Effect of Ca²⁺ complexation on oxygen partial pressure in the urinary bladder meridian of goats

Abstract

The aim of this paper was to explore the relationship between energy metabolism and the meridian phenomenon. The manner of change of oxygen partial pressure in acupoints and no-acupoints of the urinary bladder meridian of goats was observed with a needle-type tissue oxygen tension sensor after the acupuncture effect was blocked by Ca²⁺ complexation with ethylenediamine tetraacetic acid (EDTA)-Na₂. The results showed that: (1) the concentration of calcium ion in urinary bladder meridian acupoints was higher than that in no-acupoints (P<0.01 or P<0.05); (2) the tissue oxygen tension in urinary bladder meridian acupoints was higher than that in no-acupoints (P<0.01); (3) the oxygen partial pressure in acupoints decreased significantly after injection with EDTA-Na₂, compared with injection of either normal sodium or nothing (P<0.01); (4) the oxygen partial pressure in acupoints was higher than that in no-acupoints after EDTA-Na₂ injection (P<0.01). Acupuncture could reduce oxygen partial pressure in acupoints by regulating microcirculation and increasing metabolic levels.

Keywords

oxygen partial pressure / biosensor / calcium channels / acupoint

Introduction

The meridian doctrine is the core theory of traditional Chinese medicine. However, the essence of the meridian has not been elucidated up to now. It is well documented that acupoints do not comprise special tissues different from others in macroscopy. Thus, carrying out microcosmic research might be an important means to elucidate the essence of the meridian. Contemporary research has confirmed that calcium ion is an important physiologic regulatory factor in vivo. Calcium ion participates in many physiologic activities in cells as a second messenger, such as in excitation contraction coupling in myocytes and neurotransmitter secretion. Guo [1] found that the effect of needling point Zusanli (ST36) on inhibiting hypergastrokinesis would disappear as soon as Ca²⁺ was complexed. It implied that calcium ion might be connected with the meridian phenomenon. In the current experiment, we examined the manner of change of tissue oxygen partial pressure after the calcium ion was complexed with ethylenediamine tetraacetic acid (EDTA) to explore the mechanism of calcium ion in meridian signal conduction. The distribution characteristics of calcium ion was detected with a new needle-type calcium sensor Ca²⁺ in real-time and dynamically, and the manner of change of oxygen partial pressure was detected with a new needle-type tissue oxygen tension sensor.

Materials and methods

Experimental animals

Ten hybrid female Bohr goats, weighing 30-35 kg, were provided by the Animal Breeding and Veterinary College of Huazhong Agricultural University, Wuhan, China.

Materials

A new needle-type calcium sensor Ca²⁺, a needle-type tissue oxygen tension sensor, and a dual pathway oxygen partial pressure tester were provided by the Institute of Environmental Medicine of Huazhong University of Science and Technology. A PXSJ-216 Ion Analyzer was purchased from Shanghai Rex Instrument Factory. An SK-2200LH Ultrasonic Cleaning Instrument was from Shanghai Kudos Ultrasonic Instrument Co., Ltd, and the sodium chloride injection was from Wuhan BinHu Pharmaceutical Factory, and EDTA-Na₂ from Sigma.

Experimental methods

The goats were fixed on animal operation tables and injected with 25 mg chlorpromazine hydrochloride. A carbasus was set between the goat ear and normal sodium to create a salt bridge, and the calomel reference electrode was placed in a beaker filled with normal sodium.

Ganshu (BL18), Guanyuanshu (BL26), Dachangshu (BL25) and the points 3 cm left to each acupoint and the neighboring rhachis midline were chosen respectively to be the test points. The sensor was inserted into the test point at the same depth, without twirling or rotating.

The calcium ion value was recorded every 5 seconds after the calcium ion sensor was inserted in the test point 1 min later, and the recording time lasted 3 min. Because the process of transforming calcium ion electric potential values to concentration values was very complicated, calcium ion electric potential values were recorded directly.

The tissue oxygen tension value was recorded every 20 seconds after the needle-type tissue oxygen tension sensor was inserted in the test point 5 min later and when the zero drift of the oxygen tension sensor remained stable. The recording time lasted 5 min. In addition, the same test was performed after each test point was injected with 200 μL 0.05 mol/L EDTA-Na₂ and normal sodium, respectively.

The calcium ion electric potential value in the last 1 min and the tissue oxygen tension value in the last 2 min were taken as the valid data. All the values are presented as \overline{x}\pm s. The differences between acupoints and no-acupoints were evaluated by t test, and a P<0.05 was considered significant.

Results

The calcium ion electric potential value in urinary bladder meridian acupoints was higher than that in no-acupoints (Table 1).

The tissue oxygen tension value in urinary bladder meridian acupoints was higher than that in no-acupoints (Table 2). After the tissue was injected with EDTA-Na₂, the tissue oxygen tension in acupoints increased obviously compared to that with no injection and that injected with normal sodium (P<0.01). After the tissue was injected with EDTA-Na₂, the tissue oxygen tension in no-acupoints increased significantly compared to that with no injection and that injected with normal sodium (P<0.05), but it was still lower than that in acupoints (P<0.01).

Discussion

Research on the morphology of the meridian indicates that acupoints do not comprise special tissues different from others in macroscopy besides nerves, blood vessels and lymph vessels [2]. Therefore, carrying out a cytobiologic research in terms of molecular biology and biochemistry might be an important approach in elucidating the essence of the meridian.

Energy metabolism is the basic factor for maintaining vital movement, and oxygen is the fundamental material participating in the energy metabolism of the biosystem. According to traditional Chinese medicine theory, meridians refer to the routes that transport gas and blood, regulate Yin-Yang, connect the Zang-organs with the Fu-organs, and associate the external with the internal as well as the upper with the lower. This implies that meridians might be a special channel for energy metabolism and information transfer. Results from various studies suggest that acupuncture could obviously alter the tissue oxygen tension in acupoints, but the conclusions of different studies are inconsistent. Some researches found that acupuncture could obviously increase the tissue oxygen tension in acupoints, while others have the opposite results [3 ,4,5].

However, previous researches neglected the fact that the tissue oxygen tension sensor showed the effect of the acupuncture itself, which could cause a change in tissue oxygen tension after pricking into the tissues. Moreover, the zero-drift phenomenon of the sensor can not be avoided in modern techniques, and the tissue oxygen tension changes before the zero-drift reaches stable levels. Therefore, incipient tissue oxygen tension could not be detected exactly in abstracto, and it is difficult to assess the manner of change of tissue oxygen tension after the tissue is acupunctured.

Thus, observing the manner of change of tissue oxygen tension indirectly by blocking the acupuncture effect might be a more practical method. Guo [1] found that the acupuncture effect of needling point Zusanli (ST36) would be weakened obviously as soon as Ca²⁺ was complexed by EDTA. We have shown in earlier experiments that a calcium gathering phenomenon exists in the urinary bladder meridian of goats. This implies that calcium ion might play an important role in meridian signal conduction. Thus, it is theoretically practical to block the acupuncture effect by Ca²⁺ complexation with EDTA. Our results show that (1) the tissue oxygen partial pressure in urinary bladder meridian acupoints was higher than that in no-acupoints; (2) the oxygen partial pressure in acupoints increased significantly after injection of EDTA-Na₂, compared to injection with either normal sodium or nothing; (3) the oxygen partial pressure in acupoints was higher than that in no-acupoints after EDTA-Na₂ injection. These observations suggest that meridians have high tissue oxygen partial pressures, and acupuncture could lower the tissue oxygen partial pressure of acupoints.

Although morphologic research indicates that meridians do not have special structures different from other tissues, we suppose that the meridian region might have special microstructures different from others. It has been documented that meridians have more mitochondria than regions nearby the meridians. The mitochondrion is the principal structure in cells that provides energy, and its quantity is directly connected to the metabolic levels of local regions. The difference in structure could explain why meridians have a hypermetabolic tendency. Thus, we could deduce that meridians possess more oxygen supply than other regions and can meet the oxygen consumption requirement. This can explain why high tissue oxygen partial pressure exists in meridians.

We believe that the mechanism of acupuncture lowering tissue oxygen partial pressure may be associated with regulating microcirculation and enhancing metabolism. In our previous experiment, we found that the pH value of acupoints decreased obviously after acupuncture [6], and the change in pH value in tissues may be associated with an increase in carbon dioxide output. This phenomenon indicates that the increase in oxygen consumption and carbon dioxide output in acupoint regions contributes to a decrease in pH value.

Sneyd and Sandersen [7, 8] reported that oscillations and waves of increased intracellular free calcium concentration were observed in a wide range of cell types, and the spreading of a Ca²⁺ oscillation as a Ca²⁺ wave provided a mechanism by which the regulatory signal could be distributed throughout the cell via gap junctions. In our previous experiment, we confirmed that the expression of connexin 43 in subcutaneous tissue of “Zusanli (ST36)” in rats increased significantly [9]. Based on these, we propose the hypothesis that gap junctions might be the morphologic basis of meridians, and calcium oscillation might be an important functional element of meridian conduction. The mechanical stimulus caused by acupuncture could induce a change in calcium ions in cytoplasm, generating the so-called acupuncture signal, which is then transmitted to neighboring tissues via inter-cellular gap junctions along the meridian. However, this hypothesis needs to be confirmed by further investigations.

References

[1] GuoY,ZhangY J,WanY Y,XuT P,MiaoW F,ZhangC X. The effects of complexing the Ca(2+) at zusanli upon inhibiting the motion of stomach of the normal rabbits by acupuncture at Zusanli. Zhenci Yanjiu, 1998, (2): 146-148 (in Chinese)

[2] YuA S,ZhaoY X,YanZ G,LiX L,GuanX F. Space morphology observation of the Sanyinjiao acupoint. Zhongguo Zhenjiu, 1997, (1): 42-44 (in Chinese)

[3] XuX Y,HuX L,WuB H. The influence of mechanical pressing on partial pressure of oxygen in three points along the large intestine meridian during acupuncture. Zhenci Yanjiu, 2000, 25(4): 276-279 (in Chinese)

[4] WangP,ZhangH P,LiuY X. Observation of the partial pressure of oxygen in deep tissue of acupoints, no-acupoints in meridian and no-acupoints. Shanghai Zhenjiu Zazhi, 1997, 16(1): 3-5 (in Chinese)

[5] XieY J,HuX L,WuB H. Observation of the partial pressure of oxygen in deep tissue along the meridian course and the effect of acupuncture on it. Zhongguo Zhongyiyao Keji, 2000, 7(6): 353-354 (in Chinese)

[6] JiangH Z,WangQ,ZhangM M,GongP,HuangG Y. Effect of acupuncture on pH values of acupoints in the goat. Zhongguo Zhenjiu2006, 26(10): 732-734 (in Chinese)

[7] SneydJ,KeizerJ,SandersonM J. Mechanisms of calcium oscillations and waves: a quantitative analysis. FASEB J, 1995, 9(14): 1463-1472

[8] SandersenM J,CharlesA C,DirksenE R. Mechanical stimulation and intercellular communication increased intercellular Ca²⁺ in epithelial cells. Cell Regul, 1990, 1(8): 585-596

[9] ZhengC H,HuangG Y,ZhangM M. Experimental study on connexin43 expression in “Zusnali”(ST 43) with fluorescence dual-label technique in rats. Zhenci Yanjiu, 2005, 30(4): 221-224 (in Chinese)

RIGHTS & PERMISSIONS

Higher Education Press and Springer-Verlag Berlin Heidelberg