Development of polyclonal antisera selective for angiotensin type 2 (AT(2)) receptors

Lawrence Patrick Reagan, University of Pennsylvania


Angiotensin II (Ang II) is one of the most important regulators of body fluid and cardiovascular homeostasis. The physiological responses of Ang II in the periphery and the brain are mediated through the two main classes of Ang II receptors, which are referred to as Angiotensin Type 1 (AT$\sb1$) and Angiotensin Type 2 (AT$\sb2$). The study of the pharmacologic, biochemical and cellular properties of central Ang II receptor subtypes has been impeded by the fact that Ang II receptors are expressed in low densities and in very discrete neuronal populations. In order to circumvent these potential problems, our laboratory has chosen to study the properties of neuronal Ang II receptors by use of a cloned neuronal cell line, murine neuroblastoma N1E-115 cells. We previously established that N1E-115 cells express both AT$\sb1$ and AT$\sb2$ receptor subtypes and that AT$\sb2$ receptors may be solubilized from N1E-115 membranes. Heparin-Sepharose chromatography of CHAPS solubilized N1E-115 membranes produced an enriched population of AT$\sb2$ receptors which were used as an immunogen to produce polyclonal antisera selective for the AT$\sb2$ receptor subtype. In preliminary studies, it was demonstrated that these antisera were able to immunodetect proteins in solubilized N1E-115 membranes with approximate molecular weights of 66 and 110 kDa, as well as immunoprecipitate $\sp{125}$I-Ang II binding activity from solubilized N1E-115 membranes. In subsequent studies, AT$\sb2$-directed antisera were used to identify AT$\sb2$ receptor populations in peripheral tissues of neonatal and adult rat by immunoblot analysis. In addition, immunohistochemical techniques were utilized to localize AT$\sb2$ receptor populations in adult rat brain. Lastly, immunoblot and immunoprecipitation studies were performed to differentiate between two AT$\sb2$ receptor populations in N1E-115 cells. These results demonstrate that two immunologically distinct AT$\sb2$ receptors are present in solubilized N1E-115 cells. The results of these studies strengthened the hypothesis of AT$\sb2$ receptor heterogeneity. Collectively, utilization of these AT$\sb2$-directed antisera have provided evidence for the existence of multiple AT$\sb2$ receptors, and have proven to be an invaluable tool in the characterization of a subpopulation of AT$\sb2$ receptors in both the periphery and the brain.

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Recommended Citation

Reagan, Lawrence Patrick, "Development of polyclonal antisera selective for angiotensin type 2 (AT(2)) receptors" (1995). Dissertations available from ProQuest. AAI9543137.