C-peptide-stimulated nitric oxide production in a cultured pulmonary artery endothelium is erythrocyte mediated and requires Zn2+

Background C-peptide has been shown to stimulate the production of nitric oxide (NO) in aortic endothelial cells via activation of endothelial nitric oxide synthase (eNOS) through an increased calcium influx. Here, results obtained using cultured bovine pulmonary artery endothelial cells (bPAECs) suggest that C-peptide does not induce eNOS activation directly in cultured pulmonary artery endothelium. However, C-peptide has been shown to stimulate the release of ATP from erythrocytes, a well-documented stimulus of eNOS activity in the pulmonary endothelium. Therefore, studies were performed to examine if C-peptide can indirectly stimulate NO production in a cultured pulmonary endothelium that is erythrocyte mediated. Methods NO production and free intracellular calcium changes were monitored in immobilized bPAECs using specific intracellular fluorescent probes after stimulation with adenosine triphosphate (ATP), calcium ionophore A23187, or C-peptide. A microfluidic device enabled immobilized bPAECs to interact with flowing erythrocytes in the presence and absence of C-peptide to determine the role of the erythrocyte in C-peptide-stimulated NO production in cultured bPAECs. Results ATP and the calcium ionophore stimulate significant increases in both intracellular NO production and influx of free calcium in cultured bPAECs. In contrast, C-peptide, ranging from physiological to above physiological concentrations, was unable to stimulate NO production or calcium influx in the bPAECs. However, when erythrocytes were pre-incubated with a mixture containing physiological concentrations of C-peptide with Zn2+ and haemodynamically pumped beneath bPAECs cultured on a microfluidic device, an 88.6 +/- 7.5% increase in endothelial NO production was observed. Conclusions C-peptide does not affect NO production in bPAECs directly but can impact NO production through an erythrocyte-mediated mechanism. Furthermore, in the absence of Zn2+, C-peptide does not stimulate this NO production directly or indirectly. These results suggest that C-peptide, in the presence of Zn2+, may be a determinant in purinergic receptor signalling via its ability to stimulate the release of ATP from erythrocytes. Copyright (C) 2012 John Wiley & Sons, Ltd.