MODULATION OF GLYCINE-ACTIVATED CHLORIDE
CURRENTS BY SUBSTANCE P IN RAT SACRAL
DORSAL COMMISSURAL NEURONS^*

WANG　DIAN-SHI^**，XU　TIAN-LE^***,LI　JI-SHUO
(Department of Anatomy and K.K. Leung Brain Research Center,
Fourth Military Medical University, Xi′an 710032;
^***Department of Neurobiology and Biophysics, School of Life Sciences,
University of Science and Technology of China, Hefei 230027)

ABSTRACT　　The modulatory effect of substance P (SP) on strychnine-sensitive glycine (Gly) response was examined in neurons acutely dissociated from the rat sacral dorsal commissural nucleus (SDCN) using nystatin perforated patch recording configuration under voltage-clamp conditions. Application of SP potentiated 30 μmol/L Gly-activated chloride current (I_Gly) in a concentration-dependent manner over the range of 1 nmol/L to 1 μmol/L at a holding potential of －40 mV. SP neither changed the reversal potential of Gly response nor affected the affinity of Gly to its receptor. The SP potentiation effect could be blocked by spantide as well as a selective NK₁ receptor antagonist, L-668,169, but not by NK₂ receptor antagonist, L-659,877. The facilitatory action of SP on I_Gly could also be abolished by pretreatment with chelerythrine or KN-62 in different neurons, a finding suggesting that protein kinase C (PKC) or Ca²⁺/calmodulin-dependent protein kinase Ⅱ (CaMKⅡ) possibly contributes to an intracellular pathway of SP in the augmentation of I_Gly. The results imply that SP may suppress nociception in the spinal cord by potentiating Gly response.
Key words: substance P; glycine; sacral dorsal commissural nucleus; protein kinase C; Ca²⁺/calmodulin-dependent protein kinase Ⅱ

P物质对大鼠骶髓后连合核神经元
甘氨酸激活的氯电流的调控^*

王殿仕^*　*　徐天乐^*　*　*　李继硕
（第四军医大学基础部解剖学教研室, 梁?锯脑研究中心， 西安 710032;
^***中国科学技术大学生命科学学院神经生物学与生物物理学系， 合肥 230027)

摘　　要

采用制霉菌素穿孔膜片箝技术, 研究了P物质(substance P, SP)对急性分离的大鼠骶髓后连合核神经元士的宁敏感性甘氨酸(glycine, Gly)反应的调控作用。 在箝制电压为－40 mV时，SP在1 nmol/L～1 μmol/L之间呈浓度依赖性地增强30 μmol/L甘氨酸激活的氯电流。SP既不改变I_Gly的翻转电位, 也不影响Gly与其受体的亲和力。Spantide和选择性NK₁受体拮抗剂, L-668,169, 可阻断SP的增强作用, 而选择性NK₂受体拮抗剂, L-659,877则不能阻断SP对I_Gly的增强作用, 提示SP对I_Gly的增强作用是由NK₁受体介导的。 在不同的神经元上, chelerythrine或KN-62可去除SP对I_Gly的增强作用, 提示蛋白激酶C或钙离子/钙调素依赖性的蛋白激酶Ⅱ可能参与了SP对I_Gly增强作用的胞内机制。 以上结果提示, SP可能通过增强Gly的反应而抑制脊髓伤害性信息的传导。
关键词: P物质； 甘氨酸； 骶髓后连合核； 蛋白激酶C； 钙离子/钙调素依赖性的蛋白激酶Ⅱ
学科分类号: Q424

　　Substance P (SP) is a member of the tachykinin peptide family as a neurotransmitter or modulator in the central and peripheral nervous system. Mammalian tachykinins have been considered as putative transmitter peptides of primary sensory neurons that mediate nociception in the spinal cord^［1］. Molecular cloning of cDNAs shows three distinct types of tachykinin receptors, NK₁, NK₂ and NK₃ belonging to the GTP binding protein-coupled receptor superfamily^［2］. Although SP,neurokinin A(NKA), and neurokinin B (NKB) are the preferred agonists respectively for NK₁, NK₂ and NK₃ receptors, neither of these naturally occurring tachykinins is completely selective for one subtype of receptor^［3］. Of all neuropeptides, SP is perhaps best characterized in terms of distribution, sites of release, and biological actions^［1］. SP has been reported to participate in the noxious transmission in the dorsal horn of the spinal cord^［1］. It causes enhancement or depression of the noxious response by modifying both the release of a sensory transmitter and the excitability of the postsynaptic membrane in substantia gelatinosa cells of the cat dorsal horn^［4］. SP and excitatory amino acids (EAAs) can be released following noxious peripheral stimulation^［5］, and there is considerable evidence for interactions between the two neurotransmitter systems. SP has been shown to modulate both the release of endogenous EAAs^［6］ and the postsynaptic responses to EAAs in spinal neurons^［7］. However, there are relatively few reports on SP interaction with inhibitory amino acids.
　　The glycine receptors (GlyRs) are ligand-gated ion channels that mediate inhibitory transmission in the spinal cord and other regions of the central nervous system. Recent evidence has suggested that a variety of neurotransmitters that regulate intracellular second messager levels may affect GlyRs function by modulating the phosphorylation of the concerned ion channels^［8,9］. Gly has also been implicated in nociception, e.g. intrathecal application of strychnine increases nociceptive flexor reflex^［11］ and induces allodynia and receptive field expansion^［10］. Interactions between Gly and NK₁ rceptors have also previously been described. SP can raise the level of Gly in dorsal horn^［11］ and intrathecally injected Gly is capable of inhibiting SP-evoked biting and scratching behavior^［12］. Gly can also regulate the NK₁ receptor function in rat dorsal horn neurons^［13］.
　　Sacral dorsal commissural nucleus (SDCN) represents the area just dorsal to the central canal in the lower lumbar and sacral spinal cord. Previous studies have shown that the SDCN is implicated as an important site for integration, modulation and transmission of nociceptive information from the pelvic region^{［9,14,15］}. As reported previously, SDCN contains many nerve terminals showing SP-like immunoreactivity and many neurons possessing NK₁ receptor in SDCN are also Gly-immunoreactive^［16］. All these observations lead us to explore whether SDCN is the site of SP and Gly receptor interaction, and how the Gly-induced currents are affected by SP.

1　MATERIALS AND METHODS

1.1　Preparation　　The sacral dorsal commissural nucleus (SDCN) neurons were dissociated as described elsewhere^［9］. Briefly, two-week-old SD rats were decapitated under pentobarbitone sodium anesthesia (40 mg/kg body weight, i.p.). A segment of the lumbosacral (L6-S2) spinal cord was dissected out and sectioned with a vibratome tissue slicer (DTK-1000; Dosaka, Kyoto, Japan) to yield several transverse slices (400 μm-thick) containing the SDCN region. The slices were preincubated in oxygenated incubation solution (see below) for 50 min at room temperature (22～25℃). Thereafter, slices were treated enzymatically in oxygenated incubation solution containing 1 mg/（6～8） ml pronase for 20 min at 31℃, followed by exposure to 1 mg/（6～8） ml thermolysin for another 15 min in the same condition. After enzyme treatment, the slices were kept in enzyme-free incubation solution for 1 h. Then a portion of SDCN region was micro-punched out with an electrolytically polished injection needle and transferred into a culture dish filled with standard external solution (see below). Neurons were mechanically dissociated with fire-polished Pasteur pipettes under a phase contrast microscope (Olympus, IX70). Dissociated neurons adhered to the bottom of the dish within 20 min, allowing the electrophysiological studies to be conducted. The neurons that retained their original morphological features, such as the dendritic processes, were then used for experiments.
1.2　Solutions　　 The composition of incubation solution was (in mmol/L): NaCl, 124; NaHCO₃, 24; KCl, 5; KH₂PO₄, 1.2; CaCl₂, 2.4; MgSO₄, 1.3; glucose, 10; aerated with 95% O₂-5% CO₂ to a final pH of 7.4. The normal external standard solution contained (in mmol/L): NaCl, 150; KCl, 5; CaCl₂, 2; MgCl₂, 1; N-2-hydroxyethylpiperazine-N′-2-ethanesulphonic acid (HEPES), 10; glucose, 10; pH was adjusted to 7.4 with tris-(hydroxymethyl)-aminomethane (Tris-base). The patch pipette solution for nystatin perforated patch recording was (in mmol/L): CsCl, 150; HEPES, 10; pH was adjusted to 7.2 with Tris-base. A nystatin stock solution dissolved in acidified methanol at a concentration of 10 mg/ml was prepared and stored at －20℃. The stock solution was added to the patch-pipette solution just before use to give a final nystatin concentration of 400 μg/ml. When the current-voltage (I-V) relationship of I_Gly was examined, 0.3 μmol/L tetrodotoxin (TTX) and 10 μmol/L CdCl₂ were added to the standard external solution. CdCl₂ had no effect on the I_Gly at the used level of concentration.
1.3　Perforated patch recording　　 Electrical measurements were carried out by nystatin perforated patch recording configuration under voltage-clamp conditions at room temperature (22～25℃). Patch pipettes were pulled from glass capillaries with an outer diameter of 1.5 mm (Narishige) on a two-stage puller (PB-7; Narishige, Tokyo, Japan). The resistance between the recording electrode filled with pipette solution and the reference electrode was 4～6 MΩ. The patch pipette was positioned on a neuron using a hydraulic micromanipulator (WR-3; Narishige, Tokyo, Japan). The electrode was connected to a patch clamp amplifier (CEZ-2300, Nihon Koden, Tokyo, Japan). The current and voltage were monitored with a pen recorder (Omniace RT 3100, San-ei, Japan), filtered at 1 kHz and sampled and analysed using a DigiData 1200A interface and a computer with pCLAMP 6.0.2 program (Axon Instruments, USA). The membrane potential was held at -40 mV throughout the experiment, except when examining the I-V relationships. All measurements were started after stabilization of the Gly response (15～25 min after cell attachment).
1.4　Drugs and their application　　 Pronase was purchased from Calbiochem. Chelerythrine, HA-1004, KN-62, L-668,169, and L-659,877 were from Reseach Biochemicals International. Other drugs were from Sigma. All drugs were dissolved in distilled water and further diluted to their final concentrations in standard external solution just before use. Drugs were applied via a ‘Y-tube’^［9］. This system allows a complete exchange of external solution surrounding a neuron within 20 ms.
1.5　Statistics　　 Data were presented as mean±SE. The continuous curves for concentration-response relationships of Gly were drawn according to a modified Michaelis-Menten equation (1) using a least-square fitting routine (Newton-Raphson method) after normalizing the amplitude of the response:
　　　　I=I_maxCⁿ/(Cⁿ+EC_50n)

(1)　　

where I is the normalized value of the current, I_max the maximal response, C the drug concentration, EC₅₀ the concentration at half-maximal response, and n the apparent Hill coefficient. Student′s t-test was used when two groups were compared.

2　RESULTS

2.1　Potentiation of Gly response by SP
　　The morphological and elcetrophysiological features of the isolated SDCN neurons were similar to those reported previously^［9,17］. Recordings were made in the perforated-patch configuration that mai～tains intracellular Ca²⁺ and other second messengers intact^［9］. In the present experimental condition with pipette solution containing 150 mmol/L CsCl, the resting potential of the isolated SDCN neurons was in the range of －35 to -55 mV (n=21) under clamped condition. With the extra- and intracellular Cl^－ concentrations (［Cl^－］_o and ［Cl^－］_i ) of 161 and 150 mmol/L respectively, Gly elicited inward currents in all SDCN neurons tested at a holding potential (V_H) of －40 mV. The currents were inhibited by strychnine in a concentration-dependent manner^［18］.In the present experimental con～di～tions for recording Gly-induced Cl^－ current (I_Gly) in the SDCN neurons, pretreatment with SP alone induced no noticeable current at concentrations up to 10 μmol/L, but would increase Gly-induced (30 μmol/L) current (Fig.1A) in 63% of 48 tested neurons and this facilitatory effect of SP was action-time dependent, i.e. reaching a maximum at 3～5 min. Therefore, in the following ex～pe～ri～ments, 5 min pretreatment with SP was allowed to pass before Gly was added. The concentration-dependent effect of SP on potentiation of I_Gly is depicted in Fig.1B. The effect of SP was reversible 3～5 min after SP was washed out.

Fig.1　Potentiation of I_Gly by SP

A. A typical example of Gly responses potentiated by SP at concentrations of 10 nmol/L and 1 μmol/L. B. Concentration-response relationship for SP-induced augmentation of I_Gly. Ordinate indicates the enhancement ratio of the Gly response by SP. Each point is the mean of 4～5 measurements.

2.2　Concentration-response curves and current-voltage relationships of I_Gly
　　The potentiation ratio of I_Gly by 1 μmol/L SP was independent of Gly concentrations. SP increased the maximum value of the concentration-response relationship of Gly without affecting threshold concentration (Fig.2A). The EC₅₀ value (32.3 μmol/L, n=5) of Gly in the presence of SP is similar to 30.2 μmol/L (n=5) for Gly alone, indicating that SP did not change the affinity of Gly to its receptor. Figure 2B shows the current-voltage (I-V) relationships of I_Gly with or without 1 μmol/L SP. The reversal potentials of Gly response (E_Gly) were 1.76±1.1 mV (n=4) in the control and 1.85±1.3 mV (n=4) with 1 μmol/L SP (P>0.05). These E_Gly values were near the Cl^－ equilibrium potential (E_Cl) of －1.8 mV calculated by the Nernst equation. In addition, the magnitude of potentiation showed no voltage dependency.

Fig.2　Effects of SP on the concentration-response curve and I-V relationship of Gly
A. The concentration-response curves of I_Gly in the presence (●) or absence (■) of 1 μmol/L SP. All responses were normalized to the peak current induced by 30 μmol/L Gly alone (*). Each point is the average of 4～5 neurons. B. The I-V relationships of Gly response with (●) or without (■) 1 μmol/L SP. The arrow indicates the Cl^－ equilibrium potential (E_Cl). Currents were normalized to the peak current induced by 30 μmol/L Gly alone at a V_H of －40 mV (*). Each point is the mean of 4～6 neurons.

2.3　Involvement of tachykinin receptor subtypes
　　To find out which subtype of tachykinin receptor was involved in the potentiation of I_Gly, the effects of several selective antagonists of NK₁ and NK₂ receptors were examined. Both the tachykinin receptor antagonist, spantide, and the selective antagonist for NK₁ receptor, L-668,169, completely abolished SP facilitation of I_Gly (Fig.3Aa and b, B), while NK₂ receptor selective antagonist L-659,877 had no significant effect (Fig.3B). These results suggest that activation of NK₁ receptor is mainly responsible for the potentiation of I_Gly.

Fig.3　Involvement of the tachykinin receptor subtypes in SP potentiation of I_Gly
A. Effects of tachykinin receptor antagonists on SP potentiation of I_Gly. Aa. 1 μmol/L SP augmentation of 30 μmol/L Gly was abolished by spantide. Ab. SP enhancement of I_Gly was blocked by a potent selective NK₁ receptor antagonist, L-668,169. B. The average of five neurons. ^*P<0.05 compared with the peak current induced by 30 μmol/L Gly alone (taken as 1.0).

2.4　Possible intracellular mechanism of SP action
　　It has been reported that the mechanism of the action of SP at the NK₁ receptor involves phospholipase C-mediated phosphoinositol hydrolysis, which subsequently mobilizes internal Ca²⁺ stores and activates protein kinase C^［19］. To investigate possible intracellular mechanism of SP action, experiments were performed to test whether PKC or some other intracellular messengers play a role in the augmentation of I_Gly by SP. Pretreatment with 3 μmol/L chelerythrine (Chele, a PKC inhibitor) for 1～2 min abolished the SP potentiation of I_Gly in four tested cells (Fig.4A), but a protein kinase A (PKA) inhibitor, HA-1004, could not affect SP augmentation of I_Gly. In other four tested cells, pretreatment for 6～8 min with 1 μmol/L KN-62, a potent inhibitor of Ca²⁺/calmodulin-dependent protein kinase Ⅱ (CaMKⅡ), also abolished the facilitatory effect of 1 μmol/L SP on the I_Gly, but Chele had no effect. Both Chele and KN-62 produced no significant effect on the I_Gly. The complete recovery from the Chele and KN-62 blockade took 5～8 min after washing out the inhibitor. The results confirmed that the GlyRs were modulated by PKC or CaMKⅡ in the SP-induced enhancement of I_Gly in the SDCN neurons.

Fig.4　Possible intracellular mechanisms of SP enhancement of I_Gly
A. Abolishment of SP effect on I_Gly by PKC inhibitor chelerythrine (Chele), but not by PKA inhibitor HA-1004. B. Incapability of Chele in the abolishment of the enhancement effect of SP on I_Gly in contrast with Ca²⁺/calmodulin-dependent protein kinase Ⅱ inhibitor KN-62. ～^*P<0.05 compared with the control. All the above data were obtained from 4～6 neurons.

3　DISCUSSION

　　The principal finding of the present study is that SP is capable of potentiating Gly response through NK₁ receptor in a part of the neurons of rat SDCN. Activation of PKC or CaMKⅡ may be involved in this potentiation of I_Gly by SP.
3.1　Characteristics of SP potentiation of Gly response
　　It is evident from Fig.1 that the enhancement of amplitude of Gly-activated currents increases gradually with the increase of the concentration of SP from 1 nmol/L up to a maximum of 1 μmol/L. Thus the use of SP above 1 μmol/L was avoided. Comparison between the dose-response curves of I_Gly, with and without preapplication of SP as shown in Fig.2A, indicates that SP enhances I_Gly in a non-competitive manner, without changing the affinity of Gly to its receptor. I-V relationships of I_Gly show that I_Gly is carried by Cl^－ and the SP potentiation effect is not voltage-dependent. Spantide, a peptide antagonist for the tachykinin receptors (somewhat selective for the NK₁ receptor), blocks the SP-induced potentiation of I_Gly. L-668,169, which is a selective NK₁ receptor antagonist, also blocks the SP-induced potentiation of I_Gly, whereas L-659,877, a selective NK₂ receptor antagonist, has no effect on SP enhancement of I_Gly. NK₁ receptor-LI is found in SDCN by immunohistochemical method^［16］. From these results, we conclude that the NK₁ type tachykinin recptor is responsible for the SP-induced enhancement of I_Gly.
3.2　Possible mechanisms underlying the SP-induced potentiation of I_Gly
　　A number of receptors have been shown to activate complex intracellular signal transduction cascades and produce changes in the level of second messengers such as Ca²⁺, cyclic AMP, cyclic GMP, DAG, and IP₃ in the regulation of the activity of numerous target enzymes, which in turn can modulate the function of the Gly receptors^［20］. G-protein-coupled tachykinin receptors are one of the most important family of such regulatory receptors in the central nervous system (CNS). In the present study, it is worth noting that the SP effect on I_Gly needs a preapplication of 3～5 min to reach a maximum, and full recovery needs about 5 min. This implies that a direct allosteric modulatory effect of SP on the Gly receptor is excluded.
　　Biochemical studies have demonstrated that protein kinase C and cyclic AMP-dependent protein kinase can phosphorylate Gly receptor^［8］. It has been demonstrated that Gly responses are upregulated by direct phosphorylation via PKC^［9,18］. The present study showed that potentiation of I_Gly in some neurons was bolcked by PKC inhibitor Chele. In contrast, the PKA inhibitor HA-1004 did not abolish the SP-induced enhancement of I_Gly. These results suggest that phosphorylation by a diffusible messenger, probably PKC, is involved in the signal transduction pathway of this SP-induced effect. Several studies have reported that SP facilitates the turnover of inositol polyphosphate metabolism by increasing phospholipase C (PLC) activity^［20］. The phosphoinositide hydrolysis produces two second messengers, inositol-1,4,5,-trisphosphate (IP₃) and diacylglycerol (DAG), and the latter increases PKC activity^［21］. Therefore, in a part of SDCN neurons, the cascade for signal transduction would be that activation of the NK₁ receptor leads to activation of a G-protein resulting in activation of PLC. Hydrolysis of inositol phospholipeds by PLC results in the production of DAG, which in turn activates PKC and consequently phosphorylates Gly receptor-channel complex.
　　The IP₃ can trigger the release of calcium from intracellular stores and may also promote calcium influx^［22］, leading to an elevation of ［Ca²⁺］_i. The other messenger, DAG, has been suggested to mediate the SP-induced calcium influx through opening voltage-dependent Ca²⁺ channels in a rat pancreatic acinar cell line^［23］. Increase in ［Ca²⁺］_i can cause CaMKⅡ activation that might also enhance I_Gly by phosphorylation of Gly receptors. The above hypothesis was supported by our results that KN-62 could abolish SP effect on another part of neurons. Immunohistochemical^［24］ and biochemical studies^［25］ have demonstrated the presence of CaM and CaMKⅡ in the spinal dorsal horn. Recent observation that activated CaMKⅡ could potentiate I_Gly in acutely dissociated spinal dorsal horn neurons^［26］ further supports a role for this kinase in mediating SP-induced potentiation of I_Gly in SDCN neurons. In summary, PKC and CaMKⅡ might phosphorylate the Gly receptor-channel complex respectively and therefore mediates SP potentiation effect on I_Gly in acutely dissociated rat SDCN neurons. This differential mediation by PKC and CaMKⅡ may reflect somewhat different distribution of these two kinases in SDCN neurons.
3.3　Functional implications
　　SP may serve as a pain transmitter or as a modulator of noxious transmission in the dorsal horn of the spinal cord. SP causes both enhancement and depression of the noxious response in substantia gelatinosa cells of the cat dorsal horn, by modifying both the release of a sensory transmitter and the excitability of the postsynaptic membrane. An antinociceptive effect of SP has previously been reported by Masuyama et al^［27］. In behaving mice they found that low doses of intrathecal SP reduced responses to NMDA. SP also influences the function of inhibitory amino acids in dorsal horn. Gly is released following microdialysis of SP into the spinal cord in vivo^［28］ and following bath application in vitro^［29］. Gly is a primary transmitter which decreases transmitter release from primary afferent terminals in the spinal cord through an effect known as ‘presynaptic inhibition’^［30］. If SP potentiates the Gly response at the central terminal of primary afferent neurons, then potentiation of the ‘presynaptic inhibition’ would result in suppression of nociception in the spinal cord.

*Supported by the National Natural Science Foundation of China (No.39770248)
^*国家自然科学基金资助 (No.39770248)
^**To whom correspondence should be addressed: Department of Anatomy and K.K. Leung Brain Research Center, Fourth Military Medical University, Xi′an 710032. Tel: 029-337-4501, Fax: 029-324-6270, E-mail: patchclp@fmmu.edu.cn
^**通讯作者

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Received 1998-11-19　　Revised 1999-01-22