Cytodifferentiation of the interstitial cells of Cajal of mouse colonic circular muscle layer. An EM study from fetal to adult life

Cytodifferentiation of the interstitial cells of Cajal related to the plexus entericus extremus and located on the inner face of the circular muscle layer of the mouse colon was studied in fetuses at term, unfed neonates, suckling and weaning young animals. In fetuses at term, the interstitial cells of Cajal are not found and their precursor cells are not identifiable among the cells present in the submucosal area facing the circular muscle layer, i.e., undifferentiated cells (mainly close to nerve fibers) and fibroblast-like cells (contacting each other and the developing smooth-muscle cells). Cells putatively considered as precursors of interstitial cells of Cajal, fibroblast-like cells rich in mitochondria, are present in unfed neonates (in small numbers) and in suckling animals (in large numbers). Differentiating interstitial cells of Cajal are undoubtedly recognizable during the second week of postnatal life (suckling period). Some of them have mixed fibroblastic- and interstitial-cell features and some have many interstitial-cell features. Both these cell types are already in contact with each other and differentiating and differentiated smooth-muscle cells. The undifferentiated cells, still present in unfed neonates, envelop the nerve fibers and differentiate as Schwann cells during the suckling period. During this period, as the nerve endings of the plexus entericus extremus develop, they immediately contact the differentiating interstitial cells of Cajal. During the weaning period, these cells are quite well differentiated although they do not have the same morphology as in adult animals before 30 days of age. Concomitantly, the nerve endings of the plexus entericus extremus contain an increasing number of synaptic vesicles.     

Characterization of inhibitory innervation in porcine colonic circular muscle

Effects of stimulation of intramural nerves in the circular smooth muscle layer of the porcine colon (Sus scrofa domestica) were studied using the sucrose-gap technique. Electrical field stimulation of the preparation, superfused with Krebs solution at 21 degrees C, induced a transient hyperpolarization of the smooth muscle cell membrane. This hyperpolarization was an inhibitory junction potential (IJP). The responses obtained from circular muscle originating from either the centripetal or centrifugal gyri of the ascending colon did not differ significantly. The IJP was characterized as being mediated by intramural, nonadrenergic, noncholinergic (NANC) nerves. The amplitude and latency of the IJP changed linearly with temperature (15-25 degrees C: +1 mV and -0.1 s per degree Celsius, respectively) reflecting a temperature-dependent synchronization of transmitter release. The membrane resistance decreased during the IJP. The IJP amplitude decreased or increased during conditioning hyperpolarizations or depolarizations, respectively, and reversed at membrane potentials about 30 mV more negative than the resting membrane potential. Potassium conductance blocking agents, barium (1 mM), tetraethylammonium chloride (TEA, 20 mM), 4-aminopyridine (4-AP, 5 mM), apamin (1 microM), and aminacrine (10(-4) M) added to the superfusion medium increased the membrane resistance. Only barium, TEA, and apamin depolarized the smooth muscle cell membrane. The IJP amplitude decreased in the presence of aminacrine and apamin to 75 and 35%, respectively, suggesting that apamin-sensitive Ca2+-activated K+ channels are involved in this response. ATP, adenosine, and related adenine nucleotides in concentrations up to 10(-3) M did not mimic the IJP. Superfusion with ATP for 15 min revealed a gradually increasing attenuation by up to 20% of the IJP. This might suggest that the release of neurotransmitter from intramural NANC nerves is modulated presynaptically via purinoceptors. Exogenously applied vasoactive intestinal polypeptide (VIP) in concentrations of 10(-9) to 10(-4) M did not affect the preparation. Also at elevated temperatures (up to 35 degrees C), VIP (10(-7) to 10(-4) M) did not cause measurable effects. It is concluded that the inhibitory mediator of the intramural NANC nerves present in the circular muscle layers of the porcine colon is neither a purine nor VIP.     

Oligosaccharide structures of human colonic mucin

Purified human colonic mucin was separated into six distinct components by DEAE-cellulose chromatography, and the structures of oligosaccharide side chains from the three most abundant species were determined. Oligosaccharide side chains were isolated from colonic mucin species III, IV, and V after alkaline borohydride reductive cleavage in the presence of sodium borotritide. After initial separation of acidic and neutral oligosaccharides by ion exchange chromatography, individual oligosaccharides were isolated by sequential chromatography on Bio-Gel P-4 and Bio-Gel P-2 resins followed by preparative normal phase high performance liquid chromatography. Composition and structure of individual oligosaccharides were determined by combination of gas chromatography, methylation analysis, and sequential glycosidase digestion. Collectively, 21 discrete oligosaccharide structures were identified in the major human colonic mucin species including 10 acidic oligosaccharides and 11 neutral structures which ranged in size from 2 to 12 sugar residues. Although detailed structures were defined for each oligosaccharide, the majority of the structures identified were variations of a relatively small number of "basic" structures, and several generalizations pertained. First, many oligosaccharides represented variations of a biantennary structure in which branch chains arise in N-acetylglucosaminyl residues linked to C3 and C6 of a galactosyl residue linked in turn to a GlcNAc beta (1-3)GalNAc core; second, non-branched oligosaccharides appeared to be linear chain derivatives of the same core structure; third, all acidic oligosaccharides could be derived from neutral structures present in the mucin species; fourth, sialic acid substitution was limited to few sites and always included substitution in alpha 2-6 linkage to the reducing terminal N-acetylgalactosamine, and finally several structures contained both sialic acid and fucose residues. Individually, mucin species III, IV, and V were found to contain unique mixtures of 13, 14, and 10 oligosaccharide structures, respectively. These data demonstrate that human colonic mucin contain a wide range of oligosaccharides reflecting variations of common core oligosaccharide structures. The major chromatographically defined constituents of normal colonic mucin appear to possess characteristic and distinguishable combinations of oligosaccharide structures. These findings support the concept that colonic mucin contains structurally and functionally distinct subpopulations.     

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.     

Oligosaccharide structures of isolated human colonic mucin species

Purified human colonic mucin contains six distinct components which may be separated by DEAE-cellulose chromatography. Past studies defined the structure of oligosaccharide side chains from the most abundant species III, IV, and V which elute at intermediate salt concentrations. In these studies the structures of oligosaccharide side chains liberated from the remaining early and late eluting species I, II, and VI were determined after isolation by sequential conventional and high performance liquid chromatography through combination of gas chromatography, methylation analysis, and sequential glycosidase digestion. Mucin species I, II, and VI contained a less varied array of discrete oligosaccharide structures than that observed in the major mucin components. Mucin species I and II contained five and 10 structures, respectively, which account for 68 and 71% of total oligosaccharide content in these fractions. The predominant oligosaccharides of mucin species I included three neutral structures: a disaccharide GlcNAc beta (1-3)GalNAc-ol, a trisaccharide Gal beta (1-4)GlcNAc beta (1-3)GalNAc-ol, and a tetrasaccharide GlcNAc beta (1-4)Gal beta (1-4)GlcNAc beta (1-3)GalNAc-ol as well as two acidic components representing the sialylated forms of two of these oligosaccharides. Mucin species II contained these same oligosaccharides as well as four additional acidic structures, notably a disaccharide Neu alpha (2-6)GalNAc-ol and a hexasaccharide Gal beta (1-4)GlcNAc beta (1-3)Gal beta (1-4)GlcNAc beta (1-3) (NeuAc alpha (2-6))-GalNAc-ol, not identified in any other mucin species. The late eluting mucin species VI contained at least five discrete neutral oligosaccharides and six major acidic structures. While the majority of these structures had been previously isolated from the earlier eluting mucin species IV and V, species VI also contained di- and trisialylated oligosaccharides not identified in other mucin species. In conjunction with earlier studies of the major mucin species III, IV, and V, these data define the range of oligosaccharide structures present in human colonic mucin. These studies demonstrate that human colonic mucin possesses species with characteristic and distinguishable combinations of oligosaccharides which reflect variations of common core structures.     

Functional characterisation of tachykinin receptors in the circular muscle layer of the mouse ileum

OBJECTIVES: Tachykinins are important mediators in neuromuscular signalling but have not been thoroughly characterised in the mouse gut. We investigated the participation of tachykinin receptors in contractility of circular muscle strips of the mouse ileum. RESULTS: Electrical field stimulation (EFS) of excitatory nonadrenergic noncholinergic (NANC) nerves induced frequency-dependent contractions which were mimicked by substance P (SP). Desensitisation of SP and NK(1), NK(2) or NK(3) receptors significantly reduced contractions to EFS. The NK(1) receptor blocker RP67580 significantly inhibited NANC contractions to EFS. The NK(2) and NK(3) receptor blockers nepadutant and SR142801 did not affect NANC contractions per se but increased the RP67580-induced inhibition of NANC contractions to EFS. Contractions to SP were significantly reduced by RP67580 but not affected by nepadutant or SR142801. The NK(1) and NK(2) receptor agonists, septide and [beta-ala(8)]-NKA 4-10 (beta-A-NKA), respectively, but not the NK(3) receptor agonist senktide-induced dose-dependent contractions. Atropine inhibited and l-NNA augmented contractions to septide. Contractions to beta-A-NKA were insensitive to atropine but augmented by l-NNA. CONCLUSIONS: Tachykinins mediate NANC contractions to EFS in the mouse small intestine. Endogenously released tachykinins activate mainly NK(1) receptors, located on cholinergic nerves and smooth muscle cells and, to a lesser degree, NK(2) and NK(3) receptors, most likely located presynaptically.     

Inflammatory gene expression by human colonic smooth muscle cells

Intestinal mucosal cells and invading leukocytes produce inappropriate levels of cytokines and chemokines in human colitis. However, smooth muscle cells of the airway and vasculature also synthesize cytokines and chemokines. To determine whether human colonic myocytes can synthesize proinflammatory mediators, strips of circular smooth muscle and smooth muscle cells were isolated from human colon. Myocytes and muscle strips were stimulated with 10 ng/ml of IL-1beta, TNF-alpha, and IFN-gamma, respectively. Expression of mRNA for IL-1beta, IL-6, IL-8, and cyclooxygenase-2 (COX-2) was induced within 2 h and continued to increase for 8-12 h. Regulated on activation, normal T cell-expressed and -secreted (RANTES) mRNA expression was slower, appearing at 8 h and increasing linearly through 20 h. Expression of all five mRNAs was inhibited by 0.1 microM MG-132, a proteosome inhibitor that blocks NF-kappaB activation. Expression of IL-1beta, IL-6, IL-8, and COX-2 mRNA was reduced by 30 microM PP1, an Src family tyrosine kinase inhibitor, and by 25 microM SB-203580, a p38 MAPK inhibitor. MAPK/extracellular regulated kinase-1 inhibitor PD-98059 (25 microM) was much less effective. In conclusion, human colonic smooth muscle cells can synthesize and secrete interleukins (IL-1beta and IL-6) and chemokines (IL-8 and RANTES) and upregulate expression of COX-2. Regulation of cytokine, chemokine, and COX-2 mRNA depends on multiple signaling pathways, including Src-family kinases, extracellular regulated kinase, p38 MAPKs, and NF-kappaB. SB-203580 was a consistent, efficacious inhibitor of inflammatory gene expression, suggesting an important role of p38 MAPK in synthetic functions of human colonic smooth muscle.     

The effects of cholecystokinin-octapeptide and pentagastrin on electrical and motor activities of canine colonic circular muscle

The effects of cholecystokinin-octapeptide (CCK-OP) and pentagastrin on electrical and motor activities of circular muscle of the canine colon were studied with the sucrose gap technique. Additional organ bath experiments were performed to further characterize the motor response to the peptides and to elucidate their site of action. The electrical activity consisted of slow waves having an initial potential followed by a plateau potential, at a regular frequency of 4.5 cycles/min. Both peptides prolonged the duration and increased the amplitude of the plateau phase of the slow waves. Concomitantly, the slow wave frequency was reduced. In addition, CCK-OP increased spiking activity. Both spiking activity and the prolonged plateau potential generated contractile activity, prolonged phasic contraction occurring with slow waves with a prolonged plateau. In organ bath experiments, both CCK-OP and pentagastrin increased the basal tone of the muscle strips and prolonged the duration of the phasic contractions. The prolongation of the duration of the contractions was not antagonized by tetrodotoxin (TTX) and atropine. CCK-OP but not pentagastrin increased the force of contractions, this action was not affected by atropine but was reduced in the presence of TTX, suggesting that the increase in force may be partially mediated by noncholinergic excitatory nerves. The increase in basal tension by the peptides was enhanced in the presence of TTX indicating that myenteric inhibitory neurones were tonically active under our experimental conditions. The results provide the electrophysiological basis for CCK-OP and pentagastrin induced changes in colonic motility.     

Galanin induces contraction of isolated cells from circular muscle layer of pig ileum.

The effects of galanin and its interaction with cholecystokinin and acetylcholine on smooth muscle cells were studied in vitro on isolated cells obtained from pig ileum circular muscle layer. Galanin induced a concentration-dependent cell contraction with a maximal contraction (24.5% decrease in cell length from control) obtained at 1 nM. The concentration of galanin inducing a half-maximal contraction was 3 pM. Tetrodotoxin (10 microM) failed to inhibit cell contraction induced by galanin (1 nM), pentagastrin (10 nM) and acetylcholine (1 microM). Atropine abolished the contraction induced by acetylcholine (1 microM), but had no effect on galanin- and pentagastrin-induced contraction. L 364,718 inhibited the contraction induced by CCK8 but not the galanin-induced contraction. At the uneffective concentration of 10 fM, galanin had a synergistic effect with an uneffective concentration of CCK8 (1 pM). These results suggest that (i) galanin contracts smooth muscle cells from pig ileum by acting on a specific receptor; (ii) galanin and either CCK or acetylcholine may act in a synergistic way to induce cell contraction.     

Colon cancer specific nuclear matrix protein alterations in human colonic adenomatous polyps

Most colon cancers arise within preexisting adenomatous polyps or adenomas. The slow evolution from the non-invasive premalignant lesion, the adenomatous polyp, to invasive cancer supports a strategy of early detection. Recently, we identified unique nuclear matrix proteins (NMPs) specific for colon cancer (CC2, CC3, CC4, CC5). Most of the NMPs identified are common to all cell types, but several identified NMPs are tissue and cell line specific. The objective of this study is to describe and characterize the NMP profile of premalignant adenomatous colon polyps. Specifically when in the adenoma-carcinoma sequence four specific colon cancer NMPs, previously described, appear. Using two-dimensional (2-D) gel analysis 20 colon polyps (one juvenile polyp, six tubular adenoma (TA), seven tubulovillous adenoma (TVA), six TVA with focal high-grade dysplasia (HGD), were analyzed for the presence of four (CC2, CC3, CC4, CC5) specific NMPs. CC2 was not seen in any of the premalignant polyps. CC5 was present in only two premalignant TVA with HGD and in one TA. CC3 and CC4 were present in most adenomas. None of the NMPs were seen in the juvenile polyp, which is not considered to be a precursor of colon cancer. CC2 and CC5 are NMPs expressed at the junction of an advanced adenoma and invasive colorectal cancer. CC3 and CC4 are expressed earlier in the evolution of adenomatous polyps. Development of an assay to these proteins may serve as a new method for early detection of colorectal cancer.     

In vivo specific tension of human skeletal muscle.

In this study, we estimated the specific tensions of soleus (Sol) and tibialis anterior (TA) muscles in six men. Joint moments were measured during maximum voluntary contraction (MVC) and during electrical stimulation. Moment arm lengths and muscle volumes were measured using magnetic resonance imaging, and pennation angles and fascicular lengths were measured using ultrasonography. Tendon and muscle forces were modeled. Two approaches were followed to estimate specific tension. First, muscle moments during electrical stimulation and moment arm lengths, fascicular lengths, and pennation angles during MVC were used (data set A). Then, MVC moments, moment arm lengths at rest, and cadaveric fascicular lengths and pennation angles were used (data set B). The use of data set B yielded the unrealistic specific tension estimates of 104 kN/m(2) in Sol and 658 kN/m(2) in TA. The use of data set A, however, yielded values of 150 and 155 kN/m(2) in Sol and TA, respectively, which agree with in vitro results from fiber type I-predominant muscles. In fact, both Sol and TA are such muscles. Our study demonstrates the feasibility of accurate in vivo estimates of human muscle intrinsic strength.