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 Y-1 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 Y-1 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 Y-1 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 Y-1 receptor antagonist. The results suggest that NPY and Y-1 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 Y-1 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.