Stimulation of endothelial nitric oxide synthase by proinsulin C-peptide

• DOI: 10.1016/j.niox.2003.08.004
• Publication Date: 2003-10-09T01:21:58Z
• Authors: Thomas Wallerath; Thomas Kunt; Thomas Forst; Ellen I Closs; Ralf Lehmann; Thomas Flohr; Matthias Gabriel; Dirk Schäfer; Andrea Göpfert; Andreas Pfützner; Jürgen Beyer; Ulrich Förstermann
• Keywords: Aniline Compounds; C-Peptide; Nitric Oxide Synthase Type III; Reverse Transcriptase Polymerase Chain Reaction; Blotting, Western; Endothelial Cells; Nitric Oxide; Nitroarginine; 03 medical and health sciences; 0302 clinical medicine; Microscopy, Fluorescence; Xanthenes; Animals; Humans; RNA; Calcium; Cattle; Enzyme Inhibitors; Nitric Oxide Synthase; Cyclic GMP; Fluorescent Dyes

There is increasing evidence for biological functions of human C-peptide. Recently, we have described that proinsulin C-peptide increases nutritive capillary blood flow and restores erythrocyte deformability in type 1 diabetic patients, whereas it has no such effect in non-diabetic subjects. The aim of the current study was to elucidate cellular mechanisms of this vasodilator effect in vitro by measuring the nitric oxide (NO)-mediated increase of cGMP production in a RFL-6 reporter cell assay and by demonstrating endothelial calcium influx with the Fluo-3 technique. C-peptide increased the release of NO from endothelial NO synthase (eNOS) in bovine aortic endothelial cells in a concentration- and time-dependent manner. At physiological concentrations of C-peptide, endothelial NO production was more than doubled (208+/-12% vs control; p<0.001). The NO release was abolished by the inhibitor of NO synthase N(G)-nitro-L-arginine or when Ca(2+) was removed from the medium superfusing the endothelial cells. C-peptide stimulated the influx of Ca(2+) into endothelial cells. No change in Ser-1179 phosphorylation of eNOS was detected after 6.6nM C-peptide. C-peptide did not change eNOS mRNA levels after 1, 6 or 24h. These data indicate that C-peptide is likely to stimulate the activity of the Ca(2+)-sensitive eNOS by increasing the influx of Ca(2+) into endothelial cells. We suggest that this effect may contribute to the increase in skin and muscle blood flow previously demonstrated in human in vivo.