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K R Spring

Publications and source records attributed to K R Spring.

At least 37 records · Page 2Linked to original sources
PubMed1987

Ion transport by mitochondria-rich cells in toad skin.

The optical sectioning video imaging technique was used for measurements of the volume of mitochondria-rich (m.r.) cells of the isolated epithelium of toad skin. Under short-circuit conditions, cell volume decreased by about 14% in response to bilateral exposure to Cl-free (gluconate substitution) solutions, apical exposure to a sodium-free solution, or to amiloride. Serosal exposure to ouabain resulted in a large increase in volume, which could be prevented either by the simultaneous application of amiloride in the apical solution or by the exposure of the epithelium to bilateral Cl-free solutions. Unilateral exposure to a Cl-free solution did not prevent ouabain-induced cell swelling. It is concluded that m.r. cells have an amiloride-blockable Na conductance in the apical membrane, a ouabain-sensitive Na pump in the basolateral membrane, and a passive Cl permeability in both membranes. From the initial rate of ouabain-induced cell volume increase the active Na current carried by a single m.r. cell was estimated to be 9.9 +/- 1.3 pA. Voltage clamping of the preparation in the physiological range of potentials (0 to -100 mV, serosa grounded) resulted in a cell volume increase with a time course similar to that of the stimulation of the voltage-dependent Cl conductance. Volume increase and conductance activation were prevented by exposure of the tissue to a Cl-free apical solution. The steady-state volume of the m.r. cells increased with the clamping voltage, and at -100 mV the volume was about 1.15 times that under short-circuit conditions. The rate of volume increase during current passage was significantly decreased by lowering the serosal K concentration (Ki) to 0.5 mM, but was independent of whether Ki was 2.4, 5, or 10 mM. This indicates that the K conductance of the serosal membrane becomes rate limiting for the uptake of KCl when Ki is significantly lower than its physiological value. It is concluded that the voltage-activated Cl currents flow through the m.r. cells and that swelling is caused by an uptake of Cl ions from the apical bath and K ions from the serosal bath. Bilateral exposure of the tissue to hypo- or hypertonic bathing solutions changed cell volume without detectable changes in the Cl conductance. The volume response to external osmotic perturbations followed that of an osmometer with an osmotically inactive volume of 21%.(ABSTRACT TRUNCATED AT 400 WORDS)

Amiloride↗
PubMed1987

Illumination and detection systems for quantitative fluorescence microscopy.

Systems are described for the illumination and detection of light microscope images of fluorescence in living cells at low levels of excitation. The illumination system is based on the use of an acousto-optic modulator to control the wavelength and power of the excitation beam. The detection system utilizes an image intensifier coupled with a television camera to acquire images at extremely low intensities. Both the illumination and detection systems have superior performance compared to existing devices.

Animals↗
PubMed1986

Potassium induced changes in cell volume of gallbladder epithelium.

The mechanisms of transmembrane K and anion movements were investigated by measurement of the changes in cell volume, apical membrane potential difference, and intracellular K activity resulting from exposure of Necturus gallbladder to solutions with increased K concentration. Cell swelling occurred when the tissue was exposed bilaterally to 25 mmol/l K. This swelling was both Cl and HCO3 dependent, but was not blocked by DIDS or bumetanide. Unilateral tenfold increases in extracellular K concentration did not cause cell swelling; addition of 5 mmol/l Ba to the contralateral cell surface resulted in cell volume increases comparable to those seen with bilateral K increase. Complete blockage of K channels by Ba could be demonstrated electrophysiologically at normal extracellular K concentrations but not in the presence of increased K. Our results were consistent with the passive movement of K through Ba-sensitive channels in both cell membranes. We were unable to detect other mechanisms for transmembrane K movement. The cell swelling caused by exposure to 25 mmol/l K was not due to intracellular K accumulation and may be related to the effects of membrane depolarization on voltage sensitive anion transport processes.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1986

The volume of mitochondria-rich cells of frog skin epithelium.

The pathway for movement of chloride ions across frog skin is not well understood. Mitochondria-rich (MR) cells have been proposed as the route for chloride across the skin. To test this hypothesis we studied the MR cells of the skin of the frog, Rana pipiens, by quantitative light microscopic determination of cell volume. MR cell volume was influenced by changes in the chloride concentration or osmolality of the outside bathing solution. MR cells shrank about 23% when all chloride was removed from the outside (mucosal) bathing solution. MR cells were also shown to be responsive to changes in the osmolality of either the mucosal or serosal bath. Osmotically-induced swelling caused by dilution of the serosal bath resulted in volume regulatory decrease. These results are consistent with the hypothesis that MR cells constitute the pathway for chloride movement across frog skin.

Animals↗
PubMed1986

Na-K-Cl cotransport in apical membrane of rabbit renal papillary surface epithelium.

The renal papillary surface epithelium is exposed to pelvic urine on its apical surface and to inner medullary interstitium on its basolateral surface. To investigate transport in this epithelium, we dissected it free from the renal papilla of rabbits and mounted it in a chamber that allowed both sides to be bathed independently. Cell volume was measured at 25 degrees C utilizing computerized quantitative microscopy. Addition of ouabain (10(-4) M) to the basolateral solution induced a 20% volume increase. This volume increase was completely inhibited by the removal of apical bath NaCl, Na+, K+, or Cl- but not by the removal of urea. Bumetanide, down to 10(-9) M in the apical bath, completely inhibited the ouabain-induced swelling. Changes in apical bath osmolality, resulting from addition or removal of NaCl, caused cell volume changes that were greater than could be accounted for by osmotic water flow alone. This hyperresponse was blocked by bumetanide and was stimulated by vasopressin (10(-8) M). These observations are consistent with the presence of Na-K-ATPase in the basolateral membrane and a bumetanide-sensitive, vasopressin-responsive Na-K-Cl co-transporter in the apical membrane.

Animals↗
PubMed1985

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.

Animals↗
PubMed1985

Computer-based determination of size and shape in living cells.

Measurement of cell volume in living epithelial cells has become an important technique in studies of membrane transport processes that function in cell volume regulation. Planimetry of video images of optical sections enables the measurement of the cross sectional area of each section. Cell volume is calculated from the measured area of each section and the known focus displacements. In the past the measurement of cross section area has been done by manual positioning of a cursor superimposed on the video image. Each experiment generates approximately 200 images in which two or more cells may be analysed. We have developed a computer-based method that uses one image as a template, and allows automated area determination of successive images by template matching and digital image processing. This new method is comparable to the older method in speed and accuracy, but requires much less effort from the experimenter.

Animals↗
PubMed1985

Involvement of calcium and cytoskeleton in gallbladder epithelial cell volume regulation.

The importance of calcium and cellular cytoskeletal elements in the activation or control of volume regulation by epithelial cells was explored in Necturus gallbladder. Gallbladder cells have been previously shown to rapidly readjust their volumes to control size after osmotic perturbation of the mucosal bathing solution. Removal of calcium from the perfusates caused dramatic morphological changes that prevented assessment of the role of extracellular calcium in volume regulation. The regulatory volume increase (RVI) that follows shrinkage of the cell due to perfusion of a hypertonic mannitol solution is insensitive to agents that interfere with cell calcium- or calmodulin-mediated events (quinidine, trifluoperazine) and is not blocked by agents that cause changes in the cytoskeleton (colchicine, cytochalasin B). Osmotically induced cell swelling is followed by regulatory volume decrease (RVD), which is inhibited by agents that interfere with calcium-dependent processes (quinidine, trifluoperazine) and by the microfilament inhibitor, cytochalasin B. These results indicate that RVD depends on calcium, calmodulin, and an intact microfilament network, whereas RVI is independent of these factors.

Animals↗
PubMed1985

Polarity of volume-regulatory increase by Necturus gallbladder epithelium.

Necturus gallbladder epithelial cells respond to the presence of a hypertonic perfusate in either bathing solution by first shrinking due to osmotic water loss and then swelling back to their original volume (volume-regulatory increase). Previous investigations involving increases in the osmolality of the mucosal bath had suggested that volume-regulatory increase was due to the activation of ion exchangers in the apical cell membrane. In the present study the sidedness of the transport processes involved in volume-regulatory increase was investigated. The osmolality of the serosal bath was increased by 18% either in the absence of HCO3- or when an inhibitor of volume-regulatory increase, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), was added to the mucosal or serosal bath. Volume regulation was HCO3- dependent. DIDS was only effective in inhibiting volume regulation when it was added to mucosal bathing solution, suggesting that volume-regulatory increase depended on transport across the apical membrane. Volume-regulatory increase could also be activated by first swelling the cells in hypotonic solution and then returning the tissue to control Ringer solution. The volume-regulatory increase that occurred upon return to control Ringer was also shown to be sensitive to DIDS in the mucosal bath.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1985

Regulation of water permeability in toad urinary bladder at two barriers.

The effects of prostaglandin synthesis inhibition by naproxen were studied in toad bladder. Luminal membrane water permeability was evaluated both by the frequency of intramembranous particle aggregates in granular cell luminal membrane and by direct assessment of the rate of change of cell volume during perfusion of an anisosmotic solution. Total tissue water permeability was assessed by transbladder osmotic water flow. Inhibition of prostaglandin synthesis caused luminal membrane water permeability to increase much more than expected from tissue permeability measurements. The addition of a very low dose of antidiuretic hormone (ADH) (0.125 mU/ml) during prostaglandin synthesis inhibition increased luminal membrane water permeability to the same level as maximal stimulation with ADH, while tissue water permeability failed to increase proportionately. The results imply the presence of a regulatable barrier to water movement across toad bladder that is distal to the luminal membrane and subject to control by either prostaglandins or ADH.

Animals↗
PubMed1985

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.

Animals↗
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.

Animals↗
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.

Animals↗
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.

Animals↗
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.

Animals↗
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.

Animals↗