Regina Nostramo

Commercially Available Angiotensin II At2 Receptor Antibodies Are Nonspecific

Commercially available angiotensin II AT2 receptor antibodies are widely employed for receptor localization and quantification, but they have not been adequately validated. In this study, we characterized three commercially available AT2 receptor antibodies: 2818-1 from Epitomics, sc-9040 from Santa Cruz Biotechnology, Inc., and AAR-012 from Alomone Labs. Using western blot analysis the immunostaining patterns observed were different for every antibody tested, and in most cases consisted of multiple immunoreactive bands. Identical immunoreactive patterns were present in wild-type and AT2 receptor knockout mice not expressing the target protein. In the mouse brain, immunocytochemical studies revealed very different cellular immunoreactivity for each antibody tested. While the 2818-1 antibody reacted only with endothelial cells in small parenchymal arteries, the sc-9040 antibody reacted only with ependymal cells lining the cerebral ventricles, and the AAR-012 antibody reacted only with multiple neuronal cell bodies in the cerebral cortex. Moreover, the immunoreactivities were identical in brain tissue from wild-type or AT2 receptor knockout mice. Furthermore, in both mice and rat tissue extracts, there was no correlation between the observed immunoreactivity and the presence or absence of AT2 receptor binding or gene expression. We conclude that none of these commercially available AT2 receptor antibodies tested met the criteria for specificity. In the absence of full antibody characterization, competitive radioligand binding and determination of mRNA expression remain the only reliable approaches to study AT2 receptor expression.

Divergent effects of estradiol on gene expression of catecholamine biosynthetic enzymes

Within the catecholaminergic systems, there are contradictory findings regarding ability of estradiol to regulate expression of genes related to catecholamine biosynthesis. Several parameters important for effects of estradiol on the catecholamine (CA) related enzyme gene expression were examined in two CA regions. Ovariectomized (OVX) female rats were given prolonged estradiol treatments, either in a pulsatile fashion by injections or continuously by pellets. The mode affected the response of tyrosine hydroxylase (TH) and GTP cyclohydrolase I (GTPCH) mRNAs differentially in the nucleus of solitary tract (NTS) and the locus coeruleus (LC). In rostral-medial NTS, TH mRNA levels were increased with injections, but declined in rats administered estradiol by pellets. In LC, a significant change was only observed in GTPCH with injections. These differences may reflect activation of different estrogen receptors (ER). The response to estradiol in the presence of ERalpha and ER beta was examined in PC12 cell culture. Estradiol directly regulated promoter activity of TH, GTPCH and dopamine beta-hydroxylase (DBH) genes. With ERalpha, 17 beta-estradiol elevated TH promoter activity, while there was a decline with ERbeta. In contrast, both DBH and GTPCH promoters were enhanced by 17 beta-estradiol over a wide range of concentrations with either ER subtype. Thus, mode of administration, location examined and ER subtype expressed are important considerations in the overall response of catecholamine related enzymes to estradiol.

Identification of the putative tumor suppressor Nit2 as ω-amidase, an enzyme metabolically linked to glutamine and asparagine transamination

The present report identifies the enzymatic substrates of a member of the mammalian nitrilase-like (Nit) family. Nit2, which is widely distributed in nature, has been suggested to be a tumor suppressor protein. The protein was assumed to be an amidase based on sequence homology to other amidases and on the presence of a putative amidase-like active site. This assumption was recently confirmed by the publication of the crystal structure of mouse Nit2. However, the in vivo substrates were not previously identified. Here we report that rat liver Nit2 is omega-amidodicarboxylate amidohydrolase (E.C. 3.5.1.3; abbreviated omega-amidase), a ubiquitously expressed enzyme that catalyzes a variety of amidase, transamidase, esterase and transesterification reactions. The in vivo amidase substrates are alpha-ketoglutaramate and alpha-ketosuccinamate, generated by transamination of glutamine and asparagine, respectively. Glutamine transaminases serve to salvage a number of alpha-keto acids generated through non-specific transamination reactions (particularly those of the essential amino acids). Asparagine transamination appears to be useful in mitochondrial metabolism and in photorespiration. Glutamine transaminases play a particularly important role in transaminating alpha-keto-gamma-methiolbutyrate, a key component of the methionine salvage pathway. Some evidence suggests that excess alpha-ketoglutaramate may be neurotoxic. Moreover, alpha-ketosuccinamate is unstable and is readily converted to a number of hetero-aromatic compounds that may be toxic. Thus, an important role of omega-amidase is to remove potentially toxic intermediates by converting alpha-ketoglutaramate and alpha-ketosuccinamate to biologically useful alpha-ketoglutarate and oxaloacetate, respectively. Despite its importance in nitrogen and sulfur metabolism, the biochemical significance of omega-amidase has been largely overlooked. Our report may provide clues regarding the nature of the biological amidase substrate(s) of Nit1 (another member of the Nit family), which is a well-established tumor suppressor protein), and emphasizes a) the crucial role of Nit2 in nitrogen and sulfur metabolism, and b) the possible link of Nit2 to cancer biology.

Assay and purification of ω-amidase/Nit2, a ubiquitously expressed putative tumor suppressor, that catalyzes the deamidation of the α-keto acid analogues of glutamine and asparagine

omega-Amidase (omega-amidodicarboxylate amidohydrolase, EC 3.5.1.3) isolated from rat liver cytosol is a versatile enzyme that catalyzes a large number of amidase, transamidase, and ester hydrolysis reactions. omega-Amidase activity toward alpha-ketoglutaramate and alpha-ketosuccinamate (the alpha-keto acid analogues of glutamine and asparagine, respectively) is present in mammalian tissues, tumors, plants, bacteria, and fungi. Despite its versatility, widespread occurrence, and high specific activity, the enzyme has been little studied, possibly because the assay procedure previously required a substrate (alpha-ketoglutaramate) that is not commercially available. Here we report a simplified method for preparing alpha-ketoglutaramate and an assay procedure that measures alpha-ketoglutarate formation from alpha-ketoglutaramate in a 96-well plate format. We also describe a 96-well plate assay procedure that measures omega-amidase-catalyzed hydroxaminolysis of commercially available succinamic acid. The product, succinyl hydroxamate, yields a stable brown color in the presence of acidic ferric chloride that can be quantitated spectrophotometrically with negligible background interference. The two assay procedures (hydrolysis of alpha-ketoglutaramate and hydroxaminolysis of succinamate) were employed in purifying omega-amidase approximately 3600-fold from rat liver cytosol. The ratio of alpha-ketoglutaramate hydrolysis to succinamate hydroxaminolysis remained constant during the purification. omega-Amidase has recently been shown to be identical to Nit2, a putative tumor suppressor protein. It is anticipated that these new assay procedures will help to characterize the function of omega-amidase/Nit2 in tumor suppression, will provide the basis of high-throughput procedures to search for potent inhibitors and enhancers of omega-amidase, and will assist in identifying biological interactions between nitrogen metabolism and tumor biology.

Bradykinin B2 receptor in the adrenal medulla of male rats and mice: glucocorticoid-dependent increase with immobilization stress.

Bradykinin, acting via the bradykinin B2 receptor (B2R), is a potent stimulator of adrenomedullary catecholamine biosynthesis and release and likely plays an important role in the adrenomedullary stress response. However, the effects of stress on the expression of this receptor in the adrenal medulla are currently unclear. Here, we examined the changes in adrenomedullary B2R gene expression in male rats in response to single (1 time) and repeated (6 times) exposure to 2 hours immobilization stress (IMO). Immediately after 1 or 6 times IMO, B2R mRNA levels were increased by 9-fold and 7-fold, respectively, and returned to unstressed control levels 3 hours later. This large, but transient, increase in mRNA elicited a doubling of protein levels 3 hours after the stress exposure. Next, the role of the hypothalamic-pituitary-adrenocortical axis in the stress-induced upregulation of B2R gene expression was examined. Treatment with endogenous (corticosterone) and synthetic (dexamethasone) glucocorticoids dose-dependently increased B2R mRNA levels in adrenomedullary-derived PC12 cells. Furthermore, cortisol supplementation at levels mimicking stress exposure elevated B2R mRNA levels in the adrenal medulla of hypophysectomized rats. In response to 1 exposure to IMO, the stress-triggered rise in plasma corticosterone and adrenomedullary B2R mRNA levels was attenuated in CRH-knockout mice and absent in pharmacologically adrenalectomized rats, indicating a requirement for glucocorticoids in the upregulation of B2R gene expression with stress. Overall, the increase in B2R gene expression in response to the stress-triggered rise in glucocorticoids likely enhances catecholamine biosynthesis and release and may serve as an adaptive response of the adrenomedullary catecholaminergic system to stress.

Stress-induced changes in gene expression of urocortin 2 and other CRH peptides in rat adrenal medulla: involvement of glucocorticoids

The corticotropin‐releasing hormone (CRH) family regulates the endocrine stress response. Here, we examined the effect of immobilization stress (IMO) on gene expression of adrenomedullary CRH family members. Urocortin 2 (Ucn2) has the highest basal gene expression and is increased by > 30‐fold in response to single IMO and about 10‐fold after six daily repeated IMO. IMO also induced a smaller rise in CRH (six‐fold) and CRH receptor type 1 (CRHR1; two‐fold with single IMO). The influence of glucocorticoids was examined. Dexamethasone (DEX) or corticosterone greatly increased Ucn2 mRNA levels in PC12 cells in a dose‐dependent manner. The DEX elicited rise in Ucn2 was abolished by actinomycin D pre‐treatment, indicating a transcriptionally mediated response. DEX also triggered a rise in CRHR1 and lowered CRH mRNA levels. In CRH‐knockout mice, where the IMO‐induced rise in corticosterone was attenuated, the response of IMO on Ucn2, as well as CRHR2 mRNAs was absent. Overall, the results suggest that the stress‐triggered rise in glucocorticoids is involved in the large induction of Ucn2 mRNA levels by IMO, which may allow Ucn2 to act in an autocrine/paracrine fashion to modulate adrenomedullary function, or act as an endocrine hormone.

Regulation of Angiotensin II Type 2 Receptor Gene Expression in the Adrenal Medulla by Acute and Repeated Immobilization Stress

While the renin-angiotensin system is important for adrenomedullary responses to stress, the involvement of specific angiotensin II (Ang II) receptor subtypes is unclear. We examined gene expression changes of angiotensin II type 1A (AT(1A)) and type 2 (AT(2)) receptors in rat adrenal medulla in response to immobilization stress (IMO). AT(2) receptor mRNA levels decreased immediately after a single 2-h IMO. Repeated IMO also decreased AT(2) receptor mRNA levels, but the decline was more transient. AT(1A) receptor mRNA levels were unaltered with either single or repeated IMO, although binding was increased following repeated IMO. These effects of stress on Ang II receptor expression may alter catecholamine biosynthesis, as tyrosine hydroxylase and dopamine β-hydroxylase mRNA levels in PC12 cells are decreased with Ang II treatment in the presence of ZD7155 (AT(1) receptor antagonist) or with CGP42112 (AT(2) receptor agonist) treatment. Involvement of stress-triggered activation of the hypothalamic-pituitary-adrenocortical or sympathoadrenal axis in AT(2) receptor downregulation was examined. Cultured cells treated with the synthetic glucocorticoid dexamethasone displayed a transcriptionally mediated decrease in AT(2) receptor mRNA levels. However, glucocorticoids are not required for the immediate stress-triggered decrease in AT(2) receptor gene expression, as demonstrated in corticotropin-releasing hormone knockout (Crh KO) mice and hypophysectomized rats, although they can regulate basal gene expression. cAMP and pituitary adenylate cyclase-activating polypeptide also reduced AT(2) receptor gene expression and may mediate this response. Overall, the effects of stress on adrenomedullary AT(1A) and AT(2) receptor expression may contribute to allostatic changes, such as regulation of catecholamine biosynthesis.

Varied Mechanisms of Oestradiol Mediated Regulation of Dopamine β-Hydroxylase Transcription

Experiments performed in vivo and in cell culture have demonstrated that oestradiol induces dopamine β‐hydroxylase (DBH) gene transcription. In the present study, we examined oestrogen‐responsive elements of the rat DBH gene promoter aiming to characterise the mechanisms of oestradiol‐induced DBH transcription. Various mutations and deletions of DBH promoter reporter constructs were tested for responsiveness to 17β‐oestradiol (E2). Mutation of the half palindromic oestrogen response element (ERE) at position −759 reduced the response to E2 in PC12 cells co‐transfected with oestrogen receptor (ER) α, indicating a functional role for this motif. In cells co‐transfected with ERβ, mutations at the −759 site were unresponsive to E2. To characterise the additional E2 responsive elements, mediated by ERα, the DBH promoter was truncated to the proximal 249 or 200 nucleotides upstream of the transcription start site. Despite either truncation, 10 nm E2 still elicited an approximately two‐fold induction of DBH promoter activity. Mutation of a possible ERE‐like sequence at −59 had no effect. The lack of a functional ERE in the proximal region of the rat DBH promoter despite E2‐mediated DBH promoter activity, suggests regulation by a nonclassical mechanism, such as a membrane‐initiated signalling pathway. Moreover, the induction of DBH promoter activity and the rise in DBH mRNA levels were observed within hours. To determine whether membrane‐initiated E2 signalling is involved in rat DBH gene transcription, a membrane impermeable E2 conjugate, β‐oestradiol‐6‐(O‐carboxy‐methyl) oxime‐bovine serum albumin (E2BSA), was used. Incubation with E2‐BSA induced luciferase activity and elicited a significant rise in DBH mRNA levels in the ERα transfected cells. The findings indicate two different mechanisms whereby DBH transcription is regulated by E2 in the presence of ERα. The results implicate both genomic and membrane‐initiated mechanisms, mediated by ERα, in E2‐induced DBH gene transcription.

Regulation of nonclassical renin-angiotensin system receptor gene expression in the adrenal medulla by acute and repeated immobilization stress

The involvement of the nonclassical renin-angiotensin system (RAS) in the adrenomedullary response to stress is unclear. Therefore, we examined basal and immobilization stress (IMO)-triggered changes in gene expression of the classical and nonclassical RAS receptors in the rat adrenal medulla, specifically the angiotensin II type 2 (AT2) and type 4 (AT4) receptors, (pro)renin receptor [(P)RR], and Mas receptor (MasR). All RAS receptors were identified, with AT2 receptor mRNA levels being the most abundant, followed by the (P)RR, AT1A receptor, AT4 receptor, and MasR. Following a single IMO, AT2 and AT4 receptor mRNA levels decreased by 90 and 50%, respectively. Their mRNA levels were also transiently decreased by repeated IMO. MasR mRNA levels displayed a 75% transient decrease as well. Conversely, (P)RR mRNA levels were increased by 50% following single or repeated IMO. Because of its abundance, the function of the (P)RR was explored in PC-12 cells. Prorenin activation of the (P)RR increased phosphorylation of extracellular signal-regulated kinase 1/2 and tyrosine hydroxylase at Ser(31), likely increasing its enzymatic activity and catecholamine biosynthesis. Together, the broad and dynamic changes in gene expression of the nonclassical RAS receptors implicate their role in the intricate response of the adrenomedullary catecholaminergic system to stress.