Effects of exogenous hydrogen sulphide on calcium signalling, background (TASK) K channel activity and mitochondrial function in chemoreceptor cells
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It has been proposed that endogenous H2S mediates oxygen sensing in chemoreceptors; this study investigates the mechanisms by which H2S excites carotid body type 1 cells. H2S caused a rapid reversible increase in intracellular calcium with EC50 ≈ 6 μM. This [Ca2+]i response was abolished in Ca-free Tyrode. In perforated patch current clamp recordings, H2S depolarised type 1 cells from −59 to −35 mV; this was accompanied by a robust increase in [Ca2+]i. Voltage clamping at the resting membrane potential abolished the H2S-induced rise in [Ca2+]i. H2S inhibited background K+ current in whole cell perforated patch and reduced background K+ channel activity in cell-attached patch recordings. It is concluded that H2S excites type 1 cells through the inhibition of background (TASK) potassium channels leading to membrane depolarisation and voltage-gated Ca2+ entry. These effects mimic those of hypoxia. H2S also inhibited mitochondrial function over a similar concentration range as assessed by NADH autofluorescence and measurement of intracellular magnesium (an index of decline in MgATP). Cyanide inhibited background K channels to a similar extent to H2S and prevented H2S exerting any further influence over channel activity. These data indicate that the effects of H2S on background K channels are a consequence of inhibition of oxidative phosphorylation. Whilst this does not preclude a role for endogenous H2S in oxygen sensing via the inhibition of cytochrome oxidase, the levels of H2S required raise questions as to the viability of such a mechanism.
KeywordsHydrogen sulphide Calcium signalling Chemoreceptors Mitochondrial function Oxygen sensing
It has been proposed that hydrogen sulphide is a naturally occurring gassotransmitter similar to nitric oxide . It has also been hypothesised that endogenous H2S production plays a particularly important role in the process of acute oxygen sensing in blood vessels [36, 38], fish gill chemoreceptors  and the mammalian carotid body [37, 41]. Endogenous H2S is primarily produced through the metabolism of two sulphur-containing amino acids cysteine and homocysteine by the enzymes cystathionine β-synthase and cystathionine γ-lyase (γ-cystathionase) [23, 24, 26]. Its subsequent degradation is by oxidation to thiosulphate in the mitochondrion. This occurs in several stages; the first involves the formation of a persulphide with sulphide quinone oxidoreductase (SQR) together with reduction of ubiquinone; this is followed by oxidation of the persulphide by a sulphur dioxygenase to form sulphite; the final reaction is catalysed by a sulphur transferase which produces thiosulphate by transferring a second persulphide from SQR to sulphite . This process requires oxygen for two functions, firstly to re-oxidise ubiqinone via the electron transport chain and secondly in the oxidation of persulphide by sulphur dioxygenase. In the H2S hypothesis, oxygen sensing is proposed to be initiated by alteration of the balance between constitutive H2S production and oxygen-dependent H2S removal .
The ability of exogenous H2S to act as a respiratory stimulant has long been recognised, possibly as far back as the 1800s . An early contemporary account is given by Hagard and Henderson who, in 1922, reported that both inhalation of H2S gas and injection of 2 mg/kg Na2S evoked a hyperpnea in dogs . The stimulatory effects of sulphides were subsequently traced to actions upon the carotid body . The mechanisms by which H2S stimulates the carotid body are however largely undefined. It has been reported that chemoreceptor excitation by exogenous H2S can be blocked by removal of external calcium or application of cadmium [31, 41]. This suggests that H2S promotes Ca2+ influx as does hypoxia . It has also been reported that H2S inhibits large conductance Ca2+-activated K+ channels (BKCa) . Inhibition of BKCa alone however is usually insufficient to excite chemoreceptor cells as these channels are inactive under resting conditions . Excitatory responses to hypoxia are primarily mediated by the inhibition of a background potassium current believed to be carried through TWIK-related acid-sensitive potassium (TASK) channels [4, 10, 25]. The effects of H2S on these channels are unknown.
There is also uncertainty in the nature of the signalling pathways linking hydrogen sulphide to modulation of ion channel activity. In blood vessels, H2S has been reported to directly activate KATP channels leading to membrane hyperpolarisation, reduction of voltage-gated Ca2+ influx and vasodilation . H2S has also been reported to inhibit large conductance Ca2+-activated K+ channels (BKCa) in excised patches , again suggesting direct interaction between H2S and the ion channel. H2S is however also a powerful inhibitor of cytochrome oxidase . Indeed, a general criticism of the hypothesised role for H2S as a gasotransmitter is that the majority of studies demonstrating effects of exogenous H2S have employed concentrations that have the potential to poison energy metabolism. This issue is of particular importance when considering a potential role for H2S in oxygen sensing in peripheral chemoreceptors since these organs are sensitive to many inhibitors of oxidative phosphorylation [2, 8, 11, 20, 34, 44, 54]. This study therefore seeks to address two main issues in relation to the actions of H2S: (1) how does H2S excite the type 1 cell and are these mechanisms the same as those observed in hypoxia and (2) can the effects of H2S be attributed to a novel signalling pathway or are they simply due to metabolic inhibition.
Type 1 cell isolation
Carotid bodies were excised from neonatal rat pups (11–15 days) under terminal anaesthesia (2–4% halothane) in accordance with project and personal licences issued under the UK Animals (Scientific Procedures) Act 1986. Type 1 cells were isolated using enzymatic digestion with 0.3 mg/ml trypsin (Sigma) and 0.5 mg/ml collagenase (Worthington) in PBS or Ham’s F-12 for 25–30 min at 35°C followed by transfer to enzyme-free culture media (see below) and trituration through fire-polished pipettes. The resultant cell suspension was plated onto poly-l-lysine-coated coverslips and maintained in an incubator at 35°C for 2 h before the addition of further culture media. Cells were used within 8 h of isolation. Culture media comprised Ham’s F-12 or DMEM containing 10% heat-inactivated foetal bovine serum, 2 mM l-glutamine and 4 μg/ml insulin.
Measurement of [Ca2+]i, [Mg2+]i and NADH
Fluorescence measurements were performed using a microspectrofluorimeter based on a Nikon Diaphot 200 (Japan) equipped with a xenon lamp to provide an excitation light source and cooled (−20°C) photomultiplier tubes (Thorn EMI) to detect emitted fluorescence. [Ca2+]i was determined using Indo-1, [Mg2+]i using Mag-Indo-1 and NADH by cellular autofluorescence. Indo-1 and Mag-Indo-1 were loaded into cells by incubation with 2–5 μM of the acetoxymethyl ester derivatives of these dyes in culture media at room temperature for 1 h (Indo-1) or 10 min (Mag-Indo-1). Indo-1 and Mag-Indo-1 were excited at 340 nm and fluorescence intensity measured at 405 ± 16 and 495 ± 10 nm. The fluorescence emission ratio (405/495) for Indo-1 was calibrated as previously described ; the fluorescence ratio 405/495 for Mag-Indo-1 is presented without calibration. NADH autofluorescence was excited at 340 nm and emission measured at 450 ± 30 nm. Data acquisition and analysis was performed using a 1401 interface and Spike 2 software (Cambridge Electronic Design).
Both perforated patch and cell-attached patch recordings were performed using an Axopatch 200B. Electrodes were pulled from borosilicate glass tubing and were sylgarded and fire polished just before use. For perforated patch recording, pipette filling solutions contained (in millimolars) K2SO4 70, potassium chloride (KCl) 30, ethylene glycol tetraacetic acid (EGTA) 1, (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid ) (HEPES) 10 and MgCl2 2, and pH was adjusted to 7.2 at 37°C. Amphotericin was added to this solution from a stock solution in DMSO to a final concentration of 120–240 μg/ml just prior to recording. For cell- attached patch recording, pipette filling solutions contained (in millimolars) KCl 140, MgCl2 1, EGTA 1, HEPES 10, tetraethylammonium 10, 4-aminopyridine 5. pH was adjusted to 7.4 at 37°C.
Single-channel recordings were performed at a filter frequency of 2 kHz, and membrane current was digitised and recorded at 5–20 kHz. Membrane current and voltage during perforated patch recordings were sampled at 5 kHz. Data acquisition, voltage clamp control and data analysis were performed using Spike 2 software. Single-channel activity was quantified as NPopen using the main conductance state to set a 50% opening threshold. Current levels of greater than 150% of the main conductance state were counted as multiple openings.
Perforated patch recordings were performed with simultaneous measurement of [Ca2+]i. Cells failing to maintain low resting levels of [Ca2+]i following patch formation and perforation were rejected (see ). Whole-cell current–voltage (I/V) relationships were determined by using voltage ramps. In these experiments, average I/V curves (as in Fig. 4c) were constructed by averaging digitised current and voltage data first over 0.5-mV intervals, then over 5–15 successive ramps and finally I/Vs from individual cells were averaged.
It was noted that H2S frequently caused an offset at the reference electrode of between 3 and 10 mV. Raw recordings of membrane potential (i.e. Fig. 3a, b) are presented without correction for this offset, but the offset has been corrected for in the summary data presented in the text and Fig. 3c. This error has also been corrected for in recordings of membrane current and current–voltage relationships (Fig. 4). In single-channel recordings, a second potential source of voltage offset may result from depolarisation of the resting membrane potential in response to H2S or CN−. In these experiments, no explicit correction for change in membrane voltage (or voltage offset) was made (as these channels are voltage insensitive), but single-channel amplitude was determined under each condition from all-points histograms so that thresholds for determining NPopen could be adjusted to take into account any changes in membrane voltage.
Standard bicarbonate-buffered Tyrode solutions contained (in millimolars): NaCl 117, KCl 4.5, CaCl2 2.5, MgCl2 1, NaHCO3 23 and Glucose 11. In Ca2+-free solutions, CaCl2 was omitted and 100 μM EGTA was added. Twenty millimolars K+ Tyrode contained 20 mM KCl and 101.5 mM NaCl, all other constituents remained the same. High-K+ low-Ca2+ solutions for cell-attached patch recordings contained (in millimolars) NaCl 21.5, KCl 100, MgCl2 1, NaHCO3 23 and Glucose 11. Normoxic solutions were equilibrated 5% CO2 and 95% air, hypoxic solutions were equilibrated with 5% CO2 and 95% N2 (P O2 = 2 Torr); both had a pH of 7.4 at 37°C. Note that for solutions containing CN− or NaHS, Tyrodes were bubbled for >15 min prior to the addition of these compounds and thereafter maintained under an atmosphere of 5% CO2/95% air (i.e. these solutions were not continuously bubbled as both compounds are volatile in their acid form and would be rapidly lost from solution). NaCN and NaHS were added from stock solutions freshly prepared just before use. H2S concentration was estimated assuming pKa = 6.9 . Note that different conventions are used in reporting H2S levels; some authors present the amount of the sulphide salt added to solution rather than the resulting concentration of H2S. For comparison, in this study, the total NaHS added to solution is approximately four times the quoted H2S concentration. Different values for pKa have also been used, e.g. Whitfield et al.  employed a pKa of 6.6 for mammalian blood at 37°C; applying this value for pKa would give H2S concentrations approximately 4/7ths (57%) of those reported here.
Statistical analysis of data
Significance was assumed at p < 0.05 for t tests, ANOVA and post hoc comparisons against control using Dunnett’s method. For post hoc comparisons using the Holm–Sidak method, values were considered significant when p < the critical level, starting at p < 0.05. Statistical analysis and curve fitting were carried out using SigmaPlot 11 (Systat Software Inc, Germany) or Excel (Microsoft).
NaSH and EGTA were from Sigma. Indo-1-AM and Mag-Indo-1-AM were from Molecular Probes.
Effects of exogenous H2S on intracellular Ca2+ signalling
Effects of exogenous H2S on membrane potential and background K currents
Effects of H2S on background K+ currents and channels
Effects of H2S on mitochondrial function
The effects of H2S described above are identical to those of hypoxia (see “Discussion” section); they are also identical to those of many inhibitors of mitochondrial respiration [8, 54]. Since H2S is a well-known inhibitor of cytochrome oxidase , this poses the question as to whether the effects of H2S are mediated primarily through the inhibition of oxidative phosphorylation or by some other means. The effects of H2S on mitochondrial function were therefore investigated.
Inhibition of oxidative phosphorylation can also lead to a decline in cellular ATP levels. Since ATP is a chelator of intracellular Mg+, a decline in ATP levels results in the release of Mg2+ into the cytosol. Under normal conditions, cellular levels of Mg2+ are relatively low, e.g. 0.3–1.25 mM [18, 32, 45], and although [Mg2+ ]i is actively regulated, transmembrane fluxes are invariably slow [15, 42, 43]. As a consequence, resting [Mg2+]i tends to be relatively constant. For the above levels of free Mg, most ATP within the cell would be expected to be complexed with Mg2+, depletion of ATP by conversion first to ADP and then to AMP (by adenylate kinase) is therefore associated with the release of significant amounts of Mg2+ and a readily detectable rise in cytosolic [Mg2+]i [14, 19, 30]. Thus, ATP depletion can be followed by measuring [Mg2+]i. Rapid elevation of [Mg2+]i has previously been reported to occur in type 1 cells following the application of many other inhibitors of oxidative phosphorylation including cyanide, rotenone, oligomycin and 2-4-dinitrophenol . Figure 6b shows a recording of [Mg2+]i in a type 1 cell using Mag-Indo-1. H2S causes a reversible increase in [Mg2+]i (p < 0.001, n = 9) as did another inhibitor of mitochondrial energy metabolism 1 μM FCCP (Fig. 6b). This effect of H2S was dose dependent; the rise in [Mg2+]i was significant at H2S concentrations of ≥7.5 μM, and the dose–response relationship was well described by a four-parameter logistic curve with an EC50 of 9.7 μM (Fig. 6d).
A comparison of the concentration-dependent effects of H2S on [Ca2+]i, NADH levels and [Mg2+]i is presented in Fig. 6e (with each dose–response curve normalised to a 0–100% scale). It is apparent from these data that the dose dependency of the effects of H2S upon type 1 cell [Ca2+]i is comparable to the dose-dependent effects of H2S on mitochondrial function in these cells.
Lack of additive effect of H2S and other electron transport inhibitors on KB (TASK) channel activity
Effects of H2S on Ca2+ signalling in type 1 cells
Application of sulphide evoked an elevation of cytosolic calcium in all oxygen-sensitive type 1 cells studied. This effect was rapid in onset and also reversed rapidly upon removal of H2S. The EC50 for the H2S-evoked rise in [Ca2+]i was estimated to be about 6 μM H2S. This calcium response was abolished by removal of extracellular calcium. These effects are comparable to the actions of sulphides on carotid sinus nerve (CSN) activity in isolated rat carotid body which is rapidly and reversibly excited by 30 μM NaHS (approximately 7.5 μM H2S) and abolished by [Ca2+]o removal . The effects of sulphide on CSN discharge rate are also abolished by a combination of P2X and nicotinic receptor antagonists . Collectively, these data argue that the principal site of action of sulphide within the carotid body is presynaptic, i.e. at the type 1 cell, and that calcium signalling plays a prominent role in promoting this response.
Mechanisms of H2S signalling and their similarity to hypoxia
The effects of hypoxia on isolated rat type 1 cells include inhibition of background potassium channels (KB) , which are thought to be derived from TASK1 and TASK3 with a possible preponderance of the heterodimeric form TASK1–TASK3 [10, 25, 52]), and inhibition of calcium-activated large-conductance potassium channels (BKCa) . The inhibition of KB (TASK) channels leads to membrane depolarisation since these channels provide the major resting potassium conductance which maintains the negative resting membrane potential. Upon KB channel inhibition other, as yet poorly defined, background inward currents depolarise the cell until the threshold for activation of voltage-gated calcium channels is reached. At this point, Ca2+ floods into the cell, action potentials may be generated and other voltage-gated and Ca2+-activated channels may become active.
The effect of exogenous H2S, as described in this paper, is directly comparable to those described above for hypoxia, i.e. inhibition of KB (TASK) channels causing membrane depolarisation, electrical activity and voltage-gated Ca entry. Like hypoxia, H2S has also been reported to inhibit BKCa , although the inhibition of BKCa seems to require much higher levels of H2S than those needed to excite intact cells and tissues. In all major respects, therefore, exogenous H2S mimics the effects of hypoxia. It is however by no means unique in this regard, other inhibitors of cytochrome oxidase including cyanide, azide and carbon monoxide similarly mimic the effects of hypoxia [2, 3, 20, 22, 53, 54].
Effects of H2S on mitochondrial function
The similarity between the effects of H2S, hypoxia and other complex IV inhibitors raises the question as to whether the effects of H2S can simply be attributed to inhibition of complex IV. To address this issue, it is pertinent to consider whether H2S actually alters mitochondrial function over the range of concentrations for which it acts as a chemostimulant. Direct measurement of mitochondrial function in this tissue is not practical due to its small size; consequently, two indirect methods have been employed.
The first method measured NADH levels by its intrinsic fluorescence (NAD is not fluorescent). In short-term experiments, increase in autofluorescence indicates increase in NADH/NAD ratio which may arise from impaired NADH oxidation. The data (Fig. 6) show that H2S is able to elevate NADH levels from quite low concentrations. There are, however, two distinct mechanisms by which H2S could increase NADH. At low concentrations, H2S is oxidised by mitochondrial sulphide quinione reductase (SQR) with concomitant reduction of ubiquinone . The reduced ubiquinone is then oxidised by complex III of the electron transport chain. The oxidation of H2S (by SQR) could in principle compete with complex I for ubiquinone  if the flux of electrons through SQR is sufficient. Thus, low levels of H2S might cause a small increase in NADH without compromising oxidative phosphorylation. At higher levels, however, the H2S-induced rise in NADH is most likely to be due to inhibition of electron transport.
The second method evaluated the effects of H2S upon oxidative phosphorylation. This employs the measurement of free Mg2+ concentration within cells. Much of the Mg2+ within cells is bound to ATP, such that whenever there is net MgATP hydrolysis free Mg2+ is released into the cytosol and [Mg2+]i increases (see e.g. [1, 19, 30]). These experiments were conducted in a Ca-free Tyrode to prevent Ca2+ influx during exposure to H2S and possible interference with the Mag-Indo1 signal. The rise in [Ca2+]i encountered under Cao 2+-free conditions (15 nM) is not expected to have any measureable effect on Mag-Indo fluorescence which has a Kd for Ca2+ of around 34 μM . As H2S is a weak acid, it could also influence intracellular pH. The sensitivity of Mag-indo-1 to pH interference is however negligible between 7.2 and 7.0 , and the concentrations of H2S employed even at the highest level (75 μM H2S, 300 μM total sulphide) are unlikely to cause an intracellular acidification of more than about 0.003 pH units (assuming a total intrinsic  plus open system HCO3 −/CO2 buffering capacity of 45 mM/pH at pHi = 7.2).
Measurements of [Mg2+]i indicate that the dose–response curve for the effects of H2S on MgATP is right shifted compared to the effects of H2S on NADH levels (EC50 = 2.8 μM for NADH and 9.7 μM for [Mg2+]i). These data are compatible with the hypothesis that H2S may serve as a substrate for electron transport at low levels and an inhibitor of electron transport and oxidative phosphorylation at higher levels. It may also simply reflect a greater sensitivity of NADH levels to inhibition of cytochrome oxidase activity.
Comparison of the effects of H2S on [Ca2+]i with the above two indices of mitochondrial function revealed a particularly close correlation with the effects of H2S on [Mg2+]i indicating that the effects of H2S on [Ca2+]i may be linked to the decline in MgATP. The observations that the effects of H2S on channel activity are similar to those of cyanide and that H2S has no additional effect upon KB channel activity in the presence of cyanide (Fig. 7) support the conclusion that the actions of H2S are primarily mediated via inhibition of oxidative phosphorylation. The mechanisms by which mitochondrial energy metabolism regulate KB channel function in type 1 cells have not yet been fully resolved, but two candidate pathways have been proposed; these are (1) direct modulation of the TASK channels by changes in MgATP levels  and (2) indirect modulation via changes in AMP/ATP levels and an AMP kinase . Some questions have however been raised as to whether TASK channels can be directly modulated by AMP kinase .
In conclusion, the excitation of peripheral chemoreceptors by H2S can be explained by H2S’s ability to inhibit cytochrome oxidase and the type 1 cells widely reported sensitivity to anything that disrupts oxidative phosphorylation. There was no evidence for any significant involvement of non-mitochondrial signalling pathways in regulating KB channels or of effects of H2S on [Ca2+]i at concentrations less than those which interfere with mitochondrial metabolism.
Role of H2S in oxygen signalling
The above observations raise the question as to whether endogenous production of H2S could nonetheless play a role in oxygen sensing via inhibition of complex IV. Such a hypothesis would be compatible with other current theories regarding oxygen sensing, metabolic signalling and ion channel regulation in type 1 cells [48, 55]. It would also offer an alternative to cytochrome oxidase as the “oxygen sensor” in the form of sulphur dioxygenase. Serious concerns have however been expressed regarding levels of exogenous H2S that appear to be required to excite chemoreceptors and whether similar levels could realistically be generated endogenously in vivo . The same considerations must be applied to this study. In order to generate any reproducible [Ca2+]i response, H2S needed to be > = 2.5 μM; to generate a [Ca2+]i response comparable to that seen with hypoxia (Fig. 1) required > = 7.5 μM H2S. The need for such high levels of H2S to evoke even a small response represents a serious problem for the hypothesis that oxygen sensing is mediated via endogenous H2S production. H2S is highly membrane permeable (diffusion coefficient = 0.5 cm/s ), isolated type 1 cells are small (approximately 5 μm radius) and experiments are conducted in a fast flowing stream of saline which would prevent external H2S accumulation. Under these conditions, an internal concentration of just 1 μM H2S would drive a transmembrane H2S efflux of 1.6 fM/s (calculated using Fick’s first law of diffusion). For a type 1 cell to maintain an intracellular concentration of 1 μM, it would therefore need to continually generate H2S at the same rate. For a 5-μm radius cell, this would require H2S synthesis at a rate equivalent to 180 mM/min/l intracellular fluid. Estimates of the maximum capacity for tissues to generate H2S vary. Liver homogenate (which appears to have one of the highest capacities for H2S generation) can generate up to 1 mM/min/kg tissue at saturating concentrations of cysteine/homocysteine , but with the use of physiologically relevant concentrations of substrate, this falls to only 8 μM/min/kg liver tissue . These rates are over two orders of magnitude less than that required to sustain a 1-μM concentration gradient of H2S across the membrane. The problem is not limited just to a consideration of the kinetics of H2S production. There is an even more serious issue regarding the availability of substrate from which to synthesise H2S. Isolated type 1 cells can continue to respond to hypoxia for an hour or more whilst being maintained in just a simple saline. To continue to produce H2S at the above rate for an hour would require a source of cysteine/homocysteine equivalent to 11 moles/l intracellular fluid. In contrast, tissue levels of homocysteine/cysteine are only in the region of 10–1,000 μM . In conclusion, if the reported values for lipid bilayer permeability to H2S are correct, it is highly questionable whether endogenous H2S could act as a freely diffusible signalling molecule operating at the micromolar level under the conditions typically used for studying oxygen sensing in isolated cells.
Mitochondria and oxygen sensing
Finally, it should be noted that this study shows that yet another inhibitor of mitochondrial function mimics the effects of hypoxia in the type 1 cell. One of the most remarkable features of the carotid body is its extraordinary sensitivity to these agents and the fact that mitochondrial function in type 1 cells appears to be exceptionally sensitive to even moderate hypoxia [11, 12]. It is tempting therefore to speculate that, even if H2S does not act as a specific messenger in oxygen sensing, physiological levels of H2S production together with other endogenous inhibitors of cytochrome oxidase, e.g. NO and CO, might in some way contribute to the unusual oxygen sensitivity of mitochondrial function in these cells.
H2S excites carotid body type 1 cells via inhibition of background K-channels depolarisation and voltage-gated Ca entry. These effects are qualitatively similar to those of hypoxia.
Background K channel inhibition by H2S is probably secondary to inhibition of electron transport and oxidative phosphorylation.
There was no evidence for any effect of H2S on type 1 cell [Ca2+]i at levels less than those required to inhibit mitochondrial function nor was there any effect of H2S on channel activity in the presence of cyanide. This would appear to rule out a significant role for any additional non-mitochondrial H2S receptor-mediated signalling pathway.
The levels of H2S required to excite type 1 cells via complex IV inhibition make this pathway an unlikely candidate for mediating the effects of hypoxia in isolated cells. A minor role for lower levels of H2S in modulating mitochondrial O2 sensitivity cannot however be ruled out at this stage.
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