Sublethal oxidant stress induces a reversible increase in intracellular calcium dependent on NAD(P)H oxidation in rat alveolar macrophages

A concentration-dependent elevation of intracellular calcium ([Ca²⁺]_i) and oxidation of NAD(P)H occurred in alveolar macrophages during exposure to sublethal tert-butylhydroperoxide concentrations (tBOOH) (≤100 μm in 1 ml with 1 × 10⁶ cells). Oxidation of NAD(P)H preceded a rise in [Ca²⁺]_i. The elevation of [Ca²⁺]_i was reversible at <50 μm tBOOH exposure and the return to the steady state [Ca²⁺]_i correlated temporally with repletion of NAD(P)H. At >50 μm tBOOH, the changes in NAD(P)H and [Ca²⁺]_i were sustained. The relative contributions of NADPH and NADH oxidation were examined by varying the substrates supplying reducing equivalents and by inhibiting glutathione reductase activity. The results suggested that at <50 μm tBOOH, oxidation of NADPH predominated, while at >50 μm tBOOH, NADH oxidation predominated. A complex relationship between the relative roles of NADPH and NADH oxidation and the elevation of [Ca²⁺]_i was revealed: (i) reversible oxidation of NADPH is associated with the initial and reversible elevation of [Ca²⁺]_i at <50 μm tBOOH; (ii) the sustained elevation of [Ca²⁺]_i at >50 μm tBOOH correlates with the sustained oxidation of NADH; and (iii) the changes in [Ca²⁺]_i did not depend on influx of extracellular Ca²⁺. We speculate that at low tBOOH, Ca²⁺ was released from the NADPH/NADP⁺-sensitive mitochondrial Ca²⁺ pool while higher tBOOH caused additional Ca²⁺ release from GSH/GSSG-sensitive nonmitochondrial Ca²⁺ pools with sustained elevation of [Ca²⁺]_i due to decreased mitochondrial Ca²⁺ reuptake.