Absence of significant cellular dilution during ADH-stimulated water reabsorption.

Water reabsorption across many "tight" urinary epithelia is driven by large transepithelial osmotic gradients and is controlled by antidiuretic hormone (ADH). Numerous investigators have concluded that ADH-induced water reabsorption causes large apparent increases in cell volume with concomitant cytoplasmic dilution. A central question in renal physiology has been how cellular homeostasis is maintained in tight urinary epithelia during antidiuresis. Previous direct measurements of cell membrane permeability to water and the present direct measurements of cell volume in collecting tubules of rabbit kidney cortex by quantitative light microscopy show that cell volume does not change significantly during transcellular water flow. Fluid transported across the epithelium accumulated in lateral and basal intercellular spaces; the effect was an increase in cell height and tubule wall thickness accompanied by maintenance of nearly constant cell volume. The stability of cell volume is a consequence of the relatively high water permeability of the blood-facing cell membrane.

Size and shape of the lateral intercellular spaces in a living epithelium.

The lateral intercellular spaces of Necturus gallbladder epithelium were seen and measured while the living tissue was perfused in a new chamber. The compliance of the lateral cell membranes was calculated from the measured pressure-volume characteristics of the lateral intercellular spaces.

Epithelial cell volume regulation: bicarbonate dependence.

When Necturus gallbladder epithelial cells are osmotically shrunken, they rapidly return to their original volume despite the continued presence of a hypertonic bathing solution. This volume-regulatory process requires bicarbonate ions in the bathing solutions and is associated with the uptake of chloride ions. Volume-regulatory increase by epithelial cells in probable due to the parallel operation of sodium-hydrogen and chloride-bicarbonate exchangers in the apical cell membrane.

Permeability properties of the subepithelial tissues of Necturus gallbladder.

The permeability properties of the subepithelial connective tissue of Necturus gallbladder were evaluated by measurement of electrical resistance, dilution potentials and hydraulic water permeability. The gallbladder epithelial cells were removed by scraping and the underlying connective tissue placed in an Ussing chamber. The electrical resistance was 2.2 +/- 0.8 omega X cm2; the tissue was slightly cation selective relative to free solution. The subepithelial tissues restricted the rate of diffusion of small solutes to 50% of the free solution value. The hydraulic water permeability averaged 2.1 X 10(-2) cm/s per atm. We conclude that limitations of the area of subepithelium available for fluid movement are the most important factors in determining the restrictions to solute and water flow offered by the subepithelial tissues.

Sodium flux in Necturus proximal tubule under voltage clamp.

Na transport and electrical properties of Necturus renal proximal tubules were analyzed, in vivo, by a voltage clamp method which utilizes an axial electrode in the tubule lumen for passage of current and simultaneous determination of net fluid (or Na) flux by the split droplet method. When the average spontaneous transepithelial potential difference of -8 mv (lumen negative) was reduced to zero by current passage, net Na flux doubled from a mean of 107 to 227 pmoles/cm(2) per sec. The relationship between flux and potential over the range -25 to +10 mv was nonlinear, with flux equilibrium at -15 mv and droplet expansion at more negative values. Calculated Na permeability at flux equilibrium was 7.0 x 10(-6) cm/sec. Voltage transients, similar to those caused by intraepithelial unstirred layers, were observed at the end of clamping periods. Tubular electrical resistance measured by brief square or triangle wave pulses (<100 msec) averaged 43 ohm cm(2). The epithelial current-voltage relationship was linear over the range -100 to +100 mv, but displayed marked hysteresis during low frequency (<0.04 Hz) triangle wave clamps. The low transepithelial resistance and large opposing unidirectional ion fluxes suggest that passive ionic movements occur across extracellular shunt pathways, while the voltage transients and current-voltage hysteresis are consistent with the development of a local osmotic gradient within epithelium.

Fluid transport and the dimensions of cells and interspaces of living Necturus gallbladder.

The volume of the cells and lateral intercellular spaces were measured in living Necturus gallbladder epithelium. Under control conditions, the volume of the lateral spaces was 9% of the cell volume. Replacement of mucosal NaCl by sucrose or tetramethylammonium chloride (TMACl) caused intercellular spaces to collapse. During mucosal NaCl replacement, cell volume decreased to 79% of its control value. When NaCl was reintroduced into the mucosal bath, the intercellular spaces reopened and the cells returned to control volume. The NaCl active transport rate, calculated from the rate of cell volume decrease, was 266 pM/cm2.s, close to the observed rate of transepithelial salt transport. It was calculated from the decrease in cell volume that all of the intracellular NaCl was transported out of the cell during removal of mucosal NaCl. The flux of salt across the apical membrane, calculated from the rate of cell volume increase upon reintroducing mucosal NaCl, was 209 pM/cm2.s, in good agreement with estimates by other methods. The electrical resistance of the tight junctions was estimated to be 83.9% of the total tissue resistance in control conditions, suggesting that the lateral intercellular spaces normally offer only a small resistance to electrolyte movement.

Kinetics of Na+ transport in Necturus proximal tubule.

The dependence of proximal tubular sodium and fluid readsorption on the Na(+) concentration of the luminal and peritubular fluid was studied in the perfused necturus kidney. Fluid droplets, separated by oil from the tubular contents and identical in composition to the vascular perfusate, were introduced into proximal tubules, reaspirated, and analyzed for Na(+) and [(14)C]mannitol. In addition, fluid transport was measured in short-circuited fluid samples by observing the rate of change in length of the split droplets in the tubular lumen. Both reabsorptive fluid and calculated Na fluxes were simple, storable functions of the perfusate Na(+) concentration (K(m) = 35-39 mM/liter, V(max) = 1.37 control value). Intracellular Na(+), determined by tissue analysis, and open-circuit transepithelial electrical potential differences were also saturable functions of extracellular Na(+). In contrast, net reabsorptive fluid and Na(+) fluxes were linearly dependent on intracellular Na(+) and showed no saturation, even at sharply elevated cellular sodium concentrations. These concentrations were achieved by addition of amphotericin B to the luminal perfusate, a maneuver which increased the rate of Na(+) entry into the tubule cells and caused a proportionate rise in net Na(+) flux. It is concluded that active peritubular sodium transport in proximal tubule cells of necturus is normally unsaturated and remains so even after amphotericin-induced enhancement of luminal Na(+) entry. Transepithelial movement of NaCl may be described by a model with a saturable luminal entry step of Na(+) or NaCl into the cell and a second, unsaturated active transport step of Na(+) across the peritubular cell boundary.

Gallbladder epithelial cell hydraulic water permeability and volume regulation.

The hydraulic water permeability (Lp) of the cell membranes of Necturus gallbladder epithelial cells was estimated from the rate of change of cell volume after a change in the osmolality of the bathing solution. Cell volume was calculated from computer reconstruction of light microscopic images of epithelial cells obtained by the "optical slice" technique. The tissue was mounted in a miniature Ussing chamber designed to achieve optimal optical properties, rapid bath exchange, and negligible unstirred layer thickness. The control solution contained only 80% of the normal NaCl concentration, the remainder of the osmolality was made up by mannitol, a condition that did not significantly decrease the fluid absorption rate in gallbladder sac preparations. The osmotic gradient ranged from 11.5 to 41 mosmol and was achieved by the addition or removal of mannitol from the perfusion solutions. The Lp of the apical membrane of the cell was 1.0 X 10(-3) cm/s . osmol (Posm = 0.055 cm/s) and that of the basolateral membrane was 2.2 X 10(-3) cm/s . osmol (Posm = 0.12 cm/s). These values were sufficiently high so that normal fluid absorption by Necturus gallbladder could be accomplished by a 2.4-mosmol solute gradient across the apical membrane and a 1.1-mosmol gradient across the basolateral membrane. After the initial cell shrinkage or swelling resulting from the anisotonic mucosal or serosal medium, cell volume returned rapidly toward the control value despite the fact that one bathing solution remained anisotonic. This volume regulatory response was not influenced by serosal ouabain or reduction of bath NaCl concentration to 10 mM. Complete removal of mucosal perfusate NaCl abolished volume regulation after cell shrinkage. Estimates were also made of the reflection coefficient for NaCl and urea at the apical cell membrane and of the velocity of water flow across the cytoplasm.

Illumination, wavelength selection, and detection in fluorescence microscopy.

The presently available devices for the illumination, changing of wavelengths, and detection of the resultant fluorescence of biological samples viewed in the light microscope have been described and compared. The optimal choice for illumination is a xenon arc lamp with a filter wheel wavelength selector. The optimal choice for an imaging detector is an intensified CCD (charge-coupled-device) camera. These combinations produce the most rapid, stable, and reproducible results when fluorescence measurements are made on living epithelial cells or isolated renal tubules. Techniques for the simultaneous acquisition of fluorescence and differential interference contrast (DIC) images have also been described and compared.

The study of epithelial function by quantitative light microscopy.

Quantitative light microscopy can be used to analyze the mechanisms of salt and water movement across epithelial cells. Methods for light microscopic visualization and image acquisition are reviewed. Video image recording and processing are shown to be essential for the study of epithelial cell function by light microscopy.

Fluid transport by gallbladder epithelium.

The absorption of fluid by epithelial tissues is thought to be due to the existence of hypertonic regions within the epithelium. The magnitude of the required hypertonicity as well as its localization have been the subject of considerable experimental and theoretical effort. Model calculations demonstrated the need for knowledge of the water permeability of the membranes of epithelial cells for the purpose of estimation of the osmotic gradients required for fluid absorption. We measured the hydraulic water permeability of the individual cell membranes of Necturus gallbladder by quantitative light microscopy. The water permeabilities were sufficiently high so that small osmotic gradients were required to achieve normal rates of fluid transport. The cell osmolality was calculated to exceed that of the mucosal bathing solution by about 2 mosmol kg-1, and the basolateral interstitial osmolality was calculated to be about 1 mosmol kg-1 greater than that of the cell. The fluid absorbed by the epithelium must be slightly hypertonic to the bathing solutions. Knowledge of the apical cell membrane water permeability and the relative area of the cell and tight junction allow a calculation of the relative flow of fluid across both pathways. It can be readily shown that osmotically induced flow across the epithelium occurs predominantly transcellularly because of the small area of the junctional pathway and the high water permeability of the cell membranes.

Determinants of epithelial cell volume.

Epithelial cell volume is determined by the concentration of intracellular, osmotically active solutes. The high water permeability of the cell membrane of most epithelia prevents the establishment of large osmotic gradients between the cell and the bathing solutions. Steady-state cell volume is determined by the relative rates of solute entry and exit across the cell membranes. Inhibition of solute exit leads to cell swelling because solute entry continues; inhibition of solute entry leads to cell shrinkage because solute exit continues. Cell volume is then a measure of the rate and direction of net solute movements. Epithelial cells are also capable of regulation of the rate of solute entry and exit to maintain intracellular composition. Feedback control of NaCl entry into Necturus gallbladder epithelial cells is demonstrable after inhibition of the Na,K-ATPase or reduction in the NaCl concentration of the serosal bath. Necturus gallbladder cells respond to a change in the osmolality of the perfusion solution by rapidly regulating their volume to control values. This regulatory behavior depends on the transient activation of quiescent transport systems. These transport systems are responsible for the rapid readjustments of cell volume that follow osmotic perturbation. These powerful transporters may also play a role in steady-state volume regulation as well as in the control of cell pH.

Detectors for fluorescence microscopy.

The low light levels originating from living cells viewed in the fluorescence microscope place significant limitations on the spatial and temporal resolution which can be achieved. The development of intensified video cameras has enabled the detection, visualization and measurement of these low level signals. The performance characteristics of popular intensified video cameras has enabled the detection, visualization and measurement of these low level signals. The performance characteristics of popular intensified video cameras are compared and guidelines are given for the selection of the appropriate detector for various experimental requirements. Intensified or cooled CCD cameras appear to be the most suitable device for quantitative imaging at low light levels in fluorescence microscopy.

Routes and mechanism of fluid transport by epithelia.

The mechanism of fluid transport by leaky epithelia and the route taken by the transported fluid are in dispute. A consideration of current mathematical models for coupling of solutes and water, as well as the methodologies for the study of fluid transport, shows that local osmosis best accounts for water movement. Although it seems virtually certain that the tight junctions are water permeable, the fraction of absorbed fluid that crosses the tight junction cannot yet be determined with confidence.