P. Dorigo

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

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). Rings of aortae incubated in 50 mM glucose, contracted submaximally by 1 μM noradrenaline, lost endothelium‐dependent relaxation in response to acetylcholine (10 nM to 10 μM). 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 μM) whereas endothelium‐independent relaxation in response to adenosine (0.3 mM) was well preserved. The relaxation induced by A23187 or sodium nitroprusside (10 nM to 0.1 μM) did not differ between rings exposed to control (5.5 mM) or elevated glucose (50 mM) and contracted submaximally by 3 mM 4‐aminopyridine. When incubated with aortic tissue in the presence of elevated glucose, the cyclo‐oxygenase inhibitors, indomethacin (10 μM) and mefenamic acid (30 μM), or the scavenger of superoxide anions, superoxide dismutase (150 u ml−1), prevented the impairment of ATP‐mediated relaxation. 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.

3-Acetyl-5-acylpyridin-2(1H)-ones and 3-acetyl-7,8-dihydro-2,5(1H,6H)-quinolinediones: synthesis, cardiotonic activity and computational studies

A series of milrinone analogues, namely 6-substituted 3-acetyl-5-acylpyridin-2(1H)-ones 4a-c, e, f and 7-substituted or unsubstituted 3-acetyl-7,8-dihydro-2,5(1H,6H)-quinolinediones 4g-j, in which the cyano group was replaced by the acetyl function, was prepared. In a preliminary pharmacological investigation on spontaneously beating atria from reserpine-treated guinea-pigs, all new compounds did not induce any inotropic effect equivalent or higher than that of the milrinone chosen as the reference compound. In order to rationalise how the structure modifications influence the activity and the selectivity of the title compounds, a computational study has been performed. The important role of the substituents in positions-3 and -6 on the pyridone nucleus has been highlighted.

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.

Reduced lipolytic response in vitro to catecholamines, acth and cyclic adenosine monophosphate in brown fat of cold-acclimatized rats

Abstract The hormone-induced lipolysis and the effect of different antagonists have been studied in parallel in brown and white adipose tissue of normal and cold-acclimatized rats. The results seem to indicate that brown adipose tissue of cold-acclimatized rats is not responsive to exogenous catecholamines and ACTH, while an increase in spontaneous lipolysis is observed. Moreover, the free fatty acids accumulate within the tissue, in contrast to epididymal fat where FFA are largely released in the incubation medium. The absence of stimulation by adipokinetic agents is discussed in relation to the high level of FFA within the cells, which may produce a feedback control on lipolysis. Another possibility is that the absence of stimulation by adipokinetic agents depends on a deficiency of ATP, consequent to (a) a poor phosphorylating efficiency of the respiratory chain or, alternatively, (b) an energy dissipating mechanism operating in spite of a tightly coupled phosphorylating system, and represented by the FFA accumulated inside brown fat cells.

Interaction of amrinone with endogenous adenosine in guinea‐pig atria

1 In spontaneously beating atria from reserpine‐treated guinea‐pigs, amrinone (10 μm to 2 mm) induced a positive inotropic and chronotropic effect that was preceded by a transient reduction in contractile force and in frequency. Both the positive and negative effects were concentration‐dependent. 2 The inotropic action of amrinone was antagonized by low concentrations of 8‐phenyltheophylline that compete with adenosine at R‐receptors on plasma membrane without significantly influencing phosphodiesterase activity. 3 Cumulative concentrations of amrinone (1 mm) antagonized the reduction of rate of contraction and amplitude induced by dipyridamole 1 μm in spontaneously beating atria and restored the maximum contractile effect reached in the absence of dipyridamole. 4 In spontaneously beating preparations incubated in the presence of adenosine deaminase (1u ml−1), amrinone lost its positive effects on the atria and only reduction of rate and contractile force was evident. Both effects were antagonized by scopolamine 1 mm thus indicating their cholinergic nature. 5 Adenosine at 0.1 μm and 0.5 μm significantly inhibited the inotropic effect induced by amrinone (0.03 to 3 mm) and the concentration‐effect curves of amrinone obtained in the absence and presence of adenosine clearly indicate a competitive antagonism between the two drugs. Thus the contractile activity of amrinone in spontaneously beating atria from reserpine‐treated guinea‐pigs originates from a displacement of adenosine from its R‐receptor sites in the cardiac cell.

Positive inotropic effect of a stable analogue of PGI2 and of PGI2 on isolated guinea pig atria. Mechanism of action.

Fluoroprostacyclin (10,10-difluoro-13,14-dehydroprostacyclin) a stable analogue of PGI2, induced an increase of contractile tension and a positive chronotropic effect on spontaneously beating guinea-pig atria. The increase in contractile tension was proportional to the dose (10(-8) to 2.5 X 10(-7) M) and never exceeded 40% of absolute initial controls. The chronotropic positive effect was low (12 +/- 4%) and not dose-dependent. The positive inotropic effect was also evident on electrically driven atria and was greater at the slower rates of stimulation. Atria from guinea pigs pretreated with reserpine were still sensitive to fluoroprostacyclin, although to a lower degree. The use of appropriate agonists and antagonists excluded the possibility that beta and alpha receptors are involved in the positive inotropic effect of fluoroprostacyclin. PGI2 showed the same behavior as fluoro-PGI2. Prostacyclins were able to reverse the decrease of contractility induced by verapamil, nifedipine, tetrodotoxin and xylocaine. But the reversal by PGI2 was no longer evident when the atrial contractility was completely abolished by the presence of these drugs in high concentrations. The inotropic effect of prostacyclins was more evident when Ca2+ was reduced in the medium. These results demonstrate that the cardiostimulant effect of prostacyclins is not simply adrenergic in origin. A direct effect on ion movements, mainly dependent on Ca2+ transmembranes fluxes, may be largely responsible for their positive inotropic effect.

Synthesis and pharmacological characterization of functionalized 2-pyridones structurally related to the cardiotonic agent milrinone

A new class of cardiotonic agents characterized by a 2-pyridone structure was synthesized. Appropriate sym-2-dimethylaminomethylene-1,3-diones reacted with methylcyanoacetate to afford the desired compounds. These derivatives were evaluated for their ability in inducing cardiotonic response on guinea pig isolated myocardial preparations. Compound 8b increased atrial contractility to an extent which is significantly higher than that of milrinone, the parent drug used as a reference compound. The pharmacological characterization and the docking studies performed on 8b highlighted its selective mechanism of action via type 3 PDE (PDE3) inhibition.

Effect of oxidative phosphorylation inhibitors on cyclic adenosine monophosphate synthesis in rat adipose tissue

Abstract The concentration of cyclic AMP in rat epididymal fat and in its incubation medium, was determined by the use of cyclic AMP-dependent protein kinases. The noradrenaline plus theophylline stimulation effect on cyclic AMP synthesis, was studied in the absence and in the presence of some oxidative phosphorylation inhibitors, rotenone, 2,4-dinitrophenol and oligomycin, drugs whose strong inhibitory effect on hormone-induced lipolysis is well known. The concentration of cyclic AMP was reduced by all three drugs. The changes induced by oligomycin and dinitrophenol on the cyclic AMP level, were quantitatively quite similar to those induced on the rate of lipolysis. Rotenone, in contrast, decreased the cyclic AMP level to a lower extent than lipolysis. This could be correlated with the different site and mechanism of action of rotenone on oxidative phosphorylation. The evidence indicates that the alteration of energy equilibrium (or the decreased availability of ATP) interferes with the lipolytic process at different levels, both before and after the synthesis of cyclic AMP.

Effect of glycolysis inhibitors on cyclic AMP synthesis in rat adipose tissue

Abstract The effect of two glycolysis inhibitors, sodium fluoride and monoiodoacetate , on the level of cyclic AMP in rat epididymal fat was studied in vitro with cyclic AMP-dependent protein kinases. The large increase (about 200 times in respect of the control level) of cyclic nucleotide concentration induced by noradrenaline plus theophylline, was strongly inhibited by 0·04 M sodium fluoride and by 0·001 M monoiodoacetic acid. The same inhibitors decreased the rate of hormone-induced lipolysis to a similar extent. Cyclic AMP level inside the adipose tissue, not treated with noradrenaline plus theophylline, as well as spontaneous lipolysis, were modified in the same way by fluoride and iodoacetate. These data emphasize the relationship between lipolysis and energy metabolism and, particularly, the importance of the glycolytic pathway for cyclic AMP synthesis.

Antagonism towards endogenous adenosine and inhibition of cGI-PDE in the cardiac effects of amrinone, milrinone and related analogues.

1. The effect of amrinone, milrinone and of three milrinone analogues was tested on spontaneous chronotropic and inotropic activity of guinea-pig isolated atria, on the activity of cGMP-inhibited phosphodiesterase (cGI-PDE) from guinea-pig heart and on specific binding of N6-cyclohexyl[3H]adenosine ([3H]CHA) to Ri adenosine receptors in guinea-pig atria. 2. The Ki-values towards [3H]CHA binding to Ri receptors were linearly related to the EC50S for the increase in force of contraction but not to the EC50S for the increase in frequency of the atria. The Ki values towards cGI-PDE were linearly related to the EC50S for the positive chronotropic effect.