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The inhibitory effect of α-methyl-5-HT on ATP-activated currents in rat dorsal root ganglion neurons

Abstract

The purpose of this study was to explore the inhibitory effect of α-methyl-5- hydroxytryptamine (α-methyl-5-HT) on ATP-activated current (IATP) in rat dorsal root ganglion (DRG) neurons. Whole-cell patch clamp experiment was performed on cultured rat DRG neurons. One minute after treatment with α-methyl-5-HT, ATP (10-4 mol/L) activation current in rat DRG neurons was inhibited. However, this inhibitory effect was independent of the current caused by α-methyl-5-HT. The dose-response curve for IATP showed that α-methyl-5-HT significantly shifted it. The Kd values of ATP-activated currents before and after the pre-addition of α-methyl-5-HT were similar (4.23×10-5 mol/L vs. 6.81×10-5 mol/L). Furthermore, cyproheptadine (10-6 mol/L), an antagonist of 5-HT2 receptor, can reverse the inhibition of α-methyl-5-HT. After intracellular dialysis of KN93 (CaMKII inhibitor) and H7 (PKC inhibitor), this inhibition was also completely eliminated. In conclusion, our results showed that α-methyl-5-HT inhibited ATP-activated current through activating the 5-HT2 receptor and resulting in phosphorylation of the ATP receptor. It was caused by the activation of G protein coupled receptor and corresponding intracellular signaling transduction cascade.

Keywords:
ATP-activated current; α-methyl-5-HT; DRG

1 Introduction

The spinal dorsal horn is the primary center of sensory information integration. It is not only the primary gateway of peripheral nociceptive information transmission, but also the the termination site of the descending system which originates from the brainstem and inhibits the noxious information transmission. It has a significant effect on transmission and regulation of nociceptive information at the spinal level. Spinal dorsal root ganglion (DRG) is the location of primary sensory neurons. It was reported that ATP transmitters released by DRG neurons and membrane ATP receptors mediated responses were related to the transmission and modulation of pain information (Hu et al., 2017Hu, J., Qin, X., Song, Z. Y., Yang, P. P., Feng, Y., Sun, Q., Xu, G. Y., & Zhang, H. H. (2017). Alpha-lipoic Acid suppresses P2X receptor activities and visceral hypersensitivity to colorectal distention in diabetic rats. Scientific Reports, 7(1), 3928. http://dx.doi.org/10.1038/s41598-017-04283-7. PMid:28659591.
http://dx.doi.org/10.1038/s41598-017-042...
; Lü et al., 2017Lü, Y. F., Yang, Y., Li, C. L., Wang, Y., Li, Z., & Chen, J. (2017). The locus coeruleus-norepinephrine system mediates empathy for pain through selective up-regulation of P2X3 receptor in dorsal root ganglia in rats. Frontiers in Neural Circuits, 11, 66. http://dx.doi.org/10.3389/fncir.2017.00066. PMid:28979194.
http://dx.doi.org/10.3389/fncir.2017.000...
).

ATP is widely present in and outside animal cells. In addition to its important function in cell metabolism, extracellular ATP and its metabolite adenosine can have a rolein a series of biological processes, all of which are achieved through P1 and P2 receptors (Lü et al., 2017Lü, Y. F., Yang, Y., Li, C. L., Wang, Y., Li, Z., & Chen, J. (2017). The locus coeruleus-norepinephrine system mediates empathy for pain through selective up-regulation of P2X3 receptor in dorsal root ganglia in rats. Frontiers in Neural Circuits, 11, 66. http://dx.doi.org/10.3389/fncir.2017.00066. PMid:28979194.
http://dx.doi.org/10.3389/fncir.2017.000...
). ATP acts on primary sensory neurons as an excitatory neurotransmitter (Bele & Fabbretti, 2016Bele, T., & Fabbretti, E. (2016). The scaffold protein calcium/calmodulin-dependent serine protein kinase controls ATP release in sensory ganglia upon P2X3 receptor activation and is part of an ATP keeper complex. Journal of Neurochemistry, 138(4), 587-597. http://dx.doi.org/10.1111/jnc.13680. PMid:27217099.
http://dx.doi.org/10.1111/jnc.13680...
). In 1983, Krishtal first confirmed the presence of ATP receptors in primary sensory neurons (Kristal & Marchenko, 1986Kristal, O. A., & Marchenko, S. M. (1986). Pharmacological properties of the receptor for ATP in the sensory neurons of rats. Biomedical Research, 7, 79-81.). Thereafter, the pharmacological properties of ATP receptors in DRG cell membrane (Bean, 1990Bean, B. P. (1990). ATP-activated channels in rat and bullfog sensory neurons: concentration dependence and kinetics. The Journal of Neuroscience, 10(1), 1-10. http://dx.doi.org/10.1523/JNEUROSCI.10-01-00001.1990. PMid:1688928.
http://dx.doi.org/10.1523/JNEUROSCI.10-0...
), the kinetics of receptor activation (Krishtal et al., 1988Krishtal, O. A., Marchenko, S. M., & Obukhov, A. G. (1988). Cationic channels activated by extracellular ATP in rat sensory neurons. Neuroscience, 27(3), 995-1000. http://dx.doi.org/10.1016/0306-4522(88)90203-5. PMid:2855265.
http://dx.doi.org/10.1016/0306-4522(88)9...
), the ionic mechanism of ATP-activated current (Li et al., 1993Li, C., Peoples, R. W., Li, Z., & Weight, F. F. (1993). Zn2+ potentiates excitatory action of ATP on mammalian neurons. Proceedings of the National Academy of Sciences of the United States of America, 90(17), 8264-8267. http://dx.doi.org/10.1073/pnas.90.17.8264. PMid:7690146.
http://dx.doi.org/10.1073/pnas.90.17.826...
) and its modulation (Hu & Li, 1996Hu, H. Z., & Li, Z. W. (1996). Substance P potentiates ATP-activated currents in rat primary sensory neurons. Brain Research, 739(1-2), 163-168. http://dx.doi.org/10.1016/S0006-8993(96)00821-9. PMid:8955936.
http://dx.doi.org/10.1016/S0006-8993(96)...
) were studied in depth. We have done a series of work on the interaction between ATP receptors and other receptors in DRG cells (Wang et al., 2001Wang, M. J., Xiong, S. H., & Li, Z. W. (2001). Neurokinin B potentiates ATP-activated currents in rats DRG neurons. Brain Research, 923(1-2), 157-162. http://dx.doi.org/10.1016/S0006-8993(01)03211-5. PMid:11743983.
http://dx.doi.org/10.1016/S0006-8993(01)...
; Skagerberg & Lindvall, 1985Skagerberg, G., & Lindvall, O. (1985). Organization of diencephalic dopamine neurons projecting to the spinal cord in the rat. Brain Research, 342(2), 340-351. http://dx.doi.org/10.1016/0006-8993(85)91134-5. PMid:4041835.
http://dx.doi.org/10.1016/0006-8993(85)9...
).

5-hydroxytryptamine (5-HT) and ATP play a role as neurotransmitters by directly activating cationic channels in the postsynaptic membrane, which are named 5-HT3 and P2X receptors, respectively (Kamendi et al., 2008Kamendi, H. W., Cheng, Q., Dergacheva, O., Frank, J. G., Gorini, C., Jameson, H. S., Pinol, R. A., Wang, X., & Mendelowitz, D. (2008). Recruitment of excitatory serotonergic neurotransmission to cardiac vagal neurons in the nucleus ambiguus post hypoxia and hypercapnia. Journal of Neurophysiology, 99(3), 1163-1168. http://dx.doi.org/10.1152/jn.01178.2007. PMid:18184887.
http://dx.doi.org/10.1152/jn.01178.2007...
). Previous study suggested the inhibitory interactions between 5-HT3 and P2X channels in submucosal neurons (Barajas-López et al., 2002Barajas-López, C., Montaño, L. M., & Espinosa-Luna, R. (2002). Inhibitory interactions between 5-HT3 and P2X channels in submucosal neurons. American Journal of Physiology. Gastrointestinal and Liver Physiology, 283(6), G1238-G1248. http://dx.doi.org/10.1152/ajpgi.00054.2002. PMid:12388197.
http://dx.doi.org/10.1152/ajpgi.00054.20...
). It also has been reported that 5-HT regulates the desensitization kinetics of P2X1 responses by increasing their rate of recovery via the 5-HT2A metabotropic receptor (Ase et al., 2005Ase, A. R., Raouf, R., Bélanger, D., Hamel, E., & Séguéla, P. (2005). Potentiation of P2X1 ATP-gated currents by 5-hydroxytryptamine 2A receptors involves diacylglycerol-dependent kinases and intracellular calcium. The Journal of Pharmacology and Experimental Therapeutics, 315(1), 144-154. http://dx.doi.org/10.1124/jpet.105.089045. PMid:15958718.
http://dx.doi.org/10.1124/jpet.105.08904...
). Therefore, the purpose of this study was to explore the effects of α-methyl-5-HT on IATP in rat DRG neurons and and its potential mechanism.

2 Materials and methods

2.1 Cell isolation and culture

All experimental procedures were carried out in accordance with Chinese legislation and National Institutes of Health (NIH) publications. The isolation method of rat DRG neuron specimens and whole-cell patch clamp experiment was performed as mentioned earlier with minor modifications (Wang et al., 2001Wang, M. J., Xiong, S. H., & Li, Z. W. (2001). Neurokinin B potentiates ATP-activated currents in rats DRG neurons. Brain Research, 923(1-2), 157-162. http://dx.doi.org/10.1016/S0006-8993(01)03211-5. PMid:11743983.
http://dx.doi.org/10.1016/S0006-8993(01)...
). Spraque-Dawley (SD) rats, 4-5 week-old, were anesthetized with ether and put to death by dislocation. We dissected the thoracic and lumbar segments of vertebrate cylinder, and divided them into two parts longitudinally along the midline of dorsal and ventral sides. The DRG, dorsal root, ventral root and spinal nerve were removed from the inner side of each half of the dissected vertebrae and transferred to oxygen saturated Dulbecco’s Modified Eagle’s Medium (DMEM, Sigma) immediately under pH 7.4 and osmotic pressure 340 mOsm/kg. After removing the attached nerve and surrounding connective tissue, DRGs were cut up and put into the culture flask containing trypsin (type III, Sigma), collagenase (type I A, Sigma) and DNase (type III, Sigma). The culture flask was incubated in a constant temperature oscillating water bath (35 °C, 80 times/min) for 30-40 min. After adding soybean trypsin inhibitor (type II-s, Sigma) to stop trypsin digestion, the neurons were stored in a 35-mm culture dish for at least 30 min before the experiment. The neurons with diameter of 20-45 μ M were selected in this study.

2.2 Whole-cell patch clamp

The experiment was performed at 22-25 °C. The patch/whole-cell clamp amplifer (CEZ-2400, Nihon Kohden) was used to perform whole-cell patch-clamp recording. The inner liquid component of the glass microelectrode included KCl 140, CaCl2 1, MgCl2 2, HEPES 10, EGTA 11 and ATP 4 (in mmol/L). The external solution contained NaCl 150, KCl 5, CaCl2 2.5, MgCl2 1, HEPES 10 and D-glucose 10 (in mmol/L). Sucrose was used to adjust osmotic osmolarity to 340 mosM/kg and KOH or NaOH was used to adjust pH to 7.4 in inner solution and external solution. The resistance of recording electrodes was between 2 and 4 MΩ. A small patch of membrane underneath the tip of the pipette was aspirated to form a gigaseal and a larger negative pressure was applied to rupture it to establish a whole-cell mode. Before recording the membrane current, the capacitance compensation and series resistance compensation were adjusted. The holding potential was set to -60 mV unless otherwise indicated. Membrane currents were filtered at 10 Hz (3dB). The pen recorder (Nihon Kohden) was used to record.

2.3 Reagent preparation

ATP (Sigma) and α-methyl-5-HT (an agonist of 5-HT2 receptor, RBI) were formulated with an external solution in which the pH was adjusted to 7.4 with 1 M NaOH. Cyproheptadine (an antagonist of 5-HT2 receptor, Sigma), KN93 (RBI) and H7 (RBI) were formulated with an internal solution in which the pH was adjusted to 7.2 with 1 M KOH.

2.4 Statistical analysis

Statistical analysis was performed by using SPSS 20.0 (SPSS Inc., USA). All data were expressed as means ± SEM. The differences between groups were analyzed by t test. P < 0.05 was considered to be statistically significant. The graph was drawn by using SigmaPlot software.

3 Results

3.1 ATP activated currents in DRG neurons

It is well known that the response of DRG neurons to externally applied ATP with inward currents is in a concentration dependent manner. In order to facilitate patch clamp recording, the experiment was carried out on cells with the diameter of 25-45 μm in this study. A total of 141 cells were detected in this study. 88.65% of cells (125/141) were sensitive to externally applied ATP (10-5 - 3×10-3 mol/L) with a response of inward current. In addition, 10.64% cells (15/141) were unresponsive, of which only one was an outward current (1/141, 0.71%).

The dual stimulation method was applied to detect the recovery time of ATP-activated current (IATP) desensitization, that was, the interval between the first and second ATP additions was 2 min, 3 min, 4 min, and 5 min, respectively. The results showed that the current caused by the application of ATP at 5 minutes was the same as that of the first time, indicating that the desensitization recovery time of ATP receptor was 5 minutes (Figure 1).

Figure 1
Time course of the recovery of ATP-activated currents. *P<0.05 vs. control group.

3.2 α-methyl-5-HT inhibited ATP activated current

There were at least 5 min interval between the first ATP test and the 2nd one. After pre-adding α-methyl-5-HT (10-10 - 10-5 mol/L) to ATP-sensitive cells, 72.4% of cells caused inhibition of ATP-activated current, a small part was increased (22.4%) and the rest did not respond (5.2%). This inhibitory effect was independent of whether α-methyl-5-HT itself caused membrane current. In other words, IATP can be inhibited in the case of α-methyl-5-HT itself whether it causes outward, inward or not (Figure 2).

Figure 2
The relationship between α-methyl-5-HT induced response and α-methyl-5-HT inhibition on IATP. The three rows of current traces show that α-methyl-5-HT could inhibit IATP whetherα-methyl-5-HT itself induced an inward current (middle row), outward current (low row), or no response (upper row). *P<0.05 vs. control group.

As shown in Figure 3, α-methyl-5-HT inhibited IATP with a concentration-dependent manner. From 10-10 mol/L, the inhibition was gradually increased with the increase of concentration, reaching its peak at 10-7 mol/L. Interestingly, as the concentration continued to increase (10-6 and 10-5 mol/L), the inhibition was reduced, which may be a non-specific effect of high drug concentration (Figure 3).

Figure 3
Dose-response curve for the inhibition of IATP byα-methyl-5-HT. An example of single neuronal recording of IATP (10-4 mol/L, HP = -60 mV) pretreated with six different concentrations of α-methyl-5-HT was illustrated both in the top. The bottom graph dose-response curve for the inhibition byα-methyl-5-HT of ATP-activated currents. Data points are the mean ± SEM of 5-HT. *P<0.05 vs. control group.

In order to study the inhibitory effect of pre-addition time of α-methyl-5-HT (10-7 mol/L) on IATP, the inhibitory effects were recorded after 15 s, 30 s, 1 min and 2 min, respectively. The results suggested that the inhibition of α-methyl-5-HT was most pronounced at 1 min. (n=30, p<0.05) (Figure 4).

Figure 4
Time course ofα-methyl-5-HT inhibition on IATP.

In addition, cyproheptadine, an antagonist of 5-HT2 receptor, can reverse the inhibition of α-methyl-5-HT on IATP (Figure 5).

Figure 5
Reversal by cyproheptadine of α-methyl-5-HT inhibition on IATP.

3.3 Concentration–response relationship of ATP-activated current with or withoutα-methyl-5-HT

Figure 6 showed the dose-effect curve of IATP (10-5-3×10-3 mol/L) after adding 10-7 mol/L α-methyl-5-HT for 1 min. The results suggested that: (1) The dose-effect curve of α-methyl-5-HT+ATP was significantly lower than that of control ATP; (2) After pre-adding α-methyl-5-HT, the maximum amplitude of ATP-activated current was inhibited up to 70%; (3) The Kd values of ATP-activated currents before and after pre-adding α-methyl-5-HT were similar, which were 4.23×10-5 mol/L and 6.81×10-5 mol/L, respectively; (4) The threshold concentration values of them are consistent (10-5 mol/L).

Figure 6
Concentration-response relationship for ATP currents with (〇) and without (●) preapplication of α-methyl-5-HT. The graph shows the concentration-response curves for ATP currents with and without reapplication of α-methyl-5-HT(10-7mol/L). Each point represents the mean ± SEM. of IATP of 4-7 neurons. All ATP-activated currents were normalized to the peak current induced by 10-4mol/L ATP (marked with asterisk). Holding potential was set at -60 mV. The curve for ATP alone is a good fit of data to the logistic equation Y=Emax/[1+(Kd/C) n], where C is the concentration of ATP; Y, the normalized currents expressed as fraction of the maximum current response value. Kd, the dissociation constant of ATP receptor. The curve was drawn according to the equation described above assuming Hill coefficient is 0.67 (which was determined by Hill plotting). The curve for IATP withα-methyl-5-HT pretreatment was drawn by eye. *P<0.05 vs. control group.

3.4 Effect of α-methyl-5-HT on the current-voltage relationship of ATP-activated current

Figure 7 was an I-V curve of pre-addition of 10-7 mol/L α-methyl-5-HT (1 min) to inhibit ATP-activated current. The results showed that: (1) The two curves of control ATP and α-methyl-5-HT+ATP were linear in the range of -100 to +40 mV; (2) In the negative voltage (-100 to 0 mV) or positive voltage (0 to +40 mV) interval, the voltage values of voltage points of α-methyl-5-HT+ATP curve were smaller than those of control ATP curve; (3) Compared with control ATP, the reversal potential value of α-methyl-5-HT+ATP curve was basically inconvenient, both of which were about 0 mV.

Figure 7
Current-voltage (I-V) relationship for IATP without pretreatment of α-methyl-5-HT. Graph shows the I-V relationship for IATP (10-4 mol/L) with and without preapplication of α-methyl-5-HT (10-7mol/L), in which the values of reversal potentials in both cases are basically the same (around 0 mV). All currents recorded were normalized to the peak value of IATP without pre-treatment of α-methyl-5-HT voltage clamped at -60 mV (mark with asterisk). With pre-application of α-methyl-5-HT the inhibition of IATP is linear. HP: H Potential.

3.5 Intracellular transduction mechanism of α-methyl-5-HT inhibiting ATP- activated current

As shown in Figure 8, the inhibition of ATP-activated current by α-methyl-5-HT could be completely eliminated by KN93 (CaMK II inhibitor, 10 mmol/L) in comparison with the control record (n=6, p < 0.05). In addition, when combined with 200 μmol/L H7 (inhibitor of PKC) and 10-2 mmol/L BAPTA, the inhibition of α-methyl-5-HT on ATP can also be blocked.

Figure 8
Evidence for the abolishment of inhibition ofα-methyl-5-HT on IATP by intracellular dialysis of KN93. (A) The left pair of current traces show the inhibition byα-methyl-5-HT (10-7 mol/L) of ATP (10-4 mol/L) activated current with the micropipette filled with normal internal solution. Whereas, as can be seen from the right pair of current traces there is no significant difference between the amplitudes of ATP (10-4 mol/L) activated currents with and without pre-application of α-methyl-5-HT (10-7 mol/L) when the micropipette was filled with KN93 internal solution; (B) Histogram demonstrates the removal of inhibition byα-methyl-5-HT of IATP by intracellular dialysis of KN93. *P<0.05 vs. control group.

4 Discussion

In our study, 88.65% freshly isolated neurons (125/141) were sensitive to externally applied ATP. After pre-adding α-methyl-5-HT to ATP-sensitive cells, 72.4% of cells caused inhibition of ATP-activated current. The inhibitory effect of α-methyl-5-HT on IATP was concentration-dependent, and was most pronounced at 1 min.

As one of the main neurotransmitters of primary sensory neurons, ATP has an significant effect on the generation, regulation and transmission of nociceptive information (Nishida et al., 2014Nishida, K., Nomura, Y., Kawamori, K., Moriyama, Y., & Nagasawa, K. (2014). Expression profile of vesicular nucleotide transporter (VNUT, SLC17A9) in subpopulations of rat dorsal root ganglion neurons. Neuroscience Letters, 579, 75-79. http://dx.doi.org/10.1016/j.neulet.2014.07.017. PMid:25043192.
http://dx.doi.org/10.1016/j.neulet.2014....
). ATP receptors belong to purine receptors, and there are two categories: one is adenosine receptor (P1) and the other is ATP receptor (P2) (Nishida et al., 2014Nishida, K., Nomura, Y., Kawamori, K., Moriyama, Y., & Nagasawa, K. (2014). Expression profile of vesicular nucleotide transporter (VNUT, SLC17A9) in subpopulations of rat dorsal root ganglion neurons. Neuroscience Letters, 579, 75-79. http://dx.doi.org/10.1016/j.neulet.2014.07.017. PMid:25043192.
http://dx.doi.org/10.1016/j.neulet.2014....
). There are a variety of receptors that bind to ATP, and there are two known: P2Y receptor, which activates phospholipase C through G-protein coupling, P2X receptor, which is ligand-gated ion channels (Ralevic & Burnstock, 1998Ralevic, V., & Burnstock, G. (1998). Receptors for purines and pyrimidines. Pharmacological Reviews, 50(3), 413-492. PMid:9755289.). Seven subunits of P2X have been cloned (North & Surprenant, 2000North, R. A., & Surprenant, A. (2000). Pharmacology of cloned P2X recetors. Annual Review of Pharmacology, 40(1), 563-580. http://dx.doi.org/10.1146/annurev.pharmtox.40.1.563. PMid:10836147.
http://dx.doi.org/10.1146/annurev.pharmt...
; Ying et al., 2017Ying, M., Liu, H., Zhang, T., Jiang, C., Gong, Y., Wu, B., Zou, L., Yi, Z., Rao, S., Li, G., Zhang, C., Jia, T., Zhao, S., Yuan, H., Shi, L., Li, L., Liang, S., & Liu, S. (2017). Effect of artemisinin on neuropathic pain mediated by P2X<sub>4</sub> receptor in dorsal root ganglia. Neurochemistry International, 108, 27-33. http://dx.doi.org/10.1016/j.neuint.2017.02.004. PMid:28192150.
http://dx.doi.org/10.1016/j.neuint.2017....
), which contain two transmembrane domains, M1 and M2, and the extracellular loop is connected between M1 and M2, and the N and C ends are located intracellularly. The intracellular phosphorylation sites are located at the C-terminal (Van Eck et al., 2005Van Eck, M., Pennings, M., Hoekstra, M., Out, R., & Van Berkel, T. J. (2005). Scavenger receptor BI and ATP-binding cassette transporter A1 in reverse cholesterol transport and atherosclerosis. Current Opinion in Lipidology, 16(3), 307-315. http://dx.doi.org/10.1097/01.mol.0000169351.28019.04. PMid:15891392.
http://dx.doi.org/10.1097/01.mol.0000169...
).

The most noteworthy function of the P2X receptor in primary sensory neurons is related to pain perception. P2X3 receptors only exist in sensory neurons and are only expressed in small diameter DRG cells related to nociception. Therefore, ATP receptors, especially the P2X3 receptor, have an effect on the production and transmission of pain sensation (Van Eck et al., 2005Van Eck, M., Pennings, M., Hoekstra, M., Out, R., & Van Berkel, T. J. (2005). Scavenger receptor BI and ATP-binding cassette transporter A1 in reverse cholesterol transport and atherosclerosis. Current Opinion in Lipidology, 16(3), 307-315. http://dx.doi.org/10.1097/01.mol.0000169351.28019.04. PMid:15891392.
http://dx.doi.org/10.1097/01.mol.0000169...
). In our experiment, the reversal potential for activation of P2X receptors was close to 0 mV. Our results suggested that the reversal potential of ATP-activated current was close to 0 mV with or without 10-7 mol/L α-methyl-5-HT (1 min) (Liang et al., 2005Liang, S. D., Xu, C. S., Zhou, T., Liu, H. Q., Gao, Y., & Li, G. L. (2005). Tetramethylpyrazine inhibits ATP-activated currents in rat dorsal root ganglion neurons. Brain Research, 1040(1-2), 92-97. http://dx.doi.org/10.1016/j.brainres.2005.01.076. PMid:15804430.
http://dx.doi.org/10.1016/j.brainres.200...
), which indicated that ATP-activated current in our study might mainly activated P2X receptors.

The 5-TH receptor can be classified into 5-HT1-5-HT7 and other types. Except that 5-HT3 receptor is a Ligand-gated ion channel receptor superfamily, the rest are G-protein coupled receptor (Kluess et al., 2005Kluess, H. A., Buckwalter, J. B., Hamann, J. J., & Clifford, P. S. (2005). Elevated temperature decreases sensitivity of P2X purinergic receptors in skeletal muscle arteries. Journal of Applied Physiology, 99(3), 995-998. http://dx.doi.org/10.1152/japplphysiol.00319.2005. PMid:15890753.
http://dx.doi.org/10.1152/japplphysiol.0...
). In this experiment, it was observed that α-methyl-5-HT, a 5-HT2 receptor agonist, sometimes caused an obvious inward current (21.5%, 3/14), but no matter what reaction α-methyl-5-HT caused, it had nothing to do with its inhibition of ATP.

From the I-V curve of Figure 8, it can be seen that compared with the control ATP curve, the current value of α-methyl-5-HT+ATP curve was linearly decreased, that was to say, the suppression of ATP-activated current by α-methyl-5-HT had nothing to do with the change of voltage. It indicated that this inhibition was not due to channel blockage. Figure 7 showed that the dose-effect curve and Kd value of ATP-activated current after pre-addition of α-methyl-5-HT was similar to that of the control ATP curve, but the current amplitude was significantly decreased at the maximum concentration and the α-methyl-5-HT curve moved down. It suggested that the inhibitory effect of α-methyl-5-HTwas non-competitive, that was, α-methyl-5-HT was not the result of competitive action on ATP receptor agonist binding sites. At the same time, the inhibitory effect was shifted by cyproheptadine, a 5-HT2 receptor antagonist, which proved once again that α-methyl-5-HT acted through 5-HT2 receptor.

As mentioned above, α-methyl-5-HT receptor is a G-protein coupled receptor. Therefore, we can imagine that phospholipase C (PLC) can be activated by G-protein after 5-HT2 receptor activation. PLC can decompose PIP2 (phosphatidylinositol 4, 5-diphosphate) to produce two important second messengers, IP3 (inositol triphosphate) and DC (glycerol diester). DC may phosphorylate ATP receptors by activating PKC (protein kinase C). In future research, we can further explore this mechanism.

Acknowledgements

Thank to Professor Zhiwang Li, Laboratory of Neurobiology, Tongji Medical College, Huazhong University of Science and Technology, for providing financial resources and theoretical guidance, and Chief Technician Shiduan Wang for providing instrument maintenance. Thank to Professor Suming Zhang of Tongji Hospital Affiliated to Tongji Medical College of Huazhong University of Science and Technology for his teaching. This work was supported by the National Natural Science Foundation of China (nº. 39870259, nº. 39700041).

  • Practical Application: α-methyl-5-HT inhibited ATP-activated current through activating the 5-HT2 receptor and resulting in phosphorylation of the ATP receptor. This study is of importance for understanding the interaction between ATP receptors and other receptors in DRG cells.

References

  • Ase, A. R., Raouf, R., Bélanger, D., Hamel, E., & Séguéla, P. (2005). Potentiation of P2X1 ATP-gated currents by 5-hydroxytryptamine 2A receptors involves diacylglycerol-dependent kinases and intracellular calcium. The Journal of Pharmacology and Experimental Therapeutics, 315(1), 144-154. http://dx.doi.org/10.1124/jpet.105.089045 PMid:15958718.
    » http://dx.doi.org/10.1124/jpet.105.089045
  • Barajas-López, C., Montaño, L. M., & Espinosa-Luna, R. (2002). Inhibitory interactions between 5-HT3 and P2X channels in submucosal neurons. American Journal of Physiology. Gastrointestinal and Liver Physiology, 283(6), G1238-G1248. http://dx.doi.org/10.1152/ajpgi.00054.2002 PMid:12388197.
    » http://dx.doi.org/10.1152/ajpgi.00054.2002
  • Bean, B. P. (1990). ATP-activated channels in rat and bullfog sensory neurons: concentration dependence and kinetics. The Journal of Neuroscience, 10(1), 1-10. http://dx.doi.org/10.1523/JNEUROSCI.10-01-00001.1990 PMid:1688928.
    » http://dx.doi.org/10.1523/JNEUROSCI.10-01-00001.1990
  • Bele, T., & Fabbretti, E. (2016). The scaffold protein calcium/calmodulin-dependent serine protein kinase controls ATP release in sensory ganglia upon P2X3 receptor activation and is part of an ATP keeper complex. Journal of Neurochemistry, 138(4), 587-597. http://dx.doi.org/10.1111/jnc.13680 PMid:27217099.
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Publication Dates

  • Publication in this collection
    29 Jan 2021
  • Date of issue
    2022

History

  • Received
    06 Aug 2020
  • Accepted
    09 Sept 2020
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