Real-time analysis of ATP concentration in acupoints during acupuncture: a new technique combining microdialysis with patch clamp
This paper introduces a new technique combining microdialysis with patch clamp to detect the changes in ATP (adenosine triphosphate) concentration in acupoints during acupuncture. The microdialysis probe was implanted into the Zusanli acupoint (ST 36) of adult SD (Sprague Dawley) rats to sample acupoint fluid containing ATP released during acupuncture. Then, the fluid with ATP was delivered in real time to 293 T cells overexpressing P2X3 receptors, with which we could carry out patch clamp experiments. The results showed that changes in membrane currents could reflect changes in the concentration of ATP. Thus, we can successfully detect ATP released in acupoints during acupuncture in real time. This technique provides us with a new way to study the mechanism of acupuncture signal initiation.
KeywordsMicrodialysis Patch clamp ATP Online real-time analysis Acupoint
293 T cells overexpressing P2X3 receptors
Transient receptor potential vanilloid
Microdialysis has been widely used in life sciences. Compared to other sampling techniques, microdialysis has distinctive advantages. The microdialysis probe can be placed directly, without causing much damage, into the target organs or tissues such as skin [1, 5, 16], muscle [10, 17], brain [6, 9, 11], blood vessels , and other areas. The sampling process can be continuous for hours or even days without major changes in the microenvironment. Moreover, macromolecules, such as proteins, are excluded from the semipermeable membrane so that the samples can be directly used for subsequent analysis without a cumbersome purifying procedure .
Due to its ability for high temporal resolution recording of ion channel currents from cells or cell-free membrane patches, the patch clamp technique has become the primary method for studying cell signal transduction mechanisms. Different types of ion channels have been studied, such as voltage-gated K+ channels , ligand-gated 5-HT3 (5-hydroxytryptamine) receptor channels , and mechanosensitive TRPV4 (transient receptor potential vanilloid) channels . In vivo experiments have also been carried out though there are many limitations because of the difficulties associated with in vivo studies .
Acupuncture has been used to treat numerous diseases. As the starting point of acupuncture, acupoints play an important role in analgesia. Physical stimuli is changed to biological signals, after a series of complicated process, it reaches the target organs or tissues . Having a better understanding of initiation of acupuncture will undoubtedly do us a favor to know the whole mechanism. Several studies indicate that there are more mast cells around acupoints than around non-acupoints , and these cells participate in the mechanism of acupuncture analgesia . During acupuncture, mast cells are activated to degranulate. Several mediators released by mast cells, such as histamine and adenosine activate sensory nerve fibers . The interaction of nerves, mast cells and chemicals around acupoints participates in the initial regulation of acupuncture . Among the mediators, ATP activates P2X3 receptors located on sensory nerve endings [4, 20]. When ATP is degraded to adenosine, it also induces analgesia through adenosine A1 receptors . If we can detect the dynamic changes of ATP concentration in acupoints during acupuncture, we can better understand the underlying mechanism of acupuncture. However, current sampling and analyzing techniques (e.g., microdialysis and HPLC, ) do not have high enough temporal resolution. Thus, we developed a new detection technique combining microdialysis with patch clamp to make it possible to analyze the substances in acupoints in real time.
Different concentrations of ATP can be detected by the system in real time in vitro
Changes in cell membrane currents are induced by ATP action on P2X3 receptors
Changes in the concentration of ATP in acupoints during acupuncture can be reflected by the system in real time
Summarized data of current-voltage relations are shown in Fig. 3b. Current densities with 50 μM ATP or acupuncture were 143.7 ± 21.8 pA/pF (P < 0.05) or 312.6 ± 48.8 pA/pF (P < 0.05), respectively. Both had significant differences compared to the control (60.7 ± 13.0 pA/pF) (Fig. 3c).
ATP plays an important role in the starting process of acupuncture. Current studies indicate that acupuncture can prompt the mast cells around acupoints to degranulate and release ATP, which activates P2X3 receptors of sensory nerve endings. Then, acupuncture signals are transduced, and acupuncture efficacy is achieved [4, 20]. We may have a better understanding of the underlying mechanism of acupuncture if we can determine the changes in ATP during acupuncture in real time. This new technique combining microdialysis with a patch clamp makes it possible to detect ATP electronically and in real time. We used microdialysis to extract ATP from the acupoints of the rats during acupuncture and delivered it to 293 T cells overexpressing P2X3 receptors for patch clamp recording. The results showed that changes in the concentration of ATP could be reflected by cell membrane currents in real time. Compared to other detecting techniques, our technique has a higher temporal resolution. This new technique also has the advantages of a small sampling volume and causing little damage to tissues.
However, there are still some challenges we must take into consideration. First, the distance between the microdialysis probe and the acupuncture needle can previously influence the concentration of ATP dialyzed out. However, both of them are under the skin, which means it’s difficult to keep them at the same distance during acupuncture. Maybe a visualized method is needed in the future to keep the results stable. Second, the results we obtain just qualitatively reflect the changes of concentration of ATP. Since the recovery of the microdialysis tube is affected by several factors, we haven’t make a calibration to get the quantitative results yet. It has to be finished in further experiments.
By choosing different cells expressing specific receptors, we can detect different types of mediators such as histamine and 5-HT online and in real time using our technique. As we know, acupuncture signals are conducted to central nervous system firstly, then to target organs or tissues. Apart from local acupoints, our technique can also be used in brain research or other research areas. We believe that this technique will have a broader application in the future.
All animal experiments have followed ARRIVE guidelines. SD rats were provided by the Shanghai laboratory animal center of Chinese Academy of Sciences. Before the experiment, the rats were anesthetized with chloral hydrate (0.4 ml/100 g). Fur around the Zusanli acupoint (ST 36) was shaved to expose the skin. A plastic annular tube with a larger diameter was inserted into the skin so that the microdialysis tube could be easily implanted into the issue. After this manipulation, animals were allowed to rest for 30 min.
The composition of the normal bath solution was (mM): 140 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 10 D-sorbitol, and 10 HEPES and was titrated to pH 7.4 with NaOH. The pipette solution had the following composition (mM): 140 CsCl, 1 CaCl2, 1 MgCl2, 5 EGTA, and 10 HEPES and was titrated to pH 7.2 with CsOH. Ringer’s solution was used as the perfusate through the microdialysis tube. All solutions were stored at 4 °C until use.
Microdialysis and whole-cell current recordings
During the entire recording process, a microdialysis tube (BASi Instruments, USA) was placed into Ringer’s solution, ATP standard solutions or SD rats. When the tube was implanted into the SD rats, acupuncture was carried out at the Zusanli acupoint (ST 36). The acupuncture needle (Hwato, China) was Ø 0.35 X 25 mm.
Whole-cell membrane currents were recorded by the patch-clamp method . Cells were placed into the chamber attached to an inverted microscope (Nikon, Japan) and superfused with the bath solution by gravity at a rate of 1 ml/min. The temperature of the external solution was at room temperature (25 °C). Patch pipettes were made from glass capillaries with a diameter of 1,5 mm (WPI, USA) using a horizontal microelectrode puller (model P− 97, Sutter Instrument, USA). The patch pipettes were filled with the pipette solution, and their resistance was 2–5 MΩ when they entered the bath solution. The glass pipette was held by an electrode probe connected to a patch-clamp amplifier (model EPC-10, HEKA, Germany). The probe was controlled by an electronic micromanipulator (model MPC-2000, Sutter Instrument, USA). Command pulse signals were generated using PATCHMASTER software (KEKA, Germany). Current data were also acquired by PATCHMASTER and stored on the hard drive disk of a computer (Samsung, South Korea). Recording signals were filtered at 2 kHz bandwidth, and series resistance was not compensated.
Statistical analyses were performed with one-way analysis of variance (ANOVA) followed by Student’s t-test for paired values. Changes were considered significant at P < 0.05. Data were expressed as the mean ± S.E.M.
Thanks to Prof. Wolfgang Schwarz from Shanghai Research Center of Acupuncture and Meridians for guidance with patch clamp. Thanks to Prof. Günther Schmalzing from RWTH Aachen University for providing P2X3 plasmid. Thanks to Prof. Ryszard Grygorczyk from Université de Montréal for guidance with ATP fluorescence experiment. Thanks to Dr. Hongwei Yang from Fudan University for helping with cell culture. Thanks to Dr. Lina Wang and Miss. Yawen Zheng from Shanghai University of Traditional Chinese Medicine for helping with animal experiments.
YW did the experiments, analyzed the data and was a major contributor in writing the manuscript. MH designed the experiment system and did part of the experiments. YX guided the writing of the manuscript. GHD gave suggestions on experiments and manuscript writing. All authors read and approved the final manuscript.
This work was supported by the Project of National Natural Science Foundation of China [no. 8157150277]; the National Basic Research Program of China [no. 2012CB518502].
Ethics approval and consent to participate
All animal experiments have followed ARRIVE guidelines.
Consent for publication
The authors declare that they have no competing interests.
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