Second International Ringberg Conference: "Cell Biology and Molecular Basis of Liver Transport".
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Animals↗
Biomedical subjects
R K Kinne
Publications and source records attributed to R K Kinne.
Tamm-Horsfall protein as a marker of tubular maturation.
Tamm-Horsfall protein (THP), a glycoprotein with a molecular weight of 95 kilodaltons, is produced and secreted in the ascending loop of Henle. To evaluate the measurement of THP in the assessment of fetal renal development and function, we stained fetal kidney sections for THP and measured THP concentrations in 129 amniotic fluid samples from healthy pregnancies, together with other parameters such as transferrin, albumin, alpha 1- and beta 2-microglobulin. After the 16th week of gestation THP could be detected immunohistochemically in the distal tubular cells, but was not consistently detected by sandwich enzyme immunoassay until after the 20th week of gestation (detection limit 50 ng/ml). Between the 15th and 19th week of gestation THP was only detected occasionally, but after the 20th week of gestation the concentration increased significantly reaching levels of 0.4-4 mg/l at term. The THP concentration was lower in samples taken directly before birth than in the corresponding first urine after birth, indicating that THP is produced from the fetal kidney only and does not pass the placental barrier. This pattern was different from other proteins studied. Transferrin and albumin were significantly lower in the first urine voided, microglobulins remained unchanged, and the creatinine concentration increased. This indicates that maternal to fetal exchange or transport is likely for most of the other proteins. Measurement of THP concentrations, in addition to other proteins in the amniotic fluid, can improve fetal renal assessment, but because the range of THP concentrations is wide accurate predictions are still not possible.
Alpha-Globulins↗
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.
Animals↗
The human gene of a protein that modifies Na(+)-D-glucose co-transport.
Recently, a cDNA (pRS1) was cloned from pig kidney cortex that encodes a membrane-associated protein involved in Na(+)-coupled sugar transport. pRS1 alters sugar transport by SGLT1 from rabbit intestine or by SMIT from dog kidney which is homologous to SGLT1. In contrast, pRS1 does not influence transporters from other genetic families. We report the cloning of the intronless human gene hRS1 (6,743 bp), which encodes a 617-amino-acid protein with 74% amino acid identity to pRS1. By fluorescence in situ hybridization, hRS1 was localized to chromosome 1p36.1. The localization to one chromosome and Southern blot analysis of restricted genomic DNA suggest that there is only one RS1-homologous gene in humans. Functionality of hRS1 was demonstrated by co-expression experiments of hRS1 and SGLT1 from human intestine in oocytes from Xenopus laevis. They show that hRS1-protein inhibits Na(+)-D-glucose co-transport expressed by human SGLT1 by decreasing both the Vmax and the apparent Km value of the transporter. The analysis of the 5'-noncoding sequence of hRS1 revealed different enhancer consensus sequences that are absent in the SGLT1 gene, e.g., several consensus sequences for steroid-binding proteins.
Animals↗
Transport and metabolism of L-glutamate during oxygenation, anoxia, and reoxygenation of rat cardiac myocytes.
The intracellular glutamate concentration of oxygenated, isolated adult rat heart cells incubated with 0.15 mM glutamate amounts to 2.89 +/- 0.6 mM. Under these conditions the velocity of glutamate transport was 24.3 +/- 1.6 pmol.min-1.mg protein-1 and occurs via a high-affinity carrier characterized by an apparent affinity (K(m)) value of 0.18 +/- 0.03 mM. At high glutamate concentrations ( > 1mM) this high-affinity transport system is superimposed by additional uptake processes of a low affinity but a high capacity for glutamate. The 1.6-fold increased uptake of glutamate observed during 30 min of anoxic incubation of cardiomyocytes does not prevent an intracellular decrease in this amino acid to a concentration of 0.49 mM. After 15 min reoxygenation of cardiomyocytes the intracellular glutamate content increases to the control values of oxygenated cells. Only 2.4% of the glutamate increase after reoxygenation is due to the transport o glutamate from the incubation medium. The competitive inhibitor of transaminases, aminooxyacetate, prevents both the observed intracellular decrease in glutamate during anoxia and the increase in intracellular glutamate after reoxygenation of cardiomyocytes. Half of the amino groups needed for the synthesis of glutamate originate from intracellular alanine, which increases during anoxia and is metabolized during reoxygenation of cardiomyocytes. The velocity of the glutamate uptake of cardiomyocytes incubated in a medium containing 10 mM L-glutamate amounted to 728 +/- 140 pmol.min-1.mg protein-1. During anoxic incubation of cardiomyocytes at this high extracellular glutamate concentration, the intracellular glutamate breakdown may be compensated by a simultaneous uptake of this amino acid via the transport processes characterized by a high capacity
Amino Acids↗
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.
Amiloride↗
Na-Pi cotransport in flounder: same transport system in kidney and intestine.
The cloning of a renal Na-Pi contransport in system from winter flounder (P eudopleuronectes americanus) has recently been reported. We used this information to answer the questions 1) what is the distribution of the transport protein along the nephron? and 2) how are renal and intestinal transporters related? The distribution of the flounder NaPi-II protein was tested using two antisera raised against partial sequences (amino acids 1-14 and 388-441) of the transporter. Antibody-specific fluorescence was detected at the basolateral membrane of epithelial cells in the proximal tubular segment PII. Two clones corresponding to the renal Na-Pi cotransporter were isolated from a flounder intestinal cDNA library. Their functional properties were determined using Xenopus laevis oocytes. The apparent affinities for Pi [Michaelis constant (K(m)) = 0.063 mM] and Na (K(m) = 45.3 mM), as well as the pH dependency (increasing transport activity with increasing pH), showed the same characteristics in both intestinal and the renal systems. Sequence analysis revealed that the two intestinal clones were 100% homologous to the renal cDNA, Flounder NaPi-II-specific immunofluorescence was observed predominantly at the apical membrane on intestinal cross sections. We report the cloning and expression of the first intestinal Na-Pi cotransport system. This transporter belongs to the small group of proteins that exhibit the same function in the apical and the basolateral membranes of different cells.
Amino Acid Sequence↗
Taurine permeation through swelling-activated anion conductance in rat IMCD cells in primary culture.
Whole cell recordings were performed on rat inner medullary collecting duct (IMCD) cells in primary culture. With 140 mmol/l CsCl in bath and pipette we find within 10 min a 60-fold increase in membrane conductance from 0.02 +/- 0.003 to 1.2 +/- 0.1 nS/pF when bath osmolarity is decreased from 600 to 500 mosmol/l. The effect is due to the activation of an outwardly rectifying anion conductance with the anion selectivity SCN- > I- > NO-3 > Br- > Cl- > F- > isethionate > gluconate > or = aspartate > or = glutamate. A relative permeability of the organic osmolyte taurine to Cl- (Ptaurine: PCl-) of 0.15 was detected. With taurine in pipette and bath, the channel exhibits a nearly identical activation and sensitivity profile to a variety of anion channel blockers as under symmetrical Cl- conditions. Furthermore, the 50% inhibitory concentration value for the effect of 5-nitro-2-(3-phenylpropylamino)benzoate on both currents is virtually identical. We conclude that hypotonic stress increases the anion conductance of rat IMCD cells and that this anion conductance mediates taurine efflux.
Animals↗
Single-channel properties of swelling-activated anion conductance in rat inner medullary collecting duct cells.
Single-channel properties of the volume-activated outwardly rectifying Cl- conductance of rat IMCD cells were studied in primary cultures by means of the patch-clamp technique in the whole cell and in the outside-out configuration. Measurements were performed by noise analysis and in single-channel recordings during voltage-induced current inactivation and reactivation and in long-lasting experiments at constant membrane voltages. Unitary conductances could be defined for the voltage range of -100 to -50 mV and between +50 and +120 mV and chord conductances of 34.1 and 76.6 pS, respectively, can be calculated. The overall current-to-voltage relationship very much resembles that of the macroscopic Cl- conductance and the open probability of the activated channel is close to unity (Po = 0.98-0.99). The channel exhibits many similarities to volume-activated outwardly rectifying Cl- channels found in other systems although certain species differences do exist.
Animals↗
Hypotonicity-activated efflux of taurine and myo-inositol in rat inner medullary collecting duct cells: evidence for a major common pathway.
To further characterize the hypotonicity-activated efflux pathways for the organic osmolytes taurine and myo-inositol in inner medullary collecting duct (IMCD) cells tracer fluxes of taurine and myo-inositol were investigated. The time course of activation of both fluxes after exposure of cells isolated at 600 mosm to a hypotonic medium (300 mosm by omission of sucrose) was identical with a major increase of release within the first 10 min. All 'anion channel blockers' employed proved to be strong inhibitors of both fluxes. Inhibition of myo-inositol efflux by 0.5 mM NPPB and 0.1 mM dideoxyforskolin was not significantly different from that of taurine efflux (87.7 +/- 11.4 compared to 94.6 +/- 4.6% and 98.8 +/- 2.0 compared to 95.9 +/- 3.7%). However, SITS (0.5 and 0.01 mM), DIDS (0.5 and 0.01 mM), and niflumic acid (0.5 mM) inhibited myo-inositol efflux more strongly than taurine efflux. The respective values were 65.4 +/- 4 vs. 42.9 +/- 3.6% for 0.01 mM SITS, 65.7 +/- 4.2 vs. 45.8 +/- 2.0% for 0.01 mM DIDS, and 79.5 +/- 3.5 vs. 54.2 +/- 2.5% for 0.5 mM niflumic acid. Taurine as well as myo-inositol efflux were decreased to a similar extent by 10 mM extracellular ATP (26.9 +/- 6.3 vs. 29.8 +/- 17.7% inhibition), by 10 mM extracellular cAMP (52.8 +/- 9.8 vs. 60.1 +/- 17.2% inhibition) and by reduction of the intracellular ATP content employing 2-deoxy-D-glucose (31.9 +/- 5.9 vs. 40.4 +/- 13.6% inhibition). In polarized primary cell cultures taurine and myo-inositol were released during a hypotonic shock primarily across the basal-lateral membrane, the ratio of basolateral versus apical efflux was 4.1 for taurine and 3.9 for myo-inositol. Apical fluxes were more sensitive to 0.01 mM SITS or DIDS; this was particularly evident for apical myo-inositol efflux which was inhibited by 0.01 mM SITS by 84.1 +/- 5.9% compared to 43.5 +/- 13.1% inhibition of the basolateral efflux. Thus, taurine and myo-inositol efflux show to a great extent a similar cellular distribution, intracellular regulation and pharmacological inhibition profile. This similarity suggests that the two osmolytes share an efflux pathway that might be identical with the swelling-activated taurine conductance described previously. Additional minor pathways can, however, not be excluded.
Animals↗
Interferons modify in vitro proliferation of human bladder transitional cell carcinoma in the presence of doxorubicin and mitomycin C.
PURPOSE: The aim of the present study was to investigate whether interferons with their known antitumor activity modify the response of human bladder carcinoma cells to antitumor drugs. MATERIALS AND METHODS: We investigated the in vitro effect of doxorubicin, mitomycin C and the interferons alpha and gamma on cell proliferation in human bladder carcinoma cell lines as measured by 5-bromo-2'-deoxy-uridine (BrdU) incorporation. RESULTS: Exposure of RT 112 (but not EJ 28) cells for 2 hours to doxorubicin (500 ng./ml.) and mitomycin C (200 ng./ml.) reduced the proliferation rate to 85.9 +/- 3.3% (n = 4) and 89.3 +/- 4.0% (n = 4) of control. Treatment for 2 days with interferon alpha and gamma up to the highest concentration (200 U/ml.) showed no effect. The combination of 100 U/ml. interferon alpha and doxorubicin decreased proliferation significantly. At 50 ng./ml. the proliferation rate was decreased to 88.0 +/- 5.7% of control and at 500 ng./ml. to 67.7 +/- 3.1%. Thus interferon alpha seems to increase the sensitivity of the cells to doxorubicin. Cells treated with 20 ng./ml. mitomycin C after pretreatment with interferon alpha showed a dramatic decrease in cell proliferation (from 98.8 +/- 2.1% to 80.2 +/- 4.0% of control). This decrease was similar in the presence of 200 ng./ml. mitomycin C. Thus mitomycin C seems to render cells more sensitive to the antiproliferative action of interferon alpha. Interferon gamma had only minor effects on the response of the cells to doxorubicin or mitomycin C. CONCLUSIONS: These studies suggest that exposure to interferon alpha increases the efficacy of anticancer drugs in vitro, probably by several mechanisms. Potential consequences of this finding for the therapeutic regime employed for treatment of bladder carcinoma are discussed.
Antineoplastic Agents↗
Activation of a Cl(-)-conductive pathway in primary cultures of rat inner medullary collecting duct (IMCD) cells under hypotonic stress.
In intracellular recordings with conventional microelectrodes on rat IMCD cells, we find that hypotonic stress depolarizes membrane voltage and decreases cell input resistance. Ion substitution experiments reveal that these effects are largely due to the activation of a prominent Cl- conductance. After block of this conductance with dideoxyforskolin a smaller concomitant increase in K+ conductance becomes detectable.
Animals↗
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.
Animals↗
Hypertonic stress increases the Na+ conductance of rat hepatocytes in primary culture.
We studied the ionic mechanisms underlying the regulatory volume increase of rat hepatocytes in primary culture by use of confocal laser scanning microscopy, conventional and ion-sensitive microelectrodes, cable analysis, microfluorometry, and measurements of 86Rb+ uptake. Increasing osmolarity from 300 to 400 mosm/liter by addition of sucrose decreased cell volumes to 88.6% within 1 min; thereafter, cell volumes increased to 94.1% of control within 10 min, equivalent to a regulatory volume increase (RVI) by 44.5%. This RVI was paralleled by a decrease in cell input resistance and in specific cell membrane resistance to 88 and 60%, respectively. Ion substitution experiments (high K+, low Na+, low Cl-) revealed that these membrane effects are due to an increase in hepatocyte Na+ conductance. During RVI, ouabain-sensitive 86Rb+ uptake was augmented to 141% of control, and cell Na+ and cell K+ increased to 148 and 180%, respectively. The RVI, the increases in Na+ conductance and cell Na+, as well as the activation of Na+/K(+)-ATPase were completely blocked by 10(-5) mol/liter amiloride. At this concentration, amiloride had no effect on osmotically induced cell alkalinization via Na+/H+ exchange. When osmolarity was increased from 220 to 300 mosm/liter (by readdition of sucrose after a preperiod of 15 min in which the cells underwent a regulatory volume decrease, RVD) cell volumes initially decreased to 81.5%; thereafter cell volumes increased to 90.8% of control. This post-RVD-RVI of 55.0% is also mediated by an increase in Na+ conductance. We conclude that rat hepatocytes in confluent primary culture are capable of RVI as well as of post-RVD-RVI. In this system, hypertonic stress leads to a considerable increase in cell membrane Na+ conductance. In concert with conductive Na+ influx, cell K+ is then increased via activation of Na+/K(+)-ATPase. An additional role of Na+/H+ exchange in the volume regulation of rat hepatocytes remains to be defined.
Animals↗
Amino acid transporters.
This review focuses on two main aspects of amino acid transport. One aspect is the role of the basic amino acid transporter gene in causing cystinuria, its functional properties, and its potential transport functions. The other is the regulation of amino acid transporters at the levels of information processing and cellular organization.
Amino Acid Transport Systems↗
Characterization and pH dependence of L-glutamate transport in sarcolemmal vesicles from rat hearts.
Transport of L-glutamate in purified sarcolemmal vesicles from rat heart was Na+ dependent and characterized by an apparent affinity value of 0.149 +/- 0.04 mM (n = 6). Na+ binding to the carrier was not very specific, since an inwardly directed K+ gradient could also increase vesicular uptake of L-glutamate. Uptake of L-glutamate into sarcolemmal vesicles was inhibited by L-aspartate and L-cysteinate. These characteristics of glutamate uptake are typical of transport via system XAG-. Acidification of either the intravesicular or the extravesicular space led to a significant increase in L-glutamate transport. This increase was more pronounced when the intravesicular space had been acidified. Thus intracellular acidosis of hypoxic or ischemic myocardium is directly linked to an increased uptake of glutamate.
Alanine↗
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.
Animals↗
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.
Animals↗