Development of polyclonal antibodies against angiotensin type 2 receptors.

Abstract

Murine neuroblastoma N1E-115 cells are a useful system in which to study neuronal angiotensin II (AngII) receptors. NlE-115 cells possess both type 1 (AT1) and type 2 (AT2) AnglI receptor subtpes, as does mammalian brain. AT2 receptors in brain or NlE-115 cells can be solubilized in 3- [(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. In the present study, heparin-Sepharose chromatography was used to partiafly purify solubilized NlE-115 membranes to produce an enriched population of AT2 receptors. Subsequendy, an eluted peak, containng the majority ofAT2 binding activity, was used as an immunogen in the development of protein-directed polyclonal antibodies. The antibodies specifically detected immunoreactive proteins of approximately 110 and 66 kDa in both solubilized NlE-115 cells, as well as the orignal protein material that eluted from the heparin-Sepharose column, whereas no such immunoreactivity was detected in a kidney epithelial cell line that lacks any specific 12SI-labeled AngI (12Ang binding activity. Moreover, the antibodies immuno d with affinit-purified AT2 receptors. These antibodies were also able to immunoprecipitate AT2 receptors from solubilized NlE-115 cells, as revealed by the pharmacologic proffle of 12I-A binding to the precipitated protein. Simllariy, the antibodies were able to immunoprecipitate a 66-kDa protein that had been covalently crosslinked with 12'I-AnH by use ofthe homobinctional crosslinker dithiobis(succinimidyl propionate). Collectively, these results demonstrate the development of a specific AT2 receptor antibody that may be used to further characterize this receptor subtype at both the cellular and molecular levels.The renin-angiotensin system is one of the most important regulators of body fluid and cardiovascular homeostasis (1). Angiotensin II (AngII) is known to exert its diverse effects by acting at membrane-bound receptors present on several peripheral target tissues, such as kidney, adrenal cortex, heart, and vascular smooth muscle (1). In addition to its many peripheral effects, all of the components of the reninangiotensin system have also been localized in the central nervous system (2). Moreover, it is now recognized that many of the physiological, endocrinological, and behavioral actions of AngII are mediated by specific receptors present in the brain (3).The diversity of actions mediated by AnglI was among the first pieces of evidence leading to the suggestion of multiple receptor subtypes for Angll. The unequivocal demonstration of such multiple receptor subtypes, however, came with the development of subtype-specific antagonists (4). The existence of at least two receptor subtypes for AngII has initiated the search for a greater understanding of these receptors at the cellular and molecular levels. Such analyses have revealed that both receptor subtypes, referred to as AT1 andThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S....

Vessel-Specific Regulation of Angiotensin II Receptor Subtypes During Ovine Development

“…Again, the protein was predominantly in the soluble and total protein fractions. The AT 2 antisera detected only a 63-kD protein (not shown), consistent with previous reports in the rat and mouse (36,37).…”

“…We used two AT 1 antisera previously used for detecting ovine AT 1 receptors (4): N-10 (anti-rabbit from rat sequence N-terminal; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and C306 (human sequence a.a. 306 -359; Santa Cruz Biotechnology). The AT 2 antisera were raised in rabbits in the laboratories of Dr. Steven J. Fluharty and have been extensively validated (36,37). These antisera do not cross-react between the AT 1 and AT 2 receptors (data not shown).…”

Immunolocalization of Subtype 2 Angiotensin II (AT 2 ) Receptor Protein in Rat Heart

“…Our results in the present study are different from those of Reagan et al, 30 who described two bands (110 and 66 kDa) recognized by a protein-directed polyclonal AT 2 receptor antiserum against a partially purified membrane protein from murine neuroblastoma N1E-115 cells. It is not clear to which amino acid sequence of the cell membrane protein their antiserum was directed, whereas our antibody was raised against a specific peptide sequence of the cloned rat AT 2 receptor.…”

“…Positive immunoreactive signal was detected in the coronary vascular endothelium but not smooth muscle cells (E) (magnification ϫ800). Therefore, these results confirm that the antipeptide serum, directed toward a completely conserved region of the AT 2 receptor, can recognize in an effective and selective manner its appropriate peptide antigen in both transfected cells and experimental tissues.Our results in the present study are different from those of Reagan et al, 30 who described two bands (110 and 66 kDa) recognized by a protein-directed polyclonal AT 2 receptor antiserum against a partially purified membrane protein from murine neuroblastoma N1E-115 cells. It is not clear to which amino acid sequence of the cell membrane protein their antiserum was directed, whereas our antibody was raised against a specific peptide sequence of the cloned rat AT 2 receptor.…”

Meconium Increases Type 1 Angiotensin II Receptor Expression and Alveolar Cell Death

“…Blots were blocked in buffer containing powdered milk (5% wt/vol) and incubated overnight at 4°C with antiserum against AT 1 R (1:1500; N-10, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or AT 2 R (1:3000). Both have been validated (23)(24)(25). The nitrocellulose paper was incubated with donkey anti-rabbit IgG conjugated with affinity purified horseradish peroxidase diluted at 1:5000 with TTBS.…”