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PubMed1985

Neuropeptides in the gastrointestinal canal of Necturus maculosus. Distribution and effects on motility.

The presence and distribution of regulatory peptides in nerves and endocrine cells of the stomach, intestine and rectum of a urodele amphibian, the mudpuppy, Necturus maculosus, was studied immunohistochemically in sections or whole-mount preparations of the gut wall. The effect of the occurring peptides on gut motility was studied in isolated strip preparations of circular and longitudinal smooth muscle from different parts of the gut. Bombesin-, neurotensin-, substance P- and VIP-like immunoreactivity was present in abundant nerve fibres in the myenteric plexus of both stomach, intestine and rectum. Single fibres or bundles were present in the circular muscle layer and in a well-developed deep muscular plexus in the intestine and rectum. Immunoreactive nerve cells were found in the myenteric plexus of the stomach, intestine (neurotensin only) and rectum. Gastrin/CCK-like immunoreactivity was observed only in a few fibres in stomach and rectum. Endocrine cells containing bombesin-, met-enkephalin-, gastrin/CCK-, neurotensin-, somatostatin- or substance P- like immunoreactivity were present in the mucosa. The effect of bombesin was an inhibition of the rhythmic activity in circular muscle preparations and in longitudinal muscle from the rectum, while longitudinal muscle from the stomach usually responded with a weak increase in tonus. Neurotensin, like-bombesin, was inhibitory on the spontaneous rhythmic activity of circular muscle throughout the gut, while the effect on longitudinal muscle was an increase in tonus. Met-enkephalin and substance P increased the tonus of all types of preparations, and often, in addition, initiated a rhythmic activity superimposed on this maintained tonus. VIP had a general inhibitory effect on the preparations, decreasing tonus and/or abolishing rhythmic activity. It is concluded that bombesin-, neurotensin-, substance P- and VIP-like peptides are present in nerves throughout the urodele gut and may have physiological functions in regulating the motility of the gut. The gastrin/CCK-like peptide present in nerves of the stomach and rectum may affect the function of these parts of the gut. The regulatory peptides present in endocrine cells may, perhaps with the exception of the somatostatin-like peptide, affect the motility humorally.

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PubMed1996

Serotonergic modulation of the mudpuppy (Necturus maculatus) locomotor pattern in vitro.

The aims of the present study were to: (1) study the role of serotonin (5-HT) in modulating the central pattern generator (CPG) underlying locomotion in the mudpuppy (Necturus maculatus); (2) investigate whether there is an intrinsic spinal serotonergic system. These aims were achieved by the use of pharmacological and immunocytochemical methods. To study modulation of the locomotor pattern and rhythm, we applied 5-HT, its uptake blocker zimelidine, and a variety of 5-HT receptor agonists and antagonists to an in vitro brainstem-spinal cord preparation isolated from the mudpuppy. The preparation consisted of the first five segments of the spinal cord and the right forelimb attached by the brachial plexus. The spinal CPG for locomotion was activated chemically by adding NMDA to the superfusing solution. During locomotion, bipolar electromyographic (EMG) recordings were made unilaterally from flexor and extensor ulnae muscles. 5-HT on its own did not induce locomotion, but it did have a profound modulatory effect on NMDA-induced locomotion. 5-HT produced a dose-dependent increase in the overall cycle duration and enhanced the EMG burst duration. Use of zimelidine indicated that there is an endogenous release of 5-HT which modulated the locomotor rhythm. The endogenous release was antagonized by 5-HT1/5-HT2 receptor antagonist methiothepin. Immunocytochemical analysis, in which the entire spinal cord of the mudpuppy was used, revealed that there were more than one type of spinal serotonergic neuron. They were differentiated according to the cell diameter, shape, and arborization pattern of their processes. These neurons were located within the central gray matter ventrolateral to the central canal. Our results suggest that 5-HT plays an important role in modulating the locomotor CPG in the mudpuppy, by acting through a well-developed spinal serotonergic system. This is in contrast to what has been reported in higher vertebrates, where serotonergic innervation is derived from supraspinal structures.

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PubMed1993

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 approximately 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.

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PubMed1993

Necturus gallbladder epithelial cell volume regulation and inhibitors of arachidonic acid metabolism.

Inhibition of the metabolism of arachidonic acid by the epoxygenase (cytochrome P-450) pathway with the inhibitor ketoconazole results in excessive cell swelling upon exposure to hyposmolality instead of the rapid and complete regulatory volume decrease (RVD) normally observed. NaCl entry from bathing solutions to cell interior was shown to cause this swelling, with Na influx occurring across the basolateral membrane and electrically silent Cl influx across the apical membrane. Ion substitution experiments show that the KCl efflux mediating RVD was unimpaired by ketoconazole, but was overwhelmed by the NaCl influx. Measurements of transepithelial fluid flux, Cl concentration, osmolality and pH showed that gallbladders treated with ketoconazole transiently secreted fluid rather than the normal absorption. We conclude that inhibition of arachidonic acid metabolism does not directly affect RVD by Necturus gallbladder, but that blockade of the epoxygenase pathway can have a profound influence on NaCl entry into gallbladder epithelial cells.

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PubMed1981

Capacitive and inductive low frequency impedances of Necturus gallbladder epithelium.

The electrical impedance of Necturus gallbladder epithelium was analysed in the frequency range 0.24 Hz to 6,323 Hz. Under control conditions (NaCl-Ringer's on both sides), the impedance function yields a semicircle with depressed center. When serosal Na+ was replaced by K+, an inductive low frequency (LF) component appeared in the impedance locus. With KCl-Ringer on the mucosal side a second circular arc was observed at frequencies below 1 Hz. The resistive parts of the capacitive and inductive LF components increased after application of TAP+ to the mucosal side. Both LF features were abolished after application of 5 mM TEA+ to the mucosal medium as well as after acidification of the mucosal side. The LF components were depressed by addition of 5 mM Ba2+ to the mucosal solution. As TEA+ blocks apical K+ channels (Van Driessche and Gögelein 1978), it is concluded that the capacitive as well as the inductive LF components are related to transcellular K+ flow. With KCl-Ringer on the mucosal side, mucosa negative potentials increased the equivalent resistance and decreased the equivalent capacitance of the LF impedance. With serosal KCl-Ringer, negative potentials evoked a capacitive component which overlapped with the inductive component observed at open circuit conditions. Positive potentials, however, abolished the capacitive as well as the inductive LF component, elicited by mucosal or serosal KCl-Ringer, respectively. These results demonstrate that serosa to mucosa directed K+ flow causes an inductive LF feature and that mucosa negative potentials elicit a capacitive LF component.

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PubMed1985

Electrophysiological studies on lateral intercellular spaces of Necturus gallbladder epithelium.

The lateral intercellular spaces of Necturus gallbladder epithelium were punctured with double-barrelled ion selective microelectrodes in order to determine the ion concentrations of lateral space fluid and the contribution of the lateral spaces to transepithelial resistance. Neither under control conditions, nor after diluting the bathing fluids to increase the rate of volume absorption, nor during passage of direct current of 200 microA/cm2, were any reliable concentration differences observed between lateral space fluid and external bathing fluids. These observations suggest that water can follow salt transport without requiring osmotic concentration gradients of greater than 1 or 2 mosmol/l and indicate that recently observed high values of water permeability must still be considered as underestimates. After developing a test to recognize and exclude leaky punctures, the contribution of the lateral spaces to transepithelial resistance could be determined. It amounted to around 29%. This value agrees well with results from recent impedance measurements which were performed under control conditions in the same preparation.

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PubMed1982

Square wave pulse analysis of cellular and paracellular conductance pathways in Necturus gallbladder epithelium.

In search for a rapid and reliable method to identify and quantitatively determine cell membrane resistances and paracellular shunt resistances in epithelia we have developed appropriate techniques to measure transepithelial and intracellular potential transients in response to transepithelially applied square wave constant current pulses. Model considerations indicate that in a unilayered, homogeneous epithelium with open lateral spaces the transient potential response across each cell membrane should obey a single exponential function in case the tight junction resistance is high, as in a tight epithelium, whereas in a leaky epithelium it should consist of a superposition of two exponentials with equal sign at the membrane with the higher intrinsic time constant and of two exponentials of different sign (overshoot with recline) at the membrane with the lower intrinsic time constant. The latter predictions were experimentally verified in a study on Necturus gallbladder epithelium and equivalent circuit parameters for the cell membrane resistances and capacitances as well as for the resistance of the shunt path were calculated from the data by curve fitting procedures. The resistances of the apical and basal cell membrane and of the shunt path averaged 1220, 201 and 91 omega cm2 respectively while the apical and basal cell membrane capacitances were 8.0 and 26.3 micro F/cm2 respectively. The fact that the resistance values are 4-15 times lower than estimates derived previously from 2D-cable analysis relates to a better preservation of the transport function under the present incubation conditions as verified by a new series of cable analysis data. The capacitances agree well with estimates of the surface amplification of the cell membranes from electronmicrographs, thus confirming the validity of the interpretation of the observed voltage transients.

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PubMed1983

Reinvestigation of the transepithelial P.D. in the proximal tubule of Necturus kidney.

Published values of transepithelial potential differences (VTE) in the proximal tubule of Necturus vary from approximately 0 to approximately -15 mV. In view of this disparity, we reinvestigated VTE. Our measured VTE was on the average -1.0 mV in early convolutions and +0.6 mV in terminal proximal segments. In the course of this study, we considered five distinct causes of artifacts. 1) Tip and pre-tip potentials: their occurrence was minimized by using Ringer's filled microelectrodes. 2) Interstitial tip localizations: the position of the tip was ascertained by the shift in potential, resulting in response to peritubular perfusion with gluconate solutions. 3) Leaky impalements: VTE responses to gluconate, input resistance determinations and the presence of positive VTE's rule out the leak hypothesis. 4) Zeroline shifts between pre- and postimpalement stages, and 5) Spontaneous VTE drifts in the positive direction, due to gradual passage of the tip from cell to lumen, or in the negative direction, resulting from tip contact with the lower cellular layer. All five causes of artifacts may be involved in the controversy regarding past VTE estimates.

Animals↗

PubMed1982

Relations between intracellular ion activities and extracellular osmolarity in Necturus gallbladder epithelium.

The interactions between ion and water fluxes have an important bearing on osmoregulation and transepithelial water transport in epithelial cells. Some of these interactions were investigated using ion-selective microelectrodes in the Necturus gallbladder. The intracellular activities of K+ and Cl- in epithelial cells change when the epithelium is adapted to transport in solutions of a low osmolarity. In order to achieve new steady states at low osmolarities, cells lost K+, Cl- and some unidentified anions. Surprisingly, the apparent K+ concentration remained high: at an external osmolarity of 64 mOsm the intracellular K+ concentration averaged 95 mM. This imbalance was sensitive to anoxia and ouabain. The effects of abrupt changes in the external osmolarities on the intracellular activities of Na+, K+ and Cl- were also investigated. The gradients were effectuated by mannitol. The initial relative rates of change of the intracellular activities of Na+ and Cl- were equal. The data were consistent with Na+ and Cl- ions initially remaining inside the cell and a cell membrane Lp of 10(-3) cm sec-1 osm-1, which is close to the values determined by Spring and co-workers (K.R. Spring, A. Hope & B.E. Persson, 1981. In: Water Transport Across Epithelia. Alfred Benzon Symposium 15. pp. 190-200. Munskgaard, Copenhagen). The initial rate of change of the intracellular activity of K+ was only 0.1-0.2 times the change observed in Na+ and Cl- activities, and suggests that K+ ions leave the cell during the osmotically induced H2O efflux and enter with an induced H2O influx. The coupling is between 98 and 102 mmoles liter-1. Various explanations for the anomalous behavior of intracellular K+ ions are considered. A discussion of the apparent coupling between K+ and H2O, observed in nonsteady states, and its effects on the distribution of K+ and H2O across the cell membrane in the steady states, is presented.

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PubMed1984

Volume regulation by Necturus gallbladder: basolateral KCl exit.

Swelling of the epithelial cells of Necturus gallbladder caused by an 18% reduction in the osmolality of the mucosal bath is followed by rapid volume readjustment. This volume regulatory decrease requires Cl and is sensitive to the K and Cl gradients across the basolateral cell membrane. Volume regulatory decrease is not inhibited by amiloride, SITS, ouabain or bicarbonate removal. The process is blocked by bumetanide in the serosal bath. Measurement of the intracellular activities of K and Cl and the rate of volume regulation under five different experimental conditions showed that KCl exited from the cell across the basolateral membrane with a stoichiometry of 3 K to 2 Cl. This KCl exit process appears to be transiently activated following the reduction in osmolality of the mucosal perfusate.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗

PubMed1987

Regulation of K+ channels in the basolateral membrane of Necturus oxyntic cells.

Patch-clamp methods were used to study single-channel events in isolated oxyntic cells and gastric glands from Necturus maculosa. Cell-attached, excised inside-out and outside-out patches from the basolateral membrane frequently contained channels which had conductances of 67 +/- 21 pS in 24% of the patches and channels of smaller conductance, 33 +/- 6 pS in 56% of the patches. Channels in both classes were highly selective for K+ over Na+ and Cl-, and shared linear current-voltage relations. The 67-pS channel was activated by membrane depolarization, whereas the activity of the 33-pS channel was relatively voltage independent. The larger conductance channels were activated by intracellular Ca2+ in the range between 5 and 500 nM, but unaffected by cAMP. The smaller conductance channels were activated by cAMP, but not Ca2+. The presence of K+ channels in the basolateral membrane which are regulated by these known "second messengers" can account for the increase in conductance and the hyperpolarization of the membrane observed upon secretagogue stimulation.

Animals↗

PubMed1987

Protamine alters structure and conductance of Necturus gallbladder tight junctions without major electrical effects on the apical cell membrane.

Protamine is a naturally occurring basic protein (pI; 9.7 to 12.0). We have recently reported that protamine dissolved in the mucosal bath (2 to 20 microM), induces about a twofold increase in transepithelial resistance in Necturus gallbladder within 10 min. Conductance decreased concomitantly with cation selectivity. In this leaky epithelium, where greater than 90% of an applied current passes between cells, an increment in resistance of this magnitude suggests a paracellular action a priori. To confirm this, ionic conductance across the apical cell membrane was studied with microelectrodes. Protamine increased transepithelial resistance without changing apical cell membrane voltage or fractional membrane resistance. Variation in extracellular K concentration (6 to 50 mM) caused changes in apical membrane voltage not different from control. To determine if protamine-induced resistance changes were associated with structural alteration of tight junctions, gallbladders were fixed in situ at peak response and analyzed by freeze-fracture electron microscopy. According to a morphometrical analysis, the tight junctional intramembranous domain expands vertically due to incorporation of new strands (fibrils) into the main compact fibrillar meshwork. Since morphologic changes are complete within 10 min, strands are probably recycled into and out of the tight junctional membrane domain possibly by the cytoskeleton either from cytoplasmic vesicles or from intramembranous precursors. Regulation of tight junctional permeability by protamine and other perturbations may constitute a common mechanism by which leaky epithelia regulate transport, and protamine, in concentrations employed in this study, seems reasonably specific for the tight junction.

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PubMed1981

Chloride distribution in the proximal convoluted tubule of Necturus kidney.

To assess the mechanism(s) by which intraluminal chloride concentration is raised above equilibrium values, intracellular Cl- activity (alpha iCl) was studied in the proximal tubule of Necturus kidney. Paired measurements of cell membrane PD (VBL) and Cl-selective electrode PD (VBLCl) were performed in single tubules, during reversible shifts of peritubular or luminal fluid composition. Steady-state alpha iCl was estimated at 14.6 +/- 0.6 mmol/liter, a figure substantially higher than that predicted for passive distribution. To determine the site of the uphill Cl- transport into the cell, an inhibitor of anion transport (SITS) was added to the perfusion fluid. Introduction of SITS in peritubular perfusate decreased alpha iCl, whereas addition of the drug in luminal fluid slightly increased alpha iCl; both results are consistent with basolateral membrane uphill Cl- transport from interstitium to the cell. TMA+ for Na+ substitutions in either luminal or peritubular perfusate had no effect on alpha iCl. Removal of bicarbonate from peritubular fluid, at constant pH (a situation increasing HCO3- outflux), resulted in an increase of alpha iCl, presumably related to enhanced Cl- cell influx: we infer that Cl- is exchanged against HCO3- at the basolateral membrane. The following mechanism is suggested to account for the rise in luminal Cl- concentration above equilibrium values: intracellular CO2 hydration gives rise to cell HCO3- concentrations above equilibrium. The passive exit of HCO3- at the basolateral membrane energizes an uphill entry of Cl- into the cell. The resulting increase of alpha iCl, above equilibrium, generates downhill Cl- diffusion from cell to lumen. As a result, luminal Cl- concentration also increases.

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PubMed1983

Effects of acid base disturbances on basolateral membrane potential and intracellular potassium activity in the proximal tubule of Necturus.

The effects of extracellular acid-base disturbances on intracellular potential (Em) and potassium activity (aiK) in the early proximal tubule of Necturus were examined. Using conventional and double barreled potassium ion selective microelectrodes it was possible to measure both the transient and steady-state responses to various states of extracellular acidosis and alkalosis. The results show that (i) when extracellular [HCO-3] is varied at constant pCO2, Em and aiK decrease in acidosis and increase in alkalosis. The greatest sensitivity in Em is between pH 7.6 and 6.8 with apparent saturation above and below these extremes; (ii) decreased [HCO-3] at constant pH = 7.6 also causes a depolarization of Em and reduces aiK, suggesting a major effect of extracellular [HCO-3] on intracellular potential and aiK; (iii) rapid perfusions and transient delta Em analysis suggest a high basolateral conductance for K+ and HCO-3 and a low Cl- conductance; (iv) increasing extracellular [K+] decreases the response of both Em and aiK to reduced [HCO-3] at constant pCO2. The results of this study demonstrate the important role of extracellular pH and/or [HCO-3] on the maintenance of cellular K+ homeostasis.

Acid-Base Imbalance↗

PubMed1991

Diversity of K+ channels in the basolateral membrane of resting Necturus oxyntic cells.

Patch-clamp techniques have been applied to characterize the channels in the basolateral membrane of resting (cimetidine-treated, nonacid secreting) oxyntic cells isolated from the gastric mucosa of Necturus maculosa. In cell-attached patches with pipette solution containing 100 mM KCl, four major classes of K+ channels can be distinguished on the basis of their kinetic behavior and conductance: (1) 40% of the patches contained either voltage-independent (a) or hyperpolarization-activated (b), inward-rectifying channels with short mean open times (16 msec for a, and 8 msec for b). Some channels showed subconductance levels. The maximal inward conductance gmax was 31 +/- 5 pS (n = 13) and the reversal potential Erev was at Vp = -34 +/- 6 mV (n = 9). (2) 10% of the patches contained depolarization-activated and inward-rectifying channels with gmax = 40 +/- 18 pS (n = 3) and Erev was at Vp = -31 +/- 5 mV (n = 3). With hyperpolarization, the channels open in bursts with rapid flickerings within bursts. Addition of carbachol (1 mM) to the bath solution in cell-attached patches increased the open probability Po of these channels. (3) 10% of the patches contained voltage-independent inward-rectifying channels with gmax = 21 +/- 3 pS (n = 4) and Erev was at Vp = -24 +/- 9 mV (n = 4). These channels exhibited very high open probability (Po = 0.9) and long mean open time (1.6 sec) at the resting potential. (4) 20% of the patches contained voltage-independent channels with limiting inward conductance of 26 +/- 2 pS (n = 3) and Erev at Vp = -33 +/- 3 mV (n = 3). The channels opened in bursts consisting of sequential activation of multiple channels with very brief mean open times (10 msec). In addition, channels with conductances less than 6 pS were observed in 20% of the patches. In all nine experiments with K+ in the pipette solution replaced by Na+, unitary currents were outward, and inward currents were observed only for large hyperpolarizing potentials. This indicates that the channels are more selective for K+ over Na+ and Cl-. A variety of K+ channels contributes to the basolateral K+ conductance of resting oxyntic cells.

Animals↗

PubMed1988

Dependence of cell membrane conductances on bathing solution HCO3-/CO2 in Necturus gallbladder.

The effects of bathing solution HCO3-/CO2 concentrations on baseline cell membrane voltages and resistances were measured in Necturus gallbladder epithelium with conventional intracellular microelectrode techniques. Gallbladders were bathed in either low HCO3-/CO2 Ringer's solutions (2.4 mM HCO3-/air or 1 mM HEPES/air) or a high HCO3-/CO2 Ringer's (10 mM HCO3-/1% CO2). The principal finding of these studies was that the apical membrane fractional resistance (fRa) was higher in tissues bathed in the 10 mM HCO3-/CO2 Ringer's, averaging 0.87 +/- 0.06, whereas fRa averaged 0.63 +/- 0.07 and 0.48 +/- 0.08 in 2.4 mM HCO3- and 1 mM HEPES, respectively. Intraepithelial cable analysis was employed to obtain estimates of the individual apical (Ra) and basolateral membrane (Rb) resistances in tissues bathed in 10 mM HCO3-/1% CO2 Ringer's. Compared to previous resistance measurements obtained in tissues bathed in a low HCO3-/CO2 Ringer's, the higher value of fRa was found to be due to both an increase in Ra and a decrease in Rb. The higher values of fRa and lower values of Rb confirm the recent observations of others. To ascertain the pathways responsible for these effects, cell membrane voltages were measured during serosal solution K+ and Cl- substitutions. The results of these studies suggest that an electrodiffusive Cl- transport mechanism exists at the basolateral membrane of tissues bathed in a 10 mM HCO3-/1% CO2 Ringer's, which can explain in part the fall in Rb. The above observations are discussed in terms of a stimulatory effect of solution [HCO3-]/PCO2 on transepithelial fluid transport, which results in adaptive changes in the conductive properties of the apical and basolateral membranes.

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PubMed1983

Bumetanide inhibition of NaCl transport by Necturus gallbladder.

Salt transport by the Necturus gallbladder epithelium is the result of the coupled entry of NaCl into the cells across the apical membrane and the active transport of Na out of the cells across the basolateral membrane. The NaCl entry step was studied by measuring the rate of cell volume increase accompanying ouabain inhibition of the Na--K-ATPase in the basolateral membrane. When bumetanide, a diuretic analog of furosemide, was added to the mucosal bathing solution it reversibly blocked the entry of NaCl into the cells and abolished fluid transport. A dose-response relationship showed half-maximal inhibition of NaCl entry at a bumetanide concentration of 10(-9) M; complete inhibition of coupled NaCl movement occurred with as little as 10(-7) M bumetanide. Partial substitution of Na or Cl in the mucosal solution failed to demonstrate competition between bumetanide and either of the ions. The drug was also effective in blocking NaCl entry in the absence of ouabain; addition of the diuretic to the mucosal bathing solution resulted in prompt cell shrinkage and a decrease in intracellular NaCl. Cell volume decrease followed bumetanide addition to the mucosal bath because NaCl entry was blocked but active Na transport continued for several minutes until the intracellular Na transport pool was depleted.

Animals↗

PubMed1985

Water permeability of Necturus gallbladder epithelial cell membranes measured by nuclear magnetic resonance.

In order to assess the contribution of transcellular water flow to isosmotic fluid transport across Necturus gallbladder epithelium, we have measured the water permeability of the epithelial cell membranes using a nuclear magnetic resonance method. Spin-lattice (T1) relaxation of water protons in samples of gallbladder tissue where the extracellular fluid contained 10 to 20 mM Mn2+ showed two exponential components. The fraction of the total water population responsible for the slower of the two was 24 +/- 2%. Both the size of the slow component, and the fact that it disappeared when the epithelial layer was removed from the tissue, suggest that it was due to water efflux from the epithelial cells. The rate constant of efflux was estimated to be 15.6 +/- 1.0 sec-1 which would be consistent with a diffusive membrane water permeability Pd of 1.6 X 10(-3) cm sec-1 and an osmotic permeability Pos of between 0.3 X 10(-4) and 1.4 X 10(-4) cm sec-1 osmolar-1. Using these data and a modified version of the standing-gradient model, we have reassessed the adequacy of a fluid transport theory based purely on transcellular osmotic water flow. We find that the model accounts satisfactorily for near-isosmotic fluid transport by the unilateral gallbladder preparation, but a substantial serosal diffusion barrier has to be included in order to account for the transport of fluid against opposing osmotic gradients.

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