Evidence That There Are Specific Binding Sites For Estradiol In Csp Membrane Preparations

The presence of specific membrane E binding sites in the CNS has been documented previously (1,28). However, most authors, including us, have used either a crude synaptosomal preparation (29) or purified synaptosomal membranes (28,30) from different regions of the brain. The Kd reported are in the low (1-20 nM) and high (>100 nM) nanomolar range, with variation depending on the type of neural tissue. This is an indication of heterogenous sites most likely corresponding to different estrogen-binding proteins. For instance, using a crude synaptosomal preparation (P2 fraction) we reported (11,29) significantly different Kd values of 3 ± 0.7 (n = 3), 10 ± 1.5 (n = 6) and 34 ± 7 (n = 6) nM for the female rat hypothalamus, olfactory bulb, and cerebellum, respectively. These results suggest that different circulating levels of E may activate differentially these putative mERs in these three particular CNS regions and thereby controlling different brain functions.

Fig. 5. Dose-response to E-6-BSA. Note that 10 nM elicited a maximal percent dopamine release over dopamine levels at interval 3 (pre-infusion). Peak dopamine release is defined as the difference between values at interval 3 and maximal release after the infusion of the complex. N values represent number of superfusion chambers per dose. In one male rat (4 chambers) the 10 nM dose was ineffective, and in two other rats, neither the 25 (4 chambers) nor the 100 nM doses (4 chambers) were effective.

Fig. 5. Dose-response to E-6-BSA. Note that 10 nM elicited a maximal percent dopamine release over dopamine levels at interval 3 (pre-infusion). Peak dopamine release is defined as the difference between values at interval 3 and maximal release after the infusion of the complex. N values represent number of superfusion chambers per dose. In one male rat (4 chambers) the 10 nM dose was ineffective, and in two other rats, neither the 25 (4 chambers) nor the 100 nM doses (4 chambers) were effective.

These neural membrane preparations are by no means "pure" cell membranes and therefore the Kd values and specific binding sites reported are most likely a representation of several estrogen protein binders from different cellular organelles recovered in P2 fractions. Therefore, it was of interest to repeat those binding studies using a relatively enriched cellular membrane preparation obtained by differential centrifugation according to Darnell et al. (31). Herein, we report data on the so called plasmalemma-microso-mal fractions from either the CS or the entire brain (including the CS) of female rats. Plate 1 depicts the ultrastructural features of the three subcellular fractions generated by differential centrifugation as previously mentioned. There are clear-cut electron microscopical differences in these three fractions as expected. First, the so-called P1 or nuclear fraction generated after 10 min of centrifugation at 600g of a brain homogenate contains a variety of organelles indicating a highly contaminated fraction (see A). In contrast, the mP2 fraction (15,000g for 5 min) is enriched in swollen mitochondria showing several degrees of damage, though some large myelin sheaths and synaptic connections are also present (see B). The plasmalemma- microsomal P3 fraction (ultracentrifugation of the supernatant of the P2 fraction for 60-90 min at 125,000g) is enriched in those membrane components with little contamination from mitochondria or other organelles (see C). Note that at a large magnification, the P3 fraction shows membranes undergoing endoexocytosis as indicated by the arrowhead in Plate 1, (see D). The degree of purity of these fractions is also revealed by the amount of protein recovered from the initial brain

Plate 1. P1, mP2, and P3 fractions from rat brains were prepared according to Darnell et al. (31) and pelleted down by ultra centrifugation at 125,000g for 1 h. Then they were fixed in 4% glutaralde-hyde, 0.1 M cacodylate buffer, pH 7.2, overnight at 4°C, followed by washing 3 times, 15 min each. The fractions were subsequently postfixed in 1% osmium tetroxide, 0.1 M cacodylate buffer for 50 min followed by addition of KFeCN to a final concentration of 1.5% for 20 min. The materials were rinsed by the same cacodylate buffer, dehydrated in acetone, infiltrated with Polybed 812, and finally in fresh epoxy for polymerization at 60°C for 3 d. Thin sections of these fractions were then examined with a Hitachi H-600 electron microscope at different magnifications. Panels A, B, and C correspond to a typical sample from P , mP and P , respectively. Panel D is a higher magnification of the P3 pellet. The scale bar is 1 ¡¡m for A, B, and C and 0.2 ¡m for D.

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