Synthesis, characterization, crystal structure and preliminary reactivity behaviour of new heteropolytopic ligands based on the 1,3,5-triazine spacer and pyrazolyl, tris-pyrazolylmethyl and tris-pyrazolylethoxy bonding fragments.

Four new potentially polytopic nitrogen donor ligands based on the 1,3,5-triazine fragment, L(1)-L(4) (L(1) = 2-chloro-4,6-di(1H-pyrazol-1-yl)-1,3,5-triazine, L(2) = N,N'-bis(4,6-di(1H-pyrazol-1-yl)-1,3,5-triazin-2-yl)ethane-1,2-diamine, L(3) = 2,4,6-tris(tri(1H-pyrazol-1-yl)methyl)-1,3,5-triazine, and L(4) = 2,4,6-tris(2,2,2-tri(1H-pyrazol-1-yl)ethoxy)-1,3,5-triazine) have been synthesized and characterized. The X-ray crystal structure of L(3) confirms that its molecular nature consists of a 1,3,5-triazine ring bearing three tripodal tris(pyrazolyl) arms. L(1), L(2), and L(4) react with Cu(I), Cu(II), Pd(II) and Ag(I) salts yielding mono-, di-, and oligonuclear derivatives: [Cu(L(1))(Cy(3)P)]ClO(4), [{Ag(2)(L(2))}(CF(3)SO(3))(2)]·H(2)O, [Cu(2)(L(2))(NO(3))(2)](NO(3))(2)·H(2)O, [Cu(2)(L(2))(CH(3)COO)(2)](CH(3)COO)(2)·3H(2)O, [Pd(2)(L(2))(Cl)(4)]·2H(2)O, [Ru(L(2))(Cl)(OH)]·CH(3)OH, [Ag(3)(L(4))(2)](CF(3)SO(3))(3) and [Ag(3)(L(4))(2)](BF(4))(3). The interaction of L(3) with Ag(I), Cu(II), Zn(II) and Ru(II) complexes unexpectedly produced the hydrolysis of the ligand with formation, in all cases, of tris(pyrazolyl)methane (TPM) derivatives. In detail, the already known [Ag(TPM)(2)](CF(3)SO(3)) and [Cu(TPM)(2)](NO(3))(2), as well as the new [Zn(TPM)(2)](CF(3)SO(3))(2) and [Ru(TMP)(p-cymene)]Cl(OH)·2H(2)O complexes have been isolated. Single-crystal XRD determinations on the latter derivatives confirm their formulation, evidencing, for the Ru(II) complex, an interesting supramolecular arrangement of the anions and crystallization water molecules.

Electrophysiological instability in the acute phase: prognostic significance of early ventricular fibrillation in acute myocardial infarction.

BACKGROUND: In the prognostic stratification of patients affected by AMI is important to evaluate, besides the assessment of left ventricular function and residual ischemia, the presence of electrophysiological instability. METHODS: We have analysed 15 patients all affected by AMI complicated by early ventricular fibrillation. During the hospital phase we evaluated the E.F.% (ECHO) and the presence of late ventricular potentials (SAECG). After hospital discharge we followed up the patients for 6 months. RESULTS: None of the patients died during the hospital phase while the posthospital cardiac mortality was 20%. The three patients dead during the follow-up had an AMI localized in the anterolateral wall of the left ventricle, an E.F.% less than 40% and LVP positive in the hospital phase. Besides the clinical course was complicated by cardiac failure. CONCLUSIONS: We conclude that these three patients are a "high risk profile subgroup" and should be submitted to extensive evaluation with cardiac catheterization, coronary arteriography and programmed ventricular stimulation.

Prolonged inhibition of nitric oxide synthesis in Yoshida hyperlipidemic rat: aorta functional and structural properties.

To test whether inhibition of nitric oxide synthesis, associated with high levels of plasmatic lipids, can induce atherosclerotic lesions and phenotypic changes in smooth muscle cell composition in the aortic wall of an atherosclerotic-resistant species such as the rat, an inbred strain of hyperlipidemic Pittsburgh Yoshida rat was subjected to prolonged treatment (2 months) with the nitric oxide-synthase inhibitor L omega-nitro-arginine-methyl ester or with L-arginine. The two types of in vivo treatments were not able to modify in vitro aortic endothelium-mediated relaxation induced by acetylcholine or calcium-ionophore A-23187, the endothelium-independent sodium nitrite relaxation and the contractile response to serotonin. Histology and lipid infiltration of vascular specimens showed that L omega-nitro-arginine-methyl ester in vivo treatment did not induce any significant change in the aortic wall. Monoclonal antibodies to myosin isoforms and immunofluorescence procedures revealed the presence of an immature smooth muscle cell subpopulation in aortic specimens from saline-treated Pittsburgh Yoshida rats, whose expansion has been related in other species to atherogenesis. This peculiar cell phenotype disappeared in our animal model after prolonged L omega-nitro-arginine-methyl ester treatment. These data indicate that, despite interference with endothelium-mediated nitric oxide synthesis, atherosclerosis does not develop in this animal model and furnish for the first time a biological justification for atherogenesis resistance of rat, i.e., the lack of activation of an immature aortic smooth muscle cell population which in atherosclerosis-prone species is involved in lesion formation.

Mechanisms of plumbagin action on guinea pig isolated atria.

In electrically driven guinea pig left atria, plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone; 0.5-10 microM) produced a marked positive inotropic effect that was about 65% that caused by isoprenaline in the same experimental conditions. The effect was mainly not dependent on catecholamine release from adrenergic stores. An EC50 of 3 microM was calculated from the concentration-response curves. The increase in force of contraction was followed by a nonreversible contracture. Plumbagin was reduced by cardiac mitochondrial and soluble reductases with consequent generation of large amounts of superoxide anion. The assay of reduced glutathione/oxidized glutathione content in atria, treated with 10 microM plumbagin and frozen at the appearance of increase in diastolic tension, showed a significant decrease in reduced glutathione (-52% with respect to control atria) and a 5-fold increase in oxidized glutathione levels. Moreover, in the same experimental conditions a significant decrease in adenosine triphosphate (-55% with respect to the controls) and in adenylate energy charge (from 0.92-0.64) was observed. Of the enzymes and transport systems involved in the control of the cardiac contractility, the sarcoplasmic reticulum Ca2+ pump seemed to be a specific target for plumbagin. After 30 min of incubation with cardiac sarcoplasmic reticulum membrane vesicles, plumbagin inhibited Ca2+ uptake by the pump in a concentration-dependent manner (IC50 = 3 microM). On the basis of these results, the increase in diastolic tension caused by plumbagin appears to be related to intracellular Ca2+ accumulation, due both to the low availability of adenosine triphosphate for ionic pumps and direct inhibition of Ca2+ reuptake in sarcoplasmic reticulum.

Endothelial nucleotide-mediated aorta relaxation in aged Watanabe heritable hyperlipidemic rabbits.

We investigated the activity of muscarinic and purinergic endothelial receptors during atherosclerosis in Watanabe heritable hyperlipidemic (WHHL) rabbit aorta. Experiments were performed on isolated thoracic aorta from WHHL rabbits aged 1 and 2.5 years. The relaxant response to acetylcholine (ACh) was progressively reduced with aging, being almost completely abolished in 2.5-year-old rabbits. The relaxant effect of ATP was not affected by the P2-purinoceptor antagonist suramin, thus excluding any involvement of relaxant P2y purinoceptors in both considered ages. The pyrimidine UTP, acting on nucleotide (P2U) receptors, produced concentration-dependent relaxation in 1-year-old WHHL rabbit aorta only in the presence of endothelium; relaxation was reduced in older animals. In 1-year-old WHHL rabbits, the endothelium-dependent relaxant effect of UTP was not antagonized by suramin, but was by the inhibitors of nitric oxide (NO) effect, methylene blue (MB) and L-NG-nitroarginine methyl ester (L-NAME), suggesting involvement of NO in the UTP-mediated relaxation. Morphological data from electron microscopy observations indicated the presence of typical atherosclerotic lesions and extensive dystrophic changes in endothelial cells, gradually evolving at 1 and 2.5 years of age. The present data suggest that progressive atherosclerosis differentially affects the activity of endothelial receptors: The most precociously altered is the P2y-purinoceptor, followed by an impairment of the muscarinic and finally of the P2U-purinoceptor.

Phentolamine and hypoxia: modulation of contractility and alpha 1-adrenoceptors in isolated rat atria.

The hypoxia-induced effects on the binding sites and affinity constant of adrenoceptors, in the presence and absence of phentolamine, were determined for atrial membranes of hearts from normal and genetically hyperlipidaemic Yoshida (YOS) rats. Atrial function was also measured during normoxia and hypoxia, in the presence and absence of phentolamine. Hypoxia increased alpha 1-adrenoceptor density in atrial membranes of normal rats (Bmax 10.6 to 26.7 fmoles/mg protein). Phentolamine prevented the increase in the Bmax of alpha 1-adrenoceptors and increased the equilibrium dissociation constant of these receptors (KD 0.17 to 0.53 nmol/l). Beta-adrenoceptors did not change during hypoxia, but the Bmax was slightly increased (26%) in the presence of phentolamine. Thus, the alpha 1/beta ratio increased from 0.40 in normoxia to 1.06 in hypoxia. In normoxic atria from YOS rats, the alpha 1/beta ratio was already elevated (0.86) in comparison to control rats (mainly due to a higher density of alpha 1-adrenoceptors in atrial membranes from YOS rats). This ratio was not modified by hypoxia (0.84), but decreased when phentolamine was present (0.30). Hypoxia reduced the force of contraction and increased diastolic tension of atria of normal rats, while the sinus rate was not significantly modified. Phentolamine abolished the increase in diastolic tension and reduced the negative effect of hypoxia on contractile force. In YOS rat atria, functional parameters were modified by hypoxia in a qualitatively similar way to that of normal rat atria.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of calcium antagonists on isolated aorta from hypercholesterolemic Yoshida rats of different ages.

1. The in vitro thoracic aorta, precontracted with norepinephrine, KCl or PGF2 alpha of hypercholesterolemic Pittsburg-Yoshida (YOS) and normolipidemic Brown-Norway (BN) rats of two age groups (2 and 18 months), was relaxed by the calcium antagonists verapamil and nifedipine without any difference between age-matched YOS and BN rats. 2. The relaxant activity of verapamil was impaired in aged rats of both strains and with the different contractile agents. Conversely, no variation with aging of the nifedipine relaxing effect was observed on KCl-induced contraction was the nifedipine relaxant effect differently affected by age, both in YOS and BN rats. 3. In conclusion, prolonged exposure to hypercholesterolemia in YOS rat does not affect aortic response to nifedipine and verapamil. Only the aging process was able to affect vascular relaxation to calcium antagonists.

Functional and morphologic characterization of thoracic aorta in heritable hyperlipidemic Yoshida rats of different ages.

We investigated serum and aortic tissue lipid content, in vitro aortic response to drugs, and morphology of thoracic aorta in Pittsburgh Yoshida rats (YOS), a new animal model of endogenous hyperlipidemia. Experiments were performed on 2-, 6-, and 18-month-old rats. Normolipidemic Brown Norway rats (BN) were used as controls. Both serum cholesterol and triglycerides increased significantly with age in YOS rats, but remained constantly low in the control group. In YOS rats, absolute serum concentration of high density lipoprotein (HDL)-cholesterol increased significantly with age, although HDL-cholesterol/total-cholesterol ratio decreased. In contrast, no difference in cholesterol content in aortic tissue was detected between the two animal strains or among different age groups. The contractile force generation of thoracic aorta to norepinephrine (NE) and serotonin increased with age in both strains of animals. The endothelium-dependent relaxation induced by acetylcholine (ACh) was significantly reduced in 6- and 18-month-old YOS as compared with 2-month-old YOS but not in BN. ATP-induced relaxation was significantly impaired in YOS thoracic aorta. In contrast, the relaxation induced by NaNO2 acting in smooth muscle did not vary with age in either YOS or BN. Only alterations in endothelial cells, not typical atheromatous injuries in thoracic aorta wall were detected in YOS even at age 18 months. Our data indicate that despite high serum lipid levels, YOS do not develop typical atheromatous lesions or functional and morphologic damage of smooth muscle cells in thoracic aorta, whereas YOS show decreased endothelium-dependent relaxation and morphologic alteration of endothelial cells.

Purine- and nucleotide-mediated relaxation of rabbit thoracic aorta: common and different sites of action.

The mechanisms of the relaxant effect of purines and pyrimidines in New Zealand rabbit isolated aorta were investigated at endothelial and smooth muscle cell levels. Endothelium-mediated relaxation by ATP was only partially inhibited by the P2-purinoceptor antagonist suramin (0.1 mM). The pyrimidine UTP produced vasodilation by acting at the endothelial level and relaxation was not antagonized by suramin (0.1 mM). This effect was not mediated by P2 purinoceptors, indicating that UTP, like ATP to a certain extent, produces relaxation via an endothelium nucleotide (N) pyrimidinoceptor. ATP, ADP, AMP, adenosine, 5'-N-ethylcarboxamidoadenosine (NECA) and inosine were all active as relaxants on smooth muscle. The NECA relaxant effect was not antagonized by P1-purinoceptor antagonists 3,7-dimethyl-1-propargylxanthine (50 microM) or 1,3-dipropyl-8-(2-amino-4-chlorophenyl)xanthine (5 microM), excluding a P1-mediated effect. P2-related activity was excluded because adenosine-mediated relaxation was not antagonized by suramin (0.1 mM). UTP was ineffective as a relaxant at smooth muscle level, thus excluding the presence of muscular nucleotide (N) pyrimidinoceptor and suggesting a P3 purinoceptor. The rank order of potency of this muscle purinoceptor was NECA > adenosine > ATP approximately equal to ADP approximately equal to AMP approximately equal to inosine.

Protection of atrial function in hypoxia by high potassium concentration.

1. The effects of high extracellular potassium on hypoxia-induced atrial activity and metabolic charge were studied in isolated rat atria. 2. After hypoxia (30 min), contractile tension strongly decreased and diastolic tension increased, while frequency did not change. Adenine nucleotides and creatine phosphate levels did not change, although a significant increase in lactic acid content was observed. 3. High [K+] mostly countered the hypoxia-induced increase in diastolic tension. Moreover, in the presence of high [K+], the hypoxia-induced increase in lactic acid was not significantly different from normoxic controls. 4. Glibenclamide (0.1 microM), a selective K+ATP channel blocker, did not improve the hypoxia-induced depression of atrial function. 5. The physiopathological role of extracellular potassium during cardiac hypoxia is discussed.

Dual effect of ATP and UTP on rat atria: which types of receptors are involved?

The effects of adenine compounds and UTP were examined in electrically driven rat left atria. ATP, ADP, AMP, adenosine and UTP caused a dual inotropic effect: first a rapid decrease in contractility, and second an increase in contractile tension. alpha,beta-Methylene ATP caused an increase in contractile tension only, whereas 2-methylthio-ATP only induced a negative inotropic effect, 1,3-Dipropyl-8-cyclopentylxanthine inhibited the negative effects of ATP and adenosine, whereas 3,7-dimethyl-1-propargylxanthine did not influence the effects of ATP. Suramin but not reactive blue 2 antagonized the positive inotropism induced by ATP and alpha,beta-methylene ATP. Suramin also abolished the positive inotropic effect induced by UTP. These results demonstrate that ATP may induce negative inotropism directly by an action on A1-adenosine receptors and positive inotropism by an action on P2x-purinoceptors. UTP induces a positive inotropic effect mediated by suramin-sensitive receptors.

Atherosclerosis-related remodeling of aortic relaxation to purines in the Watanabe heritable hyperlipidemic rabbit.

The mechanism of the unimpaired relaxant effect of ATP in the Watanabe heritable hyperlipidemic rabbit aorta was investigated to elucidate the involvement of P2y purinoceptor at the endothelial level during atherosclerosis. Experiments were carried out on isolated thoracic aorta from such rabbits that were 12 months of age. The potent P2y purinoceptor agonist, 2-methylthio-ATP, did not induce any endothelium- or smooth muscle-dependent relaxation, thus excluding any involvement by the P2y purinoceptor. ADP, but not AMP, produced relaxation of the aorta by acting at both endothelial and smooth muscle levels. Adenosine relaxed the vessel by acting only in smooth muscle. The maintained endothelial relaxant effect of ATP and ADP is therefore not due to activation of P1 or P2y purinoceptors but may involve activation of a remodeled purinergic receptor site that emerges with the progression of atherosclerosis. This site is antagonized by methylene blue. The disorganization of the endothelial monolayer observed in the morphological analysis may be related to functional remodeling of the endothelial purinergic activity in atherosclerosis.

Platelet aggregation induced by the endoperoxide analogue U46619 is inhibited by polymorphonuclear leukocyte ADPase activity.

Platelet activation by the stable endoperoxide analogue U46619 is mediated largely by ADP released from platelet-dense granules. Polymorphonuclear leukocytes (PMNs) endowed with ecto-ADPase activity may operate as antiaggregatory cells in platelet aggregation induced by U46619. Unstimulated PMNs were effective in reducing aggregation when platelets were stimulated by threshold concentrations of U46619, whereas at higher concentrations of the stimulus, PMN activation is required. Evidence that the inhibition was mediated by PMN ecto-ADPase activity was obtained by high-performance liquid chromatography analysis, indicating that PMNs were able to efficiently metabolize platelet-active ADP into AMP. Moreover, PMN-derived supernatants were able to inhibit platelet aggregation, suggesting that under this circumstance the inhibition was exerted by an uncharacterized, releasable ADPase activity. This study supports the hypothesis that, besides nitric oxide and hydrogen peroxide, ADPase activity may represent another PMN-mediated pathway capable of regulating platelet activity in areas of reduced blood flow, such as those found in conditions of myocardial ischemia.

Species-dependent effects of increasing hypoxia on functions and energy balance in isolated atria.

1. The hypoxia-induced changes of contractile force, frequency and diastolic tension in isolated atria from guinea pig, rat and rabbit were studied together with tissue content of adenine nucleotides, creatine phosphate and lactic acid. 2. Increasing hypoxia induced a progressive reduction of contractility in guinea pig, rat and rabbit atria. Hypoxia also induced a progressive reduction of frequency in rat atria, whereas atrial rate decreased significantly only during drastic hypoxia in guinea pig and rabbit. In spontaneously beating atria, hypoxia increased diastolic tension only in rat. 3. After 30 min of drastic hypoxia, ATP decreased in guinea pig and rabbit but not in rat atria. Creatine phosphate decreased and lactic acid increased in all three species. 4. These data suggest that: (a) hypoxia-induced changes in atrial function and in metabolic content are species-dependent; (b) hypoxia-induced inhibition of atrial activity could represent protection aimed at saving energy.

L-Arginine does not improve endothelium-dependent relaxation inin vitro Watanabe rabbit thoracic aorta.

The findings that even a singlein vitro addition of L-arginine is able to normalize endothelium function in cerebral vessel from diet-induced hypercholesterolemic rabbits prompted us to investigate if similar results could be obtained on Watanabe rabbits thoracic aorta, in which we previously demonstrated low content of the amino acid.L-Arginine (1 mM) preincubated for 45 minutes before the addition of drugs for studing endothelium-dependent vascular relaxation, did not modify the effect of acetylcholine on aortic isolated preparations. The lack of any effect by L-arginine indicates that the amino acid deficiency is not main cause of the impairment of endothelium function. The muscarinic receptor functionality affected by atherosclerotic process and/or the increased synthesis of EDCFs could account for the reduced endothelium-dependent relaxation.

Pharmacological characterization of a new purinergic receptor site in rabbit aorta.

1. The pharmacological properties of a vasodilating purine-activated receptor that is not a P1 or P2-purinoceptor were investigated. 2. In rabbit isolated thoracic aorta precontracted with noradrenaline, ATP induced a 50% relaxation at 0.25 mM (EC 50%); in the absence of endothelium, EC 50% was 2.5 mM. 3. Adenosine induced a relaxation that was not different in both the presence and absence of endothelium, being EC 50% 0.48 and 0.37 mM, respectively. 4. The potent and selective P2y-purinoceptor agonist 2-methylthio-ATP (0.03-10 microM) induced a relaxation only in the presence of endothelium. 5. In de-endothelialized aorta, 8-phenyltheophilline (8-PT: P1 antagonist) and 3,7-dimethyl-1-propargylxanthine (DMPX: A2 antagonist) did not antagonize ATP- and adenosine-induced relaxation. 6. The present data support the presence of a new site of action for purines in rabbit isolated thoracic aorta. 7. A P3 subtype of purinoceptor, that may be identified in the hypothesized "nucleotide" receptor, is proposed.

Pharmacological characterization of ATP receptors mediating vasodilation on isolated rabbit aorta.

1. The effects of adenosine 5'-triphosphate (ATP), 2-methyl-thio-ATP and adenosine on rabbit aorta were examined in isolated preparations precontracted by noradrenaline, both in vessels where the endothelium was present and mechanically removed. 2. In the presence of endothelium, ATP (30 microM-3 mM) induced a relaxation that was reduced by removal of the endothelium. 3. The maximum endothelium-dependent relaxation of ATP was twice the maximum endothelial activity of the potent agonist at P2y-purinoceptors, 2-methyl-thio-ATP. 4. Adenosine which acts on P1 purinoceptors, induced a relaxant effect at 1 mM concentration, both in the vessels with and without endothelium. 5. It is concluded that relaxation by ATP is induced both via the endothelial P2y-purinoceptor and via a "nucleotide" receptor that is located on endothelium and on smooth muscle of rabbit aorta.

Protective role of heparin on in vitro functional aortic response in Watanabe heritable hyperlipidemic rabbits.

The effects of prolonged in vivo heparin treatment upon vasomotor responses and content of cholesterol and energy related compounds were studied in isolated thoracic and abdominal aortas from Watanabe heritable hyperlipidemic (WHHL) rabbits. Unfractionated heparin was administered subcutaneously (2 mg/kg twice a day) to 3-month-old WHHL rabbits for a period of 6 months. A group of WHHL rabbits was treated with saline solution and considered as control. Aortic cholesterol infiltration and serum cholesterol were not significantly decreased by the prolonged heparin treatment. In heparin-treated WHHL rabbits, the in vitro aortic endothelium-dependent relaxation produced by acetylcholine or calcimycin (A 23187) was greater than in saline-treated WHHL group. ATP-induced aorta relaxation (endothelium-dependent and endothelium-independent) did not vary significantly in the two groups of WHHL rabbits, even after mechanical removal of endothelium. Also the noradrenaline-induced aorta contraction did not vary between the two groups of WHHL rabbits. No significant variation in energy-related compounds (except for ADP) was found in the aortic arch. These results suggest that heparin produces a protective effect on aortic tissue by acting mainly at endothelial level.

Retinyl ester hydrolases in retinal pigment epithelium.

In bovine retinal pigment epithelium membranes we have found three hydrolases which were active against trans-retinyl palmitate. This was possible by assaying different subcellular fractions as a function of pH in the range 3-9. Detection of these activities has been favored by the use in the enzyme assay of Triton X-100, which has an activating effect up to a concentration of 0.03% at a detergent-protein ratio of about 1.5-3.0. Apparent kinetic parameters for the retinyl ester hydrolases have been determined after a study of the optimization of assay conditions. Vmax values for hydrolases acting at pH 4.5, 6.0, and 7.0 were, respectively, 156, 55, and 70 nmol/h/mg. To identify the subcellular site for these hydrolytic activities, assays of marker enzymes from various organelles in each subcellular preparation were carried out, demonstrating the lysosomal origin of the pH 4.5 retinyl ester hydrolase and the microsomal origin of the pH 6.0 retinyl ester hydrolase and suggesting that the pH 7.0 retinyl ester hydrolase originates from the Golgi complex.