Hydrogen sulfide (H2S) has been shown to affect gastrointestinal (GI) motility and signaling in mammals and O2-dependent H2S metabolism has been proposed to serve as an O2 ‘sensor’ that couples hypoxic stimuli to effector responses in a variety of other O2-sensing tissues. The low PO2 values and high H2S concentrations routinely encountered in the GI tract suggest that H2S might also be involved in hypoxic responses in these tissues. In the present study we examined the effect of H2S on stomach, esophagus, gallbladder and intestinal motility in the rainbow trout (Oncorhynchus mykiss) and coho salmon (Oncorhynchus kisutch) and we evaluated the potential for H2S in oxygen sensing by examining GI responses to hypoxia in the presence of known inhibitors of H2S biosynthesis and by adding the sulfide donor cysteine (Cys). We also measured H2S production by intestinal tissue in real time and in the presence and absence of oxygen. In tissues exhibiting spontaneous contractions, H2S inhibited contraction magnitude (area under the curve and amplitude) and frequency, and in all tissues it reduced baseline tension in a concentration-dependent relationship. Longitudinal intestinal smooth muscle was significantly more sensitive to H2S than other tissues, exhibiting significant inhibitory responses at 1–10 μmol l–1 H2S. The effects of hypoxia were essentially identical to those of H2S in longitudinal and circular intestinal smooth muscle; of special note was a unique transient stimulatory effect upon application of both hypoxia and H2S. Inhibitors of enzymes implicated in H2S biosynthesis (cystathionine β-synthase and cystathionine γ-lyase) partially inhibited the effects of hypoxia whereas the hypoxic effects were augmented by the sulfide donor Cys. Furthermore, tissue production of H2S was inversely related to O2; addition of Cys to intestinal tissue homogenate stimulated H2S production when the tissue was gassed with 100% nitrogen (∼0% O2), whereas addition of oxygen (∼10% O2) reversed this to net H2S consumption. This study shows that the inhibitory effects of H2S on the GI tract of a non-mammalian vertebrate are identical to those reported in mammals and they provide further evidence that H2S is a key mediator of the hypoxic response in a variety of O2-sensitive tissues.
Recent studies have shown that exogenous hydrogen sulfide (H2S) initiates pharmacophysiological responses in most, if not all, organ systems (Li et al., 2011; Olson, 2011). These studies have been instrumental in the hypothesis that hydrogen sulfide is a biologically relevant signaling molecule thereby joining nitric oxide (NO) and carbon monoxide (CO) as ‘gasotransmitters’ (Wang, 2002).
H2S is of particular interest in the gastrointestinal (GI) tract as it is both produced by GI tissues and generated in large quantities by bacterial flora in the lumen of the gut (Blachier et al., 2010; Hosoki et al., 1997; Linden et al., 2008; Martin et al., 2010; Teague et al., 2002; Wallace, 2010). Both pathological and physiological attributes have been ascribed to H2S in the GI tract (Wallace, 2010). H2S has been suggested to contribute to a variety of intestinal disorders including ulcerative colitis, inflammatory bowel disease and colorectal cancer (Attene-Ramos et al., 2010; Medani et al., 2010; Rowan et al., 2009). Conversely, H2S has been shown to have anti-inflammatory and antinociceptive actions in the GI tract and H2S ‘releasing’ compounds have been added to non-steroidal anti-inflammatory drugs (NSAIDs) to prevent gastric irritation and promote healing of ulcers (Wallace, 2010; Wallace et al., 2007). H2S has even been shown to serve as an energy source for ATP production by mucosal epithelial cells (Blachier et al., 2010; Bouillaud and Blachier, 2011; Goubern et al., 2007; Lagoutte et al., 2010).
Exogenous H2S has been observed to affect both GI smooth muscle and the enteric nervous system. H2S directly relaxes GI smooth muscle in the mouse stomach and colon (Dhaese and Lefebvre, 2009; Dhaese et al., 2010) and rat ileum (Nagao et al., 2010). In the guinea-pig stomach, ‘low doses’ of H2S (0.1–0.3 mmol l–1) enhance resting tension and slightly reduce contractile amplitude, whereas at higher concentrations H2S suppresses the amplitude of spontaneous contractions (Zhao et al., 2009). The mechanism of H2S-mediated relaxation is poorly understood and most common mediators of muscle relaxation have in fact been ruled out. In the mouse, H2S-induced relaxations are independent of potassium (K+) channels, NO, cAMP and cGMP pathways, and nerve inhibition, and do not involve membrane ATPase, calcium (Ca2+) channels, internal calcium stores or Rho-kinase; H2S may, or may not, act via activation of myosin light chain phosphatase (Dhaese and Lefebvre, 2009; Dhaese et al., 2010). In the rat ileum, H2S-mediated relaxations are independent of intrinsic, enteric and visceral afferent nerves, NO and both ATP-(KATP) and calcium-sensitive (KCa) potassium channels (Nagao et al., 2010). Low-dose activation of guinea-pig gastric smooth muscle may be mediated via inhibition of voltage-gated K+ channels and high-dose inhibition may be mediated via KATP channel activation (Zhao et al., 2009). In the mouse intestine, H2S inhibits pacemaker amplitude and frequency of interstitial cells of Cajal at high concentrations (0.5–1.0 mmol l–1), but may slightly stimulate at low concentrations (50–100 mmol l–1); these effects appear to be via modulation of intracellular calcium (Parajuli et al., 2010). H2S also stimulates proliferation of interstitial cells of Cajal (Huang et al., 2010).
Linden and colleagues critically evaluated the criteria for H2S as a biologically relevant gasotransmitter in the GI tract (Linden et al., 2010). They concluded that, while there is considerable evidence that H2S is generated by GI tissues and that H2S can affect a variety of GI functions, H2S cannot yet be considered an endogenous signaling molecule because, among other things, there is no evidence to date to show that the production of H2S by GI tissue is regulated.
An alternative to regulating H2S production to control H2S signaling would be to actively regulate H2S metabolism in the face of constitutive H2S production. We have previously shown that bovine lung tissue, cultured pulmonary arterial smooth muscle cells and isolated mitochondria inactivate H2S by a PO2-dependent process at physiologically relevant PO2 values (Olson et al., 2010). This balance between constitutive H2S production and O2-dependent inactivation in cells forms the basis for the hypothesis that H2S metabolism is the crucial step coupling hypoxia to tissue response, i.e. the ‘O2 sensor’ (Olson and Whitfield, 2010). Evidence for this mechanism has been found in systemic and respiratory vascular smooth muscle (Olson et al., 2006; Olson et al., 2008a; Olson et al., 2010), urinary bladder smooth muscle (Dombkowski et al., 2006), chromaffin cells (Perry et al., 2009) and O2-sensing chemoreceptor cells (Li et al., 2010; Olson et al., 2008b; Peng et al., 2010; Telezhkin et al., 2009; Telezhkin et al., 2010).
The GI tract is an intriguing candidate for O2 regulation of H2S signaling. Not only is H2S produced by GI tissue and luminal bacteria but also both tissue and bacterial metabolism can affect PO2. In the mouse, for example, tissue extraction and bacterial O2 consumption lower luminal PO2 from nearly 60 mmHg in the stomach to 11 mmHg in the small intestine and ultimately down to 3 mmHg at the sigmoid–rectal junction (He et al., 1999). A decrease in the tissue perfusion/metabolism ratio, the hallmark of ischemic bowel disease, can halve these values within 5 min (He et al., 1999). These PO2 values are sufficiently low to inhibit H2S oxidation and tip the scale in the intracellular environment from net H2S consumption to H2S production (Olson et al., 2010).
Although the effects of H2S on the mammalian GI tract have been examined in some detail, information on H2S effects in the GI tract of non-mammalian vertebrates is lacking. The present study was designed to examine the effects of this signaling molecule on the GI tract of fish as they are perhaps more intensively studied than any other non-mammalian vertebrate and they provide a broad phylogenetic and evolutionary perspective of vertebrate development. These studies also allowed us to examine the hypothesis that H2S is involved in O2 sensing in the GI tract. We first examined the effects of H2S and hypoxia on the intrinsic rhythmicity and contractile activity of esophagus, stomach, gall bladder, and circular and longitudinal GI smooth muscle of the rainbow trout (Oncorhynchus mykiss). Next, we compared the intestinal responses from rainbow trout with those of a much smaller and technically more manageable salmonid, the juvenile coho salmon (Oncorhynchus kisutsch). We then used the coho intestine to examine the hypothesis that H2S mediates the hypoxic responses by directly comparing the effects of these two stimuli on mechanical and/or enteric communication in the intestine and by examining the effect of inhibitors of H2S biosynthesis and a sulfide donor as a precursor of H2S synthesis on the hypoxic responses. Confirmation of the inverse relationship between H2S and O2 was obtained by direct measurement, in real time, of intestinal H2S production and its O2-dependent consumption using polarographic H2S and O2 electrodes.
MATERIALS AND METHODS
Rainbow trout [O. mykiss, Kamloops strain (Jordan 1892), 400–800 g] of either sex were purchased from local hatcheries (Harrietta Hills Trout Farm, Harrietta, MI, USA, and Sweetwater Springs Fish Farm, Peru, IN, USA) and kept in circulating 2000 l tanks containing well-water at 12–15°C, aerated with filtered room air, and exposed to 12 h:12 h light:dark cycles. The fish were fed a maintenance diet of commercial trout pellets (Purina, St Louis, MO, USA). The trout were stunned by a blow to the head and segments of the esophagus, stomach, gall bladder, anterior intestine and posterior intestine were dissected out and placed in cold (∼4°C) Cortland buffered saline (for composition, see below). Anterior intestinal segments were taken from a 2 cm portion of intestine starting ∼1 cm distal to the pyloric cecae. Posterior intestinal segments, 2 cm long, were taken 1 cm from the vent. The dissected tissues were cleaned and placed in fresh Cortland buffered saline at 4°C until use. All segments were used for experimentation within 24 h of removal.
Coho salmon [O. kisutch (Walbaum 1792), 10–20 cm, ∼10–30 g] juveniles were obtained from the Bodine State Fish Hatchery, Mishawaka, IN, USA, and kept in a 500 l rectangular tank containing continuously flowing well-water at 12–15°C for several weeks prior to experimentation. Coho were stunned by a blow to the head and the intestines were removed and prepared for myography as described above.
All procedures followed NIH guidelines and were approved by the local Institutional Animal Care and Use Committee (IACUC).
Circular smooth muscle rings (∼5 mm long) from the trout esophagus, stomach, gall bladder, anterior intestine and posterior intestine, and from the coho anterior intestine and posterior intestine were mounted on 280 mm diameter stainless steel wire hooks through the tissue lumen and suspended in 5 ml water-jacketed smooth muscle baths filled with 14°C Cortland buffered saline and aerated with room air. Segments from both the trout and coho anterior and posterior intestine were also mounted in a longitudinal direction by puncturing the intestinal walls with the same hooks. The cylindrical structure of the intestinal segments was not compromised in either setup. The bottom hooks were stationary; the upper hooks were connected to Grass model FT03C force-displacement transducers (Grass Instruments, West Warwick, RI, USA). Tension was measured on a Grass Model 7E or 7F polygraph (Grass Instruments). Polygraph sensitivity was calibrated prior to the beginning of each experiment and was able to detect changes as small as 5 μN. Data were archived on a PC computer at 1 Hz using Softwire software (Measurement Computing, Middleboro, MA, USA). The chart recorders and software were calibrated for zero tension and 2 mN loads prior to each experiment.
Baseline (resting) tension of ∼5–7 mN for trout gall bladders and ∼8–1.2 mN for the remaining tissues was applied and continuously adjusted for at least 1 h prior to experimentation as the tissues exhibited substantial stress relaxation. Gall bladders and intestinal segments from both fish established baseline resting loads ranging from 2 to 6 mN, and trout stomach and esophagus maintained baseline tension from ∼4 to 8 mN. All tissues were allowed to establish baseline activity in an undisturbed state for a minimum of 1 h prior to the beginning of experimentation. In the absence of external stimuli, trout gall bladder rings and circular and longitudinal intestinal smooth muscle from both fish usually (>90%) exhibited large spontaneous contractions that were approximately 2–5 times resting load. Spontaneous contractions were less frequently observed (<30%) in circular smooth muscle from trout stomach and esophagus. Tissues that did not exhibit spontaneous activity or stretch-induced relaxation were not examined.
Cumulative H2S concentration–response profiles were obtained for all tissues in the absence of other stimulation. NaHS was used in initial experiments whereas Na2S was used in later studies as it was more readily available and has fewer sulfur impurities (Doeller et al., 2005). Both salts form HS– and H2S when dissolved and we have not noticed any obvious differences in the effects produced by these salts (Dombkowski et al., 2006). In these studies ‘H2S’ refers to total free sulfide, i.e. dissolved H2S plus HS– based on the molarities of the dissolved salts.
H2S concentration–response profiles were obtained from circular smooth muscle of trout esophagus, stomach and gall bladder, as well as from circular and longitudinal smooth muscle of both trout and coho intestine. Concentration–response profiles were also obtained from trout esophagus, stomach, gall bladder and circular smooth muscle and coho intestinal longitudinal smooth muscle pre-contracted with 10 mmol l–1 carbamylcholine chloride (carbachol).
The effects of hypoxia, produced by gassing with 100% N2, were examined in otherwise unstimulated and carbachol (10 mmol l–1) pre-contracted anterior and posterior circular and longitudinal intestinal smooth muscle from both trout and coho. Unstimulated or carbachol pre-contracted intestinal segments were exposed to either two or three 20 min bouts of hypoxia, separated by 20 min of aeration with room air.
Relationship between H2S and hypoxia
Two protocols were employed to examine the role of H2S in hypoxic responses. In the first, the effects of hypoxia were examined in coho longitudinal intestinal smooth muscle in the presence of inhibitors of H2S biosynthesis. The cystathionine γ-lyase (CSE) inhibitor propargylglycine (PPG, 10 mmol l–1) or the cystathionine β-synthase (CBS) inhibitor amino-oxyacetic acid (AOA, 1 mmol l–1) was added to the tissues 30 min prior to hypoxia. In another group of experiments, the tissues were exposed to the inhibitor for 30 min then pre-contracted with 10 mmol l–1 carbachol and hypoxia was administered when the carbachol response plateaued. In a third group of experiments the sulfide donor cysteine (0.1, 1 or 10 mmol l–1) was added 30 min prior to the 10 mmol l–1 carbachol pre-contraction and subsequent hypoxia.
Effects of O2 on H2S production
H2S production by homogenized trout intestine was measured in real time using an amperometric (polarographic) H2S sensor constructed in the laboratory (Whitfield et al., 2008). Approximately 1 g of anterior intestine was homogenized, on ice, in 9 ml of Cortland buffered saline and gassed on a rotary tonometer with humidified 100% nitrogen to remove the oxygen. A 1.5 ml sample of homogenate was placed in a closed chamber with ports for simultaneous measurement of oxygen with a standard Clark-type electrode and H2S. H2S production was measured in the absence and in the presence of 1 and 10 mmol l–1 cysteine. Room air was administered through an injection port and the relationship between PO2 and PH2S recorded. The relationship between PH2S and H2S concentration was derived from a standard curve using Na2S; the rate of H2S production was obtained from the slope of the H2S concentration as a function of time.
Text files were converted to .acq files for analysis with Biopac Lab Pro (AcqKnowledge, Biopac Systems Inc., Goleta, CA, USA). Four different parameters were captured for each tissue segment using this software: (1) baseline tension, defined by the average tension in between spontaneous contractions; (2) spontaneous contraction amplitude, defined by the difference between the highest and lowest tension measured during each cycle; (3) spontaneous contraction frequency, if present; and (4) total contractile activity, determined as the area under the contractile curve (AUC) from zero tension, which essentially captures baseline tension and spontaneous activity combined. For comparison, all data were averaged over a 600 s (10 min) time frame immediately before treatment and during the final 600 s (10 min) of each respective H2S concentration or hypoxic exposure. Data presented are all from the initial exposure to hypoxia. For all H2S concentration–response experiments on carbachol pre-contracted segments, only the total contractile activity was examined for comparison with otherwise unstimulated tissues. All four parameters were analyzed in the coho intestinal 1 mmol l–1 Na2S bolus experiments and hypoxic experiments with PPG, AOA and cysteine. Esophageal and gastric rings infrequently displayed spontaneous contractions; therefore, only baseline tension and total contractile activity were examined for these two tissues. Because H2S and hypoxia reduced all parameters, the H2S and hypoxic responses are presented as a percentage of pretreatment values. The H2S concentration–response curves were truncated at 1 mmol l–1 NaHS or Na2S as higher concentrations are quite likely irrelevant physiologically. The effective concentration for half-maximal response (EC50) was calculated using TableCurve® (Jandel Corp., Chicago, IL, USA).
In several instances the relative responses in different intestinal segments to H2S or hypoxia were not significantly different and these data were combined. In coho, only two significant differences were found between anterior and posterior intestinal segments. The amplitude of spontaneous contractions in both anterior circular and anterior longitudinal smooth muscle was nearly twice the amplitude observed in posterior tissues (Fig. 1). This was likely due to the degree of muscularity, which was considerably greater in the anterior segments. Because the relative (as a percentage of control) responses of these segments to either H2S or hypoxia were not significantly different, the percentage change in anterior and posterior intestinal data were combined for EC50 calculations and for comparisons between circular and longitudinal smooth muscle preparations. In trout, H2S and hypoxia produced a significantly greater relaxation of baseline tension in posterior longitudinal intestinal smooth muscle than in anterior longitudinal intestinal smooth muscle (Fig. 2). However, all of the other responses to H2S and hypoxia expressed as a percentage of pretreatment values (total contractile activity, spontaneous contraction amplitude and frequency) were not significantly different and these data were pooled for calculation of EC50 values and for comparisons between circular and longitudinal intestinal smooth muscle. Student's t-tests and one-way ANOVA were used for comparisons between groups with SigmaStat® (Jandel Corp.). Results are presented as means ± s.e.m. Significance was assumed at P≤0.05.
Unless otherwise stated all chemicals were purchased from Sigma-Aldrich (St Louis, MO, USA). Na2S and NaHS were purchased from Fisher Scientific (Pittsburgh, PA, USA). Cortland buffered saline (pH 7.8) was: 124 mmol l–1 NaCl, 3 mmol l–1 KCl, 1.1 mmol l–1 MgSO4·7H2O, 2 mmol l–1 CaCl2·2H2O, 5.55 mmol l–1 glucose, 12 mmol l–1 NaHCO3, 0.09 mmol l–1 NaH2PO4 and 1.8 mmol l–1 Na2HPO4. NaHS and Na2S stock solutions were made fresh daily under nitrogen and used within 8 h of preparation. By convention, H2S is used hereafter to indicate either Na2S or NaHS.
There was no significant deterioration in any of the measured parameters in any of the control tissues for up to 4 h (not shown). All experimental protocols were completed in 4 h or less.
Effects of H2S on trout GI tract
The effects of H2S on total contractile activity (AUC), baseline tone, spontaneous contraction amplitude and frequency of spontaneous contractions for trout GI tissues are shown in Table 1 and in Figs 3, 4, 5. Longitudinal intestinal smooth muscle was the most sensitive to H2S, with significant decreases in all but spontaneous contraction amplitude occurring at 1 mmol l–1. In other tissues half of the measured parameters were significantly reduced at 10 mmol l–1 H2S and all parameters were significantly reduced at 100 mmol l–1 H2S. The effects of high concentrations of H2S (100 mmol l–1 to 1 mmol l–1) were often significantly greater in longitudinal smooth muscle than they were in circular smooth muscle. In a number of tissues, but especially noticeable in longitudinal smooth muscle of trout intestine (Fig. 4), application of 1 mmol l–1 H2S produced an initial increase in baseline tension and amplitude of spontaneous contractions which was then followed by a decrease in baseline tension and spontaneous contractions. Application of 1 mmol l–1 H2S produced one or several spontaneous contractions in anterior intestinal circular smooth muscle but did not increase baseline tension. H2S also produced a concentration-dependent relaxation of 10 mmol l–1 carbachol pre-contracted esophagus and anterior intestinal circular smooth muscle (Fig. 5), as well as stomach and gall bladder (not shown). Spontaneous activity and baseline tension were restored when the tissues were rinsed with H2S-free buffer.
The EC50 values for the H2S effect on contractile activity (AUC) for both trout and coho tissues are shown in Table 2. Longitudinal intestinal smooth muscle preparations from both fish were significantly more sensitive to H2S than the respective circular smooth muscle preparations; there were no other significant differences between or within species, tissues or unstimulated vs pre-contracted preparations.
Effects of hypoxia on trout intestine
The effects of hypoxia (gassing with 100% N2) on AUC, spontaneous contraction amplitude, baseline tone and frequency in trout circular and longitudinal intestinal smooth muscle are shown in Table 1 and Fig. 6. The effects of hypoxia were similar, if not identical, to those produced by H2S. Hypoxia significantly reduced all parameters in circular and longitudinal intestinal smooth muscle. Hypoxia was significantly more efficacious in decreasing AUC in longitudinal smooth muscle than it was in circular smooth muscle and, although not significant, there was a tendency for a greater effect on spontaneous contraction amplitude, baseline tone and frequency as well. In longitudinal smooth muscle of trout intestine, the onset of hypoxia produced an initial increase in baseline tension and spontaneous contraction amplitude followed by a subsequent decrease in these parameters (Fig. 6). These responses were strikingly similar to those produced by 1 mmol l–1 H2S (Fig. 4). Spontaneous activity returned and baseline tension partially recovered when the tissues were aerated with room air.
Effects of H2S on coho intestine
As in trout, H2S caused a concentration-dependent reduction in AUC, spontaneous contraction amplitude, baseline tone and frequency of spontaneous contractions in coho circular and longitudinal intestinal smooth muscle (Table 3, Fig. 7). Longitudinal intestinal smooth muscle was also more sensitive to H2S than circular smooth muscle (Table 2). Application of high concentrations (100 mmol l–1 to 1 mmol l–1) of H2S often resulted in a transient burst of contractile activity followed by a stable relaxation and essentially complete loss of spontaneous activity (Fig. 7). Spontaneous activity and baseline tension were restored when the tissues were rinsed with H2S-free buffer.
Effects of carbachol and inhibitors of H2S biosynthesis on coho intestine
Carbachol (10 mmol l–1) increased AUC, spontaneous contraction amplitude and frequency of spontaneous contractions in longitudinal smooth muscle of the coho intestine (Table 4). Pretreatment with the CSE inhibitor PPG had no effect on any of the variables, whereas pretreatment with the CBS inhibitor AOA significantly reduced AUC. Addition of H2S (1 mmol l–1) significantly decreased the carbachol-induced effect on AUC, spontaneous contraction amplitude and frequency of spontaneous contractions (Table 4).
Effects of hypoxia on coho intestine
Hypoxia (N2) significantly reduced AUC, spontaneous contraction amplitude, baseline tone and frequency of spontaneous contractions in otherwise unstimulated coho anterior and posterior, circular and longitudinal smooth muscle (Table 5, Fig. 8). As shown in Fig. 8, the onset of hypoxia often resulted in a transient burst of contractile activity, followed by a prolonged relaxation that persisted until normal aeration was restored. This response was similar to the effects of elevated H2S (Fig. 7). The effects of hypoxia on AUC and baseline tone were significantly more pronounced in longitudinal than in circular intestinal smooth muscle. There were no significant differences between hypoxia and 1 mmol l–1 H2S for any measured parameter in either circular or longitudinal smooth muscle.
Relationship between H2S and hypoxia in coho longitudinal intestinal smooth muscle
Incubation with the CSE inhibitor PPG had no effect on the hypoxic relaxation or inhibition of spontaneous contractions in unstimulated coho longitudinal intestinal smooth muscle, whereas incubation with the CBS inhibitor AOA significantly inhibited the hypoxic effect on AUC, spontaneous contraction amplitude and frequency (Table 5). In carbachol-precontracted longitudinal smooth muscle, PPG significantly inhibited the hypoxic effect on AUC, spontaneous contraction amplitude and baseline tone while AOA inhibited spontaneous contraction amplitude and frequency (Fig. 9).
Prior addition of 0.1 mmol l–1 cysteine did not affect the hypoxic response of carbachol pre-contracted intestinal strips whereas 1 and 10 mmol l–1 cysteine augmented the hypoxic effect on AUC, baseline tone and frequency of spontaneous contractions (Fig. 9, Table 5). The effects of 1 and 10 mmol l–1 of cysteine on the hypoxic response were not significantly different.
O2 dependency of H2S production in the trout intestine
Addition of 1 mmol l–1 cysteine to deoxygenated, homogenized trout intestine slightly stimulated H2S production (not shown). Subsequent addition of 10 mmol l–1 cysteine greatly increased the rate of H2S formation (10.5±6.3 mmol min–1 g–1 wet mass, N=5 fish). H2S production quickly reverted to net H2S consumption upon addition of room air to the reaction chamber. After the oxygen was removed, presumably by tissue consumption, H2S production returned (Fig. 10).
Our findings show that exogenous H2S relaxes intestinal smooth muscle and inhibits spontaneous contractions in multiple areas of the fish GI tract, indicative of effects on either contractile activity of GI smooth muscle or enteric signaling, or both. Longitudinal smooth muscle of intestine from both trout and coho was consistently more sensitive to H2S than circular smooth muscle, which may indicate differential regulation of function. Further evidence that H2S is the oxygen-sensitive couple linking tissue hypoxia to physiological responses was provided by observations that: (1) hypoxic responses of the GI tract exactly mimicked those produced by H2S, (2) hypoxic responses were inhibited by inhibitors of H2S biosynthesis and were augmented by the sulfide donor cysteine, and (3) H2S production by the GI tract is stimulated by cysteine and inversely related to oxygen availability.
Comparison of H2S effects in mammals and trout
Our study shows that the effects of H2S on AUC, spontaneous contractions, baseline tone and rhythmicity in the GI tract of fish are qualitatively, if not quantitatively, identical to those observed in a number of mammalian preparations. Salmonid tissues, however, appear to be more sensitive to H2S.
Although few studies have provided detailed concentration–response characterization of the effects of H2S in the mammalian GI tract, most studies suggest that the threshold for a response is 10 mmol l–1 or above and EC50 values (either provided by the authors or estimated from their data) are usually well above 300 mmol l–1. In the mouse gastric fundus pre-contracted with prostaglandin F2α (PGF2α), relaxation was first observed at 10 mmol l–1 and EC50 values ranged from 350 to 429 mmol l–1 (Dhaese and Lefebvre, 2009). In the guinea-pig stomach, 10 mmol l–1 H2S significantly enhanced resting tension and slightly reduced the contractile amplitude, whereas 300 mmol l–1 H2S suppressed spontaneous contractions (Zhao et al., 2009). EC50 values estimated from the inhibitory effect of H2S appeared to be ∼400 mmol l–1. Relaxation of the mouse distal colon was observed at 100 mmol l–1 H2S, although this was the lowest concentration tested, and the EC50 was 444 mmol l–1 (Dhaese et al., 2010). In longitudinal muscle from rat ileum, 10 mmol l–1 H2S reduced AUC, average amplitude of spontaneous activity and baseline tone by 7%, 3% and 6%, respectively (Nagao et al., 2010). These parameters were reduced 7%, 3% and 5% by 100 mmol l–1 H2S (Nagao et al., 2010). As the responses to 10 and 100 mmol l–1 H2S were not different, this does not appear to be a concentration-dependent response (each tissue served as its own control; time-matched controls were not employed, making it impossible to determine whether these effects were due to slight deterioration or stabilization of the preparation). However, 500 mmol l–1 H2S reduced these parameters by 60%, 20% and 32%, suggesting that the threshold may be above 100 mmol l–1. Based on the responses at 100, 500 and 1000 mmol l–1 (Nagao et al., 2010), the EC50 appears to be ∼500 mmol l–1. Similarly high concentrations (500 mmol l–1 H2S) were necessary to affect pacemaker currents in interstitial cells of Cajal from mouse small intestine (Parajuli et al., 2010).
Significant changes in either motility or frequency in the trout and coho GI tract were observed between 1 and 10 mmol l–1 H2S and the EC50 values for tissue response was between 78 and 171 mmol l–1 (overall mean was 120 mmol l–1). These concentrations are around 5-fold (or more) lower than those commonly necessary to achieve physiological responses in mammalian preparations. This suggests that the GI tract of the fish is exquisitely sensitive to this signaling molecule. However, the reason for this difference is not clear. Tissue (body) temperature could influence H2S metabolism or kinetics of downstream mediators, although one would expect that the intrinsically lower metabolic rate of exothermic fish would decrease responsivness, not increase it. It is also possible that there is not as much background H2S from bacterial production of H2S in the lumen of the fish GI tract as there is in mammals. To our knowledge bacterial H2S production has not been measured in the fish GI tract.
Are the effects of exogenous H2S ‘physiological’?
It is not clear whether the effects of H2S produced in this study (or others) are ‘physiological’ responses. This is due in large part to uncertainties surrounding: (1) the concentration of endogenous H2S in and around cells, (2) the actual concentration of exogenous H2S at the effector site, and (3) whether tissue H2S concentration can be regulated by physiologically relevant stimuli.
Although there are many reports of plasma and blood H2S concentration between 30 and 300 mmol l–1, these may be experimental artifacts (reviewed in Olson, 2009). In fact, H2S may not even exist in the circulation (Whitfield et al., 2008). Recent measurements have also shown that the concentration of H2S in a variety of tissues appears to be well below 1 mmol l–1 (Furne et al., 2008). Furthermore, as H2S is rapidly oxidized at H2S concentrations below 20 mmol l–1, yet higher concentrations of H2S inhibit mitochondrial cytochrome oxidase, and hence oxidative phosphorylation (Lagoutte et al., 2010), one could argue that H2S most likely does not exist in appreciable amounts in the GI tract and that it would be physiologically disadvantageous to allow H2S concentrations to even approach 20 mmol l–1. Passive loss of H2S from experimental apparatus due to volatility could be another confounding factor in the discrepancy between endogenous and physiologically effective exogenous H2S concentrations. We (DeLeon et al., 2011) recently observed that the half-time for H2S in a smooth muscle myograph of the type used in the present study was under 5 min, and this was due to volatilization not oxidation of H2S. More accurate assessment of EC50 values awaits continuous monitoring of H2S concentrations throughout an experiment.
The distal GI tract, however, may be exposed to high H2S concentrations as a result of bacterial H2S generation in the lumen. But here it is difficult to envisage how lumenal H2S production could have a signaling function. It is more likely that bacterially generated H2S is a potentially toxic metabolite to be disposed of or, perhaps, used as an energy source (Blachier et al., 2010). Collectively, this suggests that if H2S is a physiologically relevant signal, then it acts in a paracrine or more probably autocrine manner, and it is most likely of tissue, not lumenal, origin. Exact details of H2S signaling activities await detailed measurement of intracellular H2S concentrations.
Comparison of effects of H2S and hypoxia in the GI tract: the role of H2S in oxygen sensing
Rapid oxidation of H2S by the colon has been well documented (see Furne et al., 2001; Lagoutte et al., 2010; Levitt et al., 1999). This supports the argument that not only are tissue H2S concentrations minimal but also removal of H2S requires oxygen. The corollary to this is that an increase in H2S is predicated upon the reciprocal decrease in O2. Thus, the inverse relationship between H2S and O2 may be the physiological couple involved in H2S signaling.
As described above, we have proposed that O2-dependent metabolism of H2S serves as an O2 ‘sensor’ in a variety of mammalian and non-mammalian tissues (Dombkowski et al., 2006; Olson et al., 2006; Olson et al., 2008a; Olson et al., 2008b; Olson et al., 2010; Whitfield et al., 2008) (reviewed in Olson and Whitfield, 2010). Our hypothesis is based on the following observations: (1) H2S and hypoxia produce essentially identical responses in a variety of tissues, (2) tissues enzymatically generate H2S, (3) tissue production of H2S, or the concentration of exogenous H2S, is inversely related to oxygen availability, i.e. PO2, (4) inhibitors of H2S synthesis inhibit hypoxic responses, and (5) potential sulfide donors augment the hypoxic responses. Recent studies on the mammalian carotid body (Li et al., 2010; Peng et al., 2010; Telezhkin et al., 2009; Telezhkin et al., 2010) and kidney (Beltowski, 2010) have supported this hypothesis and our assertion that this is a phylogenetically ancient and ubiquitous O2-sensing mechanism. The present study extends these observations to the fish GI tract and supports the hypothesis that O2-dependent H2S metabolism is a fundamental oxygen-sensing mechanism.
In the fish GI tract essentially all of the effects of hypoxia are similar, if not identical, to those produced by H2S. Both stimuli relax visceral smooth muscle, inhibit spontaneous contractions and decrease their frequency. Furthermore, the magnitude of the effects of these two stimuli is also similar and they vary from tissue to tissue. Both hypoxia and H2S are more efficacious in longitudinal than in circular intestinal smooth muscle and, within a preparation (longitudinal or circular smooth muscle), the magnitude of inhibition produced by these two stimuli is virtually identical. Even more striking is the transient stimulatory effect observed in both trout and coho intestine at the onset of hypoxia or H2S application. This is reminiscent of the signature biphasic hypoxic constriction of rat pulmonary arteries that is uniquely mimicked by exogenous H2S (Olson et al., 2006).
Fig. 10 clearly shows that the trout intestine generates H2S in the presence of cysteine. This is consistent with the demonstrated production of H2S by CBS and CSE in the mouse colon (Linden et al., 2008). By simultaneously measuring PO2, we were also able to determine that H2S was only generated in the absence of O2 or, at the very least, at very low PO2 values. Furthermore, the reversal from net H2S production by intestinal tissue to net H2S consumption when oxygen was injected into the intestinal homogenate supports our previous observations in other tissues that H2S production and/or tissue concentrations are inversely related to oxygen availability.
Given that the effects of hypoxia and H2S on the trout and coho GI tract were virtually identical, and that the GI tissue produces H2S when hypoxic, one would expect that inhibition of H2S biosynthesis would inhibit the hypoxic response and that an increase in H2S biosynthesis by addition of the sulfide donor cysteine would enhance the hypoxic response. This is precisely what we observed (Fig. 9). Interestingly, however, the hypoxic response was equally inhibited by inhibition of CSE with PPG and of CBS with AOA. The effects of PPG on the hypoxic response appeared to be direct, whereas AOA may have an indirect effect on the hypoxic response as it also inhibited the carbachol contraction independent of hypoxia. It remains to be determined whether H2S generated by CBS has other effects on the intestine that are independent of the hypoxic response.
What is the physiological function of H2S in the GI tract?
The present results show that H2S inhibits GI activity and provide considerable evidence that this may be the mechanism through which the hypoxic response is expressed. This raises the question, what is the physiological significance of either H2S or hypoxia in GI function? As both of these stimuli would presumably decrease the GI motility and transit time through the gut, they may serve to also decrease oxygen demand by GI tissues in the face of an oxygen deficit or perhaps permit more thorough processing of lumenal contents. Alternatively, these responses may be an evolutionary relic. As H2S and hypoxia have similar effects on vascular smooth muscle in a broad range of vessels, and on smooth muscle in the urinary bladder, and as shown here in a variety of GI tissues, it may be that this response was at one time employed in a different signaling capacity during the early evolution of eukaryotes. However, as tissues became more specialized it was no longer linked to a specific physiological function. Clearly, this remains to be investigated.
This work was supported in part by the National Science Foundation [grant nos IOS 0641436 and IOS 1051627].
The authors wish to thank D. Meunick, B. Bell and the staff at the Richard Clay Bodine State Fish Hatchery, Indiana Department of Natural Resources, for their help in obtaining coho tissues.