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. CONCLUSION: 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.
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. CONCLUSION: These diverse alterations in NO production by high glucose may be involved in impaired host-defense and wound healing in the skin of diabeticpatients.