Regulation of inducible nitric oxide production by intracellular calcium.

Abstract

Because increased nitric oxide (NO) production during sepsis can be detrimental to the host, inhibition of NO synthesis by various antagonists has been proposed as a therapeutic option. However, none of these approaches has addressed the possible regulation of inducible NO synthesis (iNOS) by endogenous intracellular signaling mechanisms. The purpose of our study was to determine whether intracellular calcium ([Ca2+]i) regulates iNOS. The macrophage cell line RAW 264.7 cells stimulated to produce NO were cultured in the presence or absence of the calcium ionophore A23187, ionomycin, or the Ca(2+)-adenosine triphosphatase inhibitor thapsigargin. Supernatants and total cellular RNA were recovered for measurement of iNOS messenger RNA (Northern blot) and NO2- (stable end product of NO), respectively. Simultaneous measurement of [Ca2+]i was performed by fluorescence spectrophotometry. The calcium ionophore A23187, ionomycin, and thapsigargin all produced a dose-dependent inhibition of NO end product and iNOS messenger RNA (confirmed by densitometry) with a simultaneous increase in [Ca2+]i, confirmed by fluorescence spectrophotometry. This could not be reversed by exogenous L-arginine and was not observed if these agents were added beyond 0 hours of culture. Although iNOS is traditionally viewed as calcium independent, these data clearly show that [Ca2+]i regulates iNOS messenger RNA induction and end product synthesis. Endogenous cellular second messenger may thus be important in autoregulation of NO synthesis. Alternatives to pharmacologic or antagonist inhibition of NO deserve further investigation.