L. Pandolfo

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. α,β-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-l-propargylxanthine did not influence the effects of ATP. Suramin but not reactive blue 2 antagonized the positive inotropism induced by ATP and α,β-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.

Effect of Age on Rabbit Aortic Responses to Relaxant Endothelium-Dependent and Endothelium-Independent Agents

The aim of this study was to evaluate the effects of aging on endothelium-dependent and endothelium-independent relaxation of rabbit thoracic aorta from New Zealand white rabbits aged 4-6 and 7-12 months. The contractile response to noradrenaline (NA) decreased with increasing age, but NA [EC50] did not vary significantly. Acetylcholine (Ach)-induced relaxation of aortic rings precontracted with NA [EC50] did not change significantly with increasing age. The relaxation induced by ATP of aortic rings, precontracted with NA [EC50], was significantly greater in young than in adult rabbits. This difference between young and adult animals became more evident in aortic rings deprived of endothelium: in adult animals, the ATP-induced relaxation of aortic rings with endothelium was significantly greater than in the rings without endothelium. The endothelium-independent relaxation by sodium nitrite (NaNO2) at lower concentrations was significantly greater in young than in adult rabbit aortic rings precontracted with NA [EC50]. Concluding, the age-induced changes in vascular response in male New Zealand white rabbits are related to an impaired mechanism at smooth muscle level.

Aortic response to relaxing agents in Watanabe heritable hyperlipidemic (WHHL) rabbits of different age

Serum and aortic tissue cholesterol levels in parallel with aortic relaxation to endothelium-dependent and independent drugs were determined in Watanabe heritable hyperlipidemic (WHHL) rabbits in comparison with New Zealand (N.Z.) normocholesterolemic rabbits, aged 4-14 months. Serum cholesterol was elevated (626 +/- 99 mg/100 ml) in 4-6-month-old WHHL rabbits and significantly lower in 12-14-month-old animals (344 +/- 51 mg/100 ml). Cholesterol infiltration in thoracic aorta was high in young WHHL compared with N.Z. rabbits (0.88 +/- 0.3 mg/100 mg fresh tissue vs. 0.08 +/- 0.003 mg/100 mg, respectively) and it did not vary with age. In N.Z. rabbits, serum and aortic cholesterol levels were low from 4 to 14 months of age. The aortic relaxation to acetylcholine (0.03-3 microM) on EC50 noradrenaline precontracted rings was similar in 4-6-month-old WHHL and N.Z. rabbits of the same age. In WHHL rabbits, the relaxation to acetylcholine was significantly reduced in 7-11- (-35% at maximum) and in 12-14-month-old rabbits (-40% at maximum). In N.Z. rabbits the response to acetylcholine was not modified in the 3 age groups. The relaxation to ATP (30 microM to 3 mM) was reduced by age both in N.Z. and in WHHL rabbits, but in 12-14-month-old WHHL rabbits the maximal relaxing response was significantly more elevated than in age-matched N.Z. rabbits (50.1 +/- 2.5% vs. 35.1 +/- 3.2%, respectively). The aortic relaxation to NaNO2 (10 microM to 3 mM) was reduced by age both in N.Z. and in WHHL rabbits.(ABSTRACT TRUNCATED AT 250 WORDS)

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

Abstract— 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 μm) or 1,3‐dipropyl‐8‐(2‐amino‐4‐chlorophenyl)xanthine (5 μm), 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 ≅ ADP ≅ AMP ≅ inosine.

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

Summary: 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.

Potassium channel blockers differentially affect carbachol and (-)-N6-phenylisopropyladenosine on guinea-pig atria.

1 The effect of three different potassium channel blockers (tetraethylammonium, TEA; 4‐aminopyridine, 4‐AP; and apamin) and of variations in the concentration of K+ and Ca2+ in the medium, have been studied on the responses of guinea‐pig isolated atria to (−)‐N6‐phenylisopropyladenosine (R‐PIA), a stable adenosine A1‐receptor agonist, and to carbachol, a muscarinic agonist. R‐PIA and carbachol showed the same negative inotropic effects over a similar range of concentrations (3–300 μm), both in spontaneously beating and in electrically driven atria. 2 TEA (0.1 to 20 mm) and 4‐AP (0.3 to 3 mm), both antagonized the negative inotropic and chronotropic effects of carbachol in a concentration‐dependent manner. In contrast, these compounds failed to inhibit the effects induced by R‐PIA. Apamin, a specific blocker of a low conductance Ca2+‐activated K+ channel, was ineffective in accordance with the absence of these channels in atrial tissue. 3 TEA (0.1 to 20 mm) inhibited the negative inotropic effect of carbachol, but not that of R‐PIA, in atria paced and depolarized by a high K+ medium (22mm). In this preparation Na+ current is abolished and the contraction induced by noradrenaline and electrical stimulation is solely dependent on Ca2+ influx currents. 4 Stepwise addition of Ca2+ to a calcium‐depleted perfusing medium of electrically driven atria, induced a positive inotropic effect which was inhibited by R‐PIA. In contrast, carbachol had no effect. 5 In agreement with our previous study, the data suggest that R‐PIA acts on isolated atria by inhibiting Ca2+ influx through L‐channels.

Effect of selective agonists and antagonists on atrial adenosine receptors and their interaction with Bay K 8644 and [3H]‐nitrendipine

1 (−)‐N6‐phenylisopropyladenosine (R‐PIA) and N6‐cyclohexyladenosine (CHA), highly selective agonists at A1‐adenosine receptors, 5′‐N‐ethyl‐carboxamidoadenosine (NECA), a non‐selective agonist at A1 and A2 receptors, and 2‐phenylaminoadenosine (CV‐1808), a selective A2 agonist, were compared in spontaneously beating and electrically driven atria. R‐PIA, CHA and NECA inhibited contraction in both preparations. CV‐1808 was not effective up to 500 nM. 2 1,3‐Dipropyl‐8‐cyclopentylxanthine (DPCPX), a new selective A1 receptor antagonist, competitively inhibited the effects of the adenosine agonists, at low concentrations (IC50 < 1 nm). 3 CHA and NECA were able to inhibit the positive inotropic effect of Bay K 8644 both in spontaneously beating and in electrically driven atria. 4 R‐PIA, CHA and NECA (agonists), 8‐phenyltheophylline (PT) and DPCPX (antagonists), failed to influence [3H]‐nitrendipine binding on microsomal membranes from guinea‐pig atria and ventricles in a range of concentrations from 1 nM to 100 μm. 5 The data support the existence of A1 receptors in atrial tissue. No evidence for a direct interaction between adenosine analogues and Bay K 8644 was found at the level of slow calcium channels. Adenosine analogues appear to antagonize the effects of Bay K 8644 indirectly by activation of A1 receptors.

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 a1-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 α1-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 α1/\ ratio increased from 0.40 in normoxia to 1.06 in hypoxia. In normoxic atria from YOS rats, the α1/\ ratio was already elevated (0.86) in comparison to control rats (mainly due to a higher density of at-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.The observed increase in α1-adrenoceptor density during hypoxia is in accordance with the results of experiments with animal models of the ischaemic heart and with findings in human heart failure. The possible therapeutic significance of these data is considered.

Atrial bioenergetic variations in moderate hypoxia: danger or protective defense?

SummaryThe effects of hypoxia on contractile tension and on tissue adenylate pool content, nicotinamide adenine nucleotide, NAD, nicotinamide adenine dinucleotide phosphate, NADP, and creatine phosphate, CrP, were investigated in isolated, spontaneously beating, guinea pig atria. When two different degrees of hypoxia were induced by lowering oxygen tension from 95% O2 (control) to 40% (moderate hypoxia) and 20% (severe hypoxia) for 30 min, contractile tension slowly decreased to 60% and 40% of control, respectively. In 40% O2 hypoxic atria, ATP was not significantly decreased, AMP slightly increased, TAN (total adenylate nucleotides) and adenylate energy charge [(ATP+0.5ADP)/(ATP+ADP+AMP)] did not change and creatine phosphate was decreased down to 53%. Hypoxic atria in 20% O2 showed a significant decrease of 26% in ATP, while ADP and AMP increased four and seven times, respectively. The adenylate energy-charge value was reduced from 0.93 to 0.70. Creatine phosphate decreased to below the analytical detection limit.Moderate hypoxia (40% O2), which induced a significant decrease of contractile tension but only minor changes of energetic tissue metabolism, was further investigated 2, 5, and 10 min after low oxygen tension was applied. Two stages of variations were evident during 30 min of experimental hypoxia. Within the first 10 min, concomitantly with atrial tension decrease, ATP, NAD, NADP, ATP/AMP, ATP/ADP, and TAN decreased, CrP began to decrease, inosine and xanthine showed no significant change. During the following 20 min of hypoxia, all parameters returned to the control levels with the exception of creatine phosphate. Adenylate energy charge did not change.The electrophysiological analysis of atrial cells did not show any major change in action potential configuration and resting potential, during 40% O2 hypoxia.The differences at metabolic level between moderate and more severe hypoxia suggest that the energetic state may be extremely unbalanced, in atrial tissue, as long as hypoxia is aggravated. Moreover, the time-course study, during 30 min of 40% O2, suggests that the early decrease of contractile tension does not depend on lowered energy availability, instead it might be, at least in part, a preventive measure to maintain energy balance in myocardial tissue to counteract hypoxic damage and, in this mechanism of defense, creatine phosphate shuttle seems to play a relevant role.

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.