calcimycin and alpha-beta-methyleneadenosine-5--triphosphate

The contracting and relaxing effects of purines and UTP were investigated on rings of mouse thoracic aorta in vitro. UTP, ATP gamma S, and alpha-beta-Methyleneadenosine 5'triphosphate contracted rings with and without endothelium. On the contrary, adenosine, AMP, ADP, ATP, and 2-(methylthio)adenosine 5'-diphosphate had no effect on relaxed rings. When rings were tonically contracted by U46619 a thromboxane A2 analogue, ATP, ADP, ATP gamma S, 2-(methylthio)adenosine 5'-diphosphate, and UTP caused endothelium-dependent but not independent relaxations.I conclude that ATP acts on P2Y2 and P2Y1 receptors on the endothelial cells to cause endothelium-dependent relaxation. In this tissue, the relaxing effect of ATP dominates by endothelium-dependent ways when aorta rings are contracted by a stable thromboxane A2 analog. However receptors mediating contraction in response to purines and pyrimidines are present on smooth muscle cells. Indeed, the stimulation of P2Y receptors by UTP as well as the activation of P2X family receptors by ATP gamma S causes a contraction. The potential contractile effect of ATP seems masked by its hydrolysis by ectonucleotidases.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Acetylcholine; Adenosine; Adenosine Diphosphate; Adenosine Monophosphate; Adenosine Triphosphate; Animals; Aorta, Thoracic; Biological Factors; Calcimycin; Dose-Response Relationship, Drug; Endothelium, Vascular; Male; Mice; Mice, Inbred ICR; Muscle Contraction; Muscle Relaxation; Muscle, Smooth, Vascular; Nitroglycerin; Phenylephrine; Receptors, Purinergic P2; Uridine Triphosphate

The ATP-induced change in the tryptophan fluorescence of the Ca(2+)-ATPase was determined with sarcoplasmic reticulum vesicles at pH 7.0 in the presence of Ca2+ under various conditions by steady-state measurements and stopped-flow spectrofluorometry. Formation of the phosphoenzyme intermediate was also determined by the continuous flow-rapid quenching method. The steady-state fluorescence at 0 degrees C decreased by 1.1% on addition of ATP, whereas no fluorescence change was induced by adenosine 5'-(beta,gamma-methylene)triphosphate (a nonhydrolyzable ATP analog incapable of phosphorylating the enzyme). The time course of the ATP-induced fluorescence drop agreed well with that of the phosphoenzyme formation under all of the conditions tested, and the phosphoenzyme formed was largely sensitive to ADP. When phosphoenzyme isomerization from the ADP-sensitive form to the ADP-insensitive form was almost completely prevented by N-ethylmaleimide treatment, the time course of the ATP-induced fluorescence drop again agreed with that of the phosphoenzyme formation. These results show that the ATP-induced fluorescence drop occurs upon formation of the ADP-sensitive phosphoenzyme. The results further indicate that the tryptophan fluorescence of this enzyme is insensitive to the conformational change which was previously shown (Suzuki, H., Obara, M., Kuwayama, H., and Kanazawa, T. (1987) J. Biol. Chem. 262, 15448-15456) to occur upon formation of the calcium-enzyme-substrate complex. Thus, we conclude that the ATP-induced drop in the tryptophan fluorescence reflects a conformational change occurring upon formation of the ADP-sensitive phosphoenzyme.

Topics: Adenosine Triphosphate; Animals; Calcimycin; Calcium; Calcium-Transporting ATPases; Egtazic Acid; Fluorescence; Phosphorylation; Protein Conformation; Protein Kinases; Rabbits; Sarcoplasmic Reticulum; Tryptophan