Circulating soluble fibroblast activation protein (FAP) levels are independent of cardiac and extra-cardiac FAP expression determined by targeted molecular imaging in patients with myocardial FAP activation.

INTRODUCTION: Tissue Fibroblast Activation Protein alpha (FAP) is overexpressed in various types of acute and chronic cardiovascular disease. A soluble form of FAP has been detected in human plasma, and low circulating FAP concentrations are associated with increased risk of death in patients with acute coronary syndrome. However, little is known about the regulation and release of FAP from fibroblasts, and whether circulating FAP concentration is associated with tissue FAP expression. This study characterizes the release of FAP in human cardiac fibroblasts (CF) and analyzes the association of circulating FAP concentrations with in vivo tissue FAP expression in patients with acute (ST-segment elevation myocardial infarction, STEMI) and chronic (severe aortic stenosis, AS) myocardial FAP expression. METHODS AND RESULTS: FAP was released from CF in a time- and concentration-dependent manner. FAP concentration was higher in supernatant of TGFß-stimulated CF, and correlated with cellular FAP concentration. Inhibition of metallo- and serine-proteases diminished FAP release in vitro. Median FAP concentrations of patients with acute (77 ng/mL) and chronic (75 ng/mL, p = 0.50 vs. STEMI) myocardial FAP expression did not correlate with myocardial nor extra-myocardial nor total FAP volume (P ≥ 0.61 in all cases) measured by whole-body FAP-targeted positron emission tomography. CONCLUSION: We describe a time- and concentration dependent, protease-mediated release of FAP from cardiac fibroblasts. Circulating FAP concentrations were not associated with increased in vivo tissue FAP expression determined by molecular imaging in patients with both chronic and acute myocardial FAP expression. These data suggest that circulating FAP and tissue FAP expression provide complementary, non-interchangeable information.

The eNOS enhancer AVE 9488: a novel cardioprotectant against ischemia reperfusion injury.

Nitric oxide (NO) is an important regulator of vascular and myocardial function. Cardiac ischemia/reperfusion injury is reduced in mice overexpressing endothelial NO synthase (eNOS) suggesting cardioprotection by eNOS. Novel pharmacological substances, so called eNOS enhancers, upregulate eNOS expression and thereby increase NO production. We tested the effects of the eNOS enhancer AVE 9488 on cardiac ischemia/reperfusion injury in vivo in mice. After treatment with the eNOS enhancer AVE 9488 (30 mg/kg/day) or placebo for one week mice underwent 30 min of coronary artery ligation and 24 h of reperfusion in vivo. Ischemia-reperfusion damage was significantly reduced in mice treated with the eNOS enhancer when compared to placebo treated mice (infarct/area at risk 65.4 +/- 4.1 vs. 36.9 +/- 4.0%, placebo vs. eNOS enhancer, P = 0.0002). The protective effect was blunted in eNOS knockout mice treated with the eNOS enhancer (infarct/area at risk 64.1 +/- 6.2%, eNOS knockout + eNOS enhancer vs. WT + eNOS enhancer, P = ns). Reactive oxygen species were significantly reduced in mice treated with the eNOS enhancer as indicated by significantly lower malondialdehyde-thiobarbituric acid levels (placebo vs. eNOS enhancer, 3.2 +/- 0.5 vs. 0.8 +/- 0.07 micromol/l, P = 0.0003). Thus pharmacological interventions addressed to increase eNOS-derived NO production constitute a promising therapeutic approach to prevent myocardial ischemia/reperfusion injury.

Improvement of vascular function by acute and chronic treatment with the PDE-5 inhibitor sildenafil in experimental diabetes mellitus.

BACKGROUND AND PURPOSE: Diabetes-associated vascular dysfunction contributes to increased cardiovascular risk. We investigated whether the phosphodiesterase-5 inhibitor sildenafil would improve vascular function in diabetic rats. EXPERIMENTAL APPROACH: Male Wistar rats were injected with streptozotocin (50 mg kg(-1), i.v.) to induce insulin-deficient diabetes. Direct effects of sildenafil as well as modification of endothelium-dependent and -independent vasorelaxation were investigated in vitro. The effects of acute and chronic (2 week) treatment in vivo of sildenafil on vascular function were also characterized in isolated aortic segments in organ bath chambers 4 weeks after diabetes induction. KEY RESULTS: Sildenafil induced a concentration-dependent vasorelaxation, which was attenuated by the nitric oxide (NO) synthase inhibitor, N(G)-nitro-L-arginine. Acetylcholine-induced endothelium-dependent as well as endothelium-independent relaxation induced by the NO donor, DEA-NONOate, was significantly reduced in aortae from diabetic rats. Incubation with sildenafil in vitro normalized both endothelium-dependent and -independent relaxation in aortae from diabetic rats. Acute as well as chronic in vivo treatment with sildenafil resulted in enhanced endothelium-dependent and -independent vasorelaxation. Superoxide formation was increased in diabetes, associated with enhanced membrane expression of the NAD(P)H oxidase subunit gp91(phox) and Rac, which were both reduced by chronic treatment with sildenafil. CONCLUSIONS AND IMPLICATIONS: We demonstrate that sildenafil treatment rapidly and chronically improves vascular relaxation in diabetic rats. Treatment with sildenafil might provide a similarly beneficial effect in diabetic patients.

Role of p38 mitogen-activated protein kinase in cardiac remodelling.

BACKGROUND AND PURPOSE: Mitogen-activated protein kinases (MAPK) are centrally involved in several mechanisms important for heart failure such as apoptosis, activation of inflammatory responses and cell proliferation. We therefore evaluated the effect of the selective p38 MAPK inhibitor SB 239063 on progression of left ventricular remodelling after myocardial infarction (MI) in rats. EXPERIMENTAL APPROACH: Rats were treated for 9 weeks with placebo or SB 239063 by gavage (15 mg kg(-1)) twice daily starting 7 days after ligation of the left anterior descending artery. Serial transthoracic echocardiography was performed at days 7, 36 and 70. KEY RESULTS: Over the 9 weeks, mortality was not different between the groups. On echocardiography, animals after myocardial infarction exhibited significant left ventricular dilatation as expected (week 10, end-systolic diameter, placebo sham 5.21+/- 0.34 vs. placebo MI 8.44+/- 0.57 mm). However, there was no difference between placebo and SB 239063-treated rats (week 10, end-systolic diameter, SB MI 7.76+/- 0.74 mm, not significantly different from placebo MI). Haemodynamics changed accordingly. Moreover, SB 239063 had no effect on left ventricular hypertrophy. Treatment with SB 239063 significantly reduced cytokine expression of tumour necrosis factor and interleukin-1beta after myocardial infarction. However, collagen content was not influenced by the treatment. CONCLUSION: Despite a reduction of inflammation, treatment with the p38 inhibitor SB 239063 does not affect cardiac remodelling and cardiac function when treatment is started 7 days after myocardial infarction.

Striking increase of natriuresis by low-dose spironolactone in congestive heart failure only in combination with ACE inhibition: mechanistic evidence to support RALES.

BACKGROUND: A marked reduction of overall mortality in patients with severe congestive heart failure (CHF) has been demonstrated by addition of the mineralocorticoid receptor antagonist spironolactone to ACE inhibition. The aim of the present study was to examine a hypothesized interaction of spironolactone and ACE inhibitors in renal electrolyte and volume regulation. METHODS AND RESULTS: Wistar rats with extensive myocardial infarction or sham operation were treated with either placebo, the ACE inhibitor trandolapril, low-dose spironolactone, or a combination of the 2. Twelve weeks after infarction, rats were housed in metabolic cages. Urinary volume and sodium excretion were significantly increased in CHF rats on a combined treatment with spironolactone and trandolapril (21.2+/-2.6 mL/d, 2489+/-320 mmol/d, mean+/-SD; P<0.05 versus other experimental groups) versus placebo-treated rats (16.7+/-5.6 mL/d, 1431+/-458 mmol/d),whereas these parameters were not affected in rats on either spironolactone (16.1+/-6.6 mL/d, 1153+/-273 mmol/d) or trandolapril alone (15.9+/-4.2 mL/d, 1392+/-294 mmol/d). The effects on natriuresis coincided with a significant reduction of left ventricular end-diastolic pressure (LVEDP) in rats on trandolapril and spironolactone (10.8+/-8.2 mm Hg; P:<0.05 versus CHF placebo: 23.3+/-7.2 mm Hg; sham-operated rats: 5.1+/-0.9 mm Hg), whereas LVEDP remained elevated in rats treated with either compound alone. CONCLUSIONS: In the present study, we found an unexpected interaction of low-dose spironolactone and the ACE inhibitor trandolapril in experimental CHF leading to marked effects on renal electrolyte and volume regulation that were not apparent by treatment with either drug alone. These findings may explain the beneficial effects of spironolactone in CHF patients.

Improvement of left ventricular remodeling and function by hydroxymethylglutaryl coenzyme a reductase inhibition with cerivastatin in rats with heart failure after myocardial infarction.

BACKGROUND: Hydroxymethylglutaryl coenzyme A reductase inhibitors (statins) attenuate angiotensin II-induced cellular signaling. Because angiotensin II is involved in left ventricular (LV) remodeling after myocardial infarction (MI), we examined the effects of statin treatment in an experimental model of chronic heart failure after MI. METHODS AND RESULTS: Rats with extensive MI were treated with placebo or cerivastatin (0.3 mg/kg per day) as a dietary supplement or via gavage for 11 weeks starting on the 7th postoperative day. Infarct size and cholesterol levels were similar among all groups. LV cavity area, an index of LV dilatation, was reduced in MI rats on cerivastatin compared with placebo. LV end-diastolic pressure was increased in MI rats on placebo (24.1+/-4.1 mm Hg versus sham: 5.1+/-0.3 mm Hg; P<0.01), and it was significantly reduced by cerivastatin treatment (13.7+/-2.7 mm Hg; P<0.05 versus placebo). Cerivastatin partially normalized LV dP/dt(max) and dP/dt(min), indices of LV systolic and diastolic function, which were significantly reduced in MI rats on placebo. Improvement of LV function by cerivastatin was accompanied by a reduced expression of collagen type I and beta-myosin heavy chain. LV endothelial nitric oxide synthase was increased, whereas the nitrotyrosine protein level was decreased in MI rats by cerivastatin treatment. CONCLUSIONS: Cerivastatin improved LV remodeling and function in rats with heart failure. This effect was associated with an attenuated LV expression of fetal myosin heavy chain isoenzymes and collagen I. Statin treatment may retard the progression of chronic heart failure.

Endothelial dysfunction in chronic myocardial infarction despite increased vascular endothelial nitric oxide synthase and soluble guanylate cyclase expression: role of enhanced vascular superoxide production.

BACKGROUND: Endothelial dysfunction of the peripheral vasculature is a well-known phenomenon in congestive heart failure that contributes to the elevated peripheral resistance; however, the underlying mechanisms have not yet been clarified. METHODS AND RESULTS: Dilator responses, the expression of protein and mRNA of the endothelial nitric oxide synthase (eNOS), inducible NOS (iNOS), and soluble guanylate cyclase (sGC), and superoxide anion (O(2)(-)) and peroxynitrite production were determined in aortic rings from Wistar rats 8 weeks after myocardial infarction and compared with those in sham-operated animals. In rats with heart failure, the concentration-response curve of the endothelium-dependent vasodilator acetylcholine (after preconstriction with phenylephrine) was significantly shifted to the right, and the maximum relaxation was attenuated. Determination of expression levels of the 2 key enzymes for NO-mediated dilations, eNOS and sGC, revealed a marked upregulation of both enzymes in aortas from rats with heart failure, whereas iNOS expression was not changed. Pretreatment with exogenous superoxide dismutase partially restored the acetylcholine-induced relaxation in aortas from rats with heart failure. Aortic basal and NADH-stimulated O(2)(-) production assessed by use of lucigenin-enhanced chemiluminescence was significantly elevated in rats with chronic myocardial infarction. Peroxynitrite-mediated nitration of protein tyrosine residues was not different between the 2 groups of rats. CONCLUSIONS: These results demonstrate that endothelial dysfunction in ischemic heart failure occurs despite an enhanced vascular eNOS and sGC expression and can be attributed to an increase in vascular O(2)(-) production by an NADH-dependent oxidase. By inactivation of NO, O(2)(-) production appears to be an essential mechanism for the endothelial dysfunction observed in heart failure.

Endothelin-receptor blockade improves endothelial vasomotor dysfunction in heart failure.

OBJECTIVES: To elucidate the effect of selective endothelin ET(A)- and mixed ET(A/B)-receptor antagonists on endothelial vasomotor dysfunction in rats with heart failure after myocardial infarction (MI). METHODS: Vasoreactivity and superoxide anion formation were determined in aortic rings from Wistar rats 12 weeks after extensive MI (>46% of left ventricle) compared to sham-operated animals. Rats were either treated with the selective ET(A)-receptor antagonist LU 135252 (30 mg/kg/day), the mixed ET(A/B)-receptor antagonist Bosentan (100 mg/kg/day) or placebo. RESULTS: In MI rats, the concentration-response curve of the endothelium-dependent, nitric oxide-mediated relaxation induced by acetylcholine was significantly shifted to the right and the maximum relaxation was attenuated. Long-term treatment with both ET antagonists significantly improved acetylcholine-induced relaxation in MI rats. LU 135252 was more effective than Bosentan. Endothelium-independent relaxations induced by sodium nitroprusside as well as endothelin- and phenylephrine-induced contractions were similar in all groups of rats. Plasma renin activity and aortic superoxide formation, which were enhanced in rats with heart failure, were normalized by LU 135252, but not by Bosentan treatment. CONCLUSIONS: Long-term treatment with ET-receptor antagonists improves endothelial vasomotor dysfunction in rats with chronic MI. This mechanism may essentially contribute to the beneficial effects of ET receptor blockade in heart failure.

Prevention of endothelial dysfunction in heart failure by vitamin E: attenuation of vascular superoxide anion formation and increase in soluble guanylyl cyclase expression.

OBJECTIVES: Enhanced vascular superoxide anion generation contributes to endothelial dysfunction in heart failure. However, the effect of long-term treatment with the antioxidant vitamin E is unknown. METHODS AND RESULTS: Relaxant responses were determined in aortic rings from Wistar rats with heart failure 12 weeks after myocardial infarction (MI) and compared with responses in tissues from sham-operated animals. From the seventh post-operative day, rats were given either a standard chow or a chow enriched in vitamin E (approximate intake 100 mg/day). In rings from rats with heart failure, acetylcholine-induced relaxation was attenuated (maximum relaxation, R(max) 54 +/- 3%) when compared with rings from sham-operated animals (79 +/- 3%, n=12, P < 0.01), while endothelium-independent relaxation elicited by sodium-nitroprusside was unchanged. Aortic superoxide generation was significantly enhanced in rats with heart failure. Vitamin E supplementation significantly improved acetylcholine-induced relaxation in rats with heart failure (R(max) 75 +/- 4%, P < 0.01) and led to a leftward shift in sodium-nitroprusside-induced relaxation curve. Aortic expression of the beta(1)-subunit of soluble guanylyl cyclase was significantly enhanced by vitamin E supplementation. In addition, the elevated vascular superoxide formation was normalised by vitamin E. CONCLUSIONS: These results demonstrate that dietary supplementation with the antioxidant vitamin E restores normal endothelial function, reduces vascular superoxide anion formation and increases the expression of the soluble guanylyl cyclase in rats with heart failure.

Improvement of renal dysfunction in rats with chronic heart failure after myocardial infarction by treatment with the endothelin A receptor antagonist, LU 135252.

OBJECTIVE: To investigate the role of an activated endothelin system in the renal dysfunction observed in chronic heart failure after myocardial infarction. METHODS: In rats with heart failure after myocardial infarction and in sham-operated animals (Sham), we investigated the effect on renal function of long-term oral treatment with the selective endothelin A (ETA) receptor antagonist, LU 135252 (30 mg/kg per day; groups MI/LU and Sham/LU) or placebo (groups MI/P, Sham/P). Only animals with extensive myocardial infarction (at least 46% of the left ventricle) were included in the study. Infarct size was matched between groups MI/P and MI/LU. Endogenous creatinine clearance, fractional sodium excretion, and plasma and urinary concentrations of endothelin were determined 12 weeks after myocardial infarction. RESULTS: Endogenous creatinine clearance was significantly lower in group MI/P than in group Sham/P (MI/P: 0.64 +/- 0.05, Sham/P: 0.81 +/- 0.04 ml/min per 100 g body weight; P= 0.01 (means +/- SEM)). Treatment with LU 135252 completely prevented the decline in creatinine clearance in rats with chronic myocardial infarction (MI/LU: 0.98 +/- 0.21; Sham/LU: 0.83 +/- 0.10). Fractional sodium and protein excretion did not differ among the four groups. Group MI/P had a marked increase in plasma endothelin concentrations, which was not affected by treatment with LU 135252. Urinary endothelin excretion was significantly lower in group MI/P than in group Sham/P. In the treatment groups, no difference could be observed between animals that had suffered myocardial infarction and the sham-operated group, although LU 135252 markedly increased the urinary excretion of endothelin. CONCLUSION: Our data demonstrate a restoration of impaired renal function in chronic ischaemic heart failure by treatment with the selective ETA receptor antagonist, LU 135252. These results offer a promising therapeutic option for the treatment of renal insufficiency in patients with chronic heart failure.

A pharmacological, crystallographic, and quantum chemical study of new inotropic agents.

The cardiac activity of a series of milrinone analogues, 2-substituted 3-acyl-1,6-dihydro-6-oxo-5-pyridinecarbonitriles, 1,6,3,2,11,12-hexahydro-6,3-dioxo-5-quinolinecarbonitriles, the correlated carboxylic acids, 2-substituted 3-acyl-6(1H)-pyridones, and 7,8-dihydro-2,5(1H,6H)-quinolinediones, was evaluated in spontaneously beating and in electrically driven atria from reserpine-treated guinea pigs. Their effects were compared with those induced by amrinone and milrinone in both the atria preparations. Compounds SF28 (3-acetyl-1,6-dihydro-2-methyl-6-oxo-5-pyridinecarbonitrile) and SF40 (7,8-dihydro-7-methyl-2,5(1H,6H)-quinolinedione) were the most effective positive inotropic agents. An inhibition of the negative influence exerted by endogenous adenosine on heart preparations seems to be involved in their contractile activity. SF38 (3-benzoyl-2-phenyl-6(1H)-pyridinone), on the contrary, reduced the contractile force and the frequency rate of guinea pig atria with a mechanism not related to an activation of cholinergic or purinergic inhibitory receptors on the heart. X-ray analysis carried out on the three model compounds, SF28, SF40 (positive inotropic agents), and SF38 (negative inotropic agent), and molecular modeling evidenced that the change from phenyl (SF38) to methyl (SF28) or the introduction of a side cyclic aliphatic chain (SF40) results in a variation of conformational preference and topography which may address the different molecules toward distinct receptor pockets according to the resulting inotropic effect.

Synthesis and cardiotonic activity of novel pyrimidine derivatives: crystallographic and quantum chemical studies.

The synthesis of ethyl or methyl 4-substituted or unsubstituted 2-(dimethylamino)-5-pyrimidinecarboxylates 10-20, which is mainly carried out by reaction of ethyl or methyl 2-[(dimethylamino)methylene]-3-oxoalkanoates with 1,1-dimethylguanidine, is described. The above esters were hydrolyzed to the relative carboxylic acids 21-30, which were decarboxylated to the corresponding 2,4-disubstituted pyrimidines 31-40. All the new synthesized pyrimidines were evaluated in spontaneously beating and electrically driven atria from reserpine-treated guinea pigs. Their effects were compared to those induced by milrinone in both atria preparations. Compound 28 (4-benzyl-2-(dimethylamino)-5-pyrimidinecarboxylic acid) was the most effective positive inotropic agent, while the corresponding methyl ester 17 reduced both the contractile force and the frequency of guinea pig atria. An antagonism toward the negative influence exerted by endogenous adenosine on the heart seems to be involved in the contractile activity of compound 28. By contrast, compound 17 might be partial agonist at the purinergic inhibitory (A1) receptor. X-ray analysis carried out on 17 and 28 and molecular modeling investigations extended also to related derivatives allowed a possible rationalization between structure and inotropic activity for this series of compounds.

Effect of quinapril initiated during progressive remodeling in asymptomatic patients with healed myocardial infarction.

Approximately 20% of patients with healed myocardial infarction develop asymptomatic progressive left ventricular (LV) dilation and remodeling and are at increased risk for progression to symptomatic congestive heart failure and premature death. It was the goal of this study to test whether quinapril may interrupt this process and to analyze potential mechanisms. Of 138 patients with an average infarct age of 56 months, 25 had asymptomatic progressive LV dilation and were randomized in a prospective, double-blind study to placebo or quinapril. At baseline (mean +/- SEM) ejection fraction was reduced (35 +/- 3% and 39 +/- 3%) and end-diastolic volume (gated single-photon emission computed tomography) increased (104 +/- 9 and 117 +/- 12 ml/m(2)) with placebo (n = 13) and quinapril (n = 12), respectively. Progressive dilation continued in patients taking placebo (6 months: 9.4 +/- 5.2 ml/m(2), 12 months 24.6 +/- 5. 4 ml/m(2); change from baseline: p <0.05 vs baseline; p <0.05 vs 6 months), but not with quinapril (6 months: -0.9 +/- 4.0 ml/m(2); 12 months: 4.1 +/- 5.2 ml/m(2) [p <0.05] vs placebo). Wedge pressure during bicycle exercise was similar at baseline, but at 12 months tended to be lower with quinapril (17 +/- 1 mm Hg) than with placebo (24 +/- 4 mm Hg, p = 0.1673). Thus, quinapril prevented further progression of asymptomatic LV dilation and remodeling after remote myocardial infarction, possibly due to attenuation of an exercise-induced increase in LV filling pressure.

Impairment of endothelium-dependent but not of endothelium-independent dilatation in guinea-pig aorta rings incubated in the presence of elevated glucose.

1. Purine compounds such as ATP and adenosine, respectively endothelium-dependent and- independent vasodilators, are largely involved in the control of vascular tone and vascular reactivity to contracting stimuli. We investigated the relaxing activity of ATP and adenosine in guinea-pig aorta rings exposed for 6 h to elevated glucose concentration (50 mM), in order to mimic hyperglycaemic conditions. Guinea-pigs were reserpine-treated (2 mg kg-1, i.p., 48 and 24 h before death). 2. Rings of aortae incubated in 50 mM glucose, contracted submaximally by 1 microM noradrenaline, lost endothelium-dependent relaxation in response to acetylcholine (10 nM to 10 microM). Aortae incubated with 50 mM mannose, as a hyperosmotic control, relaxed to acetylcholine normally. Rings of aortae incubated in 50 mM glucose, contracted submaximally by 3 mM 4-aminopyridine, lost endothelium-dependent relaxation in response to ATP (30 microM) whereas endothelium-independent relaxation in response to adenosine (0.3 mM) was well preserved. 4. The relaxation induced by A23187 or sodium nitroprusside (10 nM to 0.1 microM) did not differ between rings exposed to control (5.5 mM) or elevated glucose (50 mM) and contracted submaximally by 3 mM 4-aminopyridine. 5. When incubated with aortic tissue in the presence of elevated glucose, the cyclo-oxygenase inhibitors, indomethacin (10 microM) and mefenamic acid (30 microM), or the scavenger of superoxide anions, superoxide dismutase (150 u ml-1), prevented the impairment of ATP-mediated relaxation. 6. The present results indicate that endothelium-dependent, receptor-induced relaxation in response to acetylcholine and ATP is impaired in guinea-pig aorta rings exposed to elevated glucose. The endothelial dysfunction caused by glucose might be located at a step between receptor activation and intracellular calcium increase, and might be related to an increased metabolism of arachidonic acid coupled to an increased production, or to a reduced inactivation of superoxide anions.

Endothelium is required in the vascular spasm induced by tetraethylammonium and endothelin-1 in guinea-pig aorta.

1. To investigate the role of endothelium in vascular spasm, we studied the influence of endothelin-1 (ET-1) on the contracting and spasmogenic effect of the K+-channel blocker, tetraethylammonium (TEA), in aorta rings of reserpine-treated guinea-pigs, perfused with either control (5.5 mM) or elevated (50 mM) glucose concentration. 2. Endothelium-dependent relaxation induced by acetylcholine was lost in rings contracted by noradrenaline in the presence of elevated glucose. In control medium, TEA (1-20 mM) induced a sustained tonic contraction, followed by a phasic spasm, characterized by rhythmic contractions. Elevated glucose, ET-1 (3 nM), or both, reduced the EC50 of TEA-induced tonic contraction, without modifying the maximum contractile effect. 3. In control medium, ET-1 reduced the time before TEA-induced spasm and increased the rate of rhythmic contractions. TEA-induced spasm was abolished by elevated glucose, and restored by ET-1. The spasm induced by TEA and ET-1 was amplified by the ETA antagonist, EMD94246, and suppressed by the ET(A)-ET(B) antagonist, bosentan. In endothelium-denuded vessels incubated with high glucose and ET-1, TEA evoked only a tonic contraction. 4. In control medium, L-NAME (N(G)-nitro-L-arginine methyl ester) abolished TEA-induced rhythmic contractions. L-arginine, but not D-arginine, prevented the effect of L-NAME. In the presence of elevated glucose and ET-1, TEA-induced spasm was not affected by L-NAME, whereas verapamil, indomethacin, metyrapone, glybenclamide or apamin abolished the phasic spasm, unmasking the tonic contracture. 5. In conclusion, endothelium plays a regulatory role in the genesis and maintenance of TEA-induced rhythmic contractions, through the release endothelium derived relaxing factor and vasodilating eicosanoids.

Hydralazine prevents endothelial dysfunction, but not the increase in superoxide production in nitric oxide-deficient hypertension.

Dilator responses, superoxide anion-production, endothelial nitric oxide (NO) synthase and soluble guanylyl cyclase expression were determined in aortic rings from Wistar rats treated for 5 weeks either with the NO synthase inhibitor NG-nitro-L-arginine-methylester (L-NAME), L-NAME plus hydralazine or placebo. In the L-NAME-treated group, acetylcholine-induced relaxation was significantly attenuated whereas it was nearly normal in the L-NAME/hydralazine group. This difference was even more pronounced following inhibition of the endogenous superoxide dismutase using diethyldithiocarbamate. Aortic superoxide production was significantly elevated in both L-NAME-treated groups and hydralazine had no acute effect on superoxide formation. Expression of endothelial NO synthase was similar in all three groups whereas the attenuated soluble guanylyl cyclase expression in rats treated with L-NAME was nearly normalised by concomitant hydralazine treatment. These results demonstrate that in NO-deficient hypertension hydralazine treatment improves vasodilator responses but not the increased superoxide production.

Mineralocorticoid receptor antagonism and cardiac remodeling in ischemic heart failure.

Aldosterone production in the heart as well as aldosterone plasma levels are increased after myocardial infarction and in congestive heart failure, correlating with the severity of disease. Aldosterone promotes sodium and water retention, sympathoadrenergic activation, endothelial dysfunction, and cardiovascular fibrosis and hypertrophy. Even maximally recommended doses of ACE inhibitors do not completely prevent formation of aldosterone. The Randomized Aldactone Evaluation Study (RALES) and the Eplerenone Post acute myocardial infarction Heart failure Efficacy and SUrvival Study (EPHESUS) demonstrated that aldosterone receptor blockade markedly reduces mortality among patients with heart failure. This review summarizes recent clinical and experimental data on the effect of aldosterone antagonists on left ventricular remodeling and function in ischemic heart failure with special emphasis on potential underlying mechanisms. While reduction of excessive extracellular matrix turnover leading to decreased fibrosis appears to be the most important effect of mineralocorticoid receptor antagonism in heart failure, other mechanisms such as regression of hypertrophy, improvement of endothelial function, reduction of superoxide formation, and enhanced renal sodium excretion may contribute. Recent data showed that in rats with left ventricular dysfunction after extensive myocardial infarction, eplerenone on top of ACE inhibition more effectively improved cardiac remodeling and molecular alterations than ACE inhibition alone.

Pharmacological characterization of a new milrinone analogue.

In a new series of milrinone analogues (esters of 2-substituted 5-acetyl-1,6-dihydro-6-oxo-3-pyridinecarboxylic acids), ethyl 5-acetyl-1,6-dihydro-6-oxo-2-phenyl-3-pyridinecarboxylate (compound 2f) has been found to be more potent and more effective than milrinone as a positive inotropic agent while affecting only marginally the frequency rate of guinea-pig isolated atria. This finding prompted us to study the mechanism of cardiac action of compound 2f in electrically driven left atrium from reserpine-treated guinea pigs. Compound 2f induced a statistically significant increase in the contractile force at a concentration as low as 1 microM, while the minimum effective concentration of milrinone was 10 microM. The beta-blocker propranolol (0.1 microM) caused a marked inhibition of the inotropic effect of compound 2f. Adenosine deaminase (1 and 2 U/ml) inhibited significantly and in a concentration-dependent manner the increase in inotropism induced by compound 2f and the adenosine deaminase-resistant response was abolished by 0.1 microM propranolol. In the presence of 0.1 microM propranolol, compound 2f (5 to 30 microM) antagonised in competitive manner the negative inotropic effect induced by N6-(R-phenylisopropyl) adenosine (R-PIA) (0.01-1.0 microM), a stable adenosine receptor agonist. Schild regression analysis gave in fact a slope of 1.02 +/- 0.06 and the pA2 value for compound 2f was 5.41 +/- 0.28. Compound 2f also inhibited phosphodiesterase (PDE) III isolated from calf heart, this inhibition being quantitatively significant only at the highest concentrations tested (0.5 M to 1 mM).(ABSTRACT TRUNCATED AT 250 WORDS)

Synthesis and pharmacological activities of ethyl 5-cyano-1,6-dihydro-6-oxo-2-(2,3,4-pyridyl)-3-pyridinecarboxylates and derivatives.

The synthesis of ethyl esters of 5-cyano-1,6-dihydro-6-oxo-3-pyridinecarboxylic acids carrying as 2-substituent the 2-,3- or 4-pyridyl group is described. By alkaline hydrolysis followed by acidification, these esters gave the corresponding carboxylic acids, which were decarboxylated to 1,2-dihydro-2-oxo-6-(2,3,4-pyridyl)-3-pyridinecarbonitriles. As milrinone analogues, the above compounds were tested on contractile activity and frequency rate of spontaneously beating atria from reserpine-treated guinea-pigs. Ethyl 5-cyano-1,6-dihydro-6-oxo-2-(2-pyridyl)-3-pyridinecarboxylate showed an appreciable positive inotropic activity, although inferior to that of milrinone; moreover, some other compounds bearing the above 2-substitution pattern showed interesting antiinflammatory, analgesic and hypotensive activity.

Aldosterone antagonism in addition to angiotensin-converting enzyme inhibitors in heart failure.

Although the role of the systemic renin-angiotensin-aldosterone system in the pathophysiology of heart failure is well-known for years, the impact of a local cardiac aldosterone system has been recognized recently. Aldosterone promotes cardiac hypertrophy and fibrosis in hypertension and heart failure and is involved in left ventricular remodeling after myocardial infarction. Plasma aldosterone levels in patients with heart failure are an indicator of a worse prognosis. Although ACE inhibitor therapy in these patients reduces plasma aldosterone levels, this effect is only transitory, a phenomenon referred to as "aldosterone escape". Even maximally recommended doses of ACE inhibitors do not completely prevent ACE-mediated formation of angiotensin II in chronic heart failure, and those patients with increased aldosterone levels during ACE inhibition have impaired exercise capacity. The RALES study has demonstrated convincingly that in patients with heart failure, addition of the mineralocorticoid receptor antagonist spironolactone (25 mg/d) to ACE inhibition markedly reduces mortality and prevents worsening heart failure. While reduction of excessive extracellular matrix turnover leading to decreased fibrosis appears to be the most important effect of spironolactone in heart failure, other mechanisms such as regression of hypertrophy, improvement of endothelial function, enhanced renal sodium excretion and antiarrhythmic actions may contribute. In RALES, low-dose spironolactone did not confer a substantial risk of hyperkalemia, however, with broader use of spironolactone in heart failure, cases of hyperkalemia associated with the use of this drug increase. Close control of serum potassium and creatinine and estimation of creatinine clearance are mandatory, especially in the presence of additional factors impairing renal function. The new and more selective aldosterone antagonist eplerenone which is devoid of some side effects of spironolactone, has been shown to be effective in hypertension and holds great promise as future therapeutic agent in patients with heart failure.