Ca2+ activation and pH dependence of a maxi K+ channel from rabbit distal colon epithelium

To determine if their properties are consistent with a role in regulation of transepithelial transport, Ca²⁺-activated K⁺ channels from the basolateral plasma membrane of the surface cells in the distal colon have been characterized by single channel analysis after fusion of vesicles with planar lipid bilayers. A Ca²⁺-activated K⁺ channel with a single channel conductance of 275 pS was predominant. The sensitivity to Ca²⁺ was strongly dependent on the membrane potential and on the pH. At a neutral pH, the K _0.5 for Ca²⁺ was raised from 20nm at a potential of 0 mV to 300nm at –40 mV. A decrease in pH at the cytoplasmic face of the K⁺ channel reduced the Ca²⁺ sensitivity dramatically. A loss of the high sensitivity to Ca²⁺ was also observed after incubation with MgCl₂, possibly a result of dephosphorylation of the channels by endogenous phosphatases. Modification of the channel protein may thus explain the variation in Ca²⁺ sensitivity between studies on K⁺ channels from the same tissue. High affinity inhibition (K _0.5=10nm) by charybdotoxin of the Ca²⁺-activated K⁺ channel from the extracellular face could be lifted by an outward flux of K⁺ through the channel. However, at the ion gradients and potentials found in the intact epithelium, charybdotoxin should be a useful tool for examination of the role of maxi K⁺ channels. The high sensitivity for Ca²⁺ and the properties of the activator site are in agreement with an important regulatory role for the high conductance K⁺ channel in the epithelial cells.Dr. E. Moczydlowsky, Yale University School of Medicine, New Haven, CT, and Dr. Per Stampe, Brandeis University, Waltham, MA, are thanked for introduction to the bilayer technique. Tove Soland is thanked for excellent technical assistance. This work was supported by the Novo Nordisk Foundation, the Carlsberg Foundation, the Danish Medical Research Council, and the Austrian Research Council.     

Segmental differences in short-chain fatty acid transport in rabbit colon: Effect of pH and Na

Short-chain fatty acids (SCFAs) are the predominant luminal anion in the mammalian colon. Although they are rapidly absorbed in vivo, little is known about the mechanisms of transepithelial transport in vitro. Previous studies have suggested that SCFA transport may be linked to Na absorption or an anion exchange mechanism. We compared the transport of propionate under short-circuit conditions in rabbit proximal and distal colon to determine whether there were segmental differences, how SCFAs may be linked to either Na absorption or anion transport, and whether SCFAs, as weak electrolytes, may be affected by transepithelial pH gradients. In distal colon, propionate transport was not significantly altered by stimulation of electrogenic Na absorption, epinephrine or Cl removal. However, a modest transepithelial pH gradient (luminal 6.8/serosal 7.4) stimulated propionate absorption. In proximal colon, propionate transport was significantly altered by manuevers that either stimulated (lowered [Na] in the bathing media) or inhibited (theophylline) apical Na−H exchange. Neither Cl removal, nor the anion exchange inhibitor DIDS, nor a transepithelial bicarbonate gradient, altered propionate transport. A transepithelial pH gradient inhibited propionate secretion, but not in a manner entirely consistent with the effect of pH on the distribution of a weak electrolyte. These results suggest that there is significant segmental heterogeneity in colonic SCFA transport; that transepithelial propionate fluxes are altered by changes in pH or electroneutral Na absorption (Na−H exchange), but not by chloride removal, bicarbonate gradients or electrogenic Na absorption. Regulation of SCFA transport may be an important factor in the physiology of colonic fluid balance.     

TRPV3, a thermosensitive channel is expressed in mouse distal colon epithelium

The thermo-transient receptor potential (thermoTRP) subfamily is composed of channels that are important in nociception and thermo-sensing. Here, we show a selective expression of TRPV3 channel in the distal colon throughout the gastrointestinal tract. Expression analyses clearly revealed that TRPV3 mRNA and proteins were expressed in the superficial epithelial cells of the distal colon, but not in those of the stomach, duodenum or proximal colon. In a subset of primary epithelial cells cultured from the distal colon, carvacrol, an agonist for TRPV3, elevated cytosolic Ca²⁺concentration in a concentration-dependent manner. This response was inhibited by ruthenium red, a TRPV channel antagonist. Organotypic culture supported that the carvacrol-responsive cells were present in superficial epithelial cells. Moreover, application of carvacrol evoked ATP release in primary colonic epithelial cells. We conclude that TRPV3 is present in absorptive cells in the distal colon and may be involved in a variety of cellular functions.     

Na+-dependent co-transport of α-methyl d-glucoside across the mucosal border of rabbit descending colon

(1) The uptake and bidirectional fluxes of 1-α-methyl d-glucoside were studied in isolated rabbit colonic mucosa. (2) The uptake of α-methyl d-glucoside was linear over the first 30 min and reached maximum after 1 h; was a saturable function of sugar concentration and was Na⁺-dependent. (3) An increase in sugar uptake across the mucosal border and net transepithelial sugar flux across sheets of colon was observed in the presence of 10^?4 M amiloride. (4) Phlorizin (10^?4 M) inhibited sugar uptake into the tissue water and abolished net sugar flux. Amiloride-stimulated sugar uptake was also abolished by 10^?4 M phlorizin. (5) Ouabain (10^?4 M) prevented the effect of amiloride on sugar uptake and inhibited sugar uptake into the tissue. (6) These results corroborate the findings of Henriques de Jesus et al.(Henriques de Jesus, C., Da Gracia Emilio, M. and Santos, M.A. Gastroenterol. Clin. Biol. 3, 172–173) who found a sugar-dependent increase in short-circuit current in colonic mucosa exposed to amiloride.     

The interstitial cells in the colon of the rabbit

Summary The interstitial cells associated with the myenteric plexus of the rabbit colon were studied by scanning and transmission electron microscopy. It was demonstrated that the interstitial cells were stellate or fusiform in shape and located over the ganglia, over nerve bundles and between muscle cells. They were characterized by many slender processes, and resemble fibroblasts. No basal lamina was observed between the interstitial cells and muscle cells. It was concluded that structural features of the interstitial cells are distinctly different from those of neurons, Schwann cells, or of smooth muscle cells, while they show clear similarities to those of fibroblasts. By scanning electron microscopy the shapes and the relations of these cells could be demonstrated in great detail.     

The transepithelial shunt pathway in the renal proximal tubule of newt kidney

The transepithelial shunt pathway of newt proximal tubule was examined with glass micro-electrode and electron microscopic methods. The input resistance of the peritubular (basal) membrane and tubular wall were found to be $\text{4.2 ± 0.1 · 10}{}^6$ ($\text{mean ± S.E.}\text{, n = 16}$) and $\text{11.4 ± 0.2 · 10}{}^4\text{(n = 11)}$, respectively. The input resistance of the peritubular membrane was approximately 40-times larger than that of the tubular wall. When the kidneys were perfused in a lanthanum solution, the lanthanum ions were then observed in the junctional complexes and in the intercellular spaces on both the basal and apical sides. The results indicate that the electrical shunt pathway corresponds to the apical junctional complexes and the intercellular spaces, and that the tight junctions are not truly ‘tight’ for the transepithelial movement of small ions in the proximal tubule of the newt kidney.     

Ca2+ and cAMP activate K+ channels in the basolateral membrane of crypt cells isolated from rabbit distal colon

Summary Using patch-clamp techniques, we have studied Ca²⁺-activated K⁺ channels in the basolateral membrane of freshly isolated epithelial cells from rabbit distal colon. Epithelial cell clusters were obtained from distal colon by gentle mechanical disruption of isolated crypts. Gigaohm seals were obtained on the basolateral surface of the cell clusters. At the resting potential (approximately –45 mV), with NaCl Ringer's bathing the cell, the predominant channels had a conductance of 131±25 pS. Channel activity depended on voltage as depolarization of the membrane increased the open probability. In excised inside-out patches, channels were found to be selective for K⁺ over Na⁺. Channel activity correlated directly with bath Ca²⁺ concentration in the excised patches. Channel currents were blocked by 5mm TEA⁺ and 1mm Ba²⁺. In cell-attached patches, after addition of the Ca²⁺ ionophore A23187, which increases intracellular Ca²⁺, open probability was markedly increased. Channel activity was also regulated by cAMP as addition of 1mm dibutyryl-cAMP in the bath solution in cell-attached patches increased channel open probability over 20-fold. Channels that had been activated by cAMP were further activated by Ca²⁺. We conclude that the basolateral membrane of epithelial cells from descending colon contains a class of potassium channels, which are regulated by intracellular Ca²⁺ and cAMP.     

Aldosterone regulation of active sodium chloride transport in the lizard colon (Gallotia galloti)

Summary Bioelectrical parameters and unidirectional sodium and chloride fluxes were measured under voltageclamp conditions in groups of lizards submitted to single or chronic aldosterone treatment. Both acute (AT) and chronic (CT) treatment induced significant increases in the short-circuit current (I _sc), as well as in the mucosa-to-serosa (J _m-s ^Na ) and net sodium flux (J _net ^Na ). In AT tissues, aldosterone did not change net chloride flux (J _net ^Cl ) but did so in CT tissues. Amiloride reduced the aldosterone-increased I _sc in AT and CT tissues, inhibited J _net ^Na in AT tissues and abolished it in CT colons. J _net ^Cl was also reduced by the diuretic in the group of AT colons, whereas no changes were observed in the CT tissues. Addition of luminal DIDS reduced Na⁺ absorption and totally inhibited Cl^- absorption in the AT tissues, but did not change I _sc. However, in CT tissues neither Na⁺ nor Cl^- transport were affected by DIDS. A good relationship between I _sc and J _m-s ^Na was apparent after DIDS treatment in AT tissues. In this group, simultaneous addition of DIDS and amiloride totally abolished J _net ^Na and reduced I _sc to untreated control values. Addition of serosal ouabain abolished I _sc and Na⁺ absorption in AT and CT colons, but Cl^- absorption was only altered in AT tissues. These results support the hypothesis that aldosterone induces an electrogenic, amiloride-sensitive sodium absorption, and in a dose-dependent fashion suppresses electroneutral NaCl absorption in the lizard colon.Abbreviations AT acutely treated - CT chronically treated animals - DIDS 4-4-diisothiocyanatostibene-2-2-disulfonic acid - DMSO dimethylsulphoxide - G _t tissue conductance - I _sc short circuit current - PD transepithelial potential difference - SITS 4-acetamido-4-isothiocyanatostilbene-2-2-disulfonic acid - UC untreated controls Preliminary results of this paper were presented at the X ^th meeting of the European Intestinal Transport Group (EITG), Askov Hojskole, Denmark, 16–19 September 1990     

Electroaffinity in paracellular absorption of hydrophilic d-dipeptides by sparrow intestine

We previously demonstrated size selectivity in the absorption of nonelectrolyte hydrosoluble probes in birds, presumably by the paracellular pathway. Our goal in this study was to determine the charge selectivity in the absorption of hydrosoluble d-dipeptides, because there have been no studies of the electroaffinity of this absorption pathway in birds. For this purpose isosmotic solutions with two hydrophilic d-dipeptides: serine-lysine (positive at pH 7.4) and serine-aspartic (negative at pH 7.4) were gavaged into the stomach in nonanesthetized house sparrows (Passer domesticus), and injected into the pectoralis with a syringe in different trials. Fractional absorption was calculated as F = [AUC by gavage)]/[AUC by injection] (AUC = area under the curve of plasma probe concentration vs. time). Fractional absorption was significantly higher for the positively charged than negatively charged dipeptide (respectively, F=0.30±0.05 vs. F=0.17±0.03). These findings give the first evidence of cation selectivity by the paracellular route in the absorption of hydrosoluble solutes in the small intestine in birds.     

Regulation of the paracellular Na+ and Cl- conductances by the NaCl-generated osmotic gradient in a manner dependent on the direction of osmotic gradients

In the present study, we investigated the effect of osmolality on the paracellular ion conductance (Gp) composed of the Na⁺ conductance (G_Na) and the Cl⁻ conductance (G_Cl). An osmotic gradient generated by NaCl with relatively apical hypertonicity (NaCl-absorption-direction) induced a large increase in the G_Na associated with a small increase in the G_Cl, whereas an osmotic gradient generated by NaCl with relatively basolateral hypertonicity (NaCl-secretion-direction) induced small increases in the G_Na and the G_Cl. These increases in the Gp caused by NaCl-generated osmotic gradients were diminished by the application of sucrose canceling the NaCl-generated osmotic gradient. The osmotic gradient generated by basolateral application of sucrose without any NaCl gradients had little effects on the Gp. However, this basolateral application of sucrose produced a precondition drastically quickening the time course of the action of the NaCl-generated osmotic gradient on the Gp. Further, we found that application of the basolateral hypotonicity generated by reduction of NaCl concentration shifted the localization of claudin-1 to the apical from the basolateral side. These results indicate that the osmotic gradient regulates the paracellular ion conductive pathway of tight junctions via a mechanism dependent on the direction of NaCl gradients associated with a shift of claudin-1 localization to the apical side in renal A6 epithelial cells.     

High-definition spatiotemporal mapping of contractile activity in the isolated proximal colon of the rabbit

Four types of contractile activity were identified and characterised in the isolated triple haustrated proximal colon of the rabbit using high-definition spatiotemporal mapping techniques. Mass peristalses were hexamethonium-sensitive deep circular contractions with associated taenial longitudinal contractile activity that occurred irregularly and propagated rapidly aborad, preceded by a zone of local lumen distension. They were sufficiently sustained for each event to occupy the length of the isolated colonic segment and the contraction persisted longer orally than aborally, the difference being more pronounced when lumen contents were viscous. Haustra were bounded by deep even-spaced ring contractions that progressed slowly aborad (haustral progression). Haustral formation and progression were hexamethonium-sensitive and coordinated across intertaenial domains. Ripples were hexamethonium-resistant phasic circular contractions that propagated predominantly orad at varying rates. In the presence of haustra, they were uncoordinated across intertaenial domains but were more coordinated when haustra were absent. Fast phasic contractions were relatively shallow hexamethonium-resistant contractions that propagated rapidly in a predominantly aborad direction. Fast phasic circular contractions were accompanied by taenial longitudinal muscle contractions which increased in amplitude prior to a mass peristaltic event and following the administration of hexamethonium. On the basis of the concurrence and interaction of these contractile activities, we hypothesise that dual pacemakers are present with fast phasic contractions being modulated by the interstitial cells of Cajal in the Auerbach’s plexus (ICC-MY) while ripples are due to the submucosal ICC (ICC-SM). Further, that ICC-SM mediate the enteric motor neurons that generate haustral progression, while the intramuscular ICC (ICC-IM) mediate mass peristalsis. The orad movement of watery fluid was possibly due to ripples in the absence of haustra.     

Sodium uptake mechanisms in brush-border membrane vesicles prepared from rabbit renal cortex

Amiloride-sensitive and amiloride-insensitive components of ²²Na⁺ uptake were examined in brush-border membrane vesicles prepared from rabbit renal cortex. Both components could be stimulated by interior-negative electrical potentials, demonstrating a sodium conductance pathway and an effect of electrical potential on the initial rate of Na⁺/H⁺ exchange.     

Type 2 cGMP-dependent protein kinase regulates homeostasis by blocking c-Jun N-terminal kinase in the colon epithelium

Analysis of knockout animals indicates that 3′,5′cyclic guanosine monophosphate (cGMP) has an important role in gut homeostasis but the signaling mechanism is not known. The goals of this study were to test whether increasing cGMP could affect colon homeostasis and determine the mechanism. We increased cGMP in the gut of Prkg2^+/+ and Prkg2^−/− mice by treating with the PDE5 inhibitor Vardenafil (IP). Proliferation, differentiation and apoptosis in the colon mucosa were then quantitated. Vardenafil (Vard) treatment increased cGMP in colon mucosa of all mice, but reduced proliferation and apoptosis, and increased differentiation only in Prkg2^+/+ mice. Vard and cGMP treatment also increased dual specificity protein phosphatase 10 (DUSP10) expression and reduced phospho-c-Jun N-terminal kinase (JNK) levels in the colon mucosa of Prkg2^+/+ but not Prkg2^−/− mice. Treatment of Prkg2^−/− mice with the JNK inhibitor SP600125 reversed the defective homeostasis observed in these animals. Activation of protein kinase G2 (PKG2) in goblet-like LS174T cells increased DUSP10 expression and reduced JNK activity. PKG2 also increased goblet cell-specific MUC2 expression in LS174T cells, and this process was blocked by DUSP10-specific siRNA. The ability of cGMP signaling to inhibit JNK-induced apoptosis in vivo was demonstrated using dextran sodium sulfate (DSS) to stress the colon epithelium. Vard was a potent inhibitor of DSS-induced epithelial apoptosis, and significantly blocked pathological endpoints in this model of experimental colitis. In conclusion, Vard treatment activates cGMP signaling in the colon epithelium. Increased PKG2 activity alters homeostasis by suppressing proliferation and apoptosis while promoting differentiation. The PKG2-dependent mechanism was shown to involve increased DUSP10 and subsequent inhibition of JNK activity.     

Frizzled-7 receptor ectodomain expression in a colon cancer cell line induces morphological change and attenuates tumor growth

Frizzled (FZD) receptors have a conserved N-terminal extracellular cysteine-rich domain that interacts with Wnts and co-expression of the receptor ectodomain can antagonize FZD-mediated signalling. Using the ectodomain as an antagonist we have modulated endogenous FZD7 signalling in the moderately differentiated colon adenocarcinoma cell line, SK-CO-1. Unlike the parental cell line, which grows as tightly associated adherent cell clusters, the FZD7 ectodomain expressing cells display a spread out morphology and grow as a monolayer in tissue culture. This transition in morphology was associated with decreased levels of plasma membrane-associated E-cadherin and beta-catenin, localized increased levels of vimentin and redistribution of alpha6 integrin to cellular processes in the FZD7 ectodomain expressing cells. The morphological and phenotype changes induced by FZD7 ectodomain expression in SK-CO-1 cells is thus consistent with the cells undergoing an epithelial-to-mesenchymal-like transition. Furthermore, initiation of tumor formation in a xenograft tumor growth assay was attenuated in the FZD7 ectodomain expressing cells. Our results indicate a pivotal role for endogenous FZD7 in morphology transitions that are associated with colon tumor initiation and progression.     

Novel Daple-like protein positively regulates both the Wnt/beta-catenin pathway and the Wnt/JNK pathway in Xenopus

Wnt signaling pathways are essential in various developmental processes including differentiation, proliferation, cell migration, and cell polarity. Wnt proteins execute their multiple functions by activating distinct intracellular signaling cascades, although the mechanisms underlying this activation are not fully understood. We identified a novel Daple-like protein in Xenopus and named it xDal (Xenopus Daple-like). As with Daple, xDal contains several leucine zipper-like regions (LZLs) and a putative PDZ domain-binding motif, and can interact directly with the dishevelled protein. In contrast to mDaple, injection of xDal mRNA into the dorso-vegetal blastomere does not induce ventralization and acted synergistically with xdsh in secondary axis induction. XDal also induced expression of siamois and xnr-3, suggesting that XDal functions as a positive regulator of the Wnt/beta-catenin pathway. Injection of xDal mRNA into the dorso-animal blastomere, however, induced gastrulation-defective phenotypes in a dose-dependent manner. In addition, xDal inhibited activin-induced elongation of animal caps and enhanced c-jun phosphorylation. Based on these findings, xDal is also thought to function in the Wnt/JNK pathway. Moreover, functional domain analysis with several deletion mutants indicated that xDal requires both a putative PDZ domain-binding motif and at least one LZL for its activity. These findings with xDal will provide new information on the Wnt signaling pathways.