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Title:Pharmacological, functional, and biochemical characterization of a high-affinity angiotensin receptor on differentiated NG108-15 cells
Author(s):Carrithers, Michael Dennis
Doctoral Committee Chair(s):Weyhenmeyer, James A.
Department / Program:Biology
Discipline:Biology
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):Biology, Neuroscience
Abstract:In the first part of these studies, the binding characteristics of $\sp{125}$I-angiotensin II (ANG II) to membranes prepared from undifferentiated and differentiated neuroblastoma x glioma hybrid cells (NG108-15) were investigated. Scatchard analysis revealed the existence of high and low affinity sites in differentiated cells, but only a low affinity site in undifferentiated cells. Similarly, self-displacement studies revealed competition to a single low affinity site in undifferentiated cells, and to high and low affinity sites in differentiated cells. Angiotensin III (ANG III) displaced high affinity binding in differentiated cells but did not displace low affinity binding in either differentiated or undifferentiated cells. Furthermore, GPP(NH)P inhibited $\sp{125}$I-ANG II binding to differentiated cells, in a dose dependent fashion, but had no effect on binding to undifferentiated cells.
In the second part of this work, the second messenger response of ANG II and III was investigated in differentiated NG108-15 cells. We observed that ANG II increased the synthesis of inositol monophosphates (IP$\sb1$), inositol diphosphates (IP$\sb2$), and inositol trisphosphates (IP$\sb3$) with maximal responses observed at 300, 120, and 30 sec., respectively. This effect was not attenuated by pretreatment of the cells with pertussis toxin. Furthermore, both ANG II and ANG III increased the production of inositol polyphosphates in a dose-dependent manner with ED$\sb{50}$ values of 145 nM and 11 nM, respectively.
Finally, we performed covalent crosslinking analysis to obtain a molecular weight estimate of the receptor protein. In these studies, saturation and kinetic analysis revealed a single high affinity site. Using the homobifunctional crosslinking reagent bis-(sulfosuccinimidyl) suberate (BS$\sp3$), a site with an estimated M$\sb{\rm r}$ of 78 kDa was specifically labeled with $\sp{125}$I-ANG II as determined by SDS-polyacrylamide gel electrophoresis. Both ANG II and ANG III (10$\sp{-6}$ M) inhibited specific labeling. The K$\sb{\rm i}$ for ANG III binding was similar by both pharmacologic (K$\sb{\rm i}$ = 3.33 $\pm$ 0.98 nM) and gel densitometric analyses (K$\sb{\rm i}$ = 2.65 $\pm$ 0.32 nM).
We conclude that the 78 kDa protein represents a high affinity ANG binding site linked to a functional cellular response and that differentiated NG108-15 cells are an appropriate model system to study neuronal ANG receptors.
Issue Date:1991
Type:Text
Language:English
URI:http://hdl.handle.net/2142/22826
Rights Information:Copyright 1991 Carrithers, Michael Dennis
Date Available in IDEALS:2011-05-07
Identifier in Online Catalog:AAI9136560
OCLC Identifier:(UMI)AAI9136560


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