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Biomedical subjects

K R Spring

Publications and source records attributed to K R Spring.

At least 19 recordsLinked to original sources
PubMed1992

Regulation of apical membrane ion transport in Necturus gallbladder.

Na and Cl movement through the apical membrane of Necturus gallbladder epithelium was investigated using electrophysiological and light microscopic measurements. Changes in membrane potential difference, fractional resistance of the apical membrane, and transepithelial resistance caused by changes in apical bath Cl concentration revealed the presence of a Cl conductance in the apical membrane of control tissues that was apparently not present in the preparations studied by other investigators. This Cl conductance was blocked by bumetanide (10(-5) M) or by the inhibitor of adenosine 3',5'-cyclic monophosphate (cAMP) action, the Rp isomer of adenosine 3',5'-cyclic monophosphorothioate (Rp-cAMPS; 0.5 mM). Treatment of the tissues with Rp-cAMPS also eliminated bumetanide-sensitive cell swelling in the presence of ouabain and activated an amiloride-sensitive swelling, changes consistent with inhibition of NaCl cotransport and the activation of Na-H and Cl-HCO3 exchange. We conclude that the mode of NaCl entry into Necturus gallbladder epithelial cells is determined by the level of cAMP. When cAMP levels are high, entry occurs by NaCl cotransport; when cAMP levels are low, parallel exchange of Na-H and Cl-HCO3 predominates. These observations explain the previous disagreements about the mode of NaCl entry into Necturus gallbladder epithelial cells.

Animals↗
PubMed1991

Activation of osmolyte efflux from cultured renal papillary epithelial cells.

The rabbit renal papillary epithelial cell line PAP-HT25 accumulates sorbitol and other organic osmolytes when cultured in hypertonic media. When returned to isotonic media, PAP-HT25 cells swell because of water influx and then shrink to their normal volume because of rapid osmolyte and water efflux (volume regulatory decrease, VRD). Sorbitol efflux from PAP-HT25 cells during VRD was reduced to 18% of control by incubation of the cells with 100 microM eicosatetraynoic acid (ETYA), indicating that an enzyme that metabolizes arachidonic acid (AA) is a key component of the efflux process. Sorbitol efflux was unaffected by incubation with cyclooxygenase and lipoxygenase inhibitors but was reduced to 9% by incubation with 100 microM ketoconazole and to 37% by incubation with 100 microM SKF-525A, indicating that the cytochrome P-450 limb of the AA cascade is involved in the efflux process. The efflux of other organic osmolytes betaine and myoinositol, but not glycerolphosphorylcholine, was also inhibited by incubation with ETYA and ketoconazole.

Animals↗
PubMed1991

Flow rate measurements in isolated perfused kidney tubules by fluorescence photobleaching recovery.

We have developed a new application of the fluorescence photobleaching recovery (FPR) technique for instantaneous measurement of volume flow rates at any axial position along isolated perfused kidney tubules. The method requires fast data acquisition of emitted fluorescence through a photomultiplier (time resolution, 0.5 ms) coupled with differential interference contrast microscopy to measure luminal diameters accurately. While the tubule is perfused in vitro with an impermeant fluorophore (fluorescein sulfonate), a 20-ms bleach pulse reduces the fluorescence in the observation region by 20-25%. Fluorescence recovery is a direct function of perfusate velocity; diffusion plays no significant role in the early phase of recovery. A fluid dynamics approach to data analysis shows that fractional recovery increases linearly with time until t = L/2vm, where L is the length of the observation window and vm is the mean axial velocity. Practically, a linear regression analysis of the early recovery phase allows measurement of vm of up to 0.14 cm/s, i.e., a 40-nl/min flow rate in a 25-microns-diameter tubule. Calibration experiments in small glass tubes perfused at predetermined flow rates demonstrated good accuracy (within 10%) and reproducibility (coefficient of variation, 8.7%). In rat inner medullary collecting ducts microperfused at 4-40 nl/min, the correlation with a standard fluid collection method was excellent (r2 greater than 0.97). The method should also be suitable for the direct measurement of fluid flow rate in kidney tubules or blood vessels microperfused in vivo.

Animals↗
PubMed1991

Sorbitol permease: an apical membrane transporter in cultured renal papillary epithelial cells.

The efflux of sorbitol from the rabbit papillary epithelial cell line PAP-HT25 occurs through a specific transport pathway, which we denote the "sorbitol permease." The permease was studied by measuring cell volume changes that accompanied osmotic swelling and by determination of the sorbitol efflux from plasma membrane vesicles. The cell volume studies showed that sorbitol efflux in response to hypotonicity occurred only across the apical membrane of the cells and that loss of sorbitol was the primary mechanism for regulatory volume decrease (RVD) by these cells. Quinidine, a permeant inhibitor of the sorbitol permease, was shown to prevent RVD when added to either apical or basolateral bathing solution. Cell volume experiments also showed that the permease was present only on the apical membrane of cells that had been grown in isotonic medium and did not accumulate sorbitol. The permease could be demonstrated in membrane vesicles obtained from cells exposed to a hypotonic environment before being homogenized. Quinidine blocked the sorbitol efflux from vesicles indicating that it either directly inhibited the permease or a membrane-associated activation step.

Animals↗
PubMed1991

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.

Animals↗
PubMed1991

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.

Microscopy, Fluorescence↗
PubMed1990

Identification of riboflavin transport by MDCK cells using quantitative fluorescence video microscopy.

MDCK cells, when examined by low-light level video microscopy displayed an endogenous fluorescence with two differing patterns. A low intensity emission which was punctate and associated with cell organelles was observed with emission and excitation conditions generally used to observe either fluorescein (450-500 nm excitation/greater than 510 nm emission) or rhodamine (514 nm excitation/greater than 530 emission) type dyes. A second 5- to 10-fold brighter emission for 450-500 nm excitation was observed, which was unusual in that each cell appeared to be outlined. Evidence obtained from spectroscopy and from using culture media of altered composition supported the conclusion that the water-soluble vitamin riboflavin accumulated in the basolateral spaces and fluid-filled "domes" and was the source of this fluorescent emission. Quantitative measurements showed that exposure to cultures to 10 microM riboflavin resulted in accumulation in domes of 565 +/- 80 microM. The transport rate was calculated to be 189 +/- 30 pmol/min-cm2. One mM probenecid, a known inhibitor of riboflavin transport in vivo, reduced transport to 54% of control, while 10 mM nearly abolished the uptake. The results demonstrate that removal of riboflavin reduces MDCK cell fluorescence to levels compatible with low-light level imaging. Furthermore, these cells actively transport riboflavin and provide a new in vitro model for this process.

Animals↗
PubMed1990

Effects of serosal hypertonicity on water permeability in toad urinary bladder.

We studied in toad urinary bladder the effects of serosal hypertonicity on tissue water permeability, granular cell luminal membrane water permeability, and granular cell luminal membrane particle aggregates and compared them with effects of antidiuretic hormone (ADH). In tissues challenged by a hypertonic (447 mosmol/kgH2O) serosal bath, luminal membrane aggregates were structurally similar to those caused by ADH. The tissue water permeability increase induced by serosal hypertonicity was much less than that caused by a maximally stimulating concentration of ADH on tissue in isotonic serosal baths with approximately the same transmural gradient. The difference is explained not only by a reduced incidence of luminal membrane aggregates but also by an increased resistance to water movement at a postluminal membrane site. Measurements of luminal membrane water permeability showed a close correlation with luminal membrane aggregate frequency, indicating that the calculated permeability of an individual aggregate was a constant. Thus the relation of luminal membrane aggregates to tissue osmotic permeability is modified by serosal hypertonicity. Morphological examination of these tissues suggested that luminal membrane aggregates may be less stable in the absence of hormone. This was evident by the proportionally greater number of structures interpreted as aggregates captured in the process of disassembly ("patches"). Membrane depressions containing intramembrane particles ("craters") were also observed. They corresponded in terms of frequency and size to coated pits as seen in thin sections.

Animals↗
PubMed1990

Mechanisms underlying volume regulatory decrease by Necturus gallbladder epithelium.

Volume regulatory decrease (VRD) by Necturus gallbladder epithelial cells in Cl Ringer was unaffected by the addition of 5 mM BaCl2 to apical perfusates but was inhibited by the addition of 5 mM BaCl2 and 50 or 3 microM phencyclidine (PCP) to serosal perfusates, suggesting that K channels in the basolateral membrane were activated during VRD. VRD was unaffected by replacement of Cl with NO3 or SCN, suggesting that Cl-dependent Na-K-Cl and K-Cl cotransport were not involved. In SCN Ringer, VRD was inhibited by the addition of 0.1 mM bumetanide to serosal perfusates, suggesting that bumetanide-sensitive anion channels in the basolateral membrane were also activated. A transient 10-mV hyperpolarization of the membrane potential was associated with VRD. The channel blockers that inhibited VRD had little or no effect on the hyperpolarization, suggesting that the changes in membrane potential were unrelated to the changes in cell volume. Perfusion of the apical surface of the epithelium with isotonic solutions containing 10 mM D-glucose resulted in a variable increase in cell volume followed by a variable shrinkage to normal, suggesting that VRD was also activated during organic solute absorption. The increase in cell volume was blocked by the addition of 0.01 or 1 mM phlorizin to mucosal perfusates. The reduction in cell volume was inhibited by the addition of 0.1 mM bumetanide, but not BaCl2 or PCP, to serosal perfusates, indicating the the shrinkage mechanism secondary to glucose addition differed from that seen after exposure to hypotonic perfusates.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1990

Net acid transport by isolated perfused inner medullary collecting ducts.

The isolated perfused tubule technique was used to study net acid transport in rat terminal inner medullary collecting duct (IMCD) segments. The stop-flow luminal pH [measured fluorometrically with the acidic form of the pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein in the lumen] fell 0.35 units below the bath pH in tubules from control rats and 0.53 units below the bath in tubules from deoxycorticosterone-treated rats. Tubules from control rats absorbed bicarbonate and secreted ammonium against concentration gradients, although at low rates. In control rats, 10(-8) M vasopressin added to the bath increased bicarbonate absorption almost threefold. Treatment of rats in vivo with deoxycorticosterone significantly increased the rate of bicarbonate absorption in vitro. In vivo NH4Cl loading also significantly increased bicarbonate absorption. Staining microdissected tubules with acridine orange confirmed that the perfused segments lacked intercalated cells. We conclude that the terminal IMCD spontaneously acidifies the lumen despite an absence of intercalated cells. Bicarbonate absorption appears to be regulated by the same factors that affect net acidification in other collecting duct segments.

Absorption↗
PubMed1990

Water permeability of apical and basolateral cell membranes of rat inner medullary collecting duct.

To quantify the pathways for water permeation through the kidney medulla, knowledge of the water permeability (Posmol) of individual cell membranes in inner medullary collecting duct (IMCD) is required. Therefore IMCD segments from the inner two thirds of inner medulla of Sprague-Dawley rats were perfused in vitro using a setup devised for rapid bath and luminal fluid exchanges (half time, t1/2, of 55 and 41 ms). Differential interference contrast microscopy, coupled to video recording, was used to measure volume and approximate surface areas of single cells. Volume and volume-to-surface area ratio of IMCD cells were strongly correlated with their position along the inner medullary axis. Transmembrane water flow (Jv) was measured in response to a variety of osmotic gradients (delta II) presented on either basolateral or luminal side of the cells. The linear relation between Jv and delta II yielded the cell membrane Posmol, which was then corrected for membrane infoldings. Basolateral membrane Posmol was 126 +/- 3 microns/s. Apical membrane Posmol rose from a basal value of 26 +/- 3 microns/s to 99 +/- 5 microns/s in presence of antidiuretic hormone (ADH). Because of amplification of basolateral membrane, the ADH-stimulated apical membrane remained rate-limiting for transcellular osmotic water flow, and the IMCD cell did not swell significantly. Calculated transcellular Posmol, expressed in terms of smooth luminal surface, was 64 microns/s without ADH and 207 microns/s with ADH. IMCD cells in anisosmotic media displayed almost complete volume regulatory decrease but only partial volume regulatory increase.

Animals↗
PubMed1989

A novel sorbitol transport mechanism in cultured renal papillary epithelial cells.

The renal papillary epithelial cell line, GRB-PAP1, accumulates sorbitol when grown in a hypertonic (500 mosmol/kgH2O) bathing medium. When the cells are returned to a 300 mosmol/kgH2O medium, they lose their sorbitol rapidly to the bath. Sorbitol movement across the membranes of these cells was investigated by studying the uptake of radioactive sorbitol and related compounds. Sorbitol uptake increased 71-fold when cells grown in 500 mosmol/kgH2O medium were exposed to a 300 mosmol/kgH2O test solution. The magnitude of the permeability increase was proportional to the size of the change in the osmolality of the bathing medium and not the absolute osmolality. Sorbitol uptake was a linear function of medium sorbitol concentration with no sign of saturation at sorbitol concentrations up to 315 mM. Although the permeability of other polyols was increased when the osmolality was reduced, competition between sorbitol and related sugars and polyols could not be demonstrated. Both the increased sorbitol uptake after a decrease in medium osmolality and the decrease to control permeability after return to the original osmolality were complete within 30 s. A wide variety of transport inhibitors and ion substitutions failed to alter the magnitude of the sorbitol permeability increase. The most effective inhibitor was quinidine, 1 mM reducing sorbitol uptake by 73%. The sorbitol permeability increase could also be blocked by reducing the temperature to 0 degrees C. Nonspecific uptake of sorbitol, such as endocytosis, was shown to be of only minor significance. The large increase in sorbitol permeability and subsequent sorbitol efflux enables these cells to withstand large decreases in osmolality without excessive swelling and consequent damage. A similar compensatory mechanism may operate in vivo in the renal papilla during the onset of diuresis.

Animals↗
PubMed1988

Solute transport and epithelial cell volume regulation.

1. The regulation of epithelial cell volume is an essential requirement for normal tissue function and the maintenance of cellular integrity. 2. Renal papillary epithelial cells utilize an organic to compensate for the shrinkage associated with exposure to hypertonic solutions. 3. These cells synthesize the polyol, sorbitol, to increase their intracellular solute content. 4. Sorbitol is synthesized from glucose by the enzyme aldose reductase; exposure of the cells to hypertonic media causes aldose reductase synthesis and subsequent sorbitol generation over a two or three day period. 5. The intracellular signal for the formation of aldose reductase is not yet identified.

Aldehyde Reductase↗
PubMed1988

Secretion of endolymph by semicircular canals of the shark.

The semicircular canals of the vestibular labyrinth of the dogfish shark, Squalus acanthias, may serve as a simple in vitro system for the study of the ionic transport mechanisms involved in endolymph formation. Electron microscopy showed that the epithelium was made up of at least three distinct cell types divided into separate regions running the length of the canals. Secretion of endolymph was studied in isolated canals by the split droplet method; when the lumens of the canals were filled with shark Ringer a potassium-rich fluid was secreted into the lumen at a rate of 0.34 microliter.cm-2.min-1. The K concentration of the secreted fluid averaged 112 mM; the calculated rate of K secretion was 2.3 mumol.cm-2.h-1, comparable to recent measurements in mammalian utricle. Fluid secretion was dependent on active transport and was inhibited by ouabain, bumetanide, or methazolamide in the external bathing solution. Fluid secretion was unaffected by the K channel blocker, Ba, in the luminal droplet or by the adenylate cyclase stimulator, forskolin, in the external bathing solution. For electrophysiological analysis, isolated canals were perfused in a chamber designed for voltage/current clamping; an axial wire was inserted into the canal lumen and constant-current pulses were passed to determine tissue resistance. When the luminal fluid was high-K Ringer, transepithelial potential difference was -1.1 mV (lumen negative) and resistance 37 omega.cm2. Dilution and bi-ionic potential measurements showed that the epithelium of the canals exhibited only slight cation selectivity. These results are consistent with a model for endolymph secretion involving cotransport secondary to the ion gradients created by the Na+-K+-ATPase.

Animals↗
PubMed1987

Cell membrane water permeability of rabbit cortical collecting duct.

The water permeability (Posm) of the cell membranes of isolated perfused rabbit cortical collecting ducts was measured by quantitative light microscopy. Water permeability of the basolateral membrane, corrected for surface area, was 66 microns X sec-1 for principal cells and 62.3 microns X sec-1 for intercalated cells. Apical membrane Posm values corrected for surface area, were 19.2 and 25 microns X sec-1 for principal and intercalated cells, respectively, in the absence of antidiuretic hormone (ADH). Principal and intercalated cells both responded to ADH by increasing Posm of their apical membranes to 92.2 and 86.2 microns X sec-1, respectively. The ratio of the total basolateral cell membrane osmotic water permeability to that of the apical cell membrane was approximately 27:1 in the absence of ADH and approximately 7:1 in the presence of the hormone for both cell types. This asymmetry in water permeability is most likely due to the fact that basolateral membrane surface area is at least 7 to 8 times greater than that of the apical membrane. Both cell types exhibited volume regulatory decrease when exposed to dilute serosal bathing solutions. Upon exposure to a hyperosmotic serosal bath (390 mosM), principal cells did not volume regulate while two physiologically distinct groups of intercalated cells were observed. One group of intercalated cells failed to volume regulate; the second group showed almost complete volume regulatory increase behavior.

Animals↗
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↗