NADPH Alters DUOX1 Calcium Responsiveness

Abstract

Hydrogen peroxide is a key element in redox signaling and in setting cellular redox tone. DUOX1 and DUOX2, that directly synthesize hydrogen peroxide, are the most abundant NADPH oxidase transcripts in most epithelia. DUOX1 and DUOX2 hydrogen peroxide synthesis is regulated by intracellular calcium transients and thus cells can respond to signals and initiate responses by increasing cellular hydrogen peroxide synthesis. Nevertheless, many details of their enzymatic regulation are still unexplored. DUOX1 and DUOXA1 were expressed in HEK293T cells and activity was studied in homogenates and membrane fractions. When DUOX1 homogenates or membranes were pre-incubated in NADPH and started with addition of Ca²⁺, to mimic intracellular activation, progress curves were distinctly different from those pre-incubated in Ca²⁺ and started with NADPH. The Ca²⁺ EC₅₀ for DUOX1's initial rate when pre-incubated in Ca²⁺, was three orders of magnitude lower (EC₅₀ ∼ 10^-6 M) than with preincubation in NADPH (EC₅₀ ∼ 10^-3 M). In addition, activity was several fold lower with Ca²⁺ start. Identical results were obtained using homogenates and membrane fractions. The data suggested that DUOX1 Ca²⁺ binding in expected physiological signaling conditions only slowly leads to maximal hydrogen peroxide synthesis and that full hydrogen peroxide synthesis activity in vivo only can occur when encountering extremely high concentration Ca²⁺ signals. Thus, a complex interplay of intracellular NADPH and Ca²⁺ concentrations regulate DUOX1 over a wide extent and may limit DUOX1 activity to a restricted range and spatial distribution.

Keywords: DUOX; Hydrogen peroxide; NADPH oxidase.

Graphical abstract

Fig. 1

Preincubation of homogenates in NADPH alters DUOX1 Ca²⁺ sensitivity. Panel A, Homogenates were preincubated with increasing [Ca²⁺] and reactions were started by addition of 50 μM NADPH. Resorufin fluorescence was measured at timed intervals and H₂O₂ calculated from parallel standard curves. Progress curves were fit to a double exponential (equation 3 in Ref. [35], viz. Supplemental methods) using Graphpad Prism 9. Panel B, homogenates were preincubated with 50 μM NADPH and reactions were started by addition of increasing [Ca²⁺]. Resorufin fluorescence was measured at timed intervals and H₂O₂ calculated as in panel A. Panel C, EC50's for Ca²⁺ were obtained by fitting initial rates of reactions to a 3-parameter dose response curve using Graphpad Prism 9 for reactions shown in panel A preincubated in various [Ca²⁺] and started with NADPH (1.6 μM, CI95 = 0.9–2.9 μM, black circles in panel C); using reactions shown in panel B preincubated in NADPH and started with various [Ca²⁺] (0.3 mM, CI95 = 0.4–2.6 mM, red squares panel C); and using reactions preincubated with sequential addition of 100 nM Ca²⁺ followed by NADPH and finally starting reactions with addition of various higher [Ca²⁺] (progress curves not shown) (0.96 mM, CI95 = 0.34–2.2 mM, blue triangles panel C). Panels A - C are mean values of triplicate assays ± S.D. from a single experiment. Some error bars are within the symbols. Panel D, mean EC₅₀ values from repeated experiments, as described in panel A (Ca²⁺ preincubation) and B (NADPH preincubation), were respectively, 2.3 ± 1.3 μM (SD), n = 16 and 1.9 ± 1.2 mM (SD), n = 5, p < 0.0001, Mann-Whitney test.

Fig. 2

Preincubation of PM-enriched fractions in NADPH alters DUOX1 Ca²⁺ sensitivity. Panel A, PM-enriched fractions were preincubated with increasing [Ca²⁺] and reactions were started by addition of 5 μM NADPH. Resorufin fluorescence was measured at timed intervals and H₂O₂ calculated from parallel standard curves. Progress curves were fit to a double exponential (equation 3 in Ref. [35], viz. Supplemental methods) using GraphPad Prism 9. Panel B, Homogenates were preincubated with 5 μM NADPH and reactions were started by addition of increasing [Ca²⁺]. Resorufin fluorescence was measured at timed intervals and H₂O₂ calculated as in panel A. Panel C, The EC50 was calculated from Ca²⁺ dose response curves: using reactions shown in panel A preincubated in various [Ca²⁺] and started with NADPH (2.2 μM, CI95 = 0.9–5.6 μM, red circles in panel C) and using reactions shown in panel B preincubated in NADPH and started with various [Ca²⁺] (2.5 mM, CI95 = 1.2–8.2 mM, black squares panel C). EC₅₀'s for Ca²⁺ were obtained by fitting initial rates of reactions to a 3-parameter dose response curve using GraphPad Prism 9. Panels A-C are mean values of triplicates ± S.D. from a single experiment. Some error bars are within the symbols.

Fig. 3

DUOX1 progress curves are nonlinear. Panel A, Homogenates were preincubated in 12 μM Ca²⁺ and reactions were started with 50 μM NADPH, and in Panel B, homogenates were preincubated in 50 μM NADPH and started by addition of Ca²⁺ to 12 μM. Lines represent the slope over the first three data points. Preincubation in Ca²⁺ showed an initial burst of activity followed by a slower rate. Conversely, preincubation with NADPH showed a lower initial rate that slowly increased with longer reaction times. Panel C. Homogenates (black squares) or PM-enriched fractions (red squares) were preincubated in 12 μM Ca²⁺ for 5, 25 or 150 min as indicated in the graph before starting with 50 μM NADPH addition. Preincubation in Ca²⁺ increased the activity. Plotted values are means of triplicate assays. Some error bars are within the symbols.