In recent years, it has become evident that the volume of a given cell is an important factor not only in defining its intracellular osmolality and its shape, but also in defining other cellular functions, such as transepithelial transport, cell migration, cell growth, cell death, and the regulation of intracellular metabolism. In addition, besides inorganic osmolytes, the existence of organic osmolytes in cells has been discovered. Osmolyte transport systems-channels and carriers alike-have been identified and characterized at a molecular level and also, to a certain extent, the intracellular signals regulating osmolyte movements across the plasma membrane. The current review reflects these developments and focuses on the contributions of inorganic and organic osmolytes and their transport systems in regulatory volume increase (RVI) and regulatory volume decrease (RVD) in a variety of cells. Furthermore, the current knowledge on signal transduction in volume regulation is compiled, revealing an astonishing diversity in transport systems, as well as of regulatory signals. The information available indicates the existence of intricate spatial and temporal networks that control cell volume and that we are just beginning to be able to investigate and to understand.
In examining the functional aspects of human milk oligosaccharides (HMO), it is not known whether they are digested during the passage through the infant's gastrointestinal tract. HMO were prepared from individual milk samples (n = 6) and separated into neutral and acidic compounds by chromatography. These oligosaccharide fractions were studied for their digestibility by human salivary amylase, porcine pancreatic amylase and brush border membrane vesicles (BBMV) isolated from porcine small intestine; we also examined the effect of low pH on these structures. The characterization of HMO and their digestion products was performed by high-pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) as well as TLC. It was shown that neither salivary amylase nor pancreatic amylase cleaved HMO. Only after a 2-h incubation with BBMV were slight modifications of the HMO observed. HPAEC-PAD analysis revealed two new components within the neutral oligosaccharide fractions; these were characterized by mass spectrometric analysis as lacto-N:-triose and galactose. Only lacto-N:-triose was present within digestion assays of oligosaccharides, which did not contain fucosyl or N:-acetylneuraminic acid residues. These results suggest that <5% of the HMO are digested in the intestinal tract. Hence, HMO may play a role as prebiotics or as factors influencing the local immune system of the intestine in breast-fed infants.
In order to investigate the question whether ammonium reabsorption in the thick ascending limb of Henle's loop (TALH) proceeds via the Na+,K+,Cl(-)-cotransporter, plasma membrane vesicles were prepared from TALH cells isolated from rabbit kidney outer medulla and the effect of NH+4 on their transport properties was investigated. It was found that, in the presence of a 78-mmol/liter NaCl gradient, 5 mmol/liter NH+4 inhibited bumetanide-sensitive rubidium flux by 86%; a similar decrease was observed for 5 mmol/liter, K+. Inhibition of bumetanide-sensitive rubidium uptake by NH+4 was competitive and an apparent Ki of 1.9 mmol/liter was found. Bumetanide-sensitive sodium uptake measured in the presence of a 83 mmol/liter KCl gradient was not inhibited by 5 mmol/liter NH+4. A 100-mmol/liter NH4Cl gradient was, however, capable of stimulating bumetanide-sensitive sodium uptake to the same extent as a KCl gradient. These data suggest that NH+4 is accepted by the K+ site of the Na+,K+,Cl-cotransport system and that the transporter can function in a Na+,NH+4,2Cl mode. Since the affinity of the transporter for NH+4 lies in the concentration range found in the TALH lumen in vivo, it is concluded that Na+,NH+4,2Cl-cotransport can contribute to the NH+4 reabsorption in this tubular segment.
Two different membrane fractions were obtained from a brush-border fraction of rat kidney cortex by using their different electrical surface charges in preparative free-flow electrophoresis. One membrane fraction contained only morphologically intact microvilli and was characterized by a high specific activity of alkaline phosphatase. The other fraction morphologically resembled classical plasma membranes by possessing junctional complexes and a high Na-K-ATPase activity The contamination of the isolated membrane fractions by other cell organelles was extremely low These two fractions represent the apical (luminal) and the basal (interstitial) area of the renal proximal tubule cell membrane and clearly demonstrate the polarity of this cell.
The balance of a high extracellular osmolarity in the kidney medulla is mainly based on an accumulation of organic osmolytes in the cells. The regulation of cell volume during hypotonic conditions results in a release of organic osmolytes – a process that is partly calcium-dependent. Using calcium-sensitive fluorescent dye and confocal laser scanning microscopy, we have investigated calcium signalling during regulatory volume decrease (RVD) in kidney cells. In rat inner medullary collecting duct (IMCD) cells in primary culture, hypotonic stress induced a calcium release from intracellular stores that preceded calcium entry from the extracellular milieu. Hyposmotic stress had no effect on the cellular IP3 content. Preincubation with 100 μmol/l ETYA (a non-metabolizible derivative of arachidonic acid), however, reduced the calcium response to hypotonic stress as well as the RVD. Blocker of voltage-dependent calcium channels (verapamil, diltiazem, and nifedipine) in the concentration of 40 μmol/l reduced partly the calcium response. SKF-96365, an inhibitor of receptor-mediated calcium channels, also attenuated the calcium influx. In conclusion, swelling of IMCD cells increases intracellular calcium by release from intracellular stores and entry across the cell membranes. The signalling involves arachidonic acid metabolism.
Uptake studies of D-and L-glucose were performed on vesicles derived from brush-border and basal-lateral membranes. The uptake of the sugars into the vesicles was osmotically sensitive and independent of glucose metabolism. In brush-border vesicles D-glucose but not L-glucose transport was Na-+-dependent, wn the presence of an initial Na+gradient. Basal-lateral membranes take up D-glucose faster than L-glucose, but the D-glucose uptake is significantly less sensitive to sodium removal and only moderately inhibited by phlorzin as compared to the prush-border fraction.
In order to define potential interaction sites of SGLT1 with the transport inhibitor phlorizin, mutagenesis studies were performed in a hydrophobic region of loop 13 (aa 604-610), located extracellularly, close to the C-terminus. COS 7 cells were transiently transfected with the mutants and the kinetic parameters of alpha-methyl-D-glucopyranoside (AMG) uptake into the cells were investigated. Replacement of the respective amino acids with lysine reduced the maximal uptake rate: Y604K showed 2.2%, L606K 48.4%, F607K 15.1%, C608K 13.1%, G609K 14.1%, and L610K 17.2% of control. In all mutants the apparent K(i) for phlorizin increased at least by a factor of 5 compared to the wild-type K(i) of 4.6 +/- 0.7 micromol/l; most striking changes were observed for Y604K (K(i) = 75.3 +/- 19.0 micromol/l) and C608K (K(i) = 83.6 +/- 13.9 micromol/l). Replacement of these amino acids with a nonpolar amino acid instead of lysine such as in Y604F, Y604G and C608A showed markedly higher affinities for phlorizin. In cells expressing the mutants the apparent affinity of AMG uptake for the sugar was not statistically different from that of the wild type (Km = 0.8 +/- 0.2 mmol/l). These studies suggest that the region between amino acids 604 and 610 is involved in the interaction between SGLT1 and phlorizin, probably by providing a hydrophobic pocket for one of the aromatic rings of the aglucone moiety of the glycoside.
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