内脏平滑肌Cajal间质细胞起搏功能(英文)

胃肠道的大部分区域都存在着一种特殊的间质细胞——Cajal间质细胞(interstitial cells of Cajal,ICCs)。尽管在100多年前它们的存在就已被发现,但是直到最近几十年的研究才逐渐揭示了它们的功能。在胃肠道,ICCs被认为是平滑肌自发性节律性电活动,即"基本电节律"(又称"慢波")的起搏细胞,并介导神经至平滑肌的神经信号传递活动。除胃肠道外,ICC样细胞同样存在于其它内脏平滑肌,如泌尿、生殖系统以及血管平滑肌等。本文仅就这些内脏平滑肌ICCs的功能做一简单综述。     

Uterine contractions depend on KIT-positive interstitial cells in the mouse: genetic and pharmacological evidence

In the gastrointestinal tract, interstitial cells of Cajal (ICCs) generate a pacemaker activity. They produce electric slow waves that trigger and coordinate gut smooth muscle contractions. Interstitial cells of Cajal's slender shape is revealed by KIT immunostaining. Based on several features, including KIT expression and KIT dependence, ICC-like cells were identified in nongastrointestinal tissues. Here, we investigated in the mouse whether uterine contractions depend on ICC-like cells' activity. By labeling KIT-expressing cells, we found putative ICC-like cells in the uterus, observed as KIT-positive interstitial, long spindle-shaped cells with fine branched cytoplasm processes, distributed in muscular layers and in subepithelial connective tissue. We then checked the potential KIT dependence of ex vivo contractile activity of the uterus by combining genetic and pharmacological approaches, using the Kit W-v hypomorphic mutation, and imatinib as a KIT noncompetitive inhibitor. We found a significant reduction in frequency of longitudinal uterine contractions in Kit W-v/Kit W-v compared with Kit+/+ mice, whereas amplitude was unaffected. There was no difference in frequency or amplitude of circular uterine contractions between Kit W-v/Kit W-v and Kit+/+ mice. Ex vivo treatment of Kit+/+ uterine horns with imatinib resulted in a dose-dependent reduction of the frequency and amplitude of longitudinal myometrial contractions. Amplitude and frequency of circular contractions were unaffected in presence of imatinib. These concurrent results suggest that longitudinal contractions of the uterus depend on a KIT signaling pathway of ICC-like cells. The existence of ICC-like cells in the myometrium may enhance our understanding of uterine spontaneous contractile activity and suggest new approaches for treatment of uterine contractility disorders.     

Use of a microelectrode array to record extracellular pacemaker potentials from the gastrointestinal tracts of the ICR mouse and house musk shrew (Suncus murinus)

BackgroundThe rhythmic contraction and relaxation of smooth muscles in the gastrointestinal (GI) tract is governed by pacemaker electrical potentials, also termed slow waves, which are calcium currents generated by interstitial cells of Cajal (ICCs). Malfunction of pacemaker rhythms contributes to a number of clinically challenging gastrointestinal motility disorders.MethodA microelectrode array (MEA) was used to record slow waves in vitro from intact GI tissues freshly isolated from the ICR mouse and Suncus murinus. The effects of temperature, extracellular calcium and potassium concentrations on pacemaker potentials were quantified using spatiotemporal metrics.ResultsPacemaker frequency decreased from the duodenum to the ileum in the mouse, but this phenomenon was less significant in Suncus murinus. In both the mouse and Suncus murinus, the stomach had a much lower pacemaker frequency than the intestine. Propagation velocity and amplitude were highest in the proximal intestine. Temperature significantly increased pacemaker frequency in the intestinal tissues of both species. Removal of Ca²⁺ from the medium inhibited pacemaker potential and increasing the Ca²⁺ concentration increased pacemaker frequency in the mouse ileum. Increasing K⁺ concentration decreased pacemaker frequency in the absence of nifedipine.ConclusionsThe MEA allows efficient investigation of gut pacemaker frequency and propagation.     

Loss of interstitial cells of Cajal after pulsating electromagnetic field (PEMF) in gastrointestinal tract of the rats.

Exposure to the magnetic field has remarkably increased lately due to fast urbanization and widely available magnetic field in diagnosis and treatment. However, biological effects of the magnetic field are not well recognized. The myoelectric activity recorded from the gastrointestinal and urinary systems is generated by specialized electrically active cells called interstitial cells of Cajal (ICCs). Thus it seems rational that ICC have significant vulnerability to physical factors like an electromagnetic field. The aim of this study was to evaluate the influence of pulsating electromagnetic field (PEMF) (frequency 10 kHz, 30ms, 300 muT burst, with frequency 1Hz) on ICCs density in the rat gastrointestinal tract. Rats were divided into two groups (n=32). The first group was exposed to PEMF continuously for 1, 2, 3, and 4 weeks (n = 16), and the second group (n=16) served as a control. Tissue samples of the rat stomach, duodenum and proximal colon were fixed and paraffin embedded. The tangential sections of 5 microm thickness were stained immunohistochemically with anti-c-Kit (sc-168) antibody and visualized finally by DAB as chromogen (brown end product). C-Kit positive branched ICC-like cells were detected under the light microscope, distinguished from the c-kit-negative non-branched smooth muscle cells and from the c-kit positive but non-branched mast cells and quantitatively analyzed by MultiScan computer program. Apoptosis detection was performed with rabbit anti-Bax polyclonal antibody (Calbiochem, Germany) and LSAB 2 visualization system. The surface of c-Kit immunopositive cells decreased after exposure to PEMF in each part of the gastrointestinal tract. Reduced density of ICCs was related to exposure time. The most sensitive to PEMF were ICCs in the fundus of the stomach and in the duodenum, less sensitive were ICCs in the colon and pacemaker areas of the stomach. No marked changes in ICC density in the pyloric part of the stomach were observed. We demonstrate that the PEMF induced apoptosis dependent decrease in ICC expression.     

Regional differences in signalling transduction pathways among smooth muscle cells from rabbit colon

Smooth muscle cells (SMC) from the circular muscle layer of rabbit colon, taken from the proximal and distal regions that are known to have different physiological and motor activities, were used to highlight distinct regional intrinsic myogenic properties and to investigate the correlations between receptor and signalling transduction pathways. Contractile agonists were shown to be more potent on proximal than on distal SMC in inducing contraction and intracellular Ca(2+) increase. Concentration-response curves of agonists-induced Ca(2+) increase were constantly shifted to the right, though remaining parallel, with respect to contraction curves, independently of the region analysed. Using agents activating different steps of cAMP-or cGMP-mediated intracellular cascades, main regional differences were revealed as far as relaxation was concerned. Relaxation of proximal SMC was found to be essentially cGMP mediated, while that of distal SMC was cAMP mediated. In conclusion, the motor patterns of the two regions appear to be influenced by distinct regional biochemical characteristics that are intrinsic to colonic SMC.     

Further observations on upper urinary tract smooth muscle

Summary Light and electron microscopic techniques have been employed to study the arrangement and distribution of two types of muscle in the upper urinary tract of the rat. An outer layer of cells has been identified in the wall of the renal calix and pelvis. These cells are separated by connective tissue but possess numerous processes which make close contacts with adjacent cells. A layer of similar cells has not been observed in the wall of the upper ureter. The inner layer of muscle in the calix and pelvis is composed of larger cells similar to and apparently continuous with ureteric muscle. These cells are closely related to one another without intervening connective tissue and possess numerous bundles of myofilaments which extend along the length of the cell. The two types of muscle are closely related and, in the junctional region, cells of the outer layer are arranged along the length and make close contacts with one or more of the inner smooth muscle cells. A quantitative estimation has been made of nerve bundles associated with smooth muscle forming the outer layer of the calix and pelvis and with the muscle of the ureter. The results have shown a five fold increase in nerves associated with the caliceal muscle when compared with the ureter. The results are discussed in relation to the concept of a ureteric pacemaker.The authors wish to thank Professor G. A. G. Mitchell for his useful advice and encouragement.     

Contraction of human colonic circular smooth muscle cells is inhibited by the calcium channel blocker pinaverium bromide

The effects of L-type calcium channel blockers (CCBs) selective for the gastrointestinal tract (pinaverium) or non-selective (nicardipine and diltiazem), were investigated on CCK-, CCh- or KCl-induced contraction of smooth muscle cells (SMC) isolated from the circular muscle layer of normal or of inflamed human colons. In the normal tissue colon, whatever the contractile agent used, CCK-8 (1nM), CCh (1nM) or KCl (20mM), a micromolar concentration of pinaverium significantly inhibited contraction (88.36%, 93.10%, 93.92% inhibition respectively); this effect was concentration-dependent for CCh (IC50 = 0.73 +/- 0.08nM) and for CCK (IC50 = 0.92 +/- 0.12nM). In parallel, both nicardipine and diltiazem inhibit significantly contraction of isolated SMC. In inflamed colons, pinaverium (1 microM) display a significant higher efficacy than diltiazem or nicardipine to reduce cell contraction induced by CCK-8 or by KCl. In addition, RT-PCR experiments were performed to evidence tissue specificity of the L-type calcium channel. They revealed the expression of the messenger of the a-1 subunit L-type calcium channel (binding site of such CCBs), consistent with the expression of the rbC-2 splice variant of the alpha1-C gene.In conclusion: (i) the inhibition by calcium channel blockers of agonist-induced contractile activity suggest a modulation of SMC contraction upon extracellular calcium via 'L-type' voltage-dependent calcium channel; (ii) this study provides a rationale for the clinical use of pinaverium in colonic motor disoders affecting the contractility of SMC, since it appeared to decrease the contraction even in pathological situation; and (iii) RT-PCR experiments confirms the presence in human colon SMC of the alpha-1 subunit mRNA of calcium channel.     

Close relation of arterial ICC-like cells to the contractile phenotype of vascular smooth muscle cell

This work aimed to establish the lineage of cells similar to the interstitial cells of Cajal (ICC), the arterial ICC-like (AIL) cells, which have recently been described in resistance arteries, and to study their location in the artery wall. Segments of guinea-pig mesenteric arteries and single AIL cells freshly isolated from them were used. Confocal imaging of immunostained cells or segments and electron microscopy of artery segments were used to test for the presence and cellular localization of selected markers, and to localize AIL cells in intact artery segments. AIL cells were negative for PGP9.5, a neural marker, and for von Willebrand factor (vWF), an endothelial cell marker. They were positive for smooth muscle alpha-actin and smooth muscle myosin heavy chain (SM-MHC), but expressed only a small amount of smoothelin, a marker of contractile smooth muscle cells (SMC), and of myosin light chain kinase (MLCK), a critical enzyme in the regulation of smooth muscle contraction. Cell isolation in the presence of latrunculin B, an actin polymerization inhibitor, did not cause the disappearance of AIL cells from cell suspension. The fluorescence of basal lamina protein collagen IV was comparable between the AIL cells and the vascular SMCs and the fluorescence of laminin was higher in AIL cells compared to vascular SMCs. Moreover, cells with thin processes were found in the tunica media of small resistance arteries using transmission electron microscopy. The results suggest that AIL cells are immature or phenotypically modulated vascular SMCs constitutively present in resistance arteries.     

Differences in the mechanism of collagen lattice contraction by myofibroblasts and smooth muscle cells

Both rat derived vascular smooth muscle cells (SMC) and human myofibroblasts contain α smooth muscle actin (SMA), but they utilize different mechanisms to contract populated collagen lattices (PCLs). The difference is in how the cells generate the force that contracts the lattices. Human dermal fibroblasts transform into myofibroblasts, expressing α‐SMA within stress fibers, when cultured in lattices that remain attached to the surface of a tissue culture dish. When attached lattices are populated with rat derived vascular SMC, the cells retain their vascular SMC phenotype. Comparing the contraction of attached PCLs when they are released from the culture dish on day 4 shows that lattices populated with rat vascular SMC contract less than those populated with human myofibroblast. PCL contraction was evaluated in the presence of vanadate and genistein, which modify protein tyrosine phosphorylation, and ML‐7 and Y‐27632, which modify myosin ATPase activity. Genistein and ML‐7 had no affect upon either myofibroblast or vascular SMC‐PCL contraction, demonstrating that neither protein tyrosine kinase nor myosin light chain kinase was involved. Vanadate inhibited myofibroblast‐PCL contraction, consistent with a role for protein tyrosine phosphatase activity with myofibroblast‐generated forces. Y‐27632 inhibited both SMC and myofibroblast PCL contraction, consistent with a central role of myosin light chain phosphatase. J. Cell. Biochem. 111: 362–369, 2010. © 2010 Wiley‐Liss, Inc.     

Increased gelsolin expression and retarded collagen lattice contraction with smooth muscle cells from Crohn's diseased intestine

Intestinal smooth muscle cells (SMC) produce the fibrotic tissue, strictures, that characterize Crohn's disease. These SMC change their phenotype from a contractile muscle form to an inflammation-responsive form that migrates and synthesizes a collagen matrix. It is postulated that the inflammatory responsive SMC form associates differently with its surrounding collagen matrix compared to the normal SMC form. SMC derived from Crohn's diseased and uninvolved bowel were sustained in cell culture. Cultured SMC incorporated in collagen lattices have the capacity to reduce the size of that lattice, referred to as lattice contraction. At day 2, Crohn's SMC-populated collagen lattices were reduced to 21% of their initial area, while non-Crohn's SMC collagen lattices were reduced to 8%. Crohn's SMC demonstrate retarded lattice contraction compared to non-Crohn's SMC. When grown in monolayer culture, Crohn's-derived SMC cover 30% more area than non-Crohn's SMC. By Western blot analysis Crohn's SMC express more gelsolin, an actin-binding protein found elevated in cells exhibiting increased cell motility. Was the increased expression of gelsolin related to retarded collagen lattice contraction? Intracellular levels of gelsolin were elevated by the electroporation of plasma gelsolin protein into suspended non-Crohn's SMC. When incorporated in collagen lattices, gelsolin loaded cells showed retarded lattice contraction compared to SMC loaded with albumin. Crohn's SMC show increased expression of gelsolin, which may be associated with a diminished capacity to reorganize collagen fiber bundles. It is suggested that increased concentrations of gelsolin in Crohn's SMC is consistent with enhanced cell migration as a consequence of the inflammatory state of Crohn's diseased intestine.     

Interaction between atypical microorganisms and E. coli in catheter-associated urinary tract biofilms

Most biofilms involved in catheter-associated urinary tract infections (CAUTIs) are polymicrobial, with disease causing (eg Escherichia coli) and atypical microorganisms (eg Delftia tsuruhatensis) frequently inhabiting the same catheter. Nevertheless, there is a lack of knowledge about the role of atypical microorganisms. Here, single and dual-species biofilms consisting of E. coli and atypical bacteria (D. tsuruhatensis and Achromobacter xylosoxidans), were evaluated. All species were good biofilm producers (Log 5.84–7.25 CFU cm^?2 at 192?h) in artificial urine. The ability of atypical species to form a biofilm appeared to be hampered by the presence of E. coli. Additionally, when E. coli was added to a pre-formed biofilm of the atypical species, it seemed to take advantage of the first colonizers to accelerate adhesion, even when added at lower concentrations. The results suggest a greater ability of E. coli to form biofilms in conditions mimicking the CAUTIs, whatever the pre-existing microbiota and the inoculum concentration.     

Rat cerebral microvascular smooth muscle cells in culture

This report describes the development and establishment of long-term serial cultures of adult rat vascular smooth muscle cells (SMC) derived from cerebrocortical resistance vessels (small arteries and arterioles). Electron microscopic examination of microvessels isolated off a 150 microns nylon mesh sieve clearly demonstrated the predominance of these vessel types. Initial outgrowth from collagenase-elastase-treated microvessel fragments yielded both endothelium and smooth muscle cells. However, at confluency (2-3 weeks) these cultures consisted of a homogeneous population of broad, polygonal cells that grew in a multilayered "hill and valley" pattern typical of SMC in vitro. For comparative morphological and functional studies, SMC cultures were also initiated from rat thoracic aortas utilizing ring segments as explants. The smooth muscle origin of cultures derived from both resistance vessel (RV) and aorta (RA) was further demonstrated by positive immunofluorescent staining by the specific smooth muscle alpha-actin and myosin antibodies. Ultrastructural examination of these SMC cultures revealed similar morphologic features consisting of typical cytoplasmic myofilament bundles with associated dense bodies and numerous pinocytotic vesicles. Cell growth studies on early (less than P 15)- and late (greater than P 15)-passage RV- and RA-SMC populations revealed markedly different cell growth responses. Representative growth curves of early- and late-passage RA-SMC showed a significantly higher growth rate (two- to fourfold) than RV-SMC cultures. Both cultures, however, exhibited a marked increase in growth potential at higher passage levels. Heparin, at a concentration of 100 micrograms/ml inhibited the growth of RV-SMC during the first 3 days after addition in both exponential and growth-arrested culture states, whereas RA-SMC cultures showed no inhibitory response. These studies indicate that long-term RV-SMC cultures can serve as a useful model system to study functional and metabolic properties of this cell type and provide the means to explore further the heterogeneity of SMC derived from different vasculatures in normal as well as various disease states.     

Nonmuscle Myosin motor of smooth muscle

Nonmuscle myosin can generate force and shortening in smooth muscle, as revealed by studies of the urinary bladder from mice lacking smooth muscle myosin heavy chain (SM-MHC) but expressing the nonmuscle myosin heavy chains A and B (NM-MHC A and B; Morano, I., G.X. Chai, L.G. Baltas, V. Lamounier-Zepter, G. Lutsch, M. Kott, H. Haase, and M. Bader. 2000. Nat. Cell Biol. 2:371-375). Intracellular calcium was measured in urinary bladders from SM-MHC-deficient and SM-MHC-expressing mice in relaxed and contracted states. Similar intracellular [Ca2+] transients were observed in the two types of preparations, although the contraction of SM-MHC-deficient bladders was slow and lacked an initial peak in force. The difference in contraction kinetics thus do not reflect differences in calcium handling. Thick filaments were identified with electron microscopy in smooth muscle cells of SM-MHC-deficient bladders, showing that NM-MHC can form filaments in smooth muscle cells. Maximal shortening velocity of maximally activated, skinned smooth muscle preparations from SM-MHC-deficient mice was significantly lower and more sensitive to increased MgADP compared with velocity of SM-MHC-expressing preparations. Active force was significantly lower and less inhibited by increased inorganic phosphate. In conclusion, large differences in nucleotide and phosphate binding exist between smooth and nonmuscle myosins. High ADP binding and low phosphate dependence of nonmuscle myosin would influence both velocity of actin translocation and force generation to promote slow motility and economical force maintenance of the cell.     

微通道经皮肾镜碎石术治疗感染性结石合并尿液CRPA感染两例

目的:总结一期行微通道经皮肾镜碎石术(microchannel percutaneous nephrolithotripsy,m PCNL)治疗上尿路感染性结石合并尿培养为耐碳青霉烯铜绿假单胞菌(carbapenem resistant pseudomonas aeruginosa,CRPA)的经验。方法:选择我院收治两例左肾结石合并尿培养为CRPA的患者,经积极抗感染治疗后,病例一行左侧经皮肾镜碎石术,病例二先行右肾穿刺造瘘术成功后行左侧经皮肾镜碎石术,观察分析两例患者术后结石清除情况,术中术后出现发热、腰痛、大出血、尿路损伤及肾功能衰竭等并发症情况。结果:两例患者术后复查双J管位置良好,结石基本清除;术中、术后均未出现发热、腰痛、大出血、尿路损伤及肾功能衰竭等并发症。结论:经过合适的围手术期处理,一期微通道经皮肾镜碎石术治疗感染性结石合并尿培养为耐药菌的患者是安全可行的。     

Urothelial Dysfunction and Increased Suburothelial Inflammation of Urinary Bladder Are Involved in Patients with Upper Urinary Tract Urolithiasis – Clinical and Immunohistochemistry Study

Objectives

To investigate the urothelial dysfunction and inflammation of urinary bladder in patients with upper urinary tract (UUT) urolithiasis through the results of cystoscopic hydrodistension and immunohistochemistry study.

Methods

Ninety-one patients with UUT urolithiasis underwent cystoscopic hydrodistension before the stone surgery. Immunofluorescence staining of E-cadherin, zonula occludens-1 (ZO-1), tryptase (mast cell activation), and TUNEL (urothelial apoptosis) were performed in 42 patients with glomerulations after hydrodistension, 10 without glomerulations, and 10 controls.

Results

Of the 91 patients, 62 (68.2%) developed glomerulations after hydrodistension. Lower urinary tract symptoms (LUTS) were present in 53.8% patients, in whom significantly smaller maximal anesthetic bladder capacity (MBC) was noted. Patients with middle or lower 1/3 ureteral stones had a significantly higher glomerulation rate (88.6% vs. 55.4%, p<0.01) and lower MBC (618.4±167.6 vs. 701.2±158.4 ml, p = 0.027) than those with upper 1/3 ureteral or renal stones. Patients with UUT urolithiasis had significantly lower expression of E-cadherin (26.2±14.8 vs. 42.4±16.7) and ZO-1 (5.16±4.02 vs. 11.02±5.66); and higher suburothelial mast cell (13.3±6.8 vs. 1.3±1.2) and apoptotic cell (2.6±2.5 vs. 0.1±0.3) numbers than in controls (all p<0.01).

Conclusions

Urothelial dysfunction and increased suburothelial inflammation and apoptosis are highly prevalent in the bladders of UUT urolithiasis patients, indicating inflammation cross-talk between UUT and urinary bladder. Patients with UUT urolithiaisis concomitant with LUTS had a smaller MBC, which may explain the presence of irritative bladder symptoms.     

Organization and development of pacemaker of gastrointestinal tract

Rhythmical depolarization and automatic contraction of smooth muscle of the gastrointestinal tract are consequences of pacemaker activity of c-Kit-immunoreactive cells of mesenchymal origin--interstitial Cajal cells (CC) that have a peculiar mechanism of intracellular Ca2+ exchange controlled by mitochondria. The intermuscular layer cells (ICC-MY) generate pacemaker potentials. They produce depolarization that is enhanced by unitary potentials evoked by the intermuscular population--ICC-IM. Summation of unitary potentials in time of the pacemaker ones leads to creation of the second potentials of slow waves--plateau-potentials. Due to the presence of synapse-like structures, ICC serve messengers of transmission of signals of the enteral nervous system to muscle. Long processes and tight intercellular contacts similar to the cleft ones provide transduction and coordination of excitation in the intestinal musculature. Electrical rhythmicity appears in the enteric musculature at the prenatal period in parallel with structural and functional ICC maturation, but formation of mature rhythm parameters occurs in postnatal ontogenesis. Features of similarity and differences in organization of control of heart and the gastrointestinal tract musculature by pacemakers are considered.