[Mucoprolassectomy with circular stapler: early results with a new device]. / Mucoprolassectomia con suturatrice circolare: utilizzo di un nuovo device.

AIMS: Stapled transanal mucosectomy is widely used by a lot of surgeon and in RCT, when compared to other haemorrhoidectomy techniques, it didn't show a real primacy. We describe results obtained with a new device, the EEA™ 33-mm Hemorrhoid and Prolapse Stapler Set con DST Series™ (Covidien™), used on 65 patients. PATIENTS AND METHODS: Sixty-five patients underwent a stapled transanal mucosectomy for grade III (n° 20), IV (n° 15) haemorrhois and rectal prolapse (n° 30). The operation were performed by two surgeons. Results. Resected tissue had a mean width of 4.2 cm (3.3-6.1 cm) and a mean weight of 11 g (8-21 g). Surgical time were 22 min (15-35 min). Bleeding of the suture line were observed in 5 cases (7.7%) and they required a mean of 2 haemostatic stitch (1-5) in adsorbable suture 2/0. Mucoprolapsectomy was considered excellent in 55 patients (84.7%), good in 8 patients (12.3%), bad in 2 patients (3%) by surgeons. There were no early and late complications like bleeding, abscesses, anastomotic dehiscence. We report two case of perianal haematoma that don't need any treatment and one case of anal stenosis treated with dilatation therapy. Post-operative pain, estimated with VAS, emphasized an excellent response to NSAIDs in first postoperative day, in 10 cases (15.4%) we prolonged analgesic therapy for 3 days, in one case (1.5%) for one week. After 6 month all patients were revaluated with physical examination and anoscopy. Only one patient (1.5%) had an asymptomatic recurrence of rectal prolapse. CONCLUSION: We conclude that the new device makes easy and standardized surgery. The new device reduce a lot of difficult in patients with tight pelvis and a small distance between ischiatic tubers.

Longitudinal evaluation of a fibrosis index combining MMP-1 and PIIINP compared with MMP-9, TIMP-1 and hyaluronic acid in patients with chronic hepatitis C treated by interferon-alpha and ribavirin.

We have recently described a fibrosis index combining serum procollagen type III N-terminal peptide (PIIINP) and matrix metalloproteinase 1 (MMP-1) concentrations for evaluating the amount of liver fibrosis in chronic hepatitis C patients. The aims of the present study were to validate this score in another cohort of patients and to assess its variations along those of TIMP-1, hyaluronic acid (HA) and MMP-9 during antiviral treatment. Seventy-nine patients treated by interferon-alpha and ribavirin for 24 or 48 weeks were included. A liver biopsy was performed within the 6 months before the start of treatment. Serum markers were measured in serum collected the day of the liver biopsy, at start of treatment, and every 3 months during treatment and a 6-month follow-up period. The PIIINP/MMP-1 index was significantly correlated to the METAVIR fibrosis (r = 0.68, P < 0.001). Its overall diagnostic value defined by the area under the receiver operating characteristics curves was 0.77 for discriminating F1 vs F2F3F4, and 0.81 for discriminating F1F2 vs F3F4, and was better than that observed for HA and TIMP-1. At the end of follow-up, the PIIINP/MMP-1 index significantly decreased in responders and remained stable in nonresponder patients. This decrease occurred early and continued regularly during the treatment period. This variation was because of both a decrease of PIIINP and an increase of MMP-1 concentrations. HA and TIMP-1 serum concentrations were also significantly lower at the end of follow-up in responder patients, but early changes were minimal and not influenced by the response to treatment. Our study shows that a noninvasive index combining PIIINP and MMP-1 is a useful tool to follow-up fibrosis change during and after antiviral therapy chronic hepatitis C patients.

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.

Gastric inhibitory polypeptide (GIP) stimulates cortisol secretion, cAMP production and DNA synthesis in an adrenal adenoma responsible for food-dependent Cushing's syndrome.

We studied in vitro an adrenal tumor responsible for food-dependent, ACTH independent, Cushing's's syndrome. Cortisol secretion by isolated tumor cells was stimulated by GIP and ACTH, but not by the gut hormone glucagon-like peptide-1 (GLP-1). Both GIP and ACTH stimulated production of cAMP but not inositol 1,4,5-trisphosphate IP3). In quiescent tumor cells, GIP and ACTH stimulated [3H]-thymidine incorporation and p42-p44 MAP kinase activity. In normal human adrenocortical cells cortisol secretion and [3H]-thymidine incorporation were stimulated by ACTH but not by GIP. GIP receptor mRNA, assessed by RT-PCR, was highly expressed in the tumor, but undetectable in the adjacent hypotrophic adrenal tissue, in a normal adrenal, in two adrenal tumors responsible for food-independent Cushing's syndrome and in two hyperplastic adrenals associated with ACTH hypersecretion. Low levels of ACTH receptor mRNA 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 of cAMP that may participate in stimulation of both cortisol secretion and proliferation of the tumor cells.

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.

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.

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.

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.

The 86-kDa subunit of autoantigen Ku is a somatostatin receptor regulating protein phosphatase-2A activity.

We previously reported the immunopurification of a somatostatin receptor from the human tumoral gastric cell HGT1 using the monoclonal antibody 30F3 (Reyl-Desmars, F., Le Roux, S., Linard, C., Benkouka, F., and Lewin, M. J. M. (1989) J. Biol. Chem. 264, 18789-18795). Screening of a lambda gt11 HGT1-cDNA library with 30F3 led us to isolate a cDNA encoding an 86-kDa polypeptide displaying 100% structural identity with the 86-kDa subunit (p86-Ku) of the Ku autoantigen. Recombinant p86 expressed in Escherichia coli cross-reacted with 30F3 and specifically bound [125I-Tyr11]somatstatin-14. Binding was totally displaced by somatostatin-14, somatostatin-28, and SMS 201-995, with IC50 values of 0.7, 1.0, and 1.2 nM, respectively. In a search for a biological effect associated with binding, we purified a 36-kDa, okadaic acid-sensitive phosphatase (protein phosphatase-2A (PP2A)) from rat gastric cytosol. PP2A catalyzed 32P release from p34cdc2-phosphorylated histone H1. However, PP2A-induced 32P release was concentration dependently inhibited by recombinant p86-Ku, with a decrease in maximal velocity without a change in Km. Steric exclusion high pressure chromatography indicated that the inhibition resulted from direct interaction of the enzyme with p86-Ku. Furthermore, it was antagonized by increased concentrations of somatostatin-14 and prevented by preincubating p86-Ku with 30F3. Given the key role played by PP2A in cell cycle regulation, the current findings suggest that p86-Ku could be a physiological target of somatostatin antiproliferative action.

Characterization of a membrane glycoprotein having pharmacological and biochemical properties of an AT2 angiotensin II receptor from human myometrium.

The angiotensin II receptors of human myometrial tissue were characterized using ligand binding, cross-linking with radioactive label, detergent solubilization and partial purification by lectin-affinity chromatography. Human myometrial membrane preparations contained variable amount (5-650 fmol/mg protein) of high affinity (Kd = 44-65 pM) binding sites for 125I-CGP42112, a ligand specific for the AT2 subtype of angiotensin II receptors. Competition studies with AT1-specific and AT2-specific compounds indicated that angiotensin II receptors on these membranes were exclusively of the AT2 subtype. The binding sites for 125I-CGP42112 were efficiently solubilized by the detergent Chaps, albeit with a marked decrease in affinity (Kd = 1.2 nM). The proteins in the myometrial membrane preparation were cross-linked to 125I-[Sar1, Ile8]angiotensin II (Sarile) with disuccinimidyl suberate. When low concentrations of cross-linker were used, a single radiolabelled band of about 66-70 kDa was revealed on SDS/PAGE. At higher concentrations additional bands of about 105-120 kDa and 200 kDa were labelled. The 66-70-kDa and 105-120-kDa bands were separated by electroelution of SDS/PAGE gel slices and submitted to trypsin cleavage. The tryptic-peptide maps were identical for both products, suggesting that the additional bands are homodimers and trimers of the labelled polypeptide. The Chaps-solubilized receptor was retained on wheat-germ-agglutinin-Sepharose and specifically eluted by the competing sugar triacetylchitotriose, leading to a fivefold purification factor. Treatment of the 125I-Sarile-labelled protein with N-glycanase caused a shift in its apparent molecular mass on SDS/PAGE from 66-70 kDa to 33 kDa.

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 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.

Renal actions of the selective angiotensin AT2 receptor ligands CGP 42112B and PD 123319 in the sodium-depleted rat.

The purpose of this study was to investigate the renal actions of the new selective angiotensin AT2 receptor ligands, CGP 42112B and PD 123319, in comparison to those of the AT1 receptor antagonist losartan, in the sodium-depleted, anesthetized rat. Losartan (1, 3 and 10 mg/kg i.v.) produced a dose-dependent decrease in blood pressure and renal vascular resistance that was statistically significant. Effective renal blood flow tended to increase in response to all doses of losartan while glomerular filtration rate either did not change or decreased, leading to a significant fall in filtration fraction. Losartan did not induce significant changes in urine volume, urinary sodium excretion, urinary potassium excretion or free water formation. The selective AT2 receptor ligand CGP 42112B at infusion rates of 1-100 micrograms/kg per min i.v. had no significant effect on blood pressure or any measured parameter of renal function. However, when infused at 1000 micrograms/kg per min i.v., CGP 42112B did not affect blood pressure, but significantly increased effective renal blood flow, glomerular filtration rate, urinary sodium excretion, urinary potassium excretion and free water formation, while significantly decreasing renal vascular resistance. The selective AT2 receptor ligand PD 123319 at infusion rates between 1 and 100 micrograms/kg per min i.v. also had no significant effect on blood pressure or on any measured parameter of renal function. However, at an infusion rate of 1000 micrograms/kg per min i.v., PD 123319 tended to increase renal vascular resistance, urinary sodium excretion, urinary potassium excretion and free water formation, and to decrease effective renal blood flow, although none of these changes reached a level of statistical significance.(ABSTRACT TRUNCATED AT 250 WORDS)

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.