A new morphological aspect of the brush cells of the mouse gallbladder epithelium

Summary The brush cells of the gallbladder epithelium of the mouse have microvilli not only at their luminal border but also on their lateral surface, from the level of the nucleus to the junctional complex. The lateral microvilli radiate from the brush cell in all directions, contain a core of filaments, and penetrate up to 3 m into the adjacent cells. The microvilli in these locations display small desmosomes at their base.     

Effect of cholecystokinin-pancreozymin (CCK-PZ) on glycoprotein secretion from mouse gallbladder epithelium

Summary Structural changes in the gallbladder epithelial cells of the mouse were studied following in vivo and in vitro stimulation of the gallbladder with the gastrointestinal hormone cholecystokinin-pancreozymin (CCK-PZ). Signs of increased secretory activity were observed within the first 2–3 min after hormone administration. At the ultrastructural level, best visualized with the PA-CrA-silver technique, granule discharge was observed, as was an overall increase in size of the granules. After prolonged in vitro incubation or repeated in vivo stimulation, there was an almost total depletion of secretory granules. This phenomenon is accompanied by an enhanced uptake of extracellular thorium dioxide by endocytotic vesicles at the apical cell surface. An exocytosisendocytosis coupling mechanism may be important for membrane conservation in the gallbladder epithelial cells. The findings establish that the hormone CCK-PZ stimulates the secretion of glycoproteins from the mouse gallbladder epithelium.This investigation was supported by grants from the University of Umeå and from the Swedish Medical Research Council (grant No. B76-12X-04758-01). The authors wish to thank Miss K. Karlsson and Miss M. Borg for skilful technical assistance     

A mini organ culture as a model for studying the gallbladder epithelium of mouse

Summary— A mini organ culture of mouse gallbladder was developed as an alternative to primary cultures of epithelial cells of this organ. Small pieces of tissue were prepared and maintained in minimum essential Eagle medium with 10% foetal calf serum, for as long as 7 days. Qualitative and quantitative ultrastructural studies have been performed using electron microscopy. The viability of cells was evaluated by stereological quantification of endocytotic vesicles containing horseradish peroxidase and labelling of exocytotic glycoproteins with tannic acid. The morphology of tissue pieces during the 1st h of culturing and tissue isolated directly from animals exhibited no significant differences. However, after 4 h in culture degradative changes became evident in many cells. At that time, endo- and exocytosis were both dramatically reduced. After 24 h, the morphology, as well as endo- and exocytosis recovered and were comparable to the parameters of the tissue in vivo or after 1 h in culture. The endocytotic activity remained unchanged from day 1 to 7 of culturing, while the number of exocytotic vesicles gradually decreased after 2 days in culture. Our results prove that mini organ culture of gallbladder is morphologically and functionally comparable with the tissue in vivo and for studies of epithelium in culture it is more convenient than primary cultures.     

Osmotic water permeability of Necturus gallbladder epithelium

An electrophysiological technique that is sensitive to small changes in cell water content and has good temporal resolution was used to determine the hydraulic permeability (Lp) of Necturus gallbladder epithelium. The epithelial cells were loaded with the impermeant cation tetramethylammonium (TMA+) by transient exposure to the pore-forming ionophore nystatin in the presence of bathing solution TMA+. Upon removal of the nystatin a small amount of TMA+ is trapped within the cell. Changes in cell water content result in changes in intracellular TMA+ activity which are measured with intracellular ion-sensitive microelectrodes. We describe a method that allows us to determine the time course for the increase or decrease in the concentration of osmotic solute at the membrane surface, which allows for continuous monitoring of the difference in osmolality across the apical membrane. We also describe a new method for the determination of transepithelial hydraulic permeability (Ltp). Apical and basolateral membrane Lp's were assessed from the initial rates of change in cell water volume in response to anisosmotic mucosal or serosal bathing solutions, respectively. The corresponding values for apical and basolateral membrane Lp's were 0.66 x 10(-3) and 0.38 x 10(-3) cm/s.osmol/kg, respectively. This method underestimates the true Lp values because the nominal osmotic differences (delta II) cannot be imposed instantaneously, and because it is not possible to measure the true initial rate of volume change. A model was developed that allows for the simultaneous determination of both apical and basal membrane Lp's from a unilateral exposure to an anisosmotic bathing solution (mucosal). The estimates of apical and basal Lp with this method were 1.16 x 10(-3) and 0.84 x 10(-3) cm/s.osmol/kg, respectively. The values of Lp for the apical and basal cell membranes are sufficiently large that only a small (less than 3 mosmol/kg) transepithelial difference in osmolality is required to drive the observed rate of spontaneous fluid absorption by the gallbladder. Furthermore, comparison of membrane and transepithelial Lp's suggests that a large fraction of the transepithelial water flow is across the cells rather than across the tight junctions.     

The magnitude of nonelectrolyte selectivity in the gallbladder epithelium

Summary The permeability of the rabbit gallbladder epithelium to nonelectrolytes was determinted by radioactive tracer techniques and by a rapid osmotic procedure. As expected from empirical and theoretical considerations, there was a good agreement between the selectivity sequences obtained by the two methods for the sixteen compounds used in this study. Although the permeability coefficients are directly related to their bulk-phase partition coefficients, the gallbladder behaves as if the membranes controlling selectivatity are more hydrophilic than isobutanol. The relation between permeability coefficients and molecular weight also show that these membranes are less viscous than other single cell membranes. Small polar solutes exhibit lower apparent activiation energies for permeation than larger solutes, and this is taken as support for the view that small polar molecules permeate across this tissue via a polar pathway. Inutin and sucrose permeability coefficients are in the ratio of their free-solution diffusion coefficients, and the apparent surcose activation energy is indistinguishable from that reported for diffusion in aqueous solution. These latter observations may be explained by the presence of a few large pores in the epithelium.     

Mucous granule exocytosis and CFTR expression in gallbladder epithelium

A mechanistic model of mucous granule exocytosis by columnar epithelial cells must take into account the unique physical-chemical properties of mucin glycoproteins and the resultant mucus gel. In particular, any model must explain the intracellular packaging and the kinetics of release of these large, heavily charged species. We studied mucous granule exocytosis in gallbladder epithelium, a model system for mucus secretion by columnar epithelial cells. Mucous granules released mucus by merocrine exocytosis in mouse gallbladder epithelium when examined by transmission electron microscopy. Spherules of secreted mucus larger than intracellular granules were noted on scanning electron microscopy. Electron probe microanalysis demonstrated increased calcium concentrations within mucous granules. Immunofluorescence microscopic studies revealed intracellular colocalization of mucins and the cystic fibrosis transmembrane conductance regulator (CFTR). Confocal laser immunofluorescence microscopy confirmed colocalization. These observations suggest that calcium in mucous secretory granules provides cationic shielding to keep mucus tightly packed. The data also suggests CFTR chloride channels are present in granule membranes. These observations support a model in which influx of chloride ions into the granule disrupts cationic shielding, leading to rapid swelling, exocytosis and hydration of mucus. Such a model explains the physical-chemical mechanisms involved in mucous granule exocytosis.     

The electrical potential profile of gallbladder epithelium.

In this study the relative ionic permeabilities of the cell membranes of Necturus gallbladder epithelium have been determined by means of simultaneous measurement of transmural and transmucosal membrane potential differences (PD) and by ionic substitution experiments with sodium, potassium and chloride ions. It is shown that the mucosal membrane is permeable to sodium and to potassium ions. The baso-lateral membrane PD is only sensitive to potassium ions. In both membranes chloride conductance is negligible or absent. The ratio of the resistances of the mucosal and baso-lateral membranes, RM/RS, increases upon reducing the sodium concentration in the mucosal solution. The same ratio decreases when sodium is replaced by potassium which implies a greater potassium than sodium conductance in the mucosal membrane. The relative permeability of the shunt for potassium, sodium and chloride ions is: PK/PNa/PCl=1.81:1.00:0.32. From the results obtained in this study a value for the PK/PNa ratio of the mucosal membrane could be evaluated. This ratio is 2.7. From the same data the magnitude of the electromotive forces generated across the cell membranes could be calculated. The EMF's are -15mV across the mucosal membrane and -81mV across the baso-lateral one. Due to the presence of the low resistance shunt the transmucosal membrane PD is -53.2mV (cell inside negative) and the transmural PD is +2.6mV (serosal side positive). The change in potential profile brought about by the low resistance shunt favors passive entry of Na ions into the cell across the mucosal membrane. Calculations show that this passive Na influx is maximally 64% of the net Na flux estimated from fluid transport measurements. The C-1 conductive of the baso-lateral membrane is too small to allow electrogenic coupling of C1 with Na transport across this membrane. Experiments with rabbit gallbladder epithelium indicate that the membrane properties in this tissue are qualitatively similar to those of Necturus gallbladder epithelium.     

Dimensions of polar pathways through rabbit gallbladder epithelium

Summary The permeability of rabbit gallbladder to hydrophilic nonelectrolytes, with molecular weights from 20 to 60,000, has been studied. Restriction in the diffusion of the small electrolytes is very significant up to glycerol, which suggests permeation through aqueous pores with equivalent radii of 4 Å. An extracellular pathway is responsible for the permeation of the larger solutes. This extracellular pathway shows no restriction in diffusion of molecules up to the size of inulin. Dextran (15,000 to 17,000 mol wt) is significantly restricted. Albumin permeability is <10^–8 cm sec^–1. These observations can be equated with equivalent, pore radii of 40 Å for the shunt pathway.Increasing osmolarities of the incubation medium cause decreased cell-membrane permeability and increased shunt permeability. 0.5mm phloretin induces a 60% reduction in urea permeability and a 168% increase in antipyrine permeability. No effect on the osmotic water permeability or on the shunt permeability is observed in the presence of phloretin. The apparent activation energy of urea permeation changes from values consistent with diffusion in bulk water, to values consistent with diffusion through hydrocarbon regions. This suggests that the polar route for urea permeation is blocked by phloretin.The contribution of the shunt pathway to osmotic flow induced by sucrose or NaCl gradients is smaller than 16% according to Poiseuille's flow calculations. Tetraethylammoniumchloride and albumin have been shown to be osmotically more effective than sucrose, suggesting a greater shunt contribution to the total water flow.     

Unstirred layer effects in osmotic water flow across gallbladder epithelium

Summary The standard one-dimensional model of the unstirred layer is applied in a re-examination of the experimental results of Wright, Smulders and Tormey (Wright, E.M., Smulders, A.P., Tormey, J. McD., 1972,J. Membrane Biol. 7:198) who reported large transients in the osmotic flux of water from the serosal to the mucosal side of rabbit gallbladder epithelium. They initiated osmosis by the addition of sucrose to the mucosal bathing solution (initially, approximately 300mOsm NaCl) and observed that the initial flux was more than ten times its eventual steady-state value; they interpreted this as a consequence of the piling-up of NaCl in the unstirred tissue layer on the serosal side of the epithelium. The present analysis (both steady-state and unsteady) shows that if measured values of layer thickness are used, together with reasonable values of the reduced diffusivity of NaCl in the tissue and of the fraction of tissue available for water flow, then one would predict a discrepancy of only about 10%, not tenfold, between the initial and final values of the flux. Thus the standard model is inconsistent with the observations. Furthermore, Wright et al's results cannot be used to infer that the osmotic permeability of epithelial cell membranes is much larger than steadystate measurements on whole epithelia would indicate. Mucosal-to-serosal flow is also analyzed, and in this case a considerably greater osmotic permeability is predicted; this result is consistent with the observed changes in structure of the lateral intercellular spaces when the direction of flow is reversed.     

High-conductance K+ channels in guinea pig gallbladder epithelium]

Since secretion of electrolytes may be regulated by membrane potential difference, ion channels were studied using patchclamp technique. We have identified, in cell-attached configuration, inward-rectifying channels: the zero-current potential corresponded to the K+ equilibrium potential calculated from intracellular K+ activity. Using inside-out configuration and symmetric 145 mM KCl salines, i/V curve was linear, channel conductance was about 170 pS and the reversal potential 0 mV. The channels were selective for K+ over Na+, N-methylglucamine and anions and were activated by membrane depolarization.     

Regulation of cAMP-activated apical membrane chloride conductance in gallbladder epithelium

Regulation of the cAMP-activated apical membrane Cl- conductance (GaCl) in Necturus gallbladder (NGB) epithelial cells was investigated with intracellular-microelectrode techniques. GaCl was increased by exposure to 8-Br-cAMP, theophylline or forskolin. Neither 8-Br-cGMP nor elevation of intracellular [Ca2+] using ionomycin had effects on GaCl or interfered with activation of GaCl by forskolin. N-(2- [methylamino]ethyl)-5-isoquinolinesulfonamide (H8), an inhibitor of cAMP-dependent protein kinase (PKA), slowed but did not prevent the GaCl response to 8-Br-cAMP. Phorbol 12-myristate 13-acetate (PMA), which activates protein kinase C (PKC), stimulated GaCl but had no effects on intracellular [cAMP]. GaCl was unaffected by 4 alpha- phorbol, a PMA analog which does not activate PKC. Okadaic acid (OA), an inhibitor of protein phosphatases (PP) types 1 and 2A, slowed the activation of GaCl by 8-Br-cAMP, hastened the return of GaCl to basal values following removal of 8-Br-cAMP, and significantly reduced the elevation in intracellular [cAMP] produced by forskolin. OA had no effects on the GaCl changes elicited by theophylline. We conclude that: (a) NGB GaCl can be activated by PKA-mediated phosphorylation of apical membrane Cl- channels or a regulatory protein, (b) GaCl can also be activated via PKC, by a cAMP-independent mechanism, (c) OA-sensitive PP are not required for inactivation of GaCl; OA appears to stimulate phosphodiesterase, which lowers intracellular [cAMP] and affects GaCl activation, and (d) the apical membrane of NGB epithelium lacks a Ca(2+)-activated Cl- conductance.     

Synthesis of glycoproteins in the Golgi complex of the mouse gallbladder epithelium during fasting,refeeding, and gallstone formation

Summary The mouse gallbladder epithelium was studied with light microscopic autoradiography and quantitative electron microscopy during fasting, refeeding and experimental gallstone formation. To determine the intracellular pathway of glycoproteins, H³-galactose was injected at different time intervals into the mice. At 10, 25 and 40 min after an intraperitoneal injection the gallbladders were fixed and prepared for light microscopy. As early as 10 min after injection, label was observed in supranuclear cytoplasmic regions and at 25 min, an increased radioactivity was present throughout the apical cytoplasm. At 40 min, silver grains were mainly present at the cell surface. Autoradiographs processed 25 min after an intraperitoneal H³-galactose injection after fasting for 48 h showed decreased supranuclear and apical radioactivity. After refeeding (12 h) there was an enhanced activity in both these regions. Animals fed a lithogenic diet for one month showed a marked increase of radioactive label mainly in cells of crypts and invaginations of the mouse gallbladder mucosa.Morphometric measurements of the Golgi apparatus revealed that deprivation of food significantly diminished the volume density of the Golgi apparatus. Refeeding the animals restored the volume density values to normal levels. In the course of gallstone formation there was a further significant increase in the volume density of the Golgi complexes as compared to controls.