Cell volume regulation: osmolytes, osmolyte transport, and signal transduction.

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.

Identification of a region critically involved in the interaction of phlorizin with the rabbit sodium-D-glucose cotransporter SGLT1.

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.

Different effects of cyclosporine a and FK506 on potassium transport systems in MDCK cells.

BACKGROUND: Hyperkalemia and metabolic acidosis are common manifestations in patients receiving the immunosuppressive agent cyclosporine A (CsA) and the recently introduced FK506. We compared the acute toxic and antiproliferative effects as well as the effects on the transport activity of Na(+)/K(+)-ATPase and Na(+)/K(+)/2Cl(-) cotransporter of CsA and FK506 in an established cell line of distal/collecting tubule origin (MDCK cells). METHODS: MDCK cells were exposed to various concentrations of CsA or FK506 and the effects on cell viability (MTT test and neutral red uptake), plasma membrane integrity (lactate dehydrogenase (LDH) release) and cell proliferation (bromodeoxyuridine (BrdU) incorporation) were compared. For transport studies, after confluence, MDCK cells were exposed to CsA or FK506 for 48 h in the presence and absence of aldosterone. Ouabain- and bumetanide-sensitive (86)Rubidium uptake measurements were used to study the activity of the Na(+)/K(+)-ATPase and Na(+)/K(+)/2Cl(-) cotransporter at the surface of intact cells. RESULTS: After 24 h of exposure CsA reduced the number of viable cells to 50% at 30 microM, whereas for FK506 2-3 times higher concentrations had to be employed. Similarly, LDH release was stimulated tenfold by 30 microM CsA but only fourfold by 70 microM FK506. In contrast, DNA synthesis was affected at lower concentrations of FK506 than of CsA. In cells treated for 24 h BrdU incorporation was significantly inhibited by 3 microM FK506, whereas a similar inhibition required 10 microM CsA. The transport activity of Na(+)/K(+)-ATPase and of Na(+)/K(+)/2Cl(-) cotransporter were significantly decreased (37 and 63%, respectively) on CsA administration (8 microM). In CsA-treated cells the K(+) channel blockers barium (1 mM), TEA (10 mM) and quinine (1 mM) did not further inhibit the transport activities suggesting that CsA might also act via inhibition of K(+) channels. FK506 at 8 microM had no effect on Na(+)/K(+)-ATPase transport activity but stimulated Na(+)/K(+)/2Cl(-) cotransporter activity by 59%. The stimulatory effect was abolished by K(+) channel blockers indicating that recycling of K(+) might increase by FK506. The simultaneous presence of aldosterone (5 microM) protected the cells from the inhibitory effect of CsA on Na(+)/K(+)-ATPase and Na(+)/K(+)/2Cl(-) cotransporter activity. The stimulatory effect of FK506 on the Na(+)/K(+)/2Cl(-)cotransporter activity was completely abolished in the presence of aldosterone. CONCLUSIONS: Both CsA and FK506 showed acute toxicity in MDCK cells in vitro with the effects of FK506 being less pronounced. CsA and FK506 had different effects on the in vivo transport rates of the Na(+)/K(+)-ATPase and the Na(+)/K(+)/2Cl(-) cotransporter; CsA inhibited the activity of the Na(+)/K(+)-ATPase and the Na(+)/K(+)/2Cl(-) cotransporter whereas FK506 stimulated the activity of Na(+)/K(+)/2Cl(-) cotransporter. These effects were abolished by the application of aldosterone.

Inhibition by mercuric chloride of Na-K-2Cl cotransport activity in rectal gland plasma membrane vesicles isolated from Squalus acanthias.

The rectal gland of the dogfish shark is a model system for active transepithelial transport of chloride. It has been shown previously that mercuric chloride, one of the toxic environmental pollutants, inhibits chloride secretion in this organ. In order to investigate the mechanism of action of HgCl(2) at a membrane-molecular level, plasma membrane vesicles were isolated from the rectal gland and the effect of mercury on the activity of the Na-K-2Cl cotransporter was investigated in isotope flux studies. During a 30 s exposure HgCl(2) inhibited cotransport activity in a dose-dependent manner with an apparent K(i) of approx. 50 microM. The inhibition was complete after 15 s, partly reversible by dilution of the incubation medium and completely attenuated upon addition of reduced glutathione. The extent of inhibition by mercury depended on the ionic composition of the medium. The sensitivity of the cotransporter was highest when only the high affinity binding sites for sodium and chloride were saturated. Organic mercurials such as p-chloromercuribenzoic acid and p-chloromercuriphenylsulfonic acid at 100 microM did not inhibit the cotransporter, similarly exposure of the vesicles to 10 mM H(2)O(2) or 1 mM dithiothreitol for 30 min at 15 degrees C did not change cotransport activity. Transport activity was, however, reduced by 45.9+/-2.5% after an incubation with 3 mM N-ethylmaleimide for 20 min. Blocking free amino groups by N-hydroxysuccinimide or biotinamidocapronate-N-hydroxysulfosuccinimide had no effect. Investigations on the sidedness of the plasma membrane vesicles, employing the asymmetry of the (Na+K)-ATPase, demonstrated a right-side-out orientation in which the former extracellular face of the membrane is exposed to the incubation medium. In addition, extracellular mercury (5x10(-5) M) inhibited bumetanide-sensitive rubidium uptake into T84 cells by 48.5+/-7.1% after a 2 min incubation period. This inhibition was reversible in a manner similar to that observed in the plasma membrane vesicles. These studies suggest that in isolated rectal gland plasma membrane vesicles the Na-K-2Cl cotransporter (sNKCC1) exposes functionally relevant mercury binding sites at its external surface. These sites represent probably cysteines, the accessibility and/or sensitivity of which depends on the functional state of the transporter.

Calcium signalling during RVD of kidney cells.

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 IP(3) content. Preincubation with 100 micromol/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 micromol/l reduced partly the calcium response. SKF-96365, an inhibitor of receptor-mediatedcalcium 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.

Regulation of sorbitol efflux in different renal medullary cells: similarities and diversities.

It is well accepted that organic osmolytes, including sorbitol, play a major role in the volume regulation of renal medullary cells. The signal leading to an activation of release channels during RVD is, however, poorly understood. Hypotonicity induced sorbitol efflux was investigated in freshly isolated rat inner medullary collecting duct (IMCD) cells and in rabbit medullary thick ascending limb of Henle's loop (TALH) cells biochemically or using labeled sorbitol. The time course of release was compared with changes in cell volume, measured by confocal microscopy, and alterations in cell calcium (Ca(i)) determined by Fura 2 technology. In IMCD cells sorbitol release, volume decrease and Ca(i) transients show a close temporal correlation. In addition increases in Ca(i) without volume changes stimulate sorbitol efflux. In TALH cells sorbitol release starts after a significant lag time and reaches a maximum when cell volume is already partially restored. The same discrepancy is observed with regard to changes in Ca(i) and sorbitol efflux. These studies suggest that in IMCD cells changes in Ca(i) are the main regulator for the sorbitol permeability of the plasma membrane. The sorbitol channel present in TALH cells seems to operate predominantly independently of Ca(i). Despite this diversity in signal transduction the sorbitol channels in both renal cell types appear, however, not to be stretch-activated.

Human milk oligosaccharides are minimally digested in vitro.

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.

Vitalism and synthesis of urea. From Friedrich Wöhler to Hans A. Krebs.

In 1828, Friedrich Wöhler, a German physician and chemist by training, published a paper that describes the formation of urea, known since 1773 to be a major component of mammalian urine, by combining cyanic acid and ammonium in vitro. In these experiments the synthesis of an organic compound from two inorganic molecules was achieved for the first time. These results weakened significantly the vitalistic hypothesis on the functioning of living cells, although Wöhler, at that time, was more interested in the chemical consequences of isomerism than in the philosophical implications of his finding. However, the chemical synthesis observed by Wöhler does not represent the reaction which is employed in the mammalian liver for urea synthesis. The mechanism of this process was elucidated by the German physician Hans A. Krebs and his medical student Kurt Henseleit in 1932 and was shown to include the ornithine cycle. This 'urea cycle' is only observed in living cells; this apparently vitalistic phenomenon is caused by the compartmentalization of the various enzymatic reactions in mitochondria and cytosol, respectively.

Regulation of sorbitol content in cultured porcine urinary bladder epithelial cells.

Sorbitol content was determined in porcine urinary bladder epithelial cells immediately after death of the animals and after primary culture of the cells at different osmolalities. In both instances, sorbitol content increased with urine and medium osmolality, respectively. For example, at 300 mosmol/kg the cultured cells contained 0.84 +/- 0.02 nmol/mg protein, at 600 mosmol/kg contained 21.7 +/- 0.95 nmol/mg protein, and at 900 mosmol/kg contained 59.5 +/- 2.8 nmol/mg protein. Similarly, aldose reductase activity rose from 0.27 +/- 0.04 mumol.h-1.mg protein-1 at 300 mosmol/kg to 1.81 +/- 0.16 at 600 mosmol/kg and to 3.02 +/- 0.33 at 900 mosmol/kg. These changes were, however, only observed when NaCl but not when urea was used to augment the medium osmolality, since urea equilibrated across the cell membrane. In contrast, sorbitol release from cells cultured at 900 mosmol/kg was slowest into a 900 mosmol/kg medium and fastest into a 300 mosmol/kg medium (63 +/- 16 nmol/10 min compared with 389 +/- 52 nmol/10 min). These studies demonstrate that the sorbitol content of porcine urinary bladder epithelium is regulated by changes both in sorbitol synthesis and sorbitol release. Thus the regulatory mechanisms in the urinary bladder seem to be similar to those present in the embryological related collecting duct.

Biochemistry and physiology of carbohydrates in the renal collecting duct.

Using 13C-NMR analysis of cell extracts, enzymatic determination of metabolites and cofactors as well as enzyme assays on cell homogenates aerobic and anaerobic glycolysis, sorbitol formation by aldose reductase, the pentose phosphate shunt, and gluconeogenesis could be identified as the major pathways of D-glucose metabolism in renal inner medullary collecting ducts. In flux studies it was shown that D-glucose enters the collecting duct cells via a sodium-independent, cytochalasin- and phloretin-inhibitable transport system located at the basal-lateral cell side. At the same side sorbitol leaves the cells during regulatory volume decrease in a calcium-calmodulin-dependent fashion. From cell isolation studies it is proposed that sorbitol is taken up by adjacent (interstitial) cells, converted into fructose and then recycled to the collecting duct cells. This cycle might prevent carbohydrate wasting. Thus, IMCD cells exhibit unique aspects of carbohydrate biochemistry and physiology which enable them to function in a surrounding of low oxygen tension, low substrate supply, and extreme changes in extracellular osmolality.

Sorbitol uptake in plasma membrane vesicles isolated from immortalized rabbit TALH cells: activation by a Ca2+/calmodulin-dependent protein kinase.

Apical plasma membrane vesicles were isolated from cultures of immortalized thick ascending limb of Henle's loop (TALH) cells and sorbitol uptake was investigated using a rapid filtration technique. In the presence of Mg2+, Ca2+, ATP, and GTP sorbitol equilibrated within three minutes with the intravesicular space; this uptake was reduced by 75% when the incubation temperature was decreased from 37 degrees C to 4 degrees C. A lower level of uptake was also observed in the presence of 100 microM quinidine and when Ca2+ or ATP were omitted from the medium. Membranes preincubated with Mg2+, Ca2+, ATP, and GTP showed, however, a high sorbitol uptake in ATP-free medium. Staurosporine, but only at high concentrations of 200 nm, inhibited sorbitol uptake when present during the transport experiments or during the preincubation with ATP. Similar results were obtained with 1 microM trifluoperazine. Protein kinase C inhibitory peptide was ineffective whereas 20 nm KT 5926, at low concentrations a specific inhibitor of Ca2+/calmodulin-dependent kinase, attenuated the activation. On the basis of these data we suggest that a Ca2+/calmodulin-dependent kinase is a mediator of regulation of sorbitol plasma membrane permeability in renal medullary cells.

Characteristics of renal Na(+)-D-glucose cotransport in the skate (Raja erinacea) and shark (Squalus acanthias).

We have investigated the properties of the skate (Raja erinacea) and shark (Squalus acanthias) kidney Na(+)-D-glucose cotransporters (SGLT) in uptake studies of radiolabeled substrates into isolated renal brush-border membrane vesicles (BBMV). Scatchard plot analysis of the substrate dependence revealed that the Na(+)-D-glucose cotransporter population is homogenous within each species. Skate BBMV showed a relatively high affinity for D-glucose [Michaelis constant (K(m)) = 0.12 mM] with an apparent coupling ratio of approximately 2 Na+ to 1 D-glucose, whereas the shark transporter was much lower in affinity (K(m) = 1.90 mM) and had a lower coupling ratio, more like 1 Na+ to 1 D-glucose. These characteristics resemble the properties of SGLT1 and SGLT2, which are known to coexist in the mammalian kidney. Inhibitor studies using sugar analogs and glucosides suggested structural differences of the D-glucose binding site among these transporters, whereas the hydrophobic transporter domains in the vicinity of the D-glucose binding site appeared to be similar. In the high-affinity skate system, D-glucose was recognized by hydrogen bonds to the hydroxy groups at C-2, C-3, and C-4 and by hydrophobic interaction with the C-6 methylene group. In contrast, the low-affinity shark system seemed to lack the hydrophobic recognition motif for the C-6 methylene group of D-glucose.

Effect of cyclosporine A on Na+/K(+)-ATPase, Na+/K+/2Cl- cotransporter, and H+/K(+)-ATPase in MDCK cells and two subtypes, C7 and C11.

We investigated the influence of cyclosporine A (CsA) on key plasma membrane ion transport systems Na+/K(+)-ATPase, Na+/K+/2Cl- cotransporter, and H+/K(+)-ATPase in MDCK cells and two subtypes, C7 and C11, serving as a model system to study principal (C7) and intercalated (C11) cell properties of the distal nephron. The transport activity of Na+/K(+)-ATPase was significantly decreased in all cell types on CsA administration (8 x 10(-6) M) for 2 days, whereas the protein levels of Na+/K(+)-ATPase alpha-subunit in plasma membranes isolated from MDCK, C7, and C11 cells remained unchanged. The transport activity of Na+/K+/2Cl- cotransporter was significantly inhibited by CsA only in MDCK and C11 cells, but again plasma membrane protein levels were not altered. In contrast, C7 cell plasma membranes showed an increase of transport protein content, although the Na+/K+/2Cl- cotransporter activity was not affected by CsA. The H+/K(+)-ATPase transport activity remained unchanged in all three cell types. These data indicate that in C7 cells CsA might induce insertion of transporters into the plasma membrane, thus compensating the decrease of transport activity observed in MDCK and C11 cells. Furthermore, CsA significantly inhibited cell proliferation at 4 x 10(-6) M for C7 and C11 cells and at 8 x 10(-6) M for MDCK cells. Proliferation was completely abolished at 1.6 x 10(-5) M CsA. After 48 h of CsA incubation, the intracellular sodium concentration increased in all three different cell types; however, it stayed within the physiological range of mammalian cells. We, therefore, suggest that CsA is capable of reducing Na+/K(+)-ATPase and Na+/K+/2Cl- cotransporter activities in cells of the distal nephron, thereby contributing to the hyperkalemia observed in patients treated with CsA.

Osmoregulation in the renal papilla: membranes, messengers and molecules.

This contribution summarizes recent progress in the understanding of the molecular basis of the release of organic osmolytes that occurs when inner medullary cells are confronted with a drop in osmolarity in their environment. For sorbitol release across the basolateral membrane an increase in intracellular calcium seems to be the prominent signal, initiated by G-protein activation, followed by phosphatidylcholine phospholipase activation and generation of arachidonic acid. The increase in betaine permeability is also G-protein dependent but calcium independent, and is restricted to the basal-lateral cell face. Myo-inositol and glycerophosphorylcholine efflux are calcium and G-protein independent and occur both across the apical and basolateral membrane, although to a different extent. Taurine release is also calcium and G-protein independent; a swelling-activated anion channel at the basolateral membrane represents the major efflux pathway.

Electrodiffusive transport of Mg across renal membrane vesicles of the rainbow trout Oncorhynchus mykiss.

The mechanism of tubular Mg transport was investigated in membrane vesicles (MV) of trout kidneys prepared by differential centrifugation with sucrose. MV consisted largely of brush-border membranes, as indicated by high enrichments of brush-border membrane enzymes. Although measured transport of 28 Mg included a binding component, most membrane transport was into or out of an osmotically active space. There was no evidence for amiloride-sensitive Na/Mg exchange, nor was Mg uptake affected by the carboxyl group reagents trimethyloxonium tetrafluoroborate, glycine methyl ester.HCl-1-ethyl-3- (3-dimethyl-aminopropyl)carbodiimide, and N,N'-dicyclohexyl carbodiimide or the Ca channel modulators D-600, verapamil, diltiazem, and BAY K 8644. However, Mg uptake increased in the presence of inside-negative voltages generated by inward gradients of the permeant anions NO3, SCN, and Cl or by outward gradients of K (plus valinomycin). Alkaline-earth cations displayed the selectivity sequence VII (Mg > Ca > Sr > Ba) for cis-inhibition of 28 Mg uptake. Mg efflux was trans-inhibited by La and Gd, and Mg uptake was cis-inhibited by Mn. The sulfhydryl group reagents p- chloromercuribenzoic acid and p-chloromercuriphenylsulfonate stimulated Mg uptake and efflux. These results reveal an electrodiffusive pathway for Mg transport in trout renal MV.

The effect of cadmium chloride in vitro on sodium-glutamate cotransport in brush border membrane vesicles isolated from rabbit kidney.

To further elucidate the mechanism of cadmium inhibition of renal amino acid transport, brush border membrane vesicles were isolated from rabbit renal cortex and the effect of cadmium on the uptake of L-glutamate into the vesicles was investigated. Preincubation of the membranes with CdCl2 decreased sodium-dependent L-glutamate uptake at concentrations higher than 10(-6)M. In the presence of 20 mM potassium inside the vesicles a half-maximal inhibition was observed at 0.5 to 1 x 10(-4) M. Kinetic analysis revealed a strong reduction of Vmax by cadmium but only minor changes in Km for glutamate. The inhibition required preincubation of the vesicles with cadmium, was not elicited by cadmium metallothionein, and was not reversed by ethylenediaminetetraacetic acid. These findings suggest an action of cadmium at the cytoplasmic face of the brush border membrane. Furthermore, the sensitivity of the transport system for cadmium was lower in the absence of potassium. Inhibition increased in a saturable manner when intravesicular potassium was augmented, indicating that the transporter interacts with cadmium most avidly when potassium is bound to the carrier.

Na-D-glucose cotransport in renal brush-border membrane vesicles of an early teleost (Oncorhynchus mykiss).

Brush-border membrane vesicles (BBMV) enriched with alkaline phosphatase (8.1-fold) and gamma-glutamyl transpeptidase (11.5-fold) were prepared from the rainbow trout kidney. D-[3H]glucose uptake was stimulated by inward Na gradients but not by K, choline, Li, N-methyl-D-glucamine, or mannitol gradients. Na-dependent glucose uptake displayed overshoot in voltage-polarized vesicles (VPV; negative inside) but not in short-circuited vesicles (SCV). Recognition of carbons 2 and 3 of the glucopyranose ring was essential for glucose uptake. Phlorizin inhibited Na-dependent D-glucose uptake with an inhibition constant of 11.4 microM. The Michaelis-Menten constant of glucose was 0.58 mM in VPV and increased to 1.49 mM in SCV, whereas that for sodium was 193 mM in VPV and similar in SCV. Maximum velocity of Na was reduced in SCV. The Hill coefficient was 1 for both Na and glucose in VPV and SCV. Our studies indicate a single Na-D-glucose cotransporter that transports Na and glucose with a 1:1 stoichiometry and voltage-dependent kinetics. The transporter shares functional properties with both mammalian transporters SGLT1 and SGLT2.

Urate transport in Homarus americanus hepatopancreas: studies on membrane vesicles and R cells.

[2-14C]urate uptake was studied in hepatopancreatic basolateral membrane vesicles and in R cell suspensions of the American lobster by Millipore filtration techniques. Unspecific binding of urate to the vesicular membrane was 25.5 +/- 3.0% of equilibrium. Vesicular uptake showed a diffusional and a saturable component (Km) 0.37 +/- 0.04 mM and maximal velocity (Vmax) 16.5 +/- 1.2 pmol urate.mg protein-1.s-1). [2-14C]urate uptake was significantly trans-stimulated by urate. Purine analogues, probenecid, p-aminohippuric acid, pyrazinoic, and oxonic acid cis-inhibited urate transport. Urate uptake was not affected by Na+ or K+ transmembrane gradients but stimulated by 1 mM 2-oxoglutarate at the cis-side in Na(+)-containing media. Cellular urate uptake was inhibited by pyrazinoic acid. Uptake was saturable (Km 0.53 +/- 0.11 mM and Vmax 3.7 +/- 0.4 pmol urate.mg protein-1.s-1) and Na(+)-independent. However, 2-oxoglutarate stimulated uptake in Na(+)-containing media. These results suggest that urate uptake across the basolateral membrane occurs via a specific, Na(+)-independent transport system that may operate in the exchange mode accepting 2-oxoglutarate as countertransported substrate. In vivo, urate uptake thereby would be a tertiary active system driven by a 2-oxoglutarate gradient established across the cell membrane by the operation of a Na(+)-2-oxoglutarate cotransport system.

Membrane traffic and sorbitol release during osmo- and volume regulation in isolated rat renal inner medullary collecting duct cells.

In response to hypotonic stress, cells of the inner medullary collecting duct (IMCD) undergo swelling followed by a regulatory volume decrease (RVD) and a transient release of organic osmolytes such as sorbitol. In this study, we tested the hypothesis whether membrane recycling is involved in the latter process. Therefore, the state of submembranal actin and the cellular uptake or release of the fluid-phase marker fluorescein isothiocyanate (FITC)-dextran (FD) were investigated as related to changes in membrane permeability for sorbitol. After exposure to hypotonic medium the submembranal actin web rapidly disaggregated but it started to reorganize after 5 min of incubation. The basal-lateral pole of IMCD cells showed a significant uptake of extracellular FD after 100 sec. After 5 min, part of this fluorescence intensity had moved towards the cell center but the main part remained submembranal. Disintegration of the actin network by cytochalasin D diminished the uptake of FD during hypotonicity as did a permanent increase in intracellular calcium induced by ionomycin treatment. During a second osmotic stimulation of IMCD cells preloaded with FD, FD was released in a linear time course reaching a plateau after 1 min. Isotonic ionomycin treatment of preloaded cells also generated a rapid FD release during the first minute but induced a further 2-fold increase during the next 4 min. Under both conditions initial FD release was highly correlated with the simultaneously determined increase in sorbitol permeability. A similar strong correlation was found when different incubation temperatures were used (0 degree C, 15 degrees C, 37 degrees C). These results suggest that during exposure of IMCD cells to hypotonicity the submembranal actin web rapidly disintegrates, and "reserve" vesicles, probably containing sorbitol transporter, move to and fuse with the basal-lateral plasma membrane. The fusion causes a rapid increase in sorbitol permeability. These membrane areas are recovered by internalization, and the transport systems for sorbitol are concomitantly retrieved. In parallel to this internalization the submembranal actin filament network is rearranged. This process seems to be regulated by changes in intracellular calcium.

Jacob Henle: the kidney and beyond.

The progress in science made by Henle depended on the improvements in light microscopy achieved in the 19th century. The advent of achromatic lenses in particular made it possible for Henle and his contemporary and friend Schwann to uncover the typical structures of cells, thus bringing order at the microscopic level to the world of living tissues. Henle's 'comprehensive' approach--4 basic types of tissue: epithelial, connective, muscular and nervous, contrasted with the accepted doctrine of Bichat of 21 different types of tissues that enter into different combinations in forming the organs of the body--together with rapid progress in chemistry and physics permitted the subsequent intimate probing of cellular physiology. Henle's work was not confined to the description of anatomic structures. After he had observed microorganisms in the excretions of diseased animals, he embraced the unpopular theory of 'contagion' as the source of infection, though he himself was not able to prove that microorganisms were the direct cause of diseases. His discovery of the renal tubule that now bears his name came comparatively late in his career, and although he described its structure in detail he offered no suggestions as to its function.