We report here that the rat heart is a site of oxytocin (OT) synthesis and release. Oxytocin was detected in all four chambers of the heart. The highest OT concentration was in the right atrium (2128 ؎ 114 pg͞mg protein), which was 19-fold higher than in rat uterus but 3.3-fold lower than in the hypothalamus. OT concentrations were significantly greater in the right and left atria than in the corresponding ventricles. Furthermore, OT was released into the eff luent of isolated, perfused rat heart (34.5 ؎ 4.7 pg͞min) and into the medium of cultured atrial myocytes. Reverse-phase HPLC purification of the heart extracts and heart perfusates revealed a main peak identical with the retention time of synthetic OT. Southern blots of reverse transcription-PCR products from rat heart revealed gene expression of specific OT mRNA. OT immunostaining likewise was found in atrial myocytes and fibroblasts, and the intensity of positive stains from OT receptors paralleled the atrial natriuretic peptide stores. Our findings suggest that heart OT is structurally identical, and therefore derived from, the same gene as the OT that is primarily found in the hypothalamus. Thus, the heart synthesizes and processes a biologically active form of OT. The presence of OT and OT receptor in all of the heart's chambers suggests an autocrine and͞or paracrine role for the peptide. Our finding of abundant OT receptor in atrial myocytes supports our hypothesis that OT, directly and͞or via atrial natriuretic peptide release, can regulate the force of cardiac contraction.Vasopressin and oxytocin (OT) are synthesized predominantly in the magnocellular neurons of the supraoptic nucleus and paraventricular nucleus as well as in the parvocellular neurons within the paraventricular nucleus as parts of larger precursor molecules (1). The precursors are modified posttranslationally and are transported to the posterior pituitary, where the final bioactive peptide products are stored until they are released into the blood stream. Despite being the first peptide hormone to be characterized and synthesized, the effects of oxytocin long were considered to be restricted to stimulation of uterine contractions during labor and milk ejection during lactation. However, OT is found in equivalent concentrations in the neurohypophysis and plasma of both sexes, which suggests that it also may have other physiological roles (2). Moreover, in the central nervous system, OT-containing axons terminate in several brain stem nuclei known to be involved in cardiovascular control, suggesting a potential role for OT in central cardiovascular regulation (3, 4). Indeed, decreased blood pressure may be observed in response to oxytocin given intracerebroventricularly (5), and the inhibition of brain OT synthesis by an antisense oligonucleotide increased blood pressure in rats (6). In primates or humans, the administration of oxytocin often is associated with a decrease in blood pressure (7,8). Peripherally injected OT decreases mean arterial pressure in rats by unknown mec...

BackgroundHIV proteins Nef and Vpu down-modulate various host factors to evade immune defenses. Indeed, the CD4 receptor is down-regulated by Nef and Vpu, whereas virion-tethering BST2 is depleted by Vpu. Antibody-dependent cell-mediated cytotoxicity (ADCC) is increasingly recognized as a potentially powerful anti-HIV response. Given that epitopes which are specific for ADCC-competent anti-HIV antibodies are transitionally exposed upon CD4-mediated HIV entry, we investigated whether by depleting CD4 and BST2, HIV could negatively affect ADCC function.ResultsUsing anti-envelope (Env) Abs A32 and 2G12 to trigger ADCC activity, we find that interactions between CD4 and Env within infected cells expose ADCC-targeted epitopes on cell-surface Env molecules, marking infected T cells for lysis by immune cells. We also provide evidence to show that by cross-linking nascent virions at the plasma membrane, hence increasing cell-surface Env density, BST2 further enhances the efficiency of this antiviral process. The heightened susceptibility of T cells infected with a virus lacking Nef and Vpu to ADCC was recapitulated when plasmas from HIV-infected patients were used as an alternative source of Abs.ConclusionsOur data unveil a mechanism by which HIV Nef and Vpu function synergistically to protect infected cells from ADCC and promote viral persistence. These findings also renew the potential practical relevance of ADCC function in vivo.

Previous studies demonstrated the presence of oxytocin (OT) and oxytocin receptors (OTRs) in the heart. The present work provides results supporting a potential role of OT in cardiomyogenesis. Here, we show a maximal OT and OTR protein level in the developing rat heart at day 21 of gestation and postnatal days 1-4, when cardiac myocytes are at a stage of intense hyperplasia. Between postnatal days 1 and 66, OT decreased linearly in all heart chambers (4.1-to 6.6-fold). Correspondingly, immunocytochemistry demonstrated that OTRs, which were eminent in postnatal cardiomyocytes, declined with age to low levels in adults. Interestingly, in coronary vasculature, OTRs developed in endothelial cells at postnatal days 12 and 22 and achieved a plateau in adult rats. These findings suggest that OT can be involved in developmental formation of the coronary vessels. In vivo, the OT͞OTR system in the fetal heart was sensitive to the actions of retinoic acid (RA), recognized as a major cardiac morphogen. RA treatment produced a significant increase (2-to 3-fold) both in the OT concentration and in the OT mRNA levels. Ex vivo, an OT antagonist inhibited RA-mediated cardiomyocyte differentiation of P19 embryonic stem cells. The decline of cardiac OT expression from infancy to adulthood of the rat and changes in cell types expressing OTR indicate a dynamic regulation of the OT system in the heart rather than constitutive expression. The results support the hypothesis that RA induces cardiomyogenesis by activation of the cardiac OT system. heart development ͉ oxytocin receptors ͉ retinoic acid ͉ oxytocin antagonist ͉ cardiomyocyte differentiation O xytocin (OT), recognized traditionally as a reproductive hormone with a major role in childbirth and lactation, is produced in high concentrations in the hypothalamic supraoptic nucleus and paraventricular nucleus, then transported from these source nuclei to the posterior pituitary by neurosecretion (1). Longitudinal studies of neural and hormonal circuits activated by experimental volume expansion have identified OT as a major regulator of cardiovascular functions (reviewed in refs. 2 and 3). OT, injected peripherally, causes a decrease of arterial pressure (4) while reducing both heart rate and the force of contractions in isolated atria (5). OT acts via neuroendocrine͞ endocrine͞paracrine pathways to release atrial natriuretic peptide (ANP) from the heart (6, 7). ANP is a potent diuretic, natriuretic, and vasorelaxant hormone that is also involved in cell growth regulation. In addition, we have demonstrated that in isolated, perfused hearts, an OT antagonist (OTA) blocks basal ANP release (7), suggesting the presence of local OT in the heart. Further study revealed cardiac OT synthesis (8), with OT being detected in the medium of cultured neonatal rat cardiomyocytes (9).Recently, we showed that P19 embryonic carcinoma cells, a model of mouse embryonic stem cells, express OT receptors (OTRs), and OT stimulates the differentiation of these cells into beating cell colonies expressing ...

Produced and released by the heart, oxytocin (OT) acts on its cardiac receptors to decrease the cardiac rate and force of contraction. We hypothesized that it might also be produced in the vasculature and regulate vascular tone. Consequently, we prepared acid extracts of the pulmonary artery and vena cava of male rats. OT concentrations in dog and sheep aortae were equivalent to those of rat aorta (2745 ؎ 180 pg͞mg protein), indicating that it is present in the vasculature of several mammalian species. Reverse-phase HPLC of aorta and vena cava extracts revealed a single peak corresponding to the amidated OT nonapeptide. Reverse-transcribed PCR confirmed OT synthesis in these tissues. Using the selective OT receptor ligand compound VI, we detected a high number of OT-binding sites in the rat vena cava and aorta. Furthermore, OT receptor (OTR) mRNA was found in the vena cava, pulmonary vein, and pulmonary artery with lower levels in the aorta, suggesting vessel-specific OTR distribution. The abundance of OTR mRNA in the vena cava and pulmonary vein was associated with high atrial natriuretic peptide mRNA. In addition, we have demonstrated that diethylstilbestrol treatment of immature female rats increased OT significantly in the vena cava but not in the aorta and augmented OTR mRNA in both the aorta (4-fold) and vena cava (2-fold), implying regulation by estrogen. Altogether, these data suggest that the vasculature contains an intrinsic OT system, which may be involved in the regulation of vascular tone as well as vascular regrowth and remodeling. O xytocin (OT), a neurophyseal hormone, is known to play a role in lactation and parturition and in the central nervous system as a neurotransmitter involved in sex and maternal behavior (1). The presence of equivalent amounts of plasma OT as well as a similar number of oxytocinergic neurons in males as in females suggests a more general physiological and endocrine role of this hormone. In this context, OT emerges as a regulator of natriuresis and blood volume.The mechanism of this regulation, which we proposed recently on the basis of blood volume expansion (VE) experiments in rats, involves OT release from the posterior pituitary gland into plasma, which is followed by atrial natriuretic peptide (ANP) release via activation of the OT receptors (OTRs) present in the heart (2). In effect, following blood VE, OT and ANP would produce a negative ionotropic and chronotropic response in the heart (3) that would rapidly reduce cardiac output and, thereby, effective circulating blood volume. Blood VE may also generate OT locally since the heart is a site of OT synthesis and OT secretion (4) as well as ANP secretion. Thus, the increased venous return to the heart following VE would stretch the cardiac myocytes activating OT release that would release ANP.Then, as has been shown recently, the natriuretic effects of VE in the kidney are caused in part by OT acting on its receptors in the renal tubules to generate NO, leading to increased cGMP; the increased plasma ANP acting on r...

BackgroundVpu is a multifunctional accessory protein that enhances the release of HIV-1 by counteracting the entrapment of nascent virions on infected cell surface mediated by BST2/Tetherin. Vpu-mediated BST2 antagonism involves physical association with BST2 and subsequent mislocalization of the restriction factor to intracellular compartments followed by SCF(β-TrCP) E3 ligase-dependent lysosomal degradation. Apart from BST2 antagonism, Vpu also induces down regulation of several immune molecules, including CD4 and SLAMF6/NTB-A, to evade host immune responses and promote viral dissemination. However, it should be noted that the multiple functions of Vpu have been studied in cell-based assays, and thus it remains unclear how Vpu influences the dynamic of HIV-1 infection in in vivo conditions.ResultsUsing a humanized mouse model of acute infection as well as CCR5-tropic HIV-1 that lack Vpu or encode WT Vpu or Vpu with mutations in the β-TrCP binding domain, we provide evidence that Vpu-mediated BST2 antagonism plays a crucial role in establishing early plasma viremia and viral dissemination. Interestingly, we also find that efficient HIV-1 release and dissemination are directly related to functional strength of Vpu in antagonizing BST2. Thus, reduced antagonism of BST2 due to β-TrCP binding domain mutations results in decreased plasma viremia and frequency of infected T cells, highlighting the importance of Vpu-mediated β-TrCP-dependent BST-2 degradation for optimal initial viral propagation.ConclusionsOverall, our findings suggest that BST2 antagonism by Vpu is critical for efficient early viral expansion and dissemination during acute infection and as such is likely to confer HIV-1 increased transmission fitness.

We previously reported Tie2 receptor expression on human neutrophils, which promote chemotactic activities upon activation by both angiopoietins (Ang1 and Ang2). Moreover, we observed that neutrophil pretreatment with Ang1 or Ang2 enhances interleukin-8 (IL-8) chemotactic effect. Therefore, we assessed the capacity of Ang1 and/or Ang2 to modulate neutrophil IL-8 synthesis and release. Neutrophils isolated from healthy donors were stimulated in a time- (1-6 h) and concentration-(10(-10) -10(-8)  M) dependent manner with both angiopoietins. IL-8 mRNA production was measured by RT-qPCR, whereas its protein synthesis and release from neutrophils was assessed by ELISA. Ang1 (10(-8)  M) induced a significant and maximal increase of IL-8 mRNA (4.7-fold) within 1 h, and promoted maximal IL-8 protein synthesis (3.6-fold) and release (5.5-fold) within 2 h as compared to control PBS-treated neutrophils. Treatment with Ang2 alone did not modulate IL-8 synthesis or release, and its combination to Ang1 did not affect Ang1 activity. Neutrophil pretreatment with a protein synthesis inhibitor (CHX) increased IL-8 mRNA synthesis by 18-fold, and reduced Ang1-mediated IL-8 protein synthesis and release by 96% and 92%, respectively. Pretreatment with a transcription inhibitor (ActD) reduced IL-8 mRNA synthesis by 54% and IL-8 protein synthesis and release by 52% and 79%, respectively. Using specific kinase inhibitors, we observed that Ang1-driven IL-8 mRNA and protein synthesis is p42/44 MAPK-dependent and -independent from p38 MAPK and PI3K activity. Our study is the first to report the capacity of Ang1 (as opposed to Ang2) to promote neutrophil IL-8 synthesis and release through the activation of p42/44 MAPK pathway.

We recently demonstrated that Tie2 receptor activation on human neutrophils by both angiopoietins (Ang1 and Ang2) promoted platelet-activating factor synthesis, beta(2)-integrin activation, and cell migration. Herein, we wanted to assess if human neutrophils express angiopoietins and further delineate their mechanisms of release. Employing Reverse transcriptase-polymerase chain reaction, Real time quantitative transcriptase-polymerase chain reaction, FACScan analysis and ELISA approaches, we observed that neutrophils express Ang1 but not Ang2. For each condition, vascular endothelial growth factor (VEGF) detection was performed as positive control. Using nitrogen cavitation, we observed that Ang1 is localized in the cytosolic fraction whereas VEGF is found in beta-granules. Treatment of neutrophils with phorbol myristate acetate (PMA), N-Formyl-Met-Leu-Phe (fMLP) and tumor necrosis factor-alpha (TNF-alpha) induced VEGF release. Maximal effect was observed with PMA (80 nM) stimulation inducing a complete release of VEGF content (565 +/- 100 pg/ml; 6 x 10(6) neutrophils), corresponding to a 18.9-fold increase as compared to phosphate buffer saline (PBS) treated neutrophils. By contrast, only a treatment with PMA (80 nM) induced Ang1 release. PMA treatment induced also a complete release of Ang1 (661 +/- 148 pg/ml; 6 x 10(6) neutrophils), corresponding to 2.8-fold increase as compared to PBS-treated neutrophils. In both cases, PMA-mediated release of VEGF and Ang1 was nearly maximal by 15 min. Finally, we observed that the induction of Ang1 release was calcium-independent whereas VEGF release was not. These data demonstrate the capacity of human neutrophils to synthesize Ang1, which is stored and released differently as compared to VEGF. These data suggest a different cascade of events regarding the distribution of selected growth factors during inflammation and angiogenesis.

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