Fluid Transport Across Leaky Epithelia: Central Role of the Tight Junction and Supporting Role of Aquaporins

The mechanism of epithelial fluid transport remains unsolved, which is partly due to inherent experimental difficulties. However, a preparation with which our laboratory works, the corneal endothelium, is a simple leaky secretory epithelium in which we have made some experimental and theoretical headway. As we have reported, transendothelial fluid movements can be generated by electrical currents as long as there is tight junction integrity. The direction of the fluid movement can be reversed by current reversal or by changing junctional electrical charges by polylysine. Residual endothelial fluid transport persists even when no anions (hence no salt) are being transported by the tissue and is only eliminated when all local recirculating electrical currents are. Aquaporin (AQP) 1 is the only AQP present in these cells, and its deletion in AQP1 null mice significantly affects cell osmotic permeability (by ∼40%) but fluid transport much less (∼20%), which militates against the presence of sizable water movements across the cell. In contrast, AQP1 null mice cells have reduced regulatory volume decrease (only 60% of control), which suggests a possible involvement of AQP1 in either the function or the expression of volume-sensitive membrane channels/transporters. A mathematical model of corneal endothelium we have developed correctly predicts experimental results only when paracellular electro-osmosis is assumed rather than transcellular local osmosis. Our evidence therefore suggests that the fluid is transported across this layer via the paracellular route by a mechanism that we attribute to electro-osmotic coupling at the junctions. From our findings we have developed a novel paradigm for this preparation that includes 1) paracellular fluid flow; 2) a crucial role for the junctions; 3) hypotonicity of the primary secretion; and 4) an AQP role in regulation rather than as a significant water pathway. These elements are remarkably similar to those proposed by the laboratory of Adrian Hill for fluid transport across other leaky epithelia.

Stimulation of Oxygen Consumption with Fluid Absorption in Insect Recta

The oxygen consumption of excised abdomens of cockroaches and locusts has been measured before and after the injection of fluids into the ligated recta. Fluid injection caused a transient stimulation of oxygen consumption of up to 30% of the resting rate. The extra amount of oxygen consumed is positively correlated with the osmolality of the fluid injected and the amount of fluid absorbed. Parallel experiments were carried out on the time course of fluid uptake; these experiments revealed a correlation first between a rapid increase in fluid absorption and stimulation of oxygen consumption, and secondly between the final resting rate of oxygen consumption and a slower absorption of fluid. Locusts take up fluid at double the rate of cockroaches and have double the stimulation in oxygen consumption following fluid injection. In locusts the increases in oxygen consumption can also be correlated with the net movement of Na+, K+and Cl− from the rectum. The stimulation of oxygen consumption during fluid uptake is discussed in relation to the local osmosis model for fluid uptake.

Direct Microprobe Evidence of Local Concentration Gradients and Recycling of Electrolytes During Fluid Absorption in the Rectal Papillae of Calliphora

1. The concentrations of sodium, potassium and chloride and dry mass were measured by electron-probe X-ray micro-analysis in 1 μm thick frozen-hydrated sections from Calliphora rectum in 5 different states of absorptive function. 2. In all cases the average concentrations of sodium + potassium + chloride was consistently higher in the fluid in the lateral intercellular spaces than in the cytoplasm, the average ratio being 2:1 in water-fed flies and higher in water-deprived flies. 3. The highest concentration of electrolytes was found in the extracellular channel of complex lateral membrane stacks, which is consistent with the histochemical localization of major cation pumps at these sites (Berridge & Gupta, 1968). This concentration exceeded the electrolyte concentration in other tissue compartments by some 80 m-equiv/1 H2O in water-fed flies and about 700 m-equiv/1 H2O in water-deprived flies. The potassium and sodium concentration ratio of this extracellular fluid was nearly 1:1 in water-fed flies, 3:1 in water-deprived flies with KC1 in the rectal lumen, and 0·5:1 with NaCl in the rectal lumen. 4. Results suggest that the extracellular fluid is generated in membrane infoldings along the intercellular channels, and that this fluid gains water and sodium, but loses a variable amount of potassium and chloride, as it passes to the haemolymph, thus supporting the idea of local osmosis and ion recycling.

Coupling of Transmural Flows of NaCl and Water in the Intestine of the EEl (Anguilla Anguilla)

1. An in vivo perfusion of the intestine of the yellow European eel (Anguilla anguilla) was used to measure the net absorption of NaCl and water, the osmotic permeability coefficient, the solute-linked water flow, and the osmolality difference against which the intestine could transport water as functions of the salinity of the surrounding water. The eels were adapted to fresh water, to sea water, and to 1½ strength sea water. 2. The osmolality difference against which the intestine could transport water was observed to be linearly related to the net transmural flow of NaCl; the solute-linked water flow had a constant hypertonicity in spite of differing net flows of NaCl. The findings are in agreement with the hypothesis of uphill water movement being caused by local osmosis due to the salt flow and with a shunt leak proportional to the transmural osmotic difference. 3. An important part of adaptation to waters of higher salinity is a pronounced increase in the intestinal absorption of NaCl. 4. The osmotic permeability coefficient varied from experiment to experiment without relation to the state of adaptation. An explanation for this finding may be that the osmotic permeability of the intestinal epithelium is of little importance for the total intestinal transfer of water.

Salt Regulation in the Mangroves Rhizophora Mucronata Lam. And Aegialitis Annulata Rbr

The mangrove Rhizophora mucronata grows in an intertidal region and exchfdes salt from its xylem (17 m�equiv. chloride per litre of sap) more efficiently than does the salt� secreting mangrove AegialitiB annulata (85-122 m�equiv. chloride per litre of sap). From the transpiration stream each leaf of Rhizophora receives about 17 p.�equiv. chloride each day, but the chloride concentration of the growing leaf remains approximately constant (510-560 m�equiv. chloride per litre of sap water). In Aegialiti8 input of chloride to a mature leaf is about 100 p..equiv. per day and this input is balanced by secretion (mainly of sodium chloride) from the salt glands. Secretion collected under oil contains chloride, 450 p.-equiv/ml, sodium, 355 p.-equiv/ ml, and potassium, 27 p.-equiv/ml. Secretion rates from leaves on the tree, based on leaf area, vary from 93 p-equiv. cm-2 sec-1 during the day to 3 p-equiv. cm-2 sec-1 in darkness; the secretion in light, based on an effective gland area, is about 25,000 p-equiv. cm-2 sec-I. The water potential of the secretion is close to that in the leaf suggesting that secretion involves active transport of salt and passive movement of water by local osmosis.

The Mechanism of Isotonic Water Transport

The mechanism by which active solute transport causes water transport in isotonic proportions across epithelial membranes has been investigated. The principle of the experiments was to measure the osmolarity of the transported fluid when the osmolarity of the bathing solution was varied over an eightfold range by varying the NaCl concentration or by adding impermeant non-electrolytes. An in vitro preparation of rabbit gall bladder was suspended in moist oxygen without an outer bathing solution, and the pure transported fluid was collected as it dripped off the serosal surface. Under all conditions the transported fluid was found to approximate an NaCl solution isotonic to whatever bathing solution used. This finding means that the mechanism of isotonic water transport in the gall bladder is neither the double membrane effect nor co-diffusion but rather local osmosis. In other words, active NaCl transport maintains a locally high concentration of solute in some restricted space in the vicinity of the cell membrane, and water follows NaCl in response to this local osmotic gradient. An equation has been derived enabling one to calculate whether the passive water permeability of an organ is high enough to account for complete osmotic equilibration of actively transported solute. By application of this equation, water transport associated with active NaCl transport in the gall bladder cannot go through the channels for water flow under passive conditions, since these channels are grossly too impermeable. Furthermore, solute-linked water transport fails to produce the streaming potentials expected for water flow through these passive channels. Hence solute-linked water transport does not occur in the passive channels but instead involves special structures in the cell membrane, which remain to be identified.