Acta Physiologica Sinica, April 25, 2003, 55(2): 177-182

Received 2002-07-16

Accepted 2002-10-23

This project was supported by the National Natural Science Foundation of China (No. 39860031).

Corresponding author. Tel: +86-433-2660586; Fax: +86-433-2659795; E-mail: wenxiexu@ybu.edu.cn

Research Paper

Role of actin microfilament in hyposmotic membrane stretch-induced increase in muscarinic current of guinea-pig gastric myocytes

WANG Zuo-Yu1,YU Yong-Chun1, CUI Yi-Feng1, LI Lin1, GUO Hui-Shu1, LI Zai-Liu1,XU Wen-Xie1,2,*

1Research Laboratory of Digestive Physiology and 2Research Center of Affiliated Hospital, Yanbian University College of Medicine, Yanji 133000

Abstract:To investigate the relationship between cytoskeleton and hyposmotic membrane stretch-induced increase in muscarinic current, the role of actin microfilament in hyposmotic membrane stretch-induced increase in muscarinic current was studied with the whole-cell patch clamp technique in guinea-pig gastric myocytes. In this study, the muscarinic current was induced by carbachol (50 μmol/L) or GTPγS (0.5 mmol/L). The results showed that hyposmotic superfusate (202 mOsmol/L) increased carbachol-induced current (ICCh) by 145±27% and increased GTPγS-induced current by 183±30%; but in the presence of cytochalasin-B (Cyt-B, 20 μmol/L), an actin cytoskeleton disruptor, hyposmotic membrane stretch increased ICCh by 70±6%. However, hyposmotic membrane stretch induced increase in ICCh was potentiated to 545±81% by phalloidin (20 μmol/L), an actin microfilament stabilizer. The results demonstrated that hyposmotic membrane stretch increased the muscarinic currents induced by carbachol or GTPγS and that the actin microfilament is involved in the process in guinea-pig gastric myocytes.

Key words: muscarinic currents; gastric myocytes; stretch; actin microfilament

微丝在低渗牵张诱导毒蕈碱电流增加中的作用

王佐妤1, 禹永春1, 崔艺峰1, 李林1, 郭慧淑1, 李在琉1, 许文燮1,2，*

延边大学医学院 1消化生理研究室、2附属医院中心实验室, 延吉 133000

摘要: 在急性分离的豚鼠胃窦平滑肌细胞上, 利用膜片钳技术的传统全细胞模式记录离子电流的方法, 探讨微丝在低渗牵张诱导毒蕈碱电流增加中的作用。当豚鼠胃窦平滑肌细胞的膜电位钳制在-20 mV时, 灌流液中50 μmol/L 卡巴胆碱(carbachol, CCh)或电极内液中0.5 mmol/L GTPγS均可引导毒蕈碱电流(muscarinic current ICCh), 低渗牵张(202 mOsmol/L)分别使其增加145±27%和183±30%; 当电极内液中加入20 μmol/L的细胞松弛B (一种微丝骨架的解聚剂)时, 低渗牵张使ICCh只增加70±6%; 而电极内液中加入20 μmol/L的鬼笔环肽(一种微丝骨架的稳定剂)则使ICCh增加了545±81%。结果表明, 低渗牵张可增加由卡巴胆碱或GTPγS诱导的毒蕈碱电流, 微丝参与调节低渗牵张诱导豚鼠胃窦平滑肌细胞ICCh增加的作用。

关键词: 毒蕈碱电流; 胃窦平滑肌; 牵张; 微丝

中图分类号: Q482

Gastrointestinal motility is regulated by neural, humoral and myogenic mechanisms[1]. ACh is the major excitatory neurotransmitter of mammalian gastrointestinal tract. The release of ACh from vagus nerves caused an increase in the force of phasic contractions in chick skeletal muscle[1]. Muscarinic stimulation also activates nonselective cation conductance in the isolated smooth muscle cells[2,3]. Stretch is a physiological stimulation in gut smooth muscles, and mechanical forces stimulate a number of cell signal pathways, including ion channel activation. It has been reported that membrane stretch activates the stretch-sensitive ion channel and causes sodium and calcium ion influx, and membrane potential depolarization[3,4]. Matsuda et al. also reported that osmotic cell swelling and cell inflation caused by applying positive pressure increased reversible L-type calcium current in rabbit cardiac myocytes[5]. Our previous study demonstrated that osmotic membrane stretch increased voltage-dependent Ca2+ channel current in guinea-pig gastric myocytes[6]. However, there is no report on the relationship between membrane stretch and ligand gated channels. For example, there is no report on the muscarinic channel in gastric smooth muscle. Wanish et al. reported that reduced extracellular tonicity increased the muscarinic receptor-activated inward current in ileal smooth muscle of the guinea pig[7]. In the present study, we observed that hyposmotic membrane stretch enhanced muscarinic current induced by carbachol (50 μmol/L, external perfusion) or GTPγS (0.5 mmol/L, in pipette solution) in guinea-pig gastric myocytes.

The actin microfilaments of the cytoskeleton form a complex network, providing the structural basis for simultaneous interactions between multiple cellular structures. For example, the actin-based cytoskeleton was involved in the control of ion channel activity across the plasma membrane of different cell types[8,9]. It is well established that many ion channels and transporters are anchored in the membrane by either direct or indirect association with the cytoskeleton. In addition, there was growing evidence that altering the integrity of cytoskeletal elements, in particular actin filaments, modulated the activity of a variety of ion channels[10] and receptors[11]. Our previous study demonstrated that actin microfilament played a role in the modulation of membrane stretch-induced calcium influx in guinea-pig gastric myocytes[12]. It has been proposed that cell surface proteins and extracellular matrix are linked to the cytoskeleton by transmembrane proteins and modulate ion channels and enzymes by mechanical deformation under physiological conditions[13]. For example, disruption of actin filaments with cytochalasin resulted in activation of ATP-sensitive K+ (KATP) channels in cardiac myocytes[14].

Since actin microfilament is related to ion channel which is activated by a mechanical signal, for example membrane stretch in the present study, we examined the effects of cytochalasin-B (Cyt-B) as an actin microfilament disruptor and phalloidin as a stabilizer on hyposmotic stretch-induced increase in muscarinic currents (△ICCh hypo).

1MATERIALS AND METHODS

1.1 Preparation of cells. EWG/B guinea pigs (obtained from the Experimental Animal Department of Yanbian University Medical College) of either sex (250-350 g), were exsanguinated after being stunned. The antral part of the stomach was cut immediately and cleaned in the Tyrode′s solution. The mucosal layer was separated from the muscle layer in Ca2+-free solution, and cut into small segments (1 mm×4 mm). These segments were kept in a modified Kraft-Bruhe (K-B) medium at 4℃ for 15 min, then were incubated at 36℃ in 4 ml digestion medium [Ca2+-free physiological salt solution (Ca2+-free PSS)] containing 0.1% collagenase (Ⅱ), 0.1% dithiothreitol, 0.15% trypsin inhibitor, and 0.2% bovine serum albumin for 25-35 min. The softened muscle segments were transferred into the modified K-B medium and cells were dispersed individually with a wide-bore fire-polished glass pipette. At last, the isolated gastric myocytes were kept in modified K-B medium at 4℃ until being used.

1.2 Electrophysiologic recording. The isolated cells were transferred to a small chamber (0.1 ml) on the stage of an inverted microscope (Ⅸ-70 Olympus, Japan) for 10-15 min to settle down. The cells were superfused continuously with isosmotic solution. An 8-channel perfusion system (L/M-sps-8, List Electronics, Germany) was used to change the solution. Experiments were performed at 20-25℃ and the whole-cell configuration of the patch-clamp technique was applied. Patch-clamp pipettes were manufactured from borosilicate glass capillaries (GC 150T-7.5, Clark Electromedical Instruments, UK) by a two-stage puller (PP-83, Narishige, Japan). The resistance of the patch pipette was 3-5 MΩ when being filled with pipette solution. Liquid junction potentials were compensated prior to seal formation. The whole-cell holding currents were recorded with an Axopatch 1-D patch-clamp amplifier (Axon Instrument, USA), and command pulses were applied by using the IBM-compatible 486-grade computer and pCLAMP software (Version 6.02). The data were filtered at 5 kHz and recorded by a pen recorder (LMS-2B, Chengdu, China).

1.3 Drugs and solutions. Drugs were purchased from Sigma. Tyrode′s solution contained (in mmol/L): NaCl 147, KCl 4, MgCl2 1.05, CaCl2 0.42, Na2HPO4 1.81, and glucose 5.5, and the pH was adjusted to 7.35 with NaOH. The pH of Ca2+-free solution containing NaCl 134.8, KCl 4.5, glucose 5 and HEPES 10 mmol/L, was adjusted to 7.4 with tris. The isosmotic solution (290 mOsmol/L) contained (in mmol/L): NaCl 80, KCl 4.5, MgCl2 1, CaCl2 2, glucose 5, HEPES 10, and sucrose 110, and the pH was adjusted to 7.4 with Tris. The isosmotic solution for recording carbachol current contained CsCl 85, MgCl2 1, CaCl2 2, glucose 5, HEPES 10, and sucrose 110 mmol/L, and the pH was adjusted to 7.4 with tris. Hyposmotic solution (202 mOsmol/L) was free of sucrose, and other ingredients were the same as the isosmotic solution. Modified K-B solution contained (in mmol/L): EGTA 0.5, L-glutamate 50, KCl 50, taurine 20, KH2PO4 20, MgCl2 3, glucose 10, and HEPES 10, and the pH was adjusted to 7.4 with KOH. The pipette solution for recording muscarinic current contained (in mmol/L): CsCl 135, Na2ATP 3, MgCl2 3, di-tris-creatine phosphate 2.5, disodium-creatine phosphate 2.5, HEPES 5, and EGTA 0.5, whose pH was adjusted to 7.3 with Tris. Carbachol was prepared as aqueous stock solutions (10 mmol/L). Cytochalasin-B was dissolved in DMSO (20 mmol/L) and phalloidin was dissolved in alcohol (1 mmol/L). The same amount of DMSO or alcohol as the final experimental solution was added to the pipette solution. GTPγS was dissolved in the distilled water (50 mmol/L) and diluted in the experimental pipette solution.

1.4 Data analysis. Data were expressed as mean±SE. Statistical significance was evaluated by the Student′s t test. The difference was considered to be significant when the P value was less than 0.05.

2 RESULTS

2.1 Effect of hyposmotic membrane stretch on muscarinic currents

With the conventional whole-cell patch-clamp technique, inward cationic current (ICCh) was induced by carbachol (CCh, 50 μmol/L, external perfusion) when the membrane potential was held at -20 mV. ICCh reached a steady state after the application of CCh within 1-2 min. (Fig.1A). The same cationic current was also induced by GTPγS (0.5 mmol/L, in pipette solution). About 3-5 min after dialysis of GTPγS from the pipette solution, the inward current was activated reaching a steady state at about 8 min (Fig.1B). Hyposmotic superfusing increased ICCh (Fig.2A) and GTPγS induced inward current (Fig.2B) by 145±27% and 183±30% respectively.

Fig.1.Muscarinic currents recorded from guinea-pig gastric myocytes in isosmotic solution (290 mOsm). A: The muscarinic current induced by carbachol (CCh, 50 μmol/L) when membrane potential was held at -20 mV. B: The muscarinic current induced by GTPγS (0.5 mmol/L, in the pipette solution).

Fig.2.Effect of hyposmotic membrane stretch on muscarinic currents. A: Hyposmotic membrane stretch enhanced ICCh (CCh, 50 μmol/L, n=8). B: Hyposmotic membrane stretch-enhanced muscarinic currents induced by GTPγS (0.5 mmol/L in the pipette solution, n=8).

2.2 Effect of Cyt-B on hyposmotic membrane stretch-induced increase in ICCh

To determine the possibility of actin microfilament involved in hyposmotic membrane stretch-induced increase in ICCh, the effect of Cyt-B (20 μmol/L, in pipette) on ICCh in which cells were perfused with isosmotic and hyposmotic solutions was observed respectively. Cyt-B, an actin microfilament disruptor, did not affect ICCh under the isosmotic condition (Fig.3B). However, Cyt-B inhibited the increasing effect of hyposmotic membrane stretch on ICCh (Fig.3A). Hyposmotic membrane stretch increased ICCh by 145±27% in the control group and by only 70±6% in Cyt-B group (Fig.3A， C).

Fig.3.Effect of Cyt-B on hyposmotic membrane stretch-induced increase in ICCh. A: Hyposmotic membrane stretch induced increase in ICCh in the presence of Cyt-B. B: Cyt-B did not affect ICCh in isosmotic condition (P>0.05, n=8). C: Cyt-B inhibited the increase in ICCh induced by hyposmotic membrane stretch. (a: P<0.05 vs 290 mOsm; b: P<0.05 vs 202 mOsm; n=8)．

2.3 Effect of phalloidin on hyposmotic membrane stretch-induced increase in ICCh

In this experiment, cells were dialyzed with an internal pipette solution containing phalloidin (20 μmol/L). In the isosmotic condition phalloidin decreased ICCh from 102±14.6 pA (in the control group) to 74±5.4 pA (Fig.4B). However, phalloidin potentiated the increase in ICCh induced by hyposmotic membrane stretch (Fig.4A), and it was significantly enhanced from 145±27% to 525±81% when the cells were superfused with hyposmotic solution (Fig.4A，C).

Fig.4.Effect of phalloidin on hyposmotic membrane stretch-induced increase in ICCh. A: Hyposmotic membrane stretch-induced increase in ICCh in the presence of phalloidin. B: Phalloidin decreased the amplitude of ICCh in isosmotic solution (*P<0.05, n=10). C: Phalloidin inhibited the increase in ICCh induced by hyposmotic membrane stretch. (a: P<0.05 vs 290 mOsm; b: P<0.05 vs 202 mOsm; n=10)．

3 DISCUSSION

The cytoskeleton is an intracellular superstructure that consists of microfilaments of actin and associated proteins, microtubules, and intermediate filaments. The actin cytoskeleton, in particular, is involved in structural support and a functional role in cell motility[15,16]. Recent evidence indicated, however, that cytoskeletal components also regulated membrane transport events[10]. The actin-based cytoskeleton, including actin filaments and associated proteins, is a dynamic structure which plays an essential role in the regulation of integral membrane protein[13,17]. It was proposed that cell surface proteins and extra cellular matrix were linked to the cytoskeleton by transmembrane proteins and modulate ion channels and enzymes by mechanical deformation under physiological conditions[13]. The actin cytoskeleton is implicated in the regulation of epithelial sodium channels[17,18], including ENaC[19] potassium channels in nonexcitable[20] and excitable cells[21], and anion channels such as cystic fibrosis transmembrane conductance regulator[22,23]. In neurons, for example, actin-based microfilamental and tubulin-based microtubular cytoskeletons are both implicated in the regulation of sodium[24] and calcium[25,26] channel activity, respectively.

In the present study, Cyt-B, an actin filament disruptor, suppressed markedly the hyposmotic membrane stretch-induced increase in ICCh (Fig.3A，C). And Phalloidin, an actin filaments stabilizer, potentiated significantly the increasing effect of hyposmotic membrane stretch on ICCh (Fig.4A，C). The results suggest that the actin microfilament may participate in the process of hyposmotic membrane stretch induced increase in ICCh. However, it is still unclear whether the effect of cytoskeleton on ICCh is via a direct linkage or by an indirect effect exerted on the cell membrane in the gastric myocyte. Sokabe et al.[1] measured patch geometry, capacitance, and stretch-activated channel current simultaneously, and therefore permitted the calculation of membrane stress, strain and its influence on ion channel activation. They suggested that because the patch lipid was free to flow, and hence stress-free in the steady state, the stretch-activated channel must be activated by tension in the cytoskeleton. Our previous study demonstrated that hyposmotic membrane stretch increased IKCa[27] and ICCh, and the increment was related to the influx of external calcium and triggered calcium to be released. But it is not clear whether there is an interrelationship between calcium induced calcium release (CICR) triggered by hyposmotic membrane stretch and cytoskeleton. Hyposmotic membrane stretch might directly affect the muscarinic channel which linked with actin microfilaments or indirectly affected muscarinic channel via triggering the intracellular calcium pool which linked with actin microfilaments. According to the results that phalloidin inhibited ICCh while cytochalasin-B had no effect on ICCh when superfused by with isosmotic solution, the possible reason was the nonspecific effect of phalloidin on the muscarinic current.

Under the physiological conditions when the gastric wall is distended by ingested food, this mechanical stress may be an important factor in the control of contractility. Distension of the stomach increases the force of antral contractions through long or short reflexes triggered by mechanoreceptors in the stomach wall. In this process, the vagus nerve plays an important role, and acetylcholine is the major excitatory neurotransmitter. Our results implied that distension of the stomach could potentiate the force of antral contraction via increasing muscarinic current and that actin microfilament might play a role in the mechanical modulation of muscarinic current in gastric smooth muscle.

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