Immunohistochemical detection of angiotensin receptors AT1 and AT2 in normal rat pituitary gland, estrogen-induced rat pituitary tumor and human pituitary adenomas

Male rat pituitary glands, diethylstilbestrol (DES)-induced rat pituitary tumors and 12 human pituitary adenomas were immunostained with antibodies raised against AT1 and AT2 angiotensin receptor proteins. Positive immunostaining of AT1 was observed in a subpopulation of anterior and intermediate pituitary lobe cells as well as in some nerve endings of the neurohypophysis. In the DES-induced rat pituiary tumors, the subpopulation of AT1-immunnopositive cells was smaller than in the non-tumoral anterior pituitary. In human pituitary adenomas, weak AT1 immunostaining was found in 5 tumors. In the remaining adenomas, the AT1 immunostaining was trace (doubtful) or absent. The AT1 immunostaining in the peritumoral non-neoplastic pituitary tissue was stronger than that observed in the tumors. The normal rat pituitaries and rat tumors did not show immunostaining with anti-AT2 antibody. In human pituitary adenomas, the tumoral cells were AT2- negative but moderate to strong AT2 immunostaining was observed in intratumoral blood vessel walls. The data suggest that the experimental (in rat) and spontaneous (in man) pituitary tumorigenesis is associated with the down-regulation of AT1 receptors. The expression of AT2 receptors, in turn, may be connected with the process of tumoral neo-angiogenesis.     

Immunocytochemical localization of angiotensin II receptor subtypes and angiotensin II with monoclonal antibodies in the rat adrenal gland

Angiotensin II (Ang II), a major regulator of cardiovascular function and body fluid homeostasis, mediates its biological actions via two subtypes of G protein-coupled receptors, termed AT(1) and AT(2). The primary goal of this study was to raise monoclonal anti-peptide antibodies specific to angiotensin AT(1)- and AT(2)-receptor subtypes and to Ang II itself and using these monoclonal antibodies to determine the intraadrenal localization of AT(1) and AT(2) receptors and Ang II in male adult rats. Immunocytochemistry unambiguously demonstrates a regional colocalization of Ang II and angiotensin II receptors in the adrenal gland. The novel antibodies localized Ang II and the AT(1) receptors to the zona glomerulosa of the cortex and to the medulla whereas AT(2) receptors were limited to the medulla. The specificity of immunostaining was documented by pre-adsorption of the antibody with the immunogenic peptide. Our data underscore that AT(1) appears to mediate most of the physiological actions of Ang II in adrenal. Western blot analysis of rat adrenal protein extracts using AT(1) antibody showed a predominant 73-kDa band and a weaker 97-kDa immunoreactive band corresponding to glycosylated forms of the AT(1) receptor. Immunostaining with anti-AT(2) yielded one major immunoreactive band of 73-kDa size and one additional fainter band of 120 kDa. These antibodies may prove of value in unraveling the subcellular localization and intracellular effector pathways of AT(1) and AT(2).     

Involvement of c-Src tyrosine kinase in SHP-1 phosphatase activation by Ang II AT2 receptors in rat fetal tissues

Angiotensin II (Ang II) AT(2) receptors are abundantly expressed in rat fetal tissues where they probably contribute to development. In the present study we examine the effects of Ang II type 2 receptor stimulation on SHP-1 activation. Ang II (10(-7) M) elicits a rapid and transient tyrosine phosphorylation of SHP-1, maximal at 1 min, in a dose-dependent form, blocked by the AT(2) antagonist, PD123319. SHP-1 phosphorylation is followed in time by tyrosine dephosphorylation of different proteins, suggesting a sequence of events. Ang II induces association of SHP-1 to AT(2) receptors as shown by co-immunoprecipitation, Western blot and binding assays. SHP-1 activity was determined in immunocomplexes obtained with either anti-AT(2) or anti-SHP-1 antibodies, after Ang II stimulation (1 min), in correlation with the maximal level of SHP-1 phosphorylation. Interestingly, following receptor stimulation (1 min) c-Src was associated to AT(2) or SHP-1 immunocomplexes. Preincubation with the c-Src inhibitor PP2 inhibited SHP-1 activation and c-Src association, thus confirming the participation of c-Src in this pathway. We demonstrated here for the first time the involvement of c-Src in SHP-1 activation via AT(2) receptors present in an ex vivo model expressing both receptor subtypes. In this model, AT(2) receptors are not constitutively associated to SHP-1 and SHP-1 is not constitutively activated. Thus, we clearly establish that SHP-1 activation, mediated by the AT(2) subtype, involves c-Src and precedes protein tyrosine dephosphorylation, in rat fetal membranes.     

Angiotensin II AT2 Receptors Contribute to Regulate the Sympathoadrenal and Hormonal Reaction to Stress Stimuli

Angiotensin II, through AT1 receptor stimulation, mediates multiple cardiovascular, metabolic, and behavioral functions including the response to stressors. Conversely, the function of Angiotensin II AT2 receptors has not been totally clarified. In adult rodents, AT2 receptor distribution is very limited but it is particularly high in the adrenal medulla. Recent results strongly indicate that AT2 receptors contribute to the regulation of the response to stress stimuli. This occurs in association with AT1 receptors, both receptor types reciprocally influencing their expression and therefore their function. AT2 receptors appear to influence the response to many types of stressors and in all components of the hypothalamic–pituitary–adrenal axis. The molecular mechanisms involved in AT2 receptor activation, the complex interactions with AT1 receptors, and additional factors participating in the control of AT2 receptor regulation and activity in response to stressors are only partially understood. Further research is necessary to close this knowledge gap and to clarify whether AT2 receptor activation may carry the potential of a major translational advance.     

Expression of Trophic Peptides and Their Receptors in Chromaffin Cells and Pheochromocytoma

Pheochromocytomas are catecholamine-producing tumors arising from chromaffin cells of the adrenal medulla or extra-adrenal location. Along with catecholamines, tumoral cells produce and secrete elevated quantities of trophic peptides which are normally released in a regulated manner by the normal adrenal medulla. Among these peptides, the amounts of pituitary adenylate cyclase-activating polypeptide (PACAP), adrenomedullin (AM), and neuropeptide Y (NPY) are particularly high. These peptides can exert endocrine, paracrine or autocrine effects in numerous cell types. In particular, they have been shown to be involved in cell proliferation and survival, catecholamine production and secretion, and angiogenesis. Some of these processes are exacerbated in pheochromocytomas, raising the possibility of the involvement of trophic peptides. Here, we review the expression levels of NPY, PACAP, and AM and theirs receptors in chromaffin cells and pheochromocytomas, and address their possible implication in the adrenal medulla tumorigenesis and malignant development of pheochromocytomas.     

Elevated AT1 receptor protein but lower angiotensin II-binding in adipose tissue of rats with monosodium glutamate-induced obesity.

Age-related hypertrophy of adipose tissue has been associated with a significant decrease in the number of angiotensin II receptors. The aim of this study was to investigate the characteristics of angiotensin II receptors in hypertrophic adipose tissue in animal obesity model using rats postnatally treated with monosodium glutamate. Angiotensin II is known to induce hypertrophy in several tissues of the cardiovascular system and might do the same in fat tissue. The expression and binding properties of angiotensin II AT(1) receptors in epididymal fat tissue of adult rats were studied using membrane-binding, RT-PCR, and immunoblotting. The amount of AT(1) receptor mRNA did not differ significantly between obese and control rats. Despite that glutamate-treated rats displayed approximately 4-times more AT(1) receptor immunoreactive protein content in fat tissue cell membranes than the controls did. In contrast, binding experiments showed a significant (40.3 +/- 6.2 %) decrease of (125)I-Sar(1)-Ile(8)-angiotensin II-binding to fat tissue cell membranes in obese rats compared to controls. In conclusion, the present study provides evidence for the low binding properties associated with an accumulation of AT(1) receptor protein in cell membranes of the fat tissue of rats with glutamate-induced obesity. Discrepancies among angiotensin II-binding, AT(1) receptor protein, and AT(1) receptor mRNA levels indicate a possible defect in the receptor protein, which remains to be identified. The results obtained support a role of angiotensin II and AT(1) receptors in the pathogenesis of obesity.     

Specific single chain variable fragment (ScFv) antibodies to angiotensin II AT2 receptor: evaluation of the angiotensin II receptor expression in normal and tumor-bearing mouse lung

To gain insight into the mechanism by which angiotensin II type 2 receptor (AT(2)) regulates carcinogen-induced lung tumorigenesis, we have newly developed anti-AT(2) single chain variable fragment (ScFv) antibodies using a rodent phage-displayed recombinant antibody library with various peptide fragments of the receptor protein, and investigated the expression of the AT(2) receptor protein. The specificity of the antibodies was verified using AT(2) over-expressing COS-7 cells and AT(2) naturally expressing PC12W cells. In control wild type mouse lung, a stronger immunoreactivity was observed in bronchial epithelial cells. A moderate immunoreactivity was detected in pulmonary vascular walls and vascular endothelial cells. In the lungs possessing tobacco-specific nitrosamine (NNK)-induced tumors, significantly increased AT(2) and AT(1 )immunostaining was observed in adenomatous lesions. These data suggest that the increase in both receptors' expression in the alveolar epithelial cells may be accompanied with the onset of NNK-induced tumorigenesis and hence play important roles in lung tumorigenesis.     

Immunohistochemical detection of dopamine D2 receptors in human pituitary adenomas

Thirty one pituitary adenomas and 3 samples of peritumoral anterior pituitary tissue were immunostained with an antibody raised against dopamine D2 receptor protein. The positive reactions were found in cell cytoplasm, a subpopulation of cell nuclei and the intratumoral blood vessels walls. As expected, the positive immunostaining was shown in cytoplasm and/or cell nuclei of all examined prolactinomas (7/7). In acromegaly the positive D2 staining occurred in 5/7 samples, in gonadotropinomas in 6/8 and in plurihormonal adenomas 2/4. The lowest expression was observed in corticotropinomas (1/5). These findings corroborate with the well known efficacy of D2 agonists in the treatment of prolactinomas and somatotropinomas, and support the rationale of the therapeutic trials with these compounds in gonadotropinomas. Moreover, the presence of D2 receptors in intratumoral blood vessels walls constitutes the possibility of the anti-angiogenic action of D2 agonists in pituitary adenomas.     

Immunohistochemical detection of angiotensin II receptors in mouse cerebellum and adrenal gland using “in vivo cryotechnique”

Angiotensin II (AT) receptors, including AT receptor type 1 (AT1R) and type 2 (AT2R), are expressed in the rodent central nervous system, but their distributions and activation states are still unclear. In this study, we have performed immunohistochemical analyses of AT receptors in mouse cerebellum and adrenal gland using our “in vivo cryotechnique” (IVCT). We used antibodies against amino-terminal domains of AT receptors, which are considered to undergo conformational changes upon the binding of AT. Immunoreactivity of AT1R was detected in mouse cerebellum, and was highest in the outer tissue areas of molecular layers using IVCT. The AT1R immunostaining largely overlapped with glial fibrillary acidic protein (GFAP), a marker of Bergmann glia. Surprisingly, the AT1R immunoreactivity in the cerebellar cortex was remarkably reduced following 5 and 10 min of hypoxia or direct administration of an AT1R antagonist, losartan. By contrast, in the adrenal cortex, such AT1R immunoreactivity detected at the zona glomerulosa did not change even after 15 min of hypoxia. The correlation of localization with GFAP and also hypoxia-induced decrease of its immunoreactivity were similarly observed by immunostaining of AT2R in the cerebellar specimens. These findings demonstrated that IVCT is useful to reveal dynamically changing immunoreactivities usually affected by receptor-ligand binding as well as hypoxia, and also suggested that functional activities of AT receptors are time-dependently modulated under hypoxia in the central nervous system in comparison with the adrenal glands.     

Estrogen upregulates renal angiotensin II AT1 and AT2 receptors in the rat

We studied renal AT1 and AT2 receptors in male, female, ovariectomized and ovariectomized-estrogen-treated Wistar-Hanover and Wistar-Kyoto rats. AT1 receptors and AT1A receptor mRNA predominated, with no significant differences between males and females. AT2 receptor expression was restricted in female rats to the capsule, the transition zone between outer and inner medulla, the endothelium lining the papilla, and arcuate arteries and veins. There were no AT2 receptors in male rats, while male mice express substantial numbers of estrogen-dependent AT2 receptors. Arcuate arteries and veins expressed AT1B mRNA in males and females, and AT2 mRNA in females only. AT1 receptor and AT2 receptor expression were estrogen-dependent, with increases in AT1 and AT2 receptor expression after estrogen treatment in ovariectomized rats. Estrogen treatment increased prostaglandin E2 (PGE2) and cGMP concentrations in the renal medulla, and eNOS expression in cortical arteries. In rodents, expression of renal Angiotensin II receptor types is estrogen-dependent, with significant species, strain and area differences. Our results support an important role for AT2 receptors in the regulation of renal function and in the protective effects of estrogen in the kidney.     

Agonist-independent nuclear localization of the Apelin, angiotensin AT1, and bradykinin B2 receptors

Signaling of the apelin, angiotensin, and bradykinin peptides is mediated by G protein-coupled receptors related through structure and similarities of physiological function. We report nuclear expression as a characteristic of these receptors, including a nuclear localization for the apelin receptor in brain and cerebellum-derived D283 Med cells and the AT(1) and bradykinin B(2) receptors in HEK-293T cells. Immunocytochemical analyses revealed the apelin receptor with localization in neuronal nuclei in cerebellum and hypothalamus, exhibiting expression in neuronal cytoplasm or in both nuclei and cytoplasm. Confocal microscopy of HEK-293T cells revealed the majority of transfected cells displayed constitutive nuclear localization of AT(1) and B(2) receptors, whereas apelin receptors did not show nuclear localization in these cells. The majority of apelin receptor-transfected cerebellum D283 Med cells showed receptor nuclear expression. Immunoblot analyses of subcellular-fractionated D283 Med cells demonstrated endogenous apelin receptor species in nuclear fractions. In addition, an identified nuclear localization signal motif in the third intracellular loop of the apelin receptor was disrupted by a substituted glutamine in place of lysine. This apelin receptor (K242Q) did not exhibit nuclear localization in D283 Med cells. These results demonstrate the following: (i) the apelin receptor exhibits nuclear localization in human brain; (ii) distinct cell-dependent mechanisms for the nuclear transport of apelin, AT(1), and B(2) receptors; and (iii) the disruption of a nuclear localization signal sequence disrupts the nuclear translocation of the apelin receptor. This discovery of apelin, AT(1), and B(2) receptors with agonist-independent nuclear translocation suggests major unanticipated roles for these receptors in cell signaling and function.     

Increased expression of (pro)renin receptor in aldosterone-producing adenomas

(Pro)renin receptor ((P)RR) is a specific receptor for renin and prorenin. The aim of the present study is to clarify expression and possible pathophysiological roles of (P)RR in aldosterone-producing adenomas (APAs) and other adrenal tumors. Expression of (P)RR was studied by immunocytochemistry, western blot analysis and real-time RT-PCR in adrenal tumor tissues obtained at surgery. Immunocytochemistry showed that (P)RR was expressed in normal adrenal glands and tumor tissues of adrenocortical tumors including APAs. In the normal adrenal glands, positive (P)RR immunostaining was observed in both adrenal cortex and medulla, with higher (P)RR immunostaining observed in zona glomerulosa and zona reticularis. Positive (P)RR immunostaining was also observed in the adrenocortical tumors, with elevated (P)RR immunostaining found in APAs, particularly in compact cells. By contrast, no apparent (P)RR immunostaining was observed in pheochromocytomas. Western blot analysis showed a band of (P)RR protein in normal adrenal glands and adrenocortical tumors at the position of 35 kDa. The relative expression levels of (P)RR protein were higher in tumor tissues of APAs than in attached non-neoplastic adrenal tissues of APAs. Real-time RT-PCR showed that expression levels of (P)RR mRNA were significantly increased in tumor tissues of APAs compared with other adrenal tumor tissues and attached non-neoplastic adrenal tissues of APAs. The present study has shown for the first time that expression of (P)RR is elevated in tumor tissues of APAs, raising the possibility that (P)RR may play pathophysiological roles in APAs, such as aldosterone secretion and cell proliferation.     

Angiotensin II AT2 receptor localization in cardiovascular tissues by its antibody developed in AT2 gene-deleted mice

In contrast to well-established physiological roles of the angiotensin II type 1 receptor (AT1), the significance of the type 2 receptor (AT2) remains largely unclear. AT2-knockout (AT2KO) mice have a phenotype associated with mild hypertension. This implies that AT2 has a role for the regulation of blood pressure. To gain insight into the mechanism by which AT2 regulates systemic blood pressure, we have investigated the expression of the AT2 receptor protein in adult rat cardiovascular tissues, using a newly developed polyclonal anti-AT2 antiserum that was successfully obtained in the AT2KO mice by immunizing with a peptide fragment of the receptor protein. In blood vessels, a stronger immunoreactivity was observed in endothelial cells than in the muscular media of resistant arteries. In the thoracic aorta, AT2 was observed only in muscular media. Abundant AT2 immunoreactivity was detected in perivascular nerve fibers. In the heart, positive immunostaining for AT2 was restricted to the coronary blood vessels. These data suggest that AT2 expressed in the vascular endothelial cells and muscular media in resistant arteries may play a pivotal role in systemic blood pressure regulation. AT2 was observed for the first time in the perivascular nerve fibers and may also play a role in neuronal blood pressure regulation.     

Sarcosine1,glycine8 angiotensin II is an AT1 angiotensin II receptor subtype selective antagonist

Studies predating the discovery of the two major subtypes of angiotensin II (Ang II) receptors, AT1 and AT2, revealed anomalous characteristics of sarcosine1,glycine8 Ang II (Sar1,Gly8 Ang II). It competed poorly for 125I-Ang II binding in bovine brain but potently antagonized dipsogenic responses to intracerebroventricularly administered Ang II. Subsequent recognition that bovine brain contains AT(2) receptors, while dipsogenic responses to Ang II are mediated by AT1 receptors, suggests that Sar1,Gly(8) Ang II is AT1 selective. Sar1,Gly8 Ang II competed for 125I-sarcosine1,isoleucine8 Ang II binding to AT1 receptors in pituitary, liver and adrenal (the latter with the AT2 selective antagonist PD 123,319) with Ki's of 0.66, 1.40 and 1.36 nM, respectively. In contrast, the Ki of Sar1,Gly8 Ang II for AT2 receptors in rat adrenal (with the selective AT1 antagonist losartan) was 52 nM. 125I-Sar1,Gly8 Ang II (0.5-3 nM) bound to AT1 receptors in pituitary, liver, heart, adrenal, and hypothalamic membranes with high affinity (Kd=0.43, 1.6, 2.3, 0.96 and 1.8 nM, respectively), but showed no saturable binding to the adrenal AT2 receptor. 125I-Sar1,Gly8 Ang II selectively labeled AT1 receptors in sections of adrenal using receptor autoradiography. Thus, binding studies reveal Sar1,Gly8 Ang II to be the first angiotensin peptide analog to show AT1 receptor selectivity. 125I-Sar1,Gly8 Ang II offers a new means to selectively radiolabel AT1 receptors and may help to characterize ligand docking sites and agonist switches for AT1 versus AT2 receptors.     

Anti-inflammatory effects of angiotensin II AT1 receptor antagonism prevent stress-induced gastric injury

Stress reduces gastric blood flow and produces acute gastric mucosal lesions. We studied the role of angiotensin II in gastric blood flow and gastric ulceration during stress. Spontaneously hypertensive rats were pretreated for 14 days with the AT1 receptor antagonist candesartan before cold-restraint stress. AT1 receptors were localized in the endothelium of arteries in the gastric mucosa and in all gastric layers. AT1 blockade increased gastric blood flow by 40-50%, prevented gastric ulcer formation by 70-80% after cold-restraint stress, reduced the increase in adrenomedullary epinephrine and tyrosine hydroxylase mRNA without preventing the stress-induced increase in adrenal corticosterone, decreased the stress-induced expression of TNF-alpha and that of the adhesion protein ICAM-1 in arterial endothelium, decreased the neutrophil infiltration in the gastric mucosa, and decreased the gastric content of PGE2. AT1 receptor blockers prevent stress-induced ulcerations by a combination of gastric blood flow protection, decreased sympathoadrenal activation, and anti-inflammatory effects (with reduction in TNF-alpha and ICAM-1 expression leading to reduced neutrophil infiltration) while maintaining the protective glucocorticoid effects and PGE2 release. Angiotensin II has a crucial role, through stimulation of AT1 receptors, in the production and progression of stress-induced gastric injury, and AT1 receptor antagonists could be of therapeutic benefit.     

Coexpression of AT1 and AT2 receptors by human fibroblasts is associated with resistance to angiotensin II

Angiotensin II (AngII) is considered as a cytokine-like factor displaying a variety of proinflammatory and profibrotic cellular effects. Most of these effects seem mediated by AT1 signaling, whereas AT2 expression and function in adult human cells remain unclear. We have studied AT1 and AT2 expression in different human adult fibroblasts types and analyze their response to AngII. AngII did not induce thymidine incorporation, apoptosis nor collagen gene or protein expression in human fibroblasts. Specific AT1 or AT2 inhibitors did not modify this apparent resistance to AngII. We found abundant expression of both AT1 and AT2 receptors in all human fibroblasts studied, whereas vascular smooth muscle cells (VSMC) which only expressed AT1 receptor, displayed a clear AT1-dependent proliferative response to AngII. These data demonstrate that cultured human adult fibroblasts express both AT1 and AT2 receptor types and this phenomenon is associated with a lack of growth or collagen synthesis responses to AngII.