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Angiotensin II (AngII) induces heart growth via cardiomyocyte hypertrophy, and central to this is the capacity of the type 1 AngII receptor (AT1R) to "transactivate" epidermal growth factor receptors (EGFRs)--a family with four main subtypes (HER1-4)--although the exact molecular mechanism remains unresolved. In this study, the pharmacological inhibition of AngII-stimulated ERK1/2 activation and cardiomyocyte hypertrophy by increasing concentrations of an EGFR inhibitor, AG1478, indicated that other EGFR subtypes, in addition to HER1, may be involved. We constructed expression vectors and adenoviruses expressing truncated mutant versions of HER1, HER2, and HER4 and determined their capacity to act as dominant-negative inhibitors when co-transfected with full-length EGFRs. It is surprising that adenoviral-mediated expression of these truncated EGFRs in cardiomyocytes led to paradoxical, ligand-independent increases in cardiomyocyte hypertrophy and unusual morphological changes. These results challenge our perception of AT1R-mediated EGFR transactivation and imply that truncated EGFRs may affect cell function through unconventional mechanisms.

Urotensin II and its receptor are coexpressed in the heart and up-regulated during cardiac dysfunction. In cultured neonatal cardiomyocytes, we mimicked this up-regulation using an adenovirus to increase expression of the urotensin receptor. In this model system, urotensin II promoted strong hypertrophic growth and phenotypic changes, including cell enlargement and sarcomere reorganization. Urotensin II potently activated the MAPKs, ERK1/2 and p38, and blocking these kinases with PD098059 and SB230580, respectively, significantly inhibited urotensin II-mediated hypertrophy. In contrast, urotensin II did not activate JNK. The activation of ERK1/2 and p38 as well as cellular hypertrophy was independent of protein kinase C, and calcium and phosphoinositide 3-kinase, yet dependent on the capacity of the urotensin receptor to trans-activate the epidermal growth factor receptor. Urotensin II promoted the tyrosine phosphorylation of epidermal growth factor receptors, which was inhibited by the selective epidermal growth factor receptor kinase inhibitor, AG1478. These data indicate that perturbations in cardiac homeostasis, which lead to up-regulation of urotensin II receptors, promote urotensin II-mediated cardiomyocyte hypertrophy via ERK1/2 and p38 signaling pathways in an epidermal growth factor receptor-dependent manner.

Adrenomedullin (AM) is a multifunctional peptide hormone with wide-ranging actions related to cardiovascular homeostasis. AM receptors are highly expressed in the heart and AM has antihypertrophic and antiproliferative effects on cardiac myocytes and fibroblasts, respectively. We have investigated the interaction between AM and angiotensin II (Ang II) signalling in neonatal cardiac fibroblast cultures to determine whether the antagonistic effects of AM are mediated via the modulation of Ang II receptors. Cardiac fibroblasts exclusively expressed the Ang II type 1 receptor (AT(1)R) and binding to this site was downregulated by 35% following an 18-h incubation with 100 nM AM. Levels of AT(1A)R mRNA were dose-dependently lowered by AM, with a maximal 40-50% inhibition by 6 h. The decreases in both AT(1)R binding and AT(1A)R mRNA levels were mimicked by 8-Br-cAMP or forskolin, suggesting that the effects of AM were mediated via an elevation of cAMP. In cardiac fibroblasts pretreated with AM, the Ang II induction of collagen biosynthesis was attenuated, although basal collagen synthesis was unaffected. These data suggest that AM mediates the heterologous downregulation of AT(1)R expression via a relatively rapid decrease in AT(1A)R mRNA pools. This interaction may represent a relevant pathophysiological mechanism for modulating Ang II responsiveness in the diseased heart.

Binding of the peptide hormone angiotensin II (AngII) to the type 1 (AT(1A)) receptor and the subsequent activation of phospholipase C-mediated signaling, involves specific determinants within the AngII peptide sequence. In contrast, the contribution of such determinants to AT(1A) receptor internalization, phosphorylation and activation of mitogen-activated protein kinase (MAPK) signaling is not known. In this study, the internalization of an enhanced green fluorescent protein-tagged AT(1A) receptor (AT(1A)-EGFP), in response to AngII and a series of substituted analogs, was visualized and quantified using confocal microscopy. AngII-stimulation resulted in a rapid, concentration-dependent internalization of the chimeric receptor, which was prevented by pretreatment with the nonpeptide AT(1) receptor antagonist EXP3174. Remarkably, AT(1A) receptor internalization was unaffected by substitution of AngII side chains, including single and double substitutions of Tyr(4) and Phe(8) that abolish phospholipase C signaling through the receptor. AngII-induced receptor phosphorylation was significantly inhibited by several substitutions at Phe(8) as well as alanine replacement of Asp(1). The activation of MAPK was only significantly inhibited by substitutions at position eight in the peptide and specific substitutions did not equally inhibit inositol phosphate production, receptor phosphorylation and MAPK activation. These results indicate that separate, yet overlapping, contacts made between the AngII peptide and the AT(1A) receptor select/induce distinct receptor conformations that preferentially affect particular receptor outcomes. The requirements for AT(1A) receptor internalization seem to be less stringent than receptor activation and signaling, suggesting an inherent bias toward receptor deactivation.

Arrestins bind to phosphorylated G protein-coupled receptors and participate in receptor desensitization and endocytosis. Although arrestins traffic with activated type 1 (AT(1A)) angiotensin II (AngII) receptors, the contribution of arrestins to AT(1A) receptor internalization is controversial, and the physical association of arrestins with the AT(1A) receptor has not been established. In this study, by coimmunoprecipitating AT(1A) receptors and beta-arrestin 1, we provide direct evidence for an association between arrestins and the AT(1A) receptor that was agonist- and time-dependent and contingent upon the level of beta-arrestin 1 expression. Serial truncation of the receptor carboxyl terminus resulted in a graded loss of beta-arrestin 1 association, which correlated with decreases in receptor phosphorylation. Truncation of the AT(1A) receptor to lysine(325) prevented AngII-induced phosphorylation and beta-arrestin 1 association as well as markedly inhibiting receptor internalization, indicating a close correlation between these receptor parameters. AngII-induced association was also dramatically reduced in a phosphorylation- and internalization-impaired receptor mutant in which four serine and threonine residues in the central portion of the AT(1A) receptor carboxyl terminus (Thr(332), Ser(335), Thr(336), Ser(338)) were substituted with alanine. In contrast, substitutions in another serine/threonine-rich region (Ser(346), Ser(347), Ser(348)) and at three PKC phosphorylation sites (Ser(331), Ser(338), Ser(348)) had no effect on AngII-induced beta-arrestin 1 association or receptor internalization. While AT(1A) receptor internalization could be inhibited by a dominant-negative beta-arrestin 1 mutant (beta arr1(319-418)), treatment with hyperosmotic sucrose to inhibit internalization did not abrogate the differences in arrestin association observed between the wild-type and mutant receptors, indicating that arrestin binding precedes, and is not dependent upon, receptor internalization. Interestingly, a substituted analog of AngII, [Sar(1)Ile(4)Ile(8)]-AngII, which promotes robust phosphorylation of the receptor but does not activate receptor signaling, stimulated strong beta-arrestin 1 association with the full-length AT(1A) receptor. These results identify the central portion of the AT(1A) receptor carboxyl terminus as the important determinant for beta-arrestin 1 binding and internalization and indicate that AT(1A) receptor phosphorylation is crucial for beta-arrestin docking.

Protein kinase C (PKC) phosphorylates the C-terminus of the type 1 angiotensin II receptor (AT(1)), although the exact site(s) of phosphorylation are unidentified. In the present study, we examined the phosphorylation of epitope-tagged wild-type AT(1A) receptors, transiently expressed in Chinese hamster ovary K1 cells, in response to angiotensin II (AngII) and following selective activation and inhibition of PKC. This phosphorylation was compared with mutant receptors where C-terminal serine residues (Ser(331), Ser(338) and Ser(348)) within three putative PKC consensus sites were replaced with alanine, either individually or in combination. Stimulation by AngII or the phorbol ester PMA to activate PKC induced an increase in phosphorylation of the wild-type AT(1A) receptor, which was prevented by truncation of the receptor C-terminus to remove the last 34 amino acids, including Ser(331), Ser(338) and Ser(348). Whereas single alanine mutation (Ser(331)Ala, Ser(338)Ala and Ser(348)Ala) resulted in decreased receptor phosphorylation, no single mutant completely inhibited either AngII- or PMA-induced phosphorylation. Combined mutation of the three PKC consensus sites caused an approximately 70% reduction in PMA-mediated phosphorylation. The approximately 60% reduction in AngII (1 microM)-induced phosphorylation of this triple mutant and the partial inhibition of wild-type receptor phosphorylation by bisindolylmaleimide, a specific PKC inhibitor, suggest a significant contribution of PKC to agonist-stimulated regulation. The ratio of PKC to total receptor phosphorylation was greatest at low doses of AngII (1 nM), consistent with the idea that PKC phosphorylates and regulates receptor function at low levels of stimulation, whereas phosphorylation by other kinases is more prevalent at high levels of agonist stimulation. To determine if a single PKC site is favoured when the contribution of PKC varies, the phosphorylation of wild-type and mutant receptors was examined over a range of AngII concentrations (0, 1, 10 and 100 nM). At all AngII concentrations, single mutation of Ser(331), Ser(338) or Ser(348) was incapable of completely preventing receptor phosphorylation, suggesting no clear preference for PKC consensus-site utilization. Together, these results indicate a redundancy in PKC phosphorylation of the AT(1A) receptor, whereby all three consensus sites are utilized to some degree following homologous (AngII) and heterologous (PMA) stimulation. The contribution of PKC phosphorylation to receptor regulation is unclear, but multiple PKC phosphorylation of the AT(1A) receptor may allow independent and/or complementary events to occur at the three separate sites of the C-terminus.

1. The role of the metalloendopeptidase EC 3.4.24.15 (EP 24.15) in peptide metabolism in vivo is unknown, in part reflecting the lack of a stable enzyme inhibitor. The most commonly used inhibitor, N-[1-(R,S)-carboxy-3-phenylpropyl]-Ala-Ala-Tyr-p-aminobenzoate (cFP-AAY-pAB, Ki = 16 nM), although selective in vitro, is rapidly degraded in the circulation to cFP-Ala-Ala, an angiotensin converting enzyme (ACE) inhibitor. This metabolite is thought to be generated by neutral endopeptidase (NEP; EC 3.4.24.11), as the Ala-Tyr bond of cFP-AAY-pAB is cleaved by NEP in vitro. In the present study, we have examined the role of NEP in the metabolism of cFP-AAY-pAB in vivo, and have tested a series of inhibitor analogues, substituted at the second alanine, for both potency and stability relative to the parent compound. 2. Analogues were screened for inhibition of fluorescent substrate cleavage by recombinant rat testes EP 24.15. D-Ala or Asp substitution abolished inhibitory activity, while Val-, Ser- and Leu-substituted analogues retained activity, albeit at a reduced potency. A relative potency order of Ala (1) > Val (0.3) > Ser (0.16) > Leu (0.06) was observed. Resistance to cleavage by NEP was assessed by incubation of the analogues with rabbit kidney membranes. The parent compound was readily degraded, but the analogues were twice (Ser) and greater than 10 fold (Leu and Val) more resistant to cleavage. 3. Metabolism of cFP-AAY-pAB and the Val-substituted analogue was also examined in conscious rabbits. A bolus injection of cFP-AAY-pAB (5 mg kg-1, i.v.) significantly reduced the blood pressure response to angiotensin I, indicating ACE inhibition. Pretreatment with NEP inhibitors, SCH 39370 or phosphoramidon, slowed the loss of cFP-AAY-pAB from the plasma, but did not prevent inhibition of ACE. Injection of 1 mg kg-1 inhibitor resulted in plasma concentrations at 10 s of 23.5 microM (cFP-AAY-pAB) and 18.0 microM (cFP-AVY-pAB), which fell 100 fold over 5 min. Co-injection of 125I-labelled inhibitor revealed that 80-85% of the radioactivity had disappeared from the circulation within 5 min, and h.p.l.c. analysis demonstrated that only 25-30% of the radiolabel remained as intact inhibitor at this time. Both analogues were cleared from the circulation at the same rate, and both inhibitors blunted the pressor response to angiotensin I, indicative of ACE inhibition. 4. These results suggest that both NEP and other clearance/degradation mechanisms severely limit the usefulness of peptide-based inhibitors such as cFP-AAY-pAB. To examine further EP 24.15 function in vivo, more stable inhibitors, preferably non-peptide, must be developed, for which these peptide-based inhibitors may serve as useful molecular templates.

Inflammatory pseudotumor is a rare benign lesion; the lung and conducting airways can be the seat where is often localized. The ileal localization, reported at present, can be defined unique. In our case was very difficult to perform a right diagnosis cause the absence of pathognomonic symptoms. Surgical procedure too was not able to resolve the diagnostic problem: in fact macroscopically the lesion seemed an ileal carcinoma or Crohn's disease. Only the microscopical examination with the individuation of Russell's bodies, macrophages and other inflammatory cells can perform the right diagnosis.