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Heme oxygenase–carbon monoxide pathway is involved in regulation of respiration in medullary slice of neonatal rats
Neuroscience Letters 426 (2007) 128–132
Heme oxygenase–carbon monoxide pathway is involved in regulation of respiration in medullary slice of neonatal rats Wenxing Yang a , Qilan Zhang a , Hua Zhou a , Xuechuan Sun b , Qi Chen a , Yu Zheng a,∗ a
Department of Physiology, West China School of Preclinical and Forensic Medicine, Sichuan University, 3-17 Renmin South Road, Chengdu, Sichuan 610041, PR China b Physical Education Institute of PLA of China, Guangzhou, Guangdong 510502, PR China Received 28 June 2007; received in revised form 2 August 2007; accepted 4 September 2007
Abstract Carbon monoxide (CO) is a novel biological messenger molecule. It is well known that CO can be synthesized in mammalian cells. In addition, CO is also demonstrated to participate in many physiological processes, such as vasomotion, thermoregulation and respiratory regulation. The purpose of our present study was to investigate the role of heme oxygenase–carbon monoxide (HO–CO) pathway in central regulation of respiration. The experiments were carried out on the medullary slices of neonatal Sprague–Dawley rats. The discharge activity of the hypoglossal rootlets was recorded to indicate the central rhythmic respiratory activity and its duration (DD), interval (DI), frequency (DF) and integrated amplitude (IA) were analyzed. The slices were perfused with ZnPP-9 (a potent inhibitor of heme oxygenase), CO and hemin (substrate of heme oxygenase), respectively, to observe their effects on respiratory activity. The results obtained were as follows: ZnPP-9 could decrease DD, DI and IA, and increase DF (P < 0.05); exogenous CO caused a decrease in DD and DF, and an increase in DI and IA (P < 0.05); in response to hemin, DI and IA decreased, DF increased (P < 0.05), and DD did not change significantly (P > 0.05); administration of both ZnPP-9 and hemin could decrease DI, and increase DF (P < 0.05), but did not affect DD and IA significantly (P > 0.05). It can be concluded from the results above that the HO–CO pathway may be involved in the regulation of rhythmic respiration at the level of medulla oblongata. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Carbon monoxide; Heme oxygenase; Respiration; In vitro; Neonatal rat
Carbon monoxide (CO) has been thought to be noxious and harmful to the body for a long time. More than five decades ago, Sjostrand [18] suggested that CO is formed during the breakdown of heme in the human body. In 1981, Meyer [14] reported that CO appeared to interact with iron–sulfur centers of a variety of enzymes and then to inhibit the enzymes. In 1986, two forms of heme oxygenase (HO) were characterized from rat liver [9]. In the hematological system, CO had been shown to inhibit platelet aggregation apparently due to activation of soluble guanylyl cyclase (sGC) [1]. Based on these investigations, Marks et al. [10] presumed that CO may have physiological roles. By in situ hybridization, Verma et al. [20] demonstrated a discrete neuronal localization of mRNA for the constitutive form of HO throughout the brain. Besides, in primary cultures of olfactory neurons, zinc protoporphyrin-9 (ZnPP-9), a potent selective inhibitor of HO, depletes endogenous cyclic guanosine
∗
Corresponding author. Tel.: +86 28 85503433; fax: +86 28 85503204. E-mail address: [email protected] (Y. Zheng).
0304-3940/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2007.09.007
3 , 5 -monophosphate (cGMP). Thus, CO, like nitric oxide, was believed to be a new physiological regulator of cGMP. These findings proved the presumption of Marks and suggested that CO may function as a neurotransmitter. Now, it is well known that CO can be synthesized in mammalian cells. In addition, CO has also been demonstrated to participate in many physiological processes, such as vasomotion [7], thermoregulation [4] and respiratory regulation [16]. Endogenous CO arises from the cleavage of the heme molecule yielding biliverdin, free iron and CO, a process catalyzed by HO. Some metalloporphyrins, such as ZnPP-9, SnPP-9 and ZnDPBG, are selective inhibitors of HO and can reduce formation of endogenous CO. Three distinct HO isoforms encoded by different genes have been identified to date, inducible HO-1 [9], constitutive HO-2 [17] and constitutive HO3 [13], among which HO-1 and HO-2 are the most studied. Expression of HO-2 mRNA was seen in the rostral ventrolateral medulla (RVLM) under both control and hypoxic conditions, whereas expression of HO-1 mRNA was only seen in the RVLM by induction of hypoxia. HO-2 was immunocyto-
W. Yang et al. / Neuroscience Letters 426 (2007) 128–132
chemically localized in the RVLM to the pre-B¨otzinger complex (PBC) which has been considered to be critical for the genesis of rhythmic respiration [12]. Electrophysiological studies on neurons dissociated from the RVLM showed that, in response to SnPP-9, many of these neurons increased baseline-firing frequency that is associated with depolarization of the membrane [11]. These observations indicate that the HO–CO pathway may be involved in central regulation of respiration. The present study was carried out to investigate the possible roles of HO–CO pathway in central regulation of respiratory activity on neonatal rats in vitro. The experiments were performed on medullary slices from either male or female neonatal (0–3 days) Sprague–Dawley rats. All procedures were reviewed and approved by Sichuan University Committee on the Use of Live Animals in Research, and conformed to the Principles of Laboratory Animal Care (NIH publication no. 86-23 revised 1985). The animals were anesthetized with ether by inhalation and decapitated, and 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 (in mmol/L): 129 NaCl, 3 KCl, 2 CaCl2 , 1 MgSO4 , 21 NaHCO3 , 1 KH2 PO4 , and 30 d-glucose, pH 7.4. The brainstem was positioned with the 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 1000–1500 m thick was prepared, which was at the level of around the obex and contained functional respiratory neuronal network. Then 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, pH7.4. To obtain and maintain consistent respiratory rhythmic activity, the KCl concentration of the perfusing ACSF was raised from 3 to 7 mmol/L (meanwhile the concentration of NaCl was reduced from 129 to 125 mmol/L to balance the osmotic pressure). 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. 1A). Signals were amplified, filtered (τ = 0.001 s, F = 1 kHz), and integrated with a time constant of 50 ms by BL-420E+ biological signal processing system (Taimeng Biotech. Co., China). The discharge duration (DD, Fig. 1B), discharge interval (DI, Fig. 1B), discharge frequency (DF, number of discharge in 1 min) and integrated amplitude (IA, Fig. 1B) of hypoglossal rootlets were analyzed. The slices were divided into five groups (n = 8 for each group): control, ZnPP-9, exogenous CO, hemin and ZnPP9 + hemin. In the control group, the slices were perfused with ACSF during the whole process. In the ZnPP-9 group, the slices were perfused with ZnPP-9-ACSF (20 mol/L) for 13 min. In the exogenous CO group, ACSF was bubbled with CO for 15 min just before the perfusion, then the slices were perfused with CO-ACSF [16] for 8 min. In the hemin group, the slices were perfused with hemin-ACSF (50 mol/L) for 8 min. In the ZnPP9 + hemin group, the slices were perfused with ZnPP-9-ACSF for 5 min, then with hemin-ZnPP-9-ACSF for 8 min; the concentrations of ZnPP-9 and hemin were the same as the ZnPP-9
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Fig. 1. Schematic diagram of recording technique of hypoglossal rootlets burst of medullary slices of neonatal rats. (A) The transversal view of medullary slice. IO: inferior olivary nucleus; NA: nucleus ambiguous; NTS: nucleus tractus solitarii, PBC: pre-B¨otzinger complex; XII: hypoglossal nucleus; XIIn: hypoglossal rootlets. (B) The burst activity of hypoglossal rootlets, raw recording (top) and integrated activity (bottom), respectively. DD: discharge duration; DI: discharge interval; IA: integrated amplitude of discharge.
group and the hemin group. The discharge activity of hypoglossal rootlets before chemical application was recorded for 5 min as baseline of the activity for each group. From the end of administration of chemicals, the slices were continuously perfused with ACSF for washout. All data were normalized according to the baseline. Normalized DD, DI, DF and IA of hypoglossal rootlets in each 5 min were reported as means ± S.E.M. and statistically analyzed with two-tail repeated measure ANOVA. Statistical analysis were performed by SPSS 13.0 for Windows; P values of <0.05 were considered statistically significant. Each burst of hypoglossal rootlets had a rapid rising phase that was followed by a slowly decrementing period (Fig. 1B), which is similar to those published elsewhere [19]. Analysis of the rectified and integrated signal from medullary slices revealed that rhythmic activity was stable for more than 1 h and in some instances still could be recorded up to 8 h later. The activity of the rootlets perfused with ACSF during 45 min observing time was shown in Fig. 2. ZnPP-9 can inhibit endogenous CO production through its inhibitory effect on HO. In the present experiments, ZnPP-9 was bath-applied continuously for 13 min at 20 mol/L to investigate the possible role of the HO–CO pathway in central respiratory regulation. After administration of ZnPP-9, DD shortened by 6.65% (P < 0.05), DI shortened by ∼11.84% (P < 0.05), DF increased by ∼14.27% (P < 0.05), and IA decreased by ∼3.41% (P < 0.05), as shown in Fig. 3. To further confirm the effects of CO, we investigated the effects of exogenous CO on discharge of hypoglossal rootlets of medullary slices. In response to exogenous CO, DD shortened by
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Fig. 2. Rhythmic discharge activity of hypoglossal rootlets of ACSF perfused medullary slices of neonatal rats. The time course of normalized discharge duration (DD), discharge interval (DI), discharge frequency (DF) and integrated amplitude (IA) of the control group are shown. The horizontal dotted line in the middle of each graph is the average DD, DI, DF and IA during the 5-min of baseline activity. There is no significant change in DD, DI, DF and IA during the whole 40 min of observation (P > 0.05, n = 8).
Fig. 4. Effects of exogenous CO on the rhythmic discharge activity of hypoglossal rootlets of medullary slices in neonatal rats. All data were averaged into 5-min bins and normalized to illustrate the time course of CO-dependent changes of the nerve activity. The shadow column of each graph indicates the time of CO application. *P < 0.05 vs. baseline, n = 8.
∼7.29% (P < 0.05), DI lengthened by ∼31.70% (P < 0.05), DF decreased by ∼21.90% (P < 0.05), and IA increased by ∼9.04% (P < 0.05), as shown in Fig. 4. HO–CO pathway is the most important source of endogenous CO generation in mammals. In our experiments, the slices were perfused with hemin, a substrate of HO, to evaluate the respiratory effects of CO which is generated by HO–CO pathway. As shown in Fig. 5, hemin could not affect DD significantly (P > 0.05); but similar to the effects of ZnPP-9, it produced a shortening in DI for ∼12.66% (P < 0.05), an increase in DF for ∼15.35% (P < 0.05), and a decrease in IA for ∼9.17% (P < 0.05).
Effects of combination of ZnPP-9 and hemin were also investigated in our experiments. This combination did not affect DD significantly (P > 0.05). After the drugs administration, the DI shortened by ∼13.79% (P < 0.05), DF increased by ∼15.41% (P < 0.05), and IA did not change significantly (P > 0.05). These results are shown in Fig. 6. The basic respiratory center is located in the medulla oblongata, and the PBC has been shown to be the kernel region of respiratory rhythm generation, but the underlying mechanisms of respiratory rhythm genesis and regulation are still unclear [3,23]. The hypoglossal nucleus is partially innervated by the respiratory center, so hypoglossal nerve has respiratory rhythmic discharge activity. In 1991, Smith et al. [19] developed
Fig. 3. Effects of ZnPP-9 on the rhythmic discharge activity of hypoglossal rootlets of medullary slices in neonatal rats. All data were averaged into 5-min bins and normalized to illustrate the time course of ZnPP-9-dependent changes of the nerve activity. The shadow column of each graph indicates the time of ZnPP-9 application. *P < 0.05 vs. baseline, n = 8.
Fig. 5. Effects of hemin on the rhythmic discharge activity of hypoglossal rootlets of medullary slices in neonatal rats. All data were averaged into 5-min bins and normalized to illustrate the time course of hemin-dependent changes of the nerve activity. The shadow column of each graph indicates the time of hemin application. *P < 0.05 vs. baseline, n = 8.
W. Yang et al. / Neuroscience Letters 426 (2007) 128–132
Fig. 6. Effects of ZnPP-9 + hemin on the rhythmic discharge activity of hypoglossal rootlets of medullary slices in neonatal rats. All data were averaged into 5-min bins and normalized to illustrate the time course of ZnPP-9 + hemindependent changes of the nerve activity. The shadow column of each graph indicates the time of ZnPP-9 + hemin application. *P < 0.05 vs. baseline, n = 8.
a technique for recording the respiratory rhythmic discharge of hypoglossal rootlets. This technique has been used widely, which is very helpful for investigating the genesis and regulation of respiratory rhythm. CO is a novel biological messenger molecule. Endogenously formed CO arises primarily from the degradation of heme and is generated in a reaction catalyzed by HO. In this reaction heme is degraded to release iron, biliverdin and CO. The iron is primarily recycled into the formation of new heme while the biliverdin is rapidly converted to bilirubin by biliverdin reductase. It has been proven that endogenous CO is involved in many physiological processes, such as vasomotion [7], thermoregulation [4] and respiratory regulation [16]. Investigation of the effects of endogenous CO on respiratory regulation has been focused on the carotid body for many years. Prabhakar et al. [16] found that HO-2 protein was present in glomus cells of the cat and rat carotid bodies; and ZnPP-9 could augment carotid body activity in a dosedependent manner, which could be reversed by exogenous CO. Yang et al. [22] reported that respiratory responses to hypoxia were selectively potentiated by SnPP-9; whereas responses to hypercapnia were unaffected. These findings further support the idea that CO generated by HO is a physiological modulator of respiration. HO is expressed widely in the central nervous system. In normal condition, HO-2-like immunoreactivity was found in many central areas, such as PBC [12], hypoglossal nucleus [12], facial nucleus [12,2] and nucleus tractus solitarius [5], which are closely correlated with respiratory regulation. Besides, hypoxia and oxidative stress could induce the expression of HO-1 in the areas mentioned above [12,15]. Mazza and Neubauer [11] found that CO arising from HO–CO pathway could regulate the excitability of RVLM neurons. These findings imply that endogenous CO may be involved in regulation of respiration at the level of the medulla oblongata.
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In our experiments, we found that ZnPP-9 can decrease DI, increase DF and reduce IA of hypoglossal nerve burst, and that exogenous CO can increase DI, reduce DF and increase IA. Taking these finding together, we presume that (i) endogenous CO could be produced through the HO–CO pathway by the neurons in medullary respiratory center; (ii) endogenous CO could maintain the respiratory frequency at a relatively low level through its lengthening effects on expiratory duration; and (iii) endogenous CO could maintain the respiratory magnitude at a relatively high level. CO production has been related to hypoxic response. As reported by Mazza et al. [12], chronic hypoxia could up-regulate the HO-2 immunoreactivity in rat RVLM. In the present study, we used thick medullary slices. Therefore, the possibility cannot be excluded that the relatively hypoxic experimental conditions may favor the CO production. We postulate that CO may be involved in the hypoxic respiratory reaction. Besides, CO seems to have an inhibitory effect on respiratory rhythmicity since it slowed down the DF. Whether this effect is one side of its poisonous actions is also intriguing. A paradox is found in our experiments. Hemin could decrease the DI, increase the DF and attenuate the IA, which contrary to the effects of CO. Hemin is a substrate of HO, which is degraded to release CO. Many research achievements revealed that CO is an activator of sGC, which catalyzes the conversion of GTP to the secondary messenger cGMP, and then induces the vasodilatation [21]. However, Imai et al. [6] showed that overexpression of HO in vascular smooth muscle cells attenuates NO-induced vasodilatation in transgenic mice. Koglin and Behrends [8] did some investigation on this paradox and revealed that biliverdin is an endogenous inhibitor of sGC. Putting these findings together, we speculate that the paradox in our experiments may be caused by biliverdin. Besides, we found that the effect of CO can be washed out quickly and the effect of hemin can last longer. We ascribe this difference to the physical properties of these two chemicals. CO is a gaseous molecule, which diffuses easily; hemin is pulverized, and is difficult to diffuse and to be washed out. In the end of our experiments, we found that there was no evident change in the color of the slices perfused with CO, while the color of slices perfused with hemin was darker. To further verify our presumption, spectrophotometric determination technique was used to evaluate the hemin level of slices undergoing different treatments. It could be concluded from the results that hemin could not be washed out in our experiments (data not shown). These findings may prove our presumption from their physical properties. In conclusion, the present study demonstrates that the HO–CO pathway is involved in the central control of rhythmic respiration at the level of medulla oblongata. Acknowledgements This work was supported by 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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References [1] B. Brune, V. Ullrich, Inhibition of platelet aggregation by carbon monoxide is mediated by activation of guanylate cyclase, Mol. Pharmacol. 32 (1987) 497–504. [2] J.F. Ewing, M.D. Maines, In situ hybridization and immunohistochemical localization of heme oxygenase-2 mRNA and protein in normal rat brain: differential distribution of isozyme 1 and 2, Mol. Cell. Neurosci. 3 (1992) 559–570. [3] J.L. Feldman, G.S. Mitchell, E.E. Nattie, Breathing: rhythmicity, plasticity, chemosensitivity, Annu. Rev. Neurosci. 26 (2003) 239–266. [4] M.P. Flavia, A.S. Alexandre, G.S.B. Luiz, Thermoregulatory response to hypoxia after inhibition of the central heme oxygenase–carbon monoxide pathway, J. Thermal. Biol. 26 (2001) 339–343. [5] S.R. Glaum, R.J. Miller, Zinc protoporphyrin-IX blocks the effects of metabotropic glutamate receptor activation in the rat nucleus tractus solitarii, Mol. Pharmacol. 43 (1993) 965–971. [6] T. Imai, T. Morita, T. Shindo, R. Nagai, Y. Yazaki, H. Kurihara, M. Suematsu, S. Katayama, Vascular smooth muscle cell-directed overexpression of heme oxygenase-1 elevates blood pressure through attenuation of nitric oxide-induced vasodilation in mice, Circ. Res. 89 (2001) 55–62. [7] R.A. Johnson, M. Lavesa, B. Askari, N.G. Abraham, A. Nasjletti, A heme oxygenase product, presumably carbon monoxide, mediates a vasodepressor role in rats, Hypertension 25 (1995) 166–169. [8] M. Koglin, S. Behrends, Biliverdin IX is an endogenous inhibitor of soluble guanylyl cyclase, Biochem. Pharmacol. 64 (2002) 109–116. [9] M.D. Maines, G.M. Trakshel, R.K. Kutty, Characterization of two constitutive forms of rat liver microsomal heme oxygenase, J. Biol. Chem. 261 (1986) 411–419. [10] G.S. Marks, J.F. Brien, K. Nakatsu, B.E. Mclaughlin, Does carbon monoxide have a physiological function, Trends Pharmacol. Sci. 12 (1991) 185–188. [11] E. Mazza, J.A. Neubauer, Electrophysiological response to hypoxia after inhibition of heme oxygenase in neurons cultured from the rostral ventrolateral medulla, FASEB J. 12 (1998) A495.
[12] E. Mazza, S. Thakkar-Varia, C.A. Tozzi, J.A. Neubauer, Expression of heme oxygenase in the oxygen-sensing regions of the rostral ventrolateral medulla, J. Appl. Physiol. 91 (2001) 379–385. [13] W.K. McCoubrey, T.J. Huang, M.D. Maines, Isolation and characterization of a cDNA from the rat brain that encodes hemoprotein heme oxygenase-3, Eur. J. Biochem. 247 (1997) 725–732. [14] J. Meyer, Comparison of carbon monoxide, nitric oxide, and nitrite as inhibitors of the nitrogenase from Clostridium pasteurianum, Arch. Biochem. Biophys. 210 (1981) 246–256. [15] N.R. Prabhakar, Endogenous carbon monoxide in control of respiration, Respir. Physiol. 114 (1998) 57–64. [16] N.R. Prabhakar, J.L. Dinerman, F.H. Agani, S.H. Snyder, Carbon monoxide: a role in carotid body chemoreception, Proc. Natl. Acad. Sci. U.S.A. 92 (1995) 1994–1997. [17] S. Shibahara, M. Yoshizawa, H. Suzuki, K. Takeda, K. Meguro, K. Endo, Functional analysis of cDNA for two types of human heme oxygenase and evidence for their separate regulation, J. Biochem. 113 (1993) 214–218. [18] T. Sjostrand, Endogenous formation of carbon monoxide in man under normal and pathological conditions, J. Clin. Lab. Invest. 1 (1949) 201–210. [19] J.C. Smith, H.H. Ellenberger, K. Ballanyi, D.W. Richter, J.L. Feldman, Pre-Botzinger complex: a brain stem region that may generate respiratory rhythm in mammals, Science 254 (1991) 726–729. [20] A. Verma, D.J. Hirsch, C.E. Glatt, G.V. Ronnett, S.H. Snyder, Carbon monoxide: a putative neural messenger, Science 259 (1993) 381–384. [21] L. Wu, R. Wang, Carbon monoxide: endogenous production, physiological functions, and pharmacological applications, Pharmacol. Rev. 57 (2005) 585–630. [22] T. Yang, D.D. Kline, D.R. Premkumar, R.R. Mishra, N.R. Prabhakar, Evidence for a physiological role of endogenous carbon monoxide (CO) in respiratory responses to hypoxia in rats, FASEB J. 12 (1998) A167. [23] C.W. Zhang, Y. Zheng, Pre-botzinger complex: a kernel region for respiratory rhythmogenesis, Prog. Physiol. Sci. 33 (2002) 179–181 (in Chinese).
Heme oxygenase–carbon monoxide pathway is involved in regulation of respiration in medullary slice of neonatal rats Wenxing Yang a , Qilan Zhang a , Hua Zhou a , Xuechuan Sun b , Qi Chen a , Yu Zheng a,∗ a
Department of Physiology, West China School of Preclinical and Forensic Medicine, Sichuan University, 3-17 Renmin South Road, Chengdu, Sichuan 610041, PR China b Physical Education Institute of PLA of China, Guangzhou, Guangdong 510502, PR China Received 28 June 2007; received in revised form 2 August 2007; accepted 4 September 2007
Abstract Carbon monoxide (CO) is a novel biological messenger molecule. It is well known that CO can be synthesized in mammalian cells. In addition, CO is also demonstrated to participate in many physiological processes, such as vasomotion, thermoregulation and respiratory regulation. The purpose of our present study was to investigate the role of heme oxygenase–carbon monoxide (HO–CO) pathway in central regulation of respiration. The experiments were carried out on the medullary slices of neonatal Sprague–Dawley rats. The discharge activity of the hypoglossal rootlets was recorded to indicate the central rhythmic respiratory activity and its duration (DD), interval (DI), frequency (DF) and integrated amplitude (IA) were analyzed. The slices were perfused with ZnPP-9 (a potent inhibitor of heme oxygenase), CO and hemin (substrate of heme oxygenase), respectively, to observe their effects on respiratory activity. The results obtained were as follows: ZnPP-9 could decrease DD, DI and IA, and increase DF (P < 0.05); exogenous CO caused a decrease in DD and DF, and an increase in DI and IA (P < 0.05); in response to hemin, DI and IA decreased, DF increased (P < 0.05), and DD did not change significantly (P > 0.05); administration of both ZnPP-9 and hemin could decrease DI, and increase DF (P < 0.05), but did not affect DD and IA significantly (P > 0.05). It can be concluded from the results above that the HO–CO pathway may be involved in the regulation of rhythmic respiration at the level of medulla oblongata. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Carbon monoxide; Heme oxygenase; Respiration; In vitro; Neonatal rat
Carbon monoxide (CO) has been thought to be noxious and harmful to the body for a long time. More than five decades ago, Sjostrand [18] suggested that CO is formed during the breakdown of heme in the human body. In 1981, Meyer [14] reported that CO appeared to interact with iron–sulfur centers of a variety of enzymes and then to inhibit the enzymes. In 1986, two forms of heme oxygenase (HO) were characterized from rat liver [9]. In the hematological system, CO had been shown to inhibit platelet aggregation apparently due to activation of soluble guanylyl cyclase (sGC) [1]. Based on these investigations, Marks et al. [10] presumed that CO may have physiological roles. By in situ hybridization, Verma et al. [20] demonstrated a discrete neuronal localization of mRNA for the constitutive form of HO throughout the brain. Besides, in primary cultures of olfactory neurons, zinc protoporphyrin-9 (ZnPP-9), a potent selective inhibitor of HO, depletes endogenous cyclic guanosine
∗
Corresponding author. Tel.: +86 28 85503433; fax: +86 28 85503204. E-mail address: [email protected] (Y. Zheng).
0304-3940/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2007.09.007
3 , 5 -monophosphate (cGMP). Thus, CO, like nitric oxide, was believed to be a new physiological regulator of cGMP. These findings proved the presumption of Marks and suggested that CO may function as a neurotransmitter. Now, it is well known that CO can be synthesized in mammalian cells. In addition, CO has also been demonstrated to participate in many physiological processes, such as vasomotion [7], thermoregulation [4] and respiratory regulation [16]. Endogenous CO arises from the cleavage of the heme molecule yielding biliverdin, free iron and CO, a process catalyzed by HO. Some metalloporphyrins, such as ZnPP-9, SnPP-9 and ZnDPBG, are selective inhibitors of HO and can reduce formation of endogenous CO. Three distinct HO isoforms encoded by different genes have been identified to date, inducible HO-1 [9], constitutive HO-2 [17] and constitutive HO3 [13], among which HO-1 and HO-2 are the most studied. Expression of HO-2 mRNA was seen in the rostral ventrolateral medulla (RVLM) under both control and hypoxic conditions, whereas expression of HO-1 mRNA was only seen in the RVLM by induction of hypoxia. HO-2 was immunocyto-
W. Yang et al. / Neuroscience Letters 426 (2007) 128–132
chemically localized in the RVLM to the pre-B¨otzinger complex (PBC) which has been considered to be critical for the genesis of rhythmic respiration [12]. Electrophysiological studies on neurons dissociated from the RVLM showed that, in response to SnPP-9, many of these neurons increased baseline-firing frequency that is associated with depolarization of the membrane [11]. These observations indicate that the HO–CO pathway may be involved in central regulation of respiration. The present study was carried out to investigate the possible roles of HO–CO pathway in central regulation of respiratory activity on neonatal rats in vitro. The experiments were performed on medullary slices from either male or female neonatal (0–3 days) Sprague–Dawley rats. All procedures were reviewed and approved by Sichuan University Committee on the Use of Live Animals in Research, and conformed to the Principles of Laboratory Animal Care (NIH publication no. 86-23 revised 1985). The animals were anesthetized with ether by inhalation and decapitated, and 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 (in mmol/L): 129 NaCl, 3 KCl, 2 CaCl2 , 1 MgSO4 , 21 NaHCO3 , 1 KH2 PO4 , and 30 d-glucose, pH 7.4. The brainstem was positioned with the 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 1000–1500 m thick was prepared, which was at the level of around the obex and contained functional respiratory neuronal network. Then 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, pH7.4. To obtain and maintain consistent respiratory rhythmic activity, the KCl concentration of the perfusing ACSF was raised from 3 to 7 mmol/L (meanwhile the concentration of NaCl was reduced from 129 to 125 mmol/L to balance the osmotic pressure). 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. 1A). Signals were amplified, filtered (τ = 0.001 s, F = 1 kHz), and integrated with a time constant of 50 ms by BL-420E+ biological signal processing system (Taimeng Biotech. Co., China). The discharge duration (DD, Fig. 1B), discharge interval (DI, Fig. 1B), discharge frequency (DF, number of discharge in 1 min) and integrated amplitude (IA, Fig. 1B) of hypoglossal rootlets were analyzed. The slices were divided into five groups (n = 8 for each group): control, ZnPP-9, exogenous CO, hemin and ZnPP9 + hemin. In the control group, the slices were perfused with ACSF during the whole process. In the ZnPP-9 group, the slices were perfused with ZnPP-9-ACSF (20 mol/L) for 13 min. In the exogenous CO group, ACSF was bubbled with CO for 15 min just before the perfusion, then the slices were perfused with CO-ACSF [16] for 8 min. In the hemin group, the slices were perfused with hemin-ACSF (50 mol/L) for 8 min. In the ZnPP9 + hemin group, the slices were perfused with ZnPP-9-ACSF for 5 min, then with hemin-ZnPP-9-ACSF for 8 min; the concentrations of ZnPP-9 and hemin were the same as the ZnPP-9
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Fig. 1. Schematic diagram of recording technique of hypoglossal rootlets burst of medullary slices of neonatal rats. (A) The transversal view of medullary slice. IO: inferior olivary nucleus; NA: nucleus ambiguous; NTS: nucleus tractus solitarii, PBC: pre-B¨otzinger complex; XII: hypoglossal nucleus; XIIn: hypoglossal rootlets. (B) The burst activity of hypoglossal rootlets, raw recording (top) and integrated activity (bottom), respectively. DD: discharge duration; DI: discharge interval; IA: integrated amplitude of discharge.
group and the hemin group. The discharge activity of hypoglossal rootlets before chemical application was recorded for 5 min as baseline of the activity for each group. From the end of administration of chemicals, the slices were continuously perfused with ACSF for washout. All data were normalized according to the baseline. Normalized DD, DI, DF and IA of hypoglossal rootlets in each 5 min were reported as means ± S.E.M. and statistically analyzed with two-tail repeated measure ANOVA. Statistical analysis were performed by SPSS 13.0 for Windows; P values of <0.05 were considered statistically significant. Each burst of hypoglossal rootlets had a rapid rising phase that was followed by a slowly decrementing period (Fig. 1B), which is similar to those published elsewhere [19]. Analysis of the rectified and integrated signal from medullary slices revealed that rhythmic activity was stable for more than 1 h and in some instances still could be recorded up to 8 h later. The activity of the rootlets perfused with ACSF during 45 min observing time was shown in Fig. 2. ZnPP-9 can inhibit endogenous CO production through its inhibitory effect on HO. In the present experiments, ZnPP-9 was bath-applied continuously for 13 min at 20 mol/L to investigate the possible role of the HO–CO pathway in central respiratory regulation. After administration of ZnPP-9, DD shortened by 6.65% (P < 0.05), DI shortened by ∼11.84% (P < 0.05), DF increased by ∼14.27% (P < 0.05), and IA decreased by ∼3.41% (P < 0.05), as shown in Fig. 3. To further confirm the effects of CO, we investigated the effects of exogenous CO on discharge of hypoglossal rootlets of medullary slices. In response to exogenous CO, DD shortened by
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Fig. 2. Rhythmic discharge activity of hypoglossal rootlets of ACSF perfused medullary slices of neonatal rats. The time course of normalized discharge duration (DD), discharge interval (DI), discharge frequency (DF) and integrated amplitude (IA) of the control group are shown. The horizontal dotted line in the middle of each graph is the average DD, DI, DF and IA during the 5-min of baseline activity. There is no significant change in DD, DI, DF and IA during the whole 40 min of observation (P > 0.05, n = 8).
Fig. 4. Effects of exogenous CO on the rhythmic discharge activity of hypoglossal rootlets of medullary slices in neonatal rats. All data were averaged into 5-min bins and normalized to illustrate the time course of CO-dependent changes of the nerve activity. The shadow column of each graph indicates the time of CO application. *P < 0.05 vs. baseline, n = 8.
∼7.29% (P < 0.05), DI lengthened by ∼31.70% (P < 0.05), DF decreased by ∼21.90% (P < 0.05), and IA increased by ∼9.04% (P < 0.05), as shown in Fig. 4. HO–CO pathway is the most important source of endogenous CO generation in mammals. In our experiments, the slices were perfused with hemin, a substrate of HO, to evaluate the respiratory effects of CO which is generated by HO–CO pathway. As shown in Fig. 5, hemin could not affect DD significantly (P > 0.05); but similar to the effects of ZnPP-9, it produced a shortening in DI for ∼12.66% (P < 0.05), an increase in DF for ∼15.35% (P < 0.05), and a decrease in IA for ∼9.17% (P < 0.05).
Effects of combination of ZnPP-9 and hemin were also investigated in our experiments. This combination did not affect DD significantly (P > 0.05). After the drugs administration, the DI shortened by ∼13.79% (P < 0.05), DF increased by ∼15.41% (P < 0.05), and IA did not change significantly (P > 0.05). These results are shown in Fig. 6. The basic respiratory center is located in the medulla oblongata, and the PBC has been shown to be the kernel region of respiratory rhythm generation, but the underlying mechanisms of respiratory rhythm genesis and regulation are still unclear [3,23]. The hypoglossal nucleus is partially innervated by the respiratory center, so hypoglossal nerve has respiratory rhythmic discharge activity. In 1991, Smith et al. [19] developed
Fig. 3. Effects of ZnPP-9 on the rhythmic discharge activity of hypoglossal rootlets of medullary slices in neonatal rats. All data were averaged into 5-min bins and normalized to illustrate the time course of ZnPP-9-dependent changes of the nerve activity. The shadow column of each graph indicates the time of ZnPP-9 application. *P < 0.05 vs. baseline, n = 8.
Fig. 5. Effects of hemin on the rhythmic discharge activity of hypoglossal rootlets of medullary slices in neonatal rats. All data were averaged into 5-min bins and normalized to illustrate the time course of hemin-dependent changes of the nerve activity. The shadow column of each graph indicates the time of hemin application. *P < 0.05 vs. baseline, n = 8.
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Fig. 6. Effects of ZnPP-9 + hemin on the rhythmic discharge activity of hypoglossal rootlets of medullary slices in neonatal rats. All data were averaged into 5-min bins and normalized to illustrate the time course of ZnPP-9 + hemindependent changes of the nerve activity. The shadow column of each graph indicates the time of ZnPP-9 + hemin application. *P < 0.05 vs. baseline, n = 8.
a technique for recording the respiratory rhythmic discharge of hypoglossal rootlets. This technique has been used widely, which is very helpful for investigating the genesis and regulation of respiratory rhythm. CO is a novel biological messenger molecule. Endogenously formed CO arises primarily from the degradation of heme and is generated in a reaction catalyzed by HO. In this reaction heme is degraded to release iron, biliverdin and CO. The iron is primarily recycled into the formation of new heme while the biliverdin is rapidly converted to bilirubin by biliverdin reductase. It has been proven that endogenous CO is involved in many physiological processes, such as vasomotion [7], thermoregulation [4] and respiratory regulation [16]. Investigation of the effects of endogenous CO on respiratory regulation has been focused on the carotid body for many years. Prabhakar et al. [16] found that HO-2 protein was present in glomus cells of the cat and rat carotid bodies; and ZnPP-9 could augment carotid body activity in a dosedependent manner, which could be reversed by exogenous CO. Yang et al. [22] reported that respiratory responses to hypoxia were selectively potentiated by SnPP-9; whereas responses to hypercapnia were unaffected. These findings further support the idea that CO generated by HO is a physiological modulator of respiration. HO is expressed widely in the central nervous system. In normal condition, HO-2-like immunoreactivity was found in many central areas, such as PBC [12], hypoglossal nucleus [12], facial nucleus [12,2] and nucleus tractus solitarius [5], which are closely correlated with respiratory regulation. Besides, hypoxia and oxidative stress could induce the expression of HO-1 in the areas mentioned above [12,15]. Mazza and Neubauer [11] found that CO arising from HO–CO pathway could regulate the excitability of RVLM neurons. These findings imply that endogenous CO may be involved in regulation of respiration at the level of the medulla oblongata.
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In our experiments, we found that ZnPP-9 can decrease DI, increase DF and reduce IA of hypoglossal nerve burst, and that exogenous CO can increase DI, reduce DF and increase IA. Taking these finding together, we presume that (i) endogenous CO could be produced through the HO–CO pathway by the neurons in medullary respiratory center; (ii) endogenous CO could maintain the respiratory frequency at a relatively low level through its lengthening effects on expiratory duration; and (iii) endogenous CO could maintain the respiratory magnitude at a relatively high level. CO production has been related to hypoxic response. As reported by Mazza et al. [12], chronic hypoxia could up-regulate the HO-2 immunoreactivity in rat RVLM. In the present study, we used thick medullary slices. Therefore, the possibility cannot be excluded that the relatively hypoxic experimental conditions may favor the CO production. We postulate that CO may be involved in the hypoxic respiratory reaction. Besides, CO seems to have an inhibitory effect on respiratory rhythmicity since it slowed down the DF. Whether this effect is one side of its poisonous actions is also intriguing. A paradox is found in our experiments. Hemin could decrease the DI, increase the DF and attenuate the IA, which contrary to the effects of CO. Hemin is a substrate of HO, which is degraded to release CO. Many research achievements revealed that CO is an activator of sGC, which catalyzes the conversion of GTP to the secondary messenger cGMP, and then induces the vasodilatation [21]. However, Imai et al. [6] showed that overexpression of HO in vascular smooth muscle cells attenuates NO-induced vasodilatation in transgenic mice. Koglin and Behrends [8] did some investigation on this paradox and revealed that biliverdin is an endogenous inhibitor of sGC. Putting these findings together, we speculate that the paradox in our experiments may be caused by biliverdin. Besides, we found that the effect of CO can be washed out quickly and the effect of hemin can last longer. We ascribe this difference to the physical properties of these two chemicals. CO is a gaseous molecule, which diffuses easily; hemin is pulverized, and is difficult to diffuse and to be washed out. In the end of our experiments, we found that there was no evident change in the color of the slices perfused with CO, while the color of slices perfused with hemin was darker. To further verify our presumption, spectrophotometric determination technique was used to evaluate the hemin level of slices undergoing different treatments. It could be concluded from the results that hemin could not be washed out in our experiments (data not shown). These findings may prove our presumption from their physical properties. In conclusion, the present study demonstrates that the HO–CO pathway is involved in the central control of rhythmic respiration at the level of medulla oblongata. Acknowledgements This work was supported by 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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References [1] B. Brune, V. Ullrich, Inhibition of platelet aggregation by carbon monoxide is mediated by activation of guanylate cyclase, Mol. Pharmacol. 32 (1987) 497–504. [2] J.F. Ewing, M.D. Maines, In situ hybridization and immunohistochemical localization of heme oxygenase-2 mRNA and protein in normal rat brain: differential distribution of isozyme 1 and 2, Mol. Cell. Neurosci. 3 (1992) 559–570. [3] J.L. Feldman, G.S. Mitchell, E.E. Nattie, Breathing: rhythmicity, plasticity, chemosensitivity, Annu. Rev. Neurosci. 26 (2003) 239–266. [4] M.P. Flavia, A.S. Alexandre, G.S.B. Luiz, Thermoregulatory response to hypoxia after inhibition of the central heme oxygenase–carbon monoxide pathway, J. Thermal. Biol. 26 (2001) 339–343. [5] S.R. Glaum, R.J. Miller, Zinc protoporphyrin-IX blocks the effects of metabotropic glutamate receptor activation in the rat nucleus tractus solitarii, Mol. Pharmacol. 43 (1993) 965–971. [6] T. Imai, T. Morita, T. Shindo, R. Nagai, Y. Yazaki, H. Kurihara, M. Suematsu, S. Katayama, Vascular smooth muscle cell-directed overexpression of heme oxygenase-1 elevates blood pressure through attenuation of nitric oxide-induced vasodilation in mice, Circ. Res. 89 (2001) 55–62. [7] R.A. Johnson, M. Lavesa, B. Askari, N.G. Abraham, A. Nasjletti, A heme oxygenase product, presumably carbon monoxide, mediates a vasodepressor role in rats, Hypertension 25 (1995) 166–169. [8] M. Koglin, S. Behrends, Biliverdin IX is an endogenous inhibitor of soluble guanylyl cyclase, Biochem. Pharmacol. 64 (2002) 109–116. [9] M.D. Maines, G.M. Trakshel, R.K. Kutty, Characterization of two constitutive forms of rat liver microsomal heme oxygenase, J. Biol. Chem. 261 (1986) 411–419. [10] G.S. Marks, J.F. Brien, K. Nakatsu, B.E. Mclaughlin, Does carbon monoxide have a physiological function, Trends Pharmacol. Sci. 12 (1991) 185–188. [11] E. Mazza, J.A. Neubauer, Electrophysiological response to hypoxia after inhibition of heme oxygenase in neurons cultured from the rostral ventrolateral medulla, FASEB J. 12 (1998) A495.
[12] E. Mazza, S. Thakkar-Varia, C.A. Tozzi, J.A. Neubauer, Expression of heme oxygenase in the oxygen-sensing regions of the rostral ventrolateral medulla, J. Appl. Physiol. 91 (2001) 379–385. [13] W.K. McCoubrey, T.J. Huang, M.D. Maines, Isolation and characterization of a cDNA from the rat brain that encodes hemoprotein heme oxygenase-3, Eur. J. Biochem. 247 (1997) 725–732. [14] J. Meyer, Comparison of carbon monoxide, nitric oxide, and nitrite as inhibitors of the nitrogenase from Clostridium pasteurianum, Arch. Biochem. Biophys. 210 (1981) 246–256. [15] N.R. Prabhakar, Endogenous carbon monoxide in control of respiration, Respir. Physiol. 114 (1998) 57–64. [16] N.R. Prabhakar, J.L. Dinerman, F.H. Agani, S.H. Snyder, Carbon monoxide: a role in carotid body chemoreception, Proc. Natl. Acad. Sci. U.S.A. 92 (1995) 1994–1997. [17] S. Shibahara, M. Yoshizawa, H. Suzuki, K. Takeda, K. Meguro, K. Endo, Functional analysis of cDNA for two types of human heme oxygenase and evidence for their separate regulation, J. Biochem. 113 (1993) 214–218. [18] T. Sjostrand, Endogenous formation of carbon monoxide in man under normal and pathological conditions, J. Clin. Lab. Invest. 1 (1949) 201–210. [19] J.C. Smith, H.H. Ellenberger, K. Ballanyi, D.W. Richter, J.L. Feldman, Pre-Botzinger complex: a brain stem region that may generate respiratory rhythm in mammals, Science 254 (1991) 726–729. [20] A. Verma, D.J. Hirsch, C.E. Glatt, G.V. Ronnett, S.H. Snyder, Carbon monoxide: a putative neural messenger, Science 259 (1993) 381–384. [21] L. Wu, R. Wang, Carbon monoxide: endogenous production, physiological functions, and pharmacological applications, Pharmacol. Rev. 57 (2005) 585–630. [22] T. Yang, D.D. Kline, D.R. Premkumar, R.R. Mishra, N.R. Prabhakar, Evidence for a physiological role of endogenous carbon monoxide (CO) in respiratory responses to hypoxia in rats, FASEB J. 12 (1998) A167. [23] C.W. Zhang, Y. Zheng, Pre-botzinger complex: a kernel region for respiratory rhythmogenesis, Prog. Physiol. Sci. 33 (2002) 179–181 (in Chinese).