The dogfish shark (Squalus acanthias) rectal gland (SRG) cell has served as a model experimental system for investigating the relationship between the actin cytoskeleton and cell volume regulation. Previous reports employing conventional fluorescence microscopy of tissue slices have shown that cells exposed to high external K+ and hypotonically-induced cell swelling displayed a fading of F-actin staining intensity, particularly at the basolateral cell borders. However, spectroscopic measurement of the F-actin present in similarly treated rectal gland slices failed to demonstrate a net change in F-actin amount.
View Article and Find Full Text PDFThe botanist Carl Nägeli is generally considered to have laid the basis for the cell membrane concept by his 1855 study of the osmotic properties of plant cells. It is shown here that William Hewson in 1773 presented cogent experimental evidence for the concept of a cell membrane in red blood corpuscles. Although his work was largely confirmed in subsequent studies, and a cell membrane became an attribute of the cell in T.
View Article and Find Full Text PDFMembrane vesicles of A549 lung cells accumulate choline by two pathways: the Na(+)-independent uphill uptake of choline [Michaelis-Menten constant (Km) approximately 44 microM; steady-state gradient approximately 45 at 5 microM external choline] is dependent on a transmembrane H+ gradient, is relatively insensitive to hemicholinium-3, is amiloride sensitive, and is abolished by valinomycin plus carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). The Na(+)-dependent active choline uptake (Km approximately 4 microM, inhibitor constant for hemicholinium-3 approximately 0.1 microM), is specific for Na+, is amiloride and FCCP sensitive, and is electrogenic: the overshoot using K(+)-loaded vesicles and NaCl gradient was increased by valinomycin.
View Article and Find Full Text PDFA549 cells, a lung epithelium-derived cell line, were used as a model system to study choline transport by granular pneumocytes. Intact cells accumulated free choline against a concentration gradient by a low-affinity transport system with kinetic characteristics similar to that previously described for granular pneumocytes (Am. J.
View Article and Find Full Text PDFBiochim Biophys Acta
October 1992
At least two types of glucose transporter exist in cultured renal epithelial cells, a Na(+)-glucose cotransporter (SGLT), capable of interacting with D-glucose but not 2-deoxy-D-glucose (2dglc) and a facilitated transporter (GLUT) capable of interacting with both D-glucose and 2dglc. In order to examine the polarity of transport in cultured renal epithelia, 2dglc and D-glucose uptakes were measured in confluent cultures of LLC-PK1 cells grown on collagen-coated filters that permitted access of medium to both sides of the monolayer. The rates of basolateral uptake of both 1 mM glucose (Km 3.
View Article and Find Full Text PDFAm J Respir Cell Mol Biol
April 1992
Isolated alveolar type II epithelial cells (granular pneumocytes) from rat lung accumulate free choline against a concentration gradient by an energy-dependent saturable transport process with apparent Km approximately 18 microM. In order to evaluate the structural requirements for choline transport by these cells, the inhibition of the initial rate of cellular uptake of [3H]choline (5 microM) by its analogue was measured. There was no significant inhibition of substrate uptake by analogues lacking an amino group while the presence of a quaternary nitrogen was most effective.
View Article and Find Full Text PDFHypotonic stress (reduction of external tonicity from approximately 900 mosM and 295 mM NaCl to approximately 600 mosM and 135 mM NaCl) produced a relatively slow regulatory volume decrease (RVD) in dogfish shark (Squalus acanthias) rectal gland cells. During the 5-h experiment, cell K+ content remained unchanged; cell content of Na+ and Cl- dropped in the initial swelling phase by some 50% (reflecting the corresponding reduction in medium NaCl), and then remained unchanged during volume recovery phase. Also, cellular fluxes of 86Rb+ and urea were not affected by hypotonic stress.
View Article and Find Full Text PDF(1) 0.1-1.0 mM p-chloromercuribenzene sulfonate (pCMBS) and some other organic mercurials produce a swelling of slices of dogfish shark (Squalus acanthias) rectal glands, with an uptake of cell Na+ and a loss of K+.
View Article and Find Full Text PDFThe uptake of [3H]choline was investigated using isolated perfused rat lungs and primary cultures of granular pneumocytes isolated by tryptic digestion of rat lungs. Metabolic products were separated from free choline by chloroform:methanol extraction and column chromatography. Tissue-associated [3H]choline increased progressively in the perfused lung, and estimated mean intracellular concentration at 2 h was 12 times the extracellular concentration (5 microM).
View Article and Find Full Text PDFWhen sheets of mucosa from the cecum of clinically normal horses were incubated in vitro with radiolabeled L-alanine, they could accumulate this amino acid against an apparent concentration gradient after 60 to 150 minutes of incubation. The active transport system for L-alanine was on the serosal surface of the mucosal sheet only. L-Alanine accumulation at 60 minutes was partly inhibited by 20 mM glycine (P less than 0.
View Article and Find Full Text PDFDogfish shark (Squalus acanthias) rectal gland cells swell massively when incubated in elasmobranch media in which Na+ was equivalently replaced by K+; this swelling was abolished when the impermeant gluconate replaced Cl-, while the cell depolarization was comparable in both media. The K+-effect was associated with (a) an increase of the steady-state 42K (and 86Rb) efflux (particularly of the rate constant of the fast cellular efflux component) and a rearrangement of the respective cellular pools of K+; (b) an alteration of cell morphology and the pattern of the F-actin staining along the basolateral cell membrane as revealed with fluorescent analogs of phallacidin. These changes were independent of cell volume, being identical in KCl and K-gluconate media.
View Article and Find Full Text PDFAdipocytes accumulate 2-deoxy-D-glucose (2-DG) against significant chemical gradients (J. Foley and J. Gliemann.
View Article and Find Full Text PDFSmall organic anions have been reported to induce cell solute accumulation and swelling. To investigate the mechanism of swelling, we utilized preparations of rectal gland cells from Squalus acanthias incubated in medium containing propionate. Propionate causes cells to swell by diffusing across membranes in its nonionic form, acidifying cell contents, and activating the Na+-H+ antiporter.
View Article and Find Full Text PDFThe uphill accumulation of free 2-deoxy-D-glucose and 2-deoxy-D-glucose 6-phosphate in rat adipocytes was found not to affect the steady-state distribution of 3-O-methyl-D-glucose although both hexoses share a common transport pathway. This observation argues against a possible effect of 2-deoxy-D-glucose phosphate on the equilibrating nature of the carrier. The results are discussed in light of hypotheses advanced to explain free 2-deoxy-D-glucose accumulation in a variety of cells.
View Article and Find Full Text PDFBiochim Biophys Acta
February 1989
The simple theory of a dynamic diffusion barrier is described and it is shown how this could account for the accumulation, in adipocytes, of those free sugars which are also phosphorylated. The standing concentration gradient established by this mechanism depends on the recycling of free sugar and sugar phosphate in submembrane structures which start in juxtaposition to conventional membrane hexose transporters. Although a continual expenditure of metabolic energy is involved, there can be a net gain from the potential-energy store of accumulated substrates.
View Article and Find Full Text PDFBiochim Biophys Acta
August 1988
Tissue slices of shark rectal gland are studied to examine the kinetics of the cellular fluxes of taurine, a major intracellular osmolyte in this organ. Maintenance of high steady-state cell taurine (50 mM) is achieved by a ouabain-sensitive active Na+-dependent uptake process and a relatively slow efflux. Uptake kinetics are described by two saturable taurine transport components (high-affinity, Km 60 microM; and low-affinity, Km 9 mM).
View Article and Find Full Text PDFBiochim Biophys Acta
April 1986
Glucose transport and metabolism, and the effect of insulin thereon, was studied using suspensions of rat renal tubules enriched in the proximal component. [U-14C]Glucose oxidation is a saturable process (Km 3.1 +/- 0.
View Article and Find Full Text PDFThe characteristics of methyl alpha-D-glucoside transport by the LLC-PK1 cell line are extended by a study of the interaction of this glucose analog with the basal-lateral membrane of these cells: 1 mM methyl alpha-D-glucoside enters LLC-PK1 cells across the basal-lateral membrane 10-times more slowly than when entering across the apical membrane; neither 10 mM glucose nor 10 mM methyl alpha-D-glucoside affect the rate of methyl glucoside uptake at the basal lateral membrane; 0.1 mM phlorizin in the apical hemichamber significantly decreases the rate at which methyl glucoside enter the cell when methyl glucoside is present in the basal-lateral hemichamber; the methyl glucoside transcellular flux ratio, Ja/Jb (apical to basal vs. basal to apical) is 15, whereas Ja/Jb for D-mannitol equals 1; and basal-lateral to apical fluxes (Jb) of mannitol consistently exceed those of methyl glucoside.
View Article and Find Full Text PDFDeterminants of the steady-state vol of the dogfish shark (Squalus acanthias) rectal gland cells were studied. The cellular levels of trimethylamine oxide (TMAO) in fresh tissue and slices incubated aerobically 60 min in standard (TMAO-free) elasmobranch saline were close to those in the plasma (71 +/- 5 mM S.E.
View Article and Find Full Text PDFD-Glucose (Glc) transport was studied in stripped intestinal epithelial sheets of the winter flounder Pseudopleuronectes americanus. At 1 mM Glc and 15 degrees C, Glc uptake did not occur against a concentration gradient (free cell Glc 0.78 +/- 0.
View Article and Find Full Text PDFCell solute composition and apparent intracellular ionic concentrations (subscript i) were determined in rectal gland slices of the dogfish shark (Squalus acanthias). On aerobic incubation in elasmobranch Ringer's at 15 degrees C, the steady state values were: Ki+, 151 mM, close to its apparent electrochemical equilibrium; Cai2+, 1.7 mM; Mgi2+, 7.
View Article and Find Full Text PDFGridlike patterns of differing cell density were observed in evenly seeded cell monolayers. Such patterns were obtained in five of six cell lines tested, suggesting widespread occurrence. The mechanism appears to involve small, transient temperature changes related to incubator tray structure.
View Article and Find Full Text PDFThe transport of D-mannose (Man) in flounder kidney was studied using renal clearance techniques in vivo and brush border membrane (BBM) vesicles in vitro. At plasma concentrations of 50-100 microM Man, the winter flounder (Pseudopleuronectes americanus) reabsorbed up to 70% of the filtered sugar. Man phosphates, but not free Man, accumulated in renal cells.
View Article and Find Full Text PDFLLC-PK1 renal epithelial cells do not appreciably synthesize D-glucose or glycogen from L-lactate under the conditions described. [U-14C]L-lactate is metabolized to [14C]L-alanine and [14C]L-glutamate. Synthesis of alanine but not glutamate is decreased by the transaminase inhibitor, aminooxyacetate.
View Article and Find Full Text PDFUptake of nonmetabolizable glucose analogues was investigated in isolated rat lungs ventilated with 5% CO2 in air and perfused with Krebs-Ringer bicarbonate medium. In some experiments, [5-3H]glucose, methyl(alpha-D-[U-14C]gluco)pyranoside (alpha-MG), 3-O-methyl-D-[U-14C]glucose (3-O-MG), or various inhibitors were added to the initial perfusate to determine glucose utilization by the rate of 3H2O production or to characterize the uptake of glucose analogues. [1,2-3H]polyethylene glycol (PEG) was used as an indicator of the extracellular water space and gave a mean value of 0.
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