Stimulation of endothelial nitric oxide synthase by proinsulin C-peptide.
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.
Wallerath
Thomas
T
Department of Pharmacology, Johannes Gutenberg University, Mainz, Germany.
Kunt
Thomas
T
Forst
Thomas
T
Closs
Ellen I
EI
Lehmann
Ralf
R
Flohr
Thomas
T
Gabriel
Matthias
M
Schäfer
Dirk
D
Göpfert
Andrea
A
Pfützner
Andreas
A
Beyer
Jürgen
J
Förstermann
Ulrich
U
D000814:Aniline Compounds / Q000737:chemistry; D000818:Animals; D015153:Blotting, Western; D002096:C-Peptide / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D006152:Cyclic GMP / Q000096:biosynthesis / Q000378:metabolism; D042783:Endothelial Cells / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes; D006801:Humans; D008856:Microscopy, Fluorescence; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D019335:Nitroarginine / Q000494:pharmacology; D012313:RNA / Q000737:chemistry / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014966:Xanthenes / Q000737:chemistry
D000814:Aniline Compounds; D002096:C-Peptide; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D014966:Xanthenes; D019335:Nitroarginine; C059715:Fluo-3; D009569:Nitric Oxide; D012313:RNA; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D006152:Cyclic GMP; D002118:Calcium