Divergent roles of glycolysis and the mitochondrial electron transport chain in hypoxic pulmonary vasoconstriction of the rat: identity of the hypoxic sensor

Abstract

The mechanisms responsible for sensing hypoxia and initiating hypoxic pulmonary vasoconstriction (HPV) are unclear. We therefore examined the roles of the mitochondrial electron transport chain (ETC) and glycolysis in HPV of rat small intrapulmonary arteries (IPAs). HPV demonstrated a transient constriction (phase 1) superimposed on a sustained constriction (phase 2). Inhibition of complex I of the ETC with rotenone (100 nm) or complex III with myxothiazol (100 nm) did not cause vasoconstriction in normoxia, but abolished both phases of HPV. Rotenone inhibited the hypoxia‐induced rise in intracellular Ca2+ ([Ca2+]i). Succinate (5 mm), a substrate for complex II, reversed the effects of rotenone but not myxothiazol on HPV, but did not affect the rise in NAD(P)H fluorescence induced by hypoxia or rotenone. Inhibition of cytochrome oxidase with cyanide (100 μm) potentiated phase 2 constriction. Phase 2 of HPV, but not phase 1, was highly correlated with glucose concentration, being potentiated by 15 mm but abolished in its absence, or following inhibition of glycolysis by iodoacetate or 2‐deoxyglucose. Glucose concentration did not affect the rise in [Ca2+]i during HPV. Depolarisation‐induced constriction was unaffected by hypoxia except in the absence of glucose, when it was depressed by ∼50 %. Depolarisation‐induced constriction was depressed by rotenone during hypoxia by 23 ± 4 %; cyanide was without effect. Hypoxia increased 2‐deoxy‐[3H]glucose uptake in endothelium‐denuded IPAs by 235 ± 32 %, and in mesenteric arteries by 218 ± 38 %. We conclude that complex III of the mitochondrial ETC acts as the hypoxic sensor in HPV, and initiates the rise in smooth muscle [Ca2+]i by a mechanism unrelated to changes in cytosolic redox state per se, but more probably by increased production of superoxide. Additionally, glucose and glycolysis are essential for development of the sustained phase 2 of HPV, and support an endothelium‐dependent Ca2+‐sensitisation pathway rather than the rise in [Ca2+]i.

Superoxide Generated at Mitochondrial Complex III Triggers Acute Responses to Hypoxia in the Pulmonary Circulation

“…By depleting RISP, the formation of ubisemiquinone at the Qo site is prevented, thus mimicking the response to myxothiazol. Taken together, these results are also consistent with a broader body of work indicating that hypoxia increases mitochondrial ROS signaling (10,17,18,(27)(28)(29)(36)(37)(38)(39).…”

“…Our previous work has implicated increases in reactive oxygen species (ROS) signaling during hypoxia (9,10,12). Previous studies using mitochondrial inhibitors and mitochondria-deficient (r 0 ) cells suggested that the electron transport chain (ETC) is required for hypoxia-induced ROS signaling in the pulmonary circulation (9,(11)(12)(13)(14)(15)(16)(17)(18). We subsequently assessed ROS signaling in hypoxic PASMC using roGFP, a thiol-containing, redox-sensitive reporter (19)(20)(21)(22)(23) targeted to compartments within mitochondria or the cytosol (10).…”

Oxidized-1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine induces vascular endothelial superoxide production: Implication of NADPH oxidase

“…BAEC remained viable compared to the control as reported previously [25]. 2-DOG has been widely used to study intracellular redox status in pulmonary arterial endothelial cell electron transport [63,64]. Although 2-DOG was not a specific NADPH inhibitor, others such as 6-amino-nicotinamide [65] to inhibit NADPH regeneration via the pentose phosphate pathway can also be toxic and nonspecific.…”

Classical transient receptor potential channel 6 (TRPC6) is essential for hypoxic pulmonary vasoconstriction and alveolar gas exchange

“…2A). (21) was needed and the fact that, in isolated lungs from rats as well as in isolated pulmonary arterial rings or PASMC from different species, a ''priming'' with angiotensin II, ET-1, or PGF 2a is a prerequisite to elicit any or full hypoxic vasoconstriction (22)(23)(24). The underlying mechanism of this priming is still not resolved (for review, see ref.…”