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Redox signaling in oxygen sensing by vessels
Respiratory Physiology & Neurobiology 132 (2002) 121– 130 www.elsevier.com/locate/resphysiol
Redox signaling in oxygen sensing by vessels E. Kenneth Weir a,b,*, Zhigang Hong a, Valerie A. Porter b,c, Helen L. Reeve b,c a Department of Medicine, VA Medical Center, Minneapolis, MN 55417, USA Department of Physiology, Uni6ersity of Minnesota, Minneapolis, MN 55455, USA c Guidant Corporation, A221 Clinicals, 4100 Hamline A6enue, St. Paul, MN 55112, USA b
Received 11 February 2002; received in revised form 16 April 2002; accepted 16 April 2002
Abstract In response to the increase in oxygen tension at birth, the resistance pulmonary arteries dilate, while the ductus arteriosus constricts. Although modulated by the endothelium, these opposite responses are intrinsic to the vascular smooth muscle. While still controversial, it seems likely that during normoxia the production of reactive oxygen species (ROS) increases and the smooth muscle cell cytoplasm is more oxidized in both pulmonary arteries and ductus, compared to hypoxia. However, the effect of changes in the endogenous redox status or the addition of a redox agent, oxidizing or reducing, is exactly opposite in the two vessels. A reducing agent, dithiothreitol, like hypoxia, in the pulmonary artery will inhibit potassium current, cause depolarization, increase cytosolic calcium and lead to contraction. Responses to dithiothreitol in the ductus are opposite and removal of endogenous H2O2 by intracellular catalase in the ductus increases potassium current. Oxygen sensing in both vessels is probably mediated by redox effects on both calcium influx and calcium release from the sarcoplasmic reticulum (SR). © 2002 Elsevier Science B.V. All rights reserved. Keywords: Endothelium, pulmonary; Oxygen, sensing in pulmonary vessels; Pulmonary vasculature, oxygen sensing; Redox state, pulmonary vessels; Smooth muscle, pulmonary vessels
1. Introduction The most dramatic difference in vascular responses to changes in oxygen tension is demonstrated by the pulmonary resistance arteries and the ductus arteriosus (DA). At birth, these tissues are closely related anatomically and yet behave in a diametrically opposite manner to the increase in * Corresponding author. Tel.: +1-612-725-2000x3653; fax: +1-612-727-5668 E-mail address: [email protected] (E.K. Weir).
oxygen; the resistance pulmonary arteries dilate, while the DA constricts. While all workers in the field would agree on this observation, the mechanism by which the level of oxygen is sensed and transduced into changes in vascular tone remains controversial. Most would accept that the level of cytosolic calcium in the smooth muscle is the main determinant of vascular tone, although the sensitivity of actin-myosin to a given level of calcium might change with hypoxia (Robertson et al., 1995) and endothelial factors undoubtedly modulate smooth muscle reactivity.
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2. Sources of calcium
3. Hypoxia and reactive oxygen species
If cytosolic calcium is important, is one pool within the cytoplasm particularly relevant and what is the source of calcium entering this pool? Attention has been focused on two possible sources, the influx of extracellular calcium and the release of intracellular calcium, especially from the sarcoplasmic reticulum (SR). It seems quite likely that both may be involved. In the case of the influx of extracellular calcium, the potential paths include voltage-dependent calcium channels (VDCC) and store-operated calcium channels (SOCC). Considering the possible role of the VDCC, SOCC and SR in the pulmonary arteries and DA, what could signal opposite responses to a change in oxygen tension in these two vessels? In the case of the VDCC in the sarcolemma of the pulmonary artery smooth muscle cell (PASMC), one sequence of events precipitated by hypoxia has been extensively documented (Weir and Archer 1995). Inhibition of one or more potassium channels (Post et al., 1992; Yuan et al., 1993), may lead to membrane depolarization (Madden et al., 1985) and calcium entry through the VDCC (McMurtry 1985; McMurtry et al., 1976; Tolins et al., 1986). The same sequence seems to occur in smooth muscle cells of the DA but, in this case, the sequence is initiated by an increase in oxygen tension that inhibits potassium channels, not by hypoxia (Nakanishi et al., 1993; Roulet and Coburn, 1981; Tristani-Firouzi et al., 1996). While much of the calcium required for hypoxic pulmonary vasoconstriction (HPV), during the initial 10 minutes, appears to come from outside the PASMC, it is clear that some is released from within the cell (Gelband and Gelband, 1997; Morio and McMurtry, 2002; Salvaterra and Goldman, 1993). It is also possible that the increase in cytosolic calcium itself might inhibit potassium channels in the PASMC, as a secondary event (Post et al., 1995), or that the inhibition of potassium channels might play a relatively minor role (Robertson et al., 2000; Sham et al., 2000). The underlying question remains, what signals the release of intracellular calcium, and the change in potassium channel gating?
Changes in the redox status of the pancreatic beta cell influence the level of cytosolic calcium and insulin secretion (Archer et al., 1986). By analogy with the beta cell, it was suggested that oxygen-induced changes in the redox status of the PASMC membrane might alter potassium channel gating, membrane potential and calcium influx through the VDCC. The hypothesis was that hypoxia would lead to a decrease in reactive oxygen species (ROS), that would in turn cause the cytoplasm and cell membrane to be more reduced, thus closing potassium channels and resulting in membrane depolarization and calcium entry. It seemed likely that ROS are involved in intracellular signaling throughout the body and are not merely part of the neutrophil armamentarium. In the last 15 years parts of the puzzle have been completed but new questions have arisen. Surprisingly, it is not yet agreed whether ROS go up or down during acute exposure of the lung to hypoxia at physiological levels. Chemiluminescence techniques using luminol or lucigenin enhancement indicate a decrease in ROS production during hypoxia in the rat lung (Archer et al., 1989), mouse lung (Archer et al., 1999), rabbit lung (Paky et al., 1993) and in the rabbit DA (Reeve et al., 2001b). Similarly, an increase in oxygen tension increases ROS in rat lung and lung mitochondria (Freeman and Crapo, 1981; Turrens et al., 1982). Interestingly, hypoxia has also been shown to decrease superoxide anion production by neutrophils, as measured by cytochrome c reduction (Gabig et al., 1979). On the other hand, in cultured PASMCs, superoxide anion production is found to increase during hypoxia, as measured using lucigenin-enhanced chemiluminescence (Marshall et al., 1996), and hydrogen peroxide increases, as measured by dichlorofluorescein fluorescence (Waypa et al., 2001). Other work based on the use of a variety of enzyme inhibitors, also favors a role for an increase of ROS in the genesis of HPV (Weissman et al., 2000). These differences, that are probably secondary to the techniques used, need to be resolved before the role of ROS in the mechanism of HPV can be determined. Another question that
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is seldom addressed is whether superoxide anion, under physiologic circumstances, may on occasion act as a reducing agent, rather than as an oxidizing agent. It is usually assumed that, like hydrogen peroxide (H2O2), it will always cause oxidation. However, superoxide anion production in vitro is routinely measured by its ability to reduce oxidized markers, such as potassium ferricyanide and cytochrome c. The net effect of ROS production by mitochondria or enzymes, such as NAD(P)H oxidases, on vascular tone may depend on complex interactions: the balance between superoxide anion and H2O2 controlled by superoxide dismutase, the level of H2O2 controlled by catalase, glutathione peroxidase and the availability of NADPH, and the interaction of superoxide anion and NO.
4. NADPH-oxidase The first suggestion that NADPH-oxidase might be the oxygen sensor in the pulmonary vasculature was made by Thomas et al. (Thomas et al., 1991). The hypothesis was based on the observation that the NADPH-oxidase inhibitor, diphenyleneiodonium (DPI), inhibits HPV. Unfortunately, DPI also blocks calcium channels thus making the interpretation less specific (Weir et al., 1994). It was hoped that the development of an NADPH oxidase deficient mouse, lacking the gp 91 phox subunit, might help to solve the riddle. However, HPV (Archer et al., 1999) and oxygen sensing in the carotid body (He et al., 2002) are unchanged in these mice, suggesting that NADPH oxidase, at least that containing the gp 91 phox subunit, is not involved (Fig. 1). However, recent studies on the same mouse model does indicate that NADPH oxidase is an important oxygen sensor in the pulmonary neuroepithelial body (Fu et al., 2000). The possibility that a low-output form of NADPH oxidase, lacking the gp 91 phox subunit, could be the oxygen sensor involved in HPV has not been excluded. It has also been suggested that NADH oxidase might be the oxygen sensor (Burke-Wolin and Wolin, 1989; Mohazzab-H et al., 1995). These studies found
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that H2O2 production increased in normoxia, relative to hypoxia, and caused PASMC relaxation through an increase in cGMP. The role of NAD(P)H oxidases in oxygen sensing has been extensively reviewed (Jones et al., 2000; Wolin et al., 1999).
5. Hypoxia and redox couples If the concept is correct that a change in redox status is important in HPV, should the redox status be defined by the production of ROS or by the ratio of reduced to oxidized redox couples such as GSH/GSSG and NAD(P)H/ NAD(P)? If the latter, do these couples shift during hypoxia because electrons are not passed so efficiently down the mitochondrial electron transport chain or because of a change in the production of ROS, or both? Changes in oxygen tension are known to alter the ratio of reduced to oxidized glutathione and pyridine nucleotides (Archer et al., 1986; Cross et al., 1990; Patterson et al., 1985; White et al., 1988). Presumably the redox couples could be important if the mitochondria are involved in sensing the oxygen tension (He et al., 2002; Waypa et al., 2001) but perhaps less so if the oxygen sensor is membrane-bound, such as NADPH-oxidase (Cross et al., 1990; Marshall et al., 1996; Wang et al., 1996; Weissman et al., 2000) and there is the potential for direct interaction of ROS and ion channels. Changes in cytosolic redox secondary to alteration in mitochondrial function could affect both calcium influx, related to ion channel gating, and calcium release from the SR. If NAD(P)H-oxidase were the sole oxygen sensor, at first glance it would seem more likely that it would modulate ion channel gating. However, considering the close relationship of localized calcium release from the SR (‘sparks’) to opening of the calcium-sensitive potassium channels in the cell membrane (STOCs) (Nelson et al., 1995), one could also postulate that ROS from the membrane could alter the handling of calcium in the SR that is nearby by changing the redox control of SR calcium release.
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6. Redox control of ion channels What is the evidence that redox changes can alter potassium or calcium channel gating, whether the redox change is in ROS or redox couples? In neuroepithelial bodies hypoxia in-
hibits whole cell potassium current (IK) and IK can be increased by the external application of H2O2 (Fu et al., 2000). Incidentally, this experiment alone would suggest that hypoxia does not increase ROS, at least in the neuroepithelial body. In A-type Kv channels (e.g. Kv 1.4 and 3.4)
Fig. 1. Reduced glutathione (GSH) in the patch pipette reduces whole cell potassium current and single channel open probability in rat pulmonary artery smooth muscel cells.
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Fig. 2. The isolated perfused lungs of mice lacking the gp 91 subunit of NADPH oxidase have normal HPV but there is virtually no oxygen radical production at the lung surface, measurable by luminol-enhanced chemiluminescence.
exposure to H2O2 causes oxidation of cysteine groups at the amino terminus of the channel protein and inhibits rapid inactivation (Pongs, 1998). Beta subunits, such as Kvb1 and b3, also possess an amino-terminal inactivating domain, containing a cysteine residue, removal of which eliminates rapid inactivation. Consequently oxidation would tend to increase the potassium current and reduction to decrease it. An example of the potential importance of the beta subunit in oxygen sensing was recently demonstrated. Kv 4.2 channels expressed in HEK 293 cells were not inhibited by hypoxia but, when coexpressed with Kv b 1.2, the channel was reversibly inhibited (Perez-Garcia et al., 1999). On the other hand, oxidation of a methionine group in the P-segment of Shaker Kv channels expressed in Xenopus oocytes accelerates inactivation (Chen et al., 2000). In this situation, oxidation would tend to decrease the potassium current
and reduction to increase it; the reverse of what was observed in the case of cysteine residues. The different behavior of these channels in response to redox changes might help to explain the opposite responses of the pulmonary arteries and DA to oxygen.
7. Redox control of pulmonary vascular ion channels. A variety of redox-active agents have been studied in PASMCs. Reduced glutathione (GSH) introduced into PASMCs in the patch pipette reduces IK, while oxidized glutathione increases IK (Weir and Archer, 1995). An example of the effect of GSH on whole cell K current and on single K channel opening is shown in Fig. 2. The glutathione and sulfhydryl oxidant, diamide, increases both IK and single channel KCa current
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(Post et al., 1993; Reeve et al., 1995), while also causing pulmonary vasodilatation (Weir et al., 1983). With a ‘calcium-free’ solution in the patch pipette to eliminate KCa current, the residual IKv current in PASMCs can be increased by the oxidizing agent dithio-bis (5-nitropyridine) and decreased by the reducing agent dithiothreitol (DTT) (Park et al., 1995a). Using ‘inside-out’ patches of PASMC sarcolemma, it can be demonstrated that several reducing agents (DTT, GSH, NADH) decrease KCa current, while several oxidising agents (DTNB, GSSG, NAD) increase KCa current (Park et al., 1995b). Similar observations have been made in another oxygen-sensitive tissue, the Type 1 cell of the carotid body (LopezBarneo et al., 1998). It appears that a number of Kv channels that are present in PASMCs are inhibited by hypoxia. These include Kv 1.2 and the Kv 1.2/1.5 heteromultimer, as well as Kv 2.1 and the Kv 2.1/Kv 9.3 heteromultimer (Archer et al., 1998; Hulme et al., 1999; Patel et al., 1997). Kv 3.1b may also be involved (Coppock and Tamkun, 2001; Osipenko et al., 2000). In support of a role for the Kv 1.5 channel is the observation that mice lacking this channel have diminished HPV (Archer et al., 2001). The beta subunits, mentioned earlier, Kv b 1.1, Kv b 2 and Kv b 3 are also present in PASMCs (Coppock and Tamkun, 2001; Yuan et al., 1998). These subunits show homology with NAD(P)H oxidoreductases and the redox state of the subunit may modulate the activity of the channel and, possibly, the activity of the channel may regulate the function of the oxidoreductase (Gulbis et al., 2000; McCormack and McCormack 1994). If the latter is true, changes in channel activity might, through the generation of ROS, alter calcium release from the SR as postulated earlier. The evidence relating both a and b channel subunits to oxygen sensing has recently been reviewed (Archer et al., 2000; Patel and Honore 2001; Yuan 2001).
8. Redox and potassium channel expression in chronic hypoxia It has long been known that acute HPV is
markedly diminished in rats exposed to chronic hypoxia, while pressor responses to angiotensin II, prostaglandin F2a and norepinephrine are increased (McMurtry et al., 1978). More recently it was described that in the PASMCs obtained from hypoxic rats IK is decreased and the membrane potential is depolarized, in comparison to the measurements in cells from normoxic rats (Smirnov et al., 1994). Production of ROS in the lungs, as measured by luminol-enhanced chemiluminescence, is diminished in the chronic hypoxic rats and levels of reduced glutathione are increased (Reeve et al., 2001a). Consistent with the observed reduction in current density, the levels of Kv 1.5 and Kv 2.1 channel protein are also diminished. The decrease in channel expression could be the result of direct redox control or could also be caused by prolonged depolarization (Levitan et al., 1995), initiated by the decrease in IK. In either case, it appears likely that redox changes mediate pulmonary vascular responses to chronic, as well as acute, hypoxia.
9. The role of redox in oxygen sensing by sarcoplasmic reticulum As stated earlier, it is probable that the increase in cytosolic calcium responsible for HPV comes from both outside the PASMC and from the SR (Gelband and Gelband, 1997; Morio and McMurtry, 2002; Salvaterra and Goldman, 1993). Although it is not certain which SR pool(s) are released during hypoxia, most workers at least favor release from a ryanodine/caffeine-sensitive (calcium-induced calcium release) store (Dipp et al., 2001; Jabr et al., 1997; Morio and McMurtry, 2002; Vadula et al., 1993). In this review the important question is whether the effect of hypoxia that is causing calcium release, might be mediated by a redox change. The answer is complicated by the observation that the ryanodine receptor, also termed the calcium release channel, may have four different classes of functional cysteine residues (Dulhunty et al., 2000). Oxidation, nitrosylation or alkylation can activate or inhibit calcium release. In the case of skeletal muscle, a
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transporter selective for glutathione is co-localized with the ryanodine receptor in the SR membrane (Feng et al., 2000). This maintains the local redox status. Changes in the transmembrane glutathione redox potential act as a sensor and modulate calcium release. As glutathione (GSH) and glutathione disulfide (GSSG) form the principal cellular redox bufering system and are altered by changes in oxygen tension, this control mechanism could initiate hypoxic release of SR calcium. Another redox-sensitive mechanism involves cyclic ADP-ribose (cADPR) (Wilson et al., 2001). In PASMCs during hypoxia, b-NADH accumulates and inhibits cADPR hydrolase. The increased level of cADPR activates the ryanodine receptor and leads to SR calcium release. Accumulation of NADH during hypoxia is also thought to cause release of calcium from IP3-sensitive stores in PC12 cells (Kaplin et al., 1996). In these studies it is clear that cellular redox is important in the SR response to changes in oxygen tension and that hypoxia, by producing a more reduced environment, leads to calcium release.
10. Redox control of oxygen sensing in the ductus arteriosus Normoxic contraction of the DA, such as occurs at birth, appears to be dependent upon influx of calcium into the SMCs, as it can be inhibited by nisoldipine, lanthanum and nifedipine (Michelakis et al., 2000; Tristani-Firouzi et al., 1996). The increase in oxygen reversibly inhibits a 4-aminopyridine-sensitive potassium channel and causes membrane depolarization. The oxygen-induced inhibition of IK involves the production of H2O2. If a low level of catalase (200 U/ml) is included in the patch pipette, to metabolize endogenous H2O2, IK increases markedly, indicating that H2O2 usually inhibits IK (Reeve et al., 2001b). Control solutions, such as boiled catalase, catalase buffer and albumen do not change Ik. This experiment demonstrates a role for ROS in oxygen sensing and indicates that, at least in the DA, H2O2 increases with an increase in oxygen tension. It does not tell us whether H2O2 acts directly
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on the K+ channel or if it alters the ratio of redox couples (e.g. GSH/GSSG), that then act on the channel. The importance of redox status in the control of DA tone can also be illustrated by the use of oxidizing and reducing agents. The oxidizing agent, 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB), causes contraction of DA rings, while the reducing agent dithiothreitol (DTT) and the H2O2 scavenger N-mercaptopropionylglycine (NMPG) cause relaxation (Reeve et al., 2001b).
11. Conclusions It seems likely that oxygen sensing in both the resistance PA and the DA is signalled by a change in redox status in the smooth muscle cell. A change in redox status, secondary to a change in oxygen tension, probably affects cytosolic calcium in two ways. The same redox signal can change calcium release from the SR and, through control of K+ channel gating, alter membrane potential and calcium influx (Fig. 3). The same redox signal in the PA and DA can achieve exactly opposite results in terms of constriction or relaxation, depending on the gating of the sarcolemmal or SR channels. While the importance of redox status in oxygen sensing seems clear, there are many questions still to be resolved. Is the initial change in ROS or in redox couples? Which of these interacts with the channels? What is the relevant source of endogenous ROS; mitochondria, NAD(P)H oxidase? What is the relative importance of the reducing and oxidizing functions of superoxide anion and of its interaction with nitric oxide? Does the concentration of superoxide dismutase, catalase or glutathione peroxidase alter vascular tone? Finally, do ROS go up or down with hypoxia? Until we know the answers to these questions the detailed mechanism of oxygen sensing will remain elusive.
Acknowledgements E. Kenneth Weir is supported by general medical research funds from the US Department of
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Fig. 3. Redox changes can alter cytosolic calcium and vessel diameter by changing membrane potential and calcium influx into the smooth muscle cell and by changing calcium release from the sarcoplasmic reticulum. VDCC: voltage-dependent calcium channel.
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Reeve, H., Michelakis, E., Nelson, D., Weir, E., 2001a. Alterations in redox oxygen sensing mechanism in chronic hypoxia. J. Appl. Physiol. 90, 2249 –2256. Reeve, H., Tolarova, S., Nelson, D., Archer, S., Weir, E., 2001b. Redox control of oxygen sensing in the rabbit ductus arteriosus. J. Physiol. Lond. 533, 253 –261. Robertson, T., Aaronson, P., Ward, J., 1995. Hypoxic vasoconstriction and intracellular Ca2 + in pulmonary arteries: evidence for PKC-independent Ca2 + sensitization. Am. J. Physiol. 268, H301 –H307. Robertson, T., Hague, D., Aaronson, P., Ward, J., 2000. Voltage-independent calcium entry in hypoxic pulmonary vasoconstriction of intrapulmonary arteries of the rat. J. Physiol. Lond. 525, 669 –680. Roulet, M., Coburn, R., 1981. Oxygen-induced contraction in the guinea-pig neonatal ductus arteriosus. Circ. Res. 49, 997 – 1002. Salvaterra, C., Goldman, W., 1993. Acute hypoxia increases cytosolic calcium in cultured pulmonary arterial myocytes. Am. J. Physiol. 264, L323 –L328. Sham, J., Crenshaw, B., Derg, L.-H., Shimoda, L., Sylvester, J., 2000. Effects of hypoxia in porcine arterial myocytes: roles of Kv channel and endothelin-1. Am. J. Physiol. 279, L262 – L272. Smirnov, S., Robertson, T., Ward, J., Aaronson, P., 1994. Chronic hypoxia is associated with reduced delayed rectifier K + current in rat pulmonary artery muscle cells. Am. J. Physiol. 266, H365 –H370. Thomas, H.I., Carson, R., Fried, E., Novitch, R., 1991. Inhibition of hypoxic pulmonary vasoconstriction by diphenyleneiodonium. Biochem. Pharm. 42, R9 –R13. Tolins, M., Weir, E., Chesler, E., Nelson, D., From, A., 1986. Pulmonary vascular tone is increased by a voltage-dependent calcium channel potentiator. J. Appl. Physiol. 60, 942– 948. Tristani-Firouzi, M., Reeve, H., Tolarova, S., Weir, E., Archer, S., 1996. Oxygen-induced constriction of rabbit ductus arteriosus occurs via inhibition of a 4-aminopyridine-voltage-sensitive potassium channel. J. Clin. Invest. 98, 1959 – 1965. Turrens, J., Freeman, B., Levitt, J., Crapo, J., 1982. The effect of hyperoxia on superoxide production by lung submitochondrial particles. Arch. Biochem. Biophys. 217, 401 – 410. Vadula, M., Kleinman, J., Madden, J., 1993. Effect of hypoxia and norepinephrine on cytoplasmic free Ca2 + in pulmonary and cerebral arterial myocytes. Am. J. Physiol. 265, L591 – L597.
Wang, D., Youngson, C., Wong, V., Yeger, H., Dinauer, M., Vega-Saenz de Miera, E., Rudy, B., Cutz, E., 1996. NADPH-oxidase and a hydrogen peroxide-sensitive K + channel may function as an oxygen sensor complex in airway chemoreceptors and small cell lung carcinoma cell lines. Proc. Natl. Acad. Sci. USA 93, 13182 – 13187. Waypa, G., Chandel, N., Schumacher, P., 2001. Model for hypoxic pulmonary vasoconstriction involving mitochondrial oxygen sensing. Circ. Res. 88, 1259 – 1266. Weir, E., Archer, S., 1995. The mechanism of acute hypoxic pulmonary vasoconstriction: the tale of two channels. FASEB 9, 183 – 189. Weir, E., Will, J., Lundquist, L., Eaton, J., Chesler, E., 1983. Diamide inhibits pulmonary vasoconstriction induced by hypoxia or prostaglandin F2. Proc. Soc. Exp. Biol. Med. 173, 96 – 103. Weir, E., Wyatt, C., Reeve, H., Huang, J., Archer, S., Peers, C., 1994. DPI inhibits both K + and Ca+ + currents in isolated pulmonary artery smooth muscle cells. J. Appl. Physiol. 76, 2611 – 2615. Weissman, N., Tadic, A., Hanze, J., Rose, F., Winterhalder, S., Nollen, M., Schermuly, R., Ghofrani, H., Seeger, W., Grimminger, F., 2000. Hypoxic vasoconstriction in intact lungs: a role for NADPH oxidase-derived H2O2. Am. J. Physiol. 279, L683 – L690. White, R., Jackson, J., McMurtry, I., Repine, J., 1988. Hypoxia increases glutathione redox cycle and protects lungs against oxidants. J. Appl. Physiol. 65, 2607 – 2616. Wilson, H., Dipp, M., Thomas, J., Lad, C., Galione, A., Evans, A., 2001. ADP-ribosyl cyclase and cyclic ACP-ribose hydrolase act as a redox sensor. J. Biol. Chem. 276, 11180 – 11188. Wolin, M., Burke-Wolin, T., Mohazzab-H, K., 1999. Roles for NAD(P)H oxidases and reactive oxygen species in vascular oxygen sensing mechanisms. Respir. Physiol. 115, 229 – 238. Yuan, J.X.-J., 2001. Oxygen-sensitive K + channel(s): where and what? Am. J. Physiol. 281, L1345 – L1349. Yuan, X.-J., Goldman, W., Tod, M., Rubin, L., Blaustein, M., 1993. Hypoxia reduces potassium currents in cultured rat pulmonary but not mesenteric arterial myocytes. Am. J. Physiol. 264, L116 – L123. Yuan, X., Wang, J., Juhaszova, M., Golovina, V., Rubin, L., 1998. Molecular basis and function of voltage-gated K + channels in pulmonary artery smooth muscle cells. Am. J. Physiol. 274, L621 – L635.
Redox signaling in oxygen sensing by vessels E. Kenneth Weir a,b,*, Zhigang Hong a, Valerie A. Porter b,c, Helen L. Reeve b,c a Department of Medicine, VA Medical Center, Minneapolis, MN 55417, USA Department of Physiology, Uni6ersity of Minnesota, Minneapolis, MN 55455, USA c Guidant Corporation, A221 Clinicals, 4100 Hamline A6enue, St. Paul, MN 55112, USA b
Received 11 February 2002; received in revised form 16 April 2002; accepted 16 April 2002
Abstract In response to the increase in oxygen tension at birth, the resistance pulmonary arteries dilate, while the ductus arteriosus constricts. Although modulated by the endothelium, these opposite responses are intrinsic to the vascular smooth muscle. While still controversial, it seems likely that during normoxia the production of reactive oxygen species (ROS) increases and the smooth muscle cell cytoplasm is more oxidized in both pulmonary arteries and ductus, compared to hypoxia. However, the effect of changes in the endogenous redox status or the addition of a redox agent, oxidizing or reducing, is exactly opposite in the two vessels. A reducing agent, dithiothreitol, like hypoxia, in the pulmonary artery will inhibit potassium current, cause depolarization, increase cytosolic calcium and lead to contraction. Responses to dithiothreitol in the ductus are opposite and removal of endogenous H2O2 by intracellular catalase in the ductus increases potassium current. Oxygen sensing in both vessels is probably mediated by redox effects on both calcium influx and calcium release from the sarcoplasmic reticulum (SR). © 2002 Elsevier Science B.V. All rights reserved. Keywords: Endothelium, pulmonary; Oxygen, sensing in pulmonary vessels; Pulmonary vasculature, oxygen sensing; Redox state, pulmonary vessels; Smooth muscle, pulmonary vessels
1. Introduction The most dramatic difference in vascular responses to changes in oxygen tension is demonstrated by the pulmonary resistance arteries and the ductus arteriosus (DA). At birth, these tissues are closely related anatomically and yet behave in a diametrically opposite manner to the increase in * Corresponding author. Tel.: +1-612-725-2000x3653; fax: +1-612-727-5668 E-mail address: [email protected] (E.K. Weir).
oxygen; the resistance pulmonary arteries dilate, while the DA constricts. While all workers in the field would agree on this observation, the mechanism by which the level of oxygen is sensed and transduced into changes in vascular tone remains controversial. Most would accept that the level of cytosolic calcium in the smooth muscle is the main determinant of vascular tone, although the sensitivity of actin-myosin to a given level of calcium might change with hypoxia (Robertson et al., 1995) and endothelial factors undoubtedly modulate smooth muscle reactivity.
1569-9048/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 1 5 6 9 - 9 0 4 8 ( 0 2 ) 0 0 0 5 4 - X
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2. Sources of calcium
3. Hypoxia and reactive oxygen species
If cytosolic calcium is important, is one pool within the cytoplasm particularly relevant and what is the source of calcium entering this pool? Attention has been focused on two possible sources, the influx of extracellular calcium and the release of intracellular calcium, especially from the sarcoplasmic reticulum (SR). It seems quite likely that both may be involved. In the case of the influx of extracellular calcium, the potential paths include voltage-dependent calcium channels (VDCC) and store-operated calcium channels (SOCC). Considering the possible role of the VDCC, SOCC and SR in the pulmonary arteries and DA, what could signal opposite responses to a change in oxygen tension in these two vessels? In the case of the VDCC in the sarcolemma of the pulmonary artery smooth muscle cell (PASMC), one sequence of events precipitated by hypoxia has been extensively documented (Weir and Archer 1995). Inhibition of one or more potassium channels (Post et al., 1992; Yuan et al., 1993), may lead to membrane depolarization (Madden et al., 1985) and calcium entry through the VDCC (McMurtry 1985; McMurtry et al., 1976; Tolins et al., 1986). The same sequence seems to occur in smooth muscle cells of the DA but, in this case, the sequence is initiated by an increase in oxygen tension that inhibits potassium channels, not by hypoxia (Nakanishi et al., 1993; Roulet and Coburn, 1981; Tristani-Firouzi et al., 1996). While much of the calcium required for hypoxic pulmonary vasoconstriction (HPV), during the initial 10 minutes, appears to come from outside the PASMC, it is clear that some is released from within the cell (Gelband and Gelband, 1997; Morio and McMurtry, 2002; Salvaterra and Goldman, 1993). It is also possible that the increase in cytosolic calcium itself might inhibit potassium channels in the PASMC, as a secondary event (Post et al., 1995), or that the inhibition of potassium channels might play a relatively minor role (Robertson et al., 2000; Sham et al., 2000). The underlying question remains, what signals the release of intracellular calcium, and the change in potassium channel gating?
Changes in the redox status of the pancreatic beta cell influence the level of cytosolic calcium and insulin secretion (Archer et al., 1986). By analogy with the beta cell, it was suggested that oxygen-induced changes in the redox status of the PASMC membrane might alter potassium channel gating, membrane potential and calcium influx through the VDCC. The hypothesis was that hypoxia would lead to a decrease in reactive oxygen species (ROS), that would in turn cause the cytoplasm and cell membrane to be more reduced, thus closing potassium channels and resulting in membrane depolarization and calcium entry. It seemed likely that ROS are involved in intracellular signaling throughout the body and are not merely part of the neutrophil armamentarium. In the last 15 years parts of the puzzle have been completed but new questions have arisen. Surprisingly, it is not yet agreed whether ROS go up or down during acute exposure of the lung to hypoxia at physiological levels. Chemiluminescence techniques using luminol or lucigenin enhancement indicate a decrease in ROS production during hypoxia in the rat lung (Archer et al., 1989), mouse lung (Archer et al., 1999), rabbit lung (Paky et al., 1993) and in the rabbit DA (Reeve et al., 2001b). Similarly, an increase in oxygen tension increases ROS in rat lung and lung mitochondria (Freeman and Crapo, 1981; Turrens et al., 1982). Interestingly, hypoxia has also been shown to decrease superoxide anion production by neutrophils, as measured by cytochrome c reduction (Gabig et al., 1979). On the other hand, in cultured PASMCs, superoxide anion production is found to increase during hypoxia, as measured using lucigenin-enhanced chemiluminescence (Marshall et al., 1996), and hydrogen peroxide increases, as measured by dichlorofluorescein fluorescence (Waypa et al., 2001). Other work based on the use of a variety of enzyme inhibitors, also favors a role for an increase of ROS in the genesis of HPV (Weissman et al., 2000). These differences, that are probably secondary to the techniques used, need to be resolved before the role of ROS in the mechanism of HPV can be determined. Another question that
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is seldom addressed is whether superoxide anion, under physiologic circumstances, may on occasion act as a reducing agent, rather than as an oxidizing agent. It is usually assumed that, like hydrogen peroxide (H2O2), it will always cause oxidation. However, superoxide anion production in vitro is routinely measured by its ability to reduce oxidized markers, such as potassium ferricyanide and cytochrome c. The net effect of ROS production by mitochondria or enzymes, such as NAD(P)H oxidases, on vascular tone may depend on complex interactions: the balance between superoxide anion and H2O2 controlled by superoxide dismutase, the level of H2O2 controlled by catalase, glutathione peroxidase and the availability of NADPH, and the interaction of superoxide anion and NO.
4. NADPH-oxidase The first suggestion that NADPH-oxidase might be the oxygen sensor in the pulmonary vasculature was made by Thomas et al. (Thomas et al., 1991). The hypothesis was based on the observation that the NADPH-oxidase inhibitor, diphenyleneiodonium (DPI), inhibits HPV. Unfortunately, DPI also blocks calcium channels thus making the interpretation less specific (Weir et al., 1994). It was hoped that the development of an NADPH oxidase deficient mouse, lacking the gp 91 phox subunit, might help to solve the riddle. However, HPV (Archer et al., 1999) and oxygen sensing in the carotid body (He et al., 2002) are unchanged in these mice, suggesting that NADPH oxidase, at least that containing the gp 91 phox subunit, is not involved (Fig. 1). However, recent studies on the same mouse model does indicate that NADPH oxidase is an important oxygen sensor in the pulmonary neuroepithelial body (Fu et al., 2000). The possibility that a low-output form of NADPH oxidase, lacking the gp 91 phox subunit, could be the oxygen sensor involved in HPV has not been excluded. It has also been suggested that NADH oxidase might be the oxygen sensor (Burke-Wolin and Wolin, 1989; Mohazzab-H et al., 1995). These studies found
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that H2O2 production increased in normoxia, relative to hypoxia, and caused PASMC relaxation through an increase in cGMP. The role of NAD(P)H oxidases in oxygen sensing has been extensively reviewed (Jones et al., 2000; Wolin et al., 1999).
5. Hypoxia and redox couples If the concept is correct that a change in redox status is important in HPV, should the redox status be defined by the production of ROS or by the ratio of reduced to oxidized redox couples such as GSH/GSSG and NAD(P)H/ NAD(P)? If the latter, do these couples shift during hypoxia because electrons are not passed so efficiently down the mitochondrial electron transport chain or because of a change in the production of ROS, or both? Changes in oxygen tension are known to alter the ratio of reduced to oxidized glutathione and pyridine nucleotides (Archer et al., 1986; Cross et al., 1990; Patterson et al., 1985; White et al., 1988). Presumably the redox couples could be important if the mitochondria are involved in sensing the oxygen tension (He et al., 2002; Waypa et al., 2001) but perhaps less so if the oxygen sensor is membrane-bound, such as NADPH-oxidase (Cross et al., 1990; Marshall et al., 1996; Wang et al., 1996; Weissman et al., 2000) and there is the potential for direct interaction of ROS and ion channels. Changes in cytosolic redox secondary to alteration in mitochondrial function could affect both calcium influx, related to ion channel gating, and calcium release from the SR. If NAD(P)H-oxidase were the sole oxygen sensor, at first glance it would seem more likely that it would modulate ion channel gating. However, considering the close relationship of localized calcium release from the SR (‘sparks’) to opening of the calcium-sensitive potassium channels in the cell membrane (STOCs) (Nelson et al., 1995), one could also postulate that ROS from the membrane could alter the handling of calcium in the SR that is nearby by changing the redox control of SR calcium release.
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6. Redox control of ion channels What is the evidence that redox changes can alter potassium or calcium channel gating, whether the redox change is in ROS or redox couples? In neuroepithelial bodies hypoxia in-
hibits whole cell potassium current (IK) and IK can be increased by the external application of H2O2 (Fu et al., 2000). Incidentally, this experiment alone would suggest that hypoxia does not increase ROS, at least in the neuroepithelial body. In A-type Kv channels (e.g. Kv 1.4 and 3.4)
Fig. 1. Reduced glutathione (GSH) in the patch pipette reduces whole cell potassium current and single channel open probability in rat pulmonary artery smooth muscel cells.
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Fig. 2. The isolated perfused lungs of mice lacking the gp 91 subunit of NADPH oxidase have normal HPV but there is virtually no oxygen radical production at the lung surface, measurable by luminol-enhanced chemiluminescence.
exposure to H2O2 causes oxidation of cysteine groups at the amino terminus of the channel protein and inhibits rapid inactivation (Pongs, 1998). Beta subunits, such as Kvb1 and b3, also possess an amino-terminal inactivating domain, containing a cysteine residue, removal of which eliminates rapid inactivation. Consequently oxidation would tend to increase the potassium current and reduction to decrease it. An example of the potential importance of the beta subunit in oxygen sensing was recently demonstrated. Kv 4.2 channels expressed in HEK 293 cells were not inhibited by hypoxia but, when coexpressed with Kv b 1.2, the channel was reversibly inhibited (Perez-Garcia et al., 1999). On the other hand, oxidation of a methionine group in the P-segment of Shaker Kv channels expressed in Xenopus oocytes accelerates inactivation (Chen et al., 2000). In this situation, oxidation would tend to decrease the potassium current
and reduction to increase it; the reverse of what was observed in the case of cysteine residues. The different behavior of these channels in response to redox changes might help to explain the opposite responses of the pulmonary arteries and DA to oxygen.
7. Redox control of pulmonary vascular ion channels. A variety of redox-active agents have been studied in PASMCs. Reduced glutathione (GSH) introduced into PASMCs in the patch pipette reduces IK, while oxidized glutathione increases IK (Weir and Archer, 1995). An example of the effect of GSH on whole cell K current and on single K channel opening is shown in Fig. 2. The glutathione and sulfhydryl oxidant, diamide, increases both IK and single channel KCa current
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(Post et al., 1993; Reeve et al., 1995), while also causing pulmonary vasodilatation (Weir et al., 1983). With a ‘calcium-free’ solution in the patch pipette to eliminate KCa current, the residual IKv current in PASMCs can be increased by the oxidizing agent dithio-bis (5-nitropyridine) and decreased by the reducing agent dithiothreitol (DTT) (Park et al., 1995a). Using ‘inside-out’ patches of PASMC sarcolemma, it can be demonstrated that several reducing agents (DTT, GSH, NADH) decrease KCa current, while several oxidising agents (DTNB, GSSG, NAD) increase KCa current (Park et al., 1995b). Similar observations have been made in another oxygen-sensitive tissue, the Type 1 cell of the carotid body (LopezBarneo et al., 1998). It appears that a number of Kv channels that are present in PASMCs are inhibited by hypoxia. These include Kv 1.2 and the Kv 1.2/1.5 heteromultimer, as well as Kv 2.1 and the Kv 2.1/Kv 9.3 heteromultimer (Archer et al., 1998; Hulme et al., 1999; Patel et al., 1997). Kv 3.1b may also be involved (Coppock and Tamkun, 2001; Osipenko et al., 2000). In support of a role for the Kv 1.5 channel is the observation that mice lacking this channel have diminished HPV (Archer et al., 2001). The beta subunits, mentioned earlier, Kv b 1.1, Kv b 2 and Kv b 3 are also present in PASMCs (Coppock and Tamkun, 2001; Yuan et al., 1998). These subunits show homology with NAD(P)H oxidoreductases and the redox state of the subunit may modulate the activity of the channel and, possibly, the activity of the channel may regulate the function of the oxidoreductase (Gulbis et al., 2000; McCormack and McCormack 1994). If the latter is true, changes in channel activity might, through the generation of ROS, alter calcium release from the SR as postulated earlier. The evidence relating both a and b channel subunits to oxygen sensing has recently been reviewed (Archer et al., 2000; Patel and Honore 2001; Yuan 2001).
8. Redox and potassium channel expression in chronic hypoxia It has long been known that acute HPV is
markedly diminished in rats exposed to chronic hypoxia, while pressor responses to angiotensin II, prostaglandin F2a and norepinephrine are increased (McMurtry et al., 1978). More recently it was described that in the PASMCs obtained from hypoxic rats IK is decreased and the membrane potential is depolarized, in comparison to the measurements in cells from normoxic rats (Smirnov et al., 1994). Production of ROS in the lungs, as measured by luminol-enhanced chemiluminescence, is diminished in the chronic hypoxic rats and levels of reduced glutathione are increased (Reeve et al., 2001a). Consistent with the observed reduction in current density, the levels of Kv 1.5 and Kv 2.1 channel protein are also diminished. The decrease in channel expression could be the result of direct redox control or could also be caused by prolonged depolarization (Levitan et al., 1995), initiated by the decrease in IK. In either case, it appears likely that redox changes mediate pulmonary vascular responses to chronic, as well as acute, hypoxia.
9. The role of redox in oxygen sensing by sarcoplasmic reticulum As stated earlier, it is probable that the increase in cytosolic calcium responsible for HPV comes from both outside the PASMC and from the SR (Gelband and Gelband, 1997; Morio and McMurtry, 2002; Salvaterra and Goldman, 1993). Although it is not certain which SR pool(s) are released during hypoxia, most workers at least favor release from a ryanodine/caffeine-sensitive (calcium-induced calcium release) store (Dipp et al., 2001; Jabr et al., 1997; Morio and McMurtry, 2002; Vadula et al., 1993). In this review the important question is whether the effect of hypoxia that is causing calcium release, might be mediated by a redox change. The answer is complicated by the observation that the ryanodine receptor, also termed the calcium release channel, may have four different classes of functional cysteine residues (Dulhunty et al., 2000). Oxidation, nitrosylation or alkylation can activate or inhibit calcium release. In the case of skeletal muscle, a
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transporter selective for glutathione is co-localized with the ryanodine receptor in the SR membrane (Feng et al., 2000). This maintains the local redox status. Changes in the transmembrane glutathione redox potential act as a sensor and modulate calcium release. As glutathione (GSH) and glutathione disulfide (GSSG) form the principal cellular redox bufering system and are altered by changes in oxygen tension, this control mechanism could initiate hypoxic release of SR calcium. Another redox-sensitive mechanism involves cyclic ADP-ribose (cADPR) (Wilson et al., 2001). In PASMCs during hypoxia, b-NADH accumulates and inhibits cADPR hydrolase. The increased level of cADPR activates the ryanodine receptor and leads to SR calcium release. Accumulation of NADH during hypoxia is also thought to cause release of calcium from IP3-sensitive stores in PC12 cells (Kaplin et al., 1996). In these studies it is clear that cellular redox is important in the SR response to changes in oxygen tension and that hypoxia, by producing a more reduced environment, leads to calcium release.
10. Redox control of oxygen sensing in the ductus arteriosus Normoxic contraction of the DA, such as occurs at birth, appears to be dependent upon influx of calcium into the SMCs, as it can be inhibited by nisoldipine, lanthanum and nifedipine (Michelakis et al., 2000; Tristani-Firouzi et al., 1996). The increase in oxygen reversibly inhibits a 4-aminopyridine-sensitive potassium channel and causes membrane depolarization. The oxygen-induced inhibition of IK involves the production of H2O2. If a low level of catalase (200 U/ml) is included in the patch pipette, to metabolize endogenous H2O2, IK increases markedly, indicating that H2O2 usually inhibits IK (Reeve et al., 2001b). Control solutions, such as boiled catalase, catalase buffer and albumen do not change Ik. This experiment demonstrates a role for ROS in oxygen sensing and indicates that, at least in the DA, H2O2 increases with an increase in oxygen tension. It does not tell us whether H2O2 acts directly
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on the K+ channel or if it alters the ratio of redox couples (e.g. GSH/GSSG), that then act on the channel. The importance of redox status in the control of DA tone can also be illustrated by the use of oxidizing and reducing agents. The oxidizing agent, 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB), causes contraction of DA rings, while the reducing agent dithiothreitol (DTT) and the H2O2 scavenger N-mercaptopropionylglycine (NMPG) cause relaxation (Reeve et al., 2001b).
11. Conclusions It seems likely that oxygen sensing in both the resistance PA and the DA is signalled by a change in redox status in the smooth muscle cell. A change in redox status, secondary to a change in oxygen tension, probably affects cytosolic calcium in two ways. The same redox signal can change calcium release from the SR and, through control of K+ channel gating, alter membrane potential and calcium influx (Fig. 3). The same redox signal in the PA and DA can achieve exactly opposite results in terms of constriction or relaxation, depending on the gating of the sarcolemmal or SR channels. While the importance of redox status in oxygen sensing seems clear, there are many questions still to be resolved. Is the initial change in ROS or in redox couples? Which of these interacts with the channels? What is the relevant source of endogenous ROS; mitochondria, NAD(P)H oxidase? What is the relative importance of the reducing and oxidizing functions of superoxide anion and of its interaction with nitric oxide? Does the concentration of superoxide dismutase, catalase or glutathione peroxidase alter vascular tone? Finally, do ROS go up or down with hypoxia? Until we know the answers to these questions the detailed mechanism of oxygen sensing will remain elusive.
Acknowledgements E. Kenneth Weir is supported by general medical research funds from the US Department of
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Fig. 3. Redox changes can alter cytosolic calcium and vessel diameter by changing membrane potential and calcium influx into the smooth muscle cell and by changing calcium release from the sarcoplasmic reticulum. VDCC: voltage-dependent calcium channel.
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