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Constitutive nitric oxide synthase is present in normal human keratinocytes
1L'Oréal, Centre de Recherche Charles Zviak, Sciences du Vivaut, Clichy, France.
The Journal of Investigative Dermatology
|March 1, 1996
Summary
Human skin cells, keratinocytes, possess nitric oxide synthase (NOS) activity. This constitutive enzyme is similar to neuronal NOS I and plays a role in skin physiology.
Area of Science:
- Biochemistry
- Dermatology
- Molecular Biology
Background:
- Normal human keratinocytes in culture display nitric oxide synthase (NOS) activity.
- The enzyme is cytosolic, dependent on calcium, nicotinamide adenine dinucleotide phosphate, reduced form (NADPH), and flavin adenine dinucleotide.
- Calmodulin antagonists inhibit this enzyme activity.
Purpose of the Study:
- To characterize the nitric oxide synthase (NOS) activity in normal human keratinocytes.
- To determine the properties and potential isoform of NOS present in these cells.
Main Methods:
- Enzyme assays were performed to measure NOS activity.
- Inhibitor studies were conducted using various arginine analogs.
- Partial purification was achieved using affinity chromatography.
- Protein analysis involved SDS-PAGE and Western blotting with antibodies against neuronal NOS.
Main Results:
- Human keratinocytes exhibit NOS activity between 50-150 pmol/min/mg protein.
- NG-nitro-L-arginine was a potent inhibitor, while L-canavanine was weak.
- A 152 kDa protein band cross-reacted with antibodies for neuronal constitutive NOS (NOS I).
- Kinetic analysis revealed a Vmax of 7.3 nmol/min/mg and a Km for L-arginine of 22.3 microM.
Conclusions:
- Normal human keratinocytes contain a constitutive nitric oxide synthase (cNOS).
- This enzyme is biochemically and immunologically related to the neuronal NOS I isoform.
- The findings suggest a role for NOS in keratinocyte function.