ANGIOTENSIN II AT2 RECEPTOR EXPRESSION IN THE CEREBELLUM AFTER RECEPTOR BLOCKADE

CORREA MM; SOLER GARCIA M; ARCE ME; SANCHEZ, SI ; CIUFFO GM; FUENTES LB

Estancia Grande

Congreso; Reunion Anual de la Soc. Biología de Cuyo; 2014

Angiotensin II (Ang II) exerts its physiological effects through binding to two receptor subtypes: AT1 and AT2. These are differentially blocked by Losartan or PD123319 (PD), respectively. Ang II receptor expression is highly modulated during development suggesting a role of these receptors in growth and organogenesis. In rodents, cerebellum development takes places during 15-20 days after birth. RNA probes (riboprobes) generated by transcription in vitro are much more sensitive and stables than DNA probes. With the aim to explore the expression pattern during the blockade of Ang II AT2 receptors with PD in PND8 rats, we performed an in situ hybridization study using digoxigenin riboprobes. The PCR fragments of AT2 receptors were subcloned in the p-GEM T easy vector. The identity of the subcloned inserts was verified by RFLP. The insert was extracted from the vector with restriction enzymes, and the band was purified. The riboprobes were obtained by in vitro transcription using SP6 or T7 RNA polymerases in both sense and antisense orientations to provide non-specific control and specific probes. The riboprobes were labeled with non radioactive digoxigenin. In situ hybridization was performed using the synthetized AT2 riboprobes on sagittal sections of PD treated PND8 rat cerebellum. In coincidence with our previous data by autoradiography and immunohistochemistry we observed specific signal in cerebellar Purkinje cell layer of PD treated animals. In conclusion, the riboprobes generated here allows sensitive and efficient detection of AT2 receptor gene expression in PND8 PD treated animals.