NAD(P)H oxidase-dependent intracellular and extracellular O2•− production in coronary arterial myocytes from CD38 knockout mice

Activation of NAD(P)H oxidase has been reported to produce superoxide (O2•−) extracellularly as an autocrine/paracrine regulator or intracellularly as a signaling messenger in a variety of mammalian cells. However, it remains unknown how the activity of NAD(P)H oxidase is regulated in arterial myocytes. Recently, CD38-associated ADP-ribosylcyclase has been reported to use an NAD(P)H oxidase product, NAD+ or NADP+, to produce cyclic ADP-ribose (cADPR) or nicotinic acid adenine dinucleotide phosphate, which mediates intracellular Ca2 + signaling. This study was designed to test a hypothesis that the CD38/cADPR pathway as a downstream event exerts feedback regulatory action on the NAD(P)H oxidase activity in production of extra- or intracellular O2•− in mouse coronary arterial myocytes (CAMs). By fluorescence microscopic imaging, we simultaneously monitored extra- and intracellular O2•− production in wild-type (CD38+/+) and CD38 knockout (CD38−/−) CAMs in response to oxotremorine (OXO), a muscarinic type 1 receptor agonist. It was found that CD38 deficiency prevented OXO-induced intracellular but not extracellular O2•− production in CAMs. Consistently, the OXO-induced intracellular O2•− production was markedly inhibited by CD38 shRNA or the CD38 inhibitor nicotinamide in CD38+/+ CAMs. Further, Nox4 siRNA inhibited OXO-induced intracellular but not extracellular O2•− production, whereas Nox1 siRNA attenuated both intracellular and extracellular O2•− production in CD38+/+ CAMs. Direct delivery of exogenous cADPR into CAMs markedly elevated intracellular Ca2 + and O2•− production in CD38−/− CAMs. Functionally, CD38 deficiency or Nox1 siRNA and Nox4 siRNA prevented OXO-induced contraction in isolated perfused coronary arteries in CD38 WT mice. These results provide direct evidence that the CD38/cADPR pathway is an important controller of Nox4-mediated intracellular O2•− production and that CD38-dependent intracellular O2•− production is augmented in an autocrine manner by CD38-independent Nox1-derived extracellular O2•− production in CAMs.

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