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, 96 (14), 7944-9

O2 Sensing Is Preserved in Mice Lacking the gp91 Phox Subunit of NADPH Oxidase

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O2 Sensing Is Preserved in Mice Lacking the gp91 Phox Subunit of NADPH Oxidase

S L Archer et al. Proc Natl Acad Sci U S A.

Abstract

The rapid response to hypoxia in the pulmonary artery (PA), carotid body, and ductus arteriosus is partially mediated by O2-responsive K+ channels. K+ channels in PA smooth muscle cells (SMCs) are inhibited by hypoxia, causing membrane depolarization, increased cytosolic calcium, and hypoxic pulmonary vasoconstriction. We hypothesize that the K+ channels are not themselves "O2 sensors" but rather respond to the reduced redox state created by hypoxic inhibition of candidate O2 sensors (NADPH oxidase or the mitochondrial electron transport chain). Both pathways shuttle electrons from donors, down a redox gradient, to O2. Hypoxia inhibits these pathways, decreasing radical production and causing cytosolic accumulation of unused, reduced, freely diffusible electron donors. PASMC K+ channels are redox responsive, opening when oxidized and closing when reduced. Inhibitors of NADPH oxidase (diphenyleneiodonium) and mitochondrial complex 1 (rotenone) both inhibit PASMC whole-cell K+ current but lack the specificity to identify the O2-sensor pathway. We used mice lacking the gp91 subunit of NADPH oxidase [chronic granulomatous disease (CGD) mice] to assess the hypothesis that NADPH oxidase is a PA O2-sensor. In wild-type lungs, gp91 phox and p22 phox subunits are present (relative expression: macrophages > airways and veins > PASMCs). Deletion of gp91 phox did not alter p22 phox expression but severely inhibited activated O2 species production. Nonetheless, hypoxia caused identical inhibition of whole-cell K+ current (in PASMCs) and hypoxic pulmonary vasoconstriction (in isolated lungs) from CGD vs. wild-type mice. Rotenone vasoconstriction was preserved in CGD mice, consistent with a role for the mitochondrial electron transport chain in O2 sensing. NADPH oxidase, though a major source of lung radical production, is not the pulmonary vascular O2 sensor in mice.

Figures

Figure 1
Figure 1
gp91 phox and p22 phox are present in the lung. gp91 phox (A) and p22 phox (B) are present in wild-type mice (immunohistochemistry in brown, counterstain blue). Controls lacking primary antibody are completely negative (not shown). As expected, the gp91 phox (C), but not the p22 phox (D), is absent in the CGD mouse. Lack of gp91 phox in C reveals the hematoxylin counterstain. Qualitative grading of the intensity of expression of gp91 phox by blinded observers was: alveolar macrophages (arrows), 4+; alveolar epithelium airways, 3+; pulmonary veins, 3+; large PAs, 2+; and small PAs, 1+. (E) Immunoblots show a typical band for gp91 phox at ≈60 kDa. The gp91 phox refers to the molecular weight in human tissue, where the subunit is more heavily glycosylated.
Figure 2
Figure 2
HPV is preserved and there is no pulmonary hypertension in CGD mice. (A) There are no intergroup differences in HPV or the constrictor response to AII in isolated mouse lungs (perfusate contains l-nitro arginine methylester and meclofenamate). (B) There are no intergroup differences in the pressure/flow relationship in isolated mouse lungs (perfusate contains meclofenamate). (C) There are no intergroup differences in duroquinone-induced constriction in isolated mouse lungs (perfusate contains meclofenamate and l-nitro arginine methylester). In separate experiments, rotenone vasoconstriction is enhanced in isolated lungs from CGD vs. wild-type mice (P < 0.05), (perfusate contains meclofenamate). (D) There is no medial hypertrophy of PAs on light microscopy. P, AD, and B refer to the size of PA studied as related to the associated airway (parenchymal, alveolar duct, and bronchial, respectively).
Figure 3
Figure 3
NADPH oxidase is functionally disrupted in the lungs of CGD mice. (A) Isolated lungs from CGD mice produce less AOS than wild-type mice, whether measured unenhanced or with lucigenin (∗, P < 0.05). PMA increases lucigenin chemiluminescence in lungs from wild-type, but not CGD, mice (†, P < 0.05). (B) Lucigenin-enhanced chemiluminescence is diminished by acute hypoxia and by DPI in rat PA rings (fourth division) denuded of endothelium. ∗, P < 0.05 value differs from normoxia. (C) Schematic of two competing theories for redox regulation of K+ channels. In one, a gp91 phox containing NADPH produces AOS, which modulate channel function through effects on critical channel cysteine groups. In the other, inhibition of mitochondrial complex 1 leads to accumulation of cytosolic reducing equivalents, which in turn inhibit the K+ channel by interaction with its cysteine groups.
Figure 4
Figure 4
EM and IK are similar in resistance PASMCs from wild-type and CGD mice (A) Resting EM is similar in wild-type (filled bar) and CGD (empty bar) PASMCs. (B) Normoxic current density is similar in wild-type and CGD PASMCs. (C and D) In PASMCs from CGD mice, 4-AP, a Kv channel inhibitor, reduces IK at negative potentials (−10 mV, C, ∗, P < 0.05). Tetraethylammonium (TEA) inhibits IK only at positive potentials, where KCa channels are active (∗, P < 0.05, D). (E and F) Four minutes of acute hypoxia inhibits IK (n = 4 cells/group, ∗, P < 0.01). (G and H) In separate experiments, 4 min of acute hypoxia inhibits IK even after specifically inhibiting large conductance KCa channels with IBTX. This finding suggests the hypoxia-sensitive current is at least partially Kv current (†, ∗ P < 0.05 IBTX and hypoxia inhibit IK, respectively).

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