Presence of neuropeptide Y and the Y1receptor in the plasma membrane and nuclear envelope of human endocardial endothelial cells: modulation of intracellular calcium

Abstract

The aims of the present study were to investigate the presence and distribution of NPY and the Y1receptor 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 Y1receptor. Immunofluorescence, 3-D confocal microscopy and radioimmu noassay techniques were used on 20-week-old human fetal EECs. Our results showed that NPY and the Y1receptor 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 radio immunoassay, 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]iwas completely reversible upon washout of NPY and was partially blocked by BIBP3226, a selective Y1receptor antagonist. The results suggest that NPY and Y1receptors 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 Y1receptor as well as other NPY receptors seem to participate in mediating the effects of NPY on [Ca]iin these cells. Thus, NPY released by EECs may modulate excitation–secretion coupling of these cells.Key words: neuropeptide Y (NPY), nuclear envelope receptors, endocardial endothelial cells, NPY receptors, intracellular calcium.