NTRODUCTION & IMPORTANCE: Adult Hirschsprung’s disease (AHD) is a difficult diagnosis to m, Guías, Proyectos, Investigaciones de Medicina

¡Descarga NTRODUCTION & IMPORTANCE: Adult Hirschsprung’s disease (AHD) is a difficult diagnosis to m y más Guías, Proyectos, Investigaciones en PDF de Medicina solo en Docsity! Regulation of gastrointestinal motility—insights from smooth muscle biology Kenton M. Sanders, Sang Don Koh, Seungil Ro, and Sean M. Ward Department of Physiology and Cell Biology, University of Nevada School of Medicine, Anderson Medical Sciences, Reno, NV 89557, USA Abstract Gastrointestinal motility results from coordinated contractions of the tunica muscularis, the muscular layers of the alimentary canal. Throughout most of the gastrointestinal tract, smooth muscles are organized into two layers of circularly or longitudinally oriented muscle bundles. Smooth muscle cells form electrical and mechanical junctions between cells that facilitate coordination of contractions. Excitation–contraction coupling occurs by Ca2+ entry via ion channels in the plasma membrane, leading to a rise in intracellular Ca2+. Ca2+ binding to calmodulin activates myosin light chain kinase; subsequent phosphorylation of myosin initiates cross-bridge cycling. Myosin phosphatase dephosphorylates myosin to relax muscles, and a process known as Ca2+ sensitization regulates the activity of the phosphatase. Gastrointestinal smooth muscles are ‘autonomous’ and generate spontaneous electrical activity (slow waves) that does not depend upon input from nerves. Intrinsic pacemaker activity comes from interstitial cells of Cajal, which are electrically coupled to smooth muscle cells. Patterns of contractile activity in gastrointestinal muscles are determined by inputs from enteric motor neurons that innervate smooth muscle cells and interstitial cells. Here we provide an overview of the cells and mechanisms that generate smooth muscle contractile behaviour and gastrointestinal motility. Introduction The gastrointestinal tract is a series of tubular organs that process ingested food, assimilate water and nutrients, and eliminate waste. The outer layers of the gut wall are muscular tissues (tunica muscularis) that provide the forces necessary to stir the contents of the gut and move food, water and waste through the tubular chambers. In the human gastrointestinal tract, the muscles in the proximal two-thirds of the oesophagus and in the external anal sphincter are skeletal; the rest of the tunica muscularis contains smooth muscle cells (SMCs). Gastrointestinal smooth muscles are autonomous—generating spontaneous electrical rhythmicity and contractions driven by intrinsic pacemakers, Ca2+ handling and Ca2+ sensitization mechanisms. SMCs contracting independently at their own whim would Correspondence to: K. M. Sanders ksanders@medicine.nevada.edu. Competing interests The authors declare no competing interests. Author contributions K. M. Sanders contributed to all aspects of this manuscript. S. D. Koh and S. M. Ward contributed to the discussion of content and reviewing/editing the manuscript. S. Ro contributed to writing and reviewing/editing the manuscript. HHS Public Access Author manuscript Nat Rev Gastroenterol Hepatol. Author manuscript; available in PMC 2016 March 16. Published in final edited form as: Nat Rev Gastroenterol Hepatol. 2012 November ; 9(11): 633–645. doi:10.1038/nrgastro.2012.168. A uthor M anuscript A uthor M anuscript A uthor M anuscript A uthor M anuscript not produce useful movements; however, electrical and mechanical junctions formed with neighbouring cells create a syncytium that facilitates coordination of contractions of thousands of cells. This level of cooperation is still not sufficient to produce gastrointestinal motility patterns and orderly progression of luminal contents. Multiple levels of regulatory cells and mechanisms, including interstitial cells, motor neurons, hormones, paracrine substances and inflammatory mediators, are superimposed upon myogenic activity to generate normal and abnormal contractile behaviours (Figure 1). It should also be noted that smooth muscle tissues of the gastrointestinal tract are not homogenous. Differences exist in the behaviours of circular and longitudinal muscles, and in the muscles of each of the four major organs. There are also important differences in electrical and mechanical activities in different regions of organs and in the sphincters separating the organs. Here we review the cells and general mechanisms that generate smooth muscle electrical and contractile behaviours and gastrointestinal motility. Design of the smooth muscle motor Specialized binding of actin and myosin results in cross-bridge formation and cycling, and force generation in smooth muscles. Myosin is a hexametric protein with parallel functions as a force-generating motor protein and enzymatic activity for the hydrolysis of ATP.1,2 Smooth muscle myosin is composed of two 200 kDa heavy chains that are each associated with two light chains: a 20 kDa regulatory subunit (MLC20), and a 17 kDa (MLC17) nonphosphorylatable subunit. The N-terminal ends of the heavy chains form globular heads with enzymatic activity and sites for actin binding. MLC20 and MLC17 bind to the neck region of myosin heads. Four isoforms of myosin heavy chains are found in smooth muscles, resulting from transcription of MYH11 and alternative splicing.3–5 Two isoforms, 204 kDa (SM1) and 200 kDa (SM2) result from a splice site at the COOH terminus, and two isoforms (SMB and SMA) result from a splice site in the S1 head region. Various combinations of these isoforms (for example, SM1A or SM1B) have tissue and cell specific distributions in different smooth muscles. Typically, SM1 and SM2 are about equally distributed in smooth muscle cells,6 but substantial diversity exists in the expression of SMA and SMB isoforms. In the fundus, for example, SMA is the dominant isoform, whereas SMB increases with distance from the fundus to the antrum and represents the dominant isoform in antral muscles.6 There is also diversity in myosin heavy chain isoform expression between cells within a given tissue, but the functional importance of this cellular heterogeneity is not yet understood.7 Contractions are initiated by phosphorylation of MLC20 by Ca2+/calmodulin-dependent myosin light chain kinase or Ca2+-independent kinases, including Rho-kinase, integrin- linked kinase and zipper-interacting protein kinase (ZIPK; Figure 2).8–11 An increasing level of cytoplasmic Ca2+ ([Ca2+]i) is the main physiological event that activates myosin light chain kinase. Phosphorylation of MLC20 facilitates myosin binding to actin, initiating cross- bridge cycling and force development. MLC20 phosphorylation and contraction are balanced by myosin light chain phosphatase (MLCP). MLCP is composed of three subunits.12 One of these subunits (myosin phosphatase target subunit; MYPT) anchors MLCP to phosphorylated MLC20 and targets a 37 kDa catalytic subunit (type 1 serine/ threonine phosphatase, PP1c), to myosin.13 Phosphorylation of MYPT (see below) can Sanders et al. Page 2 Nat Rev Gastroenterol Hepatol. Author manuscript; available in PMC 2016 March 16. A uthor M anuscript A uthor M anuscript A uthor M anuscript A uthor M anuscript of smooth muscles. Interactions between Ca2+ sparks and puffs can induce regenerative Ca2+ waves45 These waves seem to run along the inner surface of cell membranes without a generalized increase in [Ca2+]i, because there is typically no contractile response associated with Ca2+ waves. Ca2+ waves are thought to regulate ion channel openings and participate in setting the membrane potential and cellular excitability. Regulation of Ca2+ sensitivity As described above, activation of myosin light chain kinase and phosphorylation of MLC20 in SMCs is Ca2+ dependent, but force development is not simply a function of [Ca2+]i levels. Additional pathways modulate the Ca2+ sensitivity of the contractile apparatus, such that different agonists can elicit contractile events of different magnitudes with equivalent changes in [Ca2+]i.46 A neutral [Ca2+]i versus force curve (such as a contractile response to Ca2+ entry via voltage-dependent ion channels) might shift dramatically to the left when excitatory agonists, such as acetylcholine, stimulate muscles; in such cases, low levels of [Ca2+]i can elicit contractile responses of far greater amplitude than normal.8 Ca2+ sensitization is controlled through the activity of MLCP (Figure 2). Contraction is initiated by phosphorylation of myosin (MLC20), so dephosphorylation of myosin reduces contraction. Phosphorylation of MYPT (the regulatory subunit of MLCP) reduces the activity of the phosphatase and sustains cross-bridge cycling and contraction.47,48 Rho kinase was the first enzyme shown to phosphorylate MYPT, but ZIPK is also known to phosphorylate MYPT and enhance Ca2+ sensitization.49,50 CPI-17 is another signalling molecule that regulates MLCP. When phosphorylated by protein kinase C, Rho kinase or other kinases, CPI-17 binds to the catalytic subunit of MLCP and inhibits dephosphorylation of MLC20.8,47 As discussed above, acetylcholine is coupled to responses via M2 and M3 receptors. M3 receptors are coupled through Gq/11 to production of diacylglycerol and IP3. Diacylglycerol and increased levels of [Ca2+]i activate protein kinase C isoforms, which phosphorylate CPI-17. M3 receptors also couple to Ca2+ sensitization mechanisms through G q/11 and possibly a second population of G proteins, G12/13, to activate Rho kinase. Phosphorylation of CPI-17 and activation of Rho kinase and subsequent phosphorylation of MYPT facilitate Ca2+ sensitization and enhance contraction. Cyclic nucleotides, cGMP and cAMP, via cGMP-dependent and cAMP-dependent protein kinases (PKG and PKA), relax smooth muscles at constant [Ca2+]i.8,51,52 cGMP-dependent Ca2+ desensitization is mediated, in part, via protein kinase G phosphorylation of RhoA at serine 188 (S188), which prevents its binding to Rho kinase and decreases Ca2+ sensitization.53 This mechanism explains inhibition of Ca2+ sensitization by cyclic nucleotide-dependent mechanisms, but additional pathways are needed to fully explain a rightward shift in the Ca2+-force relationship, the hallmark of Ca2+ desensitization. Telokin, a small acidic protein with partial sequence homology to myosin light chain kinase, is phosphorylated at serine 13 (S13) in response to 8-bromo-cGMP or forskolin and tends to stabilize the unphosphorylated state of MLC20.54 Thus, cGMP-dependent and cAMP- dependent Ca2+ desensitization might be mediated through telokin. Studies on telokin null Sanders et al. Page 5 Nat Rev Gastroenterol Hepatol. Author manuscript; available in PMC 2016 March 16. A uthor M anuscript A uthor M anuscript A uthor M anuscript A uthor M anuscript mice demonstrate that telokin enhances the activity of MLCP, thus reducing Ca2+ sensitivity.55 Ca2+ sensitization mechanisms are important regulatory pathways in gastrointestinal SMCs, and might provide novel therapeutic strategies for controlling gastrointestinal motility. Whether regulation of Ca2+ sensitization mechanisms contribute to responses to neurotransmitters released from neurons is unknown. These mechanisms have been studied by adding neurotransmitters to muscles immersed in organ baths in which all cells in the muscles are exposed to agonists. As discussed below, neurotransmitter binding might be restricted to receptors in neuroeffector junctions. If this is the case, then neurotransmitter responses might not be mediated by the same receptors and pathways affected by exogenous, bath-applied agonists. Regulation of smooth muscle by motor neurons Motility patterns in the gastrointestinal tract depend upon control of muscles by enteric neurons. The motor responses of gastrointestinal muscles have been studied for many years, and considerable progress in identifying neurotransmitters and clarifying postjunctional mechanisms has been made (Table 1). Gastrointestinal muscles are innervated by inhibitory and excitatory nerves.56,57 Control by enteric neurons enables complex motor patterns, such as the peristaltic reflex, segmentation, regulation of tonic contractions (such as receptive relaxation) and control of sphincters. The peristaltic reflex, a stereotypical response of gastrointestinal muscles, consists of contraction at, or above, the site of stimulation and relaxation below the site of stimulation.58–61 A poststimulus excitatory response follows the inhibitory response (‘rebound’ excitation),62,63 and constitutes a third phase of the peristaltic reflex. Excitatory and inhibitory neurotransmitters Gastrointestinal smooth muscle responses depend upon excitatory and inhibitory neurotransmitters. These molecules are linked to complex cell signalling pathways through a variety of receptors in postjunctional cells (Table 1). Acetylcholine is the most prominent excitatory neurotransmitter, and postjunctional responses are mediated by muscarinic receptors (M2 and M3).20,32,33,64,65 Neurokinins (substance P and neurokinin A) bind to NK1 and NK2 receptors and activate pathways similar to acetylcholine.22,66–69 Nitric oxide (NO) is now considered the predominant inhibitory neurotransmitter,70,71 and it is coupled, via nontraditional postjunctional receptors (that is, cytoplasmic proteins), to activation of guanylyl cyclase leading to production of cGMP, activation of protein kinase G, activation of K+ channels and decreased Ca2+ sensitivity53,55,72,73 The purine neurotransmitter is inhibitory in gastrointestinal smooth muscles and has long been thought to be ATP. In fact, the purine neurotransmitter might be β-nicotinic adenine dinucleotide (β- NAD), because this nucleotide better fulfils the criteria for a neurotransmitter.74,75 Purines bind to P2Y1 receptors in postjunctional cells, elicit production of IP3 and diacylglycerol, and activate SK channels and hyperpolarization.76–78 In some regions of the gut, vasoactive intestinal polypeptide and/or pituitary adenylate cyclase-activating polypeptide (PACAP) also contribute to inhibitory neurotransmission, but these substances are released generally Sanders et al. Page 6 Nat Rev Gastroenterol Hepatol. Author manuscript; available in PMC 2016 March 16. A uthor M anuscript A uthor M anuscript A uthor M anuscript A uthor M anuscript at high stimulus frequencies of nerve stimulation (≥10Hz).79,80 Vasoactive intestinal polypeptide and PACAP act through VPAC1/2 receptors coupled to Gs, leading to activation of adenylyl cyclase, generation of cAMP and activation of cAMP-dependent protein kinase A.81,82 This pathway generally reduces excitability and contraction by activating K+ channels and by reducing the Ca2+ sensitivity of the contractile apparatus.8,81 Excitatory and inhibitory neurotransmitters are released from distinct populations of excitatory and inhibitory motor neurons. Postjunctional responses are determined by a series of factors, including: first, which cells are in close proximity to the sites of neurotransmitter release (once released, transmitters might be rapidly metabolized or ‘deactivated’ by diffusion as the concentration of the transmitter is diluted into the postjunctional interstitium); second, which cells express appropriate receptors for specific neurotransmitters (all cells in the postjunctional, neuroeffector field might express receptors for a given transmitter, but responses might be elicited by specific receptors expressed by only one type of cell); and third, which ionic conductances or effector mechanisms are linked to second messenger pathways in postjunctional cells. Neuropeptides in motility responses Excitatory and inhibitory neurons also express neuropeptides that regulate the motor control of gastrointestinal muscles.83 This topic is less well-described in the literature because peptidergic responses are only elicited in most gastrointestinal muscles at higher frequencies of enteric nerve firing (usually >5 Hz). At lower frequency stimulation, responses can normally be blocked entirely by a combination of M2 and M3 muscarinic receptor blockers, P2Y1 receptor blockers, and NO synthesis inhibitors.75 The physiological firing frequencies of enteric neurons during enteric reflexes are poorly documented, but the fact that the same antagonists and inhibitors can block peristaltic reflex responses, receptive or adaptive relaxation, and lower oesophageal opening, suggests that many motility responses depend on the small molecule neurotransmitters NO, acetylcholine and β-NAD/ATR The peptides seem to be reserved for more extreme conditions or possibly when other motor pathways are compromised. The role of interstitial cells ICC Many gastrointestinal smooth muscle tissues and organs display ‘autonomous’ activity. Spontaneous pacemaker activity in the stomach, small intestine and colon (electrical slow waves) organizes contractile patterns into phasic contractions that are the basis for peristaltic or segmental motility patterns.84 Pacemaker activity is intrinsic to gastrointestinal muscles and does not depend on neural or hormonal inputs, although the degree of coupling between pacemaker activity and contractions is highly dependent on neural and other regulatory inputs. Basal slow wave activity generates low amplitude contractions, and inhibitory or excitatory neural inputs modulate the amplitude of contractions during each cycle. In the 1960s and 1970s Ladd Prosser’s group at the University of Illinois suggested that longitudinal muscle was the source of pacemaker activity;85 however, only excised muscles retaining a bit of the myenteric plexus tissue were spontaneously active. Morphologists Sanders et al. Page 7 Nat Rev Gastroenterol Hepatol. Author manuscript; available in PMC 2016 March 16. A uthor M anuscript A uthor M anuscript A uthor M anuscript A uthor M anuscript effectively shield other cells from neurotransmitters. In the very small (<20 nm) regions between ICC and enteric nerve varicosities (neuro-ICC junctions), high concentrations of neurotransmitters might be achieved during neurotransmission, which could enhance the rate of metabolism of transmitters.112 Thus, when ICC are absent, transmitters might be available to bind to receptors of other postjunctional cells.121 An extensive review describing the controversy about the role of ICC in motor neurotransmission is available.121 Future studies to characterize which cell-specific effectors are activated in response to neurotransmission or which key receptor and effector genes are deactivated in specific cell- types will be needed to clarify the role of ICC in neurotransmission. For example, muscarinic agonists stimulate different ion channels in SMCs and ICC. Postjunctional responses to cholinergic nerve stimulation in the small intestine have been shown to be mediated by ion channels expressed by ICC (that is, Ca2+-activated Cl− channels) but not by SMCs.122 These data suggest that neurotransmitters released from nerve terminals might bind to receptors on ICC but not reach muscarinic receptors expressed by SMCs. In our view, low resistance, electrical coupling between ICC and SMCs is essential for the functions of ICC in gastrointestinal muscles. Ultrastructural studies have provided evidence that gap junctions exist between ICC and SMCs;86,87,111,123 however, the junctions between ICC–MY and SMCs seem to be small and relatively rare.124 Electrical coupling between these cells is clearly evident, however, from electrophysiological studies.105 Several studies have investigated the expression of connexin proteins (gap junction proteins). SMCs and ICC express connexin 43105,125,126 and connexin 40 immunoreactivity has also been observed in the dog.127 Connexin 45 might be specific to gap junctions between ICC.126 PDGFRα+ cells Purines, one of the inhibitory neurotransmitters released from enteric motor neurons, are weakly active on gastro-intestinal SMCs.128,129 A new type of interstitial cell in gastrointestinal muscles has been shown to respond to purines.76 Electron microscopy previously described a non-ICC type of interstitial cell in gastrointestinal muscles. These cells, referred to as fibroblast-like cells, are found near terminals of motor neurons and form gap junctions with SMCs.130–132 Fibroblast-like cells express small conductance Ca2+- activated K+ channels, SK3 (encoded by KCNN3),133–136 which might be activated in purinergic inhibitory responses. These fibroblast-like cells are labelled with antibodies to PDGFRα, and PDGFRα+ cells express SK3 channels and P2Y1 receptors.76,135,136 These proteins are key in purinergic inhibitory regulation of gastrointestinal motility76–78 A transgenic animal with enhanced GFP targeted to PDGFRα+ cells was used to isolate these cells and test their responsiveness to purine neurotransmitters.76 Purine neurotransmitters elicited large amplitude K+ currents in PDGFRα+ cells that were blocked by P2Y1 receptor antagonists and SK3 channel antagonists. Under the same experimental conditions (that is, ionic gradients and holding potentials equivalent to resting potentials in gastrointestinal muscles), purines failed to elicit outward currents in SMCs.76 These data suggest that the large amplitude hyperpolarization responses elicited in gastrointestinal muscles by purine neurotransmission (inhibitory junction potentials) are more likely to be meditated by PDGFRα+ cells than by SMCs. Sanders et al. Page 10 Nat Rev Gastroenterol Hepatol. Author manuscript; available in PMC 2016 March 16. A uthor M anuscript A uthor M anuscript A uthor M anuscript A uthor M anuscript Integration of behaviour in the SIP syncytium Gastrointestinal motility, resulting from coordinated contractions of thousands of SMCs, is a highly integrated phenomenon (Figure 1). Ionic channels in the plasma membrane and basal levels of contractile protein phosphorylation set the excitability and contractility of SMCs. SMCs are electrically coupled, so conductance changes in one cell or group of cells can influence the excitability of the broader syncytium of cells. Superimposed on myogenic mechanisms are the behaviours of interstitial cells (ICC and PDGFRα+ cells) that are electrically coupled to the smooth muscle syncytium. Thus, the myogenic apparatus is an electrical syncytium consisting of SMCs/ ICC/PDGFRα+ cells or the ‘SIP syncytium’. Activation of ion channels in any of these cells can affect voltage-dependent channels in SMCs. For example, activation of Ca2+-activated Cl− channels in ICC generates inward currents, producing slow waves. Slow waves conduct to SMCs producing cycles of depolarization that can activate Ca2+ channels and couple slow waves to smooth muscle contractions. Superimposed on the summed activity of smooth muscle and interstitial cells are inputs from enteric motor neurons. As we have seen, neurotransmitters can activate conductances in SMCs and interstitial cells (for example, Ca2+-activated Cl− channels or K+ conductances in ICC or SK3 channels in PDGFRα+ cells) that enhance (net inward currents) or reduce (net outward currents) the excitability of the SIP syncytium. When neurotransmitters bind to receptors of SMCs, mechanisms of Ca2+ sensitization or desensitization regulate contractile responses. In addition to these mechanisms are layers of regulation including circulating hormones, local paracrine substances and inflammatory mediators. Inputs from some endogenous bioregulatory molecules can modulate smooth muscle responses such that otherwise normal behaviours of SMCs, interstitial cells and motor neurons result in inappropriate motility (paralysis or hypercontractile states). Such dysmotilities can adversely affect movements of food, absorption of nutrients and water, and transit of wastes. New therapeutic strategies Tissue engineering of gastrointestinal muscles Motility disorders can result from developmental failures and disease processes that compromise function or from surgical interventions that remove portions of gastrointestinal muscle. Regenerative medicine raises hope that some of these problems might be corrected through engineering functional gastrointestinal muscles, and progress has been made in this field during the past decade. However, the complex organization and plasticity of cells within the SIP syncytium, vascularization, and immunological constraints are major obstacles to accomplishing this feat. Tissue engineering of gastrointestinal muscles has benefitted from attempts to develop vascular grafts. Nearly 250,000 patients per year undergo coronary bypass surgery, and up to one-quarter of these patients do not have suitable autologous vessels for the bypass.137 Thus, extensive effort has gone into engineering blood vessel segments that might be suitable for bypass procedures, and there has been considerable investigation of soluble signalling factors, mechanical stimulation, extracellular matrix, scaffolding materials, and how other cells influence smooth muscle phenotypes.138 The use of differentiated SMCs as Sanders et al. Page 11 Nat Rev Gastroenterol Hepatol. Author manuscript; available in PMC 2016 March 16. A uthor M anuscript A uthor M anuscript A uthor M anuscript A uthor M anuscript a starting point in tissue engineering is complicated by the tendency of these cells to dedifferentiate from a contractile phenotype to a synthetic or proliferative phenotype. Initially, the phenotypic plasticity is useful to expand the SMCs from a biopsy sample, but inducing proliferating SMCs to return to the contractile phenotype is problematic. Although several groups have attempted to engineer gastrointestinal muscles by a variety of techniques,139 it has been considerably more difficult to accomplish than for vascular tissue owing to the multiple cell types that contribute to postjunctional integration of neural inputs and regulation of motility. One promising area might be to grow sphincteric muscles from dispersed smooth muscle tissues because the motility in sphincters is far easier to simulate than complex motility patterns in most regions of the gastrointestinal tract. Some successes have been achieved with engineering of sphincteric tissues, and rings of muscle have been grown and grafted into animals to stimulate vascularization.140,141 The muscle rings were characterized for expression of α-actin and caldesmon. However, we believe that demonstration of only a few proteins represents a low standard for verification of a contractile smooth muscle phenotype, and we have previously suggested that more vigorous phenotyping is necessary in studies of this type (see below).27 Functional studies showed that engineered muscle rings developed low levels of spontaneous tone and responded to excitatory and inhibitory neurotransmitters.140,141 In other studies, human muscle cells have been co-cultured with neurons from imortomice donors, and cells immunopositive for vasoactive intestinal polypeptide and choline acetyl-transferase developed within the engineered muscle rings.140,141 However, in our opinion, the histological images of the engineered muscle rings provided in these papers suggest that the predominant cells grown in the muscular rings were skeletal muscles, not SMCs (for example, cells are multinucleated and much larger than expected for SMCs140). Much is now known about the ‘excitasome’ of SMCs (that is, the genes transcribed to generate proteins essential for smooth muscle function and phenotype); several essential pathways have been discussed in this Review. Modern genomic or sequencing techniques could easily assay gene expression in engineered smooth muscle tissues or cultured cells to verify the degree to which so-called SMCs and tissues recapitulate the phenotype of native SMCs. As described in this Review, gastrointestinal smooth muscle behaviours result from coordinated activity of several types of cells (for example, motor neurons, cells of the SIP syncytium, etc.). We feel that progress toward successful tissue engineering of gastrointestinal smooth muscles will be slow until more rigorous standards of evaluating tissue structure, cellular composition and SMC phenotype are employed. MicroRNAs Another therapeutic approach might be the molecular regulation of transcription factors that regulate the smooth muscle phenotype. Genes that determine the smooth muscle phenotype are regulated by serum response elements (CArG boxes) in promoter/enhancer regions, to which the transcription factor serum response factor (SRF) binds.142 The actions of SRF are regulated by myocardin (MYOCD) and ELK1.143 Studies have shown that the differentiation and growth of SMCs are regulated by microRNAs (miRNAs; for example, Sanders et al. Page 12 Nat Rev Gastroenterol Hepatol. Author manuscript; available in PMC 2016 March 16. A uthor M anuscript A uthor M anuscript A uthor M anuscript A uthor M anuscript 10. Niiro N, Ikebe M. Zipper-interacting protein kinase induces Ca2+-free smooth muscle contraction via myosin light chain phosphorylation. J. Biol. Chem. 2001; 276:29567–29574. [PubMed: 11384979] 11. Ihara E, MacDonald JA. 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A uthor M anuscript A uthor M anuscript A uthor M anuscript A uthor M anuscript Key points ▪ Gastrointestinal motility occurs by the coordinated contractions of the tunica muscularis, which forms the outer wall of the alimentary canal from the distal oesophagus to the external anal sphincter ▪ Excitation-contraction coupling results from Ca2+ entry into smooth muscle cells, Ca2+ release from the sarcoplasmic reticulum, activation of myosin light chain kinase and phosphorylation of the regulatory light chains of myosin ▪ Contractile force is tuned by Ca2+ sensitization mechanisms that balance rates of myosin phosphorylation and dephosphorylation ▪ Interstitial cells of Cajal (ICC) provide spontaneous pacemaker activity in gastrointestinal muscles; ICC and PDGFRa+ cells also contribute to mediation of inputs from enteric motor neurons ▪ Gastrointestinal motility patterns are highly integrated behaviours requiring coordination between smooth muscle cells and utilizing regulatory inputs from interstitial cells, neurons, and endocrine and immune cells ▪ Therapeutic regulation and tissue engineering of gastrointestinal motility is proving difficult Sanders et al. Page 22 Nat Rev Gastroenterol Hepatol. Author manuscript; available in PMC 2016 March 16. A uthor M anuscript A uthor M anuscript A uthor M anuscript A uthor M anuscript ganglia (not shown). Information is sent by efferent neurons from the CNS to enteric ganglia. Hormones, reaching the gut via the circulation, and paracrine and immune factors, produced by mast cells and resident macrophages, also affect motor output. Abbreviations: ICC, interstitial cells of Cajal; SMC, smooth muscle cell; SIP, syncytium of neuro-effector cells consisting of SMCs, ICC and PDGFRα+ cells. Sanders et al. Page 25 Nat Rev Gastroenterol Hepatol. Author manuscript; available in PMC 2016 March 16. A uthor M anuscript A uthor M anuscript A uthor M anuscript A uthor M anuscript Figure 2. Major cellular mechanisms controlling contraction in gastrointestinal smooth muscle cells. Mechanisms leading to enhanced contraction are depicted in red, and pathways linked to decreased contraction are shown in blue. Ca2+ required for excitation–contraction coupling can enter cells through VDCC or NSCC. The open probability of VDCC is enhanced (circle with + sign) by depolarization caused by opening of NSCC and influx of Na+ or Ca2+. Openings of VDCC are decreased by a variety of K+ channels expressed by SMCs; most inhibitory agonists regulate Ca2+ influx by activating K+ channels. Ca2+ entry can also be supplemented by release of Ca2+ from IP3 receptor-operated Ca2+ channels in the sarcoplasmic reticulum membrane. Ca2+ release from sacroplasmic reticulum can also occur through ryanodine receptors (not shown). IP3 is synthesized by PLCβ in response to agonist binding to G-protein-coupled receptors and coupling through Gα q/11. [Ca2+]i binds to calmodulin and activates myosin light chain kinase, which phosphorylates MLC20 to facilitate cross-bridge formation. Phosphorylation of MLC20 is balanced by the action of MLCP. Dephosphorylation of MLC20 reduces cross-bridge cycling and leads to muscle relaxation. Factors that lead to inhibition of MLCP increase contraction and, in effect, enhance Ca2+ sensitivity of the contractile apparatus. The opposite is true for factors that activate MLCP. A pathway that increases Ca2+ sensitization (and therefore increases contraction) occurs through binding of G-protein-coupled (Gαq/11 or Gα12/13) receptors and regulation of the GDP-GTP exchange factor (Rho-GEF), RhoA and activation of Rho Sanders et al. Page 26 Nat Rev Gastroenterol Hepatol. Author manuscript; available in PMC 2016 March 16. A uthor M anuscript A uthor M anuscript A uthor M anuscript A uthor M anuscript kinase. Rho kinase and protein kinase C can phosphorylate CPI-17 (at T38), a protein that when phosphorylated inhibits the catalytic subunit of MLCP (PPlc; circle with negative sign). Rho kinase can also phosphorylate the regulatory subunit of MLCP (MYPT) at T696 and T853. Phosphorylation of MYPT decreases the activity of MLCP, preserving phosphorylation of MLC20. ZIPK also phosphorylates CPI-17 and MYPT. Cyclic nucleotide-dependent pathways decrease Ca2+ sensitivity. NO, for example, binds to guanylyl cyclase (composed of GCα and GCβ subunits) and generates cGMR cGMP activates cGMP-dependent protein kinase (PKG), which can phosphorylate RhoA and reduce activation of Rho kinase, thus reducing Ca2+ sensitization, or it can phosphorylate telokin (S13), which stimulates MLCP (circle with + sign). Binding of receptors coupled through Gαs activates adenylate cyclase and production of cAMP. PKA can also phosphorylate telokin and increase MLCP activity. Abbreviations; [Ca2+]i, cytoplasmic Ca2+; DAG, diacylglycerol; IP3, inositol 1,4,5-trisphosphate; MLC20, 20kDA light chain of myosin; MLCP, myosin light chain phosphatase; NO, nitric oxide; NSCC, nonselective cation channels; PKA, cAMP-dependent protein kinase; PKG, cGMP-dependent protein kinase; PLCβ, phospholipase Cβ; VDCC, voltage-dependent Ca2+ channels; ZIPK, zipper- interacting protein kinase. Permission obtained Wiley © Sanders, K. M. Neurogastroenterol. Motil. 20 (Suppl. 1), 39–53 (2008). Sanders et al. Page 27 Nat Rev Gastroenterol Hepatol. Author manuscript; available in PMC 2016 March 16. A uthor M anuscript A uthor M anuscript A uthor M anuscript A uthor M anuscript