Effects of high glucose on NO synthesis in human keratinocyte cell line (HaCaT).
BACKGROUND
There is a possibility that alteration of nitric oxide (NO) synthesis by high glucose leads to a variety of diabetic complications.


OBJECTIVE
In this study, we examined whether NO synthesis is altered by high glucose in spontaneously immortalized human keratinocyte cell line (HaCaT) that have three isoforms of NO synthases (NOS).


METHODS
We measured NO end product nitrite in the culture medium using the Griess reagent and analyzed mRNA expression of three isoforms of NOS in HaCaT cells by RT-PCR.


RESULTS
High glucose enhanced constitutively produced NO production in HaCaT cells, which persisted for 10 days and was attenuated by an inhibitor of protein kinase C (PKC), without altering eNOS/nNOS mRNA levels. Cytokine stimulation induced iNOS mRNA in HaCaT cells. Pretreatment with high glucose for 24 h enhanced cytokine-induced NO production in HaCaT cells. However, when these cells were exposed to high glucose for 10 days, cytokine treatment did not induce iNOS mRNA and nitrite production.


CONCLUSIONS
These diverse alterations in NO production by high glucose may be involved in impaired host-defense and wound healing in the skin of diabetic patients.
Department of Dermatology, Kagawa Medical University, Kagawa 761-0793, Japan.
Kozo
K
Nakai
Shigemoto
S
Fujii
Akira
A
Yamamoto
Junsuke
J
Igarashi
Yasuo
Y
Kubota
Hiroaki
H
Kosaka
D002460:Cell Line; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005947:Glucose / Q000494:pharmacology*; D006801:Humans; D015603:Keratinocytes / Q000187:drug effects / Q000378:metabolism*; D009569:Nitric Oxide / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase / Q000235:genetics; D052248:Nitric Oxide Synthase Type I; D052250:Nitric Oxide Synthase Type III; D009573:Nitrites / Q000378:metabolism; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D014158:Transcription, Genetic / Q000187:drug effects
D004791:Enzyme Inhibitors; D009573:Nitrites; D009569:Nitric Oxide; C496320:NOS1 protein, human; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; D052250:Nitric Oxide Synthase Type III; D011493:Protein Kinase C; D005947:Glucose