Cyclic AMP-induced changes in membrane conductance ofNecturus gallbladder epithelial cells

Summary Enhanced cellular cAMP levels have been shown to increase apical membrane Cl^– and HCO ₃ ^– conductances in epithelia. We found that the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX) increases cAMP levels inNecturus gallbladder. We used conventional open-tip and double-barreled Cl^–-selective microelectrodes to study the effects of IBMX on membrane conductances and intracellular Cl^– activities in gallbladders mounted in a divided chamber and bathed with Ringer's solutions at 23°C and pH 7.4. In HCO ₃ ^– -free media, 0.1mM IBMX added to the mucosal medium depolarized the apical membrane potentialV _a , decreased the fractional resistanceF _R , and significantly reduced intracellular Cl^– activity (a _Cl ⁱ ). Under control conditions,a _Cl ⁱ was above the value corresponding to passive distribution across the apical cell membrane. In media containing 25mM HCO ₃ ^– , IBMX caused a small transient hyperpolarization ofV _a followed by a depolarization not significantly different from that observed in HCO ₃ ^– -free Ringer's. Removal of mucosal Cl^–, Na⁺ or Ca²⁺ did not affect the IBMX-induced depolarization inV _a . The basolateral membrane ofNecturus gallbladder is highly K⁺ permeable. Increasing serosal K⁺ from 2.5 to 80mM, depolarizedV _a . Mucosal IBMX significantly reduced this depolarization. Addition of 10mM Ba²⁺, a K⁺ channel blocker, to the serosal medium depolarizedV _a and, essentially, blocked the depolarization induced by IBMX. These results indicate that mucosal IBMX increases apical HCO ₃ ^– conductance and decreases basolateral K⁺ conductance in gallbladder epithelial cells via a cAMP-dependent mechanism. The latter effect, not previously reported in epithelial tissues, appears to be the major determinant of the IBMX-induced depolarization ofV _a .     

Interstitial Cajal-like cells in human gallbladder

We describe here an interstitial Cajal-like cell type (ICLC) in human gallbladder, resembling the archetypal enteric interstitial cells of Cajal. Gallbladder ICLC were demonstrated in fresh preparations (tissue cryosections) using methylene-blue, and fixed specimens in Epon semi-thin sections stained with toluidine blue or transmission electron microscopy (TEM). The positive diagnosis of gallbladder ICLC was further verified by immunohistochemistry: CD117/c-kit, CD34, and another 16 antigens: vimentin, desmin, nestin, α-smooth muscle actin, NK-1, S-100, PGP-9.5, tau protein, chromogranin A, NSE, GFAP, CD1a, CD62-P, CD68, estrogen and progesterone receptors. Double immunostaining was performed for CD117, CD34 and CD117 and nestin, respectively. In fresh specimens, the spatial density of gallbladder ICLC was 100–110 cells/mm². ICLC mainly appeared beneath the epithelium and in muscularis (about 7%, and ∼5%, respectively). In toto, ICLC represent in gallbladder ∼5.5% of subepithelial cells. TEM showed that diagnostic criteria were fulfilled by ICLC. Moreover, TEM indicated that the main ultrastructural distinctive feature for ICLC, the cell processes, develop into the characteristic shape at a relatively early stage of development. It remains to be established if, in humans, ICLC are involved in gallbladder (dis)functions (e.g. pace-making, secretion (auto-, juxta- and/or paracrine), intercellular signaling, or stone formation). M. E. Hinescu and C. Ardeleanu contributed equally to this study.     

Differences in Action of PACAP-27 and PACAP-38 on Guinea Pig Gallbladder Smooth Muscle Using Synthetic C-terminally Modified PACAP Peptides

Pituitary adenylate cyclase activating polypeptide (PACAP) occurs in two bioactive forms, PACAP-38 and PACAP-27 that have identical N-terminal sequences but differ by the presence of a C-terminal 11 residue elongation in the former. Although VIP and PACAP have several similar biological actions due to their amino acid sequence similarity, we have found that they evoke opposite responses in the guinea pig gallbladder smooth muscle, where PACAP induces contraction while VIP causes relaxation. In addition the response to PACAP-38 is four times lower than that of PACAP-27. In a previous study we have reported the role of the N-terminal α-helical regions of PACAP-27 which play a key role in gallbladder contraction. In the present study the biological action on the guinea pig gallbladder was investigated using a synthetic mini-library of C-terminally deleted peptides related to PACAP-38. The effects caused by residues within the C-terminus are not a result of a response via the M-receptor or Na⁺ channel, but most likely arise from a delicate balance between the differential effects of PACAP-38 on specific PAC1 and VPACs receptors.     

A mechanism for isotonic fluid flow through the tight junctions of Necturus gallbladder epithelium

During isotonic fluid flow, Necturus gallbladder epithelium mediates net fluxes of paracellular probes by a convective process. We show here that the paracellular system is modeled by permeation through three populations of channels: (i) convective parallel-sided ones of width 7.7 nm (ii) small diffusive ones of radius 0.6 nm, and (ii) large diffusive ones of radius exceeding 50 nm. The reflexion coefficient of the convective channels is very low and the calculated osmotic flow rate is close to zero when compared with the observed fluid absorptive rate of 2 x 10^–6 cm/sec. Analysis reveals that the convective channels behave as though closed to back-diffusion of probes; if this is due to solvent drag then very high fluid velocities are required, acting through minute areas. There are no transjunctional gradients that could drive the flow, and so the fluid must be propelled through the channel by components of the junction.We propose a mechanism based upon an active junctional peristalsis which allows discrimination on the basis of molecular size, in which the channels are always occluded at some point and so back-diffusion cannot occur. There is no local gradient of salt distal to the junctions and therefore the osmotic permeability of the membranes is irrelevant. High fluid velocities are not required, and the flow can occur over a substantial fraction of the junction. The mechanism must involve motile and contractile elements associated with the junction for which there is already considerable evidence.Symbols A _i filtration area of channel i;i=b (big), s (small) and c (convectional) - B constant for streamline flow - C _i concentration of probe at i - D diffusion coefficient - D ^o diffusion coefficient in free solution - d width of junction - F _i diffusive drag factor in channel i - g ionic conductivity - G _i convective drag factor in channel i - J _ij probe flux from i to j - J _net net probe flux - J _v volume flow per cm² of epithelium - l linear extent of junction per cm² epithelial plane - L length of junctional channel - L _p hydraulic conductivity - N Avogadro's number - q available filtration area fraction of channel - r _s probe molecular radius - r _c channel radius or half-width - S _i steric factor in channel i - V _w,s partial molar volume of water or salt - v _i fluid velocity in channel i - _w dynamic viscosity of water - specific conductivity - ratio of solute radius to channel radius or half-width - diffusive/pressure-driven flow ratio - reflexion coefficient     

清胆颗粒利胆作用的实验研究

目的：通过研究清胆颗粒对正常大鼠及模型动物胆汁及病理组织的影响,确定其利胆功效,为临床应用提供依据.方法：采用胆管引流法测定清胆颗粒对正常大鼠胆汁量的影响;应用石胆酸（lithocholic acid,LCA）造成豚鼠胆囊炎模型,测定其对模型动物肝重、肝指数、胆囊容积及胆囊病理组织的影响.结果：清胆颗粒能显著增加正常大鼠胆汁流量,明显减轻模型动物肝湿重和肝指数,缩小胆囊容积,能明显降低胆汁中TB、DB及UCB/TB含量,对Ca^2＋增高有明显的抑制作用,对模型动物胆囊粘膜上皮增生有明显的抑制作用.结论：清胆颗粒具有显著的利胆作用.     

Downregulated circRNA_CDKN1A promotes gallbladder cancer progression through activation of the NF-κB pathway

This study uncovered the potential clinical value and molecular driving mechanisms of circular RNAs (circRNAs) in gallbladder cancer (GBC). Differentially expressed circRNAs in GBC cells were screened by high-throughput sequencing. CircRNA_CDKN1A (circBase ID: hsa_circ_0076194) was knocked out in BGC-SD cells through transfection with sh-circRNA_CDKN1A. Then, proliferation was investigated via CCK8 and EdU assays, apoptosis via flow cytometry, migration via wound healing assays, and invasion via Transwell assays. Bioinformatics analysis of circRNA_CDKN1A-related signaling pathways was performed using MetScape and g:Profiler. Results showed that the knockdown of circRNA_CDKN1A enhanced the proliferation, migration, and invasion of GBC cells and inhibited apoptosis. In addition, knocking out circRNA_CDKN1A promoted GBC cell proliferation and enhanced the dry indices of the OCT4 protein and CD34 expression levels. The knockdown of circRNA_CDKN1A activated the epithelial–mesenchymal transition pathway. Bioinformatics analysis revealed that the biological role of circRNA_CDKN1A in GBC cells involved the NF-κB pathway. LY2409881, which is an NF-κB inhibitor, reversed the effects induced by the knockdown of circRNA_CDKN1A in GBC-SD cells. In summary, the knockdown of circRNA_CDKN1A promoted the progression of GBC by activating the NF-κB signaling pathway. For the first time, this study revealed the mechanism of circRNA_CDKN1A-mediated regulatory action in GBC and identified the newly discovered circRNA_CDKN1A–NF-κB signaling axis as a potentially important candidate for clinical therapy and prognostic diagnosis of GBC.     

Intestinal cholesterol absorption is substantially reduced in mice deficient in both ABCA1 and ACAT2

The process of cholesterol absorption has yet to be completely defined at the molecular level. Because of its ability to esterify cholesterol for packaging into nascent chylomicrons, ACAT2 plays an important role in cholesterol absorption. However, it has been found that cholesterol absorption is not completely inhibited in ACAT2-deficient (ACAT2 KO) mice. Because ABCA1 mRNA expression was increased 3-fold in the small intestine of ACAT2 KO mice, we hypothesized that ABCA1-dependent cholesterol efflux sustains cholesterol absorption in the absence of ACAT2. To test this hypothesis, cholesterol absorption was measured in mice deficient in both ABCA1 and ACAT2 (DKO). Compared with wild-type, ABCA1 KO, or ACAT2 KO mice, DKO mice displayed the lowest level of cholesterol absorption. The concentrations of hepatic free and esterified cholesterol and gallbladder bile cholesterol were significantly reduced in DKO compared with wild-type and ABCA1 KO mice, although these measures of hepatic cholesterol metabolism were very similar in DKO and ACAT2 KO mice. We conclude that ABCA1, especially in the absence of ACAT2, can have a significant effect on cholesterol absorption, although ACAT2 has a more substantial role in this process than ABCA1.     

Fluid transport and dimensions of epithelial cells and intercellular spaces in frog gallbladder

Summary Morphologic findings of widely dilated intercellular spaces in fluid transporting epithelia have been claimed as evidence for the existence of an epithelial compartment in which the coupling between solute and water fluxes takes place. The validity of using epithelial geometry in sectioned material as an argument can be questioned. The present report describes the morphological appearance of frog gallbladder epithelium — normal and ouabain-treated — in the living state in vitro and after fixation, dehydration and embedding. Gallbladder segments were photographed in the living state and at the end of each step of the preparative procedure. Direct observations of whole-mounted gallbladder segments were carried out, taking advantage of the possibility of optical sectioning and high resolution by Nomarski-microscopy. The same specimens were then sectioned and examined by conventional light and electron microscopy. The observations were quantitated and showed that the epithelial cells of normal and ouabain-treated gallbladders experienced an average linear shrinkage down to 70% of their length in Ringer's solution, which corresponds to a volume shrinkage down to 35%. Moreover, dilated lateral intercellular spaces appeared during the dehydration and embedding procedure in normal but only very moderately or not at all in ouabain-treated gallbladder specimens.