Characterization of the O-methylation of catechol oestrogens by intact rabbit thoracic aorta and subcellular fractions thereof

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Abstract
1. In the present study we investigated the O-methylation of catechol oestrogens by intact rabbit thoracic aorta and subcellular fractions thereof. 2. 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 μmol/l and 104 pmol g−1 min−1, respectively, when 2OHE2 was used as substrate; and 1.14 μmol/l and 188 pmol g−1 min−1 when 2OHE3 was used as substrate. 3. The inhibitors of the extraneuronal uptake process (viz; phenoxybenzamine 33 μmol/l; normetanephrine, 46 μmol/l; and deoxycorticosterone acetate 27 μmol/l) failed to inhibit the O-methylation of either 2OHE2 (3.4 μmol/l) or 2OHE3 (3.4 μmol/l) in intact segments of the rabbit thoracic aorta. 4. (-)-Isoprenaline (40 μmol/l) abolished the O-methylation of 2OHE2 (3.4 μmol/l) and markedly reduced that of 2OHE3 (3.4 μmol/l). Pretreatment of tissues with phenoxybenzamine (33 μmol/l) partially restored the O-methylation of 2OHE2 and 2OHE3 in the presence of (-)-isoprenaline (40 μmol/l). 5. The O-methylation of 2OHE2 (5 μmol/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. 6. 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. 7. 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 μmol/l; Vmax: 112 pmol g−1 min−1) and microsomal (Km: 0.15 μmol/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 μmol/l; Vmax: 174 pmol g−1 min−1) and membranal (Km: 0.91 μmol/l; Vmax: 105 pmol g−1 min−1) fractions were very different. 8. 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. Moreover, the catechol oestrogens gain entry to sites of O-methylation by a process independent of extraneuronal uptake; this process is assumed to be diffusion. The results high-light the role of membrane-bound COMT in the O-methylation of isoprenaline and both membrane-bound and soluble COMT in the O-methylation of catechol oestrogens.