2.2.4. Experimental Protocol

(1) Vehicle/Antagonist Infusion. Male rats receive saline (the vehicle group, group 1), AT₁R blocker; losartan (the losartan group, group 2), MasR antagonist; A779 (Bachem Bioscience Inc., King of Prussia, PA, USA) (the A779 group, group 3); and the combination of A779 and losartan (the A779+losartan group, group 4). The female rats also had the same protocol as male rats of groups 1–4. Losartan and A779 were dissolved in 0.9% w/v saline. Losartan and A779 either alone or together were administrated in bolus doses of 5 mg·kg⁻¹ and 50 µg·kg⁻¹, respectively, in relative groups, which then were followed by continuous infusions of 5 mg·kg⁻¹·h⁻¹ losartan and 50 μg·kg⁻¹ h⁻¹ A779 with a microsyringe infusion pump (New Era Pump System Inc., Farmingdale, NY, USA) during the experiment. Vehicle-treated groups followed a similar protocol but received the saline vehicle instead of antagonists during the experiment. Subsequently, 30 minutes after vehicle/antagonist infusion, the parameters were measured and considered as data to determine the vehicle/antagonist effects (antagonist phase). However, the vehicle or antagonists were continuously infused until end Ang II administration (end of experiment).

(2) Ang II Infusion. The vascular responses to graded Ang II (Sigma, St Louis MI, USA) infusion were measured in all experimental groups, while the antagonists or vehicle injection continued during the Ang II infusion. Each dose of Ang II (0, 100, 300, or 1000 ng/kg/min) was administrated for 15 min, and the last 3-minute measurements in each dose were obtained. During Ang II administration, the aortic diameter was adjusted by an aortic occluder to control RPP at constant level. Finally, the rats were killed humanly, and the right and left kidney were rapidly removed and weighed. The protocol of the experimental study is demonstrated in (Figure 1)

The protocol of experimental study (see Section 2.2.4).