The local renin-angiotensin system in rat salivary glands: pharmacological alterations

Abstract

The description of the Renin-Angiotensin System (RAS) allows the comprehension of the mechanisms on how its components can regulate the body homeostasis involving mainly vasodilatation, water intake and sodium balance. When these components are synthetized by the tissue's cells, the local RAS is characterized. Recent researches in our laboratory showed the existence of a local RAS in rat parotid, submandibular and sublingual glands. Specifically, we found angiotensinogen, ACE, ACE2, AT1a, AT2 and MAS in all the salivary glands, whereas AT1b was only found in parotid gland. The aim of the present study is to compare the expression of RAS components, in the three major salivary glands, in 3 groups with 7 rats (60 day-old) each, which will receive intraperitoneally, during 7 days: 1) saline (vehicle; control group); 2) a ²-adrenergic receptor agonist (Isoproterenol - IPR; 20 mg/kg) and 3) an antagonist for AT1 receptor of Angiotensin II (losartan; 10 mg/kg). The following techniques will be performed: quantitative polymerase chain reaction (qPCR), immunohistochemistry as well as proteomic and peptidomic analysis. For the qPCR, parotid, submandibular and sublingual glands will be harvested and maintained in freezer at -80oC. Then, following the manufacturers' protocols, this sequence will be done: extraction, quantification and qualification of the mRNAs; reverse transcription and qPCR for the RAS targets. ²-actin will be used as the reference gene for gene expression [the obtained cycle threshold (Ct) will be subtracted from the ²-actin Ct, using the formula 2-”Ct for relative quantification]. For immunostaining, slides will be prepared from serial cuts of 5 µm thickness from paraffin-embedded blocks in a rotary microtome. Mesio-distal sections will be placed onto glass non-stained slides, stained with hematoxilin-eosin for morphometric analysis, and the silanized ones will be stained with the primary antibodies for: renin, ACE, ACE2, AT1, AT2 and MAS receptors. For the proteomic and peptidomic analyses in the salivary glands, the interested molecules in the tissue will be extracted and digested, submited to liquid chromatography electrospray ionization mass spectrometry. Identification, characterization and quantification will be based on analysis of a protein database and algorithms from a specific software. The results will be expressed as mean ± standard deviation. The data will be statistically analyzed using the ANOVA test and the differences between groups will identified by Bonferroni's post-test at a significance level of p<0.05.