A transverse tubule NADPH oxidase activity stimulates calcium release from isolated triads via ryanodine receptor type 1 S-glutathionylation

Abstract

We report here the presence of an NADPH oxidase (NOX)activity both in intact and in isolated transverse tubules andin triads isolated from mammalian skeletal muscle, as establishedby immunochemical, enzymatic, and pharmacologicalcriteria. Immunohistochemical determinations with NOXantibodies showed that the gp91phox membrane subunit andthe cytoplasmic regulatory p47phox subunit co-localized intransverse tubules of adult mice fibers with the 1s subunit ofdihydropyridine receptors. Western blot analysis revealedthat isolated triads contained the integral membrane subunitsgp91phox and p22phox, which were markedly enriched inisolated transverse tubules but absent from junctional sarcoplasmicreticulum vesicles. Isolated triads and transversetubules, but not junctional sarcoplasmic reticulum, also containedvarying amounts of the cytoplasmic NOX regulatorysubunits p47phox and p67phox. NADPH or NADH elicitedsuperoxide anion and hydrogen peroxide generation by isolatedtriads; both activities were inhibited by NOX inhibitorsbut not by rotenone. NADH diminished the total thiol contentof triads by one-third; catalase or apocynin, aNOXinhibitor,prevented this effect. NADPH enhanced the activity ofryanodine receptor type 1 (RyR1) in triads, measured through[3H]ryanodine binding and calcium release kinetics, andincreased significantly RyR1 S-glutathionylation over basallevels. Preincubation with reducing agents or NOX inhibitorsabolished the enhancement of RyR1 activity produced byNADPH and prevented NADPH-induced RyR1 S-glutathionylation.We propose that reactive oxygen species generatedby the transverse tubule NOX activate via redox modificationthe neighboring RyR1 Ca2 release channels. Possible implicationsof this putative mechanism for skeletal muscle functionare discussed.