OSTEOCLAST RADICAL INTERACTIONS - NADPH CAUSES PULSATILE RELEASE OF NO AND STIMULATES SUPEROXIDE PRODUCTION

Osteoclasts have been shown to destroy calcified tissue by complex dev elopmental steps involving cell recruitment, cell attachment and deplo yment of multiple enzymes. They also appear to regulate resorption by several mechanisms. In particular, earlier investigations have indicat ed that oxygen radical metabolites may be produced by osteoclasts. The se labile reactants could accelerate destruction of calcified tissue. In addition, recent studies have suggested that nitric oxide may have an Inhibitory role in bone resorption. Previous studies of these radic al substituents have predicted that interactions of nitric oxide and o xygen radicals could explain the conflicting roles of these radicals i n the control of bone resorption. In view of the requirement of both o f the enzymes, NADPH-oxidase and NO synthase (NOS), for NADPH(beta-nic otinamide adenine dinucleotide phosphate), one level of interaction co uld be related to competition for this necessary cofactor. To test thi s hypothesis, we have investigated the ability of the osteoclast to ge nerate nitric oxide and oxygen radicals after stimulation by NADPH. Co nsistent with earlier diaphorase histochemistry, we have shown that re sorbing osteoclasts produce NO. Addition of NADPH (10 mu M) resulted i n a transient burst of NO production (measured by porphyrin coated mic rosensor) with an amplitude of 152 +/- 43 nM and a duration of 4 secon ds. Repetitive stimulation resulted in a decremental response with a p artial recovery after 30 minutes. Addition of L-NAME (N omega-nitro-L- arginine methyl ester, 100 mu M) to the cells resulted in at least 50% inhibition of the amplitude of NO peak and produced an extended peak duration. To compare the effect of the added NADPH on superoxide produ ction by osteoclast NADPH-oxidase, osteoclast oxygen radicals were det ected by EPR(electron paramagnetic resonance) spectrometer with the sp in-trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO). The production of a s pin adduct with a quadruplet signal was inhibited by SOD (superoxide d ismutase). We were not able to demonstrate an increase in superoxide p roduction after addition of L-NAME, another possible interaction of NO S and NADPH-oxidase. These results demonstrate that although osteoclas ts produce both NO and superoxide, NOS competition for NADPH is not a major site of interaction with NADPH-oxidase under these conditions. A dditionally, these initial findings set the stage for the further inve stigation of interactions of osteoclast radicals in modulating bone re sorption.