Role of Glutathione Redox State in Oxygen Sensing by Carotid Body Chemoreceptor Cells

four electrons transferred per oxidized 5-HT molecule, agreeing with the mechanismproposedforthedominantelectrochemicaloxidationreaction. 16 InNEBcells,theaverage(SEM)quantalchargeofsecretory eventsduringhypoxiawas33.12.4fC[n ¼157eventsfromsixcells, range2.3–183fC(Fig.7)].Thisvaluewasobtainedfromthetimeintegral of selected spikes with the fast-rising phase and slow decay typical of secretory events occurring at the membrane facing the amperometric electrode. Assuming that one 5-HT molecule contributes an average of four electrons, it is estimated that a single synaptic vesicle or quantum in NEB cells releasesanaverageof13,000971(n ¼157;sixcells)moleculesof 5-HT. 13 HighextracellularK þ (50mM)inducedasecretoryresponse similar to that elicited by severe hypoxia. Exocytosis was stimulated in normoxic NEB cells after exposure to tetraethylammonium (20 mM)or 4-aminopyridine (2 mM). Hypoxia-induced secretion was abolished by the nonspecific Ca 2þ channel blocker, Cd 2þ (100 M). Secretion was also largely inhibited by the L-type Ca 2þ channel blocker, nifedipine (2 M), but not by the N-type Ca 2þ channel blocker, !-conotoxin GVIA (1 M). The 5-HT 3 receptor blocker, ICS 205 930, also inhibited secretion from NEB cells under hypoxia. These results suggest that hypoxia stimulates 5-HT secretion from intact NEBs via the inhibition of K þ channels and calcium entry through L-type Ca 2þ channels, as well as by positive feedback activation of 5-HT 3 autoreceptors. 13 [3] Role of Glutathione Redox State in Oxygen Sensing by Carotid Body Chemoreceptor Cells By Constancio Gonzalez,Gloria Sanz-Alfayate, Ana Obeso, and Maria Teresa Agapito Introduction This article first presents some basic structural traits of the carotid body (CB) arterial chemoreceptors to understand the relationship between the arterial blood PO 2 and the activation of chemoreceptor cells, which are the O 2 sensing structures of the CB. Some considerations in relation to the intensity of CB blood flow and O 2 consumption of the organ would allow us to define the threshold for the detection of the hypoxic stimulus, which would lead us to the cardinal theme of the article, namely whether at the PO 2 levels detected by the CB there alterations in the genesis of reactive oxygen species (ROS). An alteration in the rate of ROS production 40 lung and the airways [3] Copyright 2004, Elsevier Inc. All rights reserved. METHODS IN ENZYMOLOGY, VOL. 381 0076-6879/04 $35.00 would impinge on the glutathione system [reduced glutathione (GSH) and oxidized glutathione (GSSG)], causing modifications in the GSH/GSSG ratio that are detected by direct measurement; the GSH/GSSG system represents the quantitatively most important mechanism to dispose ROS and to maintain the overall redox status or redox environment in mammalian cells. 1 The relationship between GSH/GSSG and oxygen chemoreception is approached from two different points of view. We will measure GSH/ GSSG levels and calculate the redox environment of the cells and correlation with the activity of chemoreceptor cells in normoxia and in hypoxia. We will also present data on pharmacological manipulation of the redox environment of the cells, as assessed by GSH/GSSG quotients, and possible correlations with the level of activity of chemoreceptor cells. The possible mechanisms of coupling between ROS and the GSH/GSSG system to the cellular effector machineries have been reviewed. 2,3 Structure, Blood Flow, Oxygen Consumption, and PO 2 of Carotid Body Tissue Carotid bodies are small paired organs, round to pear shaped, located in the proximity of the carotid artery bifurcation, which weigh 500 g in the cat and 50 g in the rat; in humans it is estimated that each CB weighs 1 and 2 mg. The CB receives sensory innervation via the carotid sinus nerve (CSN), a branch of the IX cranial nerve, by its cephalic pole. The parenchymatous cells of CB are organized in cluster-separated connective tissue converging on the surface of the organ to form the CB capsule 4 (Fig. 1). The size of the clusters varies considerably, some have 5–8 cells and others up to 20–30 cells; in any case, chemoreceptor cells, located toward the center of the clusters, exceed glial-like sustentacular cells by a factor of 3–5. In the connective tissue of the organ there is a dense net of capillaries with tortuous trajectories and variable diameters (8 to 20 m). This ample vascularization of the CB constitutes the most prominent feature in a section of a well-perfused CB, being 25–33% of the surface of the section occupied by the capillary lumens 5 (Fig. 1). The endothelium is thin, 1 F. Q. Schaffer and G. R. Buettner, Free Radic. Biol. Med.30, 1191 (2001). 2 V. J. Thannickal and B. L. Fanburg, Am. J. Physiol. Lung Cell. Mol. Physiol.279, L1005 (2000). 3 C. Gonzalez, M. T. Agapito, A. Rocher, G. Sanz-Alfayate, and A. Obeso, in ‘‘Oxygen Sensing: Responses and Adaptation to Hypoxia’’ (S. Lahiri et al., eds.), p. 489. Dekker, New York, 2003. 4 A. Verna, in ‘‘The Carotid Body Chemoreceptors’’ (C. Gonzalez, ed.), p. 1. Springer Verlag, New York, 1997. 5 C. Gonzalez, L. Almaraz, A. Obeso, and C. Gonzalez, Physiol. Rev.74, 829 (1994). [3] carotid body chemoreceptor cells 41 sometimes fenestrated, and partially surrounded by pericyte processes. The capillaries resume into venules that emerge from the organ to form a dense venous plexus in the surface of the CB. This rich vascularization determines that the distance from the center of most chemoreceptor cells to capillaries is between 10 and 20 m with a median distance of 15.77 m from the center of CB cell clusters to the nearest capillary in the adult cat CB. 6 Paralleling this ample vascularization, the CB is the organ with the highest blood flow of the organism, 1417 ml/min/100 g tissue, although with discrepancies it is estimated that the O 2 consumption in basal conditions is 1.3 ml/min/100 g tissue at a perfusing pressure and PO 2 in the perfusates (whether blood or saline) of 100 mm Hg. 7,8 These basic data on the vascularization, blood flow, and O 2 consumption show that the blood supply and distribution inside the CB have important physiological roles beyond the nutritional requirement. The aforementioned data and in vitro studies with the saline-superfused preparation of the CB show that the organ can survive, sense, and transduce O 2 and CO 2 levels just with O 2 dissolved at a PO 2 of 100 mm Hg. However, despite the apparent excess of blood flow in normal CB, there is a further increase Fig. 1. Histological sections near the equator of a rat carotid body. The section was first immunostained using a monoclonal antibody against tyrosine hydroxylase (TH) and a secondary antibody labeled with fluorescein isothiocyanate to label the TH-containing chemoreceptor cells that are grouped in clusters. The section was later counterstained with cresyl violet to show the great density of capillaries in the CB tissue. 6 D. W. Lu ¨bbers, L. Teckhaus, and E. Seidl, in ‘‘Chemoreception in the Carotid Body’’ (H. Acker et al., eds.), p. 62. Springer Verlag, Berlin, 1977. 7 W. J. Whalen and P. Nair, in ‘‘Physiology of the Peripheral Arterial Chemoreceptors’’ (H. Acker and R. G. O’Regan, eds.), p. 117. Elsevier, Amsterdam, 1983. 8 A. Obeso, A. Rocher, B. Herreros, and C. Gonzalez,in ‘‘The Carotid Body Chemoreceptors’’ (C. Gonzalez, ed.), p. 31. Springer Verlag, New York, 1997. 42 lung and the airways [3] in the vascularization of the CB with increased diameters and neoformation of the capillaries in situations of chronic hypoxia. 4 This increase in capillarity reduces the mean distance from capillaries to the border of cell clusters from 3 to 1.8 m. 4 What is the purpose of this high blood flow? The high blood flow and the increase in vascularization that occurs during chronic hypoxia (acute hypoxia produces an important vasodilatation) guarantee that the arteriovenous difference of O 2 is very small and, therefore, that CB tissue PO 2 is the optimal of the organism at any arterial PO 2 . Although there are inconsistencies in the actual tissue PO 2 in the CB, 8 Lu ¨bbers et al. 6 calculated that only about 4% of the PO 2 values in the CB would be below 40 mm Hg (in fact, the PO 2 should be higher because Lu ¨bbers et al. 6 used data for the O 2 consumption that were four to five times higher than those obtained in more recent studies). In line with those calculations, Whalen and Nair (reviewed in Refs. 7 and 8) found that mean CB tissue PO 2 is around 65 mm Hg when perfusing the CB with blood equilibrated at normal PO 2 (above 85 mm Hg). Perfused with blood at normal PO 2 , these authors found tissue PO 2 values below 40 mm Hg in some studies and, when perfused with blood at PO 2 in the range of 30–49 mm Hg, the mean CB tissue PO 2 was 20 mm Hg with very few values below 5 mm Hg. Using air-equilibrated saline solutions to perfuse, they found normal tissue PO 2 , validating the empirical observation that the CB functions normally in the saline-superfused preparation. Whalen and Nair 7 found that the hypoxic threshold for CSN discharge is a CB tissue PO 2 oscillating between 50 and 65 mm Hg, the P 50 for discharges oscillates between 10 and 32 mm Hg, and the peak CSN discharge is reached at tissues PO 2 of 3–5 mm Hg. These values correspond to arterial PO 2 of 70–75 mmHg (threshold), 40 mm Hg (P 50 ), and 10 mm Hg (peak CSN discharges). 5 To conclude, and giving an answer to the question formulated at the beginning of this paragraph, we consider that the high blood flow of the CB and the neovascularization occurring in chronic hypoxia tend to show that the CB (which constitutes the origin of a regulatory loop aimed to secure the availability of O 2 to the organism) receives an adequate O 2 supply to support its activity. Activity, however, increases in parallel to the decrease in arterial PO 2 . Genesis of ROS and Tissue PO 2 From the aforementioned data we can state that the entire range of activity of chemoreceptor cells occurs at PO 2 in their near environment at about 65 mm Hg in normoxia and above 5 mm Hg in situations of extreme hypoxia hardly compatible with life. At lower PO 2 , CBs are still able to function and, in fact, they continue functioning for long periods of time [3] carotid body chemoreceptor cells 43 after the death of experimental animals (the CB has been considered the ‘‘ultimum moriens’’). Questions to be asked include (1) is there a modification in the rate of ROS productions in the range of PO 2 where the CB is activated, (2) what is the mechanism responsible for the modification in the rate of ROS production, and (3) is the modification a cause or consequence of chemoreceptor cell activation? At the outset we must state that published experimental data related specifically to chemoreceptor cells are not available. The first question does not have an univocal answer as evidenced by the dual hypothesis put forward by Jones et al. 9 referring to hypoxic pulmonary vasoconstriction: ‘‘vasoconstrictive ROS are produced under hypoxia’’ and ‘‘a reduced production of vasodilatory ROS occurs under hypoxia.’’ We 3,10 have summarized information on the putative relationship between PO 2 and the rate of ROS production. The classical view 11,12 is that ROS production occurs in proportion to the available O 2 , except in situations of ischemia–reperfusion when xanthine dehydrogenase might be converted into xanthine oxidase by oxidative or proteolytic processes; the new enzyme transfers electrons from the purines directly to O 2 to form O 2 . According to this view, the first hypothesis on O 2 sensing in chemoreceptor cells in considering the participation of ROS assumed that hypoxia decreased the rate of ROS production (the same occurred in pulmonary artery smooth muscle cells, which like CB cells are stimulated by hypoxia). A decreased ROS would increase the GSH/GSSG ratio and determine that the O 2 sensor and additional proteins involved in the generation of the response to hypoxia were in reduced form (Prot-SH). The reduction of these proteins would produce activation of the cells and generate the hypoxic responses. Semiquantitative data giving support to this hypothesis were provided by Cross et al. 13 using dihydrorodamine 123 fluorescence in the CB and by Archer et al. 14 and Weir and Archer 15 using lucigenin chemiluminescence in the lung. The decreased ROS production during hypoxia would result from a putative decrease in the flow of electrons in the mitochondrial respiratory chain and from a decreased 9 R. D. Jones, J. T. Hancock, and A. L. Morice, Free Radic. Biol. Med.29, 416 (2000). 10 C. Gonzalez, G. Sanz-Alfayate, M. T. Agapito, A. Gomez-Nin ˜o, A. Rocher, and A. Obeso, Respir. Physiol. Neurobiol.132, 17 (2002). 11 B. Chance, H. Sies, and A. Boveris, Physiol. Rev.59, 527 (1979). 12 B. Halliwell and J. MC. Gutteridge, ‘‘Free Radicals in Biology and Medicine.’’ Oxford Univ. Press, Oxford, 1999. 13 A. R. Cross, L. Henderson, O. T. G. Jones, M. Delpiano, M. A. Hentschel, and H. Acker, Biochem. J.272, 743 (1990). 14 S. L. Archer, J. Huang, T. Henry, D. Peterson, and E. K. Weir, Cir. Res.73, 1100 (1993). 15 E. K. Weir and S. L. Archer, FASEB J.9, 183 (1995). 44 lung and the airways [3] velocity of superoxide (O 2  ) formation by a NADPH oxidase similar to the one present in phagocytes. Consistent with the classical view, it was also observed that the production of ROS in HepG-2 cells decreased with PO 2 in a very ample range of oxygen pressures. 16 More recently Archer et al. 17 found that hypoxia (36 mm Hg) decreased ROS production in endothelium-free rings of resistance rat pulmonary arteries from apparently two different sources: as hypoxia decreased the production of ROS and diphenylene iodonium (DPI; used as an inhibitor of NADPH oxidase) caused a further decrease in ROS production, ROS would be originated in mitochondria and at the level of NADPH oxidase. The same group 18 also reported that hypoxia (40 mm Hg) decreased ROS production in pulmonary artery rings, but increased ROS production in renal artery rings (measured as lucigenin chemiluminescence, but measured as 2,7-dichlorofluorescein fluorescence or by the peroxidase-based AmplexRed kit, hypoxia did not alter the production of ROS in renal artery rings). As in the previous study, rotenone (but not myxothiazol) and DPI also inhibited the production of ROS, but curiously enough, antimycin A, which blocks the respiratory chain distally to the quinone pool (the step in the respiratory chain where most ROS appear to be generated 10 ), also inhibited the production of ROS in pulmonary rings but cyanide did not alter the rate of ROS production in either pulmonary or renal artery rings (in Archer et al., 14 cyanide increased and antimycin A decreased the production of ROS in the lung). In the opposite view, where hypoxia increased ROS production, Marshall and co-workers 19 found that an NADPH oxidase-like enzyme increased ROS production in lung tissue. More recently, the view that hypoxia increases ROS production has been marshaled by Chandel and co-workers who, in different preparations, such as cardiomyocytes, 20 pulmonary artery smooth muscle cells, 21 or alveolar epithelial cells, 22 found that hypoxias of intensities in the range of PO 2 of 20–30 mm Hg (1.5–3.0% oxygenequilibrated solutions) produced increases in ROS levels, measured as 16 J. Fandrey, S. Frede, and W. Jemkelmann, Biochem. J.303, 507 (1994). 17 S. L. Archer, H. L. Reeve, E. Michelakis et al., Proc. Natl. Acad. Sci. USA 96, 7944 (1999). 18 E. D. Michelakis, V. Hampl, A. Nsair, G. Harry, A. Haromy, R. Gurtu, and S. L. Archer, Cir. Res.90, 1307 (2002). 19 C. Marshall, A. J. Mamary, A. J. Verhoeven, and B. E. Marshall, Am. J. Respir. Cell Mol. Biol.15, 633 (1996). 20 J. Duranteau, N. S. Chandel, A. Kulisz, Z. Shao, and P. T. Schumacker, J. Biol. Chem.273, 11619 (1998). 21 G. B. Waypa, N. S. Chandel, and P. T. Schumacker, Circ. Res.88, 1259 (2001). 22 L. A. Dada, N. S. Chandel, K. M. Ridge, C. Pedemonte, A. M. Bertorello, and J. I. Sznaider, J. Clin. Invest.111, 1057 (2003). [3] carotid body chemoreceptor cells 45 increased fluorescence due to the oxidation of 20,70-dichlorofluorescein, ranging from about 30 to 500–1000% above control (perfusion with 15–16% O 2 ). However, the origin of ROS according to Chandel and coworkers is mitochondrial, being produced mainly at the level of the quinone pool, because inhibitors of the respiratory chain proximal to this level (exemplified by rotenone and myxothiazol) abolished the hypoxic increase of ROS, as well as the responses elicited by hypoxia (but see Refs. 3 and 10), whereas inhibitors distal to the quinone pool (antimycin A, cyanide, azide) augmented ROS levels and mimicked hypoxia. Many of the findings of Chandel group have been contested by other groups 23 (see Gonzalez et al. 3 for additional references). Using 20,70-dichlorofluorescein fluorescence computed from the cells or the tissue sections, Kummer and co-workers also found that hypoxia increased mitochondrial ROS production in PC12 cells, 24 decreased ROS in neurons of the nodose ganglion, 25 and increased them in pulmonary artery smooth muscle cells, 26 being also this increase of mitochondrial origin. With a similar method, Killilea and co-workers 27 reported a 500% increase in ROS production in pulmonary artery smooth muscle cells exposed to 25 mm Hg for 1 h. To the unbiased reader, the collection of data presented in previous paragraphs must seem unintelligible. If it is difficult to accept that hypoxia acting at the mitochondrial level affects the rate of ROS production differently in one cell type vs another, then it is impossible to understand that hypoxia and inhibitors of the proximal vs the distal complexes of the respiratory chain decrease ROS production in some laboratories, whereas in other laboratories they act conversely. Where are the pitfalls? Available literature would indicate that the main problems may relate to the methods used to detect ROS. Serious doubts on the meaning of the information obtained with 2,7-dichlorofluorescein fluorescence have been cast by many authors. 28–33 Similar criticisms have been made as to the use of 23 N. Enomoto, N. Koshikawa, M. Gassmann, and K. Takenaga, Biochem. Biophys. Res. Commun.297, 346 (2002). 24 W. Kummer, B. Hohler, A. Goldenberg, and B. Lange, Adv. Exp. Med. Biol.475, 371 (2000). 25 Y. Yamammoto, M. Henrich, R. L. Snipes, and W. Kummer, Brain Res.961, 1 (2003). 26 R. Paddenberg, B. Ishaq, A. Goldenberg, P. Faulhammer, F. Rose, N. Weissmann, R. C. Braun-Dullaeus, and W. Kummer, Am. J. Physiol. Lung Cell. Mol. Physiol.284, L710 (2003). 27 D. W. Killilea, R. Hester, R. Balczon, P. Babal, and M. N. Gillespie, Am. J. Physiol. Lung Cell. Mol. Physiol.279, L408 (2000). 28 C. Rota, Y. C. Fann, and R. P. Mason, J. Biol. Chem.274, 28161 (1999). 29 C. Rota, C. F. Chignell, and R. P. Mason, Free Radic. Biol. Med.27, 873 (1999). 30 W. Jakubowski and G. Bartosz, Cell Biol. Int.24, 757 (2000). 31 M. j. Burkitt and P. Wardman, Biochem. Biophys. Res. Commun.282, 329 (2001). 46 lung and the airways [3] lucigenin 34 (see Janiszewski et al. 35 for additional references). In addition to those critiques, there is solid evidence that time-dependent light-induced production of ROS may affect the findings enormously. 36–38 With the use of 2,7-dichlorofluorescein, we have experienced the pitfalls mentioned earlier. The increase in fluorescence detected in short-term cultured chemoreceptor cells was mostly dependent on the parameters of stimulation (intensity of the lamp, the time of illumination per frame, and the number of frames per minute), and we could not detect any clear signal that could unequivocally be assigned to hypoxic stimulation. Those findings recommended a turn in our experimental approach to measure GSH/ GSSG as the main determinant of the redox environment of the cells. The answer to the first question is that we do not know if hypoxia increases or decreases the rate of ROS production. Regarding the second question, that is, the mechanism responsible for the alteration in ROS production, we must make a double assumption. The initial assumption would be that hypoxia decreases the rate of ROS production both at the level of mitochondria, because ROS production parallels PO 2 at the respiratory chain, 10,39 and at the level of NADPH oxidase, because the decrease in PO 2 and the K m of the enzyme would make the oxidase work at a lower rate and to produce less ROS. 13 Using dihydrorodamine 123 fluorescence, Cross et al. 13 found a decrease in fluorescence during hypoxia and favored the notion that the decrease in ROS levels was due to a decrease in the activity of NADPH oxidase. However, data from our laboratory showed that inhibition of this oxidase does not prevent the detection and the genesis of a normal response to hypoxia in chemoreceptor cells of the rat or rabbit. 40 However, a minor modulatory role for NADPH-derived ROS could not be excluded from our study. Our second assumption would be that hypoxia increases ROS. The authors concluded that the weight of the literature supports the notion that mitochondria cannot be the source of those increased levels of ROS production. In the ranges of PO 2 (and probably even at 32 S. I. Liochev and I. Fridovich, J. Biol. Chem.276, 35253 (2001). 33 J. L. Brubacher and N. C. Bols, J. Immunol. Methods 251, 81 (2001). 34 S. I. Liochev and I. Fridovich, Arch. Biochem. Biophys.337, 115 (1997). 35 M. Janiszewski, H. P. Souza, X. Liu, M. A. Pedro, J. L. Zweier, and F. R. Laurindo, Free Radic. Biol. Med.32, 446 (2002). 36 P. E. Hockberger, T. A. Skimina, V. E. Centonze, C. Levin, S. Chu, S. Dadras, J. K. Reddy, and J. G. White, Proc. Natl. Acad. Sci. USA 96, 6255 (1999). 37 M. Afzal, S. Matsugo, M. Sasai, B. Xu, K. Aoyama, and T. Takeuchi, Biochem. Biophys. Res. Commun.304, 619 (2003). 38 P. Bilski, A. G. Belanger, and C. F. Chignell, Free Radic. Biol. Med.33, 938 (2002). 39 L. E. Costa, S. Llesuy, and A. Boveris, Am. J. Physiol.264, C1395 (1993). 40 A. Obeso, A. Gomez-Nin ˜o, and C. Gonzalez, Am. J. Physiol.276, C593 (1999). [3] carotid body chemoreceptor cells 47 lower PO 2 ) where the CB works, there is no limitation in the availability of O 2 to accept the electrons flowing through the respiratory chain due to the great affinity for O 2 of cytochrome oxidase. 38–43 As stated explicitly by several authors, mitochondria with the physiologically available substrates for oxidation do not release measurable levels of ROS (even less than the 1–3% of the consumed O 2 as classically suggested 11 ) unless the respiratory chain is inhibited to build up reduced forms of the initial mitochondrial complexes and unless there is O 2 available. The most plausible explanation of the observed increased rate of ROS production by mitochondria would be the result of unspecific interactions between the dyes used to measure ROS and mitochondria. 41,43 The proposal made by Staniek and Nohl 41 that mitochondrial respiration seems not to be required as permanent sources of ROS for physiological activities, such as cell signaling, gains full support from studies of several groups showing that hypoxia-inducible factordependent gene expression 23 (see Gonzalez et al. 3 for additional references), as well as acute membrane-linked O 2 chemoreception in a model of airway chemoreceptors, 44 takes place in cells lacking functional mitochondria ( o cells). It is of special relevance for this article that the observation of the first known effector in the oxygen chemoreception cascade, represented by specificK þ currents, is inhibited in an identical percentage and with an identical time course in control and  o H146 cells. Staniek and Nohl 41 also proposed that it is more likely that any ROS involved in the physiological function of cell signaling is produced in specific compartments in the vicinity of the effector molecules. This microdomain-centered production of ROS would have two purposes: (1) to avoid inflicting unnecessary damage and activation of alternative pathways and (2) to be effective in reaching the target before the ROS are inactivated by the scavenging mechanisms of the cells. Excluding mitochondria as the source of ROS at the hypoxic levels, which activate the CB chemoreceptors physiologically, where could ROS be generated if we stay with the assumption that hypoxia increases ROS production? Potential sources of ROS during hypoxia in mammalian cells include smooth endoplasmic reticulum-oxidizing enzymes that use the cytochrome P450 and b 5 electron transport chain, microsomal cyclooxygenases and cytoplasmic lipooxygenases, and the leucocyte-type NADPH-oxidase system. 10,12 Although some suggestions have been made regarding the 41 K. Staniek and H. Nohl, Biochim. Biophys. Acta 1460, 268 (2000). 42 E. Gnaiger, G. Mendez, and S. C. Hand, Proc. Natl. Acad. Sci. USA 97, 11080 (2000). 43 J. St-Pierre, J. A. Buckingham, S. J. Roebuck, and M. D. Brand, J. Biol. Chem.277, 44780 (2002). 44 G. J. Searle, M. E. Hartness, R. Hoareau, C. Peers, and P. J. Kemp, Biochem. Biophys. Res. Commun.291, 332 (2002). 48 lung and the airways [3] of the cell response to the stimulus activates the production of ROS and the subsequent oxidation of GSH with the decrease in its redox potential. The converse would also be true. These considerations also imply that the glutathione peroxidase/glutathione reductase system, together with catalase, is responsible for the elimination of H 2 O 2 , which represents the common final path of all ROS in the cells. It should be recalled, however, that the congenital absence of catalase does not cause serious clinical problems, 12 indicating that the glutathione peroxidase system may suffice to eliminate H 2 O 2 when produced at normal physiological rates. The necessity for catalase only becomes evident when there is an extra production of H 2 O 2 . Another consideration of relevant physiological and pathophysiological significance is that all the enzyme systems involved in the disposal of ROS (superoxide dismutase, glutation and thioredoxin peroxidases and reductases, and catalase and thioredoxin itself) are upand downregulated as a function of the rate of production of ROS. 38,54–56 Additionally, in general terms, the level of activity of these enzymes in some species or strains of animals versus others or in different developmental periods correlates directly with their resistance to the oxidative insults. 57 Finally, the definition of a correct correlation between GSH levels measured experimentally and any given cellular function needs some more considerations. Figure 2 shows a 2:1 molar ratio in the utilization of GSH per mole of H 2 O 2 removed. Knowing that cells consume O 2 at a rate of 1 mmol/kg/min or less, that ROS production is around 0.01–0.03 mmol/ kg/min (i.e., 1–3% of the O 2 consumed), and that the concentration of GSH in the cells is >1 mmol/kg, it follows that to decrease GSH levels significantly in a scale of minutes, a large burst of ROS production would be required. This decrease would even be minimized by the NADPHdependent cycling of GSSG back to GSH produced by glutathione reductase. In other words, the high redox buffer power of GSH tends to prevent large changes of the redox environment of the cells to preserve the functionality of the cells. This in turn implies that when stimulation of a cell system alters the ‘‘redox status’’ or redox environment of the cells, the stimulus causes a dramatic change in the rate of production of ROS in the cells. If in a situation of increased ROS production, glutathione reductase is unable of cycling back to GSH the extra amount of GSSG produced, we would assist a time-dependent decrease in GSH and a tendency of 54 L. Frank, Fed. Proc.44, 2328 (1985). 55 D. Mustacich and G. Powis, Biochem. J.346, 1 (2000). 56 S. Hoshida, N. Yamashita, K. Otsu, and M. Hori, J. Am. Coll. Cardiol.40, 826 (2002). 57 M. A. Hass and D. Massoro, Am. J. Physiol.253, C66 (1987). [3] carotid body chemoreceptor cells 55 GSSG to increase, and therefore, to a decrease of the redox environment of cell according to the equation (at 37): Redox environmentðmVÞEGSH ¼240 30:75 log½GSH2=½GSSG (3) However, as soon as GSSG starts to increase, cells export GSSG 1 ; this release or leak of GSSG aims to avoid an intracellular accumulation of high concentrations of GSSG and therefore intends to maintain an adequate GSH/GSSG ratio and a redox environment as constant as possible inside the cells. Of course the cost of this is a loss of redox buffer capacity with a net tendency to decrease in GSH. Additional information to consider for the correct evaluation of the correlation between GSH and ROS signaling is that the turnover of GSH seems to be rather slow, as it is required to inhibit glutathione synthesis for long periods of time to deplete GSH levels significantly in the cells. For example, the inhibition of glutamylcysteine synthetase (the first enzyme in glutathione biosynthesis) with buthionine sulfoximine at concentrations of 100 and 200 Mreduced in 24 h the GSH content by the same percentage (75%) in lens cells in culture, 58 and at 200 Mdepleted at a constant rate of about 10%/h during 5 h. 59 This in turn reflects the fact that the rate (mole/min) of ROS production by cells is small. In sum, these considerations would suggest that under physiological stimulation of any cell system it should not be surprising to see only minor changes or no measurable changes in the GSH/GSSG system and in the redox environment of the cells. Additionally, imposed experimental alterations of the GSH/GSSG system aimed at mimicking a strong stimulation should not produce very dramatic changes, otherwise we would bring the redox environment of the cells to levels only observed in pathologic situations. The presumable existence of compartmentalized microdomains in the production of ROS induced by specific stimulus in the near vicinity of sensing and effector molecules 41 would impose a completely different set of considerations. Under this scope, it would be conceivable that the stimulus produces a dramatic increase in ROS production in a specific cell compartment, and therefore that there is a local dramatic alteration of the redox environment of the cells without a large change in the overall intracellular redox environment. In these physiological circumstances, we should expect only minor changes (or no changes) in the bulk levels of the GSH/GSSG pair or in its redox potential as measured, for example, 58 F. Shang, M. Lu, B. Dudek, J. Reddan, and A. Taylor, Free Radic. Biol. Med.34, 521 (2003). 59 S. Sinbandhit-Tricot, J. Cillard, M. Chevanne, I. Morel, P. Cillard, and O. Sergent, Free Radic. Biol. Med.34, 1283 (2003). 56 lung and the airways [3] in a tissue homogenate. Additionally, experimental maneuvers aimed to alter the GSH system, and through it the redox environment of the cells to mimic physiological stimulation, would tend to be rather unspecific because they would alter the entire redox environment of the cells without the precise spatial (and temporal) coordinates of the physiological stimulus. Experimental Methods Measurement of Glutathione In the present study we measured GSH and GSSG in quarters of rat diaphragm in all experimental conditions here. A previous study 51 measured GSH/GSSG in calf CB, but the experiments proved to be enormously time-consuming and inconvenient because around 40–50 mg of tissue equivalent to four to five calf CBs was needed and, in addition, the crisis of mad cows restricted access to the slaughterhouse. Selection of the rat diaphragm as the test tissue to evaluate the effects of hypoxia and other experimental treatments is due to the fact that the diaphragm is a sheet of tissue comparable in thickness to the rabbit CB (<1 mm), therefore presenting no problems for gas and drug diffusion to the interior of the tissue. Therefore, most of our experiments compare data on GSH/GSSG content in the diaphragm with data on the activity of chemoreceptor cells obtained in the rabbit CB treated identically to diaphragms. In some occasions, we also used liver and brain tissue to test the effects of specific drugs and compared findings in the diaphragm. Removal and Incubation of Tissues. Wistar rats with a 250to 300-g body weight are anesthetized with sodium pentobarbital (60 mg/kg; ip). After an incision in the abdomen, the entire diaphragm is removed carefully, clamping as needed to avoid bleeding. Animals are killed by an intracardiac overdose of sodium pentobarbital. All measures are taken to prevent distress in the animals. The Committee for Animal Care and Use at the University of Valladolid approved the protocols. The diaphragm is freed of blood by washing in ice-cold 100% O 2 -saturated Tyrode (in mM: NaCl, 140; KCl, 5; CaCl 2 , 2; MgCl 2 , 1.1; glucose, 5.5; HEPES, 10; pH 7.40 with 1 NNaOH). Under a dissecting microscope, small pieces of fat, small vessels, and phrenic tendon are eliminated, and the diaphragm is cut in to four quadrants of comparable size. The four pieces of diaphragm are transferred individually to glass scintillation vials placed in a metabolic shaker at 37containing 10 ml of bicarbonate-buffered Tyrode (composition as before except that 24 mMNaCl is eliminated and 24 mMNaHCO 3 is added) that are [3] carotid body chemoreceptor cells 57 bubbled constantly with water-saturated 5% CO 2 -containing gas mixtures. All pieces are incubated for 30 min while bubbling with 21% O 2 /5% CO 2 to allow the tissues to recover from the trauma of the surgical procedures 60 ; thereafter the incubating solutions are renewed every 10 min for up to 40 min. In control tissues, the renewing solution is 21% O 2 /5% CO 2 -equilibrated bicarbonate Tyrode, in hypoxic tissues the last 10-min incubating period the solution is equilibrated, and the vials are bubbled continuously with 7% O 2 /5% CO 2 . In drug-treated tissues the solutions are equilibrated and the vials are bubbled continuously with 21% O 2 /5% CO 2 and contain (from 0 to 40 min) 0.050 mMp-chloromercurybenzosulfonate (PCMBS) sodium salt, 0.5 mMcarmustine [1,3-bis(2-chloroethyl)- nitrosourea; BCNU], 2 mMN-acetylcysteine (NAC), or 0.2 mMdiamide (DIA). Incubation time does not modify GSH or GSSG levels for up to 80 min (Fig. 3). At the end of the incubation the tissues are transferred to new vials kept at 0–4containing 10 ml of Tyrode equilibrated with 100% O 2 for 5 min, dry blotted by touch on filter paper, weighed, and placed in Eppendorf tubes containing a solution of 5-sulfosalicylic acid (SSA) at 5% and 0.25 mMEDTA (at 4it is stable for months) whose volume is adjusted to five times the weight of the tissue. The tissues are stored at 80until the day of the assay or are immediately glass-to-glass homogenized at 0–4and centrifuged in a microfuge (4, 10 min) and the supernatant is used to measure GSH/GSSG. The assay can be performed 60 A. Obeso, L. Almaraz, and C. Gonzalez, Brain Res.371, 25 (1986). 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 GSt GSSG GSH m mol/g tissue 10 min 20 min 40 min 80 min −190 −180 −170 −160 −150 −140 Potential mV Fig. 3. Effects of duration of incubation on levels of GSt (total glutathione), GSSG, GSH, and GSH/GSSG redox potential. 58 lung and the airways [3] immediately or the supernatant can be stored at 80C until assay. No differences have been observed whether the tissues or the supernatants are stored for up to 1 month at 80(Table I). Assay for GSH and GSSG. The measurement of GSH and GSSG is made by the method of Griffith. 61 GSH reacts spontaneously with 5,50dithiobis-(2-nitrobenzoic acid) (DTNB) to generate GSSG and 5-thio2-nitrobenzoic acid (TNB; peak absorbance at 412 nm). The GSSG formed is reduced enzymatically to GSH by glutathione reductase coupled to NADPH oxidation. In this cycling assay, concentrations of DTNB, NADPH, and glutathione reductase are chosen so that the rate of color formation followed with a spectrophotometer is linear with time for 2–3 min and the slope of the line relating the increase of absorbance and time ( absorbance/time, min; A/t, min) is directly proportional to the concentration of total glutathione (GSH þGSSG; GSt) in the sample. The plot of the slopes of these lines as a function of the concentration of GSt in the samples is also linear for an ample range of concentrations and defines the standard curves used to determine the concentrations of GSt in the problem samples by interpolation. GSSG is measured identically, but first the GSH present in the samples is masked by derivatization with 2-vinylpyridine. The size of the sample is higher due to the usually much lower concentration of GSSG than GSH. In all instances (construction of standard curves and tissue homogenates), the assays are made by triplicate. The assay is highly specific, due to the enzymatic step, and is highly sensitive due to cycling; the sensitivity of this colorimetric method is in the range of 10 10 mol/assay. TABLE I Glutathione Levels in Supernatants of Homogenates of Rat Diaphragm Assayed Immediately or after Storage for 1Month at 80 a Supernatant conditions Total glutathione (M/g tissue) GSSG (M/g tissue) GSH (M/g tissue) Gltutathione redox potential (mV) Assayed immediately 0.533 0.014 (n¼41) 0.044 0.002 (n¼41) 0.489 0.014 (n¼41) 179.9 1.1 (n¼41) Assayed after frozen (80) for 1 month 0.549 0.018 (n¼23) 0.048 0.002 (n¼23) 0.500 0.015 (n¼23) 179.1 0.8 (n¼23) a The 41 samples of tissue used for the assay immediately after obtaining came from 20 different animals, whereas those used to be frozen were obtained from 11 rats. GSSG levels are expressed as GSH, i.e., the actual concentration of GSSG is half because each molecule of GSSG yields two molecules of GSH (see calculations in the text). 61 O. W. Griffith, Anal. Biochem.106, 207 (1980). [3] carotid body chemoreceptor cells 59 Solutions. The description that follows corresponds to a typical working day that includes 12–16 tissue samples to be determined by triplicate plus the standard curves for GSt and GSSG, also in triplicate. Some stock solutions can be prepared and, if stored conveniently, can be used for months. Stock solutions include .A stock buffer solution of 125 mMsodium phosphate buffer containing 6.30 mMdisodium EDTA (pH ¼7.50) is maintained at 4(1 liter). .A stock solution of 100 mMof GSH in distilled water is aliquoted (0.1 ml) in Eppendorf tubes and maintained at 20. .A stock solution of 6 mMDTNB in stock buffer and maintained at 20is aliquoted in 5-ml plastic tubes. .2-Vinyl pyridine (Aldrich) is stored at 20as provided by the supplier and can be used (usually by several months) until it starts to acquire a yellow tinge (5 ml). .Triethanolamine (Sigma) undiluted is maintained at room temperature (100 ml). The rest of the solutions are prepared daily and include .4.31% SSA solution by diluting with water the 5% SSA þ0.25 mM EDTA solution used to homogenate the tissues (2 ml of SSA at 5% þ0.32 ml water). .0.3 mMNADPH (Sigma) in the stock buffer (100 ml). .GSH standard solution: the 100 mMGSH stock solution (100 l) is diluted to 0.5 mMwith the 5% SSA þ0.25 mMEDTA solution to construct the standard curves for GSt. .GSSG standard solution: the 100 mMGSH stock solution (100 l) is diluted to 0.1 mMGSH with the 5% SSA þ0.25 mMEDTA solution to construct the standard curves for GSSG; alternatively, it can be prepared from a solution 0.05 mMGSSG in identical conditions. .1 unit of GSSG reductase (GR; Sigma)/5 l in stock buffer (1 ml). Procedure. The assay of GSt in the test samples is performed as follows. For the first sample of the run, the cuvette of the spectrophotometer is placed in a bath at 37before addition of the assay mixture (700 l of the NADPH solution þ100 l of DTNB solution þ195 lofH 2 Oþ 5l of the supernatant of the tissue homogenate). The cuvette is maintained for at least 4 min at 37and then 5 l of GSSG reductase solution is added, mixed rapidly by gentle vortexing, and introduced in the thermostatized (37) reading chamber of the spectrophotometer (Hitachi U-1100/ U1100; Hitachi Scientific Instruments, Pacisa, Madrid). The recording of absorbances starts after 30 s (to reach the 37) and proceeds for 2 min with recordings every 10 s, with the slope (absorbance/min) calculated automatically (U-1100 Data Manager Software) and stored in the computer. 60 lung and the airways [3] Additional samples are prewarmed in Eppendorf tubes, and the enzyme is added on transference of the assay mixture to the cuvette immediately prior to starting the recording of absorbances. During this warming period, the assay mixture acquires a yellowish tinge due to the reduction of a small fraction DTNB produced by the GSH present in the sample that is transformed into GSSG, but the real development of color occurs when the reductase is added and GSSG is cycled back to GSH with many cycles during the 2-min reading period. The assay of GSSG in the test samples involves an additional preparative step: to derivatize (destroy) GSH. To a 100-l aliquot of supernatant of tissue homogenate is added, under vigorous vortexing, 2 l of 2-vinyl pyridine, and the pH of the mixture is adjusted to between 6.8 and 7.2 with triethanolamine (4 l, but due to its high viscosity, the precise volume can vary with experimenter) that should be added while vortexing, preferably to the side of the tube wall to avoid sharp peaks of pH locally in the solution that will destroy GSSG. If the pH (measured with narrow-range pH paper) is higher than 7.2, derivatization should be repeated. The derivatization reaction is allowed to proceed at room temperature for 1 h and then the assay is performed with an identical assay mixture used for GSt, except for the water aliquot (175 l) and the size of the sample and enzyme solution (20 l each). The standard curves for GSt and GSSG are constructed with an assay procedure identical to the one just described for the test samples (except for the derivatization step, which is not required) with the assay mixtures shown in Table II. The slopes (absorbance/min) obtained for each amount of glutathione are plotted as a function of glutathione amount itself; the best-fit regression line is obtained by the least-square method, and the slopes (absorbance/min) of the test samples are interpolated in the correspondent standard curves. When a drug has been used in the treatment of tissues and it is suspected that it may interfere with the assay procedure (e.g., inhibiting glutathione reductase), such a possibility needs to be excluded or corrected. To exclude that possibility, a group of assays should be performed with assay mixtures containing fixed and known amounts (e.g., 1 nmol) of GSH in a fixed 1-l standard sample as the internal standard and increasing volumes of tissue homogenates (0–4l) and decreasing volumes of 4.31% SSA (4–0l). An additional assay with just 5 l of tissue homogenate should also be included. A comparable set of assays should be made with the 1-l test sample (0.2 nmol) and up to 19 l of tissue homogenate derivatized previously with 2-vinyl pyridine. If there is no interference, measurements will give additive GSH or GSSG values; however, the combination of homogenate þstandard sample will yield values less than additive, and the effect will be greater with the higher [3] carotid body chemoreceptor cells 61 volumes of homogenate. To correct the possible interference, standard curves are constructed containing the drug at concentrations equivalent to those present in the usual 5 l of the tissue supernatant used to measure GSt and in the 20 l used to measure GSSG. These equivalent concentrations are determined empirically as those concentrations producing an inhibition comparable to that observed with use of the internal standard. The assay mixtures for control tissues (that have not been treated with the drug under study) should also be supplemented with the drug at the concentration used in the standard curves. Note that the standard curve to assay GSSG can be made with standard solutions of GSH or GSSG. The reason for that is when the reading of the absorbance starts, all glutathione in the assay mixtures is in an oxidized form as GSSG due to the spontaneous reaction with DTNB. It is in the cycling process initiated by the addition of glutathione reductase where all GSSG is first transformed into GSH, which in turn reduces new molecules of DTNB generating color and becoming newly oxidized into GSSG, and so on. Therefore, it does not matter whether the construction of the standard curves is initiated with one or another form of glutathione. TABLE II Assay Mixtures used to Construct Standard Curves for GSt and GSSG a GSH (nmol) NADPH solution (l) DTNB solution (l) H 2 O (l) Standard solution (sample) (l) 4.31% SSA solution (l) GR solution (l) 0–0 700 100 195–175 0 5–20 5–20 0.5–0.1 700 100 195–175 1 4–19 5–20 1.0–0.2 700 100 195–175 2 3–18 5–20 1.5–0.3 700 100 195–175 3 2–17 5–20 2.0–0.4 700 100 195–175 4 1–16 5–20 ? 700 100 195–175 5–20 0 5–20 a Note that GSH solutions are used for both the standard curve to GSt and the standard curve to GSSG in the samples (see text for explanation). A single figure means that the same volume of that component is used for both standard curves; when there are two figures, the first one is for the GSt standard curve and the second for the GSSG standard curve. In the last row, figures correspond to the components used in the assay of the test samples. SSA is added to standard curves to maintain the same concentration of SSA that is in the test samples due to the fact that SSA tends to inhibit glutathione reductase. Note that the SSA solution used to add to the assay mixture in the standard curves is 4.31%: tissue water represents 80% of tissue weight and thereby when we homogenize in a volume of 5% SSA equivalent to five times the tissue weight, we end up diluting the SSA solution with water contained in the tissue. The final volume in the supernatant would be 5.8 volumes with a reduced concentration of SSA to 4.31%: 5 vol. 5% ¼5.8 vol. x%; x% ¼4.31%. 62 lung and the airways [3] However, it should be kept in mind that each molecule of GSSG would yield two molecules of GSH, and this would affect the calculations of the concentration of glutathione in the tissue. Calculations of Tissue Concentration of GSt,GSSG,and GSH. It should be realized that GSt in the tissue equals GSH þGSSG. Because each molecule of GSSG yields two molecules of GSH and our standard curve is made against GSH concentrations, our GSt in tissues equals GSH þ0.5 GSSG concentration in the tissue. Referring to the calculations for GSSG concentrations, two different standard curves can be used. If a standard curve made with GSH is used, we are expressing the GSSG as GSH molar units, and therefore the actual GSSG molar concentration is half the one obtained by interpolation in the standard curve. If the standard curve is made with GSSG, the actual molar concentrations in the tissues are those given directly by interpolation in the standard curve. Usually, the GSSG is expressed as GSH because it facilitates the calculation of the actual GSH concentration in the test samples obtained as a difference: GSH ¼GSt GSSG. The concentration of either GSt or GSSG in the tissue is obtained according to the following equation: GSt or GSSGðmol=g tissueÞ ¼A½tissue weightðgÞ5:8=assay volume of the test sample ðmlÞ tissue weightðgÞ (4) where Ais the amount obtained by interpolation in the standard curve, but expressed in micromoles. Simplifying the equation GSt or GSSGðmol=g tissueÞ¼ A5:8 assay volume of the test sample ðmlÞ (5) The GSH/GSSG redox potential is calculated as in Eq. (3). Assessment of CB Chemoreceptor Cell Function To assess the functionality of chemoreceptor cells of the CB, we have used an in vitro preparation of intact CB of rabbit. The rabbit CB weighs 400 g, and our group has provided ample experimental evidence of its normal functioning in vitro with the ability of chemoreceptor cells to detect a great variety of stimuli (including the natural ones hypoxia and hypercapnia/low pH) and to respond with a neurosecretory response best monitored as the release of catecholamines (CA). 62 The procedure used to monitor 62 C. Gonzalez, L. Almaraz, A. Obeso, and R. Rigual, Trends Neurosci.15, 146 (1992). [3] carotid body chemoreceptor cells 63 the neurosecretory response varies from one laboratory to the next. In our laboratory, two alternative methods are used: a radioisotopic method, which monitors the release of [ 3 H]CA synthesized from the natural precursor [ 3 H]tyrosine, which is described in some detail later, and a voltametric method that monitors continuously the release of CA from chemoreceptor cells as concentrations of endogenous CA in the extracellular space of the CB tissue. Any variation in the CA concentration associated with CB stimulation implies a response of chemoreceptor cells to such stimulus (see Rigual et al. 63 for a description of the method). Surgical Procedures. Rabbits are anesthetized with sodium pentobarbital (40 mg/kg, iv through the lateral vein of the ear) and, after a longitudinal incision in the ventral face of the neck, the carotid arteries are dissected past the carotid bifurcation. After convenient clamping, a block of tissue containing the carotid bifurcation is removed and placed in a lucite chamber filled with ice-cold/100% O 2 -equilibrated Tyrode (see earlier discussion) to dissect the CB free of surrounding connective tissue. Animals are killed by an intracardiac overdose of sodium pentobarbital. The Committee for Animal Care and Use at the University of Valladolid approved the protocols. Animals did not suffer any distress in all the experimental procedure. Labeling of CA Stores, Synthesis of [ 3 H-]CA from [ 3 H-]tyrosine, and Release of [ 3 H-]CA. The CA stores of CBs are labeled by incubating the organs in small glass vials (eight CBs/vial) and are placed in a shaker bath at 37containing 0.5 ml of a 100% O 2 -preequilibrated Tyrode solution. The incubating solution contains [3,53 H]tyrosine (30 M; 20 Ci/mmol), 100 M6-methyl-tetrahydropterine, and 1 mMascorbic acid, cofactors for tyrosine hydroxylase and dopamine--hydroxylase, respectively. 64 The incubation to label [ 3 H]CA stores lasts 2 h. After labeling the [ 3 H]CA stores, the CB are transferred individually to glass scintillation vials, containing 4 ml of precursor-free bicarbonate-buffered Tyrode solution (see earlier discussion) and are kept in a shaker bath at 37for the rest of the experiment. Solutions are bubbled continuously with 20% O 2 /5% CO 2 / 75% N 2 saturated with water vapor. When hypoxia is applied as the stimulus, the solutions are bubbled with a hypoxic gas mixture (containing 0–21% O 2 ), which in these experiments was 7% O 2 /5% CO 2 /balance N 2 . During the first hour, incubating solutions are renewed every 20 min and discarded. During the rest of the experiment, incubating solutions are collected every 10 min and are saved for ulterior analysis in their [ 3 H]CA content. Stimulus to CBs consisted in their incubation during a 10-min period 63 R. Rigual, L. Almaraz, C. Gonzalez, and D. F. Donnelly, Pflug. Arch.439, 463 (2000). 64 S. Fidone and C. Gonzalez, J. Physiol.333, 69 (1982). 64 lung and the airways [3] difficult to accept that the redox environment of the cells is critically important for oxygen sensing (see also Searle et al. 44 ) because alteration of the general redox environment of cells, which would include alteration of the putative cell compartment, does not alter the hypoxic response of CB chemoreceptor cells. Acknowledgments This work was supported by Spanish DGICYT grant BFI 2001–1713, by J. C. Y. L. Grant VA092/03, and by a FISS Grant to the Red Respira-SEPAR. [4] Determination of Signaling Pathways Responsible for Hypoxic Pulmonary Vasoconstriction: Use of the Small Vessel Myograph By Jeremy P. T. Ward and Vladimir A. Snetkov Introduction Hypoxic pulmonary vasoconstriction (HPV) was first described as an important regulatory mechanism of the ventilation–perfusion ratio by Von Euler and Liljestrand in 1946. 1 Many studies have shown that HPV can be elicited in isolated pulmonary arteries, demonstrating that both the sensor for hypoxia and effector mechanisms reside within the artery. However, the signaling pathways underlying HPV are still poorly understood. It is becoming increasingly evident that HPV is multifactorial in origin, with mechanisms in both the vascular smooth muscle (VSM) and the endothelium 2,3 ; there is, however, controversy regarding the precise identity of these mechanisms. Hypoxia sensing has been attributed to changes in redox state (e.g., NAD/NADH redox couples) and generation of reactive oxygen species (ROS) from either an NAD(P)H oxidase or the mitochondria. 4–6 The origin of the hypoxia-induced rise in VSM intracellular [Ca 2þ ] is equally controversial, with evidence for inhibition of K þ 1 U. S. Von Euler and G. Liljestrand, Acta Physiol. Scand.12, 301 (1946). 2 J. P. T. Ward and P. I. Aaronson, Respir. Physiol.115, 261 (1999). 3 P. I. Aaronson, T. P. Robertson, and J. P. Ward, Respir. Physiol. Neurobiol.132, 107 (2002). 4 R. M. Leach, H. M. Hill, V. A. Snetkov, T. P. Robertson, and J. P. Ward, J. Physiol.536, 211 (2001). 5 G. B. Waypa, N. Chandel, and P. T. Schumacker, Circ. Res.88, 1259 (2001). 6 E. K. Weir, Z. Hong, V. A. Porter, and H. L. Reeve, Respir. Physiol. Neurobiol.132, 121 (2002). [4] hypoxic pulmonary vasoconstriction 71 Copyright 2004, Elsevier Inc. All rights reserved. METHODS IN ENZYMOLOGY, VOL. 381 0076-6879/04 $35.00