Identification of metabolic pathways of brain angiotensin II and III using specific aminopeptidase inhibitors: predominant role of angiotensin III in the control of vasopressin release.

Angiotensin (Ang) II and Ang III are two peptide effectors of the brain renin-angiotensin system that participate in the control of blood pressure and increase water consumption and vasopressin release. In an attempt to delineate the respective roles of these peptides in the regulation of vasopressin secretion, their metabolic pathways and their effects on vasopressin release were identified in vivo. For this purpose, we used recently developed selective inhibitors of aminopeptidase A (APA) and aminopeptidase N (APN), two enzymes that are believed to be responsible for the N-terminal cleavage of Ang II and Ang III, respectively. Mice received [3H]Ang II intracerebroventricularly (i.c.v.) in the presence or absence of the APN inhibitor, EC33 (3-amino-4-thio-butyl sulfonate) of the APN inhibitor, EC27 (2-amino-pentan-1,5-dithiol). [3H]Ang II and [3H]Ang III levels were evaluated from hypothalamus homogenates by HPLC. EC33 increased the half-life of [3H]Ang II 2.6-fold and completely blocked the formation of [3H]Ang III, whereas EC27 increased the half-life of [3H]Ang III 2.3-fold. In addition, the effects of EC33 and EC27 on Ang-induced vasopressin release were studied in mice. Ang II was injected i.c.v. in the presence or absence of EC33, and plasma vasopressin levels were estimated by RIA. While vasopressin levels were increased 2-fold by Ang II (5 ng), EC33 inhibited Ang II-induced vasopressin release in a dose-dependent manner. In contrast, EC27 injected alone increased in a dose-dependent manner vasopressin levels. The EC27-induced vasopressin release was completely blocked by the coadministration of the Ang receptor antagonist (Sar1-Ala8) Ang II. These results demonstrate for the first time that (i) APA and APN are involved in vivo in the metabolism of brain Ang II and Ang III, respectively, and that (ii) the action of Ang II on vasopressin release depends upon the prior conversion of Ang II to Ang III. This shows that Ang III behaves as one of the main effector peptides of the brain renin-angiotensin system in the control of vasopressin release.

Tumor necrosis factor receptor associated factor 2 is a mediator of NF-kappa B activation by latent infection membrane protein 1, the Epstein-Barr virus transforming protein.

Latent infection membrane protein 1 (LMP1), the Epstein-Barr virus transforming protein, associates with tumor necrosis factor receptor (TNFR) associated factor 1 (TRAF1) and TRAF3. Since TRAF2 has been implicated in TNFR-mediated NF-kappa B activation, we have evaluated the role of TRAF2 in LMP1-mediated NF-kappa B activation. TRAF2 binds in vitro to the LMP1 carboxyl-terminal cytoplasmic domain (CT), coprecipitates with LMP1 in B lymphoblasts, and relocalizes to LMP1 plasma membrane patches. A dominant negative TRAF2 deletion mutant that lacks amino acids 6-86 (TRAF/ delta 6-86) inhibits NF-kappa B activation from the LMP1 CT and competes with TRAF2 for LMP1 binding. TRAF2 delta 6-86 inhibits NF-kappa B activation mediated by the first 45 amino acids of the LMP1 CT by more than 75% but inhibits NF-kappa B activation through the last 55 amino acids of the CT by less than 40%. A TRAF interacting protein, TANK, inhibits NF-kappa B activation by more than 70% from both LMP1 CT domains. These data implicate TRAF2 aggregation in NF-kappa B activation by the first 45 amino acids of the LMP1 CT and suggest that a different TRAF-related pathway may be involved in NF-kappa B activation by the last 55 amino acids of the LMP1 CT.

Myocardial signaling defects and impaired cardiac function of a human beta 2-adrenergic receptor polymorphism expressed in transgenic mice.

A threonine to isoleucine polymorphism at amino acid 164 in the fourth transmembrane spanning domain of the beta 2-adrenergic receptor (beta 2AR) is known to occur in the human population. The functional consequences of this polymorphism to catecholamine signaling in relevant cells or to end-organ responsiveness, however, are not known. To explore potential differences between the two receptors, site-directed mutagenesis was carried out to mimic the polymorphism. Transgenic FVB/N mice were then created overexpressing wild-type (wt) beta 2AR or the mutant Ile-164 receptor in a targeted manner in the heart using a murine alpha myosin heavy chain promoter. The functional properties of the two receptors were then assessed at the level of in vitro cardiac myocyte signaling and in vivo cardiac responses in intact animals. The expression levels of these receptors in the two lines chosen for study were approximately 1200 fmol/mg protein in cardiac membranes, which represents a approximately 45-fold increase in expression over endogenous beta AR. Myocyte membrane adenylyl cyclase activity in the basal state was significantly lower in the Ile-164 mice (19.5 +/- 2.7 pmol/min/mg) compared with wt beta 2AR mice (35.0 +/- 4.1 pmol/min/mg), as was the maximal isoproterenol-stimulated activity (49.8 +/- 7.8 versus 77.1 +/ 7.3 pmol/min/mg). In intact animals, resting heart rate (441 +/- 21 versus 534 +/- 17 bpm) and dP/dtmax (10,923 +/- 730 versus 15,308 +/- 471 mmHg/sec) were less in the Ile-164 mice as compared with wt beta 2AR mice. Similarly, the physiologic responses to infused isoproterenol were notably less in the mutant expressing mice. Indeed, these values, as well as other contractile parameters, were indistinguishable between Ile-164 mice and nontransgenic littermates. Taken together, these results demonstrate that the Ile-164 polymorphism is substantially dysfunctional in a relevant target tissue, as indicated by depressed receptor coupling to adenylyl cyclase in myocardial membranes and impaired receptor mediated cardiac function in vivo. Under normal homeostatic conditions or in circumstances where sympathetic responses are compromised due to diseased states, such as heart failure, this impairment may have important pathophysiologic consequences.

Chronic control of high blood pressure in the spontaneously hypertensive rat by delivery of angiotensin type 1 receptor antisense.

The renin-angiotensin system plays a crucial role in the development and establishment of the hypertensive state in the spontaneously hypertensive (SH) rat. Interruption of this system's activity by pharmacological means results in the lowering of blood pressure (BP) and control of hypertension. However, such means are temporary and require the continuous use of drugs for the control of this pathophysiological state. Our objective in this investigation was to determine if a virally mediated gene-transfer approach using angiotensin type 1 receptor antisense (AT1R-AS) could be used to control hypertension on a long-term basis in the SH rat model of human essential hypertension. Injection of viral particles containing AT1R-AS (LNSV-AT1R-AS) in 5-day-old rats resulted in a lowering of BP exclusively in the SH rat and not in the Wistar Kyoto normotensive control. A maximal anti-hypertensive response of 33 +/- 5 mmHg was observed, was maintained throughout development, and still persisted 3 months after administration of LNSV-AT1R-AS. The lowering of BP was associated with the expression of AT1R-AS transcript and decreases in AT1-receptor in many peripheral angiotensin II target tissues such as mesenteric artery, adrenal gland, heart, and kidney. Attenuation of angiotensin II-stimulated physiological actions such as contraction of aortic rings and increase in BP was also observed in the LNSV-AT1R-AS-treated SH rat. These observations show that a single injection of LNSV-AT1R-AS normalizes BP in the SH rat on a long-term basis. They suggest that such a gene-transfer strategy can be successfully used to control the development of hypertension on a permanent basis.

Involvement of Asn-293 in stereospecific agonist recognition and in activation of the beta 2-adrenergic receptor.

To investigate the molecular mechanism for stereospecific binding of agonists to beta 2-adrenergic receptors we used receptor models to identify potential binding sites for the beta-OH-group of the ligand, which defines the chiral center. Ser-165, located in transmembrane helix IV, and Asn-293, situated in the upper half of transmembrane helix VI, were identified as potential binding sites. Mutation of Ser-165 to Ala did not change the binding of either isoproterenol isomer as revealed after transient expression in human embryonic kidney (HEK)-293 cells. In contrast, a receptor mutant in which Asn-293 was replaced by Leu showed substantial loss of stereospecific isoproterenol binding. Adenylyl cyclase stimulation by this mutant after stable expression in CHO cells confirmed the substantial loss of stereospecificity for isoproterenol. In a series of agonists the loss of affinity in the Leu-293 mutant receptor was strongly correlated with the intrinsic activity of the compounds. Full agonists showed a 10-30-fold affinity loss, whereas partial agonists had almost the same affinity for both receptors. Stereospecific recognition of antagonists was unaltered in the Leu-293 mutant receptor. These data indicate a relationship between stereospecificity and intrinsic activity of agonists and suggest that Asn-293 is important for both properties of the agonist-receptor interaction.

Constitutive receptor activation by Crouzon syndrome mutations in fibroblast growth factor receptor (FGFR)2 and FGFR2/Neu chimeras.

Crouzon syndrome is an autosomal dominant condition primarily characterized by craniosynostosis. This syndrome has been associated with a variety of amino acid point mutations in the extracellular domain of fibroblast growth factor receptor 2 (FGFR2). FGFR2/Neu chimeras were generated by substituting the extracellular domain of Neu with that of FGFR2 containing the following Crouzon mutations: Tyr-340-->His; Cys-342-->Tyr; Cys-342-->Arg; Cys-342-->Ser; Ser-354-->Cys: and delta17 (deletion of amino acids 345-361). Each of the mutant chimeric FGFR2/Neu constructs stimulated focus formation in NIH 3T3 cells, indicating that Crouzon mutations can stimulate signal transduction through a heterologous receptor tyrosine kinase. In vitro kinase assay results indicate that FGFR2 receptors containing Crouzon mutations have increased tyrosine kinase activity and, when analyzed under nonreducing conditions, exhibited disulfide-bonded dimers. Thus the human developmental abnormality Crouzon syndrome arises from constitutive activation of FGFR2 due to aberrant intermolecular disulfide-bonding. These results together with our earlier observation that achondroplasia results from constitutive activation of the related receptor FGFR3, leads to the prediction that other malformation syndromes attributed to FGFRs, such as Pfeiffer syndrome and Thanatophoric dysplasia, also arise from constitutive receptor activation.

Matrix metalloproteinase 2 releases active soluble ectodomain of fibroblast growth factor receptor 1.

Recent studies have demonstrated the existence of a soluble fibroblast growth factor (FGF) receptor type 1 (FGFR1) extracellular domain in the circulation and in vascular basement membranes. However, the process of FGFR1 ectodomain release from the plasma membrane is not known. Here we report that the 72-kDa gelatinase A (matrix metalloproteinase type 2, MMP2) can hydrolyze the Val368-Met369 peptide bond of the FGFR1 ectodomain, eight amino acids upstream of the transmembrane domain, thus releasing the entire extracellular domain. Similar results were obtained regardless of whether FGF was first bound to the receptor or not. The action of MMP2 abolished binding of FGF to an immobilized recombinant FGFR1 ectodomain fusion protein and to Chinese hamster ovary cells overexpressing FGFR1 The released recombinant FGFR1 ectodomain was able to bind FGF after MMP2 cleavage, suggesting that the cleaved soluble receptor maintained its FGF binding capacity. The activity of MMP2 could not be reproduced by the 92-kDa gelatinase B (MMP9) and was inhibited by tissue inhibitor of metalloproteinase type 2. These studies demonstrate that FGFR1 may be a specific target for MMP2 on the cell surface, yielding a soluble FGF receptor that may modulate the mitogenic and angiogenic activities of FGF.

Cell-type and promoter-context dependent retinoic acid receptor (RAR) redundancies for RAR beta 2 and Hoxa-1 activation in F9 and P19 cells can be artefactually generated by gene knockouts.

By using RAR type (alpha, beta, or gamma)-specific synthetic retinoids and a pan-retinoic X receptor (RXR)-specific ligand, we have investigated the contribution of RARs and RXRs in the activation of RA target genes and the differentiation of embryonal carcinoma cells. We demonstrate cell-type- and promoter context-dependent functional redundancies that differ between the three RAR types for mediating the induction of RARbeta2 and Hoxa-1 in wild-type, RARgamma-/- and RARalpha-/- F9 cells and in P19 cells. The extent of redundancy between RARs is further modulated by the synergistic activation of RXRs with a pan-RXR agonist. We also demonstrate that the expression of RARbeta2 is auto-inducible in RARgamma-/- but not in wild-type F9 cells, indicating that the functional redundancies observed between RARs in gene disruption studies can be artefactually generated. Thus, even though all three RARs can functionally substitute each other for inducing the expression of RA target genes and cell differentiation, one RAR can cell-specifically override the activity of the other RARs. Interestingly, only RARgamma can mediate the retinoic acid-induced differentiation of wild-type F9 cells, whereas the differentiation of P19 cells can be mediated by either RARalpha or RARgamma.

Interleukin 2 (IL-2) and interleukin 7 (IL-7) reciprocally induce IL-7 and IL-2 receptors on gamma delta T-cell receptor-positive intraepithelial lymphocytes.

In this study, we describe the interaction between cytokine and cytokine receptor (R) for the activation and proliferation of gamma delta T-cell receptor-positive T cells (gamma delta T cells). gamma delta T cells isolated from murine intestinal intraepithelial lymphocytes (IELs) were separated into gamma delta (Dim) and gamma delta (Bright) fractions according to the intensity of gamma delta T-cell receptor expression. The gamma delta T cells express low levels of IL-2R and IL-7R as shown by flow cytometry and reverse transcriptase-PCR analysis, whereas gamma delta (Bright) T cells did not express either receptor. Our study also revealed that recombinant marine (rm)IL-2 and rmIL-7 reciprocally induced high expressions of IL-7R and IL-2R, respectively, on gamma delta (Dim) T cells but not on gamma delta (Bright) cells. Thus, treatment of gamma delta (Dim) T cells with rmIL-2 and rmIL-7 resulted in high proliferative responses, whereas gamma delta (Bright) T cells did not respond to these two cytokines. The sources of these two cytokines for gamma delta T cells were neighboring epithelial cells (IL-7) and alpha beta T cells (IL-2 and IL-7). Cytokine signaling by IL-2 and IL-7 from alpha beta T cells and epithelial cells was necessary for the expression of IL-7R and IL-2R, respectively, on a subset of gamma delta T cells (e.g., gamma delta (Dim) T cells) in mucosa-associated tissue for subsequent activation and cell division.

p56Lck and p59Fyn regulate CD28 binding to phosphatidylinositol 3-kinase, growth factor receptor-bound protein GRB-2, and T cell-specific protein-tyrosine kinase ITK: implications for T-cell costimulation.

T-cell activation requires cooperative signals generated by the T-cell antigen receptor zeta-chain complex (TCR zeta-CD3) and the costimulatory antigen CD28. CD28 interacts with three intracellular proteins-phosphatidylinositol 3-kinase (PI 3-kinase), T cell-specific protein-tyrosine kinase ITK (formerly TSK or EMT), and the complex between growth factor receptor-bound protein 2 and son of sevenless guanine nucleotide exchange protein (GRB-2-SOS). PI 3-kinase and GRB-2 bind to the CD28 phosphotyrosine-based Tyr-Met-Asn-Met motif by means of intrinsic Src-homology 2 (SH2) domains. The requirement for tyrosine phosphorylation of the Tyr-Met-Asn-Met motif for SH2 domain binding implicates an intervening protein-tyrosine kinase in the recruitment of PI 3-kinase and GRB-2 by CD28. Candidate kinases include p56Lck, p59Fyn, zeta-chain-associated 70-kDa protein (ZAP-70), and ITK. In this study, we demonstrate in coexpression studies that p56Lck and p59Fyn phosphorylate CD28 primarily at Tyr-191 of the Tyr-Met-Asn-Met motif, inducing a 3- to 8-fold increase in p85 (subunit of PI 3-kinase) and GRB-2 SH2 binding to CD28. Phosphatase digestion of CD28 eliminated binding. In contrast to Src kinases, ZAP-70 and ITK failed to induce these events. Further, ITK binding to CD28 was dependent on the presence of p56Lck and is thus likely to act downstream of p56Lck/p59Fyn in a signaling cascade. p56Lck is therefore likely to be a central switch in T-cell activation, with the dual function of regulating CD28-mediated costimulation as well as TCR-CD3-CD4 signaling.

Tonic stimulation of renin gene expression by nitric oxide is counteracted by tonic inhibition through angiotensin II.

This study was designed to examine the possible involvement of prostaglandins and nitric oxide (NO) in the renin stimulatory effect of angiotensin II (AngII) antagonists. To this end, plasma renin activities (PRAs) and renal renin mRNA levels were assayed in rats that were treated with the Ang-converting enzyme inhibitor ramipril or with the AngII AT1-receptor antagonist losartan. Ramipril and losartan increased PRA values from 7.5 +/- 1.6 to 86 +/- 6 and 78 +/- 22 ng of AngI per h per ml and renin mRNA levels from 112 +/- 9% to 391 +/- 20% and 317 +/- 10%, respectively. Inhibition of prostaglandin formation with indomethacin did not influence basal or ramipril-affected PRA. Basal renin mRNA levels also were unchanged by indomethacin, while increases in renin mRNA levels after ramipril treatment were slightly reduced by indomethacin. Inhibition of NO synthase by nitro-L-arginine methyl ester (L-NAME) reduced PRA values to 3.2 +/- 0.9, 34 +/- 13, and 12.1 +/- 2.7 ng of AngI per h per ml in control, ramipril-treated, and losartan-treated animals, respectively. Renin mRNA levels were reduced to 77 +/- 14% under basal conditions and ramipril- and losartan-induced increases in renin mRNA levels were completely blunted after addition of L-NAME. The AngII antagonists, furthermore, induced an upstream recruitment of renin-expressing cells in the renal afferent arterioles, which was also blunted by L-NAME. These findings suggest that renin mRNA levels are tonically increased by NO and that the action of NO is counteracted by AngII.

T-cell receptor (TCR) usage in Lewis rat experimental autoimmune encephalomyelitis: TCR beta-chain-variable-region V beta 8.2-positive T cells are not essential for induction and course of disease.

Predominant usage of V beta 8.2 gene segments, encoding a T-cell receptor (TCR) beta chain variable region, has been reported for pathogenic Lewis rat T cells reactive to myelin basic protein (MBP). However, up to 75% of the alpha/beta T cells in a panel of MBP-specific T-cell lines did not display TCR V beta 8.2, V beta 8.5, V beta 10, or V beta 16 elements. To further investigate TCR usage, we sorted the T-cell lines for V beta 8.2- and V beta 10-positive T cells or depleted the lines of cells with these TCRs. V beta 8.2-positive T cells and one of the depleted T-cell lines strongly reacted against the MBP peptide MBP-(68-88). The depleted T-cell line caused marked experimental autoimmune encephalomyelitis (EAE) even in Lewis rats in which endogenous V beta 8.2-positive T cells had been eliminated by neonatal treatment with anti-V beta 8.2 monoclonal antibodies. T-cell hybridomas generated from this line predominantly used V beta 3 TCR genes coexpressed with TCR V alpha 2 transcripts, which were also used by V beta 8.2-positive T cells. Furthermore, V beta 10-positive T cells reactive to MBP-(44-67) were encephalitogenic when injected immediately after positive selection. After induction of EAE by sorted V beta 8.2- or V beta 10-positive T-cell lines, immunocytochemical analysis of the spinal cord tissue showed a predominance of the injected TCR or of nontypable alpha/beta T cells after injection of the depleted line. Our results demonstrate heterogeneity of TCR beta-chain usage even for a single autoantigen in an inbred strain. Moreover, V beta 8.2-positive T cells are not essential for the induction and progression of adoptive-transfer EAE.

Calmodulin is a selective mediator of Ca(2+)-induced Ca2+ release via the ryanodine receptor-like Ca2+ channel triggered by cyclic ADP-ribose.

The ryanodine receptor-like Ca2+ channel (RyRLC) is responsible for Ca2+ wave propagation and Ca2+ oscillations in certain nonmuscle cells by a Ca(2+)-induced Ca2+ release (CICR) mechanism. Cyclic ADP-ribose (cADPR), an enzymatic product derived from NAD+, is the only known endogenous metabolite that acts as an agonist on the RyRLC. However, the mode of action of cADPR is not clear. We have identified calmodulin as a functional mediator of cADPR-triggered CICR through the RyRLC in sea urchin eggs. cADPR-induced Ca2+ release consisted of two phases, an initial rapid release phase and a subsequent slower release. The second phase was selectively potentiated by calmodulin which, in turn, was activated by Ca2+ released during the initial phase. Caffeine enhanced the action of calmodulin. Calmodulin did not play a role in inositol 1,4,5-trisphosphate-induced Ca2+ release. These findings offer insights into the multiple pathways that regulate intracellular Ca2+ signaling.

N-terminal fusion of a toll-like receptor 2-ligand to a Neospora caninum chimeric antigen efficiently modifies the properties of the specific immune response

Immunoprophylactic products against neosporosis during pregnancy should induce an appropriately balanced immune response. In this respect, OprI, a bacterial lipoprotein targeting toll like receptor (TLR)2, provides promising adjuvant properties. We report on the manipulation of the innate and the T-cell immune response through the fusion of OprI with the Neospora caninum chimeric protein Mic3-1-R. In contrast to Mic3-1-R, OprI-MIC3-1-R significantly activated bone-marrow dendritic cells from naïve mice. Mice immunized with OprI-Mic3-1-R induced an immune response with mixed T helper (Th)1 and Th2 properties (high levels of both immunoglobulin (Ig)G1 and IgG2a and of interleukin (IL)-10, IL-12(p70) and interferon-γ responses) whereas Mic3-1-R+saponin induced a clear Th2-biased response (low IgG2a and high IL-4 and IL-10). After mating and challenge with N. caninum, increased expression of interferon-γ was only found in placentas from OprI-Mic3-1-R immunized dams. However, no protection against vertical transmission and neonatal mortality was observed in either of the two groups. These results indicated that more exhaustive studies must be done to elucidate the immune mechanisms associated with transplacental transmission. Antigen linkage to TLR2-ligands, such as OprI, is a useful tool to investigate this enigma by reorienting the innate and adaptive immune responses against other candidate antigens in future studies.