved in three components: K+ depolarization-induced Ca2+ uptake, Na+/Ca2+ exchange, and passive Ca2+ entry, which were differently affected by PS depending on the amount of incorporated phospholipid. K+ depolarization-induced Ca2+ uptake was stimulated by 0.05-0.10 mumol PS/mg protein while 0.10-0.30 mumol PS/mg protein increased Na+/Ca2+ exchange activity and passive Ca2+ entry but not K+ depolarization-induced Ca2+ uptake. High amounts of incorporated PS also increased passive Rb+ uptake.

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The influence of frequency of stimulation and external calcium on the positive inotropic response of guinea-pig left atria to diamide and the inhibitory action on Na+/Ca2+ exchange activity of rat cardiomyocytes by this oxidant of sulphhydryl groups have been investigated. Diamide (50-500 microM) induces a concentration-dependent positive inotropic effect which is more pronounced when atria are driven at 1.0 Hz rather than at 0.5 and 0.1 Hz, and are bathed in 2.72 mM rather than in 1.36 mM external calcium. A decrease in the positive inotropic effect at 35 degrees C with respect to 29 degrees C is also observed. In addition, diamide in positive inotropic concentrations (100-300 microM) significantly reduces Na+/Ca2+ exchange activity and cytoplasmic glutathione levels in adult rat cardiomyocytes. The thiol reducing agent dithiothreitol either reverses or prevents diamide effects both in isolated atria and cardiomyocytes, suggesting that the actions of diamide are correlated to its property to oxidize sulphhydryl groups to disulphides. In view of the functional importance of Na+/Ca2+ exchange in myocardial contractility, it is proposed that diamide may increase the heart force of contraction by an inhibition of the sarcolemmal Na+/Ca2+ exchange activity.

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