대한내과학회지 : 제 81 권제 3 호 2011 종설 (Review) 위장관평활근수축기전과신경전달의새로운개념 : Fibroblast-like Cells 경상대학교의학전문대학원내과학교실 김현진 Smooth Muscles Contraction in Gastrointestinal Tract and New Concept of Enteric Neurotransmission: Fibroblast-like Cells Hyun Jin Kim Department of Internal Medicine, Gyeongsang National University School of Medicine, Jinju, Korea This review provides information regarding an enteric neurotransmission from enteric nerve terminals to smooth muscles. In the gastrointestinal tract, phasic contractions are caused by electrical activity termed slow waves. Slow waves are generated and actively propagated by interstitial cells of Cajal (ICC). The initiation of pacemaker activity in the ICC is caused by release of Ca 2+ from inositol 1, 4, 5-trisphosphate (IP 3) receptor-operated stores, and the development of unitary currents. Summation of unitary currents causes depolarization and activation of a dihydropyridine-resistant Ca 2+ conductance that entrains pacemaker activity in a network of ICC, resulting in the active propagation of slow waves. Slow wave frequency is regulated by a variety of physiological agonists and conditions, and shifts in pacemaker dominance can occur in response to both neural and non-neural inputs. Fibroblast-like cells (FLCs) are also closely associated with nerve varicosities and are labelled robustly with antibodies for platelet-derived growth factor receptor α (PDGFRα), and expression of this receptor may be a powerful new means of isolating and evaluating the function of FLCs and the possible contribution of these cells in disease. PDGFRα + cells share similar anatomical distributions, and FLCs in colonic smooth muscle functionally express small conductance Ca 2+ -activated K + channel (SK3). These findings are important to understand purinergic post-junctional responses. (Korean J Med 2011;81:315-321) Keywords: Smooth muscle; Muscle contraction; Interstitial cell of Cajal; Platelet-derived growth factor alpha receptors (PDGFRα); Fibroblast-like cells 서론위장관운동은자율신경에의해조절되며, 평상시에는규칙적이고, 매우섬세하게조절되고있다. 위장관운동의조절 에는평활근, 신경세포, 기질세포등의세포들이매우복잡한상호작용을통해이루어지며, 현재까지도모든기전이명확히알려져있지는않다. 중추신경과연합뉴론 (interneuron), 감각신경의복잡한신경얼기에의해발생한신경전달신호가 Correspondence to Hyun Jin Kim, M.D. Department of Internal Medicine, Gyeongsang National University School of Medicine, 90 Chiram-dong, Jinju 660-970, Korea Tel: +82-55-750-8822, Fax: +82-55-758-9122, E-mail: imdrkim@naver.com - 315 -
- The Korean Journal of Medicine: Vol. 81, No. 3, 2011 - 신경말단에서평활근으로전달되어평활근의수축이이루어진다고믿던과거의개념은카할간질세포 (interstitial cell of Cajal; ICC) 의기능이알려지면서변화하였다 [1]. 복잡한기전에의해 ICC로부터발생한주기적인수축반응은위장관운동에중요한역할을하며, 신경과의상호작용을통하여좀더정교해지고, 근위부와원위부의평활근이잘연동되도록하는역할을한다. 하지만신경말단-ICC-평활근의신호전달체계에대한많은연구에도설명할수없는부분이있다. 최근에위장관의근육층에존재하는다양한기질세포중의하나인, 섬유아세포-유사세포 (fibroblast-like cells; FLCs) 의형태학적특성과기능에대하여관심이높아지고있다 [2]. 이종설에서는위장관운동이직접적으로일어나는평활근으로부터, 신호전달체계의가장윗단계인신경말단까지의과정을생리학적인신호전달의역순으로기술하며 1) 위장관평활근의수축은어떤기전에의해나타나며, 영향을주는여러경로에는어떤것이있는가? 2) 위장관평활근과 ICC는어떤상호작용을하며, 해부학적생리학적특성은어떠한가? 3) 새로이발견된 FLCs는어떠한특성이있으며, 생리학적역할은무엇인가에대하여알아보고자한다. 위장관평활근의수축기전정상적인평활근의수축은세포막에존재하는수용체조절작용에의한, 액틴과미오신이결합하여연결고리 (actin myosin cross-bridge) 를형성하여평활근세포의길이가짧아지는것이므로, 평활근의수축정도는일차적으로, 미오신경사슬인산화 (phosphorylation of myosin light chain) 정도에의해결정되는데, 이반응의에너지원은 myosin ATPase 에서공급되어 myosin light chain kinase (MLCK) 가 20 kda 크기의미오신경사슬을인산화하여액틴과의상호작용을통해반복적인연결고리의형성이가능하게한다 [3]. 미오신경사슬인산화는평활근수축의최종단계이며, 여기에는세포내 Ca 2+ 의농도변화에의한경로인 Ca 2+ -의존수축반응 (Ca 2+ - dependent contraction) 과, Rho kinase에의한경로인 Ca 2+ -감작반응 (Ca 2+ -sensitization contraction) 이있다 [4]. Ca 2+ -의존수축반응평활근수축의첫단계는특별한자극에의해세포외 Ca 2+ 이여러가지칼슘채널 (voltage dependent, receptor-operated Ca 2+ channels) 을통해서세포내로이동하여세포내 Ca 2+ 농도가증가하는것이다. 그래서이반응을 Ca 2+ signaling cascades라고부르기도한다. 이렇게증가된 Ca 2+ 은세포내의 calmodulin과결합하여 calcium-calmodulin complex 를형성하는데, 이복합체는 MLC kinase를활성화시켜, 미오신경사슬인산화를유도한다 (Fig. 1). 세포내 Ca 2+ 증가에는세포밖으로부터의유입외에도세포내 Ca 2+ 저장소인근소포체 (sarcoplasmic reticulum; SR) 로부터의분비도포함되는데, Norepinephrine, angiotension II endothelin 등과같은평활근수축효능제는세포막의특별한수용체에결합하여 G 단백을통한 phospholipase C를활성화시켜 membrane lipid phosphatidylinositol 4, 5-bisphosphate를 inositol triphosphage (IP 3) 와 diacylglycerol (DG) 로분해한다 (Fig. 1). IP 3 는근소포체막의수용체에작용하여분비를증가시키고, DG는 protein kinase C (PKC) 를활성화하여세포내의표적단백을인산화하는역할을하게되는데 PCK 의다양한동질효소 (isozyme) 는여러장기의세포들이각각다른특별한역할을할수있게한다. 결론적으로 Ca 2+ -의존수축반응은평활근세포에전달된신호에의한세포내 Ca 2+ 농도증가를증폭하여평활근수축이일어나는기전이다. Ca 2+ - 감작반응 세포내 Ca 2+ 농도증가를통한수축반응은일차적인수축으로, 수축의정도와수축의유지및이완과같은정교한조절에필요한것이 Ca 2+ -감작반응으로, Rho kinase 경로가대표적이며 Rho/Rho kinase signaling pathway로기술하기도한다. Myosin phosphatase 의작용에의해미오신경사슬인산화 Figure 1. Regulation of smooth muscle contraction [3]. Calcium ion triggers smooth muscle contraction in gastrointestinal tract by reaction with voltage-operated and receptor operated Ca 2+ channels and modification by Rho kinase pathways. - 316 -
- Hyun Jin Kim. Smooth muscle contraction and fibroblast-like cells - 를조절한다 (Fig. 1). 이효소는인산화된미오신경사슬에서인산을분해하여수축이더이상지속되지않도록하는역할을한다. 결과적으로 myosin phosphatase의작용을억제하면수축이지속된다. 그러므로, MLC phosphatase 의조절에중요한역할을하는 Rho kinase의역할은평활근의수축과이완에중요한역할을하게되며, 이론적으로 Rho kinase는 MLC phosphatase의미오신- 결합부위 (myosin-binding subunit) 를인산화하여활성화를방해하므로, 결과적으로 MLC phosphatase 는비활성화상태를유지하게되어, 평활근의지속적수축을유지한다 (Fig. 1). 실험적으로, Rho kinase 길항제 (fasudil, Y-27632) 를사용하면, 평활근의이완이관찰된다 [5,6]. A B 카할간질세포와위장관평활근 C 위장관평활근의가장두드러진특징은규칙적인수축운동을하는것이다 (Fig. 2). 수축을유발하는세포막의탈분극기간이길고, 빈도가적어서파 (slow wave) 라고하며, 발생의기전에대하여오랫동안연구하였으나, 신경또는평활근의자동성으로는설명하기힘들었다 [7]. 기전을밝히기위한연구는자연스럽게뉴론과평활근에밀접하게위치하지만기능이정확히알려져있지않았던 ICC로이동하였다 [8]. ICC 의형태학적인연구로, ICC 네트워크를형성하고있으며, 평활근과 20 nm 이내의매우밀접한간연접 (gap junction) 을가지고있어서위장관운동의조절능력이있다는것을알았다 [9]. 지난 20년동안의 ICC의생리학적인연구의결과를정리하면, 서파를발생시키는독특한이온전류를제공하는 pacemaker의역할을하며, 전압의존성 (voltage-dependent) 기전과다른세포들과의네트워크를통한반복적인서파의발생과연동 (propagation) 을조절하고, 위장관으로전달되는신경전달신호에대한신경말단이후반응 (post-junctional response) 에관여하는수용체의발현과형질도입에관여하고, 기저막전압과신경합포체의전도에기여하여평활근의흥분성을조절하고, 서파의빈도를조절하고, 세포신축에민감한수용체를통하여평활근흥분도의조절에관여한다는것이다 [1]. ICC는위장관근층에존재하며, 그위치에따라분류하는데, 각각의부위에따라 ICC의분포에차이가있다 [10]. 위, 소장에서는근육층내에존재하는 ICC-IM (intramuscular) 과근신경총 (myenteric plexus) 에위치하는 ICC-MY (myenteric Figure 2. Examples of the diverse electrical activity recorded from GI muscles of three species [18]. Short segments of intracellular electrophysiological records from human, murine, and canine stomach (gastric antrum) (A), small intestine (B), and colon (C) are shown. There are both differences and similarities in the electrical events recorded from the different species and gut regions. Slow wave frequencies and the maximal level of polarization between slow waves (resting potential) vary significantly in different GI muscles. plexus), 또는 ICC-MP (myenteric plexus; Auerbach s plexus라고쓰여 ICC-AP로기술된논문도있다 ) 가있으며, 대장에서는 ICC-IM, ICC-MY와함께점막하신경얼기에존재하는 ICC-SM (submucosa) 이있고, 환상근 (circular muscle) 의점막하층쪽으로위치하는 ICC-DMP (deep muscular plexus) 도있다. 각각의위치에존재하는 ICC의기능은다르게나타나며, 위와소장에서는 ICC-MY 가 pacemaker 역할을하지만, 개나사람의대장에서는 ICC-SM이그역할을한다. 이와같이서파를만드는 ICC로는 ICC-MY 와 ICC-SM이, 신경전달에관여하는 ICC는 ICC-IM, ICC-DMP 등이있고, ICC 중에는평활근다발사이의격벽에존재하며근육다발의동시수축에관여하는것으로알려진 ICC-SEP (septa) 도있다. ICC-IM의 - 317 -
- 대한내과학회지 : 제 81 권제 3 호통권제 613 호 2011 - 주요기능이내장신경으로부터의정보를전달하는역할이지만, 어떤환경에서는 intrinsic pacemaker로작용하기도한다 [10-12]. ICC의형태학적인연구에는 c-kit 에대한면역현광염색이이용되고, 기능및생리학적연구에는특정부위의 ICC가소실된 W mutant (dominant white spotting locus coding) mouse A B & rat, Kit ligand 의변형이발생한 Sl/Sld mice가주로이용된다. W/W v mouse인경우, 위기저부와위전정부의 ICC-IM, 소장의 ICC-MY 가소실되어있다 [13,14]. 신체의항상성에중요한 tyrosine kinase인 c-kit 에대한중화항체 (neutralizing antibody) 를처리한신생동물 (new born animal) 에서위내우유가저류되고 ( 위배출장애 ), 장마비가생겼으며 [15], W mice 에서특정한 ICC의분포가소실된경우에서도서파가소실되었다 (Fig. 3) [16]. 조직에서의칼슘흐름을직접확인할수있는칼슘영상 (calcium imaging) 기술을통해서도, 서파의활동이 ICC에서시작하여평활근으로전도되어 [10], ICC가서파의발원지이며, pacemaker라는사실에더이상의의를제기할학자는없을것이다. Figure 3. Slow waves are lost in the small intestine of mice with compromised Kit signaling [18]. The Kit signaling pathway is essential for development of ICC and maintenance of the ICC phenotype after birth. Slow waves were recorded from wild-type control muscles. 평활근칼슘활성화기전 (smooth muscle calcium activation mechanisms) 위장관평활근의수축작용은외부자극 ( 신경으로부터의전압차, 신경전달물질, gap junction을통한전압차등 ) 에대 A B C Figure 4. The three main mechanisms responsible for generating the Ca 2+ transients that trigger smooth muscle cell (SMC) contraction [17] (A) receptor-operated channels (ROCs) or a membrane oscillator induces the membrane depolarization (ΔV) that triggers Ca 2+ entry and contraction. (B) a cytosolic Ca 2+ oscillator induces the Ca 2+ signal that drives contraction. (C) a cytosolic Ca 2+ oscillator in interstitial cells of Cajal (ICCs) or atypical SMCs induces the membrane depolarization that spreads through the gap junctions to activate neighbouring SMCs. - 318 -
- 김현진. 평활근수축기전과 fibroblast-like cells - 한평활근의반응으로나타난다. 기술한다양한외주자극에의한평활근의활성화기전이칼슘활성화기전이다. 체내에존재하는다양한평활근은직간접적으로신경과연결되어있어신경의반응에따라수축과이완상태를유지하게되지만각각의평활근은조금씩다른조절기전을가지고있어각각의특성을이해하지않고서는자극에대한반응을해석할수가없다. 특히, 위장관평활근은 ICC에의한신경전달신호의변형과 pacemaker 전달신호로더욱복잡한양상을보인다 [17]. 이러한다양한평활근수축반응은세가지로분류할수있으며각각에대하여알아보자 (Fig. 4). 첫번째기전에서는칼슘채널이중요한역할은담당한다. 칼슘채널은 Ca 2+ 을세포내로옮기는기능을하며, 이중전압의변화로구동하는경우를 L-type voltage-operated channels (VOCs) 이라고하며, 이채널에의한증가된세포내 Ca 2+ 이평활근수축을유발한다 (Fig. 4A). 정관, 방광, 자궁의평활근이이기전에해당한다. 두번째기전은주기적인수축을하는혈관, 림프관, 기관지, 해면체평활근이해당하며, cytosolic Ca 2+ oscillator에의해구동된다. 주기적으로세포내 Ca 2+ 의증가는변화된막전압이원인이지만, 이런수축은이들평활근의일차적인기능이아니고, 특수한상황에서 oscillatory 기전이증폭될때일어나는이차적인기능으로, 신경전달물질이나호르몬등에의해수축의빈도와강도가조절된다 (Fig. 4B). 세번째기전은 ICC와같은 pacemaker 역할을하는세포에의해서활성화되는평활근에서관찰되며, 요관, 위장관평활근에서관찰된다. Cytosolic oscillator를내장하고있는 pacemaker 세포에서발생한반복적인세포내 Ca 2+ 농도변화는세포내로의전류 (inward currents) 를만들어세포막을통해전파한다. 세포막과연관된 gap junction을통해서평활근으로전달된탈분극신호 (depolarizing signal; ΔV) 가평활근내에서첫번째기전과같은기전을통해수축이유발된다 (Fig. 4A and 4C) [18]. 서파발생의기전은 1 ICC 세포내의칼슘저장소인근소포체 (SR) 의막에많이분포하고있는 ryanodine receptor 3 (RYR3) 에의한 SR로부터일시적인 Ca 2+ 분비로발화한다. 2 이렇게발생한 Ca 2+ 농도변화는 Ca 2+ -induced Ca 2+ release (CICR) 반응을유발하여 ICC 세포내 Ca 2+ wave를만든다. SR내의 Ca 2+ 농도가감소하면 Ca 2+ wave도감소한다. 3 회복기에세포질내의 Ca 2+ 는세가지기전에의해농도가감소하는데 : 세포막 Ca 2+ -ATPase (PMCA) 에의한세포밖으로밀려나거나 ; sarco- endoplasmic reticulum Ca 2+ -ATPase (SERCA) 에의해칼슘저장소인 SR에다시저장되거나 ; 미토콘드리아로흡수된다 [18]. 4 세포질내 Ca 2+ 이 SR으로재흡수될때세포질내 Ca 2+ 농도가중요하며, 재흡수가계속일어나야 Ca 2+ oscillation 활동이지속적으로일어난다. Na + /Ca 2+ exchanger (NCX) 는세포질내의지속적인 Ca 2+ 농도상승에도움을주며, T-type Ca 2+ channels 도돕는다. 5 Ca 2+ wave로분비된 Ca 2+ 은 Ca 2+ -sensitive chloride channels (CLCA) 을활성화하여세포막탈분극을통한서파를발생시킨다. CLCA 의작용으로 STICs이발생한다. 6 STICs 의합은 spontaneous transient depolarization (STD) 를유발하여서파가발생한다. 7 발생한 STD는 VOC을열어세포밖의 Ca 2+ 이세포내로이동한다. VOCs opening은 ICC oscillator의작동에필수적인것은아니지만중요한역할을하는데, 세포내로들어온 Ca 2+ 은 SR로부터의 Ca 2+ 분비를도와서서파의발생속도를높이고, ICC network 의넓은지역에서서파발생이동시에일어날수있도록돕는다. 8 ICC pacemaker slow wave 는 gap junction을통한전압이수동적으로평활근에전파된다. 9 서파가평활근에도착하면, L-type Ca 2+ channel이활성화하여평활근수축이일어난다. 10 신경말단에서분비되는, 아세틸콜린 (Ach), 노에피네프린 (NA), 산화질소 (NI) 와같은효과제는 ICC pacemaker의활성도를조 Figure 5. The cytosolic Ca 2+ oscillator responsible for pacemaker activity in interstitial cells of Cajal releases periodic pulses of Ca 2+ that form a Ca 2+ wave [17]. The increase in Ca 2+ activates Cl - channels (CLCA) to give the spontaneous transient inward currents (STICs) that sum to form the spontaneous transient depolarizations (STD) resulting in the slow waves of membrane depolarization (see inset). Current flow through gap junctions allows these waves to spread into neighbouring smooth muscle cells to activate contraction. - 319 -
- The Korean Journal of Medicine: Vol. 81, No. 3, 2011 - 절한다. Ach, NA는 InsP3 형성을증가하여 Ca 2+ 농도상승을유발하고, NO는 cyclic GMP-dependent protein kinase 1β (cgk1 β) 을통하여, InsP 3 receptor를인산화하여 Ca 2+ sensitivity를감소하여 pacemaker의활성도를감소시킨다 (Fig. 5) [19]. 위장관평활근층의 fibroblast-like cells (FLCs) 의생리 평활근조직을구성하는세포는다양하여, 근육세포, 신경세포와다양한형태로밝혀지고있는간질세포 (interstitial cells) 들이있다. Fibroblast-like cells (FLCs) 로불리는간질세포는많은평활근조직내에분포하며, 해부학적으로 ICC의분포와거의일치한다 [20]. 면역조직화학염색없이는근육세포와간질세포의형태학적차이를구분할수없지만, FLCs 는 ICC와미세구조에서뚜렷한차이로, 높은전자밀도와잘발달된조면소포체 (rough endoplasmic reticulum) 를가지고있 어흥분성을내재한세포라는것을알수있다 [21]. ICC에서 c-kit 항체양성소견과같이 FLCs에서는 platelet-drived growth factor receptor α (PDGFRα) 항체양성소견을보인다 (Fig. 6). 즉, FLCs 는형태학적으로 ICC와유사하며, 분포도유사하여근육층의신경얼기와동일한방향으로주행하며, 근육세포와는 gap junction을가지고있어 ICC와유사하지만 PDGFRα + 라는특징을가진다. 기능적으로는 Ca 2+ -activated K + channel protein (SK3) 의존재가알려져있어신경전달에관여할것이다. 최근의연구에서 SK3 channel은 apamin ( 벌독소 ) 에의해차단되는위장관의 purinergic inhibitory junction potentials (IJPs) 로알려져있다 [22,23]. 다시말하면 FLCs 는 Ca 2+ -activated K + current 를만들수있으므로, gap junction을통하여주위의평활근과 FLCs로흥분성을전파할수있는충분한능력이있고, 특히퓨린성흥분을한다면, P2Y1 수용체경로를통하여, 내장신경의억제신경전달 (inhibitory neurotransmission) 을매개할것이다 [21]. A B C D E F Figure 6. Relation of PDGFRα + cells to ICC and enteric neurons [22]. (A-C) double immuno-labelling of PDGFRα (green) and c-kit (red) in circular muscle layer of murine colon. Similar anatomical spaces were occupied by PDGFRα + cells and c-kit +, but no co-labelling of cells was observed demonstrating that these are discrete populations of cells. (D-F) double immuno-labelling of PDGFRα (green) and SK3 (red) in longitudinal muscle layer of murine colon. PDGFRα + cells express small conductance Ca 2+ -activated K + channels. - 320 -
- Hyun Jin Kim. Smooth muscle contraction and fibroblast-like cells - 맺음말 위장관평활근의수축은서파에의해발생하며, 서파는 ICC에서발생한다. RYR3 수용체의점화로시작된 ICC 세포내 Ca 2+ 농도증가는세포막의 Ca 2+ -sensitive chloride channels 을활성화하여 STIC을유발하고합쳐져 STD를만들고, gap junction을통하여위장관평활근으로전달된다. 평활근세포막으로전달된신호에의한 VOD의활성화로, 평활근세포내 Ca 2+ 이증가되고, calmodulin 과결합하여결합체를형성하며, 활성화된 MLC kinase에의한미오신경사슬형성은평활근수축을유발한다. 위장관의신경은억제신경으로, 평활근세포막의수용체와연계된 G 단백을통한반응으로수축의강약과기간에영향을미치고, ICC 세포막의 InsP2, cgmp 경로를통하여 ICC pacemaking 의빈도를조절하는역할을한다. FLCs은 PDGFRα + 의새로밝혀지고있는간질세포로, 형태학적으로신경얼기와평활근과의 gap junction을가지고있으며, SK3로알려진 Ca 2+ activated K + channel을다수보유하고있어, 내장신경말단에서평활근으로의신호전달체계를밝히는중요한열쇠일것이다. 중심단어 : 평활근수축 ; 카할간질세포 ; 섬유아세포-유사세포 REFERENCES 1. Sanders KM, Hwang SJ, Ward SM. Neuroeffector apparatus in gastrointestinal smooth muscle organs. J Physiol 2010;588: 4621-4639. 2. Kurahashi M, Zheng H, Dwyer L, Ward SM, Don Koh S, Sanders KM. A functional role for the 'fibroblast-like cells' in gastrointestinal smooth muscles. J Physiol 2011;589:697-710. 3. Webb RC. Smooth muscle contraction and relaxation. Adv Physiol Educ 2003;27:201-206. 4. Meiss RA. Mechanics of smooth muscle contraction. In: Kao CY, Carsten ME, eds. Cellular Aspects of Smooth Muscle Function. New York: Cambridge Univ Press, 1997:169-201. 5. Uehata M, Ishizuki T, Satoh H, et al. Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension. Nature 1997;389:990-994. 6. Félétou M, Vanhoutte PM. Endothelium-dependent hyperpolarization of vascular smooth muscle cells. Acta Pharmacol Sin 2000;21:1-18. 7. Farrugia G. Ionic conductances in gastrointestinal smooth muscles and interstitial cells of Cajal. Annu Rev Physiol 1999; 61:45-84. 8. Cajal SR. Sur les ganglions et plexus nerveux de I'intestin. CR Soc Biol (Paris) 1893;45:217-223. 9. Thuneberg L. Interstitial cells of Cajal: intestinal pacemaker cells? Adv Anat Embryol Cell Biol 1982;71:1-130. 10. Lee HT, Hennig GW, Park KJ, et al. Heterogeneities in ICC Ca 2+ activity within canine large intestine. Gastroenterology 2009;136: 2226-2236. 11. Sanders KM. A case for interstitial cells of Cajal as pacemakers and mediators of neurotransmission in the gastrointestinal tract. Gastroenterology 1996;111:492-515. 12. Park KJ. Interstitial cells of Cajal and GI motility. Korean J Neurogastroenterol Motil 2004;10:93-99. 13. Beckett EA, Horiguchi K, Khoyi M, Sanders KM, Ward SM. Loss of enteric motor neurotransmission in the gastric fundus of Sl/Sl(d) mice. J Physiol 2002;543:871-887. 14. Beckett EA, McGeough CA, Sanders KM, Ward SM. Pacing of interstitial cells of Cajal in the murine gastric antrum: neurally mediated and direct stimulation. J Physiol 2003;553:545-559. 15. Torihashi S, Ward SM, Nishikawa S, Nishi K, Kobayashi S, Sanders KM. c-kit-dependent development of interstitial cells and electrical activity in the murine gastrointestinal tract. Cell Tissue Res 1995;280:97-111. 16. Sanders KM, Ordög T, Koh SD, Torihashi S, Ward SM. Development and plasticity of interstitial cells of Cajal. Neurogastroenterol Motil 1999;11:311-338. 17. Berridge MJ. Smooth muscle cell calcium activation mechanisms. J Physiol 2008;586:5047-5061. 18. Sanders KM, Koh SD, Ward SM. Interstitial cells of Cajal as pacemakers in the gastrointestinal tract. Annu Rev Physiol 2006; 68:307-343. 19. Aoyama M, Yamada A, Wang J, et al. Requirement of ryanodine receptors for pacemaker Ca 2+ activity in ICC and HEK293 cells. J Cell Sci 2004;117:2813-2825. 20. Iino S, Nojyo Y. Immunohistochemical demonstration of c-kitnegative fibroblast-like cells in murine gastrointestinal musculature. Arch Histol Cytol 2009;72:107-115. 21. Horiguchi K, Komuro T. Ultrastructural observations of fibroblastlike cells forming gap junctions in the W/W(nu) mouse small intestine. J Auton Nerv Syst 2000;80:142-147. 22. Kurahashi M, Zheng H, Dwyer L, Ward SM, Don Koh S, Sanders KM. A functional role for the 'fibroblast-like cells' in gastrointestinal smooth muscles. J Physiol 2011;589:697-710. 23. Mutafova-Yambolieva VN, Hwang SJ, Hao X, et al. Beta-N icotinamide adenine dinucleotide is an inhibitory neurotransmitter in visceral smooth muscle. Proc Natl Acad Sci USA 2007;104: 16359-16364. - 321 -