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Tetramethylpyrazine inhibits ATP-activated currents in rat dorsal root ganglion neurons
Brain Research 1040 (2005) 92 – 97 www.elsevier.com/locate/brainres
Research report
Tetramethylpyrazine inhibits ATP-activated currents in rat dorsal root ganglion neurons S.D. LiangT, C.S. Xu, T. Zhou, H.Q. Liu, Y. Gao, G.L. Li Department of Physiology, Jiangxi Medical College, Nanchang 330006, PR China Accepted 21 January 2005 Available online 16 March 2005
Abstract Tetramethylpyrazine (TMP) is one of the alkaloids contained in Ligustrazine which has been used in traditional Chinese medicine as an analgesic for injury and dysmenorrhea. ATP can elicit the sensation of pain. This study observed the effects of TMP on ATP-activated current (I ATP) in rat DRG neurons. TMP (0.1–1 mM) concentration-dependently inhibited ATP (100 AM)-activated current in rat DRG neurons. The inhibitory time of ATP (100 AM)-activated current appeared at 15 s after preapplication of TMP and reached its peak at about 45 s. The dose– response curves for I ATP in the absence and presence of 1 mM TMP showed that TMP (1 mM) shifted the concentration–response curve of I ATP downward markedly and the two EC50 values were very close (75 vs. 82 AM), while the threshold value remained unchanged. Therefore, the inhibitory effect of TMP on I ATP may be noncompetitive. TMP did not alter the reversal potential (0 mV) of ATP-activated current, indicating that the site of TMP action is on or near the exterior surface of channel protein and not within the channel pore. Externally applied TMP (1 mM) increases the inhibitory effect of chelerythrine (PKC inhibitor) contained in pipette solution on I ATP. The site of TMP action may be the binding of TMP to an allosteric site on the large extracellular region of ATP receptor–ion channel complex (P2X receptors) or PKC site of the N-terminus of P2X receptors. The mechanism of TMP action may be the allosteric regulation via acting on the large extracellular region of ATP receptor–ion channel complex (P2X receptors) and promoting the phosphorylation of PKC site of the N-terminus of P2X receptors. D 2005 Elsevier B.V. All rights reserved. Theme: Neurotransmitter systems and channels, Topic: Tetramethylpyrazine inhibits ATP-activated currents in rat dorsal root ganglion neurons Keywords: Tetramethylpyrazine; ATP; Purinergic receptor; DRG; Whole-cell patch-clamp technique
1. Introduction Adenosine-5V-triphosphate (ATP), in addition to its function as an intracellular energy donor, is now recognized as an important neurotransmitter or cotransmitter in both the central and peripheral nervous system [22]. The action of ATP is mediated by cell surface receptors that belong to the P2 class of purinergic receptors. P2 receptors are divided into two groups: P2X receptors function as ligand-activated Abbreviations: TMP, tetramethylpyrazine; ATP, adenosine 5V-triphosphate; I ATP, ATP-activated current; DRG, dorsal root ganglion; CNS, central nervous system; PKC, calcium-dependent protein kinase T Corresponding author. Fax: +86 791 8603113. E-mail address: [email protected] (S.D. Liang). 0006-8993/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2005.01.076
ion channels and P2Y receptors belong to the superfamily of G-protein-coupled receptors [1,22]. The dorsal root ganglion (DRG) neurons transmit various sensory information including pain to the CNSs. ATP can elicit the sensation of pain [2,9]. ATP is implicated in peripheral pain signaling by actions on P2X receptors [5]. Application of P2X agonists to acutely dissociated rat DRG neurons under voltage-clamp conditions evokes inward currents that desensitize rapidly, slowly or in a fast then slow, biphasic manner [4,21,25]. Tetramethylpyrazine (TMP) is one of the alkaloids contained in Ligustrazine which has been used in traditional Chinese medicine as an analgesic for injury and dysmenorrhea. The oral administration of TMP significantly inhibited the hindpaw edema induced by carrageen in rats [20]. Our previous studies showed that TMP attenuated ATP-induced
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depolarization in sympathetic ganglion [17] and the local administration of TMP inhibited the acute hindpaw nociception mediated by P2X receptor agonists (ATP or a,h-meATP) in rats [18]. The aim of the present study is to explore whether TMP is able to modulate ATP-activated currents (I ATP) in acutely dissociated rat DRG neurons and to try to further understand the mechanism of antinociception of TMP.
2. Materials and methods 2.1. Isolation of DRG neurons Sprague–Dawley rats, 2–3 weeks old, of both sexes were decapitated after anesthetized with urethane [1.2 g/kg, intraperitoneally (i.p.)]. The thoracic and lumbar segments of the vertebral column were dissected and longitudinally divided into two halves along the median lines on both dorsal and ventral sides. The DRGs together with dorsal and ventral roots and attached spinal nerves were taken out from the inner side of each half of the dissected vertebrae with fine dissecting forceps and transferred immediately into Dubecco’s modified Eagle’s medium (DMEM, Sigma) at pH 7.4 and 340 mosM/kg. After the removal of attached nerves and surrounding connective tissues, the DRGs were minced with dissecting spring scissors and incubated with trypsin (0.5 mg/ml; type III, Sigma), collagenase (1.0 mg/ ml; type IA, sigma), and Dnase (0.1 mg/ml; type IV, Sigma) in 5 ml DMEM at 35 8C in a shaking bath for 35–40 min, after which soybean trypsin inhibitor (1.25 mg/ml; type II-S, sigma) was added to stop the enzymatic digestion. The isolated neurons were transferred into a 35-mm culture dish and kept still for 30 min. Experiments were performed at room temperature (20–30 8C) [11,16].
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of independent reservoirs. The distance from the tubule mouth to the cell examined was approximately 100 Am. Rapid solution-exchange was achieved by shifting the tubules horizontally with a micromanipulator. 2.3. Drugs Tetramethylpyrazine (TMP) was obtained from Wuxi Seventh Reagent Factory of China. Adenosine 5V-triphosphate disodium (ATP) and chelerythrine were obtained from Sigma. TMP and ATP were dissolved and diluted in the external solution. Chelerythrine was dissolved and diluted in the internal solution. 2.4. Statistical analysis All results were expressed by mean F SEM and analyzed by t test. P value b0.05 was considered to be statistically significant.
3. Results 3.1. ATP-activated current in DRG neurons and inhibitory effects of TMP on ATP-activated current The majority of cells examined (88/93, 94.6%) responded to the external application of ATP (1–1000 AM) in a concentration-dependent manner with an inward current (I ATP). The ATP-activated currents showed a slow desensitization (Fig. 1) or a rapid desensitization (Fig. 2).
2.2. Electrophysiological recordings The whole-cell patch-clamp recording [11,16] was carried out using a patch/whole-cell clamp amplifer (CEZ-2400, Nihon Kohden). The micropipette was filled with internal solution containing (in mM): KCl 140, MgCl2 2, HEPES 10, EGTA 11, ATP 5; its osmolarity was adjusted to 340 mosM/ kg with sucrose and pH adjusted to 7.4 with KOH. The external solution contained (mM): NaCl 150, KCl 5, CaCl2 2.5, MgCl2 1, HEPES 10, d-glucose 10; its osmolarity was adjusted to 340 mosM with sucrose, pH was adjusted to 7.4 with NaOH. The resistance of recording electrodes were in the range of 1–4 MV. A small patch of membrane underneath the tip of the pipette was aspirated to form a seal (1–10 GV) and then a more negative pressure was applied to rupture it thus a whole-cell mode was established. Membrane currents were filtered at 1 kHz ( 3 dB), data were recorded by a pen recorder (LMS-2B, Chengdu). The holding potential was set at 60 mV. The drugs were dissolved in external solution and delivered by gravity flow from an array of tubules (500 Am O.D., 200 Am I.D.) connected to a series
Fig. 1. Dose-dependent inhibition of TMP on ATP-activated current. Inset, sequential current traces indicating concentration-dependent suppression of ATP (100 AM)-activated currents by preapplication of TMP (0.1–3 mM) (all records were obtained from the same neuron). Graph shows the plot of percentage decrease in amplitude of ATP-activated current (mean F SEM) vs. concentration of TMP. *P b 0.05, **P b 0.01, compared with the control.
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Fig. 2. Time course of inhibition by TMP of ATP-activated current. Records of current activated by 100 AM ATP in the absence and presence of preapplication of 1 mM TMP at different preapplication time courses. All records were obtained from the same neuron. The curve below indicates the plot of percentage amplitude of ATP-activated current vs. time (in seconds).
When TMP (0.1–3 mM) was applied for 30–45 s prior to application of ATP (100 AM), an attenuation of I ATP was observed (Fig. 1). The inhibitory effect of TMP was concentration dependent and reached its peak at 1 mM and then decreased gradually with an increase in TMP concentration. On average, the amplitude of currents activated by 100 AM ATP were inhibited by 7.99 F 1.56% (n = 7), 11.20 F 2.97% (n = 8), 35.82 F 4.62% (n = 8), 41.76 F 5.20% (n = 8), 40.45 F 7.30% (n = 8), and 38.46 F 6.73% (n = 6) by 0.1, 0.3, 0.6, 1, 2, and 3 mM TMP, respectively. In addition, TMP (1 mM) inhibited a,h-meATP (10 AM)activated current to 43.85 F 6.20% (n = 7).
Fig. 3. Concentration–response relationship for ATP-activated current with or without pretreatment of TMP. The graph shows the concentration– response curves for ATP-activated current with or without pretreatment of TMP (1 mM). Each point represents the mean F SEM of 5 neurons. All ATP-activated currents were normalized to the current activated by 100 AM ATP (marked with asterisk). Holding potential was set at 60 mV. The curve for ATP alone is a best fit of data to the logistic equation Y = I max/[1 + (K d/C)n ], where C is the concentration of ATP, Y is the maximum value, and K d, the dissociation constant of the ATP receptor, is 75 AM. The Hill coefficient (n) = 0.93.
responses were normalized to the current induced by 100 AM ATP alone. TMP (1 mM) shifted the concentration–response curve of I ATP downward markedly. The amplitude of ATPactivated current at maximum concentration was decreased by 41.3%, while the threshold value remained unchanged. The EC50 value for ATP pretreated with TMP was estimated to be 82 AM, which was very close to 75 AM of control. 3.4. I–V relationship for the inhibition by TMP of ATP-activated current Fig. 4 shows the current–voltage relationships for currents activated by 100 AM ATP in the absence and
3.2. Time course of inhibition by TMP of ATP-activated current Different time courses of preapplication of TMP from 15 s to 75 s were used to clarify whether stimulation of TMP for a longer time could result in a stronger inhibitory effect than a shorter stimulation. The inhibition of ATP (100 AM)activated current appeared at 15 s after preapplication of TMP and reached its peak at about 45 s (Fig. 2). However, when the time course of preapplication increased beyond 45 s, there was no additional increase in the inhibitory effect of TMP (1 mM) and the inhibition was kept at a steady value. 3.3. Concentration–response relationship of ATP-activated current with or without pretreatment of TMP The dose–response curves for ATP-activated current in the absence and presence of 1 mM TMP are showed in Fig. 3. All
Fig. 4. Current–voltage relationships for ATP with or without preapplication of TMP. Pretreatment with TMP inhibited ATP-activated current at all holding potentials. The values of reversal potential in both cases were around 0 mV. Each point represents the mean F SEM of 6 neurons. All current amplitudes were normalized to the current amplitude at 60 mV without TMP preapplication (marked with asterisk).
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Fig. 5. Effects of intracellular PKC inhibitor on ATP-activated current with or without externally applied TMP. Left panel: ATP (10 AM)-activated currents were decreased by TMP (1 mM) with normal internal solution in pipette. Right panel: repatch record with internal solution containing chelerythrine (10 AM) in the absence and presence of externally applied 1 mM TMP.
presence of 1 mM TMP. TMP suppressed I ATP at all holding potentials between 80 and +40 mV. I–V relationship illustrates that TMP did not alter the reversal potential of ATP-activated current. The reversal potential of current activated by 100 AM ATP was close to 0 mV in the absence and presence of 1 mM TMP. 3.5. Effects of intracellular PKC inhibitor on I ATP with or without externally applied TMP The receptor–channel complex has been reported to possess the intracellular structure that can be phosphorylated, leading to the modulation of the receptor–channel complex-mediated actions by calcium dependent protein kinase (PKC) [24,27]. We used repatch technique to explore whether suppression of I ATP by TMP was due to intracellular transduction pathways of PKC. In the control experiment with the normal internal solution, externally applied TMP (1 mM) inhibited ATP (10 AM)-activated current in rat DRG neurons. When the amplitude of I ATP returned to its original level, chelerythrine (10 AM, a PKC inhibitor) was introduced into the recording pipette in repatch test. After 20 min with pipette solution containing chelerythrine (10 AM), there was a decrease in the amplitude of I ATP. Externally applied TMP (1 mM) increases the inhibitory effect of chelerythrine (10 AM) contained in pipette solution on ATP-activated current (Fig. 5). In addition, with pipette solution containing 10 AM chelerythrine (n = 6) in normal patch test, the effects of externally applied TMP (1 mM) on ATP-activated currents were similar to that in repatch test.
4. Discussion In this experiment, the majority of freshly isolated neurons examined (94.6%, 88/93) were sensitive to ATP
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applied externally. It has been showed that there are two distinct ATP-activated currents observed in small and medium cells (but not in large cells) of rat DRG: one is a rapidly desensitizing ATP-activated current; the other is a slowly desensitizing ATP-activated current [4,16]. Smalldiameter (b30 AM) DRG neurons were sensitive to capsaicin and had ATP-activated current with a rapid desensitization; medium diameter (30–50 AM) DRG neurons were insensitive to capsaicin and had ATP-activated current with a slow desensitization [16]. The P2X3 receptor subunit had been cloned from rat DRG cDNA library [6,13] and expressed in capsaicin-sensitive, small-sized DRG neurons [6]. Additionally, it was reported that capsaicinsensitive, small-sized DRG neurons expressed mainly the homomeric P2X3 subtype and that capsaicin-insensitive, medium-sized DRG neurons expressed heteromultimeric complex of P2X2 and P2X3 subtypes [26]. In our experiment, the types of ATP-activated currents include rapidly desensitizing and slowly desensitizing. The reversal potential for activation of P2X receptors is close to 0 mV [7]. Our study indicated that the reversal potential of current activated by 100 AM ATP was close to 0 mV in the absence and presence of 1 mM TMP. Therefore, ATP-activated current in this study might mainly activated P2X receptors. a,h-meATP has the effects on P2X1, P2X3, and P2X2/3 receptors [7]. The fact that TMP inhibits a,h-meATPactivated currents supports the above opinion. It is evident from Fig. 1 that the inhibition of amplitude of ATP-activated currents increases gradually with the enhancement of the concentration of TMP from 0.1 to 1 mM. However, when the concentration of TMP increased to 1 mM, the inhibitory effect of TMP on ATP-activated currents did not increase further. The phenomenon is similar to another work which has shown that the inhibition of metEnk on ATP-activated currents was gradually increased from 0.0001 to 0.01 AM and was gradually decreased from 0.01 to 1 AM [30]. The decrease in inhibitory effect of TMP on I ATP may be nonspecific. The inhibitory effect of TMP on ATP-activated currents was both in peak value (Ip) and in steady state (Iss). This is apparent in the records demonstrated in Fig. 2. It is considered that this inhibition may result from both the rapid component of desensitizing current and the slow component of desensitizing current. From the comparison between the dose–response curves for ATP with or without pretreatment of TMP in Fig. 3, it is clear that: (1) preapplication of TMP shifts the curve downwards markedly; (2) the maximal amplitude of ATPactivated current pretreated with TMP decreases by 41.3%, while the threshold value remains unchanged; (3) the two EC50 values are very close (75 vs. 82 AM). Therefore, it is reasonable to consider that the inhibitory effect of TMP on ATP-activated current may be noncompetitive. Our work showed that chelerythrine (PKC inhibitor) contained in the pipette solution inhibited the amplitude of ATP-activated current. The result suggests that the functions of ATP receptor–ion channel complex can be modulated intracellu-
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larly by phosphorylation. A decrease in the PKC activity reduces the ATP receptor-mediated responses in rat DRG neuron. Simultaneously externally applied TMP increases the inhibitory effect of chelerythrine contained in the pipette solution on ATP-activated current. Seven P2X receptor suptypes (P2X1–7) possess two transmembrane domains, intracellular N- and C-termini and a large extracellular loop [28]. The N-terminal region is of similar size for all P2X receptor isoforms, with a conserved PKC site that appears to be phosphorylated constitutively [19]. Mutation to remove a consensus PKC site from the N-terminus (T 18) can lead to speeding of channel desensitization [8,28]. It is suggested that the intracellular N-terminus participates directly in the regulation of ionic conduction at P2X receptors, possibly contributing to the intracellular pore vestibule. The effect of PKC inhibitor in this study supports the above opinion. In addition, TMP may promote the phosphorylation of PKC site of the N-terminus to potentiate the inhibitory effect of PKC inhibitor on ATP-activated current. TMP inhibited ATP-activated current in a voltageindependent manner. The mechanism of inhibition of ATPactivated current include open-channel block. Open-channel block is usually voltage-dependent. However, open-channel block has voltage-independent when agents bind to a site within the ion channel but beyond the influence of the membrane electrical field [10,15]. In addition, the site of agent action is on or near the exterior surface of channel protein and not within the channel pore, where the membrane electrical field would not be influenced. P2X receptors can be regulated allosterically by extracellular protons, divalent cations, and a range of metals [19]. The time course of inhibition of TMP was from the onset of 15 s to the peak of 45 s. The site of TMP action may include the extracellular region and the cytoplasmic region or is mainly the extracellular region. The regulation site of the allosterism includes the action site of the intracellular signal transduction system [11] and the action site of the extracellular region [14,15,28]. It is possible that the mechanism of TMP action is the binding of TMP to an allosteric site on the large extracellular region of ATP receptor-ion channel complex and promoting the phosphorylation of PKC site of the Nterminus. This is in agreement with the effects of other P2X receptor antagonists, that is, P2X receptor antagonists act at the extracellular region of the ATP-gated receptor–channel complex [3,14,29]. It was reported that there was an expression of P2Y receptors in rat DRG [23]. In DRG, P2Y1 receptor immunoreactivity was very often coexpressed with P2X3 receptor immunoreactivity. P2Y1 and P2Y2 receptors increased the excitability of sensory neurons that were likely to be nociceptive [12]. It is not clear whether TMP may affect P2Y receptors in rat DRG. For this reason, further studies will be required to determine the precise mechanism responsible for the effects of TMP. In conclusion, TMP can inhibit ATP-activated current in rat DRG neurons. The site of TMP action may be the binding of TMP to an allosteric site on the large extracellular region
of ATP receptor–ion channel complex (P2X receptors) or PKC site of the N-terminus of P2X receptors. The mechanism of TMP action may be the allosteric regulation acting on the large extracellular region of ATP receptor–ion channel complex (P2X receptors) and promoting the phosphorylation of PKC site of the N-terminus of P2X receptors.
Acknowledgment This work was supported by the National Natural Science Foundation of China (No. 30260030) to S.D. Liang.
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[25] K. Tsuzuki, A. Ase, P. Seguela, T. Nakatsuka, C.Y. Wang, J.X. She, J.G. Gu, TNP-ATP-resistant P2X ionic currents on the central terminals and somata of rat primary sensory neurons, J. Neurophysiol. 89 (2003) 3235 – 3242. [26] S. Ueno, M. Tsuda, T. Iwanaga, K. Inoue, Cell type-specific ATPactivated responses in rat dorsal root ganglion neurons, Br. J. Pharmacol. 126 (1999) 429 – 436. [27] M.L. Vaello, A. Ruiz-Gomez, J. Lerma, F. Mayor Jr., Modulation of inhibitory glycine receptors by phosphorylation by protein kinase C and cAMP-dependent protein kinase, J. Biol. Chem. 269 (1994) 2002 – 2008. [28] C. Vial, J.A. Roberts, R.J. Evens, Molecular properties of ATPgated P2X receptor ion channels, Trends Pharmacol. Sci. 25 (2004) 487 – 493. [29] C. Virginio, G. Robertson, A. Surprenant, R.A. North, Trinitrophenylsubstituted nucleotides are potent antagonists selective for P2X1, P2X3, and heteromeric P2X2/3 receptors, Mol. Pharmacol. 53 (1998) 969 – 973. [30] B.L. Xia, Z.Z. Wu, X. Li, Q. Li, Z.W. Li, Inhibition of ATP-activated currents by met-Enk in isolated DRG neurons of the rat, Acta Physiol. Sin. 53 (2001) 205 – 208.
Research report
Tetramethylpyrazine inhibits ATP-activated currents in rat dorsal root ganglion neurons S.D. LiangT, C.S. Xu, T. Zhou, H.Q. Liu, Y. Gao, G.L. Li Department of Physiology, Jiangxi Medical College, Nanchang 330006, PR China Accepted 21 January 2005 Available online 16 March 2005
Abstract Tetramethylpyrazine (TMP) is one of the alkaloids contained in Ligustrazine which has been used in traditional Chinese medicine as an analgesic for injury and dysmenorrhea. ATP can elicit the sensation of pain. This study observed the effects of TMP on ATP-activated current (I ATP) in rat DRG neurons. TMP (0.1–1 mM) concentration-dependently inhibited ATP (100 AM)-activated current in rat DRG neurons. The inhibitory time of ATP (100 AM)-activated current appeared at 15 s after preapplication of TMP and reached its peak at about 45 s. The dose– response curves for I ATP in the absence and presence of 1 mM TMP showed that TMP (1 mM) shifted the concentration–response curve of I ATP downward markedly and the two EC50 values were very close (75 vs. 82 AM), while the threshold value remained unchanged. Therefore, the inhibitory effect of TMP on I ATP may be noncompetitive. TMP did not alter the reversal potential (0 mV) of ATP-activated current, indicating that the site of TMP action is on or near the exterior surface of channel protein and not within the channel pore. Externally applied TMP (1 mM) increases the inhibitory effect of chelerythrine (PKC inhibitor) contained in pipette solution on I ATP. The site of TMP action may be the binding of TMP to an allosteric site on the large extracellular region of ATP receptor–ion channel complex (P2X receptors) or PKC site of the N-terminus of P2X receptors. The mechanism of TMP action may be the allosteric regulation via acting on the large extracellular region of ATP receptor–ion channel complex (P2X receptors) and promoting the phosphorylation of PKC site of the N-terminus of P2X receptors. D 2005 Elsevier B.V. All rights reserved. Theme: Neurotransmitter systems and channels, Topic: Tetramethylpyrazine inhibits ATP-activated currents in rat dorsal root ganglion neurons Keywords: Tetramethylpyrazine; ATP; Purinergic receptor; DRG; Whole-cell patch-clamp technique
1. Introduction Adenosine-5V-triphosphate (ATP), in addition to its function as an intracellular energy donor, is now recognized as an important neurotransmitter or cotransmitter in both the central and peripheral nervous system [22]. The action of ATP is mediated by cell surface receptors that belong to the P2 class of purinergic receptors. P2 receptors are divided into two groups: P2X receptors function as ligand-activated Abbreviations: TMP, tetramethylpyrazine; ATP, adenosine 5V-triphosphate; I ATP, ATP-activated current; DRG, dorsal root ganglion; CNS, central nervous system; PKC, calcium-dependent protein kinase T Corresponding author. Fax: +86 791 8603113. E-mail address: [email protected] (S.D. Liang). 0006-8993/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2005.01.076
ion channels and P2Y receptors belong to the superfamily of G-protein-coupled receptors [1,22]. The dorsal root ganglion (DRG) neurons transmit various sensory information including pain to the CNSs. ATP can elicit the sensation of pain [2,9]. ATP is implicated in peripheral pain signaling by actions on P2X receptors [5]. Application of P2X agonists to acutely dissociated rat DRG neurons under voltage-clamp conditions evokes inward currents that desensitize rapidly, slowly or in a fast then slow, biphasic manner [4,21,25]. Tetramethylpyrazine (TMP) is one of the alkaloids contained in Ligustrazine which has been used in traditional Chinese medicine as an analgesic for injury and dysmenorrhea. The oral administration of TMP significantly inhibited the hindpaw edema induced by carrageen in rats [20]. Our previous studies showed that TMP attenuated ATP-induced
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depolarization in sympathetic ganglion [17] and the local administration of TMP inhibited the acute hindpaw nociception mediated by P2X receptor agonists (ATP or a,h-meATP) in rats [18]. The aim of the present study is to explore whether TMP is able to modulate ATP-activated currents (I ATP) in acutely dissociated rat DRG neurons and to try to further understand the mechanism of antinociception of TMP.
2. Materials and methods 2.1. Isolation of DRG neurons Sprague–Dawley rats, 2–3 weeks old, of both sexes were decapitated after anesthetized with urethane [1.2 g/kg, intraperitoneally (i.p.)]. The thoracic and lumbar segments of the vertebral column were dissected and longitudinally divided into two halves along the median lines on both dorsal and ventral sides. The DRGs together with dorsal and ventral roots and attached spinal nerves were taken out from the inner side of each half of the dissected vertebrae with fine dissecting forceps and transferred immediately into Dubecco’s modified Eagle’s medium (DMEM, Sigma) at pH 7.4 and 340 mosM/kg. After the removal of attached nerves and surrounding connective tissues, the DRGs were minced with dissecting spring scissors and incubated with trypsin (0.5 mg/ml; type III, Sigma), collagenase (1.0 mg/ ml; type IA, sigma), and Dnase (0.1 mg/ml; type IV, Sigma) in 5 ml DMEM at 35 8C in a shaking bath for 35–40 min, after which soybean trypsin inhibitor (1.25 mg/ml; type II-S, sigma) was added to stop the enzymatic digestion. The isolated neurons were transferred into a 35-mm culture dish and kept still for 30 min. Experiments were performed at room temperature (20–30 8C) [11,16].
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of independent reservoirs. The distance from the tubule mouth to the cell examined was approximately 100 Am. Rapid solution-exchange was achieved by shifting the tubules horizontally with a micromanipulator. 2.3. Drugs Tetramethylpyrazine (TMP) was obtained from Wuxi Seventh Reagent Factory of China. Adenosine 5V-triphosphate disodium (ATP) and chelerythrine were obtained from Sigma. TMP and ATP were dissolved and diluted in the external solution. Chelerythrine was dissolved and diluted in the internal solution. 2.4. Statistical analysis All results were expressed by mean F SEM and analyzed by t test. P value b0.05 was considered to be statistically significant.
3. Results 3.1. ATP-activated current in DRG neurons and inhibitory effects of TMP on ATP-activated current The majority of cells examined (88/93, 94.6%) responded to the external application of ATP (1–1000 AM) in a concentration-dependent manner with an inward current (I ATP). The ATP-activated currents showed a slow desensitization (Fig. 1) or a rapid desensitization (Fig. 2).
2.2. Electrophysiological recordings The whole-cell patch-clamp recording [11,16] was carried out using a patch/whole-cell clamp amplifer (CEZ-2400, Nihon Kohden). The micropipette was filled with internal solution containing (in mM): KCl 140, MgCl2 2, HEPES 10, EGTA 11, ATP 5; its osmolarity was adjusted to 340 mosM/ kg with sucrose and pH adjusted to 7.4 with KOH. The external solution contained (mM): NaCl 150, KCl 5, CaCl2 2.5, MgCl2 1, HEPES 10, d-glucose 10; its osmolarity was adjusted to 340 mosM with sucrose, pH was adjusted to 7.4 with NaOH. The resistance of recording electrodes were in the range of 1–4 MV. A small patch of membrane underneath the tip of the pipette was aspirated to form a seal (1–10 GV) and then a more negative pressure was applied to rupture it thus a whole-cell mode was established. Membrane currents were filtered at 1 kHz ( 3 dB), data were recorded by a pen recorder (LMS-2B, Chengdu). The holding potential was set at 60 mV. The drugs were dissolved in external solution and delivered by gravity flow from an array of tubules (500 Am O.D., 200 Am I.D.) connected to a series
Fig. 1. Dose-dependent inhibition of TMP on ATP-activated current. Inset, sequential current traces indicating concentration-dependent suppression of ATP (100 AM)-activated currents by preapplication of TMP (0.1–3 mM) (all records were obtained from the same neuron). Graph shows the plot of percentage decrease in amplitude of ATP-activated current (mean F SEM) vs. concentration of TMP. *P b 0.05, **P b 0.01, compared with the control.
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Fig. 2. Time course of inhibition by TMP of ATP-activated current. Records of current activated by 100 AM ATP in the absence and presence of preapplication of 1 mM TMP at different preapplication time courses. All records were obtained from the same neuron. The curve below indicates the plot of percentage amplitude of ATP-activated current vs. time (in seconds).
When TMP (0.1–3 mM) was applied for 30–45 s prior to application of ATP (100 AM), an attenuation of I ATP was observed (Fig. 1). The inhibitory effect of TMP was concentration dependent and reached its peak at 1 mM and then decreased gradually with an increase in TMP concentration. On average, the amplitude of currents activated by 100 AM ATP were inhibited by 7.99 F 1.56% (n = 7), 11.20 F 2.97% (n = 8), 35.82 F 4.62% (n = 8), 41.76 F 5.20% (n = 8), 40.45 F 7.30% (n = 8), and 38.46 F 6.73% (n = 6) by 0.1, 0.3, 0.6, 1, 2, and 3 mM TMP, respectively. In addition, TMP (1 mM) inhibited a,h-meATP (10 AM)activated current to 43.85 F 6.20% (n = 7).
Fig. 3. Concentration–response relationship for ATP-activated current with or without pretreatment of TMP. The graph shows the concentration– response curves for ATP-activated current with or without pretreatment of TMP (1 mM). Each point represents the mean F SEM of 5 neurons. All ATP-activated currents were normalized to the current activated by 100 AM ATP (marked with asterisk). Holding potential was set at 60 mV. The curve for ATP alone is a best fit of data to the logistic equation Y = I max/[1 + (K d/C)n ], where C is the concentration of ATP, Y is the maximum value, and K d, the dissociation constant of the ATP receptor, is 75 AM. The Hill coefficient (n) = 0.93.
responses were normalized to the current induced by 100 AM ATP alone. TMP (1 mM) shifted the concentration–response curve of I ATP downward markedly. The amplitude of ATPactivated current at maximum concentration was decreased by 41.3%, while the threshold value remained unchanged. The EC50 value for ATP pretreated with TMP was estimated to be 82 AM, which was very close to 75 AM of control. 3.4. I–V relationship for the inhibition by TMP of ATP-activated current Fig. 4 shows the current–voltage relationships for currents activated by 100 AM ATP in the absence and
3.2. Time course of inhibition by TMP of ATP-activated current Different time courses of preapplication of TMP from 15 s to 75 s were used to clarify whether stimulation of TMP for a longer time could result in a stronger inhibitory effect than a shorter stimulation. The inhibition of ATP (100 AM)activated current appeared at 15 s after preapplication of TMP and reached its peak at about 45 s (Fig. 2). However, when the time course of preapplication increased beyond 45 s, there was no additional increase in the inhibitory effect of TMP (1 mM) and the inhibition was kept at a steady value. 3.3. Concentration–response relationship of ATP-activated current with or without pretreatment of TMP The dose–response curves for ATP-activated current in the absence and presence of 1 mM TMP are showed in Fig. 3. All
Fig. 4. Current–voltage relationships for ATP with or without preapplication of TMP. Pretreatment with TMP inhibited ATP-activated current at all holding potentials. The values of reversal potential in both cases were around 0 mV. Each point represents the mean F SEM of 6 neurons. All current amplitudes were normalized to the current amplitude at 60 mV without TMP preapplication (marked with asterisk).
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Fig. 5. Effects of intracellular PKC inhibitor on ATP-activated current with or without externally applied TMP. Left panel: ATP (10 AM)-activated currents were decreased by TMP (1 mM) with normal internal solution in pipette. Right panel: repatch record with internal solution containing chelerythrine (10 AM) in the absence and presence of externally applied 1 mM TMP.
presence of 1 mM TMP. TMP suppressed I ATP at all holding potentials between 80 and +40 mV. I–V relationship illustrates that TMP did not alter the reversal potential of ATP-activated current. The reversal potential of current activated by 100 AM ATP was close to 0 mV in the absence and presence of 1 mM TMP. 3.5. Effects of intracellular PKC inhibitor on I ATP with or without externally applied TMP The receptor–channel complex has been reported to possess the intracellular structure that can be phosphorylated, leading to the modulation of the receptor–channel complex-mediated actions by calcium dependent protein kinase (PKC) [24,27]. We used repatch technique to explore whether suppression of I ATP by TMP was due to intracellular transduction pathways of PKC. In the control experiment with the normal internal solution, externally applied TMP (1 mM) inhibited ATP (10 AM)-activated current in rat DRG neurons. When the amplitude of I ATP returned to its original level, chelerythrine (10 AM, a PKC inhibitor) was introduced into the recording pipette in repatch test. After 20 min with pipette solution containing chelerythrine (10 AM), there was a decrease in the amplitude of I ATP. Externally applied TMP (1 mM) increases the inhibitory effect of chelerythrine (10 AM) contained in pipette solution on ATP-activated current (Fig. 5). In addition, with pipette solution containing 10 AM chelerythrine (n = 6) in normal patch test, the effects of externally applied TMP (1 mM) on ATP-activated currents were similar to that in repatch test.
4. Discussion In this experiment, the majority of freshly isolated neurons examined (94.6%, 88/93) were sensitive to ATP
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applied externally. It has been showed that there are two distinct ATP-activated currents observed in small and medium cells (but not in large cells) of rat DRG: one is a rapidly desensitizing ATP-activated current; the other is a slowly desensitizing ATP-activated current [4,16]. Smalldiameter (b30 AM) DRG neurons were sensitive to capsaicin and had ATP-activated current with a rapid desensitization; medium diameter (30–50 AM) DRG neurons were insensitive to capsaicin and had ATP-activated current with a slow desensitization [16]. The P2X3 receptor subunit had been cloned from rat DRG cDNA library [6,13] and expressed in capsaicin-sensitive, small-sized DRG neurons [6]. Additionally, it was reported that capsaicinsensitive, small-sized DRG neurons expressed mainly the homomeric P2X3 subtype and that capsaicin-insensitive, medium-sized DRG neurons expressed heteromultimeric complex of P2X2 and P2X3 subtypes [26]. In our experiment, the types of ATP-activated currents include rapidly desensitizing and slowly desensitizing. The reversal potential for activation of P2X receptors is close to 0 mV [7]. Our study indicated that the reversal potential of current activated by 100 AM ATP was close to 0 mV in the absence and presence of 1 mM TMP. Therefore, ATP-activated current in this study might mainly activated P2X receptors. a,h-meATP has the effects on P2X1, P2X3, and P2X2/3 receptors [7]. The fact that TMP inhibits a,h-meATPactivated currents supports the above opinion. It is evident from Fig. 1 that the inhibition of amplitude of ATP-activated currents increases gradually with the enhancement of the concentration of TMP from 0.1 to 1 mM. However, when the concentration of TMP increased to 1 mM, the inhibitory effect of TMP on ATP-activated currents did not increase further. The phenomenon is similar to another work which has shown that the inhibition of metEnk on ATP-activated currents was gradually increased from 0.0001 to 0.01 AM and was gradually decreased from 0.01 to 1 AM [30]. The decrease in inhibitory effect of TMP on I ATP may be nonspecific. The inhibitory effect of TMP on ATP-activated currents was both in peak value (Ip) and in steady state (Iss). This is apparent in the records demonstrated in Fig. 2. It is considered that this inhibition may result from both the rapid component of desensitizing current and the slow component of desensitizing current. From the comparison between the dose–response curves for ATP with or without pretreatment of TMP in Fig. 3, it is clear that: (1) preapplication of TMP shifts the curve downwards markedly; (2) the maximal amplitude of ATPactivated current pretreated with TMP decreases by 41.3%, while the threshold value remains unchanged; (3) the two EC50 values are very close (75 vs. 82 AM). Therefore, it is reasonable to consider that the inhibitory effect of TMP on ATP-activated current may be noncompetitive. Our work showed that chelerythrine (PKC inhibitor) contained in the pipette solution inhibited the amplitude of ATP-activated current. The result suggests that the functions of ATP receptor–ion channel complex can be modulated intracellu-
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larly by phosphorylation. A decrease in the PKC activity reduces the ATP receptor-mediated responses in rat DRG neuron. Simultaneously externally applied TMP increases the inhibitory effect of chelerythrine contained in the pipette solution on ATP-activated current. Seven P2X receptor suptypes (P2X1–7) possess two transmembrane domains, intracellular N- and C-termini and a large extracellular loop [28]. The N-terminal region is of similar size for all P2X receptor isoforms, with a conserved PKC site that appears to be phosphorylated constitutively [19]. Mutation to remove a consensus PKC site from the N-terminus (T 18) can lead to speeding of channel desensitization [8,28]. It is suggested that the intracellular N-terminus participates directly in the regulation of ionic conduction at P2X receptors, possibly contributing to the intracellular pore vestibule. The effect of PKC inhibitor in this study supports the above opinion. In addition, TMP may promote the phosphorylation of PKC site of the N-terminus to potentiate the inhibitory effect of PKC inhibitor on ATP-activated current. TMP inhibited ATP-activated current in a voltageindependent manner. The mechanism of inhibition of ATPactivated current include open-channel block. Open-channel block is usually voltage-dependent. However, open-channel block has voltage-independent when agents bind to a site within the ion channel but beyond the influence of the membrane electrical field [10,15]. In addition, the site of agent action is on or near the exterior surface of channel protein and not within the channel pore, where the membrane electrical field would not be influenced. P2X receptors can be regulated allosterically by extracellular protons, divalent cations, and a range of metals [19]. The time course of inhibition of TMP was from the onset of 15 s to the peak of 45 s. The site of TMP action may include the extracellular region and the cytoplasmic region or is mainly the extracellular region. The regulation site of the allosterism includes the action site of the intracellular signal transduction system [11] and the action site of the extracellular region [14,15,28]. It is possible that the mechanism of TMP action is the binding of TMP to an allosteric site on the large extracellular region of ATP receptor-ion channel complex and promoting the phosphorylation of PKC site of the Nterminus. This is in agreement with the effects of other P2X receptor antagonists, that is, P2X receptor antagonists act at the extracellular region of the ATP-gated receptor–channel complex [3,14,29]. It was reported that there was an expression of P2Y receptors in rat DRG [23]. In DRG, P2Y1 receptor immunoreactivity was very often coexpressed with P2X3 receptor immunoreactivity. P2Y1 and P2Y2 receptors increased the excitability of sensory neurons that were likely to be nociceptive [12]. It is not clear whether TMP may affect P2Y receptors in rat DRG. For this reason, further studies will be required to determine the precise mechanism responsible for the effects of TMP. In conclusion, TMP can inhibit ATP-activated current in rat DRG neurons. The site of TMP action may be the binding of TMP to an allosteric site on the large extracellular region
of ATP receptor–ion channel complex (P2X receptors) or PKC site of the N-terminus of P2X receptors. The mechanism of TMP action may be the allosteric regulation acting on the large extracellular region of ATP receptor–ion channel complex (P2X receptors) and promoting the phosphorylation of PKC site of the N-terminus of P2X receptors.
Acknowledgment This work was supported by the National Natural Science Foundation of China (No. 30260030) to S.D. Liang.
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