Anastrozole

This experiment determined if local application of the ARO inhibitor, anastrozole (Tocris), altered the levels of E2 recovered from microdialysis samples in OvX females. After the 60–90 min equilibration period, three 30 min samples were collected and then anastrozole (100 μM, 50 μl) was applied to the dorsal brainstem surface and simultaneously perfused through the microdialysis probe (2 μl/min for 30 min). Additional 30 min samples were collected for 150 min.

Exemestane, letrozole and anastrozole were purchased from Tocris Bioscience (Bristol, UK). The activating peptide (PAR2-AP, SLIGRL-NH₂) and its reverse peptide (PAR2-RP, LRGILS-NH₂) of the murine PAR2 receptor were synthesized from G. Cirino (University of Naples, Naples, Italy) and dissolved in distilled water. If not otherwise indicated, all other reagents were from Sigma-Aldrich (Milan, Italy). HC-030031 was synthesized as previously described45 (link).

Anastrozole and letrozole were purchased from Tocris Bioscience (Bristol, UK) and were diluted in 0.5% carboxymethylcellulose and 99.5% of NaCl (0.9%). Paclitaxel (6 mg/mL of Paclitaxel in Cremophor EL and dehydrated ethanol) was purchased from Glenmark (Buenos Aires, ARG) and was dissolved in NaCl (0.9%). Icatibant (kinin B₂ receptor peptide antagonist), des-Arg⁹-[Leu⁸]-bradykinin (DALBk; kinin B₁ receptor peptide antagonist) were purchased from Sigma-Aldrich Chemical Company (St. Louis MO, USA) and were also prepared in NaCl (0.9%). The enzyme immunoassay kit for bradykinin was obtained from Phoenix Pharmaceuticals, Inc. (California, USA). Specific anti-B₁ (bs-8675R–lot 9C20V14) or anti-B₂ (bs-2422R–lot AG08307921) antibodies were acquired from Bioss Antibodies (Massachusetts, USA) and secondary antibody (sc-2357–lot L1218) was obtained from Santa Cruz Biotechnology (California, USA). Paclitaxel from semisynthetic ≥ 97% (in vitro assays) was acquired from Sigma-Aldrich Chemical Company (St. Louis MO, USA). Annexin-V and 3- (4, 5-dimethyl-2-thiazolyl) -2, 5-diphenyl-2H-tetrazolium bromide] salt (MTT; 5 mg/mL solution) were obtained from Life Technologies (São Paulo, BR). The 7-Amino-Actinomycin D (7AAD) was acquired from BD Biosciences (California, USA). The doses of the drugs used in this study were based on previous studies^{13 (link),15 (link),23 (link),40 (link),72 (link)}.