Quantitative electron microscopical studies onin Vitro incubated rabbit gallbladder epithelium

Abstract

VitroSummary, Pieces of rabbit gallbladders were incubated in vitro for 1 hr in Ringer's solution at 37 ~ (" transporting epithelium"), or in Ringer's containing 1 mM ouabain (" inhibited epithelium"). The tissues were fixed in 2% glutaraldehyde, postfixed in OsO~ and embedded in Epon. Stereological analysis was carried out on electron micrographs; with this type of analysis it is possible to obtain quantitative data for the three-dimensional structure of the tissue. The following data were obtained for crest transporting epithelium (crypt epithelium in parentheses): Epithelial height, 28 (18) ~tm; nucleus, 16.9 (18.7) % of cell volume; mitochondria, 18.7 (14.1) % of cytoplasmic volume; paracellular channels, 28.3 (11.0) % of epithelial volume; lateral plasma membrane, 4.1 (2.9) m2/cm 3, and apical membrane, 0.5 (0.6) m2/cm 3 of cell volume. The paracellular channels appeared as interconnected tissue sheets of a highly varying width. They were broader in the basal portion of the epithelium than in the apical portion; however, immediately above the basement membrane the channels became very narrow. The arithmetic mean width of the channels was 0.9 (0.2) ~tm and their length was in the range of 40 120 gm. Our stereological data seem compatible with the standing gradient model for water transport.The ouabain inhibited cells were swollen with collapsed paracellular channels. It cannot be ruled out that volume changes occurred in the cells and the paracellular channels during the processing of the tissue. However, qualitatively the same morphology was observed by light microscopy of transporting and inhibited gallbladder epithelium fixed by freeze-drying and embedded in Epon. This constitutes a strong indication of the presence of such channels in the living, in vitro transporting rabbit gallbladder epithelium.The gallbladder has been extensively used in studies of water and electrolyte transport, and on the basis of such studies theories have been advanced which have explained transport processes across simple epithelia. Diamond and Bossert (1967) focused attention on electrolyte pumps which, by osmotic forces, drag water into paracellular channels. The capacities of the transepithelial transport rest partly on the structural characteristics of the paracellular channels-length, width, and surface area are included among the parameters discussed (Diamond & Bossert,

Paracellular non‐electrolyte permeation during fluid transport across rabbit gall‐bladder epithelium

“…Nonetheless, in order to assess this possibility quantitatively, the time constants of cellular efflux of 14C-labelled urea, ethanediol and thiourea have been determined in a series of experiments in which pieces of gall-bladder wall were loaded with the tracers for 1 or 2 hr prior to the efflux measurement. The calculated compartment volume for ethanediol and thiourea corresponds well with that expected from the stereological measurements of Blom & Helander (1977). Also, the ratio of the mean time constants for ethanediol and thiourea efflux is comparable with the inverse ratio of their olive oil: water partition coefficients, suggesting that these non-electrolytes have to cross a lipid barrier in order to leave the tissue.…”

“…If the transcellular component is to satisfy the differential equations for standinggradient osmotic flow with uniformly distributed salt transport (Diamond & Bossert, 1967) and yield a fluid isotonic with the mucosal Ringer solution to within 1 %, it can be shown (Hill & Hill, 1978a;Hill, A. E., personal communication) that the over-all velocity profile, including the paracellular component, is well approximated by 2Rlh(x) = pJv + (1-p) Jv 1 (68 ) + 4.58 5-52(X) +3-3 3()]. (A 16) Values for R and 1 have been taken as 0-465 ,tm (Blom & Helander, 1977) and 4000 cm cm-2 (Steward, 1979).…”

Intracellular chloride activities in rabbit gallbladder: Direct evidence for the role of the sodium-gradient in energizing “Uphill” chloride transport

“…This is particularly important and perhaps indispensable if (i) the epithelium is characterized by two or more distinct populations of cells and (ii) there is a wide variation in measurements made on a single tissue and among different tissues. Fortunately, rabbit gallbladder is comprised of a single layer of one cell type; histologically the only difference noted is that the columnar cells lining the crests of the mucosal folds are taller than those lining the valleys between folds (Kaye et al, 1966;Tormey & Diamond, 1967;Blom& Helander, 1977). Further, we and other investigators have noted relatively little scatter in t),, c within a given tissue and among different tissues.…”

Biochemical and morphological correlations in human gallbladder with reference to membrane permeability

“…This would lead to a hypertonic solution causing an osmosisdriven water absorption restoring an isotonic state within these intercellular spaces. This is compatible with stereologic analysis of electron micrographs showing dilated lateral intercellular spaces during maximal fluid absorption by gallbladder and spaces reduced in size when fluid transport is inhibited (Blom and Helander, 1977). However, calculations by Diamond (1978) cast doubt on the need for an osmotic gradient, suggesting instead that osmotic coupling is adequate for secre- tion of isotonic fluid throughout the intercellular space.…”