Effects of C-peptide on expression of eNOS and iNOS in human cavernosal smooth muscle cells.
OBJECTIVE
To investigate the role of C-peptide alone or in conjunction with insulin on the expression of nitric oxide synthase (NOS) in human corpus cavernosum smooth muscle cells (HCSMCs). Erectile dysfunction, among diabetic patients, is a significant health problem. The specific causes of erectile dysfunction are unknown. It has been suggested that impairment of penile relaxation is related to a reduction of penile NOS. Plasma levels of C-peptide and insulin are decreased in individuals with type 1 diabetes and late-stage type 2 diabetes.


METHODS
Primary cultures were initiated from explants of HCSMCs. Confluent cells at passages 2 to 4 were assigned to one of four groups with the following incubation conditions: (a) 27 mM glucose, (b) 27 mM glucose and insulin, (c) 27 mM glucose and human recombinant (hr)C-peptide, and (d) 27 mM glucose, insulin, and hrC-peptide. After 24 hours, total RNA and protein were extracted from cells and subjected to reverse transcriptase-polymerase chain reaction and Western blot analysis, respectively. Intracellular Ca(2+) was examined under the four conditions, using the Fura 2 method.


RESULTS
The least expression of endothelial NOS (eNOS) and inducible NOS (iNOS) in HCSMCs was observed in cells exposed to 27 mM glucose alone. Increased expression of eNOS and iNOS was found after treatment with insulin or hrC-peptide alone, and the maximal expression of eNOS and iNOS was detected in HCSMCs exposed to both insulin and hrC-peptide. Western blot analyses using eNOS and iNOS antibodies confirmed the RNA data. These effects are likely mediated by the insulin-induced and/or C-peptide-induced increase in intracellular Ca(2+).


CONCLUSIONS
Our results demonstrated that C-peptide, in the presence of insulin, increases the expression of iNOS and eNOS in HCSMCs. These results suggest that C-peptide, especially in conjunction with insulin, may have beneficial effects on cavernosal smooth muscle relaxation.
Li
Haikun
H
Department of Urology, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
Xu
Liping
L
Dunbar
Joseph C
JC
Dhabuwala
Chirpriya B
CB
Sima
Anders A F
AA
D015153:Blotting, Western; D002096:C-Peptide / Q000494:pharmacology*; D020013:Calcium Signaling / Q000187:drug effects; D002478:Cells, Cultured / Q000187:drug effects / Q000201:enzymology; D004790:Enzyme Induction / Q000187:drug effects; D005947:Glucose / Q000494:pharmacology; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D007424:Intracellular Fluid / Q000187:drug effects; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000187:drug effects* / Q000201:enzymology; D019001:Nitric Oxide Synthase / Q000096:biosynthesis* / Q000235:genetics; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; D010413:Penis / Q000098:blood supply*; D012333:RNA, Messenger / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014664:Vasodilation / Q000502:physiology
D002096:C-Peptide; D007328:Insulin; D012333:RNA, Messenger; D011994:Recombinant Proteins; C496319:NOS2 protein, human; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; D005947:Glucose