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. 2007 Jul 1;43(1):31-8.
doi: 10.1016/j.freeradbiomed.2007.03.006. Epub 2007 Mar 12.

Nox regulation of smooth muscle contraction

Darren R Ritsick  1 , William A Edens, Victoria Finnerty, J David Lambeth
Affiliations

Nox regulation of smooth muscle contraction

Darren R Ritsick et al. Free Radic Biol Med. .

Abstract

The catalytic subunit gp91phox (Nox2) of the NADPH oxidase of mammalian phagocytes is activated by microbes and immune mediators to produce large amounts of reactive oxygen species (ROS) which participate in microbial killing. Homologs of gp91phox, the Nox and Duox enzymes, were recently described in a range of organisms, including plants, vertebrates, and invertebrates such as Drosophila melanogaster. While their enzymology and cell biology are being extensively studied in many laboratories, little is known about in vivo functions of Noxes. Here, we establish and use an inducible system for RNAi to discover functions of dNox, an ortholog of human Nox5 in Drosophila. We report here that depletion of dNox in musculature causes retention of mature eggs within ovaries, leading to female sterility. In dNox-depleted ovaries and ovaries treated with a Nox inhibitor, muscular contractions induced by the neuropeptide proctolin are markedly inhibited. This functional defect results from a requirement for dNox-for the proctolin-induced calcium flux in Drosophila ovaries. Thus, these studies demonstrate a novel biological role for Nox-generated ROS in mediating agonist-induced calcium flux and smooth muscle contraction.

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Figures

Fig. 1
Fig. 1
Muscle dNox regulates ovulation. (A–C) White bars, UAS-dNox-IR; black bars, UAS-dNox-IR + TubP-GAL4. (A) Quantitative RT-PCR of dNox in adult flies. (B) Progeny produced from crosses of control and dNox RNAi females with w1118 males. (C) Eggs laid by control and dNox RNAi females mated with w1118 males. (D–H) Control, UAS-DN352-IR; dNox RNAi, UAS-DN352 + TubP-GAL4. (D–E) Photographs of 10 day old control (D) and dNox RNAi (E) females. (F–G) Light micrographs of ovaries dissected from mated control (F) and dNox RNAi (G) females. (H) Ovulation rate of control and dNox RNAi females at various time points after crossing with w1118 males. Squares, control; triangles, dNox RNAi. (I) Progeny produced from crosses of females of the indicated genotype with w1118 males.
Fig. 2
Fig. 2
dNox regulates proctolin-induced muscle contractions in ovaries. (A–D) Graphs of ovarian movement caused by muscle contractions. (A) UAS-DN352-IR ovary with no stimulus. (B) UAS-DN352-IR ovary + 1 μM proctolin. (C) UAS-DN352-IR + Nrv1-GAL4 ovary + 1 μM proctolin. (D) UAS-DN352-IR ovary + 20 μM DPI + 1 μM proctolin.
Fig. 3
Fig. 3
Proctolin activates ROS production through dNox. (A–C) L-012 luminescence after addition of 1 μM proctolin and 1 μM ionomycin. Control, UAS-DN352-IR; dNox RNAi, UAS-DN352 + TubP-GAL4. (A) Squares, control ovaries; triangles, control ovaries + 20 μM M40403. (B) Squares, control ovaries; open triangles, control ovaries + 1 μM BAPTA-AM; closed triangles, control ovaries + 5 μM BAPTA-AM. (C) Squares, control ovaries; filled triangles, control ovaries + 20 μM DPI; open triangles, dNox RNAi ovaries.
Fig. 4
Fig. 4
dNox regulates proctolin-induced calcium influx in ovarian muscle. (A–B) Aequorin luminescence after addition of 1 μM proctolin and 1 μM ionomycin. Control, UAS-DN352-IR; dNox RNAi, UAS-DN352 + TubP-GAL4. (A) Squares, control ovaries; triangles, dNox RNAi ovaries. (B) Black, control ovaries; red, control ovaries + 10 μM DPI; yellow, control ovaries + 20 μM DPI; green, control ovaries + 40 μM DPI. (B inset) Quantification of data from (A) and (B); n=5 for all samples and error bars represent standard deviation, n=5. (C) Circles, control ovaries + 1 μM proctolin; squares, control ovaries + 100 μM hydrogen peroxide; triangles, control ovaries + 1 μM proctolin + 100 μM hydrogen peroxide. (D) Schematic of mechanism for dNox regulation of Drosophila ovarian function.
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