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Mechanism of motilin-mediated inhibition on voltage-dependent potassium currents in hippocampal neurons
Neuroscience 284 (2015) 374–380
MECHANISM OF MOTILIN-MEDIATED INHIBITION ON VOLTAGE-DEPENDENT POTASSIUM CURRENTS IN HIPPOCAMPAL NEURONS Y. LU, a,b F. ZHONG, c* X. WANG, b H. LI, a* Z. ZHU, a,d X. KONG, b J. ZHAO b AND Q. WU b
INTRODUCTION Motilin, a 22-amino-acid peptide (Brown et al., 1971), is known to stimulate gastrointestinal motility (Brown et al., 1973) and mainly involved in the regulation of the migrating motor complex in the fasting state (Smet et al., 2009). Motilin mRNA and motilin receptors have been detected in the gastric body and the dodecadactylon of humans (Miller et al., 2000), as well as in the mammalian central nervous system (CNS), including the cortex, cerebellum, hippocampus, amygdala and hypothalamus (Depoortere et al., 1997a,b; Lange et al., 1986). The highest expression level of motilin receptors is found in the hippocampus (Lin and Dong, 2005). In recent years, motilin has been considered as a new treatment modality (Chapman et al., 2013), and it is reported that the motilin receptor agonist erythromycin can improve gastric emptying halftime in adult cystic fibrosis patients with gastroparesis and induce the intestinal function recovery of the anastomosis for small intestinal atresia (Tonelli et al., 2009; Lu et al., 2010a). However, the mechanism underlying the regulatory effect of motilin on gastrointestinal motility has not been clearly demonstrated so far. Additionally, the stimulatory role of motilin on the gastrointestinal motility in the hippocampus remains to be elucidated. While previous study proved that L-arginine (L-AA) can increase the excited function of gastric distension neurons in the hippocampus in rats (Lu et al., 2007), nitric oxide synthase (NOS) is newly found in the hippocampus (Lu et al., 2010b), leading to the question whether nitric oxide (NO) is involved in the regulatory effect of motilin on gastrointestinal motility. In order to determine the role of NO in motilinmediated effects on hippocampal neurons, the interaction between motilin and NO in the hippocampus using the whole-cell patch-clamp was investigated in the present study.
a Department of Neonatology, First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, Shanxi province 710061, China b Department of Physiology, Heze Medical College, Heze, Shandong Province, China c Department of Stomatology, Medical College of Qingdao University, Qingdao, Shandong Province 266003, China d Shaanxi Province Biomedicine Key Laboratory, College of Life Sciences, Northwest University, Xi’an, Shanxi province, China
Abstract—Objective: The effects of motilin on voltagedependent K+ currents in hippocampal neurons with the addition of L-arginine (L-AA), D-arginine (D-AA) and N-nitroL-arginine methyl ester (L-NAME) were investigated in this study. Methods: Mice (1–3 days old) were randomly assigned to different groups according to the addition of motilin, L-AA, + D-AA, and L-NAME. The K current signals were detected by the whole-cell patch-clamp technique. Results: Compared with the control group, the transient outward voltage-dependent K+ current was significantly inhibited by motilin added with L-AA. In contrast, the addition of motilin and L-NAME significantly increased the K+ current, while no significant change was detected by the addition of motilin accompanied with D-AA. Conclusion: The inhibiting effects of motilin on the voltagedependent K+ current in hippocampal neurons indicate that motilin acts as a regulatory factor for the nitric oxide pathway. Ó 2014 IBRO. Published by Elsevier Ltd. All rights reserved.
Key words: motilin, hippocampal neurons, whole-cell patch clamp, nitric oxide.
*Corresponding authors at: Department of Stomatology, Medical College of Qingdao University, Qingdao, Shandong Province 266003, China. Tel: +86-18053220669 (F. Zhong), Department of Neonatology, First Affiliated Hospital of Xi’an Jiaotong University, Xi’an 710061, China. Tel: +86-13991232133 (H. Li). E-mail addresses: [email protected] (Y. Lu), rayratlily@yahoo. com (F. Zhong), [email protected] (X. Wang), [email protected]. edu.cn (H. Li), [email protected] (Z. Zhu), [email protected] (X. Kong), [email protected] (J. Zhao), [email protected] (Q. Wu). Abbreviations: ACSF, Artificial Cerebrospinal Fluid; D-AA, D-arginine; EGTA, ethylene glycol tetraacetic acid; HEPES, 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid; L-AA, L-arginine; L-NAME, N-nitro-Larginine methyl ester; NO, nitric oxide; NOS, nitric oxide synthase; SD, standard deviation.
EXPERIMENTAL PROCEDURES Animals and grouping Mice of 1–3 days old (12 ± 2.3 g) were provided by Experimental Animal Center of the Heze Medical College. Motilin, L-AA, D-arginine (D-AA), and N-nitro-Larginine methyl ester (L-NAME) were all purchased from Sigma, USA. Mice were randomized into the eight groups: M (n = 6, treated with 106 M motilin only); 6 L-AA (n = 6, treated with 10 M L-AA only), D-AA (n = 6 6, treated with 10 M D-AA only), L-NAME (n = 6,
http://dx.doi.org/10.1016/j.neuroscience.2014.08.020 0306-4522/Ó 2014 IBRO. Published by Elsevier Ltd. All rights reserved. 374
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treated with 106 M L-NAME only), ML (n = 6, treated with 106 M L-AA then followed by 106 M motilin), MD (n = 6, treated with 106 M D-AA then followed by 106 M motilin), MN (n = 6, treated with 106 M 6 L-NAME then followed by 10 M motilin), and the control (n = 6, without drug treatment). All animal care and procedures were approved by the Institutional Animal Care and Use Committee of the Medical College of Qingdao University. And mice were used in accordance with the ethical guidelines of the Medical College of Qingdao University. Dissociation of hippocampal neurons cells The mice hippocampus CA1 was dissociated in the Artificial Cerebrospinal Fluid (ACSF) (NaCl 126 mM, KCl 5.0 mM, CaCl2 2 mM, MgSO4 2 mM, NaHCO3 25 mM, NaH2PO4 1.5 mM, and Glucose 10 mM, pH 7.4) at 0–4 °C for 1 min and sliced into sections with the thickness of 400–500 lm. Sections were incubated at 32 °C with 95% of O2 and 5% of CO2, then digested with 0.05 mg/ml of trypsin (Solarbio, China) for 30 min. Sections were perfused with ACSF for 2–3 times after the digestion, and further digested with 0.05 mg/ml of pronase E (Solarbio, China) for 30 min. After washing with ACSF for three times, slices were transferred to a centrifuge tube containing oxygen-saturated standard extracellular solution, and dispersed by a pipette to prepare a single-cell suspension. The upper layer of the cell suspension was transferred to a culture dish containing oxygen-saturated standard extracellular solution after 5 min, and the cells were attached to the wall after a further 20-min culture and subsequently used for whole-cell patch-clamp recording.
RESULTS Effects of arginine and motilin on the peak of outward K+ currents In order to understand the effect of motilin and NO precursor L-AA on the transient outward K+ currents, whole-cell patch clamp was performed. The membrane potential was clamped at 80 mV, and the outward currents were elicited by a step voltage command pulse from 80 mV to +30 mV for 400 ms with step of 10 mV at intervals of 30 s. In group M, motilin at the concentration of 106 M was added after recording the normal currents of the control group. Compared with the control group, motilin significantly inhibited the currents in group M (Fig. 1A, C). In group L-AA, with the addition of 106 M L-AA to the incubation dish after recording the normal currents of the control group for 10 min, a significant inhibition was also observed (Fig. 1B, C). With the further addition of 106 M motilin (group ML), the currents were sharply decreased (Fig. 1B, C). Compared with group M, the inhibition effect of group ML on the K+ currents was significantly more effective (Fig. 1C and Table 1). When adding D-AA (isolog of L-AA) at the concentration of 106 M after washing out the cells (group D-AA), outward K+ currents did not show any significant change compared to the control group (Fig. 2). In contrast, the further addition of 106 M motilin (group MD) after 5 min resulted in the significant decrease in outward K+ currents (Fig. 2), but there was no significant difference between the group M and group MD (Fig. 2 and Table 2). The above results suggest that L-AA enhances the inhibitory effect of motilin on the transient outward K+ currents, whereas no significant changes are observed by the addition of D-AA.
Whole-cell patch-clamp recording Motilin, L-AA, D-AA, and L-NAME were added directly to the incubation dish before and after the current signals were recorded. A glass capillary was pulled to form the recording electrode using a PP-830-type microelectrode puller (Narishige, Japan). The resistance at the tip was 2–4 MX when filled with the electrode solution (KCl 140 mM, EGTA 10 mM, HEPES 10 mM, MaCl2 1 mM, and Na2ATP 4 mM, pH 7.5). The current signals were saved on the computer (Lenovel, China) hard drive via a 2-kHz Digidatal 200B A/D and D/A converter. The sampling frequency was 1 kHz. During the experiment, the setting of the holding and storage was maintained with a HEKA Pulsefit 8.5 (HECK EPC-9, Germany). Statistical analysis All data were analyzed using the HEKA Pulsefit 8.5 and Origin 6.0 software, and the results were represented as mean ± standard deviation (SD). The difference caused by the drug administration was analyzed using the analysis of variance (ANOVA) and multiple comparison test. A value of P < 0.05 was considered to be statistically significant.
Effects of arginine and motilin on the current–voltage (I–V) curve of outward K+ currents To further determine the interaction between motilin and the effects of arginine and motilin on the I–V curve of outward potassium currents were observed. With the holding potential set at 80 mV, after a series of outward currents were elicited by stimulating the hippocampal neurons with depolarizing pulses from 80 mV to +30 mV in 10 mV steps, the I–V curves were generated at every level of membrane potential by plotting the peak values of the K+ currents. The outward K+ currents were gradually increased with the rise of voltage (Fig. 3A), and evident changes in the I–V curve were observed after the addition of motilin at the concentration of 106 M (Fig. 3E). Notably, although the overall shape of the I–V curves was not changed, the motilin shifted the I–V curve downward, suggesting a decrease in the outward K+ currents and an increase in the voltage as compared to the control group (Fig. 3B). When 106 M L-AA was added after 5 min, the currents were decreased significantly (Fig. 3C), and the I–V curve was also shifted downward (Fig. 3E). After motilin was added (group ML), the currents showed an obvious decrease compared with the control group and group M L-AA,
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Fig. 1. Effects of L-AA and motilin on the peak of outward K+ currents. (A) Outward K+ currents recorded by the control and addition with motilin (group M). (B) Patch-clamp recording for K+ currents obtained from the control, presence of L-AA, and subsequently the addition of motilin (group ML). (C) Comparison of outward K+ currents. Significant difference between the motilin-added groups and the control: ⁄p < 0.05, ⁄⁄p < 0.01. Compared with group M, 4p < 0.05. Table 1. The inhibition ratio of L-AA and motilin on peak of outward K+ currents Group Inhibition ratio (%)
Group M 31.38 ± 10.89
Group ML *
55.36 ± 5.99
6
Values were expressed as mean ± SD. Group M, only added with 10 M motilin; Group ML, added with 106 M L-AA followed by 106 M motilin. Significant difference between the group M and ML. * p < 0.05.
(Fig. 3D), and the I–V curve was further shifted downward (Fig. 3E). These results suggest that the inhibition effect of motilin on outward K+ currents can be significantly increased after application of L-AA. Compared to the control, the outward K+ currents showed no significant change with the addition of 106 M D-AA (Fig. 4A, C), and there was no obvious shift detected in the I–V curve (Fig. 4E). However, when motilin (106 M) was added, the outward K+ currents were significantly decreased compared with the control group (Fig. 4A, B), and D-AA addition to motilin (group MD) causes no notable shift in the I–V curves compared to the motilin-only group (group M) (Fig. 4E). These results suggest that D-AA has no significant effect on motilin.
Table 2. The inhibition ratio of D-AA and motilin on the peak of outward K+ currents Group
Group M
Group MD
Inhibition ratio (%)
31.38 ± 10.89
32.82 ± 8.93
Values were expressed as mean ± SD. Group M, only added with 106 M motilin; Group MD, added with 106 M D-AA followed by 106 M motilin.
Effects of L-NAME and motilin on the peak of outward K+ currents In order to explore the interaction between motilin and NO in modulating the transient outward K+ current, the effect of NOS inhibitor L-NAME on the ability of motilin to regulate K+ channels of hippocampal neurons was further investigated. The outward currents were inhibited by motilin (Fig. 5A), and the current peak showed no obvious change when 106 M L-NAME (group L-NAME) was added (Fig. 5A). With the further addition of motilin (group MN) (Fig. 5B), the inhibitory ratio of group MN was lower than that in the group M (Fig. 5B and Table 3). These results suggest that effect of motilin on K+ current is weakened by L-NAME.
Fig. 2. Effects of L-AA and motilin on the peak of outward K+ currents. (A) Outward K+ currents recorded by the control, group M (only added with 106 M motilin), D-AA (only added with 106 M D-AA), and MD (added with 106 M D-AA then followed by 106 M motilin). (B) Comparison of outward K+ currents. Significant difference between the motilin-added groups and the control: ⁄p < 0.05.
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Fig. 3. Effects of L-AA and motilin on outward K+ currents. Outward K+ currents recording obtained from the control group (A), group M (B), group + L-AA (C), and group ML (D). The I–V curve for outward K currents observed for all groups (E).
Fig. 4. Effects of D-AA and motilin on outward K+ currents. Outward K+ currents recording obtained from the control (A), group M only added with motilin (B), group D-AA added with D-AA (C), and group MD followed by the subsequent addition of motilin (D). The I–V curve for outward K+ currents observed for all groups (E).
Fig. 5. Effects of L-NAME and motilin on peak of outward K+ currents. (A) Outward K+ currents recorded by the control, and addition with motilin (group M), L-NAME (group L-NAME), and subsequent addition of motilin (group MLN). (B) Comparison of outward K+ currents. Significant difference between the motilin-added only group (group M) and the control: ⁄p < 0.05, and between the group M and MLN: 4p < 0.05.
Effects of L-NAME and motilin on the I–V curve of outward K+ currents To determine the interaction between motilin and the effects of L-NAME and motilin on the I–V curve of transient outward K+ currents were observed. The membrane potential was clamped at 80 mV, and
L-NAME,
the outward currents were elicited by a step voltage command pulse from 80 mV to +30 mV. Changes in the I–V curve corresponded closely with observations of the effects of L-NAME and motilin on the peak of outward K+ currents (Fig. 6). Compared with the control group, no obvious change was observed in the I–V curve of the group L-NAME. However, in group M and
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Table 3. The inhibition ratio of L-NAME and motilin on peak of outward K+ currents Group
Group M
Group MN
Inhibition ratio (%)
31.38 ± 10.89
15.69 ± 0.59*
Values were expressed as mean ± SD. Group M, only added with 106 M motilin; Group MN, added with 106 M L-NAME followed by 106 M motilin. Significant difference between the group M and MN. * p < 0.05.
group MN, the I–V curve shifted downward compared to the control group. Interestingly, the I–V curve of group MN shifted upward compared to group M. These results further confirm that the inhibition of motilin on the outward K+ current can be reduced by L-NAME. Effects of L-AA and L-NAME on the I–V curve of outward K+ currents To determine the effect of exogenous NO on K+ currents, the I–V curve of K+ currents after the addition of L-AA and L-NAME was observed. The membrane potential was clamped at 80 mV, and the outward currents were elicited by a step voltage command pulse from 80 mV to +30 mV. Compared with the control group (650.21 ± 131.62 pA), the outward peak K+ currents after treatment with L-AA alone were decreased (500.63 ± 101.74 pA) (Fig. 7A, B). The I–V curve shifted downward (Fig. 7D). However, after treatment with + L-NAME and L-AA, the outward peak K currents were increased (610.58 ± 123.51 pA) compared with those treated with L-AA only (500.63 ± 101.74 pA) (Fig. 7C, D). Additionally, the I–V curve shifted upward. Thus, these data indicate that the inhibitory effect of L-AA on the outward K+ current is reversed by L-NAME.
DISCUSSION The role of motilin in the regulation of energy activities and gastrointestinal motility has aroused extensive attention. Motilin can be detected periodically during the
interdigestive period, and can initiate the interdigestive migrating motor complex (Tack, 1992). The peripheral motilin mainly acts as the stimulatory factor for gastrointestinal motility and biliary contraction, while the intra-arterial injection of motilin can induce an antral contraction phase in a physiological dose-dependent manner (Boivin et al., 1990). It is previously reported that the motilin mRNA is expressed in the CNS of monkeys, dogs, cats, rabbits, rats and other animals (Xu et al., 2003). And, the motilin receptors are not only distributed in the human gastric antrum, gastric body, gastric fundus and duodenum, but also widely distributed in the CNS such as the hippocampus, hypothalamus, amygdala, thalamus, brain and cerebellum cortex (Lange et al., 1986). The highest expression of motilin receptor is found in the hippocampus (Guan et al., 2003). Therefore, understanding the role of motilin in the hippocampus is of great importance in illuminating the mechanism underlying the regulation of motilin on gastrointestinal motility. Voltage-dependent potassium channels in normal membrane potential generate outward currents, and play an important role in the maintenance of the basic electric rhythm and action potential (Campbell et al., 1993). The IA current is widely distributed in the dendrites of hippocampal neurons and participates in the regulation of synaptic input and action potential back propagation (Migliore et al., 1999). In a previous study, it is found that the motilin receptor agonist erythromycin can significantly inhibit the mouse hippocampal neurons (Lu et al., 2009). The study here further suggests that motilin can regulate the activity of neurons in the hippocampus. NO is a non-adrenergic and non-cholinergic neurotransmitter (Michelakis et al., 1997), and is easily diffusible across the cell membrane in the central and peripheral nervous systems. A large number of NOS-like neurons have been found in the hippocampus (O’Donohue et al., 1981). The present study showed that the suppression of the transient outward K+ current of the hippocampal neurons by motilin was attenuated by the addition of L-NAME. In contrast, the addition of the NO precursor L-AA enhanced the
Fig. 6. Effects of L-NAME and motilin on the outward K+ current. Outward K+ currents recorded by the motilin-added group M (A and B), L-NAMEadded group L-NAME (C), and subsequent addition of motilin in group MLN (D). The I–V curve for outward K+ currents observed for all groups (E).
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Fig. 7. Effects of L-AA and L-NAME on the outward K+ current. Outward K+ currents in the control group (A), after the addition of L-AA (B), and subsequent addition of L-NAME and L-AA (C). The I–V curve for outward K+ currents observed for all groups (D).
motilin-mediated inhibition of transient outward K+ currents. However, no similar effect was observed with its isomer, D-AA. Taken together, the results here indicate that motilin inhibits the transient outward K+ current of hippocampal neurons, and is closely related to the NO metabolism rather than arginine. Interestingly, although the NOS inhibitor L-NAME reduced the inhibitory effect of motilin on transient outward K+ currents, treatment with L-NAME alone did not induce an increase in transient outward K+ currents. We suppose that this may be because that in addition to NO, K+ currents in hippocampal neurons may also be regulated by other mechanisms. However, the relationship between NO and K+ currents and the specific mechanism underlying the regulation of K+ currents in hippocampal neurons still needs further investigation. Here, it is demonstrated that NO plays an important role in the regulation of gastrointestinal motility process mediated by motilin, and is involved in the biological effects of motilin. The L-AA enhances the inhibition of the K+ channel current in the hippocampal neuronal by motilin. As expected, the D-AA shows no impact on the biological function of motilin due to the lack of biological activity in itself. Since L-NAME is a NOS inhibitor and can lower the endogenous NO level, the presence of L-NAME results in the NO suppression as well as the decrease of the biological effect of motilin. Therefore, it can be deduced that the inhibiting role of motilin in voltage-dependent K+ currents in hippocampal neurons is associated with the intracellular NO levels. However, the underlying mechanism for the regulation of NO on the motilin-induced inhibiting effect in hippocampal neurons still needs further investigation. Acknowledgments—This work was supported by the Colleges and Universities’ Science and Technology Planning Project of Shandong (No. J11LC02). Special thanks are owed to Dr. Ning Li (Department of Physiology, Weifang Medical College, Weifang, China) for guidance in the patch-clamp technology.
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Lu Y, Kong X, Wang X (2010b) Mechanism of motilin’ effect on electric motility of hippocampal neurons in rats. J Epileptol Electroneurophysiol 1:4–8. Michelakis ED, Reeve HL, Huang JM, et al (1997) Potassium channel diversity in vascular smooth muscle cells. Can J Physiol Pharmacol 75:889–897. Migliore M, Hoffman DA, Magee JC, et al (1999) Role of an A2 type K+ conductance in the back-propagation of action potentials in the dendrites of hippocampal pyramidal neurons. J Comput Neurosci 7:5–15. Miller P, Roy A, St-Pierre S (2000) Motilin receptors in the human antrum. Am J Physiol Gastrointestinal Liver Physiol 278:GI8–GI23.
O’Donohue TL, Beinfeld MC, Chey WY, et al (1981) Identification, characterization and distribution of motilin immunoreactivity in the rat central nervous system. Peptides 2:467–477. Smet BD, Mitselos A, Depoortere I (2009) Motilin and ghrelin as prokinetic drug targets. Pharmacol Ther 123:207–223. Tack J (1992) Effect of Erythromycin on gastric motility in controls and in diabetic gastroparesis. Gastroenterology 103:72–79. Tonelli AR, Drane WE, Collins DP (2009) Erythromycin improves gastric emptying half-time in adult cystic fibrosis patients with gastroparesis. J Cyst Fibros 8:193–197. Xu L, Depoortere I, Thielemans L, et al (2003) Sequence, distribution and quantification of the motilin precursor in the cat. Peptides 24:1387–1395.
(Accepted 11 August 2014) (Available online 27 August 2014)
MECHANISM OF MOTILIN-MEDIATED INHIBITION ON VOLTAGE-DEPENDENT POTASSIUM CURRENTS IN HIPPOCAMPAL NEURONS Y. LU, a,b F. ZHONG, c* X. WANG, b H. LI, a* Z. ZHU, a,d X. KONG, b J. ZHAO b AND Q. WU b
INTRODUCTION Motilin, a 22-amino-acid peptide (Brown et al., 1971), is known to stimulate gastrointestinal motility (Brown et al., 1973) and mainly involved in the regulation of the migrating motor complex in the fasting state (Smet et al., 2009). Motilin mRNA and motilin receptors have been detected in the gastric body and the dodecadactylon of humans (Miller et al., 2000), as well as in the mammalian central nervous system (CNS), including the cortex, cerebellum, hippocampus, amygdala and hypothalamus (Depoortere et al., 1997a,b; Lange et al., 1986). The highest expression level of motilin receptors is found in the hippocampus (Lin and Dong, 2005). In recent years, motilin has been considered as a new treatment modality (Chapman et al., 2013), and it is reported that the motilin receptor agonist erythromycin can improve gastric emptying halftime in adult cystic fibrosis patients with gastroparesis and induce the intestinal function recovery of the anastomosis for small intestinal atresia (Tonelli et al., 2009; Lu et al., 2010a). However, the mechanism underlying the regulatory effect of motilin on gastrointestinal motility has not been clearly demonstrated so far. Additionally, the stimulatory role of motilin on the gastrointestinal motility in the hippocampus remains to be elucidated. While previous study proved that L-arginine (L-AA) can increase the excited function of gastric distension neurons in the hippocampus in rats (Lu et al., 2007), nitric oxide synthase (NOS) is newly found in the hippocampus (Lu et al., 2010b), leading to the question whether nitric oxide (NO) is involved in the regulatory effect of motilin on gastrointestinal motility. In order to determine the role of NO in motilinmediated effects on hippocampal neurons, the interaction between motilin and NO in the hippocampus using the whole-cell patch-clamp was investigated in the present study.
a Department of Neonatology, First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, Shanxi province 710061, China b Department of Physiology, Heze Medical College, Heze, Shandong Province, China c Department of Stomatology, Medical College of Qingdao University, Qingdao, Shandong Province 266003, China d Shaanxi Province Biomedicine Key Laboratory, College of Life Sciences, Northwest University, Xi’an, Shanxi province, China
Abstract—Objective: The effects of motilin on voltagedependent K+ currents in hippocampal neurons with the addition of L-arginine (L-AA), D-arginine (D-AA) and N-nitroL-arginine methyl ester (L-NAME) were investigated in this study. Methods: Mice (1–3 days old) were randomly assigned to different groups according to the addition of motilin, L-AA, + D-AA, and L-NAME. The K current signals were detected by the whole-cell patch-clamp technique. Results: Compared with the control group, the transient outward voltage-dependent K+ current was significantly inhibited by motilin added with L-AA. In contrast, the addition of motilin and L-NAME significantly increased the K+ current, while no significant change was detected by the addition of motilin accompanied with D-AA. Conclusion: The inhibiting effects of motilin on the voltagedependent K+ current in hippocampal neurons indicate that motilin acts as a regulatory factor for the nitric oxide pathway. Ó 2014 IBRO. Published by Elsevier Ltd. All rights reserved.
Key words: motilin, hippocampal neurons, whole-cell patch clamp, nitric oxide.
*Corresponding authors at: Department of Stomatology, Medical College of Qingdao University, Qingdao, Shandong Province 266003, China. Tel: +86-18053220669 (F. Zhong), Department of Neonatology, First Affiliated Hospital of Xi’an Jiaotong University, Xi’an 710061, China. Tel: +86-13991232133 (H. Li). E-mail addresses: [email protected] (Y. Lu), rayratlily@yahoo. com (F. Zhong), [email protected] (X. Wang), [email protected]. edu.cn (H. Li), [email protected] (Z. Zhu), [email protected] (X. Kong), [email protected] (J. Zhao), [email protected] (Q. Wu). Abbreviations: ACSF, Artificial Cerebrospinal Fluid; D-AA, D-arginine; EGTA, ethylene glycol tetraacetic acid; HEPES, 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid; L-AA, L-arginine; L-NAME, N-nitro-Larginine methyl ester; NO, nitric oxide; NOS, nitric oxide synthase; SD, standard deviation.
EXPERIMENTAL PROCEDURES Animals and grouping Mice of 1–3 days old (12 ± 2.3 g) were provided by Experimental Animal Center of the Heze Medical College. Motilin, L-AA, D-arginine (D-AA), and N-nitro-Larginine methyl ester (L-NAME) were all purchased from Sigma, USA. Mice were randomized into the eight groups: M (n = 6, treated with 106 M motilin only); 6 L-AA (n = 6, treated with 10 M L-AA only), D-AA (n = 6 6, treated with 10 M D-AA only), L-NAME (n = 6,
http://dx.doi.org/10.1016/j.neuroscience.2014.08.020 0306-4522/Ó 2014 IBRO. Published by Elsevier Ltd. All rights reserved. 374
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treated with 106 M L-NAME only), ML (n = 6, treated with 106 M L-AA then followed by 106 M motilin), MD (n = 6, treated with 106 M D-AA then followed by 106 M motilin), MN (n = 6, treated with 106 M 6 L-NAME then followed by 10 M motilin), and the control (n = 6, without drug treatment). All animal care and procedures were approved by the Institutional Animal Care and Use Committee of the Medical College of Qingdao University. And mice were used in accordance with the ethical guidelines of the Medical College of Qingdao University. Dissociation of hippocampal neurons cells The mice hippocampus CA1 was dissociated in the Artificial Cerebrospinal Fluid (ACSF) (NaCl 126 mM, KCl 5.0 mM, CaCl2 2 mM, MgSO4 2 mM, NaHCO3 25 mM, NaH2PO4 1.5 mM, and Glucose 10 mM, pH 7.4) at 0–4 °C for 1 min and sliced into sections with the thickness of 400–500 lm. Sections were incubated at 32 °C with 95% of O2 and 5% of CO2, then digested with 0.05 mg/ml of trypsin (Solarbio, China) for 30 min. Sections were perfused with ACSF for 2–3 times after the digestion, and further digested with 0.05 mg/ml of pronase E (Solarbio, China) for 30 min. After washing with ACSF for three times, slices were transferred to a centrifuge tube containing oxygen-saturated standard extracellular solution, and dispersed by a pipette to prepare a single-cell suspension. The upper layer of the cell suspension was transferred to a culture dish containing oxygen-saturated standard extracellular solution after 5 min, and the cells were attached to the wall after a further 20-min culture and subsequently used for whole-cell patch-clamp recording.
RESULTS Effects of arginine and motilin on the peak of outward K+ currents In order to understand the effect of motilin and NO precursor L-AA on the transient outward K+ currents, whole-cell patch clamp was performed. The membrane potential was clamped at 80 mV, and the outward currents were elicited by a step voltage command pulse from 80 mV to +30 mV for 400 ms with step of 10 mV at intervals of 30 s. In group M, motilin at the concentration of 106 M was added after recording the normal currents of the control group. Compared with the control group, motilin significantly inhibited the currents in group M (Fig. 1A, C). In group L-AA, with the addition of 106 M L-AA to the incubation dish after recording the normal currents of the control group for 10 min, a significant inhibition was also observed (Fig. 1B, C). With the further addition of 106 M motilin (group ML), the currents were sharply decreased (Fig. 1B, C). Compared with group M, the inhibition effect of group ML on the K+ currents was significantly more effective (Fig. 1C and Table 1). When adding D-AA (isolog of L-AA) at the concentration of 106 M after washing out the cells (group D-AA), outward K+ currents did not show any significant change compared to the control group (Fig. 2). In contrast, the further addition of 106 M motilin (group MD) after 5 min resulted in the significant decrease in outward K+ currents (Fig. 2), but there was no significant difference between the group M and group MD (Fig. 2 and Table 2). The above results suggest that L-AA enhances the inhibitory effect of motilin on the transient outward K+ currents, whereas no significant changes are observed by the addition of D-AA.
Whole-cell patch-clamp recording Motilin, L-AA, D-AA, and L-NAME were added directly to the incubation dish before and after the current signals were recorded. A glass capillary was pulled to form the recording electrode using a PP-830-type microelectrode puller (Narishige, Japan). The resistance at the tip was 2–4 MX when filled with the electrode solution (KCl 140 mM, EGTA 10 mM, HEPES 10 mM, MaCl2 1 mM, and Na2ATP 4 mM, pH 7.5). The current signals were saved on the computer (Lenovel, China) hard drive via a 2-kHz Digidatal 200B A/D and D/A converter. The sampling frequency was 1 kHz. During the experiment, the setting of the holding and storage was maintained with a HEKA Pulsefit 8.5 (HECK EPC-9, Germany). Statistical analysis All data were analyzed using the HEKA Pulsefit 8.5 and Origin 6.0 software, and the results were represented as mean ± standard deviation (SD). The difference caused by the drug administration was analyzed using the analysis of variance (ANOVA) and multiple comparison test. A value of P < 0.05 was considered to be statistically significant.
Effects of arginine and motilin on the current–voltage (I–V) curve of outward K+ currents To further determine the interaction between motilin and the effects of arginine and motilin on the I–V curve of outward potassium currents were observed. With the holding potential set at 80 mV, after a series of outward currents were elicited by stimulating the hippocampal neurons with depolarizing pulses from 80 mV to +30 mV in 10 mV steps, the I–V curves were generated at every level of membrane potential by plotting the peak values of the K+ currents. The outward K+ currents were gradually increased with the rise of voltage (Fig. 3A), and evident changes in the I–V curve were observed after the addition of motilin at the concentration of 106 M (Fig. 3E). Notably, although the overall shape of the I–V curves was not changed, the motilin shifted the I–V curve downward, suggesting a decrease in the outward K+ currents and an increase in the voltage as compared to the control group (Fig. 3B). When 106 M L-AA was added after 5 min, the currents were decreased significantly (Fig. 3C), and the I–V curve was also shifted downward (Fig. 3E). After motilin was added (group ML), the currents showed an obvious decrease compared with the control group and group M L-AA,
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Fig. 1. Effects of L-AA and motilin on the peak of outward K+ currents. (A) Outward K+ currents recorded by the control and addition with motilin (group M). (B) Patch-clamp recording for K+ currents obtained from the control, presence of L-AA, and subsequently the addition of motilin (group ML). (C) Comparison of outward K+ currents. Significant difference between the motilin-added groups and the control: ⁄p < 0.05, ⁄⁄p < 0.01. Compared with group M, 4p < 0.05. Table 1. The inhibition ratio of L-AA and motilin on peak of outward K+ currents Group Inhibition ratio (%)
Group M 31.38 ± 10.89
Group ML *
55.36 ± 5.99
6
Values were expressed as mean ± SD. Group M, only added with 10 M motilin; Group ML, added with 106 M L-AA followed by 106 M motilin. Significant difference between the group M and ML. * p < 0.05.
(Fig. 3D), and the I–V curve was further shifted downward (Fig. 3E). These results suggest that the inhibition effect of motilin on outward K+ currents can be significantly increased after application of L-AA. Compared to the control, the outward K+ currents showed no significant change with the addition of 106 M D-AA (Fig. 4A, C), and there was no obvious shift detected in the I–V curve (Fig. 4E). However, when motilin (106 M) was added, the outward K+ currents were significantly decreased compared with the control group (Fig. 4A, B), and D-AA addition to motilin (group MD) causes no notable shift in the I–V curves compared to the motilin-only group (group M) (Fig. 4E). These results suggest that D-AA has no significant effect on motilin.
Table 2. The inhibition ratio of D-AA and motilin on the peak of outward K+ currents Group
Group M
Group MD
Inhibition ratio (%)
31.38 ± 10.89
32.82 ± 8.93
Values were expressed as mean ± SD. Group M, only added with 106 M motilin; Group MD, added with 106 M D-AA followed by 106 M motilin.
Effects of L-NAME and motilin on the peak of outward K+ currents In order to explore the interaction between motilin and NO in modulating the transient outward K+ current, the effect of NOS inhibitor L-NAME on the ability of motilin to regulate K+ channels of hippocampal neurons was further investigated. The outward currents were inhibited by motilin (Fig. 5A), and the current peak showed no obvious change when 106 M L-NAME (group L-NAME) was added (Fig. 5A). With the further addition of motilin (group MN) (Fig. 5B), the inhibitory ratio of group MN was lower than that in the group M (Fig. 5B and Table 3). These results suggest that effect of motilin on K+ current is weakened by L-NAME.
Fig. 2. Effects of L-AA and motilin on the peak of outward K+ currents. (A) Outward K+ currents recorded by the control, group M (only added with 106 M motilin), D-AA (only added with 106 M D-AA), and MD (added with 106 M D-AA then followed by 106 M motilin). (B) Comparison of outward K+ currents. Significant difference between the motilin-added groups and the control: ⁄p < 0.05.
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Fig. 3. Effects of L-AA and motilin on outward K+ currents. Outward K+ currents recording obtained from the control group (A), group M (B), group + L-AA (C), and group ML (D). The I–V curve for outward K currents observed for all groups (E).
Fig. 4. Effects of D-AA and motilin on outward K+ currents. Outward K+ currents recording obtained from the control (A), group M only added with motilin (B), group D-AA added with D-AA (C), and group MD followed by the subsequent addition of motilin (D). The I–V curve for outward K+ currents observed for all groups (E).
Fig. 5. Effects of L-NAME and motilin on peak of outward K+ currents. (A) Outward K+ currents recorded by the control, and addition with motilin (group M), L-NAME (group L-NAME), and subsequent addition of motilin (group MLN). (B) Comparison of outward K+ currents. Significant difference between the motilin-added only group (group M) and the control: ⁄p < 0.05, and between the group M and MLN: 4p < 0.05.
Effects of L-NAME and motilin on the I–V curve of outward K+ currents To determine the interaction between motilin and the effects of L-NAME and motilin on the I–V curve of transient outward K+ currents were observed. The membrane potential was clamped at 80 mV, and
L-NAME,
the outward currents were elicited by a step voltage command pulse from 80 mV to +30 mV. Changes in the I–V curve corresponded closely with observations of the effects of L-NAME and motilin on the peak of outward K+ currents (Fig. 6). Compared with the control group, no obvious change was observed in the I–V curve of the group L-NAME. However, in group M and
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Table 3. The inhibition ratio of L-NAME and motilin on peak of outward K+ currents Group
Group M
Group MN
Inhibition ratio (%)
31.38 ± 10.89
15.69 ± 0.59*
Values were expressed as mean ± SD. Group M, only added with 106 M motilin; Group MN, added with 106 M L-NAME followed by 106 M motilin. Significant difference between the group M and MN. * p < 0.05.
group MN, the I–V curve shifted downward compared to the control group. Interestingly, the I–V curve of group MN shifted upward compared to group M. These results further confirm that the inhibition of motilin on the outward K+ current can be reduced by L-NAME. Effects of L-AA and L-NAME on the I–V curve of outward K+ currents To determine the effect of exogenous NO on K+ currents, the I–V curve of K+ currents after the addition of L-AA and L-NAME was observed. The membrane potential was clamped at 80 mV, and the outward currents were elicited by a step voltage command pulse from 80 mV to +30 mV. Compared with the control group (650.21 ± 131.62 pA), the outward peak K+ currents after treatment with L-AA alone were decreased (500.63 ± 101.74 pA) (Fig. 7A, B). The I–V curve shifted downward (Fig. 7D). However, after treatment with + L-NAME and L-AA, the outward peak K currents were increased (610.58 ± 123.51 pA) compared with those treated with L-AA only (500.63 ± 101.74 pA) (Fig. 7C, D). Additionally, the I–V curve shifted upward. Thus, these data indicate that the inhibitory effect of L-AA on the outward K+ current is reversed by L-NAME.
DISCUSSION The role of motilin in the regulation of energy activities and gastrointestinal motility has aroused extensive attention. Motilin can be detected periodically during the
interdigestive period, and can initiate the interdigestive migrating motor complex (Tack, 1992). The peripheral motilin mainly acts as the stimulatory factor for gastrointestinal motility and biliary contraction, while the intra-arterial injection of motilin can induce an antral contraction phase in a physiological dose-dependent manner (Boivin et al., 1990). It is previously reported that the motilin mRNA is expressed in the CNS of monkeys, dogs, cats, rabbits, rats and other animals (Xu et al., 2003). And, the motilin receptors are not only distributed in the human gastric antrum, gastric body, gastric fundus and duodenum, but also widely distributed in the CNS such as the hippocampus, hypothalamus, amygdala, thalamus, brain and cerebellum cortex (Lange et al., 1986). The highest expression of motilin receptor is found in the hippocampus (Guan et al., 2003). Therefore, understanding the role of motilin in the hippocampus is of great importance in illuminating the mechanism underlying the regulation of motilin on gastrointestinal motility. Voltage-dependent potassium channels in normal membrane potential generate outward currents, and play an important role in the maintenance of the basic electric rhythm and action potential (Campbell et al., 1993). The IA current is widely distributed in the dendrites of hippocampal neurons and participates in the regulation of synaptic input and action potential back propagation (Migliore et al., 1999). In a previous study, it is found that the motilin receptor agonist erythromycin can significantly inhibit the mouse hippocampal neurons (Lu et al., 2009). The study here further suggests that motilin can regulate the activity of neurons in the hippocampus. NO is a non-adrenergic and non-cholinergic neurotransmitter (Michelakis et al., 1997), and is easily diffusible across the cell membrane in the central and peripheral nervous systems. A large number of NOS-like neurons have been found in the hippocampus (O’Donohue et al., 1981). The present study showed that the suppression of the transient outward K+ current of the hippocampal neurons by motilin was attenuated by the addition of L-NAME. In contrast, the addition of the NO precursor L-AA enhanced the
Fig. 6. Effects of L-NAME and motilin on the outward K+ current. Outward K+ currents recorded by the motilin-added group M (A and B), L-NAMEadded group L-NAME (C), and subsequent addition of motilin in group MLN (D). The I–V curve for outward K+ currents observed for all groups (E).
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Fig. 7. Effects of L-AA and L-NAME on the outward K+ current. Outward K+ currents in the control group (A), after the addition of L-AA (B), and subsequent addition of L-NAME and L-AA (C). The I–V curve for outward K+ currents observed for all groups (D).
motilin-mediated inhibition of transient outward K+ currents. However, no similar effect was observed with its isomer, D-AA. Taken together, the results here indicate that motilin inhibits the transient outward K+ current of hippocampal neurons, and is closely related to the NO metabolism rather than arginine. Interestingly, although the NOS inhibitor L-NAME reduced the inhibitory effect of motilin on transient outward K+ currents, treatment with L-NAME alone did not induce an increase in transient outward K+ currents. We suppose that this may be because that in addition to NO, K+ currents in hippocampal neurons may also be regulated by other mechanisms. However, the relationship between NO and K+ currents and the specific mechanism underlying the regulation of K+ currents in hippocampal neurons still needs further investigation. Here, it is demonstrated that NO plays an important role in the regulation of gastrointestinal motility process mediated by motilin, and is involved in the biological effects of motilin. The L-AA enhances the inhibition of the K+ channel current in the hippocampal neuronal by motilin. As expected, the D-AA shows no impact on the biological function of motilin due to the lack of biological activity in itself. Since L-NAME is a NOS inhibitor and can lower the endogenous NO level, the presence of L-NAME results in the NO suppression as well as the decrease of the biological effect of motilin. Therefore, it can be deduced that the inhibiting role of motilin in voltage-dependent K+ currents in hippocampal neurons is associated with the intracellular NO levels. However, the underlying mechanism for the regulation of NO on the motilin-induced inhibiting effect in hippocampal neurons still needs further investigation. Acknowledgments—This work was supported by the Colleges and Universities’ Science and Technology Planning Project of Shandong (No. J11LC02). Special thanks are owed to Dr. Ning Li (Department of Physiology, Weifang Medical College, Weifang, China) for guidance in the patch-clamp technology.
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(Accepted 11 August 2014) (Available online 27 August 2014)