O gp91phox p67phox RaplA O p22phox p47phox Racl

Fig. 4. Antisense ODN inhibition of NADPH oxidase activity in human monocytes. Antisense ODNs can be used to inhibit NADPH oxidase activity either by directly inhibiting the expression of components of the NADPH oxidase (e.g., p41phox) or by inhibiting expression of enzymes shown to provide essential upstream regulatory signals.

oxidase. Thus, specific inhibition of cPLA2 by antisense ODN allowed us to prove that it regulates the activity of this enzyme complex and the related 02~ formation. An interesting aspect of this finding is that the inhibition of expression of cPLA2 by antisense ODNs can be completely abolished by addition of arachidonic acid (AA) at predicted physiologic levels. It seems that the requisite role for cPLA2 activity is entirely due to the requirement for the generation of AA.53

Furthermore, studies by our laboratory have indicated that the requirement for PKCa, mentioned above, is solely for phosphorylating and activating cPLA2 and generating the essential AA.60 Thus, AA can also completely restore the activity of NADPH oxidase in monocytes treated with antisense ODNs to PKCa.22'60 The identification of these pathways as requisite regulators of NADPH oxidase activity provides alternative ways to control the activity of this enzyme complex and to regulate 02~ production. The various antisense ODNs used to modulate NADPH oxidase activity in human monocytes by our laboratory are depicted in Fig. 4. Thus, direct as well as indirect targets for antisense ODN inhibition can efficiently regulate the activity of this important enzyme complex.

60 Q. Li, V. Subbulakshmi, and M. K. Cathcart, in revision (2002).

Concluding Remarks

We have devised alternative approaches for inhibiting NADPH oxidase activity in human monocytes, using antisense ODNs. Although several pharmacologic inhibitors have been suggested to inhibit the activity of this enzyme complex, antisense ODN-mediated inhibition offers greater specificity and related lower toxicity. We recommend that none of these approaches, outlined in this chapter, be used in isolation. The use of multiple complementary approaches to modulate NADPH oxidase activity, or for that matter any enzyme activity, affords the investigator confidence that the experimental observations are sound.

Acknowledgments

This work was supported by NIH Grants HL-51068 and HL-61971 (M.K.C.). Additional support was provided by Minority Research Supplement HL-51068 (E.A.B.).

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