Evaluation of the cardiovascular effect of 6-nitrodopamine in isolated atrium and perfused rat heart

Abstract

Catecholamines (CAs) are a group of compounds that have the catechol group (3,4-dihydroxybenzene) connected to an amine group by an ethyl bridge. They participate in integrative, neural and endocrine mechanisms acting on the sympathetic-adrenal system. They are synthesized through the amino acid tyrosine, which can be derived from the diet or produced by the liver, and CAs can originate from an endothelial source. These compounds act on alpha and beta-adrenergic receptors, with different responses for each receptor and tissue, being able, for example, to modulate vascular tone. 6-Nitrodopamine (6-ND) has shown prolonged and differentiated effects compared to other catecholamines, causing an increase in isolated atrium heart rate for a longer period before returning to baseline. Objectives: To investigate the physiological role of the new catecholamine 6-nitrodopamine, including: its release in the rat atrium and ventricle; the chronotropic effect of 6-ND, alpha-adrenergic, beta-adrenergic and tricyclic compounds in isolated atrium; the inotropic effect of these drugs on the isolated heart and their effects on heart rate and blood pressure in vitro. Furthermore, to isolate the receptor of this catecholamine using affinity chromatography.Methodology: All experimental protocols were submitted for approval by the Ethics Committee for the Use of Animals of UNICAMP (CEUA/UNICAMP) under No. 5942-1/2022, No. 5557-1/2020 and No.5831-1/2021. Adult male Wistar rats (280 to 320 g) will be randomly assigned to two groups: L-NAME Treated and Control. The first group will receive chronic treatment with nitric oxide synthase inhibitor (L-NAME) in drinking water for 4 weeks (20 mg/rat/day) and the second will receive tap water. The animals will be anesthetized with isoflurane and euthanasia will be performed by exsanguination, previously, for the analysis of the isolated heart, anesthetic with heparin will be administered to prevent blood clotting in the coronary arteries. Three main analyzes will be performed with the animals: 1) solid phase extraction and quantification by liquid chromatography coupled with tandem mass spectrometry (LCMS-8060, Shimadzu) of 6-nitrodopamine; 2) evaluation of concentration-response effects with different compounds, including 6-ND, on the isolated atrium of animals in a PowerLab 400 data acquisition system that records tissue tension strength; and 3) analysis of inotropism in the heart of animals with Langendorff perfusion, in which the left ventricular pressure will be recorded using a pressure transducer and the maximum rates of contraction and relaxation will be calculated. Furthermore, the isolation of the 6-nitrodopamine receptor will be assessed through the technique of affinity chromatography with an agarose column and tritiated 6-ND. (AU)