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K R Spring

Publications and source records attributed to K R Spring.

At least 55 records · Page 3Linked to original sources
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.

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PubMed1983

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.

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PubMed1982

Epithelial cell volume modulation and regulation.

Epithelial cell volume is a sensitive indicator of the balance between solute entry into the cell and solute exit. Solute accumulation in the cell leads to cell swelling because the water permeability of the cell membranes is high. Similarly, solute depletion leads to cell shrinkage. The rate of volume change under a variety of experimental conditions may be utilized to study the rate and direction of solute transport by an epithelial cell. The pathways of water movement across an epithelium may also be deduced from the changes in cellular volume. A technique for the measurement of the volume of living epithelial cells is described, and a number of experiments are discussed in which cell volume determination provided significant new information about the dynamic behavior of epithelia. The mechanism of volume regulation of epithelial cells exposed to anisotonic bathing solution is discussed and shown to involve the transient stimulation of normally dormant ion exchangers in the cell membrane.

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PubMed1982

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.

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PubMed1982

Coupled NaCl entry into Necturus gallbladder epithelial cells.

NaCl entry into Necturus maculosus gallbladder epithelial cells was studied by determination of the rate of fluid movement into the cell when the Na+-K+-ATPase was inhibited by 10(-4) M ouabain in the serosal bathing solution. The cell swelling was due to continuing entrance of NaCl into the cell across the apical membrane, which increased the solute content of the cell; the resultant rise in cell osmolality induced water flow and cell swelling. The rate of swelling was 4.3% of the cell volume per minute, equivalent to a volume flow across the apical membrane of 1.44 x 10(-6) cm/s, similar in magnitude to the normal rate of fluid absorption by the gallbladder. We determined the mechanism of NaCl entry by varying the ionic composition of the mucosal bath; when most of the mucosal Na+ or Cl- was replaced, cell volume did not increase during pump inhibition. The rate of NaCl entry was a saturable function of Na+ or Cl- in the mucosal bathing solution with K1/2 values of 26.6 mM for Na+ and 19.5 mM for Cl-. The mode of NaCl entry was probably not the parallel operation of Na+-H+ and Cl(-)-HCO-3 exchangers because of the lack of effect of bicarbonate removal or of the inhibitors amiloride and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid. NaCl entry was reversibly inhibited by bumetanide in the mucosal bathing solution. Transepithelial NaCl and water absorption is the result of the coupled, carrier-mediated movement of NaCl into the cell across the apical membrane and the active extrusion of Na+ by the Na+-K+-ATPase in the basolateral membrane.

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PubMed1982

Volume regulation by Necturus gallbladder: apical Na+-H+ and Cl(-)-HCO-3 exchange.

Necturus gallbladder epithelial cells exhibited volume regulatory swelling when exposed to a hypertonic mucosal bathing solution. The initial, osmotically induced shrinkage was followed by a rapid increase in cell volume back to the control value despite continuing hypertonicity of the mucosal perfusate. This volume regulatory increase occurred by osmotic water flow accompanying the transient cellular uptake of NaCl from the mucosal bathing solution. Volume regulatory increase required Na+ and Cl- in the mucosal bath; it was inhibited by amiloride or 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid but not by bumetanide or ouabain. The K1/2 for Na+ was 2.8 mM, the K1/2 for Cl- was 1.9 mM, and maximum velocity of fluid flow into the cell for both ions was greater than 10 x 10(-6) cm/s. Both volume regulatory increase and transepithelial fluid absorption involve NaCl flux across the apical membrane into the cells, but the nature of the NaCl fluxes differ in the two processes. During volume regulatory increase NaCl enters the cells by parallel Na+-H+ and Cl(-)-HCO-3 exchanges, whereas during transepithelial fluid absorption NaCl enters the cell by the coupled flux of NaCl.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1981

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.

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PubMed1981

Chloride movement across the basolateral membrane of proximal tubule cells.

Electrophysiologic and tracer experiments have shown that Cl- enters Necturus proximal tubule cells from the tubule lumen by a process coupled to the flow of Na+, and that Cl- entry is electrically silent. The mechanism of Cl- exit from the cell across the basolateral membrane has not been directly studied. To evaluate the importance of the movement of Cl- ions across the basolateral membrane, the relative conductance of Cl- to K+ was determined by a new method. Single-barrel ion-selective microelectrodes were used to measure intracellular Cl- and K+ as a function of basolateral membrane PD as it varied normally from tubule to tubule. Basolateral membrane Cl- conductance was about 10% of K+ conductance by this method. A second approach was to voltage clamp the basolateral PD to 20 mV above and below the spontaneous PD, while sensing intracellular Cl- activity with the second barrel of a double-barrel microelectrode. An axial wire electrode in the tubule lumen was used to pass current across the tubular wall and thereby vary the basolateral membrane PD. Cell Cl- activity was virtually unaffected by the PD changes. We conclude that Cl- leaves Necturus proximal tubule cells by a neutral mechanism, possibly coupled to the efflux of Na+ or K+.

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PubMed1979

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.

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PubMed1979

Luminal Na+ entry into Necturus proximal tubule cells.

The dependence of intracellular Na+ activity on the electrical driving force across the luminal membrane and the presence of Cl- in the luminal perfusate was studied in Necturus proximal tubule. Intracellular Na+ and K+ activities were measured with microelectrodes filled with liquid ion exchanger. Perfusion of the tubule lumen with a NaCl-free solution caused cell Na+ activity to fall from the control value of 29.7 to 6.6 mM. In the absence of luminal driving force across the luminal membrane in accordance with simple diffusion of Na+ across this membrane. When the tubule lumen contained Na+ and Cl-, an electrically neutral component of Na+ entry into cells from the lumen appeared in addition to the diffusional component of Na+ entry.

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PubMed1979

Optical techniques for the evaluation of epithelial transport processes.

Light microscopic observation of living epithelial tissues is a powerful analytical tool in the investigation of solute and water transport. New techniques in quantitative microscopy permit the determination of epithelial cell size and shape as well as accurate measurements of lateral intercellular space dimensions. Traditional techniques for the study of epithelial cell function may be used in conjunction with continuous quantitation of tissue morphology. Fluorescent probes of membrane, cell, and interspace function may be monitored at low light levels to obtain information about the dynamic behavior of the preparation. Application of some of these methods to the Necturus gallbladder epithelium has enabled determination of lateral cell membrane compliance, the hydrostatic pressure equivalent to active transport, the intracellular NaCl transport pool, the rate of active NaCl transport, and the flux of NaCl into the cell across the apical membrane. Possible future applications of optical techniques include measurement of water permeability, solute reflection coefficients, fluid flow paterns, and interspace osmolality.

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PubMed1979

Intracellular ion activities in Necturus proximal tubule.

Ion-sensitive microelectrodes were used to measure the intracellular activities of Na, K, and Cl in proximal tubules of the perfused Necturus kidney. Cell Cl was 2-3 times higher than the value predicted for passive distribution during perfusion with normal Ringer; intracellular Na was far below the level for passive distribution. Cell Na and Cl fell to very low values when the lumen was NaCl-free. Cl entry into the tubule cell from the lumen required luminal Na. Na entered the cell across the luminal membrane both by diffusion and by coupled movement with Cl.

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PubMed1979

Dimensions of cells and lateral intercellular spaces in living Necturus gallbladder.

The size and shape of the cells and lateral intercellular spaces were measured in living Necturus gallbladder epithelium. Interspace volume was determined as a function of the transepithelial hydrostatic pressure difference. The compliance of the lateral membranes of the gallbladder cells was calculated from the interspace pressure-volume curves in both the presence and absence of fluid transport. Cell and interspace volume were studied when the NaCl in the mucosal bath was substituted by equiosmolar quantities of sucrose. The cells decreased in volume after the removal of mucosal NaCl and increased to control volume when the mucosal perfusate was 100 NaCl Ringer. The interspaces collapsed when fluid transport was inhibited by the removal of mucosal NaCl and reopened when NaCl was reintroduced to the mucosal bath. The rate of change of cell volume was used to calculate the active transport rate and the flux of NaCl across the apical membrane. The magnitude of the cell volume change during NaCl replacement indicated that all intracellular NaCl was readily accessible to be transported out of the cell.

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PubMed1978

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.

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PubMed1978

Transcellular and paracellular tracer chloride fluxes in Necturus proximal tubule.

Necturus proximal tubule lumen was filled with solutions of Na36Cl or [36Cl]tetramethylammonium ([36Cl]TMA) and the tracer disappearance was measured. With these tracers it was possible to differentiate between chloride fluxes across the cellular and the extracellular shunt pathways. Since it was previously shown that chloride does not enter tubule cells from the lumen unless Na is also present in the lumen, the [36Cl]TMA disappearance rate gave the shunt flux of chloride while the Na36Cl disappearance rate gave the sum of the transcellular and the shunt fluxes. The transcellular tracer chloride flux was unaffected by changes in the transepithelial potential difference, and the rate constant for the chloride flux from lumen to cell was identical to that previously reported for luminal sodium entry. These observations support the conclusion that a coupled transport of NaCl, in an electrically silent form, occurs across the luminal membrane of the Necturus proximal tubule cell. Shunt chloride flux was directly proportional to the electrical driving force, indicating diffusional chloride movement out of the lumen into the shunt pathway.

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PubMed1977

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.

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