Making sense of oxygen sensing

Matching blood flow to lung ventilation is a tricky feat in which the tone of the pulmonary resistance vessels must be exquisitely matched to nearby oxygen tension. Without such matching of ventilation to perfusion, arterial blood oxygen tension cannot be maintained near that of the ambient atmosphere. Hypoxia-induced pulmonary vasoconstriction (HPV) shunts blood flow from poorly ventilated regions of the lung to better ventilated regions. How does the pulmonary vasculature ‘sense’ decreased oxygen tension and how is increased contractile force coupled to these sensing mechanisms? The response of the pulmonary circulation to hypoxia is opposite to that of the systemic circulation. In the systemic circulation regions of hypoxia may be underperfused relative to energy demand and hence vasodilate to increase perfusion to match oxygen supply to energy demand. The mechanisms for hypoxia-induced vasorelaxation are intuitively easier to understand and a variety of mechanisms have been proposed over the years including local adenosine and lactic acid production, endothelial nitric oxide production, and simply reduced energy charge within the hypoxic smooth muscle resulting from hypoxia and causing decreased contractile tone. In the pulmonary vasculature during hypoxia, HPV increases energy demand of the smooth muscle in the face of decreased oxygen availability for oxidative energy production. The smooth muscle cell faces a conundrum of increasing energy expenditure in the face of limits to oxidative energy production. The more poorly ventilated (oxygenated) a given pulmonary smooth muscle cell is, the more important is its constrictive response. Fortunately, vascular smooth muscle cells are highly glycolytic and normally derive 20–30 % of cell ATP production from glucose conversion to lactate which does not require oxygen (Hardin et al. 2001). By being highly glycolytic, smooth muscle can maintain force under hypoxic conditions as long as glucose is present, as would occur with poor ventilation and adequate perfusion. However, two aspects of HPV have remained mysterious and controversial over the years: (1) the nature of the oxygen sensor and the downstream signals to initially raise intracellular calcium to increase smooth muscle force generation and (2) the mechanisms by which HPV is maintained for prolonged periods in the face of attenuated intracellular free calcium concentration (calcium sensitization of contraction). Two camps have emerged over the controversy of the nature of the oxygen sensor in pulmonary smooth muscle cells responsible for initiating HPV. One camp supports the notion that NAD(P)H oxidase functions as an oxygen sensing complex which produces superoxide radicals in response to hypoxia resulting in calcium mobilization. Support for this view is based largely on the observation that the flavoprotein inhibitor diphenyleneiodonium (DPI) or the NAD(P)H oxidase inhibitor 4-(2-aminoethyl)benzenesulphonyl fluoride can abolish HPV (see Weissmann et al. 2000). However, oxygen sensing persisted in a mouse knockout model creating a deficiency in a key NAD(P)H oxidase subunit, gp91 phox (Archer et al. 1999), casting some doubt on the importance of this protein complex in oxygen sensing and HPV. A second camp supports the notion that elements of the electron transport chain function as oxygen sensors resulting in production of reactive oxygen species. Unfortunately, much of the confusion has resulted from lack of specificity of the inhibitors of NAD(P)H oxidase such as DPI and of elements of the electron transport chain such as rotenone. In this issue of the Journal of Physiology, the paper by Leach et al. (2001) helps resolve some of the controversy regarding the identity of the oxygen sensor by systematically examining the effect of inhibition of complex I and complex III on HPV. The authors report that inhibition of complex I results in inhibition of HPV which can be reversed by bypassing complex I by providing succinate as a substrate for complex II. By measuring epifluorescence, the authors found that while NAD(P)H was increased by inhibition of complex I, succinate successfully reversed the HPV inhibition but without decreasing NAD(P)H. Therefore the role of the electron transport chain as an oxygen sensor in HPV was independent of NAD(P)H levels and thus likely to be independent of NAD(P)H oxidase. Inhibiting complex III abolished HPV. Therefore, these studies clearly demonstrate a role for the electron transport chain in oxygen sensing and initiation of HPV. A second element to the puzzle of HPV is to determine how the smooth muscle maintains contractile force over prolonged periods even though cytoplasmic free calcium levels return towards baseline after the rapid constriction phase. For a wide variety of smooth muscles it has been proposed that agonist-induced contractions can be sensitized by coupling through Rho and Rho kinase with Rho kinase phosphorylating myosin phosphatase (see Somlyo & Somlyo, 2000). Fascinating observations were made by Leach et al. (2001) demonstrating that the second, or prolonged, phase of HPV depends on the presence of glucose and on the glucose concentration. Leach et al. measured cytoplasmic free calcium concentration and the dependence of the prolonged phase of HPV on glycolysis was not due to changes in free calcium concentration. Therefore, glycolysis seemed to be necessary for the calcium sensitization via the activity of Rho kinase. This intriguing proposal that glycolysis may be coupled to a cell signalling kinase is an important addition to the growing body of evidence that glycolysis is specifically coupled to a variety of ATP-requiring processes within the smooth muscle cell (Hardin et al. 2001). This study by Leach et al. (2001) suggests an interesting division of duties for oxidative metabolism and glycolysis in pulmonary vascular smooth muscle. The electron transport chain appears to play a key role in sensing hypoxia and initiating signals that result in increased cytoplasmic calcium and contraction while glycolysis specifically allows for calcium sensitization to allow a prolonged vasoconstriction in response to hypoxia. The possibility of a specific coupling of glycolysis to Rho kinase and other signalling kinases may have implications well beyond the scope of the study by Leach et al. The energetic support of cell signalling is poorly understood but studies such as this may uncover new levels of cell regulation.