Differences in the H2S-induced quantal release of catecholamine in adrenal chromaffin cells of neonatal and adult rats

ARTICLE IN PRESS

G Model TOX 51250 1–6

Toxicology xxx (2013) xxx–xxx

Contents lists available at SciVerse ScienceDirect

Toxicology journal homepage: www.elsevier.com/locate/toxicol

Differences in the H2 S-induced quantal release of catecholamine in adrenal chromaffin cells of neonatal and adult rats

1

2

3 4

Q1

Kai Wang, Dexiao Zhu, Wei Yao ∗ Department of Physiology, Shandong University School of Medicine, Jinan 250012, China

5

6

a r t i c l e

i n f o

a b s t r a c t

7 8 9 10 11

Article history: Received 20 May 2013 Received in revised form 26 June 2013 Accepted 27 June 2013 Available online xxx

12

17

Keywords: Hydrogen sulfide Chromaffin cell Catecholamine Amperometry

18

1. Introduction

13 14 15 16

19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37

Both catecholamine (CA) release from adrenal chromaffin cells and hydrogen sulfide (H2 S) have been shown to play critical roles in the regulation of hypoxic stress response. Our previous study has demonstrated that exogenous H2 S directly induced quantal CA released from adult rat adrenal chromaffin cells (ARACCs) by inhibiting Ca2+ -activated K+ current [IK(Ca) current]. However, it is not clear now whether H2 S can also directly induce quantal CA release from neonatal rat adrenal chromaffin cells (NRACCs). In the present study, we investigated whether exogenous H2 S can stimulate quantal CA release from NRACCs, and whether there were differences in the kinetics of H2 S-induced quantal CA release between ARACCs and NRACCs. Using carbon-fiber amperometry and whole-cell patch clamping techniques, our experimental results showed: (1) H2 S can directly induce quantal CA release from NRACCs; (2) H2 S induced the depolarization of membrane potential and inhibited IK(Ca) current; (3) compared with ARACCs, much smaller quantal size and faster quantal release were showed in NRACCs through the kinetic analysis of the single-vesicle secretion induced by H2 S. Our results may not only help to further understand the H2 S-induced CA release from adrenal chromaffin cells in the aspect of development, but also provide the insights for the clinical prevention and therapy for hypoxic stress-induced injury in neonates at birth. © 2013 Elsevier Ireland Ltd. All rights reserved.

Hydrogen sulfide (H2 S) is an important gaseous signaling molecule involved in the regulation of physiological functions such as respiration, reproduction, digestion, cardiovascular activity and the stress response in mammals (Guo et al., 2012; Li et al., 2012; Telezhkin et al., 2010; Zhou et al., 2013; Zhu et al., 2011). H2 S can act on many types of ionic channels in the cell membrane. H2 S induces hyperpolarization by activating ATP-sensitive K+ channels and depolarization by inhibiting Ca2+ -dependent K+ channels and background K+ channels (Buckler, 2012; Jiang et al., 2010; Li et al., 2010; Zhu et al., 2012). The enzymes synthesizing H2 S, cystathionine ␥-lyase (CSE) and cystathionine ␤-synthase (CBS), are expressed in various tissues (Olson, 2011). CBS is mainly distributed in the brain while CSE predominates in peripheral tissues (Hu et al., 2010; Perry et al., 2009). Catecholamine (CA) secreted from chromaffin cells of the adrenal medulla plays an important role in the regulation of stress response (Perez-Alvarez et al., 2010). At birth, CA is involved in the initiation of breathing and cardiovascular functions, and helps the newborn to adjust to the environment outside the uterus.

∗ Corresponding author. Tel.: +86 531 88383902; fax: +86 531 88382502. E-mail address: [email protected] (W. Yao).

During the delivery, the newborn experiences hypoxic stress. If CA release is impaired, hypoxic stress-induced brain injury may occur (Bournaud et al., 2007; Seidler and Slotkin, 1985). It has been reported that CSE is expressed in neonatal rat adrenal chromaffin cells (NRACCs) and synthesizes H2 S under hypoxic stimulation (Peng et al., 2010). Our previous study showed that exogenous H2 S induced quantal CA release from adult rat adrenal chromaffin cells (ARACCs) (Zhu et al., 2012). However, it is not clear whether H2 S can also induce CA release from NRACCs. This prompted us to further investigate: (1) whether H2 S can directly trigger quantal CA release from NRACCs and the underlying ionic mechanism; (2) if H2 S induced quantal CA release from NRACCs, whether there are differences of secretory kinetics in the H2 S-induced quantal CA release between ARACCs and NRACCs.

38 39 40 41 42 43 44 45 46 47 48 49 50 51

2. Materials and methods

52

All experimental procedures were approved by the Animal Care Committee of Shandong University School of Medicine.

53

2.1. Chromaffin cell isolation

55

Six adrenal glands were removed from 3 neonatal Wistar rats (P0–P7) and immediately immersed in ice-cold, Ca2+ - and Mg2+ -free D-Hanks solution containing (in mM): 109.5 NaCl, 5.7 KCl, 23.8 NaHCO3 , 10.1 NaH2 PO4 , 7.3 Na HEPES, 17.3 H-HEPES, 10 d-glucose, 250 ␮g/ml streptomycin SO4 , and 250 ␮g/ml penicillin G. The solution was adjusted to pH = 7.4. Then, the cortex of the adrenal gland was removed and the medulla was isolated in D-Hanks solution. The isolated medullas were digested for

56

0300-483X/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tox.2013.06.014

Please cite this article in press as: Wang, K., et al., Differences in the H2 S-induced quantal release of catecholamine in adrenal chromaffin cells of neonatal and adult rats. Toxicology (2013), http://dx.doi.org/10.1016/j.tox.2013.06.014

54

57 58 59 60 61

G Model TOX 51250 1–6

ARTICLE IN PRESS K. Wang et al. / Toxicology xxx (2013) xxx–xxx

2

Fig. 1. H2 S induces quantal CA release from NRACCs. (A) 100 ␮M NaHS evoked amperometric spikes (bottom). High-K+ (top) and standard extracellular solutions (control, middle) were applied as positive and negative controls, respectively. Asterisk indicates a typical single amperometric spike expanded in the inset. (B) Normalized amount of CA secretion evoked by high K+ and standard extracellular solution or 100 ␮M NaHS (n = 8, ***P < 0.001). (C) Representative image showing a carbon fiber electrode (black bar) and neonatal rat adrenal chromaffin cells. Scale bar, 10 ␮M.

67

20 min at 37 ◦ C in enzyme solution containing (in mg/ml): 3 collagenase type I, 3 BSA, 0.2 DNase I and 2.4 hyaluronidase I-S. After digestion, the medullas were triturated by 200 ␮l pipette tip. Finally, the dispersed NRACCs were placed on glass coverslips in a CO2 incubator at 37 ◦ C with high-glucose DMEM containing 250 ␮g/ml streptomycin SO4 , 250 ␮g/ml penicillin G and 10% heat-inactivated FBS. The NRACCs were used within 12 h.

68

2.2. Amperometric measurement of quantal CA release

69

For amperometric CA measurements, the NRACCs were transferred to a recording chamber and continuously superfused with the standard extracellular solution. We made amperometric measurements using an Axon700B amplifier and pClamp software (Axon Instruments, CA, USA). Five-micrometer glass carbon fiber electrode (CFE) was used to measure quantal CA release from the cultured NRACCs. The amperometric current (Iamp ) was recorded at a holding potential of 780 mV. Amperometric signals were low-pass filtered at 0.3 kHz, and digitized at 1 kHz before storage in a computer. The CFE surface was positioned in contact with the membrane of clean cells. The close proximity of the electrode to the cell surface was confirmed by a slight deformation in the outline of the cell. The recorded CA oxidation current fusion of (amperometric spike) reflects vesicle release close to the electrode from the exocytotic vesicles with the cell membrane. Iamp and its total charge (QCA = Iamp dt)

62 63 64 65 66

70 71 72 73 74 75 76 77 78 79 80

93

were compared among different groups. The solution was puffed using a perfusion system with a fast exchange time (PCR-2B, INBIO, Wuhan, PR China). For the kinetic analysis of single amperometric spike, including the half height duration (HHD), rise time (RT), and quantal size (Q), only the amperometric spikes with S/N > 3 (signal/noise) were used. Among all amperometric spikes, only vesicles under CFE sensitive surface give fast events, whereas vesicles distant from the surface give slow events. The fastest (RT) 25% of amperometric spikes are those induced immediately under the sensor surface (Zhou and Misler, 1995, 1996). In our kinetic analysis, we only used the fastest 25% of all the data in ARACCs and NRACCs, respectively. Data were analyzed using Igor software (WaveMetrix) with a custom-made macro program (Zhou and Misler, 1996). Data of the H2 S-induced quantal CA release in ARACCs were gained from our previous study in which the kinetic analysis of amperometric spikes was not performed (Zhu et al., 2012).

94

2.3. Whole cell recording

95

Whole-cell currents and membrane potential were recorded using an Axon700B amplifier (Axon Instruments, CA, USA) and pipettes with a resistance of 3–5 M. The

81 82 83 84 85 86 87 88 89 90 91 92

96

holding potential was −70 mV. The whole-cell currents and membrane potential were recorded under voltage clamp and current modes, respectively. The output signals from the amplifier were digitized with a DigiData 1400 interface (Axon Instruments, CA, USA) and stored on the hard disk of an IBM-PC compatible computer using pClamp software version 10.03 (Axon Instruments, CA, USA).

97 98 99 100 101

2.4. Solutions and drugs

102

In the amperometric experiments, the standard (2Ca) extracellular solution contained (in mM): 135 NaCl, 5 KCl, 2 MgCl2 , 2 CaCl2 , 10 HEPES, and 10 glucose; Ca2+ -free (0Ca) extracellular solution contained (in mM): 145 NaCl, 2.8 KCl, 1 MgCl2 , 10 HEPES, 1 EGTA; KCl solution was composed of (in mM): 78 NaCl, 70 KCl, 2 CaCl2 , 1 MgCl2 , 10 glucose and 10 HEPES. In the whole-cell patch-clamp experiments, the same standard extracellular solution was employed; the pipette solution contained (in mM): 145 KCl, 8 NaCl, 1 MgCl2 , 10 HEPES, 2 Mg-ATP and 0.4 GTP. All the solutions were adjusted to pH = 7.4 except the pipette solution (pH = 7.2). NaHS (100 ␮M), the H2 S donor, was dissolved in the standard extracellular solution. Only for the experiments performed in the Ca2+ -free extracellular solution, NaHS (100 ␮M) was dissolved in the Ca2+ -free extracellular solution. All solutions were delivered to the cells via a multiple-channel micropuffer system (RCP-2B; Inbio Inc., Wuhan, China). The tip of the puffer pipette was about 100 ␮m from the target cell. All experiments were performed at room temperature (20–23 ◦ C). All drugs and chemicals in the solutions were purchased from Sigma.

103

2.5. Data analysis

118

All the data are shown as mean ± SEM. Differences were analyzed by one-way ANOVA and Student’s t test. Differences were considered significant when P < 0.05.

104 105 106 107 108 109 110 111 112 113 114 115 116 117

119 120

3. Results

121

3.1. H2 S induced quantal CA release from NRACCs

122

To investigate the effect of H2 S on quantal CA release, we performed amperometric recordings using CFE on the surfaces of cultured NRACCs. Puff application of 100 ␮M NaHS resulted in a burst of amperometric spikes (Fig. 1A). It is well established

Please cite this article in press as: Wang, K., et al., Differences in the H2 S-induced quantal release of catecholamine in adrenal chromaffin cells of neonatal and adult rats. Toxicology (2013), http://dx.doi.org/10.1016/j.tox.2013.06.014

123 124 125 126

G Model TOX 51250 1–6

ARTICLE IN PRESS K. Wang et al. / Toxicology xxx (2013) xxx–xxx

3

3.2. H2 S induced depolarization of membrane potential and inhibited IK+ Ca in NRACCs

Fig. 2. H2 S-induced quantal CA release is voltage dependent in NRACCs. 100 ␮M H2 S induced quantal CA release at a holding potential of 780 mV, and not at 0 mV (n = 5, ***P < 0.001).

Resting membrane potential of tested chromaffin cells was −56.0 ± 1.5 mV (n = 19) in the standard extracellular solution under current-clamp recording. When the standard extracellular solution was exchanged to the 100 ␮M NaHS solution, the cell depolarization occurred. The average amplitude of this H2 S-induced membrane depolarization was 5.6 ± 0.6 mV (n = 19) (Fig. 4A). It has been showed that H2 S induced membrane depolarization by inhibiting IK(Ca) current in ARACCs. We deduced that H2 S had the same inhibitory effect in NRACCs as in ARACCs. To prove whether this deduction holds true for NRACCs, we examined the effect of H2 S on K+ currents in standard (2Ca) extracellular solution and Ca2+ free (0 Ca) extracellular solution, respectively. Fig. 4B compares the recordings of whole-cell K+ outward currents obtained in a cell under the standard and NaHS extracellular solutions. These wholecell K+ outward currents are elicited by16 depolarizing pulses of 1-s duration at 10-mV increments over the voltage range of −80 mV to +70 mV. Fig. 4D shows that H2 S inhibits whole-cell K+ outward currents over the voltage range of −10 mV to +70 mV in the standard extracellular solution. This inhibition disappears in Ca2+ -free extracellular solution (Fig. 4C and E). 3.3. Kinetic differences of the H2 S-induced quantal CA release of NRACCs and ARACCs

127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148

that these spikes reflect vesicles released close to the electrode (Mosharov, 2008). As a positive control, high-K+ (70 mM) was applied and evoked reliable amperometric responses (Fig. 1A). As a negative control, the standard extracellular solution was applied. No amperometric signals were recorded in the negative control group (Fig. 1A). To confirm that the amperometric spikes induced by NaHS were due to CA oxidation, we measured the chromaffin cell response to NaHS using CFE at different holding potentials. CA is known to be oxidized at 780 mV but not at 0 mV. We found that 100 ␮M NaHS induced no amperometric spikes when the CFE was held at 0 mV, whereas the same concentration of NaHS induced spikes at 780 mV (Fig. 2). These results demonstrated that the H2 S-induced oxidation current indeed reflected CA release from NRACCs. Both the extracellular Ca2+ influx and the intracellular Ca2+ release may increase the CA release. We investigated which pathway would be responsible for the H2 S-induced CA release from NRACCs. When we replaced the standard (2 Ca) extracellular solution with Ca2+ -free solution containing 1 mM EGTA for at least 3 min, H2 S-induced secretion was completely blocked (Fig. 3). These results clearly demonstrated that H2 S triggered quantal CA release from NRACCs mainly through extracellular Ca2+ influx.

Amperometric recording is a very effective electrochemical method to record the quantal release of hormones and neurotransmitter in various types of cells (Chow et al., 1992; Wightman et al., 1991). With higher temporal and spatial resolution, amperometry can exactly record a vesicle release, namely, quantal release. By analyzing the kinetics of quantal release, neurotransmission efficiency can be deeply understood. Therefore, we first investigated the kinetics of the H2 S-induced quantal CA release. The three most important parameters of amperometric spikes were analyzed: RT and HHD, which reflects the fusion kinetics; and Q, which reflects the vesicle content released in each spike (Fig. 5A). Compared with the ARACCs, the amperometric spike in NRACCs showed a shorter HHD (NRACCs, 3.80 ± 0.24 ms; ARACCs, 5.13 ± 0.73 ms), a shorter RT (NRACCs, 0.62 ± 0.02 ms; ARACCs, 0.68 ± 0.03 ms) and a smaller Q (NRACCs, 0.33 ± 0.05 pC; ARACCs, 0.59 ± 0.10 pC) (Fig. 5B and C). These data suggest that the H2 S induced the much faster quantal release and smaller quantal size in NRACCs. 4. Discussion H2 S has been familiar for many years as a toxic gas with a smell of rotten eggs in the world of nature. However, in recent years,

Fig. 3. H2 S-induced quantal CA release is dependent on the extracellular Ca2+ influx in NRACCs. 100 ␮M NaHS induced quantal CA release in the standard extracellular solution containing 2 mM Ca2+ . Application of Ca2+ -free extracellular solution containing 1 mM EGTA for at least 3 min completely blocked secretion induced by 100 ␮M NaHS (n = 4, ***P < 0.001).

Please cite this article in press as: Wang, K., et al., Differences in the H2 S-induced quantal release of catecholamine in adrenal chromaffin cells of neonatal and adult rats. Toxicology (2013), http://dx.doi.org/10.1016/j.tox.2013.06.014

149 150

151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170

171 172

173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189

190

191 192

G Model TOX 51250 1–6 4

ARTICLE IN PRESS K. Wang et al. / Toxicology xxx (2013) xxx–xxx

Fig. 4. H2 S induces the depolarization of membrane potential and suppression of whole-cell K+ outward currents in NRACCs. (A) 100 ␮M NaHS reversibly depolarized the resting membrane potential of NRACCs (n = 19, **P < 0.001). (B and C) Effect of H2 S on the superimposed whole-cell K+ outward currents in the standard extracellular solution (B) and in Ca2+ -free solution (C). The K+ currents were elicited by 16 depolarizing pulses of 1-s duration over the voltage range from −80 mV to +70 mV at 10 mV increments. (D and E) Normalized I–V curve in the standard extracellular solution (D, n = 14) and in Ca2+ -free solution (E, n = 8).

193 194 195 196 197 198 199 200 201 202

it has been found that H2 S can be synthesized and released in human body. This discovery prompted the investigations of H2 S for the regulation of various physiological functions in various tissues and organs. H2 S is now recognized as the third gaseous signaling molecule. H2 S not only can be produced in the brain, but also in the peripheral tissues and organs. CA can be released from the chromaffin cells in the adrenal medulla and plays a very important role in the response to various kinds of stress stimulation. It is well established that hypoxic tolerance is closely associated with CA release from chromaffin cells in the adrenal medulla (Bournaud

et al., 2007). Delivery is a process involved in the hypoxic stress. It has been proved that normal CA release is very critical for the newborn to survive the hypoxic stress. At birth, the initiation of breathing, cardiovascular functions and the maintenance of basic metabolism depends on CA release from adrenal chromaffin cells. In 2010, Peng et al. (2010) reported that hypoxia increased H2 S generation in NRACCs in a dose-dependent manner. Blackstone and Roth (2007) reported that H2 S pretreatment greatly increased the tolerance of hypoxia in the mouse. Based on these reports, we hypothesized that H2 S may contribute to the hypoxic tolerance by

Please cite this article in press as: Wang, K., et al., Differences in the H2 S-induced quantal release of catecholamine in adrenal chromaffin cells of neonatal and adult rats. Toxicology (2013), http://dx.doi.org/10.1016/j.tox.2013.06.014

203 204 205 206 207 208 209 210 211 212

G Model TOX 51250 1–6

ARTICLE IN PRESS K. Wang et al. / Toxicology xxx (2013) xxx–xxx

5

Fig. 5. H2 S induces much faster quantal CA release and smaller quantal size in NRACCs. (A) Three kinetic parameters of quantal CA release, HHD, RT, and Q are defined. PA stands for peak amplitude. (B) Representative traces displaying the difference in the shape of amperometric spikes in ARACCs versus NRACCs. (C) Quantitative analysis of HHD, RT, and Q of amperometric spikes from ARACCs (n = 15 spikes, 7 cells) and NRACCs (n = 32 spikes, 8 cells).

213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242

modulating CA release from the adrenal chromaffin cells. In our previous study, we found that exogenous H2 S directly induced quantal CA release from ARACCs. However, it is still not clear whether exogenous H2 S also directly causes NRACCs to secrete CA. In most animals including rats and humans, the adrenal chromaffin cells lack control by the splanchnic innervation at birth (Slotkin and Seidler, 1988). This has resulted in lots of investigations focusing on the modulation of CA release from neonatal chromaffin cells. At present, it is still not completely clear how the secretory activity of neonatal chromaffin cells is modulated and how they contribute to the adapting transition from uterine life to the external environment. In this study, we investigated whether H2 S induced quantal CA release from NRACCs. Amperometric experiments showed that exogenous H2 S directly induced quantal CA release from NRACCs. It is well known that CA secretion is entirely dependent on increased intracellular Ca2+ concentration by either extracellular Ca2+ influx or intracellular Ca2+ release in the adrenal chromaffin cells. Our results demonstrated that the H2 S-induced CA release was mainly dependent on the extracellular Ca2+ influx in NRACCs. In our previous study, we found in ARACCs that exogenous H2 S can make the depolarization of membrane potential by inhibiting IK(Ca) current. This depolarization can lead to the quantal CA release. In our present research, we deduced that the same inhibitory effect existed in NRACCs. Our results indicated in NRACCs that exogenous H2 S induced the depolarization of membrane potential and inhibited IK(Ca) current. This displays the similarity for the ionic mechanism in the H2 S-induced depolarization of membrane potential during the development of rat after birth. Compared with other methods of recording CA release, amperometry is an effective tool

with high temporal and spatial resolution (Huang et al., 2012). The signal of quantal CA release, namely, a single amperometric spike recorded by amperometry, can be used for the kinetic analysis of secretion. This analysis can show the efficiency and mechanism of synaptic transmission and neurotransmitter release. Combined with our previous study, we compared the kinetic difference of quantal CA release between ARACCs and NRACCs. Our analysis indicated that there was much faster quantal CA release and smaller quantal size in NRACCs than in ARACCs. In conclusion, exogenous H2 S can directly induce quantal CA released from NRACCs. Compared with ARACCs, kinetic analysis shows much faster quantal CA release and smaller quantal size in NRACCs. Our research may indicate that the H2 S-induced CA release possibly helps the newborn to avoid the hypoxic stressinduced injury. Although H2 S has been recognized as a toxic gas for a long time, our results show that H2 S owns the potential of therapy in the adrenal CA release-related diseases in the newborns.

Conflict of interest statement The authors declare that there are no conflicts of interest.

Acknowledgements This work was funded by grant to Wei Yao from the Excellent Young Scientist Foundation of Shandong Province in China (BS2012SW008).

Please cite this article in press as: Wang, K., et al., Differences in the H2 S-induced quantal release of catecholamine in adrenal chromaffin cells of neonatal and adult rats. Toxicology (2013), http://dx.doi.org/10.1016/j.tox.2013.06.014

243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260

261

262

263

264 265 266

G Model TOX 51250 1–6

267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298

ARTICLE IN PRESS K. Wang et al. / Toxicology xxx (2013) xxx–xxx

6

References Blackstone, E., Roth, M.B., 2007. Suspended animation-like state protects mice from lethal hypoxia. Shock 27, 370–372. Bournaud, R., Hidalgo, J., Yu, H., Girard, E., Shimahara, T., 2007. Catecholamine secretion from rat foetal adrenal chromaffin cells and hypoxia sensitivity. Pflugers Arch. 454, 83–92. Buckler, K.J., 2012. Effects of exogenous hydrogen sulphide on calcium signalling, background (TASK) K channel activity and mitochondrial function in chemoreceptor cells. Pflugers Arch. 463, 743–754. Chow, R.H., von Ruden, L., Neher, E., 1992. Delay in vesicle fusion revealed by electrochemical monitoring of single secretory events in adrenal chromaffin cells. Nature 356, 60–63. Guo, X., Huang, X., Wu, Y.S., Liu, D.H., Lu, H.L., Kim, Y.C., Xu, W.X., 2012. Downregulation of hydrogen sulfide biosynthesis accompanies murine interstitial cells of cajal dysfunction in partial ileal obstruction. PLoS One 7, e48249. Hu, L.F., Lu, M., Tiong, C.X., Dawe, G.S., Hu, G., Bian, J.S., 2010. Neuroprotective effects of hydrogen sulfide on Parkinson’s disease rat models. Aging Cell 9, 135–146. Huang, H.P., Zhu, F.P., Chen, X.W., Xu, Z.Q., Zhang, C.X., Zhou, Z., 2012. Physiology of quantal norepinephrine release from somatodendritic sites of neurons in locus coeruleus. Front. Mol. Neurosci. 5, 29. Jiang, B., Tang, G., Cao, K., Wu, L., Wang, R., 2010. Molecular mechanism for H(2) Sinduced activation of K(ATP) channels. Antioxid. Redox Signal. 12, 1167–1178. Li, Q., Sun, B., Wang, X., Jin, Z., Zhou, Y., Dong, L., Jiang, L.H., Rong, W., 2010. A crucial role for hydrogen sulfide in oxygen sensing via modulating large conductance calcium-activated potassium channels. Antioxid. Redox Signal. 12, 1179–1189. Li, G.F., Luo, H.K., Li, L.F., Zhang, Q.Z., Xie, L.J., Jiang, H., Li, L.P., Hao, N., Wang, W.W., Zhang, J.X., 2012. Dual effects of hydrogen sulphide on focal cerebral ischaemic injury via modulation of oxidative stress-induced apoptosis. Clin. Exp. Pharmacol. Physiol. 39, 765–771. Mosharov, E.V., 2008. Analysis of single-vesicle exocytotic events recorded by amperometry. Methods Mol. Biol. 440, 315–327.

Olson, K.R., 2011. The therapeutic potential of hydrogen sulfide: separating hype from hope. Am. J. Physiol. Regul. Integr. Comp. Physiol. 301, R297–R312. Peng, Y.J., Nanduri, J., Raghuraman, G., Souvannakitti, D., Gadalla, M.M., Kumar, G.K., Snyder, S.H., Prabhakar, N.R., 2010. H2 S mediates O2 sensing in the carotid body. Proc. Natl. Acad. Sci. U.S.A. 107, 10719–10724. Perez-Alvarez, A., Hernandez-Vivanco, A., Albillos, A., 2010. Past, present and future of human chromaffin cells: role in physiology and therapeutics. Cell. Mol. Neurobiol. 30, 1407–1415. Perry, S.F., McNeill, B., Elia, E., Nagpal, A., Vulesevic, B., 2009. Hydrogen sulfide stimulates catecholamine secretion in rainbow trout (Oncorhynchus mykiss). Am. J. Physiol. Regul. Integr. Comp. Physiol. 296, R133–R140. Seidler, F.J., Slotkin, T.A., 1985. Adrenomedullary function in the neonatal rat: responses to acute hypoxia. J. Physiol. 358, 1–16. Slotkin, T.A., Seidler, F.J., 1988. Adrenomedullary catecholamine release in the fetus and newborn: secretory mechanisms and their role in stress and survival. J. Dev. Physiol. 10, 1–16. Telezhkin, V., Brazier, S.P., Cayzac, S.H., Wilkinson, W.J., Riccardi, D., Kemp, P.J., 2010. Mechanism of inhibition by hydrogen sulfide of native and recombinant BKCa channels. Respir. Physiol. Neurobiol. 172, 169–178. Wightman, R.M., Jankowski, J.A., Kennedy, R.T., Kawagoe, K.T., Schroeder, T.J., Leszczyszyn, D.J., Near, J.A., Diliberto Jr., E.J., Viveros, O.H., 1991. Temporally resolved catecholamine spikes correspond to single vesicle release from individual chromaffin cells. Proc. Natl. Acad. Sci. U.S.A. 88, 10754–10758. Zhou, Z., Misler, S., 1995. Action potential-induced quantal secretion of catecholamines from rat adrenal chromaffin cells. J. Biol. Chem. 270, 3498–3505. Zhou, Z., Misler, S., 1996. Amperometric detection of quantal secretion from patchclamped rat pancreatic beta-cells. J. Biol. Chem. 271, 270–277. Zhou, K., Gao, Q., Zheng, S., Pan, S., Li, P., Suo, K., Simoncini, T., Wang, T., Fu, X., 2013. 17Beta-estradiol induces vasorelaxation by stimulating endothelial hydrogen sulfide release. Mol. Hum. Reprod. 19, 169–176. Zhu, X.Y., Gu, H., Ni, X., 2011. Hydrogen sulfide in the endocrine and reproductive systems. Expert Rev. Clin. Pharmacol. 4, 75–82. Zhu, D., Yu, X., Sun, J., Li, J., Ma, X., Yao, W., 2012. H2 S induces catecholamine secretion in rat adrenal chromaffin cells. Toxicology 302, 40–43.

Please cite this article in press as: Wang, K., et al., Differences in the H2 S-induced quantal release of catecholamine in adrenal chromaffin cells of neonatal and adult rats. Toxicology (2013), http://dx.doi.org/10.1016/j.tox.2013.06.014

299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332