Abstract:
:Evidence continues to accumulate that strengthens the proposal of heterogeneity within both the AT1 and the AT2 receptor subtypes. Pharmacologic, biochemical and immunological studies of AT2 receptors expressed in N1E-115 cells strengthen the hypothesis of AT2 receptor heterogeneity. However, it is important to reassess these studies, especially in terms of how these results correlate with other reports of AT2 receptor heterogeneity. For example, AT2 receptor immunoreactivity was absent in some neuronal regions which have previously been proposed to express the AT2 receptor subtype. In particular, AT2 receptor staining was not seen in the inferior olive, a region which is known to express a high density of AT2 receptors. Upon first examination, these results were somewhat troubling. However, when compared with earlier reports, these results should not have been unexpected. For instance, Tsutsumi and Saaverdra previously have shown that AT2 receptors in the locus coeruleus are sensitive to the actions of guanine nucleotides, while AT2 receptors in the inferior olive are insensitive (21). These antisera were raised against a population of AT2 receptors which are sensitive to GTP gamma S and therefore, the lack of AT2 receptor staining in the inferior olive, as well as the presence of AT2 receptor immunoreactivity in the locus coeruleus, confirms and extends these earlier reports. In addition the AT2 receptors expressed in the locus coeruleus have been shown to be functionally distinct from AT2 receptors in the inferior olive. In this regard, Ang II has been shown to depress glutamate-induced EPSPs in the locus coeruleus, an effect which is mediated through the AT2 receptor (19). Conversely, AT2 receptors have been shown to increase the firing rate of neurons in the inferior olive (20). Collectively, these results would predict that staining should be absent in the inferior olive using these AT2-directed antisera. Indeed, in view of these earlier physiological and pharmacological studies, the presence of AT2 receptor immunoreactivity in the inferior olive would have been surprising. The most convincing example of AT2 receptor heterogeneity is the characterization of AT2 receptors present in N1E-115 cells. Separation of solubilized N1E-115 membranes by heparin-Sepharose chromatography generates two populations of AT2 receptors which are pharmacologically and biochemically distinct. In particular, CGP42112A was approximately 2 orders of magnitude more selective for Peak III AT2 receptors than was PD123319. Binding activity of Peak I and Peak III AT2 receptor populations also differed in their responses to GTP gamma S and DTT treatment. Lastly, the AT2-directed antisera, raised against the Peak I population of AT2 receptors, were not able to immunodetect the Peak III population of AT2 receptors in immunoblot analysis, or immunoprecipiatate AT2 binding activity from Peak III material. Pharmacological, biochemical and immunological analysis of the AT2 receptor clone isolated from N1E-115 cells revealed that it has the identical characteristics or properties of the Peak III receptor. The AT2 receptor isolated from N1E-115 cells exhibited a similar pharmacology as the Peak III AT2 receptor, in that CGP42112A was more effective at displacing 125I-Ang II binding activity than was PD123319. The AT2 receptor clone was also shown to be insensitive to the actions of GTP gamma S, as well as demonstrated increased binding activity in the presence of DTT, identical to the Peak III AT2 receptor. Lastly, immunoblot analysis of membranes prepared from COS-1 cells transfected with the AT2 receptor cDNA from N1E-115 cells did not demonstrate any immune-specific bands with the AT2-directed antisera. Characterization of an AT2 receptor cDNA isolated from N1E-115 cells reveals that this clone is identical to the Peak III type of AT2 receptor.
journal_name
Adv Exp Med Bioljournal_title
Advances in experimental medicine and biologyauthors
Reagan LP,Yee DK,He PF,Fluharty SJdoi
10.1007/978-1-4899-1376-0_21subject
Has Abstractpub_date
1996-01-01 00:00:00pages
199-208eissn
0065-2598issn
2214-8019journal_volume
396pub_type
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