Reduction of no-reflow and reperfusion injury with the synthetic 17ß-aminoestrogen compound Prolame is associated with PI3K/Akt/eNOS signaling cascade.

A high proportion of primary percutaneous coronary interventions performed in the setting of acute myocardial infarction, concur with inadequate myocardial perfusion at the microvascular level. This phenomenon, known as "no-reflow" contributes to reperfusion injury, poor prognosis and to unfavorable clinical outcome. In this study, we evaluated the hypothesis that the synthetic 17ß-aminoestrogen Prolame, may confer cardioprotection and prevent against no-reflow. In an open-chest model of 30-min ischemia and 90-min reperfusion, male Wistar rats were randomly assigned to different groups: Control, Prolame, Prolame followed by the nitric oxide synthase inhibitor (L-NAME), and 17ß-estradiol. Areas of risk, infarct size and no-reflow were determined by planimetry with triphenyltetrazolium chloride and thioflavin-S stains. Structural damage of the vasculature was measured as capillary compression in clarified tissue after intra-atrial injection of Microfil. Hemodynamic function was obtained at the end of stabilization, ischemia and reperfusion; nitric oxide (NO·) content was determined indirectly using the Griess reaction. Activation of the eNOS signaling cascade was determined by western blot. Prolame reduced the infarcted area, decreased the zones of no-reflow and capillary compression by activating the PI3K/Akt/eNOS signaling pathway in correlation with NO· increase. Prolame also activated endothelial cells augmenting NO· production, which was inhibited by ICI182780 (a selective estrogen receptor down-regulator), supporting the notion that the cardioprotective effect of Prolame involves the preservation of endothelium through the activation of estrogen receptor downstream signaling. Our results provide evidence that Prolame has potential therapeutic application in patients with AMI, as it prevents from both vascular and cardiac tissue damage.

Platelet aggregation in whole blood, a new approach for understanding the antiplatelet effect of N-(3-hydroxy-1,3,5(10)-estratrien-17b-yl) butylamine (buame).

We have previously reported the effect of a compound derived from estradiol containing a radical amino butyl at the 17-beta position which has shown anticoagulant effects in whole blood and antiplatelet effects in light transmission aggregometry where platelets are isolated from other blood cells. In contrast, whole blood aggregometry includes the platelet interactions with blood elements such as erythrocytes and leukocytes. We examined the cooperative effect between leukocytes, erythrocytes and platelets and the antiplatelet effect of Buame in whole blood aggregometry, a tool to assess platelet function in its physiological environment. Buame (5-500 microM) dissolved in DMSO was tested in platelet aggregation induced by ADP (1.25 microM) or collagen (1 microg/mL) and the response recorded over 5 min. Controls were run with DMSO and the average control aggregation was taken as 100%. Results were obtained in both whole blood and platelet aggregometry. Buame was able to inhibit the secondary aggregation induced with ADP suggesting impairment in thromboxane A2 production. Also the first and second aggregation phases were inhibited when collagen-induced platelet activation was employed. This concentration-dependent pattern was shown in both whole blood and platelet aggregometry assays. When tested in light transmission aggregometry, a higher concentration of Buame was required in order to inhibit to the same degree ADP- or collagen-induced platelet aggregation (30 microM ,114 microM) than that required in the whole blood assay (IC50 84 microM, 191 microM). Interactions among different cell types in whole blood may modify the response of Buame-treated platelets to agonists suggesting a cooperative mechanism.

Intracellular Ca2+ stimulates the binding to androgen receptors in platelets.

In this study, we demonstrated that ADP-induced platelet aggregation activates the binding of testosterone (T) to its receptor. It is well known that binding of ADP to its receptors induced the release of Ca2+ ions from dense bodies into the cytosol of platelets. In this work, we compared the binding of testosterone or dihydrotestosterone to their receptors using cytosol obtained from ADP-treated and non-treated platelets. These experiments were repeated using EGTA (a calcium chelator) or U73122 (a phospholipase C enzymatic activity inhibitor) to the ADP-treated platelets. In addition, we also developed a competition analysis for the androgen receptors (AR) using [3H]DHT, non-radioactive T, DHT or cyproterone acetate from ADP-treated platelets cytosol. The results from this study indicate that the cytosol obtained from non-ADP-treated platelets did not show any binding to [3H]T or [3H]DHT, whereas cytosol from ADP-treated platelets binds to the radio-labeled androgens. Furthermore cytosol from ADP plus U73122-treated platelets did not show binding to [3H]T or [3H]DHT. These data suggest that intracellular Ca2+ ions stimulates the binding of androgens to their receptors in platelets cytosol. The competition analysis shows that T and DHT have high affinities for the androgen receptors with similar IC50 values, whereas cyproterone acetate shows a lower affinity. The results from these data clearly indicate the presence of androgen receptors in platelets.