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Comparison of the metabolic and ventilatory response to hypoxia and H2S in unsedated mice and rats
Respiratory Physiology & Neurobiology 167 (2009) 316–322
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Comparison of the metabolic and ventilatory response to hypoxia and H2 S in unsedated mice and rats Philippe Haouzi a,b,∗ , Harold J. Bell a , Veronique Notet c , Bernard Bihain c a b c
Pennsylvania State University, College of Medicine, Heart and Vascular Institute, Hershey, USA Division of Pulmonary Medicine, Department of Medicine, Penn State Milton Hershey Medical Center, Hershey, USA Laboratoire de Médecine et Thérapeutique Moléculaire, Genclis SAS, Vandoeuvre les Nancy, France
a r t i c l e
i n f o
Article history: Accepted 9 June 2009 Keywords: Arterial chemoreception Thermoregulation Oxygen transduction
a b s t r a c t Hypoxia alters the control of breathing and metabolism by increasing ventilation through the arterial chemoreflex, an effect which, in small-sized animals, is offset by a centrally mediated reduction in metabolism and respiration. We tested the hypothesis that hydrogen sulfide (H2 S) is involved in transducing these effects in mammals. The rationale for this hypothesis is twofold. Firstly, inhalation of a 20–80 ppm H2 S reduces metabolism in small mammals and this effect is analogous to that of hypoxia. Secondly, endogenous H2 S appears to mediate some of the cardio-vascular effects of hypoxia in nonmammalian species. We, therefore, compared the ventilatory and metabolic effects of exposure to 60 ppm H2 S and to 10% O2 in small and large rodents (20 g mice and 700 g rats) wherein the metabolic response to hypoxia has been shown to differ according to body mass. H2 S and hypoxia produced profound depression in metabolic rate in the mice, but not in the large rats. The depression was much faster with H2 S than with hypoxia. The relative hyperventilation produced by hypoxia in the mice was replaced by a depression with H2 S, which paralleled the drop in metabolic rate. In the larger rats, ventilation was stimulated in hypoxia, with no change in metabolism, while H2 S affected neither breathing nor metabolism. When mice were simultaneously exposed to H2 S and hypoxia, the stimulatory effects of hypoxia on breathing were abolished, and a much larger respiratory and metabolic depression was observed than with H2 S alone. H2 S had, therefore, no stimulatory effect on the arterial chemoreflex. The ventilatory depression during hypoxia and H2 S in small mammals appears to be dependent upon the ability to decrease metabolism. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Over the last decade, solid evidence has identified hydrogen sulfide (H2 S) as being a gasotransmitter (Abe and Kimura, 1996; Wang, 2002; Dominy and Stipanuk, 2004; Kamoun, 2004b). Endogenous production of H2S uses l-cysteine as the main substrate and is mainly under the control of the 2 different enzymes: cystathionine ß-synthase (CBS) in the central nervous system and cystathionine gama lyase (CSE) in the vessels or other tissues (Wang, 2002; Kamoun, 2004a). Relatively high concentration of endogenous H2 S has been reported in many tissues including the central nervous system (for review Wang, 2002). Although the regulation of the production and degradation of endogenous H2 S in mammalian species is still not fully understood, beneficial as well as adverse effects of endogenous H2 S accumulation have been reported (Wang, 2002;
∗ Corresponding author at: Pennsylvania State University, Penn State Hershey Medical Center, Heart and Vascular Institute, 500 University Dr., Hershey, PO Box 850, MC H047, PA, 17033-0850, Tel.: +1 717 531 0003x287518; fax: +1 717 531 0224. E-mail address: [email protected] (P. Haouzi). 1569-9048/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.resp.2009.06.006
Cheng et al., 2004; Dominy and Stipanuk, 2004; Kimura and Kimura, 2004; Bhatia, 2005; Bhatia et al., 2005). Finally, H2 S has been shown to play the role of gaseous neuromodulator (Abe and Kimura, 1996). We have recently suggested that H2 S could also be important in mediating the well-described effects of hypoxia on ventilatory and metabolic control in small mammals (Haouzi et al., 2008). For example, the response to breathing a non-toxic concentration of H2 S mimics the metabolic depression which is triggered by hypoxia in mice (Blackstone et al., 2005). Interestingly, this metabolic depression in response to H2 S is not observed in larger mammals (Haouzi et al., 2008), and this is also consistent with the metabolic response to hypoxia (Frappell et al., 1991; Frappell et al., 1992; Mortola, 1993; Gautier, 1996). Hypoxia has been shown to increase H2 S concentration in the pulmonary vessels (Olson et al., 2006), and during hypoxemia sulfide concentration increases in the blood (Olson et al., 2006; Olson et al., 2008a). The pharmacological blockade of CSE activity, the enzyme allowing the transformation of cysteine to H2 S in the vessels, is associated with a severely blunted hypoxia-induced vasoactive response in various types of vessels (Olson et al., 2006; Olson, 2008; Olson et al., 2008a). A similar vascular effect of blocking CSE activity has been reported in the trout
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(Dombkowski et al., 2004). The vascular effect of hypoxia was virtually abolished during a simultaneous exposure to exogenous H2 S, which in turn depresses the activity of CSE and thus endogenous H2 S production. Finally, H2 S has stimulatory effect on the activity of the gill chemoreceptors in fish (Olson et al., 2008b). Collectively, these data suggest that hypoxia increases the concentration of endogeneous H2 S, and that this increase in endogenous H2 S levels mediates the vacular effects of hypoxia (Olson, 2008). Whether the phenomena observed in fish or in-vitro models are applicable to other mammalian tissues and directly apply to the respiratory and metabolic response to hypoxia in mammals remains unknown. The goal of this study is to determine whether H2 S is involved in transducing the known ventilatory effects of hypoxia in mammals. More specifically, we investigated the role of H2 S in two of the most predominant physiological responses to acute hypoxia in mammals: (1) the increase in breathing mediated by the arterial chemoreceptors, and (2) the reduction in ventilation associated with the hypoxia-induced depression of metabolic rate. While the stimulation of breathing during hypoxia is common to all mammals, regardless of body size, the metabolic depression induced by hypoxia is a strategy developed early in evolution but which is only observed in small mammals (Frappell et al., 1992; Mortola et al., 1994). This latter response is associated with a relative reduction of breathing proportional to the reduction in metabolic rate, which counteracts the stimulatory effects of hypoxia-induced chemoreceptor stimulation. Nevertheless, V˙ E/V˙ O2 ratio increases by approximately the same amount in all species during hypoxia, regardless of their body size (Mortola, 1993). We therefore compared the ventilatory response to 10% hypoxia and 60 ppm H2 S in two rodent species of different body mass in the very same experimental conditions. 20 g unsedated mice were studied as a model of small-sized mammals, where both hypoxia and H2 S have been shown to depress metabolism. A similar approach was applied in a group of 700 g unsedated rats in which the response to hypoxia consists almost exclusively in a ventilatory stimulation with no or little depressive effect of hypoxia on metabolism (Mortola et al., 1994). We speculate that if H2 S is involved in transducing the effects of hypoxia, through an increase in H2 S production, one should expect the ventilatory and metabolic responses to H2 S and hypoxia to be similar. In addition, if, as previously suggested, exogenous H2 S can inhibit both the CBS and CSE by exerting a negative feedback effect on the activity of these enzymes (Kredich et al., 1973; Simpson and Freedland, 1976; Wang, 2002), the response to hypoxia should be blunted during a simultaneous exposure to H2 S. This has been demonstrated for other effects of hypoxia, which appear to be mediated through the CSE activity in the vessels (Olson et al., 2006).
2. Methods 2.1. Animals and equipment The experimental procedures were performed on five adult female C57BL/6J mice (24.05 ± 1.09 g, 45–47 weeks old) and on five adult male Sprague–Dawley rats (701 ± 46 g, 40–42 weeks old). All experiments using mice were performed in the Laboratoire de Médecine et Thérapeutique Moléculaire, Genclis, Univeristy of Nancy, France, according to the recommendations of the Council of European Communities and with the authorization from the French Ministry of Agriculture and Fisheries (Authorization number 54-42). The experiments and procedures performed using rats were performed in the facilities of the Penn State University College of Medicine, and were approved under the Penn State Hershey Institutional Animal Care and Use Committee, protocol # 2007–168.
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The metabolic and respiratory variables were measured via unrestrained whole body open-flow plethysmography in customdesigned animal chambers and air control circuits as previously described (Haouzi et al., 2008; Bell et al., 2009) The animal chambers were sealed, leak-proof acrylic cylinders designed according to the animal size. The size and shape of the chambers were defined to minimize the mixing time constant of the chamber for ventilation and gas exchange determination. For the mice, the chamber consisted of a plastic tube of low volume (90 cm3 ) with a relatively long length (11 cm). For the rats, we used a chamber with internal volume of 1400 cm3 (diameter 120 mm). The chamber was of sufficient volume that air could pass freely around all sides of the animal without interference as previously described. Animals were acclimatized to the chambers through routinely spending 2–3 h several times a week in them without any signs of anxiety or stress. Various compositions of fresh gas from dry, pre-mixed, high pressure tanks (20.9% O2 , 10% O2 , 60 ppm H2 S balanced with air or 10% O2 ) were passed through regulators and delivered to the animal chamber through the inlet port which was also equipped with a diffuser to distribute fresh gas evenly throughout the crosssectional area. The flow of gas that was typically used averaged 300–400 ml min−1 for the mice and 2.5 l min−1 for the rats. The chamber was exhausted through the outlet port which passed gas into low-pressure non-compliant CPVC tubing. The flow of air through the animal chamber was continuously monitored via a Fleisch 000 pneumotachograph which was interfaced to a pressure transducer (Sensym, DCLX O1DN, Honeywell, Morristown, NJ, USA) housed in a custom-designed electronic demodulator. CO2 and O2 levels were continuously measured in the air exiting the animal chamber (infrared, and fuel cell analysers respectively, Vacumed, Ventura CA, USA) using sampling catheters distal to the outlet of the chamber. The composition of the gas entering the chamber was measured sequentially as needed using a circuit which bypassed the animal chamber. Temperature in the animal chamber (Tc ) was continuously monitored via a fast-responding thermocouple (BAT12, Physiotemp instruments Inc, Clifton, NJ, USA for the mice, and Thermalert TH5, Physiotemp, Clifton, NJ, USA for rats). Temperature was maintained at ∼25.% C for the mice and ∼27.5 ◦ C for the rats. For the mice, analog signals from the pneumotachograph, CO2 and O2 analyzers, temperature sensor were fed into a Powerlab system (ADInstruments, Colorado Springs, CO, USA). For the rats, analog signals were fed to a 14-bit A/D converter (USB6009, National Instruments, Austin, TX, USA), which was interfaced with an Intel/Windows Vista based computer system (Compaq 8510w, Hewlett Packard, Palo Alto, CA, USA) running custom-written data acquisition software (LabView, National Instruments, Austin, TX, USA – source code available upon request). Analog signals were sampled at 200 s−1 and displayed in raw form on the monitor while being streamed to storage (1 block every 5 s) for subsequent analysis. ASCII data files were converted to ‘.adicht’ format for later visualization and analysis using Chart software (version 5.5.4, ADInstruments, Colorado Springs, CO, USA). To calculate respiratory variables from the raw flow trace, the signal was treated using a high-pass filter (>0.5 Hz) to determine and subtract the DC component. The resulting plethysmographic trace, without the DC component, contained the respiratory signal which was used for calculations of breathing frequency (f), and to obtain an index of minute ventilation (V˙ E). To obtain an index of V˙ E from the filtered flow signal, the positive deflections in the plethysmographic trace were integrated over 15 s intervals. The result of this integration was then temperature corrected based upon the difference between ambient temperature in the chamber as determined continuously throughout the experiments, and the estimated body core temperature (Tb ) of the animal as determined via rectal placement of a digital thermometer immediately after the completion of the experiment. Due to the complexity
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of factors involved in quantitative interpretation of an open flow plethysmographic signal, this determination of V˙ E provided a semiquantitative index represented in the same units as are appropriate for direct measurements of ventilation (Bell et al., 2009).V˙ O2 and V˙ CO2 . were computed in STPD conditions as:
The effects of each gas were analyzed using an analysis of variance for each of the following parameters V˙ O2 , ventilation and breathing frequency. The a priori value for acceptability of a type I (a) error in any statistical comparison was set to 0.05. 3. Results
V˙ O2 = V˙ inlet.FIO2 − V˙ outlet.FEO2
3.1. Metabolic and ventilatory response to H2 S and hypoxia in the mice
and V˙ CO2 = V˙ outlet.FECO2 − V˙ inlet.FICO2 Since only V˙ outlet was measured, V˙ inlet was determined as: V˙ inlet = V˙ outlet.(1 − FEO2 − FECO2 )/(1 − FIO2 − FICO2 ) to take into account the difference between V˙ inlet and V˙ outlet that would result from the gas exchange ratio (soda lime could not be used to eliminate FICO2 , since soda lime traps H2 S). V˙ O2 and V˙ CO2 were determined and expressed in STPD conditions 2.2. Protocol and data analysis Mice were placed in the open chamber for about half an hour before starting data acquisition. Thirty minutes of baseline condition were then recorded before the animals were exposed to either H2 S or hypoxia for 30 min. As previously discussed (Haouzi et al., 2008), criteria for baseline values are not simple to establish but are essential to discuss when determining whether H2 S or hypoxia can decrease the rate of gas exchange and ventilation. For instance, mice displayed unpredictable patterns of activity. Therefore it becomes crucial to define as precisely as possible the nature of the baseline. We used for baseline a period that could be sustained for at least 5 min during which the mice remained quiet and still, based upon visual examination and trivial spontaneous variations in metabolic rate (Haouzi et al., 2008). Rats were also placed in the chamber for approximately 30 min before the start of data collection. Typically, rats displayed much less fluctuation in their metabolic and ventilatory activity and so for rat experiments, baseline data were simply obtained using a 5 min observation window beginning between 5 and 15 min after the start of data collection in room air conditions (Bell et al., 2009). A specific observation window was selected such that unwanted sniffing and grooming behaviors were largely absent in the respiratory traces. Data were averaged and expressed as mean ± SD. V˙ O and V˙ CO 2
2
were expressed in ml min−1 or ml min−1 kg−1 and their changes were expressed in absolute and in% changes.
3.1.1. Metabolic response The metabolic response to 60 ppm H2 S consisted of a major and rapid reduction in V˙ O2 . As shown in Fig. 1, V˙ O2 reached its nadir 295 ± 15 s into the H2 S exposure, decreasing from 1.37 ± 0.09 to 0.57 ± 0.07 ml min−1 (−58 ± 5%, p < 0.001). V˙ O2 had then the tendency to slightly rise over time reaching a steady value averaging 0.94 ± 0.04 ml min−1 (−31 ± 7%, p < 0.001) by the end of the 30 min exposure. Inhalation of hypoxia (10% O2 ) also produced a reduction in V˙ O2 , however with a slower time course than was observed during H2 S exposure. Indeed V˙ O2 decreased in all tests, with an exponential-like pattern from 1.52 ± 0.15 to 1 ± 0.17 ml min−1 (−34 ± 8%, p < 0.001) at the nadir of the response to H2 S, but V˙ O2 continued to decrease reaching 0.71 ± 0.135 ml min−1 (−53 ± 12%, p < 0.001) at the end of the 30 min exposure. An example is shown in Fig. 1, while average changes are displayed in Fig. 2. 3.1.2. Ventilatory response In contrast to the V˙ O2 response which consisted of a decrease in response to either H2 S or hypoxia, ventilation was affected differently by exposure to the two gases. During H2 S exposure, ventilation decreased by 64 ± 3%, from 34.12 ± 1.51 to 12.41 ± 1.07 ml min−1 (p < 0.01) at the nadir of the metabolic depression, reaching 16.14 ± 1.62 ml min−1 (−53 ± 4%, p < 0.01) at the end of the test. As shown in Fig. 1, breathing frequency was dramatically reduced, decreasing from 204 ± 10 to 53 ± 5 then 70 ± 8 br min−1 at 5 and 30 min, respectively (p < 0.01). In marked contrast, during hypoxia ventilation increased from 29.4 ± 3.72 to 36.06 ± 5.67 ml min−1 (+23 ± 19%, p < 0.01) at 5 min then return to baseline ventilatory values 26.39 ± 2.03 ml min−1 , when the metabolic rate was at the lowest point (Fig. 2). Breathing frequency paralleled the change in total ventilation as f increased from 176 ± 35 to 256 ± 43 br min−1 at 5 min (p < 0.01) reaching 200 ± 55 breaths min−1 at 30 min.
Fig. 1. Representative example of the time course of ventilatory flow (V˙ ), breathing frequency (f), minute ventilation (V˙ E, averaged every 15 s), and oxygen uptake (V˙ O2 ) responses to inhalation of 60 ppm H2 S (left panels) and 10% O2 (right panels) in the same mouse. Note during H2 S exposure, the large, rapid depression in metabolic rate is associated with a similarly rapid decrease in breathing frequency and minute ventilation. In this example breathing frequency fell to 40 br min−1 during H2 S exposure. During hypoxic exposure, metabolism decreases as well but less rapidly than that during H2 S exposure: there was a progressive exponential-like reduction in metabolic rate. In marked contrast to H2 S, neither ventilation nor breathing frequency was depressed.
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Fig. 2. Average response expressed in% of change from baseline in the group of mice (open bars) and rats (black bars). Data are shown at the nadir of the response to H2 S in the mice (∼300 s into exposure). In the mice the metabolic rate decreased significantly during both hypoxia and H2 S exposures. No effect was observed in the rats. Hypoxia stimulated breathing in the mice and the rats. The much smaller increase in ventilation in the mice yielded to the same V˙ E/V˙ O2 as in the rats. During H2 S exposure, ventilation appeared to follow the changes in metabolism. In the absence of metabolic effects in rats no modification of breathing was observed, while ventilation dropped dramatically in the mice.
As a result, the V˙ E/V˙ O2 ratio markedly differs during exposure to the two gases. V˙ E/V˙ O2 increased significantly during exposure to hypoxia (from 20 ± 2 to 36 ± 2) reflecting the relative stimulation of breathing. V˙ E/V˙ O2 did not rise during exposure to H2 S, a result of the parallel depression V˙ E and V˙ O2 (21 ± 3). Finally the animal core temperature at the end of the test decreased by 1.2 ± 0.5 vs. 1.1 ± 0.6 degrees during H2 S and hypoxia exposures, respectively. 3.2. Metabolic and ventilatory response to H2S and hypoxia in rats 3.2.1. Metabolic response In contrast to the 20 g mice, the 700 g rats did not display any significant changes in V˙ O2 during exposure to 10% O2 or 60 ppm H2 S, as shown in Figs. 2 and 3 (p = 0.180). V˙ O2 averaged 8.3 ± 2.0 ml min−1 in air breathing, while during exposure to hypoxia alone, V˙ O2 was 7.6 ± 1.2 ml min−1 (a change of −7 ± 11%, NS). This lack of effect of
hypoxia on metabolism in rats provided a striking contrast to the nearly 50% decrease in metabolism we observed in mice. Similarly, during H2 S exposure, the rats demonstrated no significant change in V˙ O2 (7.3 ± 1.2 ml/min vs. 7.0 ± 1.8 ml min−1 , a change of −4 ± 11%, NS). Body core temperature was not different immediately after any of the conditions, and averaged 34.7 ◦ C.
3.2.2. Ventilatory response As illustrated in Fig. 3, during hypoxia ventilation increased in all tests and in a square-like manner from 308 ± 46 ml min−1 to 501 ± 86 ml min−1 (an increase of +64 ± 27%). Breathing frequency increased from 81 ± 10 to 136 ± 17 br min−1 (p < 0.01) with a very similar temporal profile (Fig. 3). V˙ E/V˙ O2 increased from 26.4 ± 4.7 to 46. 5 ± 8.9. During H2 S exposure, in marked contrast with the stimulatory effect of hypoxia, no changes in either ventilation (253 ± 63 vs 258 ± 73 ml min−1 ; 1 ± 9%, NS) or breathing frequency (72 ± 8 vs 77 ± 8 br min−1 ; NS) occurred.
Fig. 3. Example of the time course of ventilatory flow (V˙ ), breathing frequency (f) minute ventilation (V˙ E, averaged every 15 s) and oxygen uptake (V˙ O2 ) responses to inhalation of 60 ppm H2 S (left panels) and 10% O2 (right panels) in the same rat. In marked contrast to the effects observed in the mice, there was no effect of H2 S on metabolism or on breathing. During hypoxia, breathing frequency and ventilation were stimulated in a square like manner without any visible depression in metabolic rate.
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Fig. 4. Representative example ventilatory flow (V˙ ), breathing frequency (f), minute ventilation (V˙ E, averaged every 15 s) and oxygen uptake (V˙ O2 ) in the baseline condition (extreme left panels), in response to the inhalation of 60 ppm H2 S, 10% O2, a mixture of 60 ppm H2 S and 10% O2 and recovery (extreme right panels) in the same mouse. A, B, and C panels show the enlarged flow trace indicated in brackets. As in Fig. 1, H2 S provoked a large depression in metabolic rate, breathing frequency, and minute ventilation, while only V˙ O2 was reduced in hypoxia with a relative hyperventilation. When both conditions were applied simultaneously (10% O2 + 60 ppm H2 S), the reduction in metabolic rate was more pronounced than in hypoxia alone, and the relative hyperventilation was replaced by a depression in breathing which was even larger than during H2 S alone.
Fig. 5. Left panel: Responses in metabolic rate (mean ± SD) during hypoxia, H2 S and hypoxia + H2 S mixture in the mice. Note that the metabolic depression induced by hypoxia was not reduced when the animals were also exposed to H2 S. Right panel: Ventilatory responses (mean ± SD) in the same condition as in the left panel. Note that the ventilatory stimulation (relative to the reduction in V˙ O2 ) was abolished and replaced by a ventilatory depression when H2 S was added. Intriguingly, the ventilatory depression was much larger than that occurred during H2 S exposure alone.
3.3. Effect of combining H2 S and hypoxia in mice A simultaneous combination of H2 S and hypoxia provoked an additional depression in V˙ O2 when compared to hypoxia alone, as illustrated in Figs. 4 and 5. For instance, when comparisons are made at the time when the reduction in V˙ O2 is similar during both hypoxia and H2 S, that is 15 min into exposure, V˙ O2 averaged 0.99 ± 0.12 ml min−1 during hypoxia exposure, 0.94 ± 0.08 ml min−1 during H2 S alone and 0.46 ± 0.07 ml min−1 (P < 0.01) with the two gases combined (Fig. 5). The ventilatory component of the response to both gases consisted of a suppression of the relative ventilatory stimulation observed during hypoxia alone, and a paradoxically more pronounced ventilatory depression (Fig. 4). Not only was the increase in breathing frequency normally provoked by hypoxia (from 198 ± 73 br min−1 to 235 ± 86 br min−1 )
abolished, but f was lower with the mixture of gases than with H2 S alone (37 ± 12 vs 76 ± 13 br min−1 , p < 0.01). As a result, the V˙ E/V˙ O2 ratio, which increased during hypoxic exposure returned to baseline values when H2 S was combined with hypoxia (23 ± 5). 4. Discussion This study was designed to clarify the possible link between H2 S and hypoxia with respect to respiratory and metabolic control in mammals. Endogenous H2 S is produced in most tissues, including the brain (Wang, 2002; Kamoun, 2004a). More recent experimental data suggest that some of the effects of hypoxia may be related to an increase in endogenous H2 S concentration. In the trout, these responses include the hypoxic relaxation of the urinary bladder, the hypoxic vasoactive response of pulmonary artery vessels, and the
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cardiovascular response to gill chemoreceptors (Dombkowski et al., 2004; Dombkowski et al., 2006; Olson et al., 2006; Olson, 2008; Olson et al., 2008a; Olson et al., 2008b). These conclusions were based on a series of observations that can be summarized as follows: Firstly, similar vascular response can be produced by hypoxia and H2 S. Secondly, H2 S concentration increases in hypoxia. Thirdly, blocking H2 S production, though the blockade of CSE activity by exogenous H2 S, blunts or inhibits the vascular response to hypoxia. The question is, therefore, whether or not H2 S contributes to O2 sensing in mammals.
lation. The cessation of breathing occurs well before cardiac arrest (apnea appears after no more than few tens of seconds of exposure to H2 S at concentrations at or above 500 ppm) (Haggarg and Henderson, 1922). H2 S at high concentrations affects pulmonary receptors and the central pattern generator for breathing (Greer et al., 1995; Almeida and Guidotti, 1999), in contrast, at low concentration, we did not see any changes in the breathing pattern in the rats.
4.1. The arterial chemoreflex and H2 S
H2 S depresses both metabolism and ventilation (Blackstone et al., 2005; Haouzi et al., 2008), but this effect is only observed in systems wherein hypoxia depresses metabolism and ventilation as well. This effect of H2 S appears to occur at inhaled concentrations that would not impede oxidative phosphorylation (Dorman et al., 2002). The mediator of such a highly sensitive O2 (and H2 S) sensing mechanism essential to metabolic regulation is still not known. Does the fact that H2 S and hypoxia elicit the same depressive effect reflect some type of causal link? Or do H2 S and hypoxia activate a common final mechanism? As presented in the first paragraph of the discussion, the hypothesis that hypoxia increases H2 S production and that the latter can account for the physiological effects of hypoxia has some experimental basis. One of our present results is, however, not supportive of a causal relationship that could involve an increase in endogenous production of H2 S: exogenous H2 S exposure, does not blunt the metabolic response to hypoxia. This appears to dramatically contrast the effects of hypoxia reported for other tissues (Olson et al., 2006), where exogenous H2 S, thought to depress the endogenous enzymatic synthesis of H2 S, abolished the vascular response to hypoxia. Surprisingly, we found that combining H2 S and hypoxia 1- blocked the stimulatory effect of hypoxia, typically mediated by the arterial chemoreceptors, 2-produced a larger ventilatory depression than occurred during normoxic exposures to H2 S. Therefore, our original hypothesis that it is the increase in H2 S synthesis which can transduce the effect of hypoxia through an activation of the brain CBS, is not validated by the present results. This contention is, however,only valid if we assume that the synthesis of H2S is indeed impeded by exogenous H2S (Olson et al., 2006). In addition, if H2 S is to be regarded as an O2 ‘sensor’ through an increase in its concentration during hypoxia, it could also operate through a reduction in the rate of H2 S elimination and/or enhancement of H2 S production but through a “non-CBS pathway” (Kamoun, 2004a).
Our present results show that H2 S does not contribute towards hypoxia-induced arterial chemoreceptor stimulation. In neither species did we observe a stimulation of breathing with H2 S at concentrations which were able to mimic the central effects of hypoxia. Indeed, in mice the response to H2 S consisted solely of a depression of breathing proportional to the drop in metabolic rate, without any increase in V˙ E/V˙ O2 ratio in marked contrast to hypoxia. In the rats, ventilation and metabolism were not affected at all while breathing 60 ppm H2 S. 4.2. Depressive effect of hypoxia on metabolic and ventilatory control and H2 S In small-sized mammals hypoxia reduces the metabolic rate, which in turn opposes the stimulatory effects of breathing mediated by the carotid bodies (Mortola, 1993). Hypoxia-induced hypometabolism is thought to rely in part upon a neural mechanism originating in subthalamic brain regions (Hinrichsen et al., 1998), which activates the efferent autonomic nervous system to the brown adipose tissue (Gautier, 1996). The net effect is, however, an increase in V˙ E/V˙ O2 ratio, due to a relative hyperventilation produced by the arterial chemoreflex. In other words, as well documented by numerous studies, small mammals display a large increase in V˙ E/V˙ O2 ratio by mainly decreasing the V˙ O2 component of this ratio, whereas larger animals increase V˙ E without affecting V˙ O2 (Mortola, 1993; Mortola, 2004). At baseline, the specific metabolic rate (V˙ O2 kg−1 ) was six times larger in the mice (63 ml kg−1 min−1 ) than in our large rats (11.9 ± 1.7 ml kg−1 min−1 ). The ability to depress non-ATP related metabolic rate during acute hypoxia has long been recognized and has been shown to be related to the baseline metabolism per unit of weight (Frappell et al., 1992; Frappell and Butler, 2004). The latter increases dramatically in the smallest mammals in keeping with the law of Keller (Nagy, 2005; White and Seymour, 2005). Mice are not the only species which significantly depresse their metabolic rate in hypoxia (Frappell et al., 1992); neonates and premature human infants display the same type of response (Hill, 1959). Exposure to H2 S in the mice did reproduce the central depressive effect of hypoxia on both metabolism and breathing but without any stimulatory component of the chemoreceptors, so that the V˙ E/V˙ O2 ratio was not affected. In the rats, however, the absence of depressive effects of hypoxia was associated with a lack of effect of H2 S. This agrees with our previous data showing that a low concentration of H2 S does depress metabolism but only in small-sized mammals, as does hypoxia (Haouzi et al., 2008). In large mammals, including humans, the effect of H2 S on ventilatory control has mostly been investigated in the context of accidental exposure to lethal concentrations (Haggarg and Henderson, 1922; Greer et al., 1995; Almeida and Guidotti, 1999), i.e. when H2 S blocks cytochrome C oxidase activity (Reiffenstein et al., 1992). Special attention was given to the effects on breathing, since death typically occurs by an irreversible cessation of venti-
4.3. Respiratory effects of Hypoxia and H2 S: is there a causal link?
4.4. Final remarks Hypoxia and H2 S share a common effect on metabolism and temperature regulation in small mammals, both of them reduce the metabolic rate and ventilation. The mechanisms depressing breathing was capable of opposing hypoxia-induced hyperventilation, when H2 S was also present. The striking similarity between the “central” effects of these two gases requires a careful analysis of their common cellular and molecular targets, if the mechanisms transducing the central effect of hypoxia are to be clarified. This descriptive study was unable to really address the role of H2 S and CBS-O2 interaction as a possible key mechanism in the control of metabolism. We only found that exogenous H2 S did not reduce the “central” response to hypoxia, but blocked the “peripheral” one. We must acknowledge that this does not fully address this question on the possible contribution of CBS in these responses. CBS which allows the formation of H2 S in the central nervous system is a large and complex protein (Ignoul and Eggermont, 2005) which intriguingly comprises hem motifs (Meier et al., 2001), which physiological functions are far from clear. Carbon monoxide has a strong affinity with the CBS hem motif and does alter
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the enzymatic property of CBS (for review, Kamoun, 2004b). Do O2 and H2 S (Wang, 2002) have a similar effect on CBS activity? Whether other properties of CBS than those related to the formation of H2 S can be involved in temperature and metabolism regulation is an outstanding question that we should now clarify. In conclusion, we found that unlike during hypoxia, ventilation was not stimulated in small or large rodents by exposure to exogenous H2 S. During H2 S exposure, depressive effects on metabolism which was observed only in mice, and not in the larger rats, were associated with a major depression in ventilation and breathing frequency. Finally, in the mice, when hypoxia was combined with H2 S, the ventilatory stimulus component of the hypoxic response was abolished while there was an additive effect on the metabolic depression. H2 S does not mediate the effect of hypoxia on the arterial chemoreceptors. The interaction between H2 S, hypoxia and CBS activity should be clarified, since the depressive effects of H2 S on breathing appears to be linked to the acute reduction metabolic rate of small-sized mammals through a powerful and very sensitive mechanism, as is the case with hypoxia. Finally, it remains to be determined as to whether or not our present results are applicable to humans in the neonatal period and to other species capable of hypoxia-induced metabolic depression. Acknowledgements The authors are grateful to Valerie Kehoe and Carrie Fergusson for her precious technical help. References Abe, K., Kimura, H., 1996. The possible role of hydrogen sulfide as an endogenous neuromodulator. J. Neurosci. 16, 1066–1071. Almeida, A.F., Guidotti, T.L., 1999. Differential sensitivity of lung and brain to sulfide exposure: a peripheral mechanism for apnea. Toxicol. Sci. 50, 287–293. Bell, H.J., Ferguson, C., Kehoe, V., Haouzi, P., 2009. Hypocapnia increases the prevalence of hypoxia-induced augmented breaths. Am. J. Physiol. Regul. Integr. Comp. Physiol. 296, R334–344. Bhatia, M., 2005. Hydrogen sulfide as a vasodilator. IUBMB Life 57, 603–606. Bhatia, M., Sidhapuriwala, J., Moochhala, S.M., Moore, P.K., 2005. Hydrogen sulphide is a mediator of carrageenan-induced hindpaw oedema in the rat. Br. J. Pharmacol. 145, 141–144. Blackstone, E., Morrison, M., Roth, M.B., 2005. H2S induces a suspended animationlike state in mice. Science 308, 518. Cheng, Y., Ndisang, J.F., Tang, G., Cao, K., Wang, R., 2004. Hydrogen sulfide-induced relaxation of resistance mesenteric artery beds of rats. Am. J. Physiol. Heart Circ. Physiol. 287, H2316–2323. Dombkowski, R.A., Doellman, M.M., Head, S.K., Olson, K.R., 2006. Hydrogen sulfide mediates hypoxia-induced relaxation of trout urinary bladder smooth muscle. J. Exp. Biol. 209, 3234–3240. Dombkowski, R.A., Russell, M.J., Olson, K.R., 2004. Hydrogen sulfide as an endogenous regulator of vascular smooth muscle tone in trout. Am. J. Physiol. Regul. Integr. Comp. Physiol. 286, R678–685. Dominy, J.E., Stipanuk, M.H., 2004. New roles for cysteine and transsulfuration enzymes: production of H2S, a neuromodulator and smooth muscle relaxant. Nutr. Rev. 62, 348–353. Dorman, D.C., Moulin, F.J., McManus, B.E., Mahle, K.C., James, R.A., Struve, M.F., 2002. Cytochrome oxidase inhibition induced by acute hydrogen sulfide inhalation:
correlation with tissue sulfide concentrations in the rat brain, liver, lung, and nasal epithelium. Toxicol. Sci. 65, 18–25. Frappell, P., Lanthier, C., Baudinette, R.V., Mortola, J.P., 1992. Metabolism and ventilation in acute hypoxia: a comparative analysis in small mammalian species. Am. J. Physiol. 262, R1040–1046. Frappell, P., Saiki, C., Mortola, J.P., 1991. Metabolism during normoxia, hypoxia and recovery in the newborn kitten. Respir. Physiol. 86, 115–124. Frappell, P.B., Butler, P.J., 2004. Minimal metabolic rate, what it is, its usefulness, and its relationship to the evolution of endothermy: a brief synopsis. Physiol. Biochem. Zool. 77, 865–868. Gautier, H., 1996. Interactions among metabolic rate, hypoxia, and control of breathing. J. Appl. Physiol. 81, 521–527. Greer, J.J., Reiffenstein, R.J., Almeida, A.F., Carter, J.E., 1995. Sulfide-induced perturbations of the neuronal mechanisms controlling breathing in rats. J. Appl. Physiol. 78, 433–440. Haggarg, H.W., Henderson, Y., 1922. The influence of hydrogen sulphide upon respiration. Am. J. Physiol. 41, 289–297. Haouzi, P., Notet, V., Chenuel, B., Chalon, B., Sponne, I., Ogier, V., Bihain, B., 2008. H2S induced hypometabolism in mice is missing in sedated sheep. Respir. Physiol. Neurobiol. 160, 109–115. Hill, J.R., 1959. The oxygen consumption of new-born and adult mammals Its dependence on the oxygen tension in the inspired air and on the environmental temperature. J. Physiol. 149, 346–373. Hinrichsen, C.F., Maskrey, M., Mortola, J.P., 1998. Ventilatory and metabolic responses to cold and hypoxia in conscious rats with discrete hypothalamic lesions. Respir. Physiol. 111, 247–256. Ignoul, S., Eggermont, J., 2005. CBS domains: structure, function, and pathology in human proteins. Am. J. Physiol. Cell Physiol. 289, C1369–1378. Kamoun, P., 2004a. Endogenous production of hydrogen sulfide in mammals. Amino Acids 26, 243–254. Kamoun, P., 2004b. H2S, a new neuromodulator. Med. Sci. (Paris) 20, 697–700. Kimura, Y., Kimura, H., 2004. Hydrogen sulfide protects neurons from oxidative stress. Faseb. J. 18, 1165–1167. Kredich, N.M., Foote, L.J., Keenan, B.S., 1973. The stoichiometry and kinetics of the inducible cysteine desulfhydrase from Salmonella typhimurium. J. Biol. Chem. 248, 6187–6196. Meier, M., Janosik, M., Kery, V., Kraus, J.P., Burkhard, P., 2001. Structure of human cystathionine beta-synthase: a unique pyridoxal 5 -phosphate-dependent heme protein. Embo. J. 20, 3910–3916. Mortola, J.P., 1993. Hypoxic hypometabolism in mammals. News Physiol. Sci. 8, 79–82. Mortola, J.P., 2004. Implications of hypoxic hypometabolism during mammalian ontogenesis. Respir. Physiol. Neurobiol. 141, 345–356. Mortola, J.P., Matsuoka, T., Saiki, C., Naso, L., 1994. Metabolism and ventilation in hypoxic rats: effect of body mass. Respir. Physiol. 97, 225–234. Nagy, K.A., 2005. Field metabolic rate and body size. J. Exp. Biol. 208, 1621–1625. Olson, K.R., 2008. Hydrogen sulfide and oxygen sensing: implications in cardiorespiratory control. J. Exp. Biol. 211, 2727–2734. Olson, K.R., Dombkowski, R.A., Russell, M.J., Doellman, M.M., Head, S.K., Whitfield, N.L., Madden, J.A., 2006. Hydrogen sulfide as an oxygen sensor/transducer in vertebrate hypoxic vasoconstriction and hypoxic vasodilation. J. Exp. Biol. 209, 4011–4023. Olson, K.R., Forgan, L.G., Dombkowski, R.A., Forster, M.E., 2008a. Oxygen dependency of hydrogen sulfide-mediated vasoconstriction in cyclostome aortas. J. Exp. Biol. 211, 2205–2213. Olson, K.R., Healy, M.J., Qin, Z., Skovgaard, N., Vulesevic, B., Duff, D.W., Whitfield, N.L., Yang, G., Wang, R., Perry, S.F., 2008b. Hydrogen sulfide as an oxygen sensor in trout gill chemoreceptors. Am. J. Physiol. Regul. Integr. Comp. Physiol. 295, R669–680. Reiffenstein, R.J., Hulbert, W.C., Roth, S.H., 1992. Toxicology of hydrogen sulfide. Annu. Rev. Pharmacol. Toxicol. 32, 109–134. Simpson, R.C., Freedland, R.A., 1976. Fractors affecting the rate of gluconeogenesis from L-cysteine in the perfused rat liver. J. Nutr. 106, 1272–1278. Wang, R., 2002. Two’s company three’s a crowd: can H2S be the third endogenous gaseous transmitter? Faseb. J. 16, 1792–1798. White, C.R., Seymour, R.S., 2005. Allometric scaling of mammalian metabolism. J. Exp. Biol. 208, 1611–1619.
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Respiratory Physiology & Neurobiology journal homepage: www.elsevier.com/locate/resphysiol
Comparison of the metabolic and ventilatory response to hypoxia and H2 S in unsedated mice and rats Philippe Haouzi a,b,∗ , Harold J. Bell a , Veronique Notet c , Bernard Bihain c a b c
Pennsylvania State University, College of Medicine, Heart and Vascular Institute, Hershey, USA Division of Pulmonary Medicine, Department of Medicine, Penn State Milton Hershey Medical Center, Hershey, USA Laboratoire de Médecine et Thérapeutique Moléculaire, Genclis SAS, Vandoeuvre les Nancy, France
a r t i c l e
i n f o
Article history: Accepted 9 June 2009 Keywords: Arterial chemoreception Thermoregulation Oxygen transduction
a b s t r a c t Hypoxia alters the control of breathing and metabolism by increasing ventilation through the arterial chemoreflex, an effect which, in small-sized animals, is offset by a centrally mediated reduction in metabolism and respiration. We tested the hypothesis that hydrogen sulfide (H2 S) is involved in transducing these effects in mammals. The rationale for this hypothesis is twofold. Firstly, inhalation of a 20–80 ppm H2 S reduces metabolism in small mammals and this effect is analogous to that of hypoxia. Secondly, endogenous H2 S appears to mediate some of the cardio-vascular effects of hypoxia in nonmammalian species. We, therefore, compared the ventilatory and metabolic effects of exposure to 60 ppm H2 S and to 10% O2 in small and large rodents (20 g mice and 700 g rats) wherein the metabolic response to hypoxia has been shown to differ according to body mass. H2 S and hypoxia produced profound depression in metabolic rate in the mice, but not in the large rats. The depression was much faster with H2 S than with hypoxia. The relative hyperventilation produced by hypoxia in the mice was replaced by a depression with H2 S, which paralleled the drop in metabolic rate. In the larger rats, ventilation was stimulated in hypoxia, with no change in metabolism, while H2 S affected neither breathing nor metabolism. When mice were simultaneously exposed to H2 S and hypoxia, the stimulatory effects of hypoxia on breathing were abolished, and a much larger respiratory and metabolic depression was observed than with H2 S alone. H2 S had, therefore, no stimulatory effect on the arterial chemoreflex. The ventilatory depression during hypoxia and H2 S in small mammals appears to be dependent upon the ability to decrease metabolism. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Over the last decade, solid evidence has identified hydrogen sulfide (H2 S) as being a gasotransmitter (Abe and Kimura, 1996; Wang, 2002; Dominy and Stipanuk, 2004; Kamoun, 2004b). Endogenous production of H2S uses l-cysteine as the main substrate and is mainly under the control of the 2 different enzymes: cystathionine ß-synthase (CBS) in the central nervous system and cystathionine gama lyase (CSE) in the vessels or other tissues (Wang, 2002; Kamoun, 2004a). Relatively high concentration of endogenous H2 S has been reported in many tissues including the central nervous system (for review Wang, 2002). Although the regulation of the production and degradation of endogenous H2 S in mammalian species is still not fully understood, beneficial as well as adverse effects of endogenous H2 S accumulation have been reported (Wang, 2002;
∗ Corresponding author at: Pennsylvania State University, Penn State Hershey Medical Center, Heart and Vascular Institute, 500 University Dr., Hershey, PO Box 850, MC H047, PA, 17033-0850, Tel.: +1 717 531 0003x287518; fax: +1 717 531 0224. E-mail address: [email protected] (P. Haouzi). 1569-9048/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.resp.2009.06.006
Cheng et al., 2004; Dominy and Stipanuk, 2004; Kimura and Kimura, 2004; Bhatia, 2005; Bhatia et al., 2005). Finally, H2 S has been shown to play the role of gaseous neuromodulator (Abe and Kimura, 1996). We have recently suggested that H2 S could also be important in mediating the well-described effects of hypoxia on ventilatory and metabolic control in small mammals (Haouzi et al., 2008). For example, the response to breathing a non-toxic concentration of H2 S mimics the metabolic depression which is triggered by hypoxia in mice (Blackstone et al., 2005). Interestingly, this metabolic depression in response to H2 S is not observed in larger mammals (Haouzi et al., 2008), and this is also consistent with the metabolic response to hypoxia (Frappell et al., 1991; Frappell et al., 1992; Mortola, 1993; Gautier, 1996). Hypoxia has been shown to increase H2 S concentration in the pulmonary vessels (Olson et al., 2006), and during hypoxemia sulfide concentration increases in the blood (Olson et al., 2006; Olson et al., 2008a). The pharmacological blockade of CSE activity, the enzyme allowing the transformation of cysteine to H2 S in the vessels, is associated with a severely blunted hypoxia-induced vasoactive response in various types of vessels (Olson et al., 2006; Olson, 2008; Olson et al., 2008a). A similar vascular effect of blocking CSE activity has been reported in the trout
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(Dombkowski et al., 2004). The vascular effect of hypoxia was virtually abolished during a simultaneous exposure to exogenous H2 S, which in turn depresses the activity of CSE and thus endogenous H2 S production. Finally, H2 S has stimulatory effect on the activity of the gill chemoreceptors in fish (Olson et al., 2008b). Collectively, these data suggest that hypoxia increases the concentration of endogeneous H2 S, and that this increase in endogenous H2 S levels mediates the vacular effects of hypoxia (Olson, 2008). Whether the phenomena observed in fish or in-vitro models are applicable to other mammalian tissues and directly apply to the respiratory and metabolic response to hypoxia in mammals remains unknown. The goal of this study is to determine whether H2 S is involved in transducing the known ventilatory effects of hypoxia in mammals. More specifically, we investigated the role of H2 S in two of the most predominant physiological responses to acute hypoxia in mammals: (1) the increase in breathing mediated by the arterial chemoreceptors, and (2) the reduction in ventilation associated with the hypoxia-induced depression of metabolic rate. While the stimulation of breathing during hypoxia is common to all mammals, regardless of body size, the metabolic depression induced by hypoxia is a strategy developed early in evolution but which is only observed in small mammals (Frappell et al., 1992; Mortola et al., 1994). This latter response is associated with a relative reduction of breathing proportional to the reduction in metabolic rate, which counteracts the stimulatory effects of hypoxia-induced chemoreceptor stimulation. Nevertheless, V˙ E/V˙ O2 ratio increases by approximately the same amount in all species during hypoxia, regardless of their body size (Mortola, 1993). We therefore compared the ventilatory response to 10% hypoxia and 60 ppm H2 S in two rodent species of different body mass in the very same experimental conditions. 20 g unsedated mice were studied as a model of small-sized mammals, where both hypoxia and H2 S have been shown to depress metabolism. A similar approach was applied in a group of 700 g unsedated rats in which the response to hypoxia consists almost exclusively in a ventilatory stimulation with no or little depressive effect of hypoxia on metabolism (Mortola et al., 1994). We speculate that if H2 S is involved in transducing the effects of hypoxia, through an increase in H2 S production, one should expect the ventilatory and metabolic responses to H2 S and hypoxia to be similar. In addition, if, as previously suggested, exogenous H2 S can inhibit both the CBS and CSE by exerting a negative feedback effect on the activity of these enzymes (Kredich et al., 1973; Simpson and Freedland, 1976; Wang, 2002), the response to hypoxia should be blunted during a simultaneous exposure to H2 S. This has been demonstrated for other effects of hypoxia, which appear to be mediated through the CSE activity in the vessels (Olson et al., 2006).
2. Methods 2.1. Animals and equipment The experimental procedures were performed on five adult female C57BL/6J mice (24.05 ± 1.09 g, 45–47 weeks old) and on five adult male Sprague–Dawley rats (701 ± 46 g, 40–42 weeks old). All experiments using mice were performed in the Laboratoire de Médecine et Thérapeutique Moléculaire, Genclis, Univeristy of Nancy, France, according to the recommendations of the Council of European Communities and with the authorization from the French Ministry of Agriculture and Fisheries (Authorization number 54-42). The experiments and procedures performed using rats were performed in the facilities of the Penn State University College of Medicine, and were approved under the Penn State Hershey Institutional Animal Care and Use Committee, protocol # 2007–168.
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The metabolic and respiratory variables were measured via unrestrained whole body open-flow plethysmography in customdesigned animal chambers and air control circuits as previously described (Haouzi et al., 2008; Bell et al., 2009) The animal chambers were sealed, leak-proof acrylic cylinders designed according to the animal size. The size and shape of the chambers were defined to minimize the mixing time constant of the chamber for ventilation and gas exchange determination. For the mice, the chamber consisted of a plastic tube of low volume (90 cm3 ) with a relatively long length (11 cm). For the rats, we used a chamber with internal volume of 1400 cm3 (diameter 120 mm). The chamber was of sufficient volume that air could pass freely around all sides of the animal without interference as previously described. Animals were acclimatized to the chambers through routinely spending 2–3 h several times a week in them without any signs of anxiety or stress. Various compositions of fresh gas from dry, pre-mixed, high pressure tanks (20.9% O2 , 10% O2 , 60 ppm H2 S balanced with air or 10% O2 ) were passed through regulators and delivered to the animal chamber through the inlet port which was also equipped with a diffuser to distribute fresh gas evenly throughout the crosssectional area. The flow of gas that was typically used averaged 300–400 ml min−1 for the mice and 2.5 l min−1 for the rats. The chamber was exhausted through the outlet port which passed gas into low-pressure non-compliant CPVC tubing. The flow of air through the animal chamber was continuously monitored via a Fleisch 000 pneumotachograph which was interfaced to a pressure transducer (Sensym, DCLX O1DN, Honeywell, Morristown, NJ, USA) housed in a custom-designed electronic demodulator. CO2 and O2 levels were continuously measured in the air exiting the animal chamber (infrared, and fuel cell analysers respectively, Vacumed, Ventura CA, USA) using sampling catheters distal to the outlet of the chamber. The composition of the gas entering the chamber was measured sequentially as needed using a circuit which bypassed the animal chamber. Temperature in the animal chamber (Tc ) was continuously monitored via a fast-responding thermocouple (BAT12, Physiotemp instruments Inc, Clifton, NJ, USA for the mice, and Thermalert TH5, Physiotemp, Clifton, NJ, USA for rats). Temperature was maintained at ∼25.% C for the mice and ∼27.5 ◦ C for the rats. For the mice, analog signals from the pneumotachograph, CO2 and O2 analyzers, temperature sensor were fed into a Powerlab system (ADInstruments, Colorado Springs, CO, USA). For the rats, analog signals were fed to a 14-bit A/D converter (USB6009, National Instruments, Austin, TX, USA), which was interfaced with an Intel/Windows Vista based computer system (Compaq 8510w, Hewlett Packard, Palo Alto, CA, USA) running custom-written data acquisition software (LabView, National Instruments, Austin, TX, USA – source code available upon request). Analog signals were sampled at 200 s−1 and displayed in raw form on the monitor while being streamed to storage (1 block every 5 s) for subsequent analysis. ASCII data files were converted to ‘.adicht’ format for later visualization and analysis using Chart software (version 5.5.4, ADInstruments, Colorado Springs, CO, USA). To calculate respiratory variables from the raw flow trace, the signal was treated using a high-pass filter (>0.5 Hz) to determine and subtract the DC component. The resulting plethysmographic trace, without the DC component, contained the respiratory signal which was used for calculations of breathing frequency (f), and to obtain an index of minute ventilation (V˙ E). To obtain an index of V˙ E from the filtered flow signal, the positive deflections in the plethysmographic trace were integrated over 15 s intervals. The result of this integration was then temperature corrected based upon the difference between ambient temperature in the chamber as determined continuously throughout the experiments, and the estimated body core temperature (Tb ) of the animal as determined via rectal placement of a digital thermometer immediately after the completion of the experiment. Due to the complexity
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of factors involved in quantitative interpretation of an open flow plethysmographic signal, this determination of V˙ E provided a semiquantitative index represented in the same units as are appropriate for direct measurements of ventilation (Bell et al., 2009).V˙ O2 and V˙ CO2 . were computed in STPD conditions as:
The effects of each gas were analyzed using an analysis of variance for each of the following parameters V˙ O2 , ventilation and breathing frequency. The a priori value for acceptability of a type I (a) error in any statistical comparison was set to 0.05. 3. Results
V˙ O2 = V˙ inlet.FIO2 − V˙ outlet.FEO2
3.1. Metabolic and ventilatory response to H2 S and hypoxia in the mice
and V˙ CO2 = V˙ outlet.FECO2 − V˙ inlet.FICO2 Since only V˙ outlet was measured, V˙ inlet was determined as: V˙ inlet = V˙ outlet.(1 − FEO2 − FECO2 )/(1 − FIO2 − FICO2 ) to take into account the difference between V˙ inlet and V˙ outlet that would result from the gas exchange ratio (soda lime could not be used to eliminate FICO2 , since soda lime traps H2 S). V˙ O2 and V˙ CO2 were determined and expressed in STPD conditions 2.2. Protocol and data analysis Mice were placed in the open chamber for about half an hour before starting data acquisition. Thirty minutes of baseline condition were then recorded before the animals were exposed to either H2 S or hypoxia for 30 min. As previously discussed (Haouzi et al., 2008), criteria for baseline values are not simple to establish but are essential to discuss when determining whether H2 S or hypoxia can decrease the rate of gas exchange and ventilation. For instance, mice displayed unpredictable patterns of activity. Therefore it becomes crucial to define as precisely as possible the nature of the baseline. We used for baseline a period that could be sustained for at least 5 min during which the mice remained quiet and still, based upon visual examination and trivial spontaneous variations in metabolic rate (Haouzi et al., 2008). Rats were also placed in the chamber for approximately 30 min before the start of data collection. Typically, rats displayed much less fluctuation in their metabolic and ventilatory activity and so for rat experiments, baseline data were simply obtained using a 5 min observation window beginning between 5 and 15 min after the start of data collection in room air conditions (Bell et al., 2009). A specific observation window was selected such that unwanted sniffing and grooming behaviors were largely absent in the respiratory traces. Data were averaged and expressed as mean ± SD. V˙ O and V˙ CO 2
2
were expressed in ml min−1 or ml min−1 kg−1 and their changes were expressed in absolute and in% changes.
3.1.1. Metabolic response The metabolic response to 60 ppm H2 S consisted of a major and rapid reduction in V˙ O2 . As shown in Fig. 1, V˙ O2 reached its nadir 295 ± 15 s into the H2 S exposure, decreasing from 1.37 ± 0.09 to 0.57 ± 0.07 ml min−1 (−58 ± 5%, p < 0.001). V˙ O2 had then the tendency to slightly rise over time reaching a steady value averaging 0.94 ± 0.04 ml min−1 (−31 ± 7%, p < 0.001) by the end of the 30 min exposure. Inhalation of hypoxia (10% O2 ) also produced a reduction in V˙ O2 , however with a slower time course than was observed during H2 S exposure. Indeed V˙ O2 decreased in all tests, with an exponential-like pattern from 1.52 ± 0.15 to 1 ± 0.17 ml min−1 (−34 ± 8%, p < 0.001) at the nadir of the response to H2 S, but V˙ O2 continued to decrease reaching 0.71 ± 0.135 ml min−1 (−53 ± 12%, p < 0.001) at the end of the 30 min exposure. An example is shown in Fig. 1, while average changes are displayed in Fig. 2. 3.1.2. Ventilatory response In contrast to the V˙ O2 response which consisted of a decrease in response to either H2 S or hypoxia, ventilation was affected differently by exposure to the two gases. During H2 S exposure, ventilation decreased by 64 ± 3%, from 34.12 ± 1.51 to 12.41 ± 1.07 ml min−1 (p < 0.01) at the nadir of the metabolic depression, reaching 16.14 ± 1.62 ml min−1 (−53 ± 4%, p < 0.01) at the end of the test. As shown in Fig. 1, breathing frequency was dramatically reduced, decreasing from 204 ± 10 to 53 ± 5 then 70 ± 8 br min−1 at 5 and 30 min, respectively (p < 0.01). In marked contrast, during hypoxia ventilation increased from 29.4 ± 3.72 to 36.06 ± 5.67 ml min−1 (+23 ± 19%, p < 0.01) at 5 min then return to baseline ventilatory values 26.39 ± 2.03 ml min−1 , when the metabolic rate was at the lowest point (Fig. 2). Breathing frequency paralleled the change in total ventilation as f increased from 176 ± 35 to 256 ± 43 br min−1 at 5 min (p < 0.01) reaching 200 ± 55 breaths min−1 at 30 min.
Fig. 1. Representative example of the time course of ventilatory flow (V˙ ), breathing frequency (f), minute ventilation (V˙ E, averaged every 15 s), and oxygen uptake (V˙ O2 ) responses to inhalation of 60 ppm H2 S (left panels) and 10% O2 (right panels) in the same mouse. Note during H2 S exposure, the large, rapid depression in metabolic rate is associated with a similarly rapid decrease in breathing frequency and minute ventilation. In this example breathing frequency fell to 40 br min−1 during H2 S exposure. During hypoxic exposure, metabolism decreases as well but less rapidly than that during H2 S exposure: there was a progressive exponential-like reduction in metabolic rate. In marked contrast to H2 S, neither ventilation nor breathing frequency was depressed.
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Fig. 2. Average response expressed in% of change from baseline in the group of mice (open bars) and rats (black bars). Data are shown at the nadir of the response to H2 S in the mice (∼300 s into exposure). In the mice the metabolic rate decreased significantly during both hypoxia and H2 S exposures. No effect was observed in the rats. Hypoxia stimulated breathing in the mice and the rats. The much smaller increase in ventilation in the mice yielded to the same V˙ E/V˙ O2 as in the rats. During H2 S exposure, ventilation appeared to follow the changes in metabolism. In the absence of metabolic effects in rats no modification of breathing was observed, while ventilation dropped dramatically in the mice.
As a result, the V˙ E/V˙ O2 ratio markedly differs during exposure to the two gases. V˙ E/V˙ O2 increased significantly during exposure to hypoxia (from 20 ± 2 to 36 ± 2) reflecting the relative stimulation of breathing. V˙ E/V˙ O2 did not rise during exposure to H2 S, a result of the parallel depression V˙ E and V˙ O2 (21 ± 3). Finally the animal core temperature at the end of the test decreased by 1.2 ± 0.5 vs. 1.1 ± 0.6 degrees during H2 S and hypoxia exposures, respectively. 3.2. Metabolic and ventilatory response to H2S and hypoxia in rats 3.2.1. Metabolic response In contrast to the 20 g mice, the 700 g rats did not display any significant changes in V˙ O2 during exposure to 10% O2 or 60 ppm H2 S, as shown in Figs. 2 and 3 (p = 0.180). V˙ O2 averaged 8.3 ± 2.0 ml min−1 in air breathing, while during exposure to hypoxia alone, V˙ O2 was 7.6 ± 1.2 ml min−1 (a change of −7 ± 11%, NS). This lack of effect of
hypoxia on metabolism in rats provided a striking contrast to the nearly 50% decrease in metabolism we observed in mice. Similarly, during H2 S exposure, the rats demonstrated no significant change in V˙ O2 (7.3 ± 1.2 ml/min vs. 7.0 ± 1.8 ml min−1 , a change of −4 ± 11%, NS). Body core temperature was not different immediately after any of the conditions, and averaged 34.7 ◦ C.
3.2.2. Ventilatory response As illustrated in Fig. 3, during hypoxia ventilation increased in all tests and in a square-like manner from 308 ± 46 ml min−1 to 501 ± 86 ml min−1 (an increase of +64 ± 27%). Breathing frequency increased from 81 ± 10 to 136 ± 17 br min−1 (p < 0.01) with a very similar temporal profile (Fig. 3). V˙ E/V˙ O2 increased from 26.4 ± 4.7 to 46. 5 ± 8.9. During H2 S exposure, in marked contrast with the stimulatory effect of hypoxia, no changes in either ventilation (253 ± 63 vs 258 ± 73 ml min−1 ; 1 ± 9%, NS) or breathing frequency (72 ± 8 vs 77 ± 8 br min−1 ; NS) occurred.
Fig. 3. Example of the time course of ventilatory flow (V˙ ), breathing frequency (f) minute ventilation (V˙ E, averaged every 15 s) and oxygen uptake (V˙ O2 ) responses to inhalation of 60 ppm H2 S (left panels) and 10% O2 (right panels) in the same rat. In marked contrast to the effects observed in the mice, there was no effect of H2 S on metabolism or on breathing. During hypoxia, breathing frequency and ventilation were stimulated in a square like manner without any visible depression in metabolic rate.
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Fig. 4. Representative example ventilatory flow (V˙ ), breathing frequency (f), minute ventilation (V˙ E, averaged every 15 s) and oxygen uptake (V˙ O2 ) in the baseline condition (extreme left panels), in response to the inhalation of 60 ppm H2 S, 10% O2, a mixture of 60 ppm H2 S and 10% O2 and recovery (extreme right panels) in the same mouse. A, B, and C panels show the enlarged flow trace indicated in brackets. As in Fig. 1, H2 S provoked a large depression in metabolic rate, breathing frequency, and minute ventilation, while only V˙ O2 was reduced in hypoxia with a relative hyperventilation. When both conditions were applied simultaneously (10% O2 + 60 ppm H2 S), the reduction in metabolic rate was more pronounced than in hypoxia alone, and the relative hyperventilation was replaced by a depression in breathing which was even larger than during H2 S alone.
Fig. 5. Left panel: Responses in metabolic rate (mean ± SD) during hypoxia, H2 S and hypoxia + H2 S mixture in the mice. Note that the metabolic depression induced by hypoxia was not reduced when the animals were also exposed to H2 S. Right panel: Ventilatory responses (mean ± SD) in the same condition as in the left panel. Note that the ventilatory stimulation (relative to the reduction in V˙ O2 ) was abolished and replaced by a ventilatory depression when H2 S was added. Intriguingly, the ventilatory depression was much larger than that occurred during H2 S exposure alone.
3.3. Effect of combining H2 S and hypoxia in mice A simultaneous combination of H2 S and hypoxia provoked an additional depression in V˙ O2 when compared to hypoxia alone, as illustrated in Figs. 4 and 5. For instance, when comparisons are made at the time when the reduction in V˙ O2 is similar during both hypoxia and H2 S, that is 15 min into exposure, V˙ O2 averaged 0.99 ± 0.12 ml min−1 during hypoxia exposure, 0.94 ± 0.08 ml min−1 during H2 S alone and 0.46 ± 0.07 ml min−1 (P < 0.01) with the two gases combined (Fig. 5). The ventilatory component of the response to both gases consisted of a suppression of the relative ventilatory stimulation observed during hypoxia alone, and a paradoxically more pronounced ventilatory depression (Fig. 4). Not only was the increase in breathing frequency normally provoked by hypoxia (from 198 ± 73 br min−1 to 235 ± 86 br min−1 )
abolished, but f was lower with the mixture of gases than with H2 S alone (37 ± 12 vs 76 ± 13 br min−1 , p < 0.01). As a result, the V˙ E/V˙ O2 ratio, which increased during hypoxic exposure returned to baseline values when H2 S was combined with hypoxia (23 ± 5). 4. Discussion This study was designed to clarify the possible link between H2 S and hypoxia with respect to respiratory and metabolic control in mammals. Endogenous H2 S is produced in most tissues, including the brain (Wang, 2002; Kamoun, 2004a). More recent experimental data suggest that some of the effects of hypoxia may be related to an increase in endogenous H2 S concentration. In the trout, these responses include the hypoxic relaxation of the urinary bladder, the hypoxic vasoactive response of pulmonary artery vessels, and the
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cardiovascular response to gill chemoreceptors (Dombkowski et al., 2004; Dombkowski et al., 2006; Olson et al., 2006; Olson, 2008; Olson et al., 2008a; Olson et al., 2008b). These conclusions were based on a series of observations that can be summarized as follows: Firstly, similar vascular response can be produced by hypoxia and H2 S. Secondly, H2 S concentration increases in hypoxia. Thirdly, blocking H2 S production, though the blockade of CSE activity by exogenous H2 S, blunts or inhibits the vascular response to hypoxia. The question is, therefore, whether or not H2 S contributes to O2 sensing in mammals.
lation. The cessation of breathing occurs well before cardiac arrest (apnea appears after no more than few tens of seconds of exposure to H2 S at concentrations at or above 500 ppm) (Haggarg and Henderson, 1922). H2 S at high concentrations affects pulmonary receptors and the central pattern generator for breathing (Greer et al., 1995; Almeida and Guidotti, 1999), in contrast, at low concentration, we did not see any changes in the breathing pattern in the rats.
4.1. The arterial chemoreflex and H2 S
H2 S depresses both metabolism and ventilation (Blackstone et al., 2005; Haouzi et al., 2008), but this effect is only observed in systems wherein hypoxia depresses metabolism and ventilation as well. This effect of H2 S appears to occur at inhaled concentrations that would not impede oxidative phosphorylation (Dorman et al., 2002). The mediator of such a highly sensitive O2 (and H2 S) sensing mechanism essential to metabolic regulation is still not known. Does the fact that H2 S and hypoxia elicit the same depressive effect reflect some type of causal link? Or do H2 S and hypoxia activate a common final mechanism? As presented in the first paragraph of the discussion, the hypothesis that hypoxia increases H2 S production and that the latter can account for the physiological effects of hypoxia has some experimental basis. One of our present results is, however, not supportive of a causal relationship that could involve an increase in endogenous production of H2 S: exogenous H2 S exposure, does not blunt the metabolic response to hypoxia. This appears to dramatically contrast the effects of hypoxia reported for other tissues (Olson et al., 2006), where exogenous H2 S, thought to depress the endogenous enzymatic synthesis of H2 S, abolished the vascular response to hypoxia. Surprisingly, we found that combining H2 S and hypoxia 1- blocked the stimulatory effect of hypoxia, typically mediated by the arterial chemoreceptors, 2-produced a larger ventilatory depression than occurred during normoxic exposures to H2 S. Therefore, our original hypothesis that it is the increase in H2 S synthesis which can transduce the effect of hypoxia through an activation of the brain CBS, is not validated by the present results. This contention is, however,only valid if we assume that the synthesis of H2S is indeed impeded by exogenous H2S (Olson et al., 2006). In addition, if H2 S is to be regarded as an O2 ‘sensor’ through an increase in its concentration during hypoxia, it could also operate through a reduction in the rate of H2 S elimination and/or enhancement of H2 S production but through a “non-CBS pathway” (Kamoun, 2004a).
Our present results show that H2 S does not contribute towards hypoxia-induced arterial chemoreceptor stimulation. In neither species did we observe a stimulation of breathing with H2 S at concentrations which were able to mimic the central effects of hypoxia. Indeed, in mice the response to H2 S consisted solely of a depression of breathing proportional to the drop in metabolic rate, without any increase in V˙ E/V˙ O2 ratio in marked contrast to hypoxia. In the rats, ventilation and metabolism were not affected at all while breathing 60 ppm H2 S. 4.2. Depressive effect of hypoxia on metabolic and ventilatory control and H2 S In small-sized mammals hypoxia reduces the metabolic rate, which in turn opposes the stimulatory effects of breathing mediated by the carotid bodies (Mortola, 1993). Hypoxia-induced hypometabolism is thought to rely in part upon a neural mechanism originating in subthalamic brain regions (Hinrichsen et al., 1998), which activates the efferent autonomic nervous system to the brown adipose tissue (Gautier, 1996). The net effect is, however, an increase in V˙ E/V˙ O2 ratio, due to a relative hyperventilation produced by the arterial chemoreflex. In other words, as well documented by numerous studies, small mammals display a large increase in V˙ E/V˙ O2 ratio by mainly decreasing the V˙ O2 component of this ratio, whereas larger animals increase V˙ E without affecting V˙ O2 (Mortola, 1993; Mortola, 2004). At baseline, the specific metabolic rate (V˙ O2 kg−1 ) was six times larger in the mice (63 ml kg−1 min−1 ) than in our large rats (11.9 ± 1.7 ml kg−1 min−1 ). The ability to depress non-ATP related metabolic rate during acute hypoxia has long been recognized and has been shown to be related to the baseline metabolism per unit of weight (Frappell et al., 1992; Frappell and Butler, 2004). The latter increases dramatically in the smallest mammals in keeping with the law of Keller (Nagy, 2005; White and Seymour, 2005). Mice are not the only species which significantly depresse their metabolic rate in hypoxia (Frappell et al., 1992); neonates and premature human infants display the same type of response (Hill, 1959). Exposure to H2 S in the mice did reproduce the central depressive effect of hypoxia on both metabolism and breathing but without any stimulatory component of the chemoreceptors, so that the V˙ E/V˙ O2 ratio was not affected. In the rats, however, the absence of depressive effects of hypoxia was associated with a lack of effect of H2 S. This agrees with our previous data showing that a low concentration of H2 S does depress metabolism but only in small-sized mammals, as does hypoxia (Haouzi et al., 2008). In large mammals, including humans, the effect of H2 S on ventilatory control has mostly been investigated in the context of accidental exposure to lethal concentrations (Haggarg and Henderson, 1922; Greer et al., 1995; Almeida and Guidotti, 1999), i.e. when H2 S blocks cytochrome C oxidase activity (Reiffenstein et al., 1992). Special attention was given to the effects on breathing, since death typically occurs by an irreversible cessation of venti-
4.3. Respiratory effects of Hypoxia and H2 S: is there a causal link?
4.4. Final remarks Hypoxia and H2 S share a common effect on metabolism and temperature regulation in small mammals, both of them reduce the metabolic rate and ventilation. The mechanisms depressing breathing was capable of opposing hypoxia-induced hyperventilation, when H2 S was also present. The striking similarity between the “central” effects of these two gases requires a careful analysis of their common cellular and molecular targets, if the mechanisms transducing the central effect of hypoxia are to be clarified. This descriptive study was unable to really address the role of H2 S and CBS-O2 interaction as a possible key mechanism in the control of metabolism. We only found that exogenous H2 S did not reduce the “central” response to hypoxia, but blocked the “peripheral” one. We must acknowledge that this does not fully address this question on the possible contribution of CBS in these responses. CBS which allows the formation of H2 S in the central nervous system is a large and complex protein (Ignoul and Eggermont, 2005) which intriguingly comprises hem motifs (Meier et al., 2001), which physiological functions are far from clear. Carbon monoxide has a strong affinity with the CBS hem motif and does alter
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the enzymatic property of CBS (for review, Kamoun, 2004b). Do O2 and H2 S (Wang, 2002) have a similar effect on CBS activity? Whether other properties of CBS than those related to the formation of H2 S can be involved in temperature and metabolism regulation is an outstanding question that we should now clarify. In conclusion, we found that unlike during hypoxia, ventilation was not stimulated in small or large rodents by exposure to exogenous H2 S. During H2 S exposure, depressive effects on metabolism which was observed only in mice, and not in the larger rats, were associated with a major depression in ventilation and breathing frequency. Finally, in the mice, when hypoxia was combined with H2 S, the ventilatory stimulus component of the hypoxic response was abolished while there was an additive effect on the metabolic depression. H2 S does not mediate the effect of hypoxia on the arterial chemoreceptors. The interaction between H2 S, hypoxia and CBS activity should be clarified, since the depressive effects of H2 S on breathing appears to be linked to the acute reduction metabolic rate of small-sized mammals through a powerful and very sensitive mechanism, as is the case with hypoxia. Finally, it remains to be determined as to whether or not our present results are applicable to humans in the neonatal period and to other species capable of hypoxia-induced metabolic depression. Acknowledgements The authors are grateful to Valerie Kehoe and Carrie Fergusson for her precious technical help. References Abe, K., Kimura, H., 1996. The possible role of hydrogen sulfide as an endogenous neuromodulator. J. Neurosci. 16, 1066–1071. Almeida, A.F., Guidotti, T.L., 1999. Differential sensitivity of lung and brain to sulfide exposure: a peripheral mechanism for apnea. Toxicol. Sci. 50, 287–293. Bell, H.J., Ferguson, C., Kehoe, V., Haouzi, P., 2009. Hypocapnia increases the prevalence of hypoxia-induced augmented breaths. Am. J. Physiol. Regul. Integr. Comp. Physiol. 296, R334–344. Bhatia, M., 2005. Hydrogen sulfide as a vasodilator. IUBMB Life 57, 603–606. Bhatia, M., Sidhapuriwala, J., Moochhala, S.M., Moore, P.K., 2005. Hydrogen sulphide is a mediator of carrageenan-induced hindpaw oedema in the rat. Br. J. Pharmacol. 145, 141–144. Blackstone, E., Morrison, M., Roth, M.B., 2005. H2S induces a suspended animationlike state in mice. Science 308, 518. Cheng, Y., Ndisang, J.F., Tang, G., Cao, K., Wang, R., 2004. Hydrogen sulfide-induced relaxation of resistance mesenteric artery beds of rats. Am. J. Physiol. Heart Circ. Physiol. 287, H2316–2323. Dombkowski, R.A., Doellman, M.M., Head, S.K., Olson, K.R., 2006. Hydrogen sulfide mediates hypoxia-induced relaxation of trout urinary bladder smooth muscle. J. Exp. Biol. 209, 3234–3240. Dombkowski, R.A., Russell, M.J., Olson, K.R., 2004. Hydrogen sulfide as an endogenous regulator of vascular smooth muscle tone in trout. Am. J. Physiol. Regul. Integr. Comp. Physiol. 286, R678–685. Dominy, J.E., Stipanuk, M.H., 2004. New roles for cysteine and transsulfuration enzymes: production of H2S, a neuromodulator and smooth muscle relaxant. Nutr. Rev. 62, 348–353. Dorman, D.C., Moulin, F.J., McManus, B.E., Mahle, K.C., James, R.A., Struve, M.F., 2002. Cytochrome oxidase inhibition induced by acute hydrogen sulfide inhalation:
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