EVIDENCE FOR SOLUBLE AND NUCLEAR SITE-I BINDING OF ESTROGENS IN HUMANAORTA

The purpose of this study was to establish the estrogen receptor (ER) expression and content in human aorta fragments removed at the time of by-pass surgery. To this end, we adopted a radioligand binding assay to evaluate either soluble (S) or nuclear (N) ER using dextran-coated charcoal (DCC) and filtration methods, respectively. To better define the intratissular distribution and content of ER, we also measured the presence of a 27 kDa heat shock protein (HSP27), a well established E R-associated protein, using D5 monoclonal antibody. Finally, we analys ed the different molecular isoforms of both S and N ER using size excl usion-high performance liquid chromatography (SE-HPLC). High affinity (type I) sites of estrogen binding were detected in 17 out of 19 sampl es in either S or N fraction, although only 9 out of 19 cases displaye d site I ER in both cell compartments. ER levels in aortic tissues, de tected by radioligand method, compare well with those we have found in other hormone-sensitive human cancer tissues and cells. SE-HPLC analy sis revealed two main receptor isoforms in the soluble fraction, havin g 65 kDa and 18 kDa molecular mass, while a minor component of 29 kDa was also found; the nuclear fraction displayed again two major compone nts of 38 and 23 kDa. Using the HSP27 immunohistochemistry we observed a major staining occurring in smooth muscle cells (SMC), with an incr easing intensity towards the lumen. All samples, including the ER nega tive ones, exhibited some degree of histochemical staining. Using an a rbitrary cut-off value, 7 out of 12 samples displayed a highly positiv e staining, 6 of which showed nuclear ER. Furthermore, SE-HPLC separat ion indicated the presence of a 64.9 kDa component in the soluble frac tion, according to the well known relative molecular mass of ER. Follo wing HSP27 immunohistochemistry, the overall staining intensity in aor tic SMC approaches that seen in endometrial and breast epithelia, whil st the muscle ER content is generally lower. Although our data are com patible with a direct role of estrogens in arterial function, the exte nt of the link with arterial disease remains to be established.