Epithelial cell dynamics in rabbit cecum and proximal colon P1

The large intestine of mammals has long been viewed as an osmoregulatory organ, and evidence suggests that fluid and solute transport mechanisms within the intestine are heterogenous, varying depending on the particular segment involved. Variations in function are often matched by morphological correlates, but despite the widespread use of rabbit large intestine as an experimental model, there is a lack of knowledge about the cellular makeup and dynamics in the colonic mucosal epithelium. The presence of mitotic figures and immunohistochemical localization of proliferating cell nuclear antigen (PCNA) were used to identify the proliferative zone(s). Cellular migration patterns were determined through the use of the thymidine analog 5‐bromo‐2‐deoxyuridine (BrdU) over a 24‐, 48‐, and 72‐hr period. Apoptotic nuclei were identified utilizing terminal deoxynucleotidyl transferase d‐UTP nick‐end labeling (TUNEL). Both cecum and the initial portion of the proximal colon (P1) exhibited a proliferative zone at or near the crypt base, and migration proceeded upwards toward the surface epithelium lining the intestinal lumen, where apoptosis occurred Turnover time of crypt columnar cells was determined to be about 3 days; that of mucous cells was estimated to be about 5 weeks. Rabbit cecum and proximal colon P1 are similar in their cellular morphology and epithelial cell kinetics. In both, the major proliferative zone is located at or near the crypt base, from which crypt columnar cells migrate toward the lumenal surface epithelium over a period of 3 days. Goblet cell turnover rate is much slower than that of columnar cells. Anat Rec 264:427–437, 2001. © 2001 Wiley‐Liss, Inc.     

Epithelial cell nodules developing in the cecum of irradiated mice. Some comparisons with jejunal nodules

A method for producing macroscopic epithelial nodules was developed in order to investigate the properties of epithelial stem cells in the large intestine of mice. The cecum was exteriorized and exposed to various doses of X-ray radiation. Numbers of nodules subsequently developing were counted and plotted against radiation dose. From the logarithmic regression line, the susceptibility to irradiation of nodule-forming stem cells (NFSC) was determined. The susceptibility of cecal NFSC was comparable to the value reported for jejunal NFSC. Mice of (C57BL/6 x DS) F1-Pgk-1b/Pgk-1a that carried X-chromosome inactivation mosaicism for the phosphoglycerate kinase gene were used for examination of nodule clonality. Most cecal nodules contained only 1 type of phosphoglycerate kinase, suggesting a monoclonal origin of the nodules. Histochemical studies showed the presence of absorptive epithelial, goblet and entero-endocrine cells in 17-day-old nodules, implying multipotentiality of the NFSC. In spite of these similarities between cecal and jejunal NFSC, the macroscopic appearance of cecal nodules was quite different from that of jejunal nodules. Only crypt-like structures were observed in the former, whereas both crypt-like and villus-like structures were present in the latter. A comparison between cecal and jejunal nodules may be useful for understanding the morphogenesis of the intestinal mucosa.     

Ion transport and electrophysiology of the early proximal colon of rabbit

The ion transport properties of the mammalian descending colon have been the subject of numerous investigations during the last decade. In contrast, relatively few studies have investigated proximal segments of this organ. In the present study, we assessed transepithelial transport of Na⁺, K⁺ and Cl^– in the isolated initial segment (P1) of rabbit colon in vitro using radioisotopic tracer fluxes and electrophysiological techniques. Like the rabbit descending colon, the proximal colon actively absorbs sodium and chloride, howeveer, its transport systems are markedly different. In vivo, this segment absorbs potassium, however in vitro active potassium secretion was observed. Unlike the descending colon, Na⁺ absorption is relatively insensitive to amiloride and only a slight inhibition was obtained even at 1 mM concentrations of this drug. Na⁺ and Cl^– absorption appeared to be coupled (directly or indicrectly) since the absorption of each ion was inhibited by the removal of the other. Serosal ouabain also inhibited Na⁺ and Cl^– absorption and net K⁺ secretion. Unlike the descending colon, the proximal P1 segment did not have a net absorptive K⁺ transport system that was detectable in the presence of ouabain. Electrically, the early proximal colon has a low transepithelial resistance compared to descending colon (R _T=133±7 cm²) but a larger short-circuit current (l _sc=178±12 A/cm²). The transepithelial potential averaged –21±1 mV, in excellent agreement with values measured in vivo. The apical and basolateral membrane potentials averaged –21±1 mV and –42±1 mV and intracellular potassium activity was 70±2 mM. The findings indicate active K⁺ uptake across the basolateral membrane and passive exit across the apical membrane. The basolateral membrane conductance may be a potassium conductance that is blockable by barium. It is likely that K⁺ transport normally occurs by both cellular and paracellular routes in this epithelium. Because of the numerous differences between this segment and the descending colon, we conclude that the P1 segment of proximal colon has a distinct function in colonic electrolyte transport     

Characteristics of the transport of oxalate and other ions across rabbit proximal colon

In order to characterize oxalate handling by the P2 segment of the rabbit proximal colon, the fluxes of [¹⁴C]oxalate, ²²Na⁺, and ³⁶Cl^– were measured in vitro using conventional short-circuiting techniques. In standard buffer the proximal colon exhibited net secretion of Na⁺ (–2.31±0.64 equiv cm^–2 h^–1), negligible net Cl^– transport, and net secretion of oxalate (–12.7±1.6 pmol cm^–2 h^–1). Replacement of buffer Na⁺ or Cl^– abolished net oxalate secretion, while HCO ₃ ^– -free media revealed a net absorption of oxalate (19.3±4.2 pmol cm^–2 h^–1) and stimulated NaCl absorption. Mucosal amiloride and dimethylamiloride (1 mM) significantly reduced the unidirectional fluxes of oxalate and enhanced sodium secretion by decreasing J _ms ^Na . The anion exchange inhibitor 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS; 0.1 mM, both sides) reduced the unidirectional fluxes of oxalate and chloride. Serosal epinephrine (50 M) stimulated oxalate absorption (21.3±6.3 pmol cm^–2 h^–1) and sodium absorption (5.71±1.20 equiv cm^–2 h^–1), whereas dibutyryl-cAMP enhanced oxalate secretion (–43.4±6.9 pmol cm^–2 h^–1) and stimulated chloride secretion (–7.27 ±0.64 equiv cm^–2 h^–1). These results indicate that the P2 segment of the proximal colon possesses (a) secretory as well as absorptive capacities, (b) oxalate fluxes that are mediated by pathways involving Na⁺, Cl^–, HCO ₃ ^– transport and (c) a net oxalate flux that is sensitive to absorptive and secretory stimuli.     

Segmental differences in electrical properties and Na-transport of rabbit caecum,proximal and distal colon in vitro

Electrical potential difference, short circuit current, tissue conductance, and unidirectional Na-fluxes were measured in four segments of the rabbit large intestine in vitro. Compared to the relatively tight, low conductance distal colon, caecum and proximal parts of the colon are leaky epithelia with high conductances. Net Na-absorption was highest in caecum, and then decreased gradually towards the distal colon, whereas potential and short circuit current where high in the caecum and proximal colon, low in the middle part of the colon, and high again in the distal colon. Unidirectional Na-fluxes of all four segments were different.The discrepancy between the short circuit current and net Na-absorption in the two segments of the proximal colon indicates electrogenic transport of other ions. 0.1 mM ouabain virtually abolished short circuit current and Na-absorption in all segments, whereas 0.1 mM amiloride was not effective in the caecum and the proximal colonic parts. The present study focuses on the comparative aspects of Na-transport. It demonstrates the marked segmental heterogeneity of the basic electrical properties and suggests four different segmental organizations of large intestinal electrolyte transport.     

Mutations induced by 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP) in cecum and proximal and distal colon of lacI transgenic rats

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a food-borne mutagen and carcinogen that induces tumors of the colon and the prostate gland in male rats and of the mammary gland in female rats. In this study we describe the frequency and specificity of PhIP-induced mutations in the cecum, proximal colon and distal colon of male and female lacI transgenic rats. This is the first report of mutational data from discrete regions of the colon. After 61 days of treatment with 200 p.p.m. PhIP mixed into the diet, PhIP-induced mutant frequencies were elevated 7-fold in the cecum and 14- to 21-fold in the colon of male and female rats compared with untreated controls. PhIP-induced mutant frequencies increased significantly (overall trend, P < 10(-4)) along the length of the colon of both males and females, with cecum < proximal colon < distal colon. A total of 754 PhIP mutants (363 male, 391 female) were sequenced to provide the mutational spectra for each of the three tissue sections from males and females. These mutational spectra consisted predominantly of G:C-->T:A and G:C-->C:G transversions and deletions of G:C base pairs. There were no significant differences between the mutational spectra with respect to sex or position in the colon. Therefore, we surmise that following induction of mutations by PhIP in male and female colons, non-mutagenic factors, possibly hormonal, preferentially influence the formation of tumors in the colon of male rats.     

Endothelin-1 stimulates chloride and potassium secretion in rabbit descending colon

The vasoactive peptide endothelin-1 (ET-1) which is present in high concentrations in the colon, causes concentration-dependent electrogenic Cl^– secretion in rabbit descending colon. This effect is half-maximal at 0.11 mol/l. Like other secretagogues, ET-1 also stimulates K⁺ secretion. The secretory effect of ET-1 is associated with increased release of prostaglandin E₂ from the serosal surface of the mucosa. ET-1-induced Cl^– secretion is completely inhibited by the loop diuretic bumetanide and by indomethacin and quinacrine, inhibitors of prostaglandin synthesis. Neuronal mechanisms do not seem to be involved, as tetrodotoxin did not affect the secretory response to ET-1 significantly. On the other hand, neither the catalytic activity nor the transport function of the Na⁺/K⁺-ATPase of rabbit colon epithelium is affected by endothelin-1 (ET-1) in concentrations up to 10 mol/l. It is concluded that ET-1 causes Cl^– and K⁺ secretion by stimulating phospholipase A₂ and release of prostaglandins, whereas Na⁺ transport is not altered.Parts of this study were presented at the 10th meeting of the European Intestinal Transport Group [26]     

Neuropeptide distributions in the colon,cecum, and jejunum of the horse

The pelvic flexure portion of the equine large colon is the proposed location of a pacemaker mechanism. This study was conducted to ascertain whether the distribution of certain putative neurotransmitters differs at the pelvic flexure compared to other sampling sites. Tissue samples were collected from the intestinal tracts of six horses. Serial sections from these samples were reacted with primary antisera specific for substance P, vasoactive intestinal polypeptide (VIP), methionine-Enkephalin, and calcitonin gene-related peptide (CGRP). The regional distribution of immunoreactive neuronal elements was uniform for each of the neuropeptides except VIP. Although neurons exhibiting VIP-like immunoreactivity were abundant throughout the colon, they were somewhat more plentiful near the apex of the pelvic flexure and the left dorsal colon. These neurons may participate in the initiation and propagation of the propulsive/retropulsive contraction waves, which emanate from this location and are believed to lend a sphincter-like capacity to the pelvic flexure. The submucosal plexus was replete with neurons with intense substance P and VIP-like reactivity. Reactive fibers left submucosal ganglia to project to the intestinal mucosa, reflecting a possible secretogogic role for these neurons. This role may be especially important for the horse as a hindgut fermenter. There were abundant methionine-Enkephalin and substance P-like reactive varicosities throughout the myenteric plexus, many of which established a pericellular plexus of varicose fibers. The abundance of these varicosities, which may correlate with a high degree of neuronal integration, did not vary regionally. These data may enhance our understanding of both normal colonic peristalsis and motility disorders caused by a depletion of these neuropeptides. © 1993 Wiley-Liss, Inc.     

Mucin isolated from rabbit colon inhibits in vitro binding of Escherichia coli RDEC-1.

The rabbit enteric pathogen Escherichia coli RDEC-1 (serotype O15:H-) mediates attaching and effacing binding to colonic epithelium in a manner morphologically identical to that observed in both human enteropathogenic E. coli and enterohemorrhagic E. coli infections. The aim of this study was to determine if colonic mucus and its constituents, including mucin derived from goblet cells, inhibited RDEC-1 adherence in vitro. Crude mucus was prepared from mucosal scrapings of rabbit colon and separated by buoyant density into eight fractions. Purified mucin was characterized by gel electrophoresis, dot immunoblotting, indirect immunofluorescence, and amino acid composition. RDEC-1 bacteria were grown to promote and suppress the expression of mannose-resistant, hydrophobic pili. A nonpiliated mutant, strain M34, was also used as a negative control. Binding of radiolabeled RDEC-1 expressing pili was quantitated in the presence of crude mucus, purified mucin, and nonmucin fractions. Binding of piliated RDEC-1 to hydrophobic polystyrene wells was greater than for both nonpiliated RDEC-1 and strain M34 (P less than 0.05). Both crude mucus and purified mucin mediated a concentration-dependent inhibition of piliated-RDEC-1 binding. Fractions of mucus without immunoreactive mucin did not inhibit the binding of RDEC-1 expressing hydrophobic pili. We conclude that colonic goblet cell-derived mucin mediates inhibition of piliated RDEC-1 attachment in vitro. Inhibition of bacterial adherence could prevent access of attaching and effacing E. coli enteric pathogens to the colonic mucosa in vivo.     

Comparison of proximal and distal colonic muscle of the rabbit.

Mechanical length-tension properties and response to neurohumoral agents were compared for proximal and distal colonic muscle. Resting tension during stretch, acetylcholine-stimulated tension, and the total tension were determined. Proximal circular muscle developed a maximum total tension of 0.96 +/- 0.18 kg/cm2 (mean +/- SE) compared to 0.86 +/- 0.06 kg/cm2 for the distal colon (P greater than 0.05). Resting tension was 0.33 +/- 0.03 kg/cm2 for the proximal colon and 0.05 +/- 0.01 kg/cm2 for the distal colon at the length of optimal acetylcholine-stimulated tension (Lo) (P less than 0.01). Longitudinal muscle showed a similar difference for the proximal and distal colon. The high resting tension in the proximal colonic muscle was reduced by nitroprusside or calcium-free Krebs with EGTA. Dose-response curves to acetylcholine, histamine, phenylephrine, and isoproterenol were similar for the muscle of either part of the colon. Gastrin or cholecystokinin had no effect on the muscle. In summary, the circular or longitudinal muscles of the proximal and distal colon have different length-tension properties but only minimal differences in response to neurohumoral agents.     

Peristalsis in the rabbit distal colon

1. The motility of the distal colon of the rabbit has been examined by the conventional Trendelenburg method and by an isometric, isovolumic modification of this method.2. The colon shows a range of movements, and tetrodotoxin and cold-storage have been used in an attempt to differentiate between myogenic and neurally integrated activities.3. The observable myogenic movements are pendulum movements, ;tone rings' and ;tone waves'; the last of these can be weakly propulsive. The rabbit colon also shows a neurally organized and powerfully propulsive movement which corresponds to the peristaltic wave.4. The implication of a role for nervous structures in the propulsive activity of the rabbit distal colon is at variance with the view of Lee (1960).5. The myenteric reflex arc in the colon has been examined pharmacologically. Cholinergic neural transmission has been implicated at the ganglionic-synaptic and neuroeffector junctions, but additional noncholinergic mechanisms of chemical transmission have not been excluded.     

Tension and cyclic GMP changes in potassium depolarized rabbit colon muscle.

Potassium depolarization of rabbit colon muscle elicited a contraction consisting of 2 distinct phases, an initial rapid phasic contraction and a tonic contracture. The tonic contraction was, in contrast to the phasic contraction, dependent on the extracellular calcium for its development. There was a correlation between the tension development and the increase of the cyclic GMP level in the K+-depolarized muscle. Experimental conditions which abolished the tonic contracture, viz glucose omission and treatment with Ca2+-antagonists (verapamil, SKF 525A) also inhibited the cyclic GMP response. The changes of the cyclic GMP levels were Ca2+-dependent. K+-ions also changes the cyclic AMP content an effect which was atropine sensitive. From the experimental data obtained in this investigation we suggest that the co-variation of the tension and the cyclic GMP level in the depolarized colon muscle might depend on oscillations in a common intracellular factor, probably Ca2+.     

Time-dependent effects of aldosterone on sodium transport and cell membrane resistances in rabbit distal colon

Aldosterone stimulates Na⁺-absorption in rabbit distal colon. Due to circadian variations in plasma aldosterone level, Na⁺-transport varies in this epithelium. In vitro measurements (Ussing-chambers) yielded a transepithelial voltage (V _t ) of 13±1.6 mV for low-transporting epithelia (LT) and 25.7±2 mV for high-transporting epithelia (HT). However, the comparison of transepithelial conductance (G _t ) in LT epithelia (2.73±0.21 mS/cm²) and HT epithelia (2.96±0.41 mS/cm²) revealed no difference. Colons from both groups were stimulated by exogenous aldosterone (4 h prior to experiment). The transepithelial values changed as follows: LT epithelia showed a significantly increased V _t (26.1±4 mV) and G _t (3.74±0.23 mS/cm²), whereas in HT epithelia both parameters remained unchanged. Transepithelial amiloride-sensitive conductance was higher in HT than in LT. However, only in LT epithelia aldosterone increased this conductance. To get a more detailed view of the action of aldosterone, we used intracellular microelectrodes to calculate the resistances of apical (R _a ), basolateral (R _bl ) and paracellular (R _p ) pathway. The calculation of the resistances was based on a lumped equivalent circuit model and changes in R _a were induced by 50 M/l mucosal amiloride. Comparison of the control tissues revealed a lower R _bl only in HT tissues. In both groups stimulation by exogenous aldosterone led to a marked decrease of R _a . Furthermore R _bl was reduced to the same value as in HT control tissues. A leak resistance (R _l) was found, which was modulated by aldosterone in LT- and in HT epithelia. Differences in amiloride-sensitive transepithelial conductance between both epithelia groups could be explained by a regulation of r _l . Along with the regulation of the R _p the results indicate that the effects of exogenous aldosterone depended on the transport state of the rabbit distal colon. In LT epithelia aldosterone only influenced the resistances of the transcellular pathway. In HT epithelia aldosterone decreased cellular resistances and increased the paracellular resistance. Possible reasons of the augmented R _p are discussed.Abbreviations V _t transepithelial potential difference (mV) - R _r transepithelial resistance ( · cm²) - G _t transepithelial conductance (mS/cm²) - I _sc calculated short circuit current (A/cm²) - V _a apical membrane potential difference (mV) - V _bl basolateral membrane potential difference (mV) - voltage divider ratio ( = R _a /R _bl ) - voltage divider ratio ( = R _a /R _bl ) after amiloride - R _a apical membrane resistance ( · cm²) R _a = R _l · R _a ^/Na /(R _l + R _a ^/Na ) - R _a ^/Na apical membrane resistance to Na, ( · cm²) - R _bl basolateral membrane resistance ( · cm²) - R _c cellular resistance ( of apical and basolateral resistance) ( · cm²) - R _l leak resistance located in the apical membrane ( · cm²) - R _p resistance of the paracellular pathway ( · cm²) - G _a apical membrane conductance (mS/cm²) - G _bl basolateral membrane conductance (mS/cm²) - G _l apical leak conductance (compare R _l ) (mS/cm²) - G _p paracellular conductance (mS/cm²) - G _t transepithelial conductance (mS/cm²) - HF hard feces period - SF soft feces period - HT_contr high transporting control epithelia - LT_contr low transporting control epithelia - HT_aldo aldosterone pretreated high transporting epithelia - LT_aldo aldosterone pretreated low transporting epithelia; the prime designates data obtained after addition of amiloride     

Distribution of ‘dome’ type lymphoid follicles and morphology of microfold cells (M cells) in the human Bauhin's valve,cecum and proximal ascending colon

The distribution of ‘dome’ type lymphoid follicles and the morphology of their M cells were studied in human Bauhin's valves, cecums and proximal ascending colons obtained during surgery performed on 11 patients. The lymphoid follicles existed as solitary follicles, and their number was remarkably large in the Bauhin's valve than in the cecum and proximal ascending colon. Scanning electron microscopy (SEM) disclosed lymphoid follicles forming domes that were surrounded by villi or crypts and M cells with microfolds in the dome epithelium. Transmission electron microscopy (TEM) disclosed microfolds, enfolded lymphocytes and the other typical characteristics of M cells in the Bauhin's valve, cecum and proximal ascending colon. These morphological findings were compatible with previously reported findings regarding the M cells in human Peyer's patches.     

Activated BRAF targets proximal colon tumors with mismatch repair deficiency and MLH1 inactivation

BRAF, a serine/threonine kinase of the RAF family, is a downstream transducer of the RAS-regulated MAPK pathway and signals upstream of MEK1/2 kinases. Recently, activating mutations within BRAF have been reported in a high percentage of melanomas and colorectal carcinomas and shown to have oncogenic capabilities. Further, their association to mismatch-repair-deficient tumors has suggested the involvement of the RAS/RAF pathway in the tumorigenesis of microsatellite-unstable colon cancers, and that RAS and RAF mutations are alternative genetic events. We determined whether colorectal mismatch-repair-deficient tumors with BRAF mutations show a specific genotype when compared with tumors with wild-type BRAF, and whether they can be associated with a particular clinicopathological feature. Here, we report a striking association of BRAF, but not of APC, KRAS2, AXIN2, and TP53 mutations, with proximal mismatch-repair-deficient colon tumors and MLH1 hypermethylation. Our results support the hypothesis that proximal and distal colorectal tumors with mismatch repair deficiency harbor different genetic alterations, and we suggest that the involvement of the RAS/RAF pathway in colorectal tumorigenesis is differentially modulated according to tumor location and MLH1 inactivation.     

Epithelial membrane antigen in normal and proteinuric glomeruli and in damaged proximal tubules

Epithelial membrane antigen was detected in normal glomeruli by a polyclonal antiserum to the antigen and by the monoclonal antibodies Ca 1, DAKO-EMA and HMFG 2, but not HMFG 1, using an indirect immunoperoxidase method. The antigen was in the form of a thin ring or collar at the junction of glomerulus and tubule. In a series of 47 renal biopsies from patients with proteinuria, the antigen could still be seen in glomeruli, provided that there were adequate numbers of glomeruli in the sections. The main object of study was the glomerular tip lesion, in which tip adhesions were seen to be just adjacent to the patch of epithelial membrane antigen. This suggested that the antigen may be important in pathogenesis of the lesion. Normal proximal tubules did not express epithelial membrane antigen but it was detected on the luminal border of acutely damaged proximal tubules. Thus the distribution of epithelial membrane antigen in the kidney is more complex than was previously thought.     

Epithelial cell invasion and survival of Bordetella bronchiseptica.

Wild-type Bordetella bronchiseptica and a bvg mutant strain were used for invasion and survival experiments in human Caco-2 and A549 epithelial cells. Both bacterial strains were able to enter and persist within the host cells for at least a week. A significant proportion of the bacteria from both B. bronchiseptica strains but not from Bordetella pertussis were found free in the cytoplasm, suggesting different invasion and survival strategies of the two species in epithelial cells.     

Rhythmic spontaneous contractions in the rat proximal colon

C-kit immunoreactive cells are known to be interstitial cells of Cajal (ICCs), and they generate pacemaker activity of the gastrointestinal tract. Recently a large number of special smooth muscle cells corresponding to c-kit immunoreactive cells were found in the proximal colon of the guinea pig. We learned that the rat proximal colon showed tetrodotoxin-insensitive regular rhythmic spontaneous contractions (RSCs) and hypothesized that RSCs are generated and/or regulated by ICCs. To prove our hypothesis, we investigated whether RSCs are absent in homozygous Ws/Ws mutant rats, since c-kit positive ICCs along the submucosal surface of the circular muscle (ICC(SM)) and myenteric plexus (ICC(MY)) are lacking. In contrast to our hypothesis, we found that RSCs were still present in the proximal colon of the Ws/Ws mutant rats. A recent study has reported that c-kit negative ICC(SM) remains in Ws/Ws mutant rats. Taken together, RSCs may be generated by c-kit negative ICC(SM) in the rat proximal colon. The blockade of sarcoplasmic reticulum Ca(2+)-ATPase by cyclopiazonic acid (CPA) (10(-6)M) or by thapsigargin (10(-6)M) increased the frequency of RSCs. The increasing effects of CPA on the frequency of RSCs were more prominent in Ws/Ws mutant rats than in +/+ rats. We concluded that the functional coordination between c-kit negative ICC(SM) and other mutationally impaired c-kit positive ICC(MY) and ICC(SM) may be required for moderate regulation in the frequency of spontaneous activity.