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Endogenous hydrogen sulfide is involved in regulation of respiration in medullary slice of neonatal rats
Neuroscience 156 (2008) 1074 –1082
ENDOGENOUS HYDROGEN SULFIDE IS INVOLVED IN REGULATION OF RESPIRATION IN MEDULLARY SLICE OF NEONATAL RATS H. HU, Y. SHI, Q. CHEN, W. YANG, H. ZHOU, L. CHEN, Y. TANG AND Y. ZHENG*
the hippocampus, cerebellum, brainstem and cerebral cortex (Abe and Kimura, 1996; Eto et al., 2002b). H2S could not be detected in the brain of mice with gene knockout of CBS (Eto et al., 2002b). H2S production could be suppressed by CBS inhibitor, hydroxylamine (NH2OH), and enhanced by its activator, S-adenosyl-L-methionine (SAM) (Abe and Kimura, 1996). It could also be greatly enhanced by activating glutamate receptors and Ca2⫹/calmodulin in the hippocampus (Eto et al., 2002b). H2S in physiological concentration specifically enhanced the activity of NMDA receptor and facilitated the long-term potentiation in the hippocampus (Abe and Kimura, 1996), while pathways mediated by cAMP may be involved in the modulation of NMDA receptors by H2S (Kimura, 2000). It has been proposed therefore that H2S can be produced in the brain and may play an important role in probably acting as a neuromodulator or an intracellular messenger (Abe and Kimura, 1996). Inhalation of exogenous H2S within moderate concentration increased the respiratory rate, whereas high concentration of the gas functioned exactly opposite, suggesting that the respiratory activity would be affected by H2S (Beauchamp et al., 1984; Reiffenstein et al., 1992). However whether H2S functions in the central regulation of respiratory activity has been unknown although CBS was found highly expressed and endogenous H2S produced in the brainstem of rats as mentioned above. It is well known that the medulla oblongata is essential for the rhythmogenesis of respiration. We hypothesize therefore that H2S would participate in the central regulation of respiration at the level of medulla oblongata. The purpose of the present study was to investigate the effects of exogenous H2S on and the possible roles of endogenous H2S in the rhythmic respiratory activity and the mechanisms underlying the central respiratory actions of H2S of the in vitro preparations of the medullary slices of neonatal rats.
Department of Physiology, West China School of Preclinical and Forensic Medicine, Sichuan University, 3-17 Renmin South Road, Chengdu, Sichuan 610041, PR China
Abstract—The purpose of the present study was to verify our assumption that rhythmic respiratory activity may be regulated by endogenous hydrogen sulfide (H2S) in the medullary slices of neonatal rats. We found that a moderate concentration of donor of H2S, NaHS, mainly induced diphasic respiratory responses indicated by changes of discharge frequency (DF) of hypoglossal rootlets, an initial inhibitory stage followed by a later excitatory one. Cystathionine -synthase (CBS) substrate, cysteine (CYS), exerted similar effects. CBS inhibitor, NH2OH, could eliminate both inhibitory and excitatory effects in the two stages induced by CYS. KATP channel blocker, glibenclamide (Gl), could eliminate the decrease in DF in the initial stage, but not the increase in the later one. On the other hand, adenyl cyclase (AC) inhibitor, SQ-22536 (SQ) could eliminate the increase in DF in the later stage, but not the decrease in the initial one, of the rootlets caused by NaHS. Co-application of Gl and SQ eliminated both inhibitory and excitatory effect induced by NaHS. The cAMP level was increased in the later stage but not in the initial one by NaHS, and the increase in the cAMP level could be eliminated by SQ. It can be concluded that the endogenous H2S could be produced through the CBS–H2S pathway and could be involved in the control of the central rhythmic respiration in the in vitro medullary slices of neonatal rats by opening KATP channels and activating AC– cAMP pathway of the neurons. © 2008 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: respiratory regulation, medullary slices, discharge of hypoglossal rootlets, KATP channel, cAMP.
Hydrogen sulfide (H2S) is a well-known toxic gas and its toxicology has been extensively studied (Reiffenstein et al., 1992). However, a relatively high level of endogenous H2S has been detected in the brains of rats, humans and bovine (Goodwin et al., 1989; Warenycia et al., 1989; Savage and Gould, 1990), suggesting that H2S may have certain physiological functions. Endogenous H2S in the brain comes from decomposing the substrates, L-cysteine (CYS) and homocysteine, by cystathionine -synthase (CBS) (Stipanuk and Beck, 1982; Griffith, 1987). CBS was found highly expressed in
EXPERIMENTAL PROCEDURES Agents The main agents were purchased from Sigma (St. Louis, MO, USA), including NaHS, CYS, NH2OH, SAM, glibenclamide (Gl) and SQ-22536 (SQ). The solutions containing NaHS were prepared just before use.
*Corresponding author. Tel: ⫹86-28-8550-3433; fax: ⫹86-28-8550-3204. E-mail address: [email protected] (Y. Zheng). Abbreviations: AC, adenyl cyclase; ACSF, artificial cerebrospinal fluid; CBS, cystathionine -synthase; CYS, cysteine; DF, discharge frequency; Gl, glibenclamide; IA, integrated amplitude; PKA, protein kinase A; RIA, radioimmunoassay; SAM, S-adenosyl-L-methionine; SQ, SQ-22536.
Preparation of medullary slices The experiments were performed on medullary slices of male or female neonatal (0 – 4 days) Sprague–Dawley rats. All procedures were reviewed and approved by the Sichuan University Committee on the Use of Live Animals in Research, and conformed to the
0306-4522/08 © 2008 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2008.08.025
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Principles of Laboratory Animal Care (NIH publication No. 86-23 revised in 1985). The number of animals used and their suffering were minimized. Medullary slices were prepared as described elsewhere (Yang et al., 2007). In brief, the animals were anesthetized with ether by inhalation and then decapitated. The isolated brainstem was placed in the slicing chamber which was filled with ice-cold artificial cerebrospinal fluid (ACSF) bubbled with carbogen (95% O2 and 5% CO2). The ACSF contained (mM): 125 NaCl, 5 KCl, 2 CaCl2, 1 MgSO4, 22 NaHCO3, 1 KH2PO4 and 30 dglucose and pH was regulated to 7.4 by Hepes. The brainstem was positioned dorsal side upward, facing to the slicing blade at 20° to the rostral end of the block. In ice-cold ACSF, a single transverse slice of 1000 –1200 m thick from one brainstem was prepared. The obex was taken as the reference of the cutting levels under an optical microscopy. The brainstem was cut at the levels of about 100 m caudal and 1000 m rostral to the obex. The slice in such a thickness would contain the pre-Bötzinger complex (Smith et al., 1991), rostrally the parafacial respiratory group (Onimaru and Homma, 2003) and caudally the traditionally recognized rostral ventral respiratory group, and certainly the entire neuronal circuits to produce rhythmic respiratory outputs. Fig. 2. Schematic diagram of recording technique of hypoglossal rootlets discharge of medullary slice preparation of neonatal rats. (A) The transversal view of medullary slice. ION: inferior olivary nucleus; NTS: nucleus tractus solitarius; PBC: pre-Bötzinger complex; SP5: spinal trigeminal tract; XII: hypoglossal nucleus; XIIn: hypoglossal rootlets. (B) Discharge activity of hypoglossal rootlets, raw recording (XIIn) and integrated activity (兰XIIn), respectively.
Fig. 1 provides three representative levels of the medulla oblongata, around most caudal and most rostral cutting levels and a middle level between them, to show the histological evidence of the structures the slice contains. The slices were transferred to a recording chamber and continuously perfused with oxygenated ACSF at a rate of 4 – 6 mL/min at 29 °C. The slices were incubated for 30 min before starting the experiments. Glass suction electrodes filled with ACSF were used to record the rhythmic respiratory activity from the cut ends of the hypoglossal rootlets (Fig. 2A). Signals were amplified, filtered (⫽0.001 s, F⫽1 kHz) and integrated with a time constant of 50 ms by BL-420 E⫹ biological signal processing system (Taimeng Biotech. Co., China). The discharge frequency (DF, number of discharge per min) and integrated amplitude (IA, Fig. 2B) of hypoglossal rootlets were analyzed. In the present study, a total of 176 slices were used.
Perfusion of slices with different concentration of NaHS
Fig. 1. Photographs (left) and corresponding schematic diagrams (right) of three representative levels of the medullary slice of neonatal rat, including around most caudal (lower) and most rostral (upper) cutting levels and a middle level (middle) between them to show the histological evidence of the structures the slice contains, with HE staining. AP: area postrema; BC: Bötzinger complex; ION: inferior olivary nucleus; NA: nucleus ambiguous; NTS: nucleus tractus solitarius; PBC: pre-Bötzinger complex; PY: pyramidal tract; SP5: spinal trigeminal tract; XII: hypoglossal nucleus.
NaHS was used as a substitute of H2S to perfuse the slices in the present experiments because the quantity or concentration of H2S is difficult to control when the slices are perfused with bubbled H2S. NaHS can dissociate to deliver Na⫹ and HS⫺ in solution and then HS⫺ is associated with H⫹ to produce H2S. Approximately one-third of the H2S exists as the undissociated form, H2S, while the majority exists as HS⫺ in physiological saline (Reiffenstein et al., 1992). The slices were divided into eight groups (n⫽8 for each) including one control and seven tests. In the control group, the slices were perfused with ACSF and the discharge of hypoglossal rootlets was recorded during the whole experimental process. In each of the test groups, the discharge of rootlets was recorded for 5 min in ACSF as the baseline activity before NaHS application and then the slices were perfused with different concentration of NaHS at 10, 20, 50, 100, 200, 300 and 400 M, respectively, for 10 min following washout with ACSF. Discharge of the rootlets was recorded to observe the effects of different concentration of NaHS on the central respiratory activity.
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Analysis of existence of CBS–H2S pathway in medullary slices The slices were divided into five groups (n⫽8 for each): CYS (substrate of CBS), NH2OH (inhibitor of CBS), SAM (activator of CBS), NH2OH⫹CYS, and SAM⫹CYS. Discharge of the rootlets was recorded for 5 min in ACSF as the baseline activity for each group before application of chemicals. Three groups of slices were perfused with CYS (200 M), NH2OH (1 mM) and SAM (100 M), respectively, for 10 min following washout with ACSF. The remaining two groups were perfused with NH2OH and SAM, respectively, for 5 min and then continuously for 10 min once more after CYS (200 M) was added, followed by washout with ACSF. Discharge of rootlets was recorded to observe whether endogenous H2S produced through the CBS–H2S pathway participates in the central regulation of respiratory activity in the medullary slices.
Analysis of cellular signaling pathways of H2S actions on the central respiratory activity of medullary slices To analyze whether the KATP channels and cAMP mediate the H2S actions on respiration, the slices were divided into five groups (n⫽8 for each): Gl (blocker of KATP channels), SQ (inhibitor of adenyl cyclase, AC), Gl⫹NaHS, SQ⫹NaHS and Gl⫹SQ⫹NaHS. The slices were perfused with ACSF for 5 min and the discharge of hypoglossal rootlets was recorded as the baseline of the activity for each group before application of chemicals. Two groups were perfused with Gl (100 M) and SQ (100 M), respectively, for 10 min following washout with ACSF. The remaining three groups were perfused with Gl, SQ and Gl⫹SQ with the same concentration as above, respectively, for 5 min and then continuously for 10 min again after NaHS (200 M) was added followed by washout with ACSF. In order to further investigate the possible mechanisms of the H2S effects on the central regulation of respiratory activity, the slices were divided into four groups (n⫽8 for each): control (ACSF), NaHS (200 M) 3 min, NaHS (200 M) 10 min, and NaHS (200 M)⫹SQ 10 min. The slices of each group were perfused at first as described above. Then the slices were homogenated and centrifuged at 3600⫻g for 15 min at 4 °C. The supernatant was used to measure the amount of cAMP by radioimmunoassay (RIA) technique (RIA kit was purchased from Shanghai Traditional Chinese Medicine University, China).
Statistical analysis All the electrophysiological data at each time point were compared with the baseline before applying chemicals. Normalized DF and IA of the activity of hypoglossal rootlets in each minute were reported as means⫾S.E.M. and statistically analyzed with repeated-measures ANOVA. The data of RIA were reported also as means⫾S.E.M. and statistically analyzed with one-way ANOVA. P values ⬍0.05 were considered statistically significant.
RESULTS Effects of different concentrations of exogenous H2S on discharge activity of hypoglossal rootlets of medullary slices Analysis of the rectified and integrated signals from hypoglossal rootlets of medullary slices revealed that stable rhythmic activity could be recorded for more than 1 h and still could be done up to 5 h in some cases. The discharge activity of the rootlets of the slices perfused with ACSF
during 30 min is shown in Fig. 3A. In the present experiments, the different concentrations of NaHS were bathapplied continuously for 10 min to investigate the possible roles of H2S in the central respiratory regulation of the medullary slices. The change of discharge activity of rootlets in the groups with lower concentration of NaHS (10 and 20 M) was not significantly different from that of the control group (P⬎0.05, data not shown). In a same group with lower concentration of NaHS (10 and 20 M), compared with the mean of control discharge in the first 5 min perfused with ACSF, the changes of DF and IA were all insignificant (P⬎0.05, data not shown). Compared with the control group, the changes of discharge activity of the rootlets in the groups with moderate (50⬃200 M) and high (300⬃400 M) concentration of NaHS were significantly different (P⬍0.05). The moderate concentration of NaHS (50⬃200 M), defined as physiologically relevant concentration according to a previous report (Abe and Kimura, 1996), could prominently affect the discharge activity of the rootlets. A diphasic response of DF was induced, a decrease in DF in accompany with an increase in IA in the initial stage followed by an increase in DF with an insignificant change of IA in the later stage. The inhibitory response in the initial stage was more sensitive to NaHS than was the excitatory response in the later stage. The concentration threshold value of the inhibitory effect was 50 M NaHS while that of the excitatory effect was 100 M NaHS in the present study. In the 50 M NaHS group, DF was decreased by 12.9% (P⬍0.05) only at the third minute and IA did not change significantly (P⬎0.05) during the whole process as shown in Fig. 3B. In the 100 M NaHS group, DF was decreased by 35.6% at the third minute and increased by more than 23.4% from the sixth to the tenth minute (P⬍0.05), and the change of IA was insignificant during the whole application of NaHS (P⬎0.05) (Fig. 3C). In the 200 M NaHS group, DF was decreased by 20.4% at the second minute and 20.9% at the third (P⬍0.05) and followed by more than 21.7% rise from the fifth to the tenth minute (P⬍0.05 or P⬍0.01, Fig. 3D). Excepting for the IA increased by 3.3% at the third minute (P⬍0.05), the changes of IA at the remaining time points were insignificant (P⬎0.05). The discharge activity of the rootlets in these groups was recovered after washout with ACSF for 10 min. After administration of 300 M NaHS, the change of DF was similar to but less than that of 100 M NaHS group, as shown in Fig. 3E. IA was decreased by 3.9% at the ninth minute and 4.3% at the tenth minute (P⬍0.05). DF and IA were not recovered completely after washout. After 400 M NaHS used, DF was decreased from the third to the tenth minute (P⬍0.05) except for the sixth and seventh and IA was decreased from the sixth to the tenth minute (P⬍0.05) as shown in Fig. 3F; DF and IA were not recovered after washout. The results suggested that high concentration of NaHS inhibits irreversibly the discharge of hypoglossal rootlets.
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Fig. 3. Effects of the physiological relevant and high concentration NaHS on the rhythmic discharge activity of hypoglossal rootlets. All data were normalized according to the baseline which is average of 5 min before application of NaHS. The change of DF (left) and IA (right) in each minute after application of NaHS were shown (A: control, B: 50 M NaHS, C: 100 M NaHS, D: 200 M NaHS, E: 300 M NaHS, F: 400 M NaHS). * P⬍0.05, # P⬍0.01 vs. baseline, n⫽8.
Effects of endogenous H2S on discharge activity of hypoglossal rootlets of medullary slices CBS as a critical enzyme to catalyze substrates to produce H2S expressed in the brainstem gave rise to a possibility of the production of H2S from CYS. In the present part of the experiments, CYS, NH2OH and SAM were, respectively, bath-applied to the slices to observe if the endogenous H2S could be produced and participate in the regulation of the respiratory activity of the medullary slices.
Compared with the control group, the changes of discharge activity of the rootlets in the five groups, except for CYS⫹NH2OH, were significantly different (P⬍0.05). CYS (200 M) as a substrate of CBS was bath-applied continuously for 10 min to investigate the effects of H2S generated by the CBS–H2S pathway on the discharge of hypoglossal rootlets. After administration of CYS, DF was decreased by 12.1% at the third minute (P⬍0.05) and inversely increased by more than 24.6% from the fifth to
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Fig. 4. Effects of CYS, NH2OH and SAM on the rhythmic discharge activity of hypoglossal rootlets, respectively. All data were normalized according to the baseline which is average of 5 min before application chemicals. The change of DF (left) and IA (right) in each minute after application is shown (A: 200 M CYS, B: 1 mM NH2OH, C: 100 M SAM, D: CYS⫹NH2OH, E: CYS⫹SAM). * P⬍0.05, # P⬍0.01 vs. baseline, n⫽8.
the tenth minute (P⬍0.05 or P⬍0.01), qualitatively similar to that of NaHS (200 M) (Fig. 3D), whereas the change of IA was insignificant during the administration (P⬎0.05), as shown in Fig. 4A. The discharge activity of the rootlets recovered completely after washout with ACSF. NH2OH (1 mM) as an inhibitor of CBS was bath-applied continuously for 10 min to observe the effects of endogenous H2S decreased by the chemical on DF and IA. After administration of NH2OH from the eighth to the tenth minute, DF was decreased by more than 12.0% (P⬍0.05) and IA more than 3.4% (P⬍0.05, Fig. 4B). The discharge activity of the hypoglossal rootlets recovered completely after washout with ACSF. SAM (100 M) as an activator of CBS was also bath-applied continuously for 10 min to evaluate the effects of endogenous H2S increased by the chemical on DF and IA. As a result, DF increased by 21.6% at the second minute and by 27.8%, 16.9% and 26.1% from the eighth to the tenth minute, respectively, (P⬍0.05),
whereas IA was not changed significantly (Fig. 4C). The discharge activity of rootlets recovered completely after washout with ACSF. In the group of NH2OH⫹CYS, after bath-applied NH2OH for 5 min, the slices were perfused simultaneously with CYS for 10 min. The change of DF and IA was insignificant (P⬎0.05, Fig. 4D). The results revealed that NH2OH suppressed CBS activity and decreased the production of H2S, in other words, NH2OH inhibited the effect of CYS on the discharge activity. The discharge activity of the rootlets recovered completely after washout with ACSF. In the group of SAM⫹CYS, after bath-applied SAM for 5 min, the slices were perfused simultaneously with CYS for 10 min. The DF was increased prominently at the first minute and from the fourth to the tenth minute (P⬍0.05) and the IA did not change significantly (P⬎0.05) as shown in Fig. 4E. The results suggested that the decrease in DF induced by CYS in the initial stage was eliminated by SAM.
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The discharge activity of hypoglossal rootlets recovered completely after washout with ACSF. The results above indicated that endogenous H2S produced through the CBS–H2S pathway definitely plays a role in the central respiratory regulation in the medullary slices. Effects of Gl and SQ on discharge activity of hypoglossal rootlets of medullary slices As described above, both exogenous and endogenous H2S played a role in the regulation of the rhythmic respiratory activity of the medullary slices. It was reported that physiological concentration of H2S specifically enhanced the activity of the NMDA receptor probably through cAMPmediated pathways (Abe and Kimura, 1996; Kimura, 2000). Another investigation revealed that H2S could induce hyperpolarization of the neuronal membrane in hippocampus CA1 region by opening KATP channels (Baldelli
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et al., 1990). In the present experiments, Gl (KATP channels blocker) and SQ (AC inhibitor) were applied to investigate the mechanism of the H2S-mediated central regulation of respiration. Compared with the control group, the changes of discharge activity of the rootlets in the groups Gl, SQ, and Gl⫹SQ⫹NaHS were not significantly different (P⬎0.05) while those of the groups Gl⫹NaHS and SQ⫹NaHS were significantly different (P⬍0.05). DF and IA did not change significantly during the whole process of administration of Gl alone (P⬎0.05, Fig. 5A). In the group of Gl⫹NaHS, the slices were perfused with Gl for 5 min and then simultaneously with NaHS for 10 min. Compared with the data in Fig. 3D showing the effects of NaHS alone, both the decrease in DF and the increase in IA induced by NaHS in the initial stage were eliminated while the increase in DF induced by NaHS in the later stage was sustained (Fig. 5B).
Fig. 5. Effects of Gl and SQ on the rhythmic discharge activity and the change of discharge activity mediated by NaHS. All data were normalized according to the baseline which is average of 5 min before application of chemicals. The change of DF (left) and IA (right) in each minute after application is shown (A: 100 M Gl, B: Gl⫹NaHS, C: 100 M SQ, D: SQ⫹NaHS, E: Gl⫹SQ⫹NaHS). * P⬍0.05, # P⬍0.01 vs. baseline, n⫽8.
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DISCUSSION
Fig. 6. Effects of different concentrations and different time perfused with NaHS and combination of NaHS with SQ on the level of cAMP of medullary slices (pmol/mg) of neonatal rats. * P⬍0.05 vs. ACSF control, # P⬍0.01 vs. 200 M NaHS 10 min, n⫽8.
Likewise, DF and IA did not change significantly during the whole process of administration of SQ (Fig. 5C). In the group of SQ⫹NaHS, the slices were perfused with SQ for 5 min and then simultaneously with NaHS for 10 min. Compared with the data in Fig. 3D showing the effects of NaHS alone, the increase in DF induced by NaHS in the later stage was eliminated, whereas the decrease in DF and the increase in IA induced by NaHS in the initial stage was sustained (Fig. 5D). In the group of Gl⫹SQ⫹NaHS, the slices were perfused with both Gl and SQ for 5 min and then simultaneously NaHS for 10 min. The changes of DF and IA both in the initial stage and in the later stage were eliminated completely (Fig. 5E). The results above indicated that H2S inhibited first and then excited the respiratory activity of hypoglossal rootlets through opening KATP channels and increasing cAMP levels, respectively. Effects of NaHS and SQ on cAMP level of medullary slices The cAMP amount in the slice homogenate was measured with RIA technique to further determine whether the cAMP pathway played a role in the central regulation of respiratory activity mediated by H2S. The cAMP amount of the NaHS (200 M) 3 min group (0.57⫾0.04 pmol/mg) was not changed significantly versus control group (0.51⫾0.05pmol/mg) (P⬎0.05) while that of the NaHS (200 M) 10 min group (0.81⫾0.07 pmol/mg) was enhanced significantly versus control (P⬍0.05). The amount of cAMP in the NaHS⫹SQ 10 min group was decreased significantly versus the NaHS (200 M) 10 min group (0.46⫾0.02 pmol/mg, P⬍0.01). The data were shown in Fig. 6. These results further revealed that the excitation in the later stage, but not the inhibition in the initial stage, of the medullary respiratory center by physiological concentration of H2S might be attributed to indirectly raising the level of cAMP by activating AC.
In the present study we found that in the in vitro preparations of medullary slices of neonatal rats (i) exogenous H2S could affect the respiratory activity in a diphasic mode, a decrease in the respiratory frequency in the initial stage followed by its increase in the later stage, (ii) the endogenous H2S could be produced through the CBS–H2S pathway and could be involved in the control of rhythmic respiration of the slices, and (iii) the two opposite stages of effects of H2S on the respiratory activity might be induced by opening KATP channels and activating the AC– cAMP pathway, respectively. The discharge activity of the preparation did not change significantly during the whole process of experiments in the control groups and could recover from actions of the testing agents by washout with ACSF in the drug groups. Therefore the perfusion of the slices in the study would be effective although the possibility of anoxia in the core of the relatively thicker slices used could not be completely excluded. Although H2S was generally thought to be a toxic gas and its toxicology was extensively investigated in the past, recent observations showed that relatively high endogenous levels of H2S (50⬃160 M) could be observed in the tissues of mammals, especially in the brains of rat, human and bovine (Goodwin et al., 1989; Warenycia et al., 1989; Savage and Gould, 1990) and CBS was highly expressed in hippocampus as well as brainstem of rats (Abe and Kimura, 1996; Kimura, 2000). Endogenous H2S has been shown to be involved in many physiological processes such as regulation of synaptic activity in the brain (Eto et al., 2002b), relaxation of vascular smooth muscle in the circulatory system (Zhao et al., 2001), and scavenging free radicals (Searcy et al., 1995; Kimura and Kimura, 2004). In addition, traces of H2S were also found in multiple diseases such as Alzheimer disease with a decrease in endogenous H2S and Down syndrome disease with an increase in H2S (Eto et al., 2002a; Kamoun et al., 2003). Inhalation of exogenous H2S within the moderate concentration may enhance the respiratory activity, whereas inhalation of high concentration H2S would inhibit the activity of respiratory centers (Beauchamp et al., 1984; Reiffenstein et al., 1992). These findings implied that H2S may be involved in regulation of respiration. In the present experiments, we verified that the endogenous H2S could be produced through the CBS–H2S pathway and participate in the control of rhythmic respiration of the medullary slices. We observed that NaHS (50⬃200 M, the concentration of NaHS in which approximately the same amount of H2S as endogenously in the brain of animals can be produced) (Abe and Kimura, 1996) could induce a diphasic response of central respiratory activity, an inhibitory response followed by an excitatory one in regard to the changes of DF of hypoglossal rootlets. The same result could be obtained from the slices perfused with CYS. Both NaHS and CYS exert their effects on respiration through producing H2S. However the mechanisms to produce H2S
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are different. NaHS can be dissociated to deliver Na⫹ and HS⫺ in solution and then HS⫺ is associated with H⫹ to form H2S. But CYS as a substrate must be catalyzed by CBS to produce H2S. As reported previously, CBS knockout mice did not produce detectable amounts of H2S (Eto et al., 2002b) and the CBS inhibitor NH2OH suppressed H2S production in the brain (Abe and Kimura, 1996). We observed that NH2OH not only reversibly suppressed the activity of hypoglossal rootlets, but also eliminated the action of CYS on the respiratory activity. Therefore, it is suggested that CYS exerted its effects on respiration by forming H2S. On the other hand, CYS⫹NH2OH did not reduce the respiratory frequency in the later phase, it could be due to a direct action of CYS on the neurons of the slice. Furthermore, the possibility of NH2OH acting on some other enzymes to produce other amidated substances and hence to change the respiratory activity could not be completely excluded. It was also demonstrated that there exists an endogenous CBS–H2S pathway to produce H2S and the endogenous H2S participates in the control of the central respiratory activity of the medullary slices. Under physiological conditions, H2S production depends on the activity of CBS. The activity of CBS can be regulated by Ca2⫹/calmodulin, e.g. enhanced by Ca2⫹ influx which occurs with neuronal excitation (Eto et al., 2002b). So we speculate that H2S production could be increased due to activation of CBS by rhythmical discharge of the neurons of the slices. H2S may interact with other neurotransmitters and play an important role in maintenance of the excitatory/inhibitory balance in neurotransmission in the CNS (Han et al., 2005; Qu et al., 2008), presumably in the respiratory centers as well. SAM can enhance H2S production through activating CBS in the brain (Abe and Kimura, 1996; Kamoun et al., 2003) and a model for CBS regulation has been proposed in which the C-terminal domain of the enzyme bends to and covers its own catalytic domain to suppress the enzymatic activity (Kamoun, 2004). Once SAM binds to the regulatory domain of CBS, a conformational change occurs that frees the catalytic domain and CBS becomes active (Kamoun, 2004; Miles and Kraus, 2004; Shan et al., 2001). It is suggested therefore from our present experiments that SAM could activate CBS and increase endogenous H2S production. In addition, it might directly excite the neurons simultaneously because the decrease in DF of the rootlets induced by CYS in the initial stage did not occur when the slices were perfused with SAM. The presence of CBS, coupled with the identification of H2S in the brains of several species including human, suggested a role for this gas in CNS functions (Kimura, 2002). The studies about its underlying mechanisms revealed that the NMDA receptor is directly phosphorylated in specific sites by protein kinase A (PKA) (Leonard and Hell, 1997; Tingley et al., 1997). The increased activity of AC and PKA could enhance NMDA currents in the neurons of the dorsal horn (Cerne et al., 1993) and neostriatum (Gu et al., 2007), indicating that the NMDA receptor may be modulated through the cAMP cascade. Besides, H2S also hyperpolarizes CA1 pyramidal cells in the hippocampus of
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rats most probably by activating K⫹ATP channels (Baldelli et al., 1990; Qu et al., 2008). The evidence obtained from the present study shows that the two cellular signaling pathways, K⫹ATP channels and cAMP, are possibly involved in the central respiratory regulation of H2S. H2S may open directly the KATP channels and hyperpolarize the neurons and then suppress the activity of the slices at the initial stage, rather than through raising the level of cAMP to activate the KATP channels since the level of cAMP was not elevated significantly in this stage as demonstrated by RIA. The excitatory effect of H2S on the rootlets discharge in the later stage was eliminated by the AC inhibitor SQ and the high level of cAMP induced by NaHS in the later stage was also depressed by SQ as indicated by RIA. It is suggested therefore that H2S could elevate the excitability of the neurons in the respiratory centers of the medullary slices through increasing the level of cAMP. The inhibition of high concentration H2S on the central respiratory activity observed in the present study might be due to a decrease in the level of cAMP as it was reported that AC could be inhibited by high concentration H2S (Kimura, 2000). Moreover high concentration H2S can directly inhibit the cytochrome oxidase (Dorman et al., 2002) and then exert its toxic effect on the neurons of the slices. Although the precise mechanisms of respiratory rhythm genesis and regulation are still unclear (Feldman et al., 2003; Zhang and Zheng, 2002), the basic respiratory center is thought to be located in the medulla oblongata (Smith et al., 1991; Rekling and Feldman, 1998). In the present study, we found that both exogenous and endogenous H2S could change the activity of the hypoglossal rootlets of the medullary slices which were relatively thicker and would contain such respiratory nuclei as preBötzinger complex (Smith et al., 1991), parafacial respiratory group (Onimaru and Homma, 2003), rostral ventral respiratory group and so on. Therefore, we suppose that the respiratory responses to H2S observed in our experiments were induced by H2S widely acting on the respiratory centers in the medulla oblongata. The precise sites as well as neuronal mechanisms of actions of H2S on the rhythmic respiratory activity of the medullary slices remain to be investigated. Whether the present data can be used for intact animals also needs to be verified.
CONCLUSION It can be concluded from the present study that the endogenous H2S could be produced by the neurons via the CBS–H2S pathway in the medullary respiratory center and could be involved in the control of rhythmic respiration of the medullary slices of neonatal rats and that the diphasic effects of H2S on the respiratory activity might be induced by opening KATP channels in the initial inhibitory stage and activating the AC– cAMP pathway in the later excitatory stage, respectively. Acknowledgments—This work was supported by grants from the National Natural Science Foundation of China (No. 30370530 and No. 30770798). The authors are grateful to Dr. Alan D. Miller in New York for his English revision and scientific comments.
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Leonard AS, Hell JW (1997) Cyclic AMP-dependent protein kinase and protein kinase C phosphorylate N-methyl-d-aspartate receptors at different sites. J Biol Chem 272:12107–12115. Miles ED, Kraus JP (2004) Cystathionine-synthase: structure, function, regulation, and location of homocystinuria-causing mutations. J Biol Chem 279:29871–29874. Onimaru H, Homma I (2003) A novel functional neuron group for respiratory rhythm generation in the ventral medulla. J Neurosci 23:1478 –1486. Qu K, Lee SW, Bian JS, Low CM, Wong PTH (2008) Hydrogen sulfide: neurochemistry and neurobiology. Neurochem Int 52:155–165. Reiffenstein RJ, Hulbert WC, Roth SH (1992) Toxicology of hydrogen sulfide. Annu Rev Pharmacol Toxicol 32:109 –134. Rekling JC, Feldman JL (1998) Pre-Bötzinger complex and pacemaker neurons: hypothesized site and kernel for respiratory rhythm generation. Annu Rev Physiol 60:385– 405. Savage JC, Gould DH (1990) Determination of sulfide in brain tissue and rumen fluid by ion-interaction reversed-phase high-performance liquid chromatography. J Chromatogr 526:540 –545. Searcy DG, Whitehead JP, Maroney MJ (1995) Interaction of Cu, Zn superoxide-dismutase with hydrogen-sulfide. Arch Biochem Biophys 318:251–263. Shan X, Roland L, Dunbrack J, Christopher SA, Kruger WD (2001) Mutations in the regulatory domain of cystathionine -synthase can functionally suppress patient-derived mutations in cis. Hum Mol Genet 10:635– 643. Smith JC, Ellenberger HH, Ballanyi K, Richter DW, Feldman JL (1991) Pre-Bötzinger complex: a brainstem region that may generate respiratory rhythm in mammals. Science 254:726 –729. Stipanuk MH, Beck PW (1982) Characterization of the enzymic capacity for cysteine desulphhydration in liver and kidney of the rat. J Biochem 206:267–277. Tingley WG, Ehlers MD, Kameyama K, Doherty C, Ptak JB, Riley CT, Huganir RL (1997) Characterization of protein kinase A and protein kinase C phosphorylation of the N-methyl-d-aspartate receptor NR1 subunit using phosphorylation site-specific antibodies. J Biol Chem 272:5157–5166. Warenycia MW, Goodwin LR, Benishin CG, Reiffenstein RJ, Francom DM, Taylor J, Dieken FP (1989) Acute hydrogen sulfide poisoning: demonstration of selective uptake of sulfide by the brainstem by measurement of brain sulfide levels. Biochem Pharmacol 38:973–981. Yang WX, Zhang QL, Zhou H, Zheng Y (2007) Heme oxygenasecarbon monoxide pathway is involved in regulation of respiration in medullary slice of neonatal rats. Neurosci Lett 426:128 –132. Zhang CW, Zheng Y (2002) Pre-Bötzinger complex: a kernel region for respiratory rhythmogenesis [in Chinese]. Prog Physiol Sci 33: 179 –181. Zhao WJ, Zhang J, Lu YJ, Wang R (2001) The vasorelaxant effect of H2S as a novel endogenous gaseous KATP channel opener. EMBO J 20:6008 – 6016.
(Accepted 11 August 2008) (Available online 22 August 2008)
ENDOGENOUS HYDROGEN SULFIDE IS INVOLVED IN REGULATION OF RESPIRATION IN MEDULLARY SLICE OF NEONATAL RATS H. HU, Y. SHI, Q. CHEN, W. YANG, H. ZHOU, L. CHEN, Y. TANG AND Y. ZHENG*
the hippocampus, cerebellum, brainstem and cerebral cortex (Abe and Kimura, 1996; Eto et al., 2002b). H2S could not be detected in the brain of mice with gene knockout of CBS (Eto et al., 2002b). H2S production could be suppressed by CBS inhibitor, hydroxylamine (NH2OH), and enhanced by its activator, S-adenosyl-L-methionine (SAM) (Abe and Kimura, 1996). It could also be greatly enhanced by activating glutamate receptors and Ca2⫹/calmodulin in the hippocampus (Eto et al., 2002b). H2S in physiological concentration specifically enhanced the activity of NMDA receptor and facilitated the long-term potentiation in the hippocampus (Abe and Kimura, 1996), while pathways mediated by cAMP may be involved in the modulation of NMDA receptors by H2S (Kimura, 2000). It has been proposed therefore that H2S can be produced in the brain and may play an important role in probably acting as a neuromodulator or an intracellular messenger (Abe and Kimura, 1996). Inhalation of exogenous H2S within moderate concentration increased the respiratory rate, whereas high concentration of the gas functioned exactly opposite, suggesting that the respiratory activity would be affected by H2S (Beauchamp et al., 1984; Reiffenstein et al., 1992). However whether H2S functions in the central regulation of respiratory activity has been unknown although CBS was found highly expressed and endogenous H2S produced in the brainstem of rats as mentioned above. It is well known that the medulla oblongata is essential for the rhythmogenesis of respiration. We hypothesize therefore that H2S would participate in the central regulation of respiration at the level of medulla oblongata. The purpose of the present study was to investigate the effects of exogenous H2S on and the possible roles of endogenous H2S in the rhythmic respiratory activity and the mechanisms underlying the central respiratory actions of H2S of the in vitro preparations of the medullary slices of neonatal rats.
Department of Physiology, West China School of Preclinical and Forensic Medicine, Sichuan University, 3-17 Renmin South Road, Chengdu, Sichuan 610041, PR China
Abstract—The purpose of the present study was to verify our assumption that rhythmic respiratory activity may be regulated by endogenous hydrogen sulfide (H2S) in the medullary slices of neonatal rats. We found that a moderate concentration of donor of H2S, NaHS, mainly induced diphasic respiratory responses indicated by changes of discharge frequency (DF) of hypoglossal rootlets, an initial inhibitory stage followed by a later excitatory one. Cystathionine -synthase (CBS) substrate, cysteine (CYS), exerted similar effects. CBS inhibitor, NH2OH, could eliminate both inhibitory and excitatory effects in the two stages induced by CYS. KATP channel blocker, glibenclamide (Gl), could eliminate the decrease in DF in the initial stage, but not the increase in the later one. On the other hand, adenyl cyclase (AC) inhibitor, SQ-22536 (SQ) could eliminate the increase in DF in the later stage, but not the decrease in the initial one, of the rootlets caused by NaHS. Co-application of Gl and SQ eliminated both inhibitory and excitatory effect induced by NaHS. The cAMP level was increased in the later stage but not in the initial one by NaHS, and the increase in the cAMP level could be eliminated by SQ. It can be concluded that the endogenous H2S could be produced through the CBS–H2S pathway and could be involved in the control of the central rhythmic respiration in the in vitro medullary slices of neonatal rats by opening KATP channels and activating AC– cAMP pathway of the neurons. © 2008 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: respiratory regulation, medullary slices, discharge of hypoglossal rootlets, KATP channel, cAMP.
Hydrogen sulfide (H2S) is a well-known toxic gas and its toxicology has been extensively studied (Reiffenstein et al., 1992). However, a relatively high level of endogenous H2S has been detected in the brains of rats, humans and bovine (Goodwin et al., 1989; Warenycia et al., 1989; Savage and Gould, 1990), suggesting that H2S may have certain physiological functions. Endogenous H2S in the brain comes from decomposing the substrates, L-cysteine (CYS) and homocysteine, by cystathionine -synthase (CBS) (Stipanuk and Beck, 1982; Griffith, 1987). CBS was found highly expressed in
EXPERIMENTAL PROCEDURES Agents The main agents were purchased from Sigma (St. Louis, MO, USA), including NaHS, CYS, NH2OH, SAM, glibenclamide (Gl) and SQ-22536 (SQ). The solutions containing NaHS were prepared just before use.
*Corresponding author. Tel: ⫹86-28-8550-3433; fax: ⫹86-28-8550-3204. E-mail address: [email protected] (Y. Zheng). Abbreviations: AC, adenyl cyclase; ACSF, artificial cerebrospinal fluid; CBS, cystathionine -synthase; CYS, cysteine; DF, discharge frequency; Gl, glibenclamide; IA, integrated amplitude; PKA, protein kinase A; RIA, radioimmunoassay; SAM, S-adenosyl-L-methionine; SQ, SQ-22536.
Preparation of medullary slices The experiments were performed on medullary slices of male or female neonatal (0 – 4 days) Sprague–Dawley rats. All procedures were reviewed and approved by the Sichuan University Committee on the Use of Live Animals in Research, and conformed to the
0306-4522/08 © 2008 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2008.08.025
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Principles of Laboratory Animal Care (NIH publication No. 86-23 revised in 1985). The number of animals used and their suffering were minimized. Medullary slices were prepared as described elsewhere (Yang et al., 2007). In brief, the animals were anesthetized with ether by inhalation and then decapitated. The isolated brainstem was placed in the slicing chamber which was filled with ice-cold artificial cerebrospinal fluid (ACSF) bubbled with carbogen (95% O2 and 5% CO2). The ACSF contained (mM): 125 NaCl, 5 KCl, 2 CaCl2, 1 MgSO4, 22 NaHCO3, 1 KH2PO4 and 30 dglucose and pH was regulated to 7.4 by Hepes. The brainstem was positioned dorsal side upward, facing to the slicing blade at 20° to the rostral end of the block. In ice-cold ACSF, a single transverse slice of 1000 –1200 m thick from one brainstem was prepared. The obex was taken as the reference of the cutting levels under an optical microscopy. The brainstem was cut at the levels of about 100 m caudal and 1000 m rostral to the obex. The slice in such a thickness would contain the pre-Bötzinger complex (Smith et al., 1991), rostrally the parafacial respiratory group (Onimaru and Homma, 2003) and caudally the traditionally recognized rostral ventral respiratory group, and certainly the entire neuronal circuits to produce rhythmic respiratory outputs. Fig. 2. Schematic diagram of recording technique of hypoglossal rootlets discharge of medullary slice preparation of neonatal rats. (A) The transversal view of medullary slice. ION: inferior olivary nucleus; NTS: nucleus tractus solitarius; PBC: pre-Bötzinger complex; SP5: spinal trigeminal tract; XII: hypoglossal nucleus; XIIn: hypoglossal rootlets. (B) Discharge activity of hypoglossal rootlets, raw recording (XIIn) and integrated activity (兰XIIn), respectively.
Fig. 1 provides three representative levels of the medulla oblongata, around most caudal and most rostral cutting levels and a middle level between them, to show the histological evidence of the structures the slice contains. The slices were transferred to a recording chamber and continuously perfused with oxygenated ACSF at a rate of 4 – 6 mL/min at 29 °C. The slices were incubated for 30 min before starting the experiments. Glass suction electrodes filled with ACSF were used to record the rhythmic respiratory activity from the cut ends of the hypoglossal rootlets (Fig. 2A). Signals were amplified, filtered (⫽0.001 s, F⫽1 kHz) and integrated with a time constant of 50 ms by BL-420 E⫹ biological signal processing system (Taimeng Biotech. Co., China). The discharge frequency (DF, number of discharge per min) and integrated amplitude (IA, Fig. 2B) of hypoglossal rootlets were analyzed. In the present study, a total of 176 slices were used.
Perfusion of slices with different concentration of NaHS
Fig. 1. Photographs (left) and corresponding schematic diagrams (right) of three representative levels of the medullary slice of neonatal rat, including around most caudal (lower) and most rostral (upper) cutting levels and a middle level (middle) between them to show the histological evidence of the structures the slice contains, with HE staining. AP: area postrema; BC: Bötzinger complex; ION: inferior olivary nucleus; NA: nucleus ambiguous; NTS: nucleus tractus solitarius; PBC: pre-Bötzinger complex; PY: pyramidal tract; SP5: spinal trigeminal tract; XII: hypoglossal nucleus.
NaHS was used as a substitute of H2S to perfuse the slices in the present experiments because the quantity or concentration of H2S is difficult to control when the slices are perfused with bubbled H2S. NaHS can dissociate to deliver Na⫹ and HS⫺ in solution and then HS⫺ is associated with H⫹ to produce H2S. Approximately one-third of the H2S exists as the undissociated form, H2S, while the majority exists as HS⫺ in physiological saline (Reiffenstein et al., 1992). The slices were divided into eight groups (n⫽8 for each) including one control and seven tests. In the control group, the slices were perfused with ACSF and the discharge of hypoglossal rootlets was recorded during the whole experimental process. In each of the test groups, the discharge of rootlets was recorded for 5 min in ACSF as the baseline activity before NaHS application and then the slices were perfused with different concentration of NaHS at 10, 20, 50, 100, 200, 300 and 400 M, respectively, for 10 min following washout with ACSF. Discharge of the rootlets was recorded to observe the effects of different concentration of NaHS on the central respiratory activity.
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Analysis of existence of CBS–H2S pathway in medullary slices The slices were divided into five groups (n⫽8 for each): CYS (substrate of CBS), NH2OH (inhibitor of CBS), SAM (activator of CBS), NH2OH⫹CYS, and SAM⫹CYS. Discharge of the rootlets was recorded for 5 min in ACSF as the baseline activity for each group before application of chemicals. Three groups of slices were perfused with CYS (200 M), NH2OH (1 mM) and SAM (100 M), respectively, for 10 min following washout with ACSF. The remaining two groups were perfused with NH2OH and SAM, respectively, for 5 min and then continuously for 10 min once more after CYS (200 M) was added, followed by washout with ACSF. Discharge of rootlets was recorded to observe whether endogenous H2S produced through the CBS–H2S pathway participates in the central regulation of respiratory activity in the medullary slices.
Analysis of cellular signaling pathways of H2S actions on the central respiratory activity of medullary slices To analyze whether the KATP channels and cAMP mediate the H2S actions on respiration, the slices were divided into five groups (n⫽8 for each): Gl (blocker of KATP channels), SQ (inhibitor of adenyl cyclase, AC), Gl⫹NaHS, SQ⫹NaHS and Gl⫹SQ⫹NaHS. The slices were perfused with ACSF for 5 min and the discharge of hypoglossal rootlets was recorded as the baseline of the activity for each group before application of chemicals. Two groups were perfused with Gl (100 M) and SQ (100 M), respectively, for 10 min following washout with ACSF. The remaining three groups were perfused with Gl, SQ and Gl⫹SQ with the same concentration as above, respectively, for 5 min and then continuously for 10 min again after NaHS (200 M) was added followed by washout with ACSF. In order to further investigate the possible mechanisms of the H2S effects on the central regulation of respiratory activity, the slices were divided into four groups (n⫽8 for each): control (ACSF), NaHS (200 M) 3 min, NaHS (200 M) 10 min, and NaHS (200 M)⫹SQ 10 min. The slices of each group were perfused at first as described above. Then the slices were homogenated and centrifuged at 3600⫻g for 15 min at 4 °C. The supernatant was used to measure the amount of cAMP by radioimmunoassay (RIA) technique (RIA kit was purchased from Shanghai Traditional Chinese Medicine University, China).
Statistical analysis All the electrophysiological data at each time point were compared with the baseline before applying chemicals. Normalized DF and IA of the activity of hypoglossal rootlets in each minute were reported as means⫾S.E.M. and statistically analyzed with repeated-measures ANOVA. The data of RIA were reported also as means⫾S.E.M. and statistically analyzed with one-way ANOVA. P values ⬍0.05 were considered statistically significant.
RESULTS Effects of different concentrations of exogenous H2S on discharge activity of hypoglossal rootlets of medullary slices Analysis of the rectified and integrated signals from hypoglossal rootlets of medullary slices revealed that stable rhythmic activity could be recorded for more than 1 h and still could be done up to 5 h in some cases. The discharge activity of the rootlets of the slices perfused with ACSF
during 30 min is shown in Fig. 3A. In the present experiments, the different concentrations of NaHS were bathapplied continuously for 10 min to investigate the possible roles of H2S in the central respiratory regulation of the medullary slices. The change of discharge activity of rootlets in the groups with lower concentration of NaHS (10 and 20 M) was not significantly different from that of the control group (P⬎0.05, data not shown). In a same group with lower concentration of NaHS (10 and 20 M), compared with the mean of control discharge in the first 5 min perfused with ACSF, the changes of DF and IA were all insignificant (P⬎0.05, data not shown). Compared with the control group, the changes of discharge activity of the rootlets in the groups with moderate (50⬃200 M) and high (300⬃400 M) concentration of NaHS were significantly different (P⬍0.05). The moderate concentration of NaHS (50⬃200 M), defined as physiologically relevant concentration according to a previous report (Abe and Kimura, 1996), could prominently affect the discharge activity of the rootlets. A diphasic response of DF was induced, a decrease in DF in accompany with an increase in IA in the initial stage followed by an increase in DF with an insignificant change of IA in the later stage. The inhibitory response in the initial stage was more sensitive to NaHS than was the excitatory response in the later stage. The concentration threshold value of the inhibitory effect was 50 M NaHS while that of the excitatory effect was 100 M NaHS in the present study. In the 50 M NaHS group, DF was decreased by 12.9% (P⬍0.05) only at the third minute and IA did not change significantly (P⬎0.05) during the whole process as shown in Fig. 3B. In the 100 M NaHS group, DF was decreased by 35.6% at the third minute and increased by more than 23.4% from the sixth to the tenth minute (P⬍0.05), and the change of IA was insignificant during the whole application of NaHS (P⬎0.05) (Fig. 3C). In the 200 M NaHS group, DF was decreased by 20.4% at the second minute and 20.9% at the third (P⬍0.05) and followed by more than 21.7% rise from the fifth to the tenth minute (P⬍0.05 or P⬍0.01, Fig. 3D). Excepting for the IA increased by 3.3% at the third minute (P⬍0.05), the changes of IA at the remaining time points were insignificant (P⬎0.05). The discharge activity of the rootlets in these groups was recovered after washout with ACSF for 10 min. After administration of 300 M NaHS, the change of DF was similar to but less than that of 100 M NaHS group, as shown in Fig. 3E. IA was decreased by 3.9% at the ninth minute and 4.3% at the tenth minute (P⬍0.05). DF and IA were not recovered completely after washout. After 400 M NaHS used, DF was decreased from the third to the tenth minute (P⬍0.05) except for the sixth and seventh and IA was decreased from the sixth to the tenth minute (P⬍0.05) as shown in Fig. 3F; DF and IA were not recovered after washout. The results suggested that high concentration of NaHS inhibits irreversibly the discharge of hypoglossal rootlets.
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Fig. 3. Effects of the physiological relevant and high concentration NaHS on the rhythmic discharge activity of hypoglossal rootlets. All data were normalized according to the baseline which is average of 5 min before application of NaHS. The change of DF (left) and IA (right) in each minute after application of NaHS were shown (A: control, B: 50 M NaHS, C: 100 M NaHS, D: 200 M NaHS, E: 300 M NaHS, F: 400 M NaHS). * P⬍0.05, # P⬍0.01 vs. baseline, n⫽8.
Effects of endogenous H2S on discharge activity of hypoglossal rootlets of medullary slices CBS as a critical enzyme to catalyze substrates to produce H2S expressed in the brainstem gave rise to a possibility of the production of H2S from CYS. In the present part of the experiments, CYS, NH2OH and SAM were, respectively, bath-applied to the slices to observe if the endogenous H2S could be produced and participate in the regulation of the respiratory activity of the medullary slices.
Compared with the control group, the changes of discharge activity of the rootlets in the five groups, except for CYS⫹NH2OH, were significantly different (P⬍0.05). CYS (200 M) as a substrate of CBS was bath-applied continuously for 10 min to investigate the effects of H2S generated by the CBS–H2S pathway on the discharge of hypoglossal rootlets. After administration of CYS, DF was decreased by 12.1% at the third minute (P⬍0.05) and inversely increased by more than 24.6% from the fifth to
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Fig. 4. Effects of CYS, NH2OH and SAM on the rhythmic discharge activity of hypoglossal rootlets, respectively. All data were normalized according to the baseline which is average of 5 min before application chemicals. The change of DF (left) and IA (right) in each minute after application is shown (A: 200 M CYS, B: 1 mM NH2OH, C: 100 M SAM, D: CYS⫹NH2OH, E: CYS⫹SAM). * P⬍0.05, # P⬍0.01 vs. baseline, n⫽8.
the tenth minute (P⬍0.05 or P⬍0.01), qualitatively similar to that of NaHS (200 M) (Fig. 3D), whereas the change of IA was insignificant during the administration (P⬎0.05), as shown in Fig. 4A. The discharge activity of the rootlets recovered completely after washout with ACSF. NH2OH (1 mM) as an inhibitor of CBS was bath-applied continuously for 10 min to observe the effects of endogenous H2S decreased by the chemical on DF and IA. After administration of NH2OH from the eighth to the tenth minute, DF was decreased by more than 12.0% (P⬍0.05) and IA more than 3.4% (P⬍0.05, Fig. 4B). The discharge activity of the hypoglossal rootlets recovered completely after washout with ACSF. SAM (100 M) as an activator of CBS was also bath-applied continuously for 10 min to evaluate the effects of endogenous H2S increased by the chemical on DF and IA. As a result, DF increased by 21.6% at the second minute and by 27.8%, 16.9% and 26.1% from the eighth to the tenth minute, respectively, (P⬍0.05),
whereas IA was not changed significantly (Fig. 4C). The discharge activity of rootlets recovered completely after washout with ACSF. In the group of NH2OH⫹CYS, after bath-applied NH2OH for 5 min, the slices were perfused simultaneously with CYS for 10 min. The change of DF and IA was insignificant (P⬎0.05, Fig. 4D). The results revealed that NH2OH suppressed CBS activity and decreased the production of H2S, in other words, NH2OH inhibited the effect of CYS on the discharge activity. The discharge activity of the rootlets recovered completely after washout with ACSF. In the group of SAM⫹CYS, after bath-applied SAM for 5 min, the slices were perfused simultaneously with CYS for 10 min. The DF was increased prominently at the first minute and from the fourth to the tenth minute (P⬍0.05) and the IA did not change significantly (P⬎0.05) as shown in Fig. 4E. The results suggested that the decrease in DF induced by CYS in the initial stage was eliminated by SAM.
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The discharge activity of hypoglossal rootlets recovered completely after washout with ACSF. The results above indicated that endogenous H2S produced through the CBS–H2S pathway definitely plays a role in the central respiratory regulation in the medullary slices. Effects of Gl and SQ on discharge activity of hypoglossal rootlets of medullary slices As described above, both exogenous and endogenous H2S played a role in the regulation of the rhythmic respiratory activity of the medullary slices. It was reported that physiological concentration of H2S specifically enhanced the activity of the NMDA receptor probably through cAMPmediated pathways (Abe and Kimura, 1996; Kimura, 2000). Another investigation revealed that H2S could induce hyperpolarization of the neuronal membrane in hippocampus CA1 region by opening KATP channels (Baldelli
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et al., 1990). In the present experiments, Gl (KATP channels blocker) and SQ (AC inhibitor) were applied to investigate the mechanism of the H2S-mediated central regulation of respiration. Compared with the control group, the changes of discharge activity of the rootlets in the groups Gl, SQ, and Gl⫹SQ⫹NaHS were not significantly different (P⬎0.05) while those of the groups Gl⫹NaHS and SQ⫹NaHS were significantly different (P⬍0.05). DF and IA did not change significantly during the whole process of administration of Gl alone (P⬎0.05, Fig. 5A). In the group of Gl⫹NaHS, the slices were perfused with Gl for 5 min and then simultaneously with NaHS for 10 min. Compared with the data in Fig. 3D showing the effects of NaHS alone, both the decrease in DF and the increase in IA induced by NaHS in the initial stage were eliminated while the increase in DF induced by NaHS in the later stage was sustained (Fig. 5B).
Fig. 5. Effects of Gl and SQ on the rhythmic discharge activity and the change of discharge activity mediated by NaHS. All data were normalized according to the baseline which is average of 5 min before application of chemicals. The change of DF (left) and IA (right) in each minute after application is shown (A: 100 M Gl, B: Gl⫹NaHS, C: 100 M SQ, D: SQ⫹NaHS, E: Gl⫹SQ⫹NaHS). * P⬍0.05, # P⬍0.01 vs. baseline, n⫽8.
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DISCUSSION
Fig. 6. Effects of different concentrations and different time perfused with NaHS and combination of NaHS with SQ on the level of cAMP of medullary slices (pmol/mg) of neonatal rats. * P⬍0.05 vs. ACSF control, # P⬍0.01 vs. 200 M NaHS 10 min, n⫽8.
Likewise, DF and IA did not change significantly during the whole process of administration of SQ (Fig. 5C). In the group of SQ⫹NaHS, the slices were perfused with SQ for 5 min and then simultaneously with NaHS for 10 min. Compared with the data in Fig. 3D showing the effects of NaHS alone, the increase in DF induced by NaHS in the later stage was eliminated, whereas the decrease in DF and the increase in IA induced by NaHS in the initial stage was sustained (Fig. 5D). In the group of Gl⫹SQ⫹NaHS, the slices were perfused with both Gl and SQ for 5 min and then simultaneously NaHS for 10 min. The changes of DF and IA both in the initial stage and in the later stage were eliminated completely (Fig. 5E). The results above indicated that H2S inhibited first and then excited the respiratory activity of hypoglossal rootlets through opening KATP channels and increasing cAMP levels, respectively. Effects of NaHS and SQ on cAMP level of medullary slices The cAMP amount in the slice homogenate was measured with RIA technique to further determine whether the cAMP pathway played a role in the central regulation of respiratory activity mediated by H2S. The cAMP amount of the NaHS (200 M) 3 min group (0.57⫾0.04 pmol/mg) was not changed significantly versus control group (0.51⫾0.05pmol/mg) (P⬎0.05) while that of the NaHS (200 M) 10 min group (0.81⫾0.07 pmol/mg) was enhanced significantly versus control (P⬍0.05). The amount of cAMP in the NaHS⫹SQ 10 min group was decreased significantly versus the NaHS (200 M) 10 min group (0.46⫾0.02 pmol/mg, P⬍0.01). The data were shown in Fig. 6. These results further revealed that the excitation in the later stage, but not the inhibition in the initial stage, of the medullary respiratory center by physiological concentration of H2S might be attributed to indirectly raising the level of cAMP by activating AC.
In the present study we found that in the in vitro preparations of medullary slices of neonatal rats (i) exogenous H2S could affect the respiratory activity in a diphasic mode, a decrease in the respiratory frequency in the initial stage followed by its increase in the later stage, (ii) the endogenous H2S could be produced through the CBS–H2S pathway and could be involved in the control of rhythmic respiration of the slices, and (iii) the two opposite stages of effects of H2S on the respiratory activity might be induced by opening KATP channels and activating the AC– cAMP pathway, respectively. The discharge activity of the preparation did not change significantly during the whole process of experiments in the control groups and could recover from actions of the testing agents by washout with ACSF in the drug groups. Therefore the perfusion of the slices in the study would be effective although the possibility of anoxia in the core of the relatively thicker slices used could not be completely excluded. Although H2S was generally thought to be a toxic gas and its toxicology was extensively investigated in the past, recent observations showed that relatively high endogenous levels of H2S (50⬃160 M) could be observed in the tissues of mammals, especially in the brains of rat, human and bovine (Goodwin et al., 1989; Warenycia et al., 1989; Savage and Gould, 1990) and CBS was highly expressed in hippocampus as well as brainstem of rats (Abe and Kimura, 1996; Kimura, 2000). Endogenous H2S has been shown to be involved in many physiological processes such as regulation of synaptic activity in the brain (Eto et al., 2002b), relaxation of vascular smooth muscle in the circulatory system (Zhao et al., 2001), and scavenging free radicals (Searcy et al., 1995; Kimura and Kimura, 2004). In addition, traces of H2S were also found in multiple diseases such as Alzheimer disease with a decrease in endogenous H2S and Down syndrome disease with an increase in H2S (Eto et al., 2002a; Kamoun et al., 2003). Inhalation of exogenous H2S within the moderate concentration may enhance the respiratory activity, whereas inhalation of high concentration H2S would inhibit the activity of respiratory centers (Beauchamp et al., 1984; Reiffenstein et al., 1992). These findings implied that H2S may be involved in regulation of respiration. In the present experiments, we verified that the endogenous H2S could be produced through the CBS–H2S pathway and participate in the control of rhythmic respiration of the medullary slices. We observed that NaHS (50⬃200 M, the concentration of NaHS in which approximately the same amount of H2S as endogenously in the brain of animals can be produced) (Abe and Kimura, 1996) could induce a diphasic response of central respiratory activity, an inhibitory response followed by an excitatory one in regard to the changes of DF of hypoglossal rootlets. The same result could be obtained from the slices perfused with CYS. Both NaHS and CYS exert their effects on respiration through producing H2S. However the mechanisms to produce H2S
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are different. NaHS can be dissociated to deliver Na⫹ and HS⫺ in solution and then HS⫺ is associated with H⫹ to form H2S. But CYS as a substrate must be catalyzed by CBS to produce H2S. As reported previously, CBS knockout mice did not produce detectable amounts of H2S (Eto et al., 2002b) and the CBS inhibitor NH2OH suppressed H2S production in the brain (Abe and Kimura, 1996). We observed that NH2OH not only reversibly suppressed the activity of hypoglossal rootlets, but also eliminated the action of CYS on the respiratory activity. Therefore, it is suggested that CYS exerted its effects on respiration by forming H2S. On the other hand, CYS⫹NH2OH did not reduce the respiratory frequency in the later phase, it could be due to a direct action of CYS on the neurons of the slice. Furthermore, the possibility of NH2OH acting on some other enzymes to produce other amidated substances and hence to change the respiratory activity could not be completely excluded. It was also demonstrated that there exists an endogenous CBS–H2S pathway to produce H2S and the endogenous H2S participates in the control of the central respiratory activity of the medullary slices. Under physiological conditions, H2S production depends on the activity of CBS. The activity of CBS can be regulated by Ca2⫹/calmodulin, e.g. enhanced by Ca2⫹ influx which occurs with neuronal excitation (Eto et al., 2002b). So we speculate that H2S production could be increased due to activation of CBS by rhythmical discharge of the neurons of the slices. H2S may interact with other neurotransmitters and play an important role in maintenance of the excitatory/inhibitory balance in neurotransmission in the CNS (Han et al., 2005; Qu et al., 2008), presumably in the respiratory centers as well. SAM can enhance H2S production through activating CBS in the brain (Abe and Kimura, 1996; Kamoun et al., 2003) and a model for CBS regulation has been proposed in which the C-terminal domain of the enzyme bends to and covers its own catalytic domain to suppress the enzymatic activity (Kamoun, 2004). Once SAM binds to the regulatory domain of CBS, a conformational change occurs that frees the catalytic domain and CBS becomes active (Kamoun, 2004; Miles and Kraus, 2004; Shan et al., 2001). It is suggested therefore from our present experiments that SAM could activate CBS and increase endogenous H2S production. In addition, it might directly excite the neurons simultaneously because the decrease in DF of the rootlets induced by CYS in the initial stage did not occur when the slices were perfused with SAM. The presence of CBS, coupled with the identification of H2S in the brains of several species including human, suggested a role for this gas in CNS functions (Kimura, 2002). The studies about its underlying mechanisms revealed that the NMDA receptor is directly phosphorylated in specific sites by protein kinase A (PKA) (Leonard and Hell, 1997; Tingley et al., 1997). The increased activity of AC and PKA could enhance NMDA currents in the neurons of the dorsal horn (Cerne et al., 1993) and neostriatum (Gu et al., 2007), indicating that the NMDA receptor may be modulated through the cAMP cascade. Besides, H2S also hyperpolarizes CA1 pyramidal cells in the hippocampus of
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rats most probably by activating K⫹ATP channels (Baldelli et al., 1990; Qu et al., 2008). The evidence obtained from the present study shows that the two cellular signaling pathways, K⫹ATP channels and cAMP, are possibly involved in the central respiratory regulation of H2S. H2S may open directly the KATP channels and hyperpolarize the neurons and then suppress the activity of the slices at the initial stage, rather than through raising the level of cAMP to activate the KATP channels since the level of cAMP was not elevated significantly in this stage as demonstrated by RIA. The excitatory effect of H2S on the rootlets discharge in the later stage was eliminated by the AC inhibitor SQ and the high level of cAMP induced by NaHS in the later stage was also depressed by SQ as indicated by RIA. It is suggested therefore that H2S could elevate the excitability of the neurons in the respiratory centers of the medullary slices through increasing the level of cAMP. The inhibition of high concentration H2S on the central respiratory activity observed in the present study might be due to a decrease in the level of cAMP as it was reported that AC could be inhibited by high concentration H2S (Kimura, 2000). Moreover high concentration H2S can directly inhibit the cytochrome oxidase (Dorman et al., 2002) and then exert its toxic effect on the neurons of the slices. Although the precise mechanisms of respiratory rhythm genesis and regulation are still unclear (Feldman et al., 2003; Zhang and Zheng, 2002), the basic respiratory center is thought to be located in the medulla oblongata (Smith et al., 1991; Rekling and Feldman, 1998). In the present study, we found that both exogenous and endogenous H2S could change the activity of the hypoglossal rootlets of the medullary slices which were relatively thicker and would contain such respiratory nuclei as preBötzinger complex (Smith et al., 1991), parafacial respiratory group (Onimaru and Homma, 2003), rostral ventral respiratory group and so on. Therefore, we suppose that the respiratory responses to H2S observed in our experiments were induced by H2S widely acting on the respiratory centers in the medulla oblongata. The precise sites as well as neuronal mechanisms of actions of H2S on the rhythmic respiratory activity of the medullary slices remain to be investigated. Whether the present data can be used for intact animals also needs to be verified.
CONCLUSION It can be concluded from the present study that the endogenous H2S could be produced by the neurons via the CBS–H2S pathway in the medullary respiratory center and could be involved in the control of rhythmic respiration of the medullary slices of neonatal rats and that the diphasic effects of H2S on the respiratory activity might be induced by opening KATP channels in the initial inhibitory stage and activating the AC– cAMP pathway in the later excitatory stage, respectively. Acknowledgments—This work was supported by grants from the National Natural Science Foundation of China (No. 30370530 and No. 30770798). The authors are grateful to Dr. Alan D. Miller in New York for his English revision and scientific comments.
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(Accepted 11 August 2008) (Available online 22 August 2008)