Neuropeptide Y (NPY) and NPY receptors in the cardiovascular system: implication in the regulation of intracellular calcium

The 3-dimensional confocal microscopy technique has allowed us to identify the presence of yet another cardioactive factor and its receptor, namely neuropeptide Y (NPY) and its Y1 receptor, at the level of vascular smooth muscle cells and heart cells including endocardial endothelial cells (EECs). Using this technique, we also demonstrated that NPY is able to induce an increase in both cytosolic and nuclear calcium in all these cell types. Furthermore, besides being expressed at the level of EECs, NPY is also released from these cells following a sustained increase of intracellular Ca2+. This suggests the ability of NPY to contribute to the regulation of the excitation-secretion coupling of EECs and the excitation-contraction coupling of cardiomyocytes and vascular smooth muscle cells.     

NPY regulates human endocardial endothelial cell function

Growing evidence suggests that endocardial endothelial cells (EECs) may play an important role in the regulation of cardiac function by releasing several cardioactive factors such as endothelin-1 (ET-1), Angiotensin II (Ang II) and nitric oxide (NO). In our laboratory, we demonstrated that similar to ET-1, EECs do possess different types of NPY receptors, specifically Y(1) and Y(2) receptors. Furthermore, activation of these receptors was found to increase the steady-state level of intracellular free Ca(2+) in EECs and the frequency of beating of cardiomyocytes. In addition, NPY was also found to be present in EECs, and an increase of steady-state intracellular free Ca(2+) induced the release of this peptide from these cells. Thus, similar to ET-1, NPY seems to be released from EECs and this peptide seems to regulate excitation-secretion of these cells as well as excitation-contraction coupling of ventricular cardiomyocytes.     

Presence of neuropeptide Y and the Y1 receptor in the plasma membrane and nuclear envelope of human endocardial endothelial cells: modulation of intracellular calcium

The aims of the present study were to investigate the presence and distribution of NPY and the Y1 receptor in endocardial endothelial cells (EECs), to verify if EECs can release NPY, and to determine if the effect of NPY on intracellular calcium is mediated via the Y1 receptor. Immunofluorescence, 3-D confocal microscopy and radioimmunoassay techniques were used on 20-week-old human fetal EECs. Our results showed that NPY and the Y1 receptor are present in human EECs (hEECs) and that their distributions are similar, the fluorescence labelling being higher in the nucleus and more particularly at the level of the nuclear envelope when compared with the cytosol. Using radioimmunoassay, we demonstrated that EECs are a source of NPY and can secrete this peptide upon a sustained increase of intracellular calcium ([Ca]i). Using fluo-3 and 3-D confocal microscopy technique, superfusion of hEECs as well as EECs isolated from rat adult hearts with increasing concentrations of NPY induced a dose-dependent, sustained increase in free cytosolic and nuclear Ca2+ levels. This effect of NPY on EEC [Ca]i was completely reversible upon washout of NPY and was partially blocked by BIBP3226, a selective Y1 receptor antagonist. The results suggest that NPY and Y1 receptors are present in the EECs of 20-week-old human fetal heart and they share the same distribution and localization inside the cell. In addition, EECs are able to secrete NPY in response to an increase in [Ca]i, and the Y1 receptor as well as other NPY receptors seem to participate in mediating the effects of NPY on [Ca]i in these cells. Thus, NPY released by EECs may modulate excitation-secretion coupling of these cells.     

NPY, ET-1, and Ang II nuclear receptors in human endocardial endothelial cells

Neuropeptide Y (NPY), endothelin-1 (ET-1), and angiotensin II (Ang II) are peptides that are known to play many important roles in cardiovascular homeostasis. The physiological actions of these peptides are thought to be primarily mediated by plasma membrane receptors that belong to the G-protein-coupled receptor superfamily. However, there is increasing evidence that suggests the existence of functional G-protein-coupled receptors at the level of the nucleus and that the nucleus could be a cell within a cell. Here, we review our work showing the presence in the nucleus of the NPY Y(1) receptor, the ET(A) and ET(B) receptors, as well as the AT(1) and AT(2) receptors and their respective ligands. This work was carried out in 20-week-old fetal human endocardial endothelial cells. Our results demonstrate that nuclear Y1, AT(1), and ET(A) receptors modulate nuclear calcium in these cells.     

NPY, NPY receptors, and DPP IV activity are modulated by LPS, TNF-alpha and IFN-gamma in HUVEC

Since NPY increases endothelial cell (EC) stickiness for leukocytes, we studied the effects of LPS, TNF-alpha and IFN-gamma on its expression and action in HUVEC. Cytokines raised NPY and pro-NPY intracellular content and dipeptidyl peptidase IV (DPP IV) activity. Y1 and Y2 receptors were expressed in basal conditions, and LPS, TNF-alpha and IFN-gamma induced Y5 receptor expression with a concomitant extinction of Y2 receptor expression. NPY induced an intracellular calcium increase mainly mediated by Y2 and Y5 receptors in basal conditions. After stimulation with LPS, TNF-alpha and IFN-gamma, calcium increase was mainly caused by Y5 receptor. The modulation of the NPY system by LPS, TNF-alpha and IFN-gamma, and the NPY-induced calcium signaling suggest a role for NPY during the inflammatory response.     

Treatment with TNF-α or bacterial lipopolysaccharide attenuates endocardial endothelial cell-mediated stimulation of cardiac fibroblasts

Background

The endocardial endothelium that lines the inner cavity of the heart is distinct from the microvascular endothelial cells and modulates cardiac muscle performance in a manner similar to the vascular endothelial modulation of vascular structure and vasomotor tone. Although the modulatory effects of endocardial endothelium (EE) on cardiomyocytes are firmly established, the regulatory effects of endocardial endothelium on the cardiac interstitium and its cellular components remain ill defined.

Methods and Results

We investigated whether the stimulatory effect of EE on cardiac fibroblasts would be altered when EECs are activated by the cytokine tumor necrosis factor-α (TNF-α) or the endotoxin bacterial lipopolysaccharide (LPS). Both TNF-α and LPS were found to independently attenuate the stimulatory effect of EE on cardiac fibroblasts. These agents lowered the synthesis or release of ET-1 and increased the secretion of TGF-β and NO.

Conclusion

The findings of this study using endocardial endothelial cells (EECs) and neonatal cardiac fibroblasts demonstrate that pro-inflammatory cytokines cause altered secretion of paracrine factors by EECs and inhibit proliferation and lower collagen synthesis in fibroblasts. These changes may influence fibroblast response and extra cellular matrix remodeling in pathological conditions of the heart.     

Neuropeptide Y (NPY) suppresses experimental autoimmune encephalomyelitis: NPY1 receptor-specific inhibition of autoreactive Th1 responses in vivo

Prior studies have revealed that the sympathetic nervous system regulates the clinical and pathological manifestations of experimental autoimmune encephalomyelitis (EAE), an autoimmune disease model mediated by Th1 T cells. Although the regulatory role of catecholamines has been indicated in the previous works, it remained possible that other sympathetic neurotransmitters like neuropeptide Y (NPY) may also be involved in the regulation of EAE. Here we examined the effect of NPY and NPY receptor subtype-specific compounds on EAE, actively induced with myelin oligodendrocyte glycoprotein 35-55 in C57BL/6 mice. Our results revealed that exogenous NPY as well as NPY Y(1) receptor agonists significantly inhibited the induction of EAE, whereas a Y(5) receptor agonist or a combined treatment of NPY with a Y(1) receptor antagonist did not inhibit signs of EAE. These results indicate that the suppression of EAE by NPY is mediated via Y(1) receptors. Furthermore, treatment with the Y(1) receptor antagonist induced a significantly earlier onset of EAE, indicating a protective role of endogenous NPY in the induction phase of EAE. We also revealed a significant inhibition of myelin oligodendrocyte glycoprotein 35-55-specific Th1 response as well as a Th2 bias of the autoimmune T cells in mice treated with the Y(1) receptor agonist. Ex vivo analysis further demonstrated that autoimmune T cells are directly affected by NPY via Y(1) receptors. Taken together, we conclude that NPY is a potent immunomodulator involved in the regulation of the Th1-mediated autoimmune disease EAE.     

The distribution and density of ET-1 and its receptors are different in human right and left ventricular endocardial endothelial cells

Evidence suggests that endocardial endothelial cells (EECs) may play a role in the regulation of cardiac function by releasing ET-1. Furthermore, reports in the literature suggested that differences may exist in peptide receptor distribution between the left and right EECs. In this study, we verified if the distribution and density of ET-1 and its receptors could be different in right as compared to left ventricular EECs, and whether this difference may affect ET-1-induced increase of intracellular calcium. Using immunofluorescence and 3D confocal microscopy, our results showed that in both cell types, the ET(A) receptor is present and is homogeneously distributed throughout the two cell types. The relative density of the ET(A) receptor is similar in both right and left ventricular EECs. The ET(B) receptor is also present in right and left ventricular EECs, however, the relative density of the ET(B) receptor is higher in the nucleus as compared to the cytosol. In addition, the ET(B) receptor density was found to be higher in left EECs as compared to right EECs. In addition, our results showed that ET-1 is present in the cytosol and the nucleus of both types of cells and that the relative density of ET-1 is higher in right as compared to left ventricular EECs. Moreover, using the Fura-2 calcium measurement technique, our results showed that in left ventricular EECs, both ET(A) and ET(B) receptor activation mediated the effect of ET-1 on intracellular calcium, whereas in right ventricular EECs, this effect was solely mediated by the ET(A) receptor. In conclusion, our results showed that ET-1 and its receptors are present in both right and left ventricular EECs. However, the distribution and relative density of ET-1 and its receptors seem to be different in right EECs as compared to left EECs.     

NPY and pain as seen from the histochemical side

The expression of neuropeptide tyrosine (NPY) and two of its receptors (Y1- and Y2Rs) in different types of rodent dorsal root ganglion (DRG) and spinal cord neurons, and their regulation by peripheral nerve injury, have suggested a role in neuropathic pain. Here we present the spinal NPYergic system from an immunohistochemical perspective based on recent studies using two specific antibodies recognizing the Y1- and Y2Rs, respectively, as well as on data from a study on a Y1R knock-out mouse. We have, for example, defined seven different neuron populations of Y1R-expressing neurons in the rat spinal cord, representing multiple targets for spinally released NPY. The differential distribution of NPY receptors probably explains both the pro- and antinociceptive effects of NPY previously reported in the literature. One system possibly responsible for antinociception is a group of Y1R-positive, presumably glutamatergic interneurons in the superficial dorsal horn laminae. We also discuss the possibility that NPY released within DRGs can act in a paracrine fashion on NPY receptors on adjacent neurons, perhaps contributing to the so-called cross excitation, a concept advanced by Devor, Amir and collaborators. Taken together with behavioral and electrophysiological results summarized by Smith et al. in this volume, histochemical analyses have advanced the knowledge on the role of NPY in pain processing.     

The Y1 receptor for NPY: a key modulator of the adaptive immune system

Growing evidence suggests that the neuropeptide Y (NPY) system plays an important role in the immune system. Yet, little is known about the expression of NPY and receptors in the immune system. Moreover, original contradicting results have confused the picture and hampered a clear understanding of its role in the immune system. The use of Y(1) receptor-deficient mice, combined with advanced methods to investigate immune functions, have provided the solution to the problem raised by previous disparities. From results obtained using Y(1)-deficient mice (Y(1)(-/-)), we uncovered a bimodal role for Y(1) on immune cells. Y(1) expression on antigen-presenting cells (APC) is essential for their function as T cell priming elements. Conversely, Y(1) signaling in T cells plays a regulatory role without which T cells are hyper-responsive. The opposite role of Y(1) on APC and T cells has reconciled previous disparities by showing that signaling via Y(1) protects against inflammation by inhibiting T cell responses, whereas Y(1)(-/-) mice are protected in the same inflammatory models due to defective APCs.     

NPY promotes chemokinesis and neurogenesis in the rat subventricular zone

The subventricular zone (SVZ) is a major reservoir for stem cells in the adult mammalian brain. Neural stem cells supply the olfactory bulb with new interneurons and provide cells that migrate towards lesioned brain areas. Neuropeptide Y (NPY), one of the most abundant neuropeptides in the brain, was previously shown to induce neuroproliferation on mice SVZ cells. In the present study, performed in rats, we demonstrate the endogenous synthesis of NPY by cells in the SVZ that suggests that NPY could act as an autocrine/paracrine factor within the SVZ area. We observed that NPY promotes SVZ cell proliferation as previously reported in mice, but does not affect self-renewal of SVZ stem cells. Additionally, this study provides the first direct evidence of a chemokinetic activity of NPY on SVZ cells. Using pharmacological approaches, we demonstrate that both the mitogenic and chemokinetic properties of NPY involve Y1 receptor-mediated activation of the ERK1/2 MAP kinase pathway. Altogether, our data establish that NPY through Y1 receptors activation controls chemokinetic activity and, as for mice, is a major neuroproliferative regulator of rat SVZ cells.     

神经肽Y(NPY)的生理功能研究进展

神经肽Y(NPY)是机体内的一种重要且保守的神经递质,一般以前体形式存在,释放的有活性的NPY主要通过与其受体结合发挥作用。NPY受体包含了亚型Y1、Y2、Y3、Y4、Y5、Y6、Y7、Y8。Y1和Y2是NPY发挥收缩血管作用的关键受体;Y1、Y2和Y5是NPY调节动物摄食行为的关键受体;Y1、Y2和Y4是NPY调控动物焦虑、沮丧行为的必要受体。着重对NPY与其各种受体结合后如何行使动物的相关生理功能的情况进行了阐述。     

Shear stress enhances prostacyclin release from endocardial endothelial cells

The effect of shear stress on the release of prostacyclin (PGI2) from cultured endocardial endothelial cells (EECs) was investigated. EECs were harvested from the right ventricle (RV) and the left ventricle (LV) of porcine heart. Confluent EECs were incubated under various degrees of shear stress (0.2, 1, 4 and 6 dyne/cm2) and PGI2 release from each cell was measured. PGI2 release from LV-EECs and RV-EECs was enhanced by the elevation of shear stress in a shear-dependent manner with a rapid increase at the onset of flow; however, there was no significant difference in PGI2 production between RV-EECs and LV-EECs. production of PGI2 was significantly inhibited from cells exposed to 8-(dimetilamino) octyl 3,4,5-trymethoxybenzoate hydrochloride (10 and 100 microM: an inhibitor of intracellular calcium mobilization) or cyclopiazonic acid (10 microM: an endoplasmic reticulum Ca2+-ATPase inhibitor). These results indicate that shear stress enhances PGI2 release from cultured EECs and that mechanotransduction of shear stress depends on calcium mobilization in EECs.     

Endocardial endothelium in the rat: cell shape and organization of the cytoskeleton

The cytoskeleton in endocardial endothelium of rat heart was examined by en face confocal scanning laser microscopy. In the ventricular cavity, endocardial endothelial cells had a polygonal shape and F-actin staining was generally restricted to the peripheral junctional actin band. Central F-actin bundles, or stress fibers, in endocardial endothelial cells were found on the tendon end of papillary muscles, especially in the right ventricle, and frequently in the outflow tract of both ventricles; elsewhere, stress fibers were scarce. Many endocardial endothelial cells were elongated in areas of endothelium with stress fibers, but no correlation was found between cell elongation and the number of stress fibers. An inverse correlation was found between the number of stress fibers and the surface area of endocardial endothelial cells. Shear stress as well as mechanical deformation of the surface of the ventricular wall during the cardiac cycle may affect cell shape and the organization of actin filaments in endocardial endothelial cells. Vimentin in endocardial endothelial cells formed a filamentous network with some distinct cytoplasmic and juxtanuclear vimentin bundles. No perinuclear ring of vimentin filaments was observed in endocardial endothelium. Microtubules in endocardial endothelial cells were, in contrast to endothelial cells of rat aorta, not aligned, less closely packed and originated from randomly distributed centriolar regions. The cytoskeleton has been suggested to play an important role in cellular functions of vascular endothelial cells. Accordingly, differences in the cytoskeletal organization between endocardial and vascular endothelial cells may relate to differences in functional properties.     

NPY in rat retina is present in neurons, in endothelial cells and also in microglial and Müller cells

NPY is present in the retina of different species but its role is not elucidated yet. In this work, using different rat retina in vitro models (whole retina, retinal cells in culture, microglial cell cultures, rat Müller cell line and retina endothelial cell line), we demonstrated that NPY staining is present in the retina in different cell types: neurons, macroglial, microglial and endothelial cells. Retinal cells in culture express NPY Y(1), Y(2), Y(4) and Y(5) receptors. Retina endothelial cells express all NPY receptors except NPY Y(5) receptor. Moreover, NPY is released from retinal cells in culture upon depolarization. In this study we showed for the first time that NPY is present in rat retina microglial cells and also in rat Müller cells. These in vitro models may open new perspectives to study the physiology and the potential pathophysiological role of NPY in the retina.     

Neuropeptide Y Y5-receptor activation on breast cancer cells acts as a paracrine system that stimulates VEGF expression and secretion to promote angiogenesis

Accumulating data implicate a pathological role for sympathetic neurotransmitters like neuropeptide Y (NPY) in breast cancer progression. Our group and others reported that NPY promotes proliferation and migration in breast cancer cells, however the angiogenic potential of NPY in breast cancer is unknown. Herein we sought to determine if NPY promotes angiogenesis in vitro by increasing vascular endothelial growth factor (VEGF) expression and release from 4T1 breast cancer cells. Western blot analysis revealed that NPY treatment caused a 52 ± 14% increase in VEGF expression in the 4T1 cells compared to non-treated controls. Using selective NPY Y-receptor agonists (Y1R, Y2R and Y5R) we observed an increase in VEGF expression only when cells were treated with Y5R agonist. Congruently, using selective Y1R, Y2R, or Y5R antagonists, NPY-induced increases in VEGF expression in 4T1 cells were attenuated only under Y5R antagonism. Endothelial tube formation assays were conducted using conditioned media (CM) from NPY treated 4T1 cells. Concentration-dependent increases in number of branch points and complete endothelial networks were observed in HUVEC exposed to NPY CM. CM from Y5R agonist treated 4T1 cells caused similar increases in number of branch points and complete endothelial networks. VEGF concentration was quantified in CM (ELISA) from agonist experiments; we observed a 2-fold and 2.5-fold increase in VEGF release from NPY and Y5R agonist treated 4T1 cells respectively. Overall these data highlight a novel mechanism by which NPY may promote breast cancer progression, and further implicate a pathological role of the NPY Y5R.     

Bis(31/31')[[Cys(31), Nva(34)]NPY(27-36)-NH(2)]: a neuropeptide Y (NPY) Y(5) receptor selective agonist with a latent stimulatory effect on food intake in rats

The actions of neuropeptide Y (NPY) are mediated by at least six G-protein coupled receptors denoted as Y(1), Y(2), Y(3), Y(4), Y(5), and y(6). Investigations using receptor selective ligands and receptor knock-out mice suggest that NPY effects on feeding are mediated by both Y(1) and Y(5) receptors. We have previously shown that Cys-dimers of NPY C-terminal peptides exhibit Y(1) selectivity relative to Y(2) receptors. Re-investigation of their selectivity with respect to the newly cloned receptors, has identified bis(31/31') [[Cys(31), Nva(34)]NPY(27-36)-NH(2)] (BWX-46) as a Y(5) receptor selective agonist. BWX-46 selectively bound Y(5) receptors, and inhibited cAMP synthesis by Y(5) cells with potencies comparable to that of NPY. Moreover, BWX-46 (10 microM) exhibited no significant effect on the cAMP synthesis by Y(1), Y(2), and Y(4) cells. Thus, BWX-46 constitutes the lowest molecular weight Y(5) selective agonist reported to date. Intrahypothalamic (i.h.t)-injection of 30 and 40 microg of BWX-46 stimulated the food intake by rats in a gradual manner, reaching maximal level 8 h after injection. This response was similar to that exhibited by other Y(5) selective agonists, but differed from that of NPY, which exhibited a rapid orexigenic stimulus within 1 h. It is suggested that the differences in the orexigenic stimuli of NPY and Y(5) agonists may be due to their differences in the signal transduction mechanisms.     

Neuropeptide Y modulates functions of inflammatory cells in the rat: distinct role for Y1, Y2 and Y5 receptors

Neuropeptide Y (NPY) has been reported to be a potent anti-inflammatory peptide with ability to directly modulate activity of granulocytes and macrophages. The present study aimed to correlate the effects of NPY in vivo on lipopolysaccharide-induced air-pouch exudates cells and in vitro on peripheral blood leukocytes functions. The role of different Y receptors was examined using NPY-related peptides and antagonists with diverse subtype specificity and selectivity for Y receptors. Y1, Y2 and Y5 receptors were detected on air-pouch exudates cells (flow cytometry) and peripheral blood granulocytes (immunocitochemistry). NPY in vivo reduced inflammatory cells accumulation into the air pouch, and decreased their adherence and phagocytic capacity via Y2/Y5 and Y1/Y2 receptors, respectively. Quite the opposite, NPY in vitro potentiated adhesiveness and phagocytosis of peripheral blood granulocytes and monocytes by activating Y1 receptor. The differences between in vivo and in vitro effects of NPY on rat inflammatory cells functions are mostly due to dipeptidyl peptidase 4 activity. In addition, suppressive effect of NPY in vivo is highly dependent on the local microenvironment, peptide truncation and specific Y receptors interplay.