Nitric oxide plays a critical role in diverse physiological functions including regulation of cell growth, respiration, gastric motility, wound healing, renal function, blood pressure and neurotransmission. This reactive nitrogen intermediate is also implicated in the pathogenesis of toxicity; various xenobiotics alter nitric oxide metabolism. Our laboratory is interested in the role of nitric oxide in inflammation in the skin. It is known that chemical-induced skin injury involves a complex array of growth factors and inflammatory mediators. We have determined that nitric oxide is released by the skin during injury and that cultured keratinocytes can produce nitric oxide in response to various cytokines including gamma interferon and tumor necrosis factor alpha. Epidermal growth factor and insulin-like growth factor-1 antagonize cytokine- stimulated nitric oxide production by keratinocytes. Skin irritants including the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate and ultraviolet light also stimulate nitric oxide release by keratinocytes. Our research efforts are focused on understanding mechanisms by which nitric oxide production is regulated in keratinocytes. We have found that cytokines induce nitric oxide production in differentiating, but not proliferating keratinocytes. Transformed keratinocyte cell lines also readily produce nitric oxide. In these cells, nitric oxide production is due to expression of the inducible form of nitric oxide synthase. In almost all cellular systems, nitric oxide produced by cells is not measured directly. Most often, the oxidation products nitrite and nitrate are measured. It is still uncertain as to whether nitric oxide is directly released from cells and if so, its activity at a distance from cells once released is not known. The development of a nitric oxide probe has offered us an opportunity to perform real time measurements of nitric oxide production by keratinocytes and macrophages, an important component of the cellular immune system. The probe is several orders of magnitude more sensitive than assays for nitrite and nitrate and allows measurements on a single cells basis. This is important for examining production of nitric oxide in heterogenous cell populations. Using this probe we have been able to directly measure nitric oxide release by both keratinocytes and macrophages. Nitric oxide can be detected at a distance of 3-4 cell diameters before it is either diluted or oxidized. Nitric oxide production in both cell types is dependent on l-arginine in the culture medium. In fact, in arginine-depleted cells, l- arginine supplementation allows for nitric oxide production within 30 seconds indicating that this amino acid is rapidly taken up and utilized for nitric oxide biosynthesis. Approximately one minute is required for the nitric oxide synthase inhibitor nitro-arginine to inhibit nitric oxide production suggesting that this inhibitor is also readily taken up by the cells and interacts with l- arginine pools. The effects of the nitric oxide synthase inhibitor were reversed when the cells were rinsed free of the compound indicating that this inhibitor does not accumulate in the cells. Another guanidine analog required at least four hours to inhibit nitric oxide production possible due to slow uptake of the drug under our cell culture conditions. In both keratinocytes and macrophages, oxyhemoglobin, a nitric oxide trapping agent readily inhibited detection of nitric oxide; an effect that was reversed in wash out experiments. Cells are maintained in an extracellular matrix containing both proteins and lipids. We found that supplementing the cell culture medium with protein (bovine serum albumin) or lipids (cationic liposomes) readily suppressed detection of nitric oxide by the probe. Proteins are known to contain sulfhydryl groups which presumably bind nitric oxide and inhibit its activity. Nitric oxide is more soluble in lipids and this may account for decreased detection of the free radical. Future experiments are planned to examine mechanisms by which nitric oxide is released from keratinocytes and macrophages. We also plan to characterize extracellular components in the culture medium that affect the distance from the cells at which nitric oxide can be detected.
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