Vasodilation and radical-scavenging activity of imperatorin and selected coumarinic and flavonoid compounds from genus Casimiroa.

Hypertension is a very widespread condition which is not strictly considered as an illness but if not countered, progressively causes damage to all tissues and loss in their functionality. For this reason the find of new antihypertensive agents is prominent and medicinal plants and their derivatives are valuable for the purpose. The genus Casimiroa (Rutaceae) includes plants from Central America and Mexico; among these, Casimiroa edulis Llave et Lex. and Casimiroa pubescens Ramirez are the most relevant species, even for their medicinal uses. The decoction of leaves and seeds is traditionally taken as a tea mainly to lower blood pressure. The object of this research was the study of vascular activity of coumarinic and flavonoid compounds isolated from seeds of Casimiroa spp. in comparison with Casimiroa edulis and Casimiroa pubescens extracts. The phenolic compounds isolated from Casimiroa were herniarin (Her), imperatorin (Imp), 8-geranyloxypsoralen (GOP) and 5,6,2',3',4'-pentamethoxyflavone (PMF). All these compounds induced vasorelaxation on rat arterial tissues although with different effectiveness. To study the cellular mechanisms of the vasorelaxation exhibited by imperatorin, we used selective inhibitors of different receptors and enzymes, such as atropine, pyrilamine, nifedipine, L-NAME and DETC. In a further step of this research, we evaluated the radical-scavenging activity of Casimiroa extracts and isolated compounds by means of DPPH assay. In general, we observed that the scavenging activities increased in a concentration-dependent manner for all substances. The phenolic compounds highlight a synergism of vasodilation and antioxidant activity which may be very useful in the management of cardiovascular diseases. Among the evaluated compounds, imperatorin shows a significant vasorelaxant activity even higher than acetylcholine and similar to nitrite, and also useful antiradical capabilities. All these properties suggest its possible role against hypertension and vasculopathies, even if in vivo studies are needed to determine the actual applications.

A case of urinary incontinence by hydroxychloroquine in a geriatric patient.

WHAT IS KNOWN AND OBJECTIVE: Rheumatoid arthritis is an autoimmune disorder characterized by persistent synovitis and systemic inflammation. Genetic factors account for approximately 50% of cases of rheumatoid arthritis and environmental factors include smoking. Urinary incontinence may occur as a medication adverse effect. We present the first report of a case of hydroxychloroquine-induced urinary incontinence in rheumatoid arthritis. DETAILS OF THE CASE: A 71-year-old female with a history of rheumatoid arthritis developed urinary incontinence as an adverse drug reaction to hydroxychloroquine administered at therapeutic doses. Urinary incontinence remitted with drug withdrawal and reappeared on rechallenge. The Naranjo's algorithm indicated that hydroxychloroquine was a probable cause of this adverse drug reaction. The likely mechanism of this adverse drug is a direct action of the quinolone on the urinary system. WHAT IS NEW AND CONCLUSION: This is the first report of hydroxychloroquine-induced urinary incontinence. The absence of previous reports suggest that the drug rarely causes this adverse effect. Methotrexate is most often used as first-line treatment, and several other drugs are now available to act as Disease-Modifying Antirheumatic Drugs (DMARDs). These drugs may be used alone or combined with methotrexate, most often to increase efficacy and reduce toxicity. The introduction of new biological agents, such as abatacept, rituximab, tocilizumab and inhibitors of tumour necrosis factor, has opened new therapeutic perspectives but are restricted by high costs and risk of infections. Thus, antimalarial drugs, especially the quinolones chloroquine (CQ) and hydroxychloroquine (HCQ), are still in use, and the latter is very efficacious. An advantage of HCQ is its low toxicity compared with other antimalarial drugs. Common side-effects of HCQ and the other antimalarial drugs include gastrointestinal effects such as nausea and vomiting, as well as skin rashes and headache, whereas their most common and severe side-effect is retinopathy. No case of urinopathy has been reported previously with HCQ.

"Metabolic reprogramming" in ovarian cancer cells resistant to cisplatin.

The way cancer cells escape cisplatin-induced apoptosis has not been completely elucidated yet. We questioned the relevance of "metabolic reprogramming" in cisplatin-resistance by studying mitochondrial function and metabolism in human ovarian carcinoma cell lines, both cisplatin-sensitive (2008) and resistant (C13). C13 cells, in comparison to 2008 cells, showed lower apoptotic response to cisplatin exposure, lower basal oxygen consumption (4.2±0.2 vs 6.5±0.7 fmol/cell/min, p<0.005) and a lower basal transmembrane mitochondrial potential (Δψm) (18.7±1.5 vs 32.2±1 MFI p<0.001). Moreover, C13 cells showed a lower sensitivity to rotenone and oligomycin, two mitochondrial respiratory chain inhibitors. To further investigate the impact of mitochondria on cisplatin-resistance, 2008 and C13 cells were depleted of their mitochondrial DNA (rho(0)-clones). The cytotoxicity of cisplatin was lower in 2008-rho(0)clones than in 2008 cells (IC(50) of 3.56 µM and 0.72 µM, respectively) but similar between C13-rho(0) and C13 cells (IC(50) of 5.49 µM and 6.49 µM, respectively). The time-course of cell viability in glucose-free galactose medium indicated that C13 cells are more strictly dependent on glucose than 2008 cells. (1)H-NMR spectroscopy showed a higher basal content of intracellular glutathione (GSH) and mobile lipids (MLs) in C13 cells as compared to 2008 cells, with higher lipid accumulation mainly within cytoplasmic droplets of the C13 cells. These findings allow us to propose a "metabolic remodelling" of ovarian carcinoma cells to a lipogenic phenotype, which includes alteration of mitochondrial function, as an advantageous mechanism to escape cisplatin-induced apoptosis. This hypothesis is of interest to exploit new pharmacological targets to improve the clinical impact of platinum drugs.

Activity of sap from Croton lechleri on rat vascular and gastric smooth muscles.

The effects of red sap from Croton lechleri (SdD), Euphorbiaceae, on vascular and gastric smooth muscles were investigated. SdD, from 10 to 1000 microg/ml, induced concentration-dependent vasoconstriction in rat caudal arteries, which was endothelium-independent. In arterial preparations pre-constricted by phenylephrine (0.1 microM) or KCl (30 mM), SdD also produced concentration-dependent vasoconstriction. To study the mechanisms implicated in this effect we used selective inhibitors such as prazosin (0.1 microM), an antagonist of alpha(1)-adrenoceptors, atropine (0.1 microM), an antagonist of muscarinic receptors, and ritanserin (50 nM), a 5-HT(2A) antagonist; none of these influenced vasoconstriction caused by SdD. Likewise, nifedipine (50 nM), an inhibitor of L-type calcium channels, did not modify the action of SdD. Capsaicin (100 nM), an agonist of vanilloid receptors, also did not affect vasoconstriction by SdD. We also investigated the action of SdD (10-1000 microg/ml) on rat gastric fundus; per se the sap slightly increased contractile tension. When the gastric fundus was pre-treated with SdD (100 microg/ml) the contraction induced by carbachol (1 microM) was increased, whereas that by KCl (60mM) or capsaicin (100 nM) were unchanged. The data shows that SdD increased contractile tension in a concentration-dependent way, both on vascular and gastric smooth muscles. The vasoconstriction is unrelated to alpha(1), M, 5-HT(2A) and vanilloid receptors as well as L-type calcium channels. SdD increased also contraction by carbachol on rat gastric fundus. Thus for the first time, experimental data provides evidence that sap from C. lechleri owns constricting activity on smooth muscles.

Cell-cycle inhibition and apoptosis induced by curcumin and cisplatin or oxaliplatin in human ovarian carcinoma cells.

UNLABELLED: Alteration of appropriate cell-cycle progression and of closely related apoptotic process is a basic feature of tumour cells, and development of new tumour-targeted agents focus on apoptosis, either during cell-cycle arrest or following premature cell-cycle checkpoint exit. Increasingly, epidemiological and experimental studies suggest that curcumin protects against cancer, not only because of its well-known antioxidant properties, but also because it modulates intracellular signalling, which is related to cell proliferation and apoptosis. Cisplatin and oxaliplatin are first-line drugs in treatment of many types of epithelial cancer and their combination with other cytostatics are under investigation to limit their side effects and resistance to them. OBJECTIVES: The aim of this study was to evaluate effects of a combined treatment using curcumin with cisplatin or with oxaliplatin, in a human ovarian cancer cell line (2008) and in its cisplatin-resistant variant (C13). RESULTS: Curcumin per se caused concentration-dependent (0.1-100 microm) and time-persistent (24-72 h) reduction in cell proliferation, as well as altered cell cycle parameters and induced apoptosis, in both cell lines. When carcinoma cells were simultaneously exposed to curcumin and to cisplatin or oxaliplatin (at concentrations lower than IC(50)) cell viability was reduced more than with single-drug treatment. Moreover, dose and time related effects of curcumin, when combined with platinum drugs, were linked to consistent reduction in cell cycling and increased apoptosis, in comparison with single-drug treatment. These effects were significant both in wild type and in cisplatin-resistant cells, indicating that curcumin was also able to increase sensitivity of resistant ovarian cancer cells to cisplatin. CONCLUSIONS: The data suggests that curcumin is an interesting natural compound capable of limiting cell proliferation and possibly increasing clinical impact of platinum drugs, in ovarian cancer patients.

Do ATP and UTP involve cGMP in positive inotropism on rat atria?

ATP and UTP induced a dual inotropic effect in rat left atria: first a decrease and then an increase in contractile tension were observed. PPADS, an antagonist of P2X receptors, inhibited positive inotropism induced by ATP and alpha,beta-meATP. Chiefly, we investigated intracellular mechanisms responsible for the positive inotropism. We tested cromakalim and glibenclamide, an activator and an inhibitor, respectively, of ATP-sensitive K(+) channels. These compounds did not influence the effects of ATP. IBMX, a phosphodiesterase inhibitor, and H-7, an inhibitor of protein kinase C and cAMP-dependent protein kinase, did not modify the inotropic effects of ATP. Instead, H-8, an inhibitor of cAMP- and cGMP-dependent protein kinases, strongly inhibited the positive effects of both ATP and UTP, suggesting the possible involvement of cGMP in the inotropism. Also, LY 83583, an inhibitor of cGMP production, reduced positive inotropism by alpha,beta-meATP, ATP and UTP. Moreover, 8-Br-cGMP (50 microM), a stable analogue of cGMP, inhibited positive inotropism by all nucleotides. Lastly, we determined intracellular cGMP levels by RIA; the cyclic nucleotide increased during positive inotropism induced by ATP and UTP. The results regarding positive inotropism suggest that: (a) ATP acts through P2X receptors, while UTP may act by P2X, but also through PPADS-insensitive receptors; and (b) changes in intracellular cGMP concentration are involved in this inotropic effect.

6-Benzylaminopurine: a plant derived cytokinin inducing positive inotropism by P2-purinoceptors.

Positive inotropic effects induced by 6-benzylaminopurine (6-BAP), kinetin and zeatin were studied in rat atria. The potency order observed was 6-BAP > or = kinetin > zeatin. Suramin, a P2-purinoceptor antagonist, inhibited the positive effect of 6-BAP suggesting the involvement of P2-purinoceptors in the positive effect of this cytokinin. In order to elucidate this point, 6-BAP was used against R-PIA (a P1-purinoceptor agonist) and ATP and UTP (both P2-purinoceptor agonists). 6-BAP did not influence negative inotropism by R-PIA whereas both nucleotides were inhibited after pretreatment with the cytokinin. LY 83583, an inhibitor of cGMP production, reduced the inotropic effect by cytokinin whereas L-NAME, an inhibitor of the L-arginine/nitric oxide pathway, did not influence the effect induced by 6-BAP. Indomethacin, an inhibitor of cyclooxygenase, and neomycin, an inhibitor of phospholipase C, did not significantly modify positive inotropism by 6-BAP. Verapamil, an inhibitor of L-type calcium channels, did not change the positive effect of 6-BAP while TMB-8 and dantrolene, two inhibitors of intracellular calcium release, reduced the increase of contractile tension induced by cytokinin. Our data on rat atria suggest that 6-BAP causes a positive inotropism through activation of P2-purinoceptors, involving modification of cGMP and of intracellular calcium.

Pharmacological characterization of endothelial cell nitric oxide synthase inhibitors in isolated rabbit aorta.

Different receptors mediating the release of endothelium-derived nitric oxide (EDNO) have been identified at endothelial level. In the present study we aimed to characterise, on rabbit aorta by means of pharmacological tools, the generation of EDNO by receptors located on endothelial cell membrane (M3, P2u, P2y) and by direct activation of Ca2+ entry into the endothelial cell. Four vasodilating drugs were tested (acetylcholine, UTP, A23187 and 2-methyl-thio-ATP); they were active only if the endothelial layer was intact, suggesting that they act through endothelial receptors. The effect of different nitric oxide synthase (NOS) inhibitors (0.1 mM: L- and D-NAME, L-NMMA, L-NIO and 7-NI) was investigated on NO-mediated relaxation induced by the relaxants in vessels with intact endothelium. NOS inhibitors differently affected relaxation mediated by the vasoactive drugs in isolated rabbit aorta. Reversibility of the inhibition by using a fixed concentration of L-arginine (0.1 mM) was different depending on the relaxing drug and NOS-inhibitor. The data obtained support the coexistence in aortic vessel of more than one endothelial cell NOS isoform, each provided with different receptor coupling.

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.

P2X-purinoceptors in the heart: actions of ATP and UTP.

Positive inotropic effects of ATP and UTP (1 microM - 1mM) were studied in isolated rat and guinea pig cardiac tissues. The potency order obtained was ATP>UTP in both species, suggesting possible interaction with P2X-purinoceptors. Binding studies using [(3)H]alpha,beta-methylene ATP as marker of P2X-purinoceptors revealed two receptor sites: one high-, the other low-affinity, in atria and ventricles from rat and guinea pig. Both ATP and UTP were found to bind high-affinity sites of [(3)H]alpha,beta-methylene ATP. The effects of various calcium inhibitors such as nifedipine, dantrolene, ryanodine and TMB-8 on positive inotropic effects induced by ATP and UTP were also studied. The results suggest that ATP and UTP may increase inotropism by interaction with P2X-purinoceptors by means of a calcium-dependent mechanism.

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.

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.