Functional rescue of a defective angiotensin II AT1 receptor mutant by the Mas protooncogene.

Earlier studies with Mas protooncogene, a member of the G-protein-coupled receptor family, have proposed this gene to code for a functional AngII receptor, however further results did not confirm this assumption. In this work we investigated the hypothesis that a heterodimeration AT(1)/Mas could result in a functional interaction between both receptors. For this purpose, CHO or COS-7 cells were transfected with the wild-type AT(1) receptor, a non-functional AT(1) receptor double mutant (C18F-K20A) and Mas or with WT/Mas and C18F-K20A/Mas.

Determination of angiotensin I-converting enzyme activity in cell culture using fluorescence resonance energy transfer peptides.

An assay using fluorescence resonance energy transfer peptides was developed to assess angiotensin I-converting enzyme (ACE) activity directly on the membrane of transfected Chinese hamster ovary cells (CHO) stably expressing the full-length somatic form of the enzyme. The advantage of the new method is the possibility of using selective substrates for the two active sites of the enzyme, namely Abz-FRK(Dnp)P-OH for somatic ACE, Abz-SDK(Dnp)P-OH for the N domain, and Abz-LFK(Dnp)-OH for the C domain. Hydrolysis of a peptide bond between the donor/acceptor pair (Abz/Dnp) generates detectable fluorescence, allowing quantitative measurement of the enzymatic activity.

Mice deficient for both kinin receptors are normotensive and protected from endotoxin-induced hypotension.

Kinins play a central role in the modulation of cardiovascular function and in the pathophysiology of inflammation. These peptides mediate their effects by binding to two specific G-protein coupled receptors named B1 and B2. To evaluate the full functional relevance of the kallikrein-kinin system, we generated mice lacking both kinin receptors (B1B2-/-).

S-phase reduction in T47D human breast cancer epithelial cells induced by an S100P antisense-retroviral construct.

S100P is expressed in several malignant neoplasms. It was previously demonstrated that S100P is involved in the very early stages of breast carcinogenesis. In the present study we used a retrovirus-mediated transfer of antisense-S100P in order to check whether the decrease in expression of this protein could lead to alterations in the cell cycle of epithelial cells of human breast cancer.

Participation of transmembrane proline 82 in angiotensin II AT1 receptor signal transduction.

Most of the classical physiological effects of the octapeptide angiotensin II (AngII) are produced by activating the AT1 receptor which belongs to the G-protein coupled receptor family (GPCR). Peptidic GPCRs may be functionally divided in three regions: (i) extracellular domains involved in ligand binding; (ii) intracellular domains implicated in agonist-induced coupling to G protein and (iii) seven transmembrane domains (TM) involved in signal transduction. The TM regions of such receptors have peculiar characteristics such as the presence of proline residues.

Leptin deficiency leads to the regulation of kinin receptors expression in mice.

Kinins are vasoactive and pro-inflammatory peptides generated by the cleavage of the kininogen by kallikreins. Two kinin receptors have been described and denominated B1 and B2. Obesity frequently accompanies other pathologies, such as diabetes and hypertention.

Influence of bradykinin B1 and B2 receptors in the immune response triggered by renal ischemia-reperfusion injury.

Bradykinin B1 receptors are exclusively expressed in inflamed tissues. For this reason, they have been related with the outcomes of several pathologies. Ischemia-reperfusion injury is caused by the activation of inflammatory and cytoprotective genes, such as macrophage chemoattractant protein-1 and heme oxygenase-1, respectively.

Purification and partial characterization of two phospholipases A2 from Bothrops leucurus (white-tailed-jararaca) snake venom.

Two proteins with phospholipase A(2) (PLA(2)) activity were purified to homogeneity from Bothrops leucurus (white-tailed-jararaca) snake venom through three chromatographic steps: Conventional gel filtration on Sephacryl S-200, ion-exchange on Q-Sepharose and reverse phase on Vydac C4 HPLC column. The molecular mass for both enzymes was estimated to be approximately 14 kDa by SDS-PAGE. The N-terminal sequences (48 residues) show that one enzyme presents lysine at position 48 and the other an aspartic acid in this position, and therefore they were designated blK-PLA(2) and blD-PLA(2) respectively.

Kinin B1 and B2 receptors are overexpressed in the hippocampus of humans with temporal lobe epilepsy.

Molecular biology tools have been employed to investigate the participation of peptides in human temporal lobe epilepsy (TLE). Active polypeptides and their receptors have been related to several brain processes, such as inflammation, apoptosis, brain development, K(+) and Ca(2+) channels' activation, cellular growth, and induction of neuronal differentiation. Previous works have shown a neuroprotector effect for kinin B2 receptor and a deleterious, pro-epileptogenic action for kinin B1 receptor in animal models of TLE.

Disruption of the kinin B1 receptor gene affects potentiating effect of captopril on BK-induced contraction in mice stomach fundus.

A transgenic mouse model, deficient in kinin B(1) receptor (B(1)(-/-)) was used to evaluate the role of B(2) receptor in the smooth muscle stomach fundus. The results showed that the potency of bradykinin (BK) to induce contraction in the gastric tissue was maintained whereas the efficacy was markedly reduced. The angiotensin converting enzyme (ACE) inhibitor captopril potentiated BK-induced effect in wild type (WT) but not in B(1)(-/-) fundus.

Cooperative activation of TLR2 and bradykinin B2 receptor is required for induction of type 1 immunity in a mouse model of subcutaneous infection by Trypanosoma cruzi.

We have previously reported that exogenous bradykinin activates immature dendritic cells (DCs) via the bradykinin B(2) receptor (B(2)R), thereby stimulating adaptive immunity. In this study, we show that these premises are met in a model of s.c.

Modulation of kinin B1 receptor expression by endogenous angiotensin II in hypertensive rats.

We investigated the expression and localization of B1 receptor in tissues of rats submitted to a renin-dependent model of hypertension (2K-1C), and analyzed the influence of endogenous Ang II in modulating the in vivo expression of these receptors. B1 mRNA levels in the heart, kidney and thoracic aorta were quantified by real time PCR, B1 receptor protein expression was assessed by immunohistochemistry, plasma Ang II levels were analyzed by radioimmunoassay and the effects of AT1 receptor blockade were determined after losartan treatment. 2K-1C rats presented a marked increase in Ang II levels when compared to sham-operated rats.

Bradykinin-related peptides from Phyllomedusa hypochondrialis.

Bradykinin related peptides (BRPs) present in the water-soluble secretion and freshly dissected skin fragments of Phyllomedusa hypochondrialis were investigated by mass spectrometry techniques. Eighteen BRPs, along with their post-translational modifications, were characterized in the secretion by de novo MS/MS sequencing and direct MALDI imaging experiments of the frog skin. These molecules revealed strong sequence similarities to the main plasma kinin of some mammals and reptiles.

Effects of transforming growth factor-beta in the development of inflammatory pseudotumour-like lesions in a murine model.

Alterations in transforming growth factor (TGF)-beta signalling have been frequently implicated in human cancer, and an important mechanism underlying its pro-oncogenic nature is suppression of the host antitumour immune response. Considering the immunosuppressive effect of TGF-beta, we asked whether human tumour cells, known to secrete TGF-beta in culture, would survive and grow when implanted into the peritoneal cavity of immunocompetent mice. Therefore, we developed a xenogeneic model where mice were intraperitoneally (i.

Role of the Cys18-Cys274 disulfide bond and of the third extracellular loop in the constitutive activation and internalization of angiotensin II type 1 receptor.

An insertion of residues in the third extracellular loop and a disulfide bond linking this loop to the N-terminal domain were identified in a structural model of a G-protein coupled receptor specific to angiotensin II (AT1 receptor), built in homology to the seven-transmembrane-helix bundle of rhodopsin. Both the insertion and the disulfide bond were located close to an extracellular locus, flanked by the second extracellular loop (EC-2), the third extracellular loop (EC-3) and the N-terminal domain of the receptor; they contained residues identified by mutagenesis studies to bind the angiotensin II N-terminal segment (residues D1 and R2). It was postulated that the insertion and the disulfide bond, also found in other receptors such as those for bradykinin, endothelin, purine and other ligands, might play a role in regulating the function of the AT1 receptor.

Role of the kinin B1 receptor in insulin homeostasis and pancreatic islet function.

Kinins are potent vasoactive peptides generated in blood and tissues by the kallikrein serine proteases. Two distinct kinin receptors have been described, one constitutive (subtype B2) and one inducible (subtype B1), and many physiological functions have been attributed to these receptors, including glucose homeostasis and control of vascular permeability. In this study we show that mice lacking the kinin B1 receptor (B1-/- mice) have lower fasting plasma glucose concentrations but exhibit higher glycemia after feeding when compared to wild-type mice.

Renal gene expression profiling using kinin B1 and B2 receptor knockout mice reveals comparable modulation of functionally related genes.

The kinin B2 receptor, which is constitutively expressed in a large number of tissues, mediates most of the known effects of bradykinin (BK). Normally undetectable in healthy tissues, the B1 receptor is strongly over-expressed under pathological conditions. BK is an important mediator in renal homeostasis and is mainly known for its natriuretic and vasodilatory effects.

Genetically altered animal models in the kallikrein-kinin system.

Transgenic and gene-targeting technologies allowing the generation of genetically altered animal models have greatly advanced our understanding of the function of specific genes. This is also true for the kallikrein-kinin system (KKS), in which some, but not yet all, components have been functionally characterized using such techniques. The first genetically altered animal model for a KKS component was supplied by nature, the brown Norway rat carrying an inactivating mutation in the kininogen gene.

Activation of P2Y1 receptor triggers two calcium signaling pathways in bone marrow erythroblasts.

In this study, we describe the presence of P2 receptor subtypes and Ca2+ signaling in erythroblasts. ATP and ADP produced a biphasic increase of intracellular Ca2+ concentration ([Ca2+]i), with an initial transient phase followed by a sustained phase. Reverse transcription polymerase chain reaction (RT-PCR) showed the expression of P2Y1, P2Y2 and P2Y12.

High expression of human carboxypeptidase M in Pichia pastoris: purification and partial characterization.

Carboxypeptidase M (CPM) is an extracellular glycosylphosphatidyl-inositol-anchored membrane glycoprotein, which removes the C-terminal basic residues, lysine and arginine, from peptides and proteins at neutral pH. CPM plays an important role in the control of peptide hormones and growth factor activity on the cell surface. The present study was carried out to clone and express human CPM in the yeast Pichia pastoris in order to evaluate the importance of this enzyme in physiological and pathological processes.

Interplay between parasite cysteine proteases and the host kinin system modulates microvascular leakage and macrophage infection by promastigotes of the Leishmania donovani complex.

Kinins, the vasoactive peptides proteolytically liberated from kininogens, were recently recognized as signals alerting the innate immune system. Here we demonstrate that Leishmania donovani and Leishmania chagasi, two etiological agents of visceral leishmaniasis (VL), activate the kinin system. Intravital microscopy in the hamster cheek pouch showed that topically applied promastigotes induced macromolecular leakage (FITC-dextran) through postcapillary venules.

Expression and localization of N-domain ANG I-converting enzymes in mesangial cells in culture from spontaneously hypertensive rats.

The angiotensin-converting enzyme (ACE) profile in urine of hypertensive patients and spontaneously hypertensive rats (SHR; 90- and 65-kDa N-domain ACEs) is different from that of healthy subjects and Wistar rats (190 and 65 kDa). In addition, four ACE isoforms were purified from mesangial cells (MC) of Wistar rats in the intracellular compartment (130 and 68 kDa) and as secreted forms (130 and 60 kDa). We decided to characterize ACE forms from SHR MC in culture.

Cytokines and neutrophils as important mediators of platelet-activating factor-induced kinin B1 receptor expression.

PAF injection into the rat paw is accompanied by the concomitant activation of NF-kappaB and neutrophil influx, which appears to be relevant to the up-regulation of kinin B1 receptors. Herein, we analyse the role of TNF-alpha and IL-1beta production for PAF-induced B1 receptor upregulation in the rat paw. Additionally, we evaluate how cytokine production and neutrophil migration fit into the temporal sequence of events leading to PAF-induced B1 receptor upregulation.

Molecular structure and transcriptional regulation by nuclear factor-kappaB of the mouse kinin B1 receptor gene.

Kinins are important mediators in cardiovascular homeostasis, inflammation, and nociception. Two kinin receptors have been described, B 1 and B 2 . The B 1 receptor is normally absent in healthy tissues, but is highly induced under pathological conditions.

Angiotensin II AT1 receptor mutants expressed in CHO cells caused morphological change and inhibition of cell growth.

To assess the importance of the leucine residues in positions 262 and 265 of the angiotensin AT(1) receptor for signaling pathways and receptor expression and regulation, we compared the properties of CHO cells transfected with the wild type or the L262D or L265D receptor point mutants. It was found that the two mutants significantly increased the basal intracellular cyclic AMP (cAMP) formation in an agonist-independent mode. The morphology transformation of CHO cells was correlated with the increased cAMP formation, since forskolin, a direct activator of adenylate cyclase mimicked this effect on WT-expressing CHO cells.