Luminal sulfide and large intestine mucosa: friend or foe?

Hydrogen sulfide (H₂S) is present in the lumen of the human large intestine at millimolar concentrations. However, the concentration of free (unbound) sulfide is in the micromolar range due to a large capacity of fecal components to bind the sulfide. H₂S can be produced by the intestinal microbiota from alimentary and endogenous sulfur-containing compounds including amino acids. At excessive concentration, H₂S is known to severely inhibit cytochrome c oxidase, the terminal oxidase of the mitochondrial electron transport chain, and thus mitochondrial oxygen (O₂) consumption. However, the concept that sulfide is simply a metabolic troublemaker toward colonic epithelial cells has been challenged by the discovery that micromolar concentration of H₂S is able to increase the cell respiration and to energize mitochondria allowing these cells to detoxify and to recover energy from luminal sulfide. The main product of H₂S metabolism by the colonic mucosa is thiosulfate. The enzymatic activities involved in sulfide oxidation by the colonic epithelial cells appear to be sulfide quinone oxidoreductase considered as the first and rate-limiting step followed presumably by the action of sulfur dioxygenase and rhodanese. From clinical studies with human volunteers and experimental works with rodents, it appears that H₂S can exert mostly pro- but also anti-inflammatory effects on the colonic mucosa. From the available data, it is tempting to propose that imbalance between the luminal concentration of free sulfide and the capacity of colonic epithelial cells to metabolize this compound will result in an impairment of the colonic epithelial cell O₂ consumption with consequences on the process of mucosal inflammation. In addition, endogenously produced sulfide is emerging as a prosecretory neuromodulator and as a relaxant agent toward the intestinal contractibility. Lastly, sulfide has been recently described as an agent involved in nociception in the large intestine although, depending on the experimental design, both pro- and anti-nociceptive effects have been reported.     

Hydrogen sulfide attenuates homocysteine-induced osteoblast dysfunction by inhibiting mitochondrial toxicity

Homocysteine (Hcy) is detrimental to bone health in a mouse model of diet-induced hyperhomocysteinemia (HHcy). However, little is known about Hcy-mediated osteoblast dysfunction via mitochondrial oxidative damage. Hydrogen sulfide (H₂S) has potent antioxidant, anti-inflammatory, and antiapoptotic effects. In this study, we hypothesized that the H₂S mediated recovery of osteoblast dysfunction by maintaining mitochondrial biogenesis in Hcy-treated osteoblast cultures in vitro. MC3T3-E1 osteoblastic cells were exposed to Hcy treatment in the presence or absence of an H₂S donor (NaHS). Cell viability, osteogenic differentiation, reactive oxygen species (ROS) production were determined. Mitochondrial DNA copy number, adenosine triphosphate (ATP) production, and oxygen consumption were also measured. Our results demonstrated that administration of Hcy increases the intracellular Hcy level and decreases intracellular H₂S level and expression of the cystathionine β-synthase/Cystathionine γ-lyase system, thereby inhibiting osteogenic differentiation. Pretreatment with NaHS attenuated Hcy-induced mitochondrial toxicity (production of total ROS and mito-ROS, ratio of mitochondrial fission (DRP-1)/fusion (Mfn-2)) and restored ATP production and mitochondrial DNA copy numbers as well as oxygen consumption in the osteoblast as compared with the control, indicating its protective effects against Hcy-induced mitochondrial toxicity. In addition, NaHS also decreased the release of cytochrome c from the mitochondria to the cytosol, which induces cell apoptosis. Finally, flow cytometry confirmed that NaHS can rescue cells from apoptosis induced by Hcy. Our studies strongly suggest that NaHS has beneficial effects on mitochondrial toxicity, and could be developed as a potential therapeutic agent against HHcy-induced mitochondrial dysfunction in cultured osteoblasts in vitro.     

Studies on the orientations of the mitochondrial redox carriers II. Orientation of the mitochondrial chromophores with respect to the plane of the membrane in hydrated,oriented mitochondrial multilayers

Orientations of the active site chromophores of the mitochondrial redox carriers have been investigated in hydrated, oriented multilayers of mitochondrial membranes using optical and EPR spectroscopy. The hemes of cytochrome c oxidase, cytochrome c₁, and cytochromes b were found to be oriented in a similar manner, with the normal to their heme planes lying approximately in the plane of the mitochondrial membrane. The heme of cytochrome c was either less oriented in general or was oriented at an angle closer to the plane of the mitochondrial membrane than were the hemes of the “tightly bound” mitochondrial cytochromes. EPR spectra of the azide, sulfide and formate complexes of cytochrome c oxidase in mitochondria in situ obtained as a function of the orientation of the applied magnetic field relative to the planes of the membrane multilayers showed that both hemes of the oxidase were oriented in such a way that the angle between the heme normal and the membrane normal was approx. 90°.     

Oxidation of hydrogen sulfide remains a priority in mammalian cells and causes reverse electron transfer in colonocytes

Sulfide (H₂S) is an inhibitor of mitochondrial cytochrome oxidase comparable to cyanide. In this study, poisoning of cells was observed with sulfide concentrations above 20 µM. Sulfide oxidation has been shown to take place in organisms/cells naturally exposed to sulfide. Sulfide is released as a result of metabolism of sulfur containing amino acids. Although in mammals sulfide exposure is not thought to be quantitatively important outside the colonic mucosa, our study shows that a majority of mammalian cells, by means of the mitochondrial sulfide quinone reductase (SQR), avidly consume sulfide as a fuel. The SQR activity was found in mitochondria isolated from mouse kidneys, liver, and heart. We demonstrate the precedence of the SQR over the mitochondrial complex I. This explains why the oxidation of the mineral substrate sulfide takes precedence over the oxidation of other (carbon-based) mitochondrial substrates. Consequently, if sulfide delivery rate remains lower than the SQR activity, cells maintain a non-toxic sulfide concentration (< 1 µM) in their external environment. In the colonocyte cell line HT-29, sulfide oxidation provided the first example of reverse electron transfer in living cells, such a transfer increasing sulfide tolerance. However, SQR activity was not detected in brain mitochondria and neuroblastoma cells. Consequently, the neural tissue would be more sensitive to sulfide poisoning. Our data disclose new constraints concerning the emerging signaling role of sulfide.     

The inhibition of mitochondrial cytochrome oxidase by the gases carbon monoxide, nitric oxide, hydrogen cyanide and hydrogen sulfide: chemical mechanism and physiological significance

The four gases, nitric oxide (NO), carbon monoxide (CO), hydrogen sulfide (H₂S) and hydrogen cyanide (HCN) all readily inhibit oxygen consumption by mitochondrial cytochrome oxidase. This inhibition is responsible for much of their toxicity when they are applied externally to the body. However, recently these gases have all been implicated, to greater or lesser extents, in normal cellular signalling events. In this review we analyse the chemistry of this inhibition, comparing and contrasting mechanism and discussing physiological consequences. The inhibition by NO and CO is dependent on oxygen concentration, but that of HCN and H₂S is not. NO and H₂S are readily metabolised by oxidative processes within cytochrome oxidase. In these cases the enzyme may act as a physiological detoxifier of these gases. CO oxidation is much slower and unlikely to be as physiologically important. The evidence for normal physiological levels of these gases interacting with cytochrome oxidase is equivocal, in part because there is little robust data about their steady state concentrations. A reasonable case can be made for NO, and perhaps CO and H₂S, inhibiting cytochrome oxidase in vivo, but endogenous levels of HCN seem unlikely to be high enough.     

Studies on the orientations of the mitochondrial redox carriers I. Orientation of the hemes of cytochrome c oxidase with respect to the plane of a cytochrome oxidase-lipid model membrane

The liganded derivatives of mitochondrial cytochrome c oxidase have been prepared in hydrated oriented multilayers of membranous cytochrome c oxidase. The optical spectra of the liganded derivatives recorded at an angle of 45° between the incident light beam and the normal to the planes of the membranes in the multilayers show dichroic ratios of almost 2 in the visible region and 1.2–1.4 in the Soret region. The dichroic ratios were found to be similar for both cytochromes a and a₃. Electron paramagnetic resonance spectra of the azide, sulfide, and formate complexes of cytochrome c oxidase obtained as a function of the orientation of the applied magnetic field relative to the planes of the membranes in the multilayer confirm the optical data and demonstrate that both hemes of cytochrome c oxidase are oriented such that the angle between the heme normal and the membrane normal is approximately 90°.     

Exogenous hydrogen sulfide attenuates diabetic myocardial injury through cardiac mitochondrial protection

In the study, we investigated how exogenous H₂S (hydrogen sulfide) influenced streptozotocin (STZ)-induced diabetic myocardial injury through cardiac mitochondrial protection and nitric oxide (NO) synthesis in intact rat hearts and primary neonatal rat cardiomyocytes. Diabetes was induced by STZ (50?mg/kg) and the daily administration of 100?μM NaHS (sodium hydrosulfide, an H₂S donor) in the diabetes?+?NaHS treatment group. At the end of 4, 8, and 12?weeks, the morphological alterations and functions of the hearts were observed using transmission electron microscopy and echocardiography system. The percentage of apoptotic cardiomyocytes, the mitochondrial membrane potential, the production of reactive oxygen species (ROS) and the level of NO were measured. The expressions of cystathionine-γ-lyase (CSE), caspase-3 and -9, the mitochondrial NOX4 and cytochrome c were analyzed by western blotting. The results showed the cardiac function injured, morphological changes and the apoptotic rate increased in the diabetic rat hearts. In the primary neonatal rat cardiomyocytes of high glucose group, ROS production was increased markedly, whereas the expression of CSE and the level of NO was decreased. However, treatment with NaHS significantly reversed the diabetic rat hearts function, the morphological changes and decreased the levels of ROS and NO in the primary neonatal rat cardiomyocytes administrated with high glucose group. Furthermore, NaHS down-regulated the expression of mitochondrial NOX4 and caspase-3 and -9 and inhibited the release of cytochrome c from mitochondria in the primary neonatal rat cardiomyocytes. In conclusion, H₂S is involved in the attenuation of diabetic myocardial injury through the protection of cardiac mitochondria.     

Methylene Blue as a Cerebral Metabolic and Hemodynamic Enhancer

By restoring mitochondrial function, methylene blue (MB) is an effective neuroprotectant in many neurological disorders (e.g., Parkinson’s and Alzheimer’s diseases). MB has also been proposed as a brain metabolic enhancer because of its action on mitochondrial cytochrome c oxidase. We used in vitro and in vivo approaches to determine how MB affects brain metabolism and hemodynamics. For in vitro, we evaluated the effect of MB on brain mitochondrial function, oxygen consumption, and glucose uptake. For in vivo, we applied neuroimaging and intravenous measurements to determine MB’s effect on glucose uptake, cerebral blood flow (CBF), and cerebral metabolic rate of oxygen (CMRO₂) under normoxic and hypoxic conditions in rats. MB significantly increases mitochondrial complex I–III activity in isolated mitochondria and enhances oxygen consumption and glucose uptake in HT-22 cells. Using positron emission tomography and magnetic resonance imaging (MRI), we observed significant increases in brain glucose uptake, CBF, and CMRO₂ under both normoxic and hypoxic conditions. Further, MRI revealed that MB dramatically increased CBF in the hippocampus and in the cingulate, motor, and frontoparietal cortices, areas of the brain affected by Alzheimer’s and Parkinson’s diseases. Our results suggest that MB can enhance brain metabolism and hemodynamics, and multimetric neuroimaging systems offer a noninvasive, nondestructive way to evaluate treatment efficacy.     

Aging-related decrease in hepatic cytochrome oxidase of the Fischer 344 rat

The effect of aging on the hepatic mitochondrial population has been determined using a rigorously defined group of Fischer 344 rats with known survivorship data. The age groups studied included mature adult controls (8.5 months; 100% survivorship), an intermediate aged group (17.5 months; 90% survivorship), and an aged group (29 months; 20% survivorship). Cytochrome oxidase activity and content were determined in homogenates and mitochondrial fractions. The mitochondrial fractions were characterized by determination of respiratory activity, and monoamine oxidase activity as well as evaluation of the polypeptide composition by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional electrophoresis. The yield of protein in the isolated mitochondrial fraction as well as the mitochondrial specific content decreased significantly as a function of aging. Mitochondrial specific content was determined from the specific activities of cytochrome oxidase in the homogenate (per gram liver) and in the isolated mitochondrial fraction (per mg protein). Specific activity of hepatic cytochrome oxidase decreased approximately 15% (P = 0.035) in homogenates from the 17.5-month animals with a further, highly significant (P = 0.0002) decrease (29%) in the 29-month animals. In contrast, there was no statistically significant difference among the age groups in the cytochrome oxidase specific activity in the isolated hepatic mitochondrial fractions. However, the percentage of the total homogenate cytochrome oxidase activity recovered in the isolated mitochondrial fraction decreased significantly in the 29-month animals (P = 0.0063 vs the 8.5-month controls; P = 0.022 vs the 17.5-month group). Cytochrome aa₃ content of total liver homogenates from aged animals decreased (P = 0.00064) which is in agreement with the decline in cytochrome oxidase specific activity in this age group. In the mitochondrial fraction from the aged animals, cytochrome aa₃ content was essentially unchanged which is consistent with the lack of aging-related change in mitochondrial cytochrome oxidase specific activity. In freshly isolated mitochondrial fractions, no aging-related alterations were observed in respiratory control and $\text{ADP}\text{O}$ ratios. The addition of exogenous NADH and cytochrome c did not change significantly the respiratory rate of hepatic mitochondria from control or aged animals. These results demonstrate the integrity of freshly isolated mitochondrial preparations from both control and aged Fischer 344 rats. In addition, there was no aging-related alteration in either monoamine oxidase specific activity or polypeptide composition. The similarities observed in the specific activities of cytochrome oxidase and monoamine oxidase, as well as in the cytochrome aa₃ content and polypeptide composition of the isolated mitochondrial fraction, suggest a generalized decrease in hepatic mitochondrial content as a function of aging rather than a selective loss of mitochondrial components.     

Decreased cytochrome oxidase activity in hepatic mitochondria after chronic ethanol consumption and the possible role of decreased cytochrome aa3 content and changes in phospholipids

In ethanol-fed baboons, hepatic mitochondrial cytochrome oxidase activity and cytochrome aa₃ content were significantly decreased by 58.3 and 50.5%, respectively, compared to their pair-fed controls. However, there was no significant correlation between the two, suggesting that other factors in addition to cytochrome aa₃ may be responsible for the depression in cytochrome oxidase activity. The total phospholipid content of the mitochondrial membranes was significantly decreased (0.24 ± 0.03 μmol of phospholipid phosphorus/mg of protein vs. 0.32 ± 0.04 in controls). This change was accounted for, in part, by the significant decrease in the levels of phosphatidylcholine and cardiolipin. In addition, the fatty acid pattern of the phospholipids was changed. There was a marked increase in the relative amounts of oleic and linoleic acid and a decrease in arachidonic acid. These changes were associated with an increase in the activity of phospholipase A₂. The reactivation rate of phospholipid-depleted cytochrome oxidase by endogenous phospholipids from ethanol-fed baboons was significantly lower than that by phospholipid from pair-fed controls, when measured at an optimal phospholipid to protein ratio. Thus, it appears that alterations in the phospholipid composition of the mitochondrial membranes are responsible, at least in part, for the depression of cytochrome oxidase activity produced by chronic ethanol consumption.     

Formamide probes a role for water in the catalytic cycle of cytochrome c oxidase

Formamide is a slow-onset inhibitor of mitochondrial cytochrome c oxidase that is proposed to act by blocking water movement through the protein. In the presence of formamide the redox level of mitochondrial cytochrome c oxidase evolves over the steady state as the apparent electron transfer rate from cytochrome a to cytochrome a₃ slows. At maximal inhibition cytochrome a and cytochrome c are fully reduced, whereas cytochrome a₃ and Cu_B remain fully oxidized consistent with the idea that formamide interferes with electron transfer between cytochrome a and the oxygen reaction site. However, transient kinetic studies show that intrinsic rates of electron transfer are unchanged in the formamide-inhibited enzyme. Formamide inhibition is demonstrated for another member of the heme-oxidase family, cytochrome c oxidase from Bacillus subtilis, but the onset of inhibition is much quicker than for mitochondrial oxidase. If formamide inhibition arises from a steric blockade of water exchange during catalysis then water exchange in the smaller bacterial oxidase is more open. Subunit III removal from the mitochondrial oxidase hastens the onset of formamide inhibition suggesting a role for subunit III in controlling water exchange during the cytochrome c oxidase reaction.     

The deleterious metabolic and genotoxic effects of the bacterial metabolite p-cresol on colonic epithelial cells

p-Cresol that is produced by the intestinal microbiota from the amino acid tyrosine is found at millimolar concentrations in the human feces. The effects of this metabolite on colonic epithelial cells were tested in this study. Using the human colonic epithelial HT-29 Glc^–/+ cell line, we found that 0.8 mM p-cresol inhibits cell proliferation, an effect concomitant with an accumulation of the cells in the S phase and with a slight increase of cell detachment without necrotic effect. At this concentration, p-cresol inhibited oxygen consumption in HT-29 Glc^–/+ cells. In rat normal colonocytes, p-cresol also inhibited respiration. Pretreatment of HT-29 Glc^–/+ cells with 0.8 mM p-cresol for 1 day resulted in an increase of the state 3 oxygen consumption and of the cell maximal respiratory capacity with concomitant increased anion superoxide production. At higher concentrations (1.6 and 3.2 mM), p-cresol showed similar effects but additionally increased after 1 day the proton leak through the inner mitochondrial membrane, decreasing the mitochondrial bioenergetic activity. At these concentrations, p-cresol was found to be genotoxic toward HT-29 Glc^–/+ and also LS-174T intestinal cells. Lastly, a decreased ATP intracellular content was observed after 3 days treatment. p-Cresol at 0.8 mM concentration inhibits colonocyte respiration and proliferation. In response, cells can mobilize their “respiratory reserve.” At higher concentrations, p-cresol pretreatment uncouples cell respiration and ATP synthesis, increases DNA damage, and finally decreases the ATP cell content. Thus, we have identified p-cresol as a metabolic troublemaker and as a genotoxic agent toward colonocytes.     

Involvement of ERK in NMDA receptor-independent cortical neurotoxicity of hydrogen sulfide

Hydrogen sulfide (H₂S), a gasotransmitter, exerts both neurotoxicity and neuroprotection, and targets multiple molecules including NMDA receptors, T-type calcium channels and NO synthase (NOS) that might affect neuronal viability. Here, we determined and characterized effects of NaHS, an H₂S donor, on cell viability in the primary cultures of mouse fetal cortical neurons. NaHS caused neuronal death, as assessed by LDH release and trypan blue staining, but did not significantly reduce the glutamate toxicity. The neurotoxicity of NaHS was resistant to inhibitors of NMDA receptors, T-type calcium channels and NOS, and was blocked by inhibitors of MEK, but not JNK, p38 MAP kinase, PKC and Src. NaHS caused prompt phosphorylation of ERK and upregulation of Bad, followed by translocation of Bax to mitochondria and release of mitochondrial cytochrome c, leading to the nuclear condensation/fragmentation. These effects of NaHS were suppressed by the MEK inhibitor. Our data suggest that the NMDA receptor-independent neurotoxicity of H₂S involves activation of the MEK/ERK pathway and some apoptotic mechanisms.     

Enhanced cytochrome oxidase activity and modification of lipids in heart mitochondria from hyperthyroid rats

In order to further investigate the mechanism regulating the control of mitochondrial respiration by thyroid hormones, the effect of the hyperthyroidism on the kinetic characteristics of cytocrome c oxidase in rat heart mitochondria was studied. Mitochondrial preparations from both control and hyperthyroid rats had equivalent K_m values for cytochrome c, while the maximal activity of cytochrome oxidase was significantly increased (by around 30%) in mitochondrial rats. This enhanced activity of cytochrome oxidase was associated to a parallel increases in mitochondrial State 3 respiration. The hormone treatment resulted in a decrease in the flux control coefficient of the oxidase. The enhanced activity of cytochrome oxidase in hyperthyroid rats does not appear to be dependent on an increases in the mass of this enzyme complex in that the heme aa₃ content was equivalent in both hyperthyroid and control preparations. The Arrhenius plot characteristics differ for cytochrome oxidase activity in mitochondria from hyperthyroid rats as compared with control rats in the breakpoint of the biphasic plot is shifted to a lower temperature. Cardiolipin content was significantly increased in mitochondrial preparations from hyperthyroid rats, while there were no significant alterations in the fatty acid composition of cardiolipin of control and hyperthyroid preparations. The results support the conclusion that the enhanced cytochrome oxidase activity in heart mitochondrial preparations from hyperthyroid rats is due to a specific increase in the content of cardiolipin.     

The Participation of calcium ions and reactive oxygen species in the induction of antioxidant enzymes and heat resistance in plant cells by hydrogen sulfide donor

The effect of hydrogen sulfide (H₂S) donor sodium hydrosulfide (NaHS) on the heat resistance of wheat (Triticum aestivum L.) coleoptile cells, the formation of reactive oxygen species (ROS), and the activity of the antioxidant enzymes in them was investigated. The treatment of coleoptiles with 100 µM NaHS caused transient enhancement of the generation of the superoxide anion radical (O₂ ^?) and an increased hydrogen peroxide content. The activities of antioxidant enzymes—superoxide dismutase, catalase, and guaiacol peroxidase— and coleoptile resistance to damaging heat was later found to have increased. The biochemical and physiological effects of the hydrogen sulfide donor described above were inhibited by the treatment of wheat coleoptiles with the hydrogen peroxide scavenger dimethylthiourea, the NADPH oxidase inhibitor imidazole, the extracellular calcium chelator EGTA, and the phosphatidylinositol-specific phospholipase C inhibitor neomycin. A conclusion was made on the role of ROS generation, which is dependent on the activity of NADPH oxidase and calcium homeostasis, in the transduction of the H₂S signal, which induces antioxidant enzymes and the development of plant cell heat resistance.     

Sulfide-Resistant Respiration in Leaves of Elodea canadensis Michx: Comparison with Cyanide-Resistant Respiration

The rate of dark O₂ uptake of Elodea canadensis leaves was titrated with either cyanide or sulfide in the presence and in the absence of 5 millimolar salicylhydroxamic acid (SHAM), an inhibitor of the alternative oxidase. The inhibition of O₂ uptake by SHAM alone was very small (3-6%), suggesting that actual respiration mainly occurred through the cytochrome pathway. O₂ uptake was slightly stimulated by cyanide at concentrations of 50 micromolar or higher, but in the presence of SHAM respiration was strongly suppressed. The effects of sulfide on O₂ uptake were similar to those of cyanide, except that the percent stimulation of O₂ uptake by sulfide alone was somewhat higher than that of cyanide. However, the estimates of the capacity of the alternative pathway were similar with both inhibitors. Another difference is that maximal inhibition of respiration in the presence of SHAM was observed with lower concentrations of sulfide (50 micromolar) than cyanide (250 micromolar). The results suggest that sulfide can be used as a suitable inhibitor of cytochrome c oxidase in studies with intact plant tissues, and that sulfide does not apparently inhibit the alternative oxidase.     

Neuroprotective effects of hydrogen sulfide on Parkinson’s disease rat models

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by a progressive loss of dopaminergic neurons in the substantia nigra (SN). The present study was designed to examine the therapeutic effect of hydrogen sulfide (H₂S, a novel biological gas) on PD. The endogenous H₂S level was markedly reduced in the SN in a 6‐hydroxydopamine (6‐OHDA)‐induced PD rat model. Systemic administration of NaHS (an H₂S donor) dramatically reversed the progression of movement dysfunction, loss of tyrosine‐hydroxylase positive neurons in the SN and the elevated malondialdehyde level in injured striatum in the 6‐OHDA‐induced PD model. H₂S specifically inhibited 6‐OHDA evoked NADPH oxidase activation and oxygen consumption. Similarly, administration of NaHS also prevented the development of PD induced by rotenone. NaHS treatment inhibited microglial activation in the SN and accumulation of pro‐inflammatory factors (e.g. TNF‐α and nitric oxide) in the striatum via NF‐κB pathway. Moreover, significantly less neurotoxicity was found in neurons treated with the conditioned medium from microglia incubated with both NaHS and rotenone compared to that with rotenone only, suggesting that the therapeutic effect of NaHS was, at least partially, secondary to its suppression of microglial activation. In summary, we demonstrate for the first time that H₂S may serve as a neuroprotectant to treat and prevent neurotoxin‐induced neurodegeneration via multiple mechanisms including anti‐oxidative stress, anti‐inflammation and metabolic inhibition and therefore has potential therapeutic value for treatment of PD.     

Hydrogen Sulfide Regulates Cardiovascular Function by Influencing the Excitability of Subfornical Organ Neurons

Hydrogen sulfide (H₂S), a gasotransmitter endogenously found in the central nervous system, has recently been suggested to act as a signalling molecule in the brain having beneficial effects on cardiovascular function. This study was thus undertaken to investigate the effect of NaHS (an H₂S donor) in the subfornical organ (SFO), a central nervous system site important to blood pressure regulation. We used male Sprague-Dawley rats for both in vivo and in vitro experiments. We first used RT-PCR to confirm our previous microarray analyses showing that mRNAs for the enzymes required to produce H₂S are expressed in the SFO. We then used microinjection techniques to investigate the physiological effects of NaHS in SFO, and found that NaHS microinjection (5 nmol) significantly increased blood pressure (mean AUC = 853.5±105.7 mmHg*s, n = 5). Further, we used patch-clamp electrophysiology and found that 97.8% (88 of 90) of neurons depolarized in response to NaHS. This response was found to be concentration dependent with an EC₅₀ of 35.6 µM. Coupled with the depolarized membrane potential, we observed an overall increase in neuronal excitability using an analysis of rheobase and action potential firing patterns. This study has provided the first evidence of NaHS and thus H₂S actions and their cellular correlates in SFO, implicating this brain area as a site where H₂S may act to control blood pressure.     

Inactivation of nitric oxide by cytochrome c oxidase under steady-state oxygen conditions

We have developed a respiration chamber that allows intact cells to be studied under controlled oxygen (O₂) conditions. The system measures the concentrations of O₂ and nitric oxide (NO) in the cell suspension, while the redox state of cytochrome c oxidase is continuously monitored optically. Using human embryonic kidney cells transfected with a tetracycline-inducible NO synthase we show that the inactivation of NO by cytochrome c oxidase is dependent on both O₂ concentration and electron turnover of the enzyme. At a high O₂ concentration (70 μM), and while the enzyme is in turnover, NO generated by the NO synthase upon addition of a given concentration of l-arginine is partially inactivated by cytochrome c oxidase and does not affect the redox state of the enzyme or consumption of O₂. At low O₂ (15 μM), when the cytochrome c oxidase is more reduced, inactivation of NO is decreased. In addition, the NO that is not inactivated inhibits the cytochrome c oxidase, further reducing the enzyme and lowering O₂ consumption. At both high and low O₂ concentrations the inactivation of NO is decreased when sodium azide is used to inhibit cytochrome c oxidase and decrease electron turnover.