Efficient transcription of the human angiotensin II type 2 receptor gene requires intronic sequence elements.

To investigate mechanisms of human angiotensin II type 2 receptor (hAT2) gene regulation we functionally characterized the promoter and downstream regions of the gene. 5'-Terminal deletion mutants from -1417/+100 to -46/+100 elicited significant but low functional activity in luciferase reporter gene assays with PC12W cells. Inclusion into the promoter constructs of intron 1 and the transcribed region of the hAT2 gene up to the translation start enhanced luciferase activity 6.7+/-1.6-fold and 11.6+/-1.7-fold (means+/-S.E.M.) respectively, whereas fusion of the promoter to the spliced 5' untranslated region of hAT2 cDNA did not, which indicated an enhancement caused by intronic sequence elements. Reverse transcriptase-mediated PCR confirmed that the chimaeric hAT2-luciferase mRNA was regularly spliced in PC12W cells. A Northern blot analysis of transfected cells showed levels of luciferase mRNA expression consistent with the respective enzyme activities. Mapping of intron 1 revealed that a 12 bp sequence in the centre of the intron was required for the increase in promoter activity, whereas the 5' adjacent intronic region mediated a decrease in luciferase activity. Mutation of the 12 bp region led to altered protein binding and markedly decreased luciferase activity. Cloned into a promoterless luciferase vector, a 123 bp intron 1 fragment was able to direct reporter gene expression to the same activity as occurred in conjunction with the 5' flanking region. These results indicate that sequence elements in intron 1 are necessary for efficient transcription of hAT2. In reporter gene assays, intron 1 might by itself function as a promoter and initiate transcription from an alternative start point.

Cushing's syndrome due to a gastric inhibitory polypeptide-dependent adrenal adenoma: insights into hormonal control of adrenocortical tumorigenesis.

We studied a patient with food-induced, ACTH-independent, Cushing's syndrome and a unilateral adrenocortical adenoma. In vivo cortisol secretion was stimulated by mixed, glucidic, lipidic, or proteic meals. Plasma ACTH levels were undetectable, but iv injection of ACTH stimulated cortisol secretion. Unilateral adrenalectomy was followed by hypocortisolism with loss of steroidogenic responses to both food and ACTH. In vitro, cortisol secretion by isolated tumor cells was stimulated by the gut hormone gastric inhibitory polypeptide (GIP) and ACTH, but not by another gut hormone, glucagon-like peptide-1 (GLP-1). Both peptides stimulated the production of cAMP but not of inositol 1,4,5-trisphosphate. In quiescent cells, GIP and ACTH stimulated [3H]thymidine incorporation and p42-p44 mitogen-activated protein kinase activity. GIP receptor messenger ribonucleic acid (RNA), assessed by RT-PCR, was highly expressed in the tumor, whereas it was undetectable in the adjacent hypotrophic adrenal tissue, in two adrenal tumors responsible for food-independent Cushing's syndrome, and in two hyperplastic adrenals associated with ACTH hypersecretion. In situ hybridization demonstrated that expression of GIP receptor RNA was confined to the adrenocortical tumor cells. Low levels of ACTH receptor messenger RNA were also detectable in the tumor. We conclude that abnormal expression of the GIP receptor allows adrenocortical cells to respond to food intake with an increase in cAMP that may participate in the stimulation of both cortisol secretion and proliferation of the tumor cells.

Repression of c-fos and c-jun gene expression is not part of AT2 receptor coupled signal transduction.

The signal transduction mechanism coupled to angiotensin AT2 receptors is still a matter of debate. Based on the findings that AT2 receptor stimulation causes inhibition of proliferation, and that other antiproliferative agents such as transforming growth factor-beta, retinoic acid, and MyoD act via repression of immediate early gene (IEG) expression, this study was aimed at elucidating whether downregulation of IEG expression is also part of the AT2 receptor coupled signaling mechanism. Stimulation of angiotensin AT2 receptors in the rat pheochromocytoma cell line PC12 W following pretreatment with growth factors was able to counteract growth factor induced proliferation but not to repress growth factor induced c-fos and c-jun expression; neither did AT2 receptor stimulation cause an induction of c-fos expression. We conclude that, in contrast to other growth-inhibiting agents, the antiproliferative effect of angiotensin II via the AT2 receptor is not mediated by repression of the immediate early genes c-fos and c-jun.

ANG II AT1 and AT2 receptors both inhibit bFGF-induced proliferation of bovine adrenocortical cells.

Angiotensin II (ANG II) has long been known for its pressor and growth-promoting effects, which are both mediated by the AT1 receptor. By contrast, the AT2 receptor has recently been reported to mediate inhibition of proliferation through as yet undefined mechanisms. We report here that in bovine adrenal fasciculata cells ANG II by itself does not affect growth but inhibits basic fibroblast growth factor (bFGF)-induced DNA synthesis and blocks the cells in G1 phase. Consistent with this, ANG II inhibits cyclin D1 expression and cyclin D1-associated kinase activity. The antimitogenic effect of ANG II is partly mimicked by the AT2-selective agonist CGP-42112. It is also blocked partly and in an additive fashion by the AT1- and AT2-selective antagonists losartan and PD-123319, indicating the contribution of both receptor subtypes to this response. AT1-dependent antiproliferation is selectively blocked by the cyclooxygenase inhibitor indomethacin and restored by prostaglandin E2, whereas AT2-receptor-mediated inhibition of growth is suppressed by the tyrosine phosphatase inhibitors orthovanadate and bpV(pic). Both pathways are, however, pertussis toxin sensitive. We hypothesize that, in fasciculata cells, the AT1 receptor inhibits bFGF-induced proliferation by stimulating prostaglandin synthesis, whereas the AT2 receptor mediates its effect through a pathway that requires protein tyrosine phosphatase activation.

The angiotensin II AT2 receptor inhibits proliferation and promotes differentiation in PC12W cells.

Angiotensin II (ANG II) has been implicated in cell growth and differentiation. We investigated the effect of AT2 receptor stimulation on proliferation and morphological differentiation in cells of neuronal origin by using the pheochromocytoma derived cell line, PC12W. ANG II (10(-8)-10(-6) M) inhibited fetal calf serum (FCS)-induced cell proliferation in a concentration dependent manner. In half of the experiments, the epidermal growth factor (EGF) exerted a mitogenic action which was concentration-dependently inhibited by ANG II. In the other half of the experiments, EGF had an antimitogenic effect which was further enhanced by ANG II (maximally at 10(-6) M). Treatment with nerve growth factor (NGF) induced an inhibition of [3H]thymidine incorporation, which was enhanced by ANG II, maximally 25% at the highest concentration. The effects of ANG II on [3H]thymidine incorporation were reflected by those on cell number and were prevented by the AT2 receptor antagonist, PD123177, but not influenced by the AT1 receptor antagonist, losartan. The ANG II-induced inhibition of cell proliferation was paralleled by morphological differentiation in response to daily treatment with ANG II. ANG II also enhanced low-dose NGF-induced neurite formation. Again, these effects of ANG II were abolished by the AT2 receptor antagonist, PD123177. Our data in PC12W cells show that the AT2 receptor not only inhibits growth factor-induced proliferation and enhances the NGF-mediated growth arrest but also induces morphological differentiation in cells of neuronal origin. These findings strongly support the hypothesis that the AT2 receptor promotes differentiation in neuronal cells.

Protein tyrosine kinase activity in 350 T1/T2, N0/N1 breast cancer. Preliminary results.

Protein tyrosine kinases (PTKs) are a family of enzymes sharing a highly conserved catalytic domain which phosphorylates substrate proteins on tyrosine residues. PTKs play a major role in the transduction of the mitogenic signal and are involved in the control of cell proliferation, differentiation, and transformation processes. PTKs can be subdivided into two major types: membrane associated PTKs consisting essentially of growth factor receptors (receptor tyrosine kinases or RTKs) and cytosolic PTKs involved in the intracellular transduction of mitogenic and differentiation signals. From January 1988 to January 1992, PTK activity was assayed in cytosolic fractions prepared from 350 T1-T2, N0-N1 M0, breast carcinomas. Enzymatic activity was measured using phosphate transfer from [32P]-ATP to poly-Glu-Tyr as an artificial substrate. According to our previously reported pilot study, we chose a cut-off value of 12 pmol 32P incorporated min-1 mg-1 protein, corresponding to the median value. We found positive PTK levels (> or = 12 pmol/min/mg) to be correlated with a loss of differentiation according to Scarff-Bloom grade (p < 0.001), negative PR (p = 0.03) and ER status (p = 0.04). With a median follow-up of 30 months (0-82), patients with a positive PTK level presented a smaller 3-year disease free survival than in the PTK negative group of patients (p = 0.07). In Cox multivariate analysis including pT, pN, Scarff-Bloom grade, PR and ER, PTK activity does not emerge as a significant prognostic factor.

Hormonal regulation of mitogen-activated protein kinase activity in bovine adrenocortical cells: cross-talk between phosphoinositides, adenosine 3',5'-monophosphate, and tyrosine kinase receptor pathways.

Angiotensin-II (AII), which stimulates steroidogenesis in bovine adrenocortical (BAC) cells through the phosphoinositides pathway, activates p42-p44 mitogen-activated protein kinases (MAPKs) after 5 min of treatment (EC50 = 0.1 nM). This activation is 1) completely inhibited by the AII receptor AT1 subtype antagonist Dup 753 (10 microM), but unaffected by the AT2 antagonist PD 123177; 2) not reproduced by the AT2 agonist CGP 42112A; 3) insensitive to pretreatment with pertussis toxin; and 4) abolished by a 48-h preexposure of the cells to the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA; 1 microM), which down-regulates protein kinase-C activity. Fibroblast growth factor-2, a potent mitogen for BAC cells, which acts through its tyrosine kinase receptor, also activates MAPK (EC50 = 0.3 in a TPA-insensitive manner, while exhibiting no detectable effect on BAC cell steroidogenesis. In contrast, ACTH, which stimulates steroidogenesis via cAMP and inhibits BAC cell proliferation, does not stimulate MAPK. Indeed, ACTH completely blocks (IC50 = 0.01 nM) the stimulation of MAPK by AII, fibroblast growth factor-2, or TPA. Therefore, bovine adrenocortical cells provide an example of positive and negative hormonal regulation of MAPK activity through a cross-talk between the inositide-, cAMP-, and growth factor-activated tyrosine kinase pathways.

Stimulation of cortisol production through angiotensin AT2 receptors in bovine fasciculata cells.

Bovine fasciculata cells in culture (BAC) express both AT1 and AT2 angiotensin receptors. The role and signaling pathways of this latter receptor are still the subject of debate. We found that in BAC stimulation of cortisol (F) production by angiotensin II (A II) is accounted for by both receptor subtypes. We have investigated the potential AT2 signalling pathways involved in this response. As previously described in other cells, we found this receptor to mediate inhibition of ANP stimulated cGMP production through a phosphodiesterase independent pathway. This phenomenon does however not appear to be involved in cortisol production as this response was not affected by the addition of 8-Br-cGMP or ANP. It was however abolished after down-regulation of PKC by phorbol esters, but not by Gi inhibition with pertussis toxin. Moreover and as opposed to the AT1 mediated response, AT2 receptor stimulation potentiated K+ induced F production. In conclusion, these observations suggest that the AT2 pathway which mediates F production requires intact PKC and might involve a Gi independent stimulation of Ca++ or K+ channels.

The angiotensin AT2-receptor mediates inhibition of cell proliferation in coronary endothelial cells.

Angiotensin II (ANG II) is known to be a potent growth promoting factor for vascular smooth muscle cells and fibroblasts but little is known about its influence on growth in endothelial cells. We studied the effects of ANG II on endothelial growth and the role of the angiotensin receptor subtypes involved. Proliferation of rat coronary endothelial cells (CEC) and rat vascular smooth muscle cells (VSMC) was determined by [3H]thymidine incorporation, the MTT-test and by directly counting cells in a coulter counter. Angiotensin AT1- and AT2-receptors were demonstrated by binding studies and by the presence of their respective mRNA through reverse transcription polymerase chain reaction (RT-PCR). In contrast to VSMC, which in culture only express the AT1-receptor, CEC express both, AT1- and AT2-receptors simultaneously up to the third passage. Whereas ANG II stimulated growth of quiescent VSMC, an effect abolished by pretreatment with the AT1-receptor antagonist, losartan, ANG II did not induce proliferation in quiescent CEC. However, after pretreatment of quiescent endothelial cells (< passage 4) with the AT2-receptor antagonist, PD 123177, ANG II induced proliferation. This effect was reversed by additional pretreatment with losartan. ANG II significantly inhibited the proliferation of bFGF-stimulated CEC in a dose-dependent manner by maximally 50%. This effect was prevented by PD 123177 while losartan was ineffective. The AT2-receptor agonist, CGP 42112, mimicked the antiproliferative actions of ANG II, confirming the specificity of the effect. Our results show that the growth modulating actions of ANG II depend on the type of angiotensin receptor present on a given cell. In coronary endothelial cells, the antiproliferative actions of the AT2-receptor offset the growth promoting effects mediated by the AT1-receptor.

A G protein is involved in the angiotensin AT2 receptor inhibition of the T-type calcium current in non-differentiated NG108-15 cells.

In non-differentiated NG108-15 cells, both angiotensin II (Ang II) (100 nM) and CGP 42112 (100 nM) decreased the T-type calcium current amplitude by 24 +/- 2% and 21 +/- 3%, respectively. cGMP is not a mediator of the Ang II effect, since loading of cells with 50 microM cGMP did not prevent the inhibitory effects of Ang II. The effects of Ang II involves a non-identified GTPase activity since incubation with GDP beta S (3 mM) completely reversed the inhibitory effect of Ang II while GTP gamma S mimicked its effect. However, Ang II binding was not affected by GTP gamma S, and the effect of Ang II was not modified in pertussis toxin-treated cells. The inhibitory effect of Ang II on the T-type Ca2+ current involves a phosphotyrosine phosphatase activity since sodium orthovanadate prevented the effects of Ang II, although microcystin-LR, a selective Ser/Thr phosphatase 1 and 2A inhibitor, did not modify the effect of Ang II. These results provide the first evidence of a modulation of membrane conductance by Ang II through the AT2 receptor and demonstrate the involvement of a phosphotyrosine phosphatase and a G protein in the AT2 transduction mechanism in NG108-15 cells. Moreover, our data suggest that phosphotyrosine phosphatase activation is proximal to receptor occupation, since sodium orthovanadate inhibits both GTPase activity and T-type current blockage induced by Ang II or CGP 42112, while GTP gamma S inhibition of the T-type calcium current is not impaired.

Angiotensin II stimulates protein tyrosine phosphatase activity through a G-protein independent mechanism.

Most of angiotensin II's (Ang II) documented effects have been attributed to the interaction of this peptide with a G-protein coupled receptor termed AT1. The role and the signalling mechanisms of the more recently characterized AT2 receptor, which does not appear to interact with G-proteins, are however still unclear. We report here that this receptor mediates the rapid dephosphorylation of tyrosine residues of specific proteins in the 60 to 150 KDa range in PC12W cells which express only AT2 receptors. We further characterized this phosphatase activity using the synthetic substrate para-nitrophenyl phosphate. Dephosphorylation of this substrate in response to Ang II is not affected by Ser/Thr phosphatase inhibitors, but is completely prevented by the protein tyrosine phosphatase (PTPase) inhibitor sodium orthovanadate. This effect is mimicked by the AT2 selective agonist CGP42112 and is not affected by the AT1 antagonist losartan, In contrast to the recently reported PTPase stimulation by somatostatin and dopamine, PTPase stimulation by Ang II is not affected by the guanyl nucleotides GTP gamma S and GDP beta S. Moreover, depletion of solubilized membrane preparations from G-proteins by lectin affinity chromatography does not alter Ang II stimulation of the measured PTPase activity. These findings indicate that Ang II stimulates a PTPase activity through AT2 receptors via G-protein independent pathways. This signalling mechanism may be involved in AT2 receptor mediated actions of Ang II such as particulate guanylate cyclase inhibition, modulation of T-type Ca++ channels and regulation of cell proliferation and differentiation.

Angiotensin AT2 receptor mediated inhibition of particulate guanylate cyclase: a link with protein tyrosine phosphatase stimulation?

Ever since the identification of two distinct Ang II receptor subtypes, the function of the AT2 receptor has been a subject of debate. As opposed to the AT1 subtype, this receptor does not interact with G-proteins in most cell lines and tissues. We show here that, in intact PC12W cells which express only AT2 receptors, Ang II significantly decreases basal and atrial natriuretic peptide (ANP)-stimulated cGMP concentration. This effect is mimicked by the AT2 selective agonist CGP 42112, and is not prevented by the AT1 selective antagonist losartan, indicating that this is an AT2 receptor mediated response. The lack of effect of the phosphodiesterase (PDE) inhibitor IBMX shows that this mechanism does not involve PDE stimulation. This is confirmed by the finding that neither Ang II or CGP 42112 affect the Ca++/calmodulin dependent cGMP PDE activity. Furthermore Ang II and CGP 42112 have no effect on nitroprusside-stimulated cGMP levels in these cells, thus ruling out interactions between the AT2 receptor and soluble guanylate cyclase. These data indicate that the AT2 receptor mediated decrease of cGMP is due to the selective inhibition of particulate guanylate cyclase (pGC) activity. In an accompanying paper we report that interaction of Ang II with the AT2 receptor in the same cells results in the stimulation of phosphotyrosine phosphatase (PTPase) activity. Interestingly, the PTPase inhibitors sodium orthovanadate and phenylarsine oxyde, but not the Ser/Thr phosphatase inhibitor okadiac acid, inhibitthe Ang II and CGP 42112 induced decreases in cellular cGMP concentration. These findings suggest that stimulation of PTPase activity may be involved in the regulation of pGC activity via AT2 receptors.

Angiotensin II receptor subtypes: characterization, signalling mechanisms, and possible physiological implications.

Thanks to the recent discovery of angiotensin II (ANG II) receptor subtypes linked to different signalling pathways, research in the different areas related to this peptide has regained a strong interest. In the following review, we first describe the biochemistry and actions of angiotensin peptides formed both in the circulation and locally at the tissue and organ level. Evidence for the existence and distribution of ANG II receptor subtypes in mammalian as well as in nonmammalian species and lower organisms is presented. The changes in receptor subtype expression during development and disease are described. The signal transduction mechanisms and biological actions of ANG II mediated by the recently cloned AT1 receptor are reviewed and the recent data concerning the signalling pathways linked to the AT2 receptor are discussed. Finally, based upon their molecular pharmacology, we present evidence and also speculate upon the physiological function of the ANG II receptor subtypes.

Agonistic and antagonistic properties of angiotensin analogs at the AT2 receptor in PC12W cells.

Despite some recent reports describing the effects of AT2 receptor selective ligands in vitro and in vivo, the physiological function of this receptor is still a matter of debate. This problem stems amongst others from the difficulty in interpreting results from in vivo experiments with drugs of which it is not known whether they act as agonists or antagonists. We reported earlier that angiotensin II inhibits basal and atrial natriuretic peptide stimulated particulate guanylate cyclase activity through AT2 receptors in PC12W cells. We have used this parameter in intact PC12W cells in order to determine the pharmacological properties of different widely used angiotensin receptor ligands. We found CGP 42112 to behave as a full agonist in this system, whereas PD 123319 and Sar Ile angiotensin II act as antagonists. As expected, the AT1 antagonist losartan did not affect this response.

Could the pharmacological differences observed between angiotensin II antagonists and inhibitors of angiotensin converting enzyme be clinically beneficial?

Over the past several years, angiotensin I converting enzyme (ACE) inhibitors, compounds that block the formation of angiotensin II (ANG II), have become widely used in the treatment of cardiovascular disease. Recently, a new class of orally active, non-peptide inhibitors of the renin-angiotensin system, the ANG II receptor antagonists have also become available. Since both classes of compounds block the renin-angiotensin system, although at different sites, it remains to be determined whether blockade of ANG II receptors will have any specific advantage over inhibition of ACE. The following review assesses the actions of ANG II antagonists and suggests ways in which blockade of ANG II receptors may differ both pharmacologically and clinically from inhibition of ACE.

The angiotensin AT2 receptor modulates T-type calcium current in non-differentiated NG108-15 cells.

We report here that angiotensin II (AII) and the AT2 receptor-selective ligand, CGP 42112, modulate the T-type calcium current in non-differentiated NG108-15 cells, which express only AT2 receptors. Both peptides decrease the T-type calcium current at membrane potentials above -40 mV and shift the current-voltage curve at lower potentials with maximal effect between 5 and 10 min after application. These data describe a new cellular response to AII and suggest that the AT2 receptor mediates certain neurophysiological actions of this hormone.

Angiotensin receptor subtypes in the brain.

Development of specific angiotensin II receptor ligands has recently provided evidence for the existence of two angiotensin II receptor subtypes, termed AT1 and AT2, which differ in their signal transduction mechanisms and in the effects they mediate. In brain, both receptor subtypes are present. Most of the known central actions of angiotensin II, for example the regulation of blood pressure and of electrolyte and water balance, seem to be mediated by the AT1 receptor, while the role of the AT2 receptor is still an enigma. This review by Thomas Unger and colleagues summarizes the current knowledge and latest hypotheses in this rapidly developing field.

Characterization and distribution of angiotensin II binding sites in fetal and neonatal astrocytes from different rat brain regions.

Although angiotensin II (Ang II) binding sites have been extensively investigated in brain, revealing the presence of both AT1 and AT2 subtypes in various areas, the question as to which cells express AT1 and AT2 sites is still open. We report here that primary cultures of astrocytes obtained from various brain regions of fetal (F17) and one-day-old rats express Ang II binding sites belonging only to the AT1 subtype. The binding sites have the same binding profile in all regions tested; however, much less binding was observed in membranes of astrocytes derived from cortical than from subcortical regions and almost none were found in neonatal cortex. In addition, the dispersion method used at the onset of culture affects the number of binding sites present at the end of the culture period.