New Page 1

Nitric Oxide

Nitric oxide (NO) is generated by many cell types, including keratinocytes, macrophages and dermal fibroblasts, and often functions as a molecular mediator for a number of biological functions. NO is formed by the oxidation of a guanidino nitrogen of L-arginine, a reaction which is catalyzed by the nitric oxide synthetase enzyme. Its mechanism of action is often the activation of soluble intracellular guanylyl cylclases that in turn will generate the second messenger cGMP. While NO is a very short-lived compound, it degrades into very stable end products: nitrite and nitrate. While the nitrite end product can be easily measured, the nitrate must be enzymatically converted to nitrite before it can be measured. Total nitrite can then be assayed either using a spectrophotometric technique based on the reaction of the nitrite and a Griess Reagent (micromolar range) or fluorometrically for extremely low levels of nitrite (nanomolar range).

NO can be generated by keratinocytes in response to UV irradiation or by macrophages in response to LPS stimulation. The use of either of these models allows for the testing of materials that either inhibit NO formation or act as NO scavengers.

cGMP

When activated by nitric oxide (NO), guanylyl cyclase can catalyze the conversion of GTP to cGMP. Cyclic GMP acts as an intracellular second messenger and can in turn activate cGMP dependent protein kinases. Cyclic GMP formation is transient and the signal is primarily ended by the breakdown of cGMP by phosphodiesterases.

Cultured dermal fibroblasts can respond to NO, generated by NO donating compounds, and form cGMP. Typically, cultured dermal fibroblasts are pretreated with 3-isobutyl-1-methylxanthine (IBMX), a phosphodiesterases inhibitor, to prevent the subsequent breakdown of any cGMP formed. After this pretreatment the fibroblasts are exposed to either test materials or spermine NONOate (which breaks down into NO), or spermine NONOate + test materials. The cells are then lysed open to release any cGMP that was formed. Cyclic GMP is assayed via a competitive fluorescent EIA assay.


		In Vitro Tests Nitric Oxide/cGMP Nitric Oxide Nitric oxide (NO) is generated by many cell types, including keratinocytes, macrophages and dermal fibroblasts, and often functions as a molecular mediator for a number of biological functions. NO is formed by the oxidation of a guanidino nitrogen of L-arginine, a reaction which is catalyzed by the nitric oxide synthetase enzyme. Its mechanism of action is often the activation of soluble intracellular guanylyl cylclases that in turn will generate the second messenger cGMP. While NO is a very short-lived compound, it degrades into very stable end products: nitrite and nitrate. While the nitrite end product can be easily measured, the nitrate must be enzymatically converted to nitrite before it can be measured. Total nitrite can then be assayed either using a spectrophotometric technique based on the reaction of the nitrite and a Griess Reagent (micromolar range) or fluorometrically for extremely low levels of nitrite (nanomolar range). NO can be generated by keratinocytes in response to UV irradiation or by macrophages in response to LPS stimulation. The use of either of these models allows for the testing of materials that either inhibit NO formation or act as NO scavengers. cGMP When activated by nitric oxide (NO), guanylyl cyclase can catalyze the conversion of GTP to cGMP. Cyclic GMP acts as an intracellular second messenger and can in turn activate cGMP dependent protein kinases. Cyclic GMP formation is transient and the signal is primarily ended by the breakdown of cGMP by phosphodiesterases. Cultured dermal fibroblasts can respond to NO, generated by NO donating compounds, and form cGMP. Typically, cultured dermal fibroblasts are pretreated with 3-isobutyl-1-methylxanthine (IBMX), a phosphodiesterases inhibitor, to prevent the subsequent breakdown of any cGMP formed. After this pretreatment the fibroblasts are exposed to either test materials or spermine NONOate (which breaks down into NO), or spermine NONOate + test materials. The cells are then lysed open to release any cGMP that was formed. Cyclic GMP is assayed via a competitive fluorescent EIA assay.