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Effects of Lizhong Tang on cultured mouse small intestine interstitial cells of Cajal.

AIM
To investigate the effects of Lizhong Tang, an herbal product used in traditional Chinese medicine, on mouse small intestine interstitial cells of Cajal (ICCs).


METHODS
Enzymatic digestions were used to dissociate ICCs from mouse small intestine tissues. The ICCs were morphologically distinct from other cell types in culture and were identified using phase contrast microscopy after verification with anti c-kit antibody. A whole-cell patch-clamp configuration was used to record potentials (current clamp) from cultured ICCs. All of the experiments were performed at 30-32 °C.


RESULTS
ICCs generated pacemaker potentials, and Lizhong Tang produced membrane depolarization in current-clamp mode. The application of flufenamic acid (a nonselective cation channel blocker) abolished the generation of pacemaker potentials by Lizhong Tang. Pretreatment with thapsigargin (a Ca²⁺-ATPase inhibitor in the endoplasmic reticulum) also abolished the generation of pacemaker potentials by Lizhong Tang. However, pacemaker potentials were completely abolished in the presence of an external Ca²⁺-free solution, and under this condition, Lizhong Tang induced membrane depolarizations. Furthermore, When GDP-β-S (1 mmol/L) was in the pipette solution, Lizhong Tang still induced membrane depolarizations. In addition, membrane depolarizations were not inhibited by chelerythrine or calphostin C, which are protein kinase C inhibitors, but were inhibited by U-73122, an active phospholipase C inhibitors.


CONCLUSION
These results suggest that Lizhong Tang might affect gastrointestinal motility by modulating pacemaker activity in interstitial cells of Cajal.


INTRODUCTION
Lizhong Tang, was first reported 1800 years ago in "Sh anghan Lun", and it remains a classical herbal product in traditional Chinese medicine. Lizhong Tang is composed of Radix Ginseng (Panax ginseng C.A. Meyer), Rhizoma Zingiberis (Zingiber officinale Roscoe), Rhizoma Atracty lodis Macrocephalae (Atractylodes macrocephala Koidz.) and Radix Glycyrrhizae (Glycyrrhiza uralensis Fisch) [1] , and it is widely used in traditional medicine to treat spleen deficiency patterns in many diseases with common symp toms, such as vomiting, diarrhea, stomach pain, poor ap petite, cold limbs, and stomach bleeding which, are caused by cold and weak organs [1,2] . However, little is known of the molecular basis of the effects of Lizhong Tang on gastrointestinal (GI) motility.
Interstitial cells of Cajal (ICCs) are pacemaker cells in GI muscles that generate rhythmic oscillations in mem brane potentials known as slow waves [35] . Slow waves propagate within ICC networks and are conducted into smooth muscle cells via gap junctions. Furthermore, they initiate phasic contractions by activating Ca 2+ entry through Ltype Ca 2+ channels. Pacemaker activity in the murine small intestine is mainly due to periodic activa tions of nonselective cation channels [6,7] or Cl chan nels [8,9] . ICCs also mediate or transduce inputs from the enteric nervous system. However, the effects of Lizhong Tang in mouse small intestine ICCs have not been inves tigated, and therefore, we undertook this study to inves tigate the characteristics of Lizhong Tang in mouse small intestine ICCs.

Preparation of cells and cell cultures
Balb/c mice (37 d old) of either sex were anesthetized with ether and sacrificed by cervical dislocation. The small intestines, from 1 cm below the pyloric ring to the cecum, were removed and opened along the mesenteric border. The luminal contents were removed by washing with KrebsRinger bicarbonate solution. The tissues were then pinned to the base of a Sylgard dish, and the muco sae were removed by sharp dissection. Small tissue strips of intestinal muscle (consisting of both circular and lon gitudinal muscles) were equilibrated in Ca 2+ free Hanks solution (containing the following in mmol/L: KCl 5.36, NaCl 125, NaOH 0.34, Na2HCO3 0.44, glucose 10, su crose 2.9, and HEPES 11) for 30 min, and then, the cells were dispersed using an enzyme solution containing col lagenase (Worthington Biochemical Co., Lakewood, NJ, United States) 1.3 mg/mL, bovine serum albumin (Sigma Chemical Co., St. Louis, MO, United States) 2 mg/mL, trypsin inhibitor (Sigma) 2 mg/mL and ATP 0.27 mg/ mL. The cells were plated onto sterile glass coverslips coated with murine collagen (2.5 μg/mL, Falcon/BD, Franklin Lakes, NJ, United States) in a 35mm culture dish and then cultured at 37 ℃ in a 95% O2, 50 mL/L CO2 incubator in smooth muscle growth medium (Clo netics Corp., San Diego, CA, United States) supplement ed with 2% antibiotics/antimycotics (Gibco, Grand Is land, NY, United States) and murine stem cell factor (SCF 5 ng/mL, Sigma). ICCs were identified immunologically using an antickit antibody (phycoerythrinconjugated rat antimouse ckit monoclonal antibody; eBioscience, San Diego, CA, United States) at a dilution of 1:50 for 20 min [10] . The ICCs were morphologically distinct from other cell types in culture and were identified using phase contrast microscopy after verification with anti ckit anti body.

Patch-clamp experiments
A wholecell patchclamp setup was used to record the membrane potentials (current clamp) of cultured ICCs. An axopatch ID (Axon Instruments, Foster, CA, United States) was used to amplify membrane currents and po tentials. The command pulse was applied using an IBM compatible personal computer running pClamp software (version 6.1; Axon Instruments). The data obtained were filtered at 5 kHz displayed on an oscilloscope and a com puter monitor, and printed using a pen recorder (Gould 2200, Gould, Valley View, OH, United States). The re sults were analyzed using pClamp and Origin (version 6.0) software. All of the experiments were performed at 3032 ℃.

Solutions and drugs
The physiological salt solution used to bathe cells (Na + Tyrode) contained the following (in mmol/L): KCl 5, NaCl 135, CaCl2 2, glucose 10, MgCl2 1.2 and HEPES 10, adjusted to pH 7.4 with NaOH. The pipette solution contained the following (in mmol/L): KCl 140, MgCl2 5, K2ATP 2.7, NaGTP 0.1, creatine phosphate disodium 2.5, HEPES 5 and EGTA 0.1, adjusted to pH 7.2 with KOH. Lizhong Tang was purchased from IWORLD Phar maceuticals (South Korea). Lizhong Tang is composed of Radix Ginseng (Panax ginseng C.A. Meyer), Rhi zoma Zingiberis (Zingiber officinale Roscoe), Rhizoma Atractylodis Macrocephalae (Atractylodes macrocephala Koidz.) and Radix Glycyrrhizae (Glycyrrhiza uralensis Fisch.). The adult dosage is 1015 g (crude material) per day. More information about Lizhong Tang can be obtained at the IWORLD Pharmaceuticals Homepage (http://ipharm.koreasme.com). The pills were dissolved with distilled water at a concentration of 0.5 g of crude drug/ml and stored in the refrigerator. All of the drugs were obtained from Sigma (Sigma Chemical Co., United States). The drugs were dissolved in distilled water, and added to the bathing solution to the desired concentra tions immediately prior to use. The addition of these chemicals to the bathing solution did not alter its pH. Thapsigargin, U73122, and U73343 were dissolved in dimethyl sulfoxide (DMSO) to produce 50 and 100 mmol/L stock solutions and added at 1000 times dilu tions to the bathing solution on the day of the experi ment. The final concentration of DMSO in the bathing solution was always < 0.1%, and we confirmed that this concentration did not affect the results.

Statistical analysis
All of the data are expressed as the mean ± SE. Student's ttest for unpaired data was used to compare the control and experimental groups. Statistical significance was ac cepted for P values < 0.05.

Effect of Lizhong Tang on the pacemaker potentials of cultured ICCs
The patchclamp technique was tested on ICCs, which had formed networklike structures in culture after 24 d. Spontaneous rhythms were routinely recorded from cul tured ICCs under current and voltageclamp conditions; ICCs within networks displayed more robust electrical rhythms. Tissuelike spontaneous slow waves have been previously recorded from these cells [11] . To understand the relationship between Lizhong Tang and the modu lation of pacemaker activity in ICCs, we examined the effects of Lizhong Tang on pacemaker potentials. Re cordings from cultured ICCs under currentclamp mode (I = 0) showed spontaneous pacemaker potentials. The mean resting membrane potential was 52 ± 1.3 mV, and the mean amplitude was 23 ± 2 mV. In the presence of Lizhong Tang (050 mg/mL), the membrane potentials were depolarized to 0.6 ± 0.5 mV at 5 mg/mL, 6.3 ± 1.5 mV at 10 mg/mL, 15.2 ± 1.3 mV at 30 mg/mL, and 22.1 ± 1.2 mV at 50 mg/mL ( Figure 1AE). Summarized values and a bar graph of the effects of Lizhong Tang on pacemaker potentials are provided in Figure 1F (n = 4).

Effects of non-selective cation channel blocker or Clchannel blocker on Lizhong Tang-induced pacemaker potentials in cultured ICCs
To determine the characteristics of the membrane de polarizations induced by Lizhong Tang, flufenamic acid (a nonselective cation channel blocker) [12,13] and niflumic acid (a Cl channel blocker) [12,14] were used. In the pres ence of flufenamic acid (5 μmol/L), pacemaker poten tials were abolished and the subsequent application of Lizhong Tang (30 mg/mL) did not produce membrane depolarization (Figure 2A). In the presence of flufenamic acid, the membrane depolarizations produced by Lizhong Tang were 0.6 ± 0.4 mV, which was significantly differ ent from the control values obtained in the absence of flufenamic acid (n = 4, Figure 2C). Pacemaker potentials were also abolished in the presence of niflumic acid (5 μmol/L), but Lizhong Tang still produced membrane de polarization ( Figure 2B). In the presence of niflumic acid, the mean membrane depolarization produced by Lizhong Tang was 15.3 ± 0.4 mV, which was not significantly dif ferent from the control condition (n = 4, Figure 2C).

Effects of external Ca 2+ -free solution and Ca 2+ -ATPase inhibitor in the endoplasmic reticulum on Lizhong Tanginduced pacemaker potentials in cultured ICCs
External Ca 2+ influx is necessary for GI smooth muscle contractions and is essential for the generation of pace maker potentials by ICCs. The generation of pacemaker currents is known to be dependent on intracellular Ca 2+ oscillations [15] . To investigate the roles of external and of internal Ca 2+ , Lizhong Tang was tested under external Ca 2+ free conditions and in the presence of thapsigargin, an inhibitor of Ca 2+ ATPase in the endoplasmic reticu Lizhong Tang on ICCs. When GDPβS (1 mmol/L) was in the pipette solution, Lizhong Tang (30 mg/mL) still in duced membrane depolarizations ( Figure 4A). However, the membrane depolarizations induced by Lizhong Tang were not significantly affected by the presence of GDP βS (1 mmol/L) in the pipette solution (n = 4, Figure  4C).

Effects of phospholipase C inhibitor on Lizhong Tanginduced pacemaker potentials in cultured ICCs
Because membrane depolarizations induced by Lizhong Tang are related to intracellular Ca 2+ mobilization, we examined whether the effects of Lizhong Tang on pacemaker potentials required phospholipase C (PLC) activation. To test this possibility, Lizhong Tanginduced membrane depolarizations were measured in the absence or presence of U73122 (an active PLC inhibitor [17] ). Pacemaker membrane depolarizations currents were completely abolished by U73122 (5 μmol/L), and under these conditions, Lizhong Tanginduced (30 mg/mL) lum [6,12] . Pacemaker potentials were completely abolished in the presence of an external Ca 2+ free solution, and under this condition, Lizhong Tang induced membrane depolarizations (n = 4, Figure 3A). However, under ex ternal Ca 2+ free conditions, membrane depolarizations by Lizhong Tang (30 mg/mL) were not significantly differ ent from the depolarizations induced by Lizhong Tang (30 mg/mL) under normal Ca 2+ conditions (n = 4, Figure  3C). In addition, Lizhong Tanginduced membrane de polarizations were inhibited by thapsigargin pretreatment ( Figure 3B). Furthermore, the membrane depolarizations induced by Lizhong Tang were significantly affected by the presence of thapsigargin (n = 4, Figure 3C).

The involvement of G protein on Lizhong Tang-induced pacemaker potentials in cultured ICCs
The effects of GDPβS (a nonhydrolysable guanosine 5'diphosphate analogue that permanently inactivates Gprotein binding proteins [16] ) were examined to deter mine whether Gproteins are involved in the effects of membrane depolarizations were suppressed (n = 4, Fig  ure 5A). In the presence of U73122, the mean mem brane depolarization produced by Lizhong Tang was 0.5 ± 0.3 mV, and this was significantly different than the depolarization observed in the absence of U73122 (n = 4, Figure 5C). Treatment with U73343 (5 μmol/L; an inactive analog of U73122) had no influence on Lizhong Tanginduced pacemaker potentials, and Lizhong Tang induced (30 mg/mL) membrane depolarizations were not suppressed by U73343 ( Figure 5C).

Effects of protein kinase C inhibitor on Lizhong Tanginduced pacemaker potentials in cultured ICCs
We tested the effects of chelerythrine and of calphostin C (both inhibitors of protein kinase C (PKC) [12,18] ) to investigate whether Lizhong Tanginduced pacemaker potential responses are mediated by the activation of PKC. Neither chelerythrine (1 μmol/L) nor calphostin C (1 μmol/L) had any effect on membrane depolarizations induced by Lizhong Tang (30 mg/mL; Figure 6) and the value was also not significantly different when compared with the membrane depolarizations induced by Lizhong Tang in the absence of chelerythrine or calphostin C (n = 5, Figure 6C).

DISCUSSION
The GI tract exhibits spontaneous mechanical con tractions that are mediated by the periodic generation of electrical pacemaker potentials, which are the basic determinant of GI smooth muscle activity [3] . Recent studies have shown that the ICCs act as the pacemakers and conductors of electrical slow waves in GI smooth muscles [35] . Moreover, evidence indicates that endog enous agents, such as, neurotransmitters, hormones, and paracrine substances modulate GI tract motility by influencing ICCs [57,19,20] . Therefore, one of the best ways to investigate the role of GI motility is to use ICCs. Many types of ICCs with different immunohistochemi cal and electrical properties, including myenteric ICCs (ICCMY), intramuscular ICCs (ICCIM), deep muscular plexus ICCs (ICCDMP), and submucosal ICCs (ICC SM), are distributed throughout the GI tract [16] . In animal models lacking ICCMY, slow waves in the small intestine are strongly attenuated, which shows that these cells are indeed essential for pacemaker activity in the GI tract [21] . Furthermore, ICCs are involved in physiological GI mo tility and are therefore clinically important in many bowel disorders, including inflammatory bowel disease, chronic idiopathic intestinal pseudoobstruction, intestinal ob struction with hypertrophy, achalasia, Hirschsprung's dis ease, juvenile pyloric stenosis, juvenile intestinal obstruc tion, and anorectal malformation [16] .
Lizhong Tang warms the liver and spleen and stren gthens the spleen and stomach. It has been widely used as treatment from deficiency, diarrhea with watery stool, nausea and vomiting. In addition, it also has ameliora tive effects on loss of appetite, abdominal pain, acute or chronic gastritis gastric or duodenal ulcers, irritable bowel syndrome, chronic colitis, chronic bronchitis, oral herpes, and functional uterine bleeding [1,22,23] . However, the effects of Lizhong Tang on GI tract motility and ICCs have not been investigated.
In this study, Lizhong Tang produced membrane depolarization in currentclamp mode, and the applica tion of flufenamic acid (a nonselective cation channel blocker), but not niflumic acid (a Cl channel blocker), abolished the generation of the pacemaker potentials induced by Lizhong Tang, suggesting that the Lizhong Tanginduced membrane depolarizations may be medi ated by nonselective cationic channels. In addition, pre treatment with a Ca 2+ free solution or with thapsigargin (a Ca 2+ ATPase inhibitor in the endoplasmic reticulum), abolished the generation of pacemaker potentials. Un der Ca 2+ free conditions, Lizhong Tang also showed membrane depolarization; however, in the presence of thapsigargin, Lizhong Tang did not show membrane de polarization, suggesting that intracellular calcium release is necessary. Furthermore, pacemaker membrane depo larizations were inhibited by U73122 (PLC inhibitor), but not by GDPβS, which permanently binds Gbinding proteins. In addition, the PKC inhibitors chelerythrine and calphostin C did not block Lizhong Tanginduced pacemaker potentials, suggesting that PLC is involved in the induction of the pacemaker potentials, but that PKC is not. In summary, Lizhong Tang affects GI motility by modulating pacemaker activity in ICCs, and this activa tion is associated with nonselective cationic channels via phospholipase C activation, and Ca 2+ release from inter nal storage in an external Ca 2+ , Gprotein, and PKC independent manner.
Taken together, our data suggest that the gastroproki netic effects of Lizhong Tang are mediated by the induc tion of pacemaker potentials in ICCs. Considering the effects of this drug on ICCs, further research is required to identify the compounds responsible for the effects of Lizhong Tang and to determine their mechanisms of ac tion.

Background
Interstitial cells of Cajal (ICCs) are the pacemaker cells that generate slow waves in the gastrointestinal (GI) tract. Lizhong Tang is a classic herbal product in traditional Chinese medicine. However, the effects of Lizhong Tang in mouse small intestine ICCs have not been investigated.

Research frontiers
The gastroprokinetic effects of Lizhong Tang are mediated by the induction of pacemaker potentials in ICCs.

Innovations and breakthroughs
Lizhong Tang affects GI motility by modulating pacemaker activity in ICCs, and this activation is associated with non-selective cationic channels via phospholipase C activation, and Ca 2+ release from internal storage in an external Ca 2+ -, G-protein-, and protein kinase C-independent manner.

Applications
Lizhong Tang may be a new target for pharmacological treatment of GI motility disorders.

Peer review
In their manuscript, authors studies the effect of Lizhong Tang, an herbal product used in traditional Chinese medicine, on the pacemaking activity of mouse small ICCs. It was well written.