Characterization of the O-methylation of catechol oestrogens by intact rabbit thoracic aorta and subcellular fractions thereof.
In the present study we investigated the O-methylation of catechol oestrogens by intact rabbit thoracic aorta and subcellular fractions thereof. The O-methylation of 2-hydroxyoestradiol (2OHE2) and 2-hydroxyoestriol (2OHE3) displayed saturation kinetics in the intact tissue. The apparent Km and Vmax values for the O-methylation of 2OHE2 were determined to be 0.91 mumol/l and 104 pmol g-1 min-1, respectively, when 2OHE2 was used as substrate; and 1.14 mumol/l and 188 pmol g-1 min-1 when 2OHE3 was used as substrate. The inhibitors of the extraneuronal uptake process (viz; phenoxybenzamine 33 mumol/l; normetanephrine, 46 mumol/l; and deoxycorticosterone acetate 27 mumol/l) failed to inhibit the O-methylation of either 2OHE2 (3.4 mumol/l) or 2OHE3 (3.4 mumol/l) in intact segments of the rabbit thoracic aorta. (-)-Isoprenaline (40 mumol/l) abolished the O-methylation of 2OHE2 (3.4 mumol/l) and markedly reduced that of 2OHE3 (3.4 mumol/l). Pretreatment of tissues with phenoxybenzamine (33 mumol/l) partially restored the O-methylation of 2OHE2 and 2OHE3 in the presence of (-)-isoprenaline (40 mumol/l). The O-methylation of 2OHE2 (5 mumol/l) was significantly reduced in segments of aorta in which the endothelium was removed. The latter reduction could not be attributed to damage to components of the vessel media. The O-methylation of 2OHE2 and (-)-isoprenaline by subcellular fractions of the rabbit aorta also was examined. Both the microsomal and cytosolic fractions were shown to O-methylate 2OHE2 and (-)-isoprenaline, providing evidence for the existence of membrane-bound and soluble forms of COMT in the rabbit aorta. The O-methylation of 2OHE2 by cytosolic and microsomal fractions of the aorta was determined and compared to that of (-)-isoprenaline. The kinetic constants for the O-methylation of 2OHE2 by cytosolic (Km: 0.27 mumol/l; V max: 112 pmol g-1 min-1) and microsomal (Km: 0.15 mumol/l; Vmax: 161 pmol g-1 min-1) fractions were similar. In contrast, the kinetic constants for the O-methylation of isoprenaline by cytosolic (Km: 121 mumol/l; Vmax: 174 pmol g-1 min-1) and membranal (Km: 0.91 mumol/l; Vmax: 105 pmol g-1 min-1) fractions were very different. It is concluded that catechol oestrogens are excellent substrates for catechol-O-methyltransferase (COMT) in the rabbit aorta. Their O-methylation can occur in endothelial structures as well as in the smooth muscle-containing medial sections of the vessel.(ABSTRACT TRUNCATED AT 400 WORDS)
D000818:Animals; D001013:Aorta, Thoracic / Q000378:metabolism; D002394:Catechol O-Methyltransferase / Q000378:metabolism; D004727:Endothelium / Q000378:metabolism; D004967:Estrogens / Q000378:metabolism*; D002393:Estrogens, Catechol / Q000378:metabolism*; D066298:In Vitro Techniques; D007545:Isoproterenol / Q000494:pharmacology; D007700:Kinetics; D008745:Methylation; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D010643:Phenoxybenzamine / Q000494:pharmacology; D011817:Rabbits; D013347:Subcellular Fractions / Q000378:metabolism*
D004967:Estrogens; D002393:Estrogens, Catechol; D010643:Phenoxybenzamine; D002394:Catechol O-Methyltransferase; D007545:Isoproterenol