Forty male rats divided into four groups of 10 animals were used: (1) 4-week-old SHR, (2) 12-week-old this website SHR, (3) 4-week-old Wistar, and (4) 12-week-old Wistar. The

animals were kept in an environment with controlled temperature (22–24 °C) and light cycle (12 h/light and 12 h/darkness), receiving standard food and water “ad libitum”. Systolic blood pressure (SBP) of SHR and Wistar rats was recorded by tail plethysmography (Plethysmograph Physiograph® MK-III-S/NBS, Narco Bio-Systems, TX, USA). Only 12-week-old Wistar rats with SBP of approximately 112 mmHg and 12-week-old SHRs with SBP equal to or higher than 150 mmHg were used in the experiments. After 12-h fasting, rats were anaesthetized with ketamine (45 mg/kg, im) and xylazine (5 mg/kg, im) and the salivary flow was stimulated by pilocarpine nitrate (5 mg/kg BW, ip, Sigma, MO, USA). Saliva collection was performed according to Bernarde’s method.5 After the pilocarpine injection, the animals were placed in an inclined bed. The stimulated saliva was collected in flasks kept on ice for 15 min after the

first drop, in temperature-controlled room (20 °C). The saliva volume was calculated from the difference in weight of full and empty flasks, considering the saliva density as 1 mg/mL. As we observed that rat body weight was altered at different ages, the SFR was normalized and expressed as mL/min/100 g body weight. Saliva samples were stored in tubes at −70 °C until biochemical experiments were conducted. The pH and salivary buffering capacity (SBC) were evaluated in fresh GSK-J4 saliva. Immediately

after collection, the salivary pH was measured in saliva samples (200 μL) with a specific electrode (Analyzer) connected to a pH meter (Thermo Fischer, Orion 720A, MA, USA), previously calibrated. The SBC was calculated by titulometric method, according to the volume of lactic acid (0.1 mol/L) used to reduce the salivary pH to 4.0 and was expressed as mL of lactic acid. The saliva protein concentration was determined by Lowry method.6 Briefly, Teicoplanin four different solutions were used: (A) 2% Na2CO3 in 0.1 M NaOH; (B) 0.5% CuSO4·5H2O and 1% sodium citrate; (C) 50 mL of solution A and 1 mL solution B and (D) Folin Ciocalteu diluted with deionized water. A standard solution of 0.1% bovine serum albumin (BSA) in 1% NaOH, was used to the calibration curve with eight different concentrations of protein (5, 10, 20, 40, 50, 80, 100, 200 μg/mL). The volume of saliva per sample used was 10 μL. To this volume, 190 μL of deionized water and 3 mL of solution C were added. After 10 min, 300 μL of solution D was added to the samples and agitated. After 30 min period, the absorbance readings were done at 660 nm in a spectrophotometer (Hitachi U-1100 Spectrophotometer). Salivary amylase activity was quantified by kinetic method at 405 nm, using 2-chloro-p-nitrophenyl-α-d-maltotrioside (CNPG3) as a substrate (Kit Amilasa 405, Wiener Lab.