Na-dependent Uptake Research Articles

Fluorescein-methotrexate transport in dogfish shark (Squalus acanthias) choroid plexus

The vertebrate choroid plexus removes potentially toxic metabolites and xenobiotics from cerebrospinal fluid (CSF) to blood for subsequent excretion in urine and bile. We used confocal microscopy and quantitative image analysis to characterize the mechanisms driving transport of the large organic anion, fluorescein-methotrexate (FL-MTX), from bath (CSF-side) to blood vessels in intact lateral choroid plexus from dogfish shark, Squalus acanthias, an evolutionarily ancient vertebrate. With 2 microM FL-MTX in the bath, steady-state fluorescence in the subepithelium/vascular space exceeded bath levels by 5- to 10-fold, and fluorescence in the epithelial cells was slightly below bath levels. FL-MTX accumulation in both tissue compartments was reduced by NaCN, Na removal, and ouabain, but not by a 10-fold increase in medium K. Certain organic anions, e.g., probenecid, MTX, and taurocholate, reduced FL-MTX accumulation in both tissue compartments; p-aminohippurate and estrone sulfate reduced subepithelial/vascular accumulation, but not cellular accumulation. At low concentrations, digoxin, leukotriene C4, and MK-571 reduced fluorescence in the subepithelium/vascular space while increasing cellular fluorescence, indicating preferential inhibition of efflux over uptake. In the presence of 10 microM digoxin (reduced efflux, enhanced cellular accumulation), cellular FL-MTX accumulation was specific, concentrative, and Na dependent. Thus transepithelial FL-MTX transport involved the following two carrier-mediated steps: electroneutral, Na-dependent uptake at the apical membrane and electroneutral efflux at the basolateral membrane. Finally, FL-MTX accumulation in both tissue compartments was reduced by phorbol ester and increased by forskolin, indicating antagonistic modulation by protein kinase C and protein kinase A.

Quercetin glucosides inhibit glucose uptake into brush-border-membrane vesicles of porcine jejunum.

Recent experimental data point to an interaction of dietary flavonol monoglucosides with the intestinal Na-dependent glucose transporter 1 (SGLT1). To investigate this interaction in more detail, we performed experiments with SGLT1-containing brush-border-membrane vesicles (BBMV) from pig jejunum. The flavonol quercetin-3-O-glucoside (Q3G) concentration-dependently inhibited Na-dependent uptake of radioactively labelled d-glucose into BBMV. Uptake of l-leucine was not inhibited by Q3G, indicating a specific interaction of the glucoside with SGLT1. Whereas the maximal transport rate of concentration-dependent initial glucose uptake was not altered in the presence of Q3G, the constant for half-maximal glucose uptake was increased, suggesting a competitive type of inhibition of glucose uptake by Q3G. Trans-stimulation experiments suggested the transport of Q3G via SGLT1. In addition, Q3G decreased the Na-independent diffusive uptake of glucose into BBMV. Other flavonoids were also tested for their inhibitory effect on d-glucose uptake. Among the tested quercetin glycosides, only the 4'-O-glucoside (Q4G) also inhibited Na-dependent glucose uptake into BBMV, whereas the 3-O-galactoside, the 3-O-glucorhamnoside and the aglycone quercetin itself were ineffective. Glucosides of some other flavonoid classes such as naringenin-7-O-glucoside, genistein-7-O-glucoside and cyanidin-3,5-O-diglucoside were ineffective as well. Thus, dietary quercetin monoglucosides, for example, Q3G and Q4G, have an impact on intestinal nutrient transporters such as SGLT1 and related systems.

Fluorescein-methotrexate transport in rat choroid plexus analyzed using confocal microscopy.

One function of the vertebrate choroid plexus (CP) is removal of potentially toxic metabolites and xenobiotics from cerebrospinal fluid (CSF) to blood for subsequent excretion in urine and bile. We have used confocal microscopy and quantitative image analysis to follow transport of the large organic anion fluorescein-methotrexate (FL-MTX) from bath (CSF side) to blood vessels in intact rat CP and found concentrative transport from CSF to blood. With 2 microM FL-MTX in the bath, steady-state fluorescence in the subepithelium and vascular spaces exceeded bath levels by 5- to 10-fold, but fluorescence in epithelial cells was below bath levels. FL-MTX accumulation in subepithelium and vascular spaces was reduced by NaCN, Na removal, and by other organic anions, e.g., MTX, probenecid, and estrone sulfate. Increasing medium K 10-fold had no effect. None of these treatments affected cellular accumulation. However, two observations indicated that apical FL-MTX uptake was indeed mediated: first, cellular accumulation was a saturable function of medium substrate concentration; and second, digoxin and MK-571 reduced FL-MTX accumulation in the subepithelial/vascular spaces but also increased cellular accumulation severalfold. In the presence of digoxin and MK-571, cellular accumulation was concentrative, specific, and Na dependent. Thus transepithelial FL-MTX transport involved the following two mediated steps: Na-dependent uptake at the apical membrane and electroneutral efflux at the basolateral membrane, possibly on Oatp2 and Mrp1.

Dynamic regulation of NKCC1 surface expression by protein kinase C epsilon in chloride secretory epithelia

cells. L-NIL also reversed the inhibition of Na-K-ATPase In villus cells during chronic enteritis (Na-K-ATPase activity was 14.0+_ 1.1 nmols Pi/mg protein/minute in normal, 4 .9_ + 1.0 in inflamed and 12.3+_ 1.0 in inflamed + L-NIL, n = 3 , p<0.01). However, L-NIL reversed the inhibition of SGLT-1 in villus cell BBMV from the chronically inflamed intestine which suggested a direct effect on the co-transporter itself. (Na-dependent uptake of 3-OMG in BBMV was 75.4+2.4 pmols/mg protein 9 seconds in normal, 11.2+_ 1.0 in inflamed and 69.1 + 3.3 in inflamed + L-NIL, n = 4, p<0.05). KInetic studies demonstrated that L-NIL reversed the inhibition of SGLT-1 by restoring the maximal rate of uptake of glucose (Vmax)without an effect on the affinity (1/Kin) of the co-transporter for glucose. Western blot studies also demonstrated that L-NIL treatment restored the immuno-reactive levels on SGLT-1 in BBM. Conclusions. When iNO production is abolished in the chronically inflamed intestine, inhibition of SGLT-I is alleviated. The mechanism of reversal of SGLT-1 inhibition is secondary to the restoration of co-transporter numbers. Thus, iNO likely mediates the inhibition of SGLT-1 in the chronically inflamed intestine.

Hepatocellular transport proteins and their role in liver disease

TRANSPORT PROTEINS Basolateral transport systems Na-dependent bile salt uptake Uptake of bile salts into the liver was first characterized in experimental models such as the isolated perfused rat liver, isolated hepatocyte cultures and basolateral plasma membrane vesicles. These studies indicated that more than 80% of taurocholate uptake but less than 50% of cholate uptake into hepatocytes is sodiumdependent. Whereas unconjugated bile salts are uncharged molecules that can traverse membranes by passive nonionic diffusion, conjugation with glycine or taurine decreases their pKa values and necessitates the presence of a specific carrier protein for hepatocellular uptake. The chief uptake system for conjugated bile salts in mammalian liver was isolated by expression and molecular cloning strategies and has been called the Na + taurocholate cotransporting polypeptide (gene symbol: SLC10A1). Rat Ntcp consists of 362 amino acids with an apparent molecular mass of 51 kD and is expressed exclusively at the basolateral membrane of hepatocytes (Figure 1). Ntcp mediates sodium-dependent uptake of taurocholate and other bile salts when expressed in stably transfected COS-7, Chinese hamster ovary (CHO) and hepatoblastoma (HepG2) cells or in cRNA injected Xenopus laevis oocytes, with apparent Km values between 17-42 μmol/L. The only non-bile salt substrates that are transported by Ntcp are selected sulfated steroid conjugates such as estrone-3sulfate and dehydroepiandrosterone sulfate (DHEAS) . In human liver, NTCP represents a 349-amino acid protein. NTCP is structurally related to the intestinal bile salt transporter (IBAT), that also mediates the Nadependent uptake of bile saltsand that is expressed not only in ileum, but also in the kidney and in cholangiocytes. Na-dependent taurocholate uptake is reduced in experimental models of cholestasis such as bile duct l igat ion , endotoxinemia and par t ia l hepatectomy, is reduced in primary hepatocyte cultures and is absent in various hepatoma cell lines . These changes in hepatic Na dependent bile salt uptake correlate with expression levels of Ntcp. Thus, Ntcp mRNA and protein levels are decreased in bile duct ligation, endotoxinemia and ethinyl estradiol induced cholestasis. In patients with a diagnosis of extrahepatic biliary atresia and clinical evidence of cholestasis, NTCP mRNA levels are also decreased.

Glutamine Uptake at the Blood‐Brain Barrier Is Mediated by N‐System Transport

The mechanism of unidirectional transport of glutamine from blood to brain in pentobarbital-anesthetized rats was examined using in situ perfusion. Amino acid uptake into brain across the blood-brain barrier (BBB) is classically thought to be via the Na-independent large neutral (L-system), acidic and basic amino acid transporters. In the presence of physiological concentrations of amino acids in the perfusate, which should saturate the known amino acid transporters at the BBB, the cortical transfer constant (Ki) for L-[14C]glutamine was 11.6 +/- 1.1 microl/g/min. The addition of either 10 mM 2-amino-2-norbornanecarboxylic acid or 10 mM 2-amino-2-norbornanecarboxylic acid and 5 mM cysteine had no effect on the cortical Ki for L-[14C]glutamine, indicating that glutamine transport under these conditions does not occur by the L-, A-, or ASC-systems. Decreasing perfusate Na from 140 to 2.4 mM by Tris substitution reduced the cortical Ki for L-[14C]glutamine by 62% (p < or = 0.001). The Na-dependent uptake has the characteristics of N-system transport. It was inhibited by L-histidine and L-glutamine, both N-system substrates, and it was pH sensitive and moderately tolerant of Li substitution for Na. This putative N-system transporter at the luminal membrane of the BBB plays an important role in mediating brain glutamine uptake.

Expression of glucose transporters in human peritoneal mesothelial cells

Glucose containing solutions, the basis of peritoneal dialysis fluids, affect the proliferation and regeneration of peritoneal mesothelial cells (MsC). The aim of this study was to examine mechanisms of glucose transport into MsC, that is, the expression of facilitative glucose transporters (GLUT) and the Na(+)-dependent glucose transporter (SGLT1) in human primary MsC and a transfected MsC line. Since expression of both transporters is differentiation dependent, we investigated the effects of cell differentiation induced by culturing MsC on membranes or by addition of hexamethylene bisacetamide (HMBA; 6 mM), which enhances SGLT1 expression in LLC-PK1 cells. Levels of mRNA for GLUT1 through GLUT4 and SGLT1 were evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR). The presence of the corresponding proteins was examined by Western blotting and localized by immunofluorescence. Active, Na(+)-dependent glucose transport was assessed by alpha-methyl-D-[14C]glucopyranoside (AMG) with and without the SGLT1-specific inhibitor phlorizin and by patch clamp experiments in NaCl or choline-chloride, For Na(+) dependent glucose uptake choline chloride instead of NaCl served as negative control. Facilitative transport was assessed using 2-fluoro-2-deoxy-[14C]-D-glucose (FDG) with and without the inhibitors cytochalasin B or phloretin. Primary and transfected MsC express GLUT1 and GLUT3 mRNA while no transcripts were found for GLUT2 and GLUT4. No SGLT1 transcript was detectable in subconfluent cells. Semiquantitative RT-PCR analysis documented that the addition of the differentiation inducer HMBA to confluent cultures or growth of MsC on membranes for seven days produced a down-regulation of mRNA for GLUT1, no change for GLUT3, and a substantial increase for SGLT1 mRNA. Under these conditions MsC express SGLT1 protein and possess a Na(+)-dependent glucose uptake as assessed by AMG. Phlorizin (1 mM) inhibits AMG uptake by 30 to 40%. In patch clamp experiments the addition of extracellular glucose depolarized the membrane potential only in the presence of sodium. These results indicate that differentiated MsC express GLUT1, GLUT3, and SGLT1. Further characterization of these transport mechanisms and their regulation may help to understand the cellular effects of glucose on MsC in peritoneal dialysis.

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.

L-Carnitine uptake into primary rat cortical cultures: Interaction with GABA

The ability of the primary rat cortical cells to take up l-carnitine increased with the age of the cultures and plateaued at around day 11 up to 25 days in vitro (DIV) when a slight decline was evident and by 32 DIV there was a major decrease in l-carnitine uptake. The uptake of l-carnitine displayed complex components. Elimination of mitochondrial energy supply by NaCN (1 mM), rotenone (1.25 μM) and DNP (50 μM), caused a small but significant decrease in the uptake (21, 11 and 16%, respectively). The uptake was highly dependent on the Na gradient, since ouabain (0.5 mM) and Na free buffer (replaced by 250 mM sucrose), reduced uptake by 54 and 63%, respectively. There was competition of l-carnitine uptake by molecules resembling its structure, e.g. γ-aminobutyric acid (GABA), acetyl- l-carnitine (ALC), d-carnitine, l-aminocarnitine and l-choline, with GABA being the most potent inhibitor (57% at 50 μM) and l-choline not being significantly active. The Na-dependent uptake of l-carnitine was saturable with a high K m (692 μM) and V max (839 pmol/min/mg). This Na-dependent component was not further additive with the GABA (500 μM) or the DNP (50 μM) inhibitable component, suggesting that it represented the same phenomenon, probably the Na gradient dependent transport of l-carnitine. The results indicate that the uptake of l-carnitine occurs by Na-dependent saturable process as well as non-saturable, Na-independent processes. At least the former uptake mechanism is potently inhibited by GABA.

Transepithelial phosphate transport in rabbit proximal tubular cells adapted to phosphate deprivation.

Both renal and nonrenal cells in culture adapt to deprivation of Pi by increasing Na-dependent Pi uptake. We studied whether this change in uptake is reflected in an increased renal transepithelial Pi transport. We grew primary cultures of rabbit renal cortical cells in plastic flasks and subcultured them onto Millicell-HA filters. This produced cell monolayers, which structurally and functionally resembled proximal tubule. These cells performed Na-dependent net transepithelial transport of 32Pi in the apical-to-basolateral direction that was inhibited by phosphonoformic acid in the apical fluid or by ouabain in the basolateral fluid or by preincubation with parathyroid hormone. Overnight incubation at low Pi concentrations led to a progressive increase in 5-min Na-dependent Pi uptake into cell monolayers. Na-dependent Pi uptake was threefold higher following overnight incubation at 25 microM Pi, compared with 3 mM Pi, and the increase was one-half maximal with incubation at an extracellular Pi concentration ([Pi]) of 300 microM. This was associated with a decrease in Na-dependent transepithelial Pi flux to the basolateral fluid by the same cells, which fell dramatically following incubation at < or = 300 microM Pi. There was no change in Na-dependent uptake or transepithelial transport of L-glutamine. This adaptation to Pi deprivation in vitro appears to serve to restore depleted cell stores of Pi rather than to regulate transepithelial Pi transport.

Electrogenicity of Na-coupled bile salt transport in isolated rat hepatocytes.

The importance of membrane voltage in uptake of bile salts into hepatocytes is not known. Electrogenicity of the primary bile salt transport process, Na-bile salt cotransport, has been difficult to determine because the large K and Cl conductances of the sinusoidal membrane (GK and GCl, respectively) obscure any transport associated currents. In the present study hepatocytes were treated to reduce these membrane conductances and electrogenic entry of taurocholate and glycocholate was demonstrated. Intracellular voltage and resistance changes resulting from bile salt transport were measured in hepatocytes in which GK and GCl were blocked by impalement with Na acetate microelectrodes and external exposure to quinine (400 microM). This increased the cell input resistance from 153 +/- 17 to 230 +/- 17 M omega (n = 14, P < 0.001). Under these conditions, exposure to 100 microM of taurocholate or glycocholate produced Na-dependent depolarizations of 3.0 +/- 0.5 and 4.2 +/- 0.8 mV, respectively. These correspond to transport currents of 13.9 and 7.6 pA/cell, which are comparable to those predicted from known [3H]taurocholate uptake rates if one positive charge enters the cell with each bile salt molecule. Although uptake of these two bile salts was electrogenic, this was not the case for all bile salts. Na-dependent transport of taurodehydrocholate, which occurs at similar rates to that for taurocholate, produced no voltage change. The unconjugated bile salts cholate and ursodeoxycholate also produced no measurable voltage or resistance changes. In conclusion, Na-dependent uptake of taurocholate and glycocholate is electrogenic, whereas uptake of taurodehydrocholate, ursodeoxycholate, and cholate is predominantly electroneutral.(ABSTRACT TRUNCATED AT 250 WORDS)

Dopamine uptake in five structures of the brain: Comparison of rate, sodium dependence and sensitivity to cocaine

The rate of uptake of dopamine (DA) per μg protein and the sensitivity of uptake to sodium ([Na] o) and cocaine, have been measured in synaptosomes from five structures in the brain of the rat: striatum, nucleus accumbens, neocortex, limbic cortex and thalamus. Probably reflecting the number of DA terminals, there was a wide variation in the rate of uptake in the different structures: uptake was far greater in the striatum and nucleus accumbens (10–20-fold) than in the neocortex, limbic cortex or thalamus. Uptake in all structures was inhibited by cocaine. With high [Na] o, the IC 50's varied from 1.10 μM for the thalamus to 3.32 μM for the nucleus accumbens. Maximum percentage inhibition varied from 76.0 for limbic cortex to 96.8 for nucleus accumbens and 97.7 for striatum; thus most uptake was carried-mediated. With low [Na] o, IC 50 for the nucleus accumbens was unchanged, while the IC 50's for the striatum and limbic cortex were less. Maximum percentage inhibition was similar to that found for high [Na] o. Although the sensitivity to [Na] varied, synaptosomes from all areas showed Na-dependent uptake; lowering [Na] in the incubation medium from 133.2 to 9.5 mM reduced the uptake by a minimum of 36.9% in the neocortex to a maximum of 78.0% in striatum. Neither the rate of uptake nor the Na-dependence correlated precisely with the sensitivity to cocaine but the two structures which showed the greatest rate of uptake (nucleus accumbens and striatum) also showed the greatest sensitivity to [Na] o. Generally, the rate of uptake correlated with the density of DA terminals and number of cocaine binding sites as reported by others. The results are consistent with the conclusion that the nucleus accumbens and striatum are particularly important in the cocaine abuse syndrome because of two factors: (1) the high rate of uptake as a result of the high density of DA terminals predisposes the functions of these structures to a greater impact by cocaine; (2) as a consequence, the overall cocaine abuse syndrome depends to a great extent on the specific functions that these structures subserve.

Sphingomyelin and cholesterol modulate sodium coupled uptakes in proximal tubular cells

Sphingomyelin (SM) and cholesterol are major lipid species of apical membranes in renal proximal tubular cells and confer to these membranes a low fluidity. Changes in membrane fluidity and/or lipidic composition were shown to affect the activity of cotransport systems of renal apical membranes. We evaluated the effect of decreasing membrane SM content on lipidic composition, membrane fluidity and sodium (Na)coupled uptakes in rabbit proximal tubular cells in primary culture. Sphingomyelinase (SMase) (30 to 250 mU/ml) decreased [3H]choline-labeled SM content, decreased cholesterol content, and increased cholesterol esterification. SMase did not modify membrane fluidity on isolated brush border membranes. SMase decreased Vmax of Na-dependent uptake of phosphate and alpha-methyl-D-glucoside, but not of alanine. SMase did not influence protein kinase C-induced inhibition of phosphate and glucose uptake. Increasing membrane cholesterol content with cholesterol-enriched liposomes subsequently to SMase action restored in part glucose uptake, but not phosphate uptake. In conclusion, SM degradation affected Na-phosphate and Na-glucose cotransports through changes in both SM and cholesterol contents of apical proximal membranes; these changes seemed to occur independently from changes in bulk membrane fluidity. These results suggest that SM and cholesterol have distinct and intricated roles in accessibility and/or activity of apical cotransport systems.

Influence of phospholipid/amphotericin B ratio and phospholipid type on in vitro renal cell toxicities and fungicidal activities of lipid-associated amphotericin B formulations.

We studied the influence of the lipid/amphotericin B (AMB) ratio and the phospholipid type on the in vitro renal cell toxicity and antifungal efficacy of lipid-associated AMB (L-AMB). L-AMB was prepared at one of two different lipid/AMB ratios (1 and 40) by incubating AMB with empty small unilamellar vesicles, made from one of three different phospholipids: dipalmitoyl-, dimirystoyl-, and distearoylphosphatidylcholine (DPPC, DMPC, and DSPC, respectively). Renal cell toxicity, investigated through an assessment of the Na-dependent uptake of phosphate by proximal tubular cells, and fungicidal effect against Candida albicans were studied after 1 h of treatment at 37 degrees C. The amount of unbound AMB present in each L-AMB formulation was studied by use of circular dichroism. At a lipid/AMB ratio of 40, the three lipidic formulations were not toxic for renal cells but were less effective against C. albicans than AMB; however, DSPC-AMB, which contained 50% unbound AMB, was more effective against C. albicans than DPCC-AMB or DMPC-AMB, containing 0 and 13% unbound AMB, respectively. At a lipid/AMB ratio of 1, the antifungal effects of L-AMB and AMB were similar, whatever the phospholipid used, but only DMPC-AMB remained highly protective against AMB renal cell toxicity, despite the presence of the same amount of unbound AMB (50%) in DMPC-AMB and DPPC-AMB. We conclude that the in vitro activities and renal cell toxicities of different L-AMB formulations are influenced by the phospholipid type and the lipid/AMB ratio. The optimal ratio depends on the phospholipid itself. At a lipid/AMB ratio of 40, the antifungal activity depends mainly on the amount of unbound AMB in the formulation. At a lipid/AMB ratio of 1, the renal cell toxicity also depends on the fluidity of the phospholipid.

Renal adaptation to dietary phosphate deprivation: role of proximal tubule brush-border membrane fluidity

With dietary phosphate (Pi) restriction, fluidity of renal proximal tubule brush-border membranes (BBM) and Na-dependent Pi transport (Na-Pi) are increased, suggesting that changes in BBM fluidity are critical for adaptation to Pi restriction. To test this hypothesis, the temporal relationship between Na-Pi transport and changes in BBM fluidity was assessed after Pi deprivation in rats. Renal cortex was obtained from rats fed either a 0.03% (-P) or a 0.6% (+P) Pi diet for 4 h or 7 days, and BBM were prepared. Na-Pi uptake by BBM was measured by use of rapid filtration, and BBM fluidity was assessed by use of the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH). After 4 h on the diets, Na-Pi uptake was 439 +/- 142 (SD) and 984 +/- 184 pmol.mg protein-1.5 s-1 in +P and -P, respectively (P less than 0.01, n, = 8). Na-dependent proline uptake was unchanged. DPH anisotropy and total cholesterol were similar between groups: 0.204 +/- 0.025 and 0.401 +/- 0.047 nmol/mg protein, respectively, in +P and 0.205 +/- 0.015 and 0.392 +/- 0.037 in -P (P greater than 0.05, n = 8-10). After 7 days, Na-Pi uptake was 841 +/- 291 in +P and 2,168 +/- 848 pmol.mg protein-1.5 s-1 in -P, P less than 0.01, n = 8. DPH anisotropy and BBM cholesterol were 0.175 +/- 0.019 and 443 +/- 132 nmol/mg protein, respectively, in +P and 0.162 +/- 0.020 (n = 8) and 341 +/- 128 (n = 3) in -P (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

High-affinity binding sites for VIP in renal cortical membranes: Possible role of VIP in renal transport

We studied binding and degradation of vasoactive intestinal peptide (VIP) by highly purified brush border and basolateral membranes from rabbit kidney cortex. Brush border and basolateral membranes were capable of 73 and 49% degradation of VIP after 20 minutes, and the degradation was totally prevented by bacitracin. There was 66 and 87% specific binding of 125I-VIP to brush border and basolateral membranes, respectively. 125I-VIP binding to renal membrane was displaced in a dose dependent fashion by unlabeled VIP with half maximal displacement at 2 x 10(-7) M. Other related peptides failed to displace VIP. Scatchard analysis showed one single class of receptors for VIP in both membranes with similar Kd (0.5 x 10(-7) M), but higher number of binding sites (Bmax) in the basolateral membranes than in the brush border membranes (22.0 vs. 4.4 pmol/mg protein), respectively. Forty-eight percent of VIP binding to brush border membranes could be explained by cross contamination of these membranes with basolateral membranes. We examined the effect of VIP on Na-H antiporter, Na-dependent glucose uptake and Na-dependent phosphate uptake by isolated proximal tubule suspension. In acid loaded proximal tubules VIP (10(-6) M) inhibited total and amiloride-sensitive 22Na uptake by 35 and 75%, respectively, as compared to control. On the other hand VIP failed to inhibit Na-dependent methyl alpha-14C-glucopyranoside and Na-dependent 32phosphate uptake. VIP failed to stimulate cyclic AMP generation by proximal tubule suspension while PTH showed the expected stimulation. Our results demonstrate the presence of specific binding for VIP in highly purified cortical membranes and suggest an effect of VIP to inhibit the Na-H antiporter by a mechanism independent of cyclic AMP.

Renal Na-myo-inositol cotransporter mRNA expression in Xenopus oocytes: regulation by hypertonicity

Canine renal cells in culture (MDCK cells) accumulate organic osmolytes, including myo-inositol (MI), in response to hypertonic stress. When medium tonicity is increased, intracellular concentration of MI rises because hypertonicity elicits increased uptake of MI via Na-MI cotransporter(s). To study the mechanism for this increase in cotransporter activity, poly(A)+ RNA isolated from MDCK cells maintained in hypertonic or isotonic medium was injected into Xenopus oocytes, and Na-dependent MI uptake was measured 3-5 days later. Poly(A)+ RNA from hypertonic cells induced clear expression of the cotransporter. In contrast, oocytes injected with poly(A)+ RNA isolated from MDCK cells maintained in isotonic medium exhibited cotransporter activity like oocytes injected with water. Upon size fractionation of RNA, peak activity appeared in a fraction that contained poly(A)+ RNA with median size of approximately 4 kilobases. Na-dependent MI uptake by poly(A)+ RNA-injected oocytes was inhibited by both phlorizin and phloretin. We suggest that hypertonicity-induced upregulation of the Na-MI cotransporter involves an increase in mRNA and synthesis of cotransporter protein(s).

Expression of high affinity sodium dependent choline transport in Xenopus oocytes injected with Torpedo electric lobe mRNA

Abstract Cholinergic nerve terminals have been shown to capture choline by a high affinity. Na-dependent mechanism which is inhibited by low (1 μM) concentrations of hemicholinium-3 (HC-3). Xenopus laevis oocytes injected with poly(A) + RNA from the cholinergic soma of the electric lobe of Torpedo marmorata brain expressed a specific component of choline uptake that was sensitive to a low concentration of HC-3, and whose presence was best revealed in the presence of a high concentration of sodium. This mechanism thus resembles choline transport by the cholinergic transporter from electric organ nerve terminals, and was clearly distinguishable from the endogenous Na-independent and HC-3 insensitive choline accumulation by non-injected oocytes. Furthermore, Na-dependent choline uptake was not found in oocytes injected with poly(A) + RNA from the electric organ itself, an innervated but non-nervous tissue. Following size-fractionation of electric lobe mRNA by ultracentrifugation in a sucrose gradient, fractions enriched in specific mRNA induced higher levels of Na-dependent choline transport when injected into oocytes. These results suggest that the Na-dependent choline transporter is formed by a single peptide chain, and that the dependence on sodium is an integral property of the protein.

12-HETE modulates Na-coupled uptakes in proximal tubular cells: role of diacylglycerol kinase inhibition

Inhibition of diacylglycerol (DAG) kinase, an alternative way to increase the cellular DAG level, was shown to reproduce, in renal proximal tubular cells, the inhibitory effect of protein kinase C (PKC) activators on Na-Pi and Na-alpha-methyl-D-glucopyranoside (MGP) cotransport. To evaluate whether 12S-hydroxyeicosatetraenoic acid (12S-HETE) or 12R-HETE, a DAG kinase inhibitor in endothelial cells, has a similar effect in proximal tubular cells, we studied the influence of this lipoxygenase product on Na-dependent uptake of Pi, MGP, and alanine, as well as on [14C]arachidonate-DAG content and [32P]phosphatidic acid (PA) content in rabbit proximal tubular cells grown as a primary culture. 12-HETE (1-10 microM) decreased [32P]PA content and stimulated [14C]DAG accumulation in a concentration-dependent manner. The labeled phosphatidylcholine, lysophosphatidylcholine, and sphingomyelin contents were not modified. 12-HETE also decreased DAG kinase activity of cell membranes. 12-HETE (10 microM) decreased the maximum velocity of Pi uptake by 36% and that of MGP uptake by 44% but did not affect alanine uptake. The effect of 12-HETE on transport was potentiated by calcium ionophore A23187 and was blunted by PKC downregulation. The effects of 12-HETE on lipid composition and transport were mimicked by R 59022, a pharmacological DAG kinase inhibitor. Neither arachidonic acid nor prostaglandin E2 reproduced the effects of 12-HETE. We conclude that in the proximal tubule, 12-HETE affected Na-dependent Pi and MGP cotransport through stimulation of PKC and that 12-HETE-induced activation of PKC is mediated by the inhibition of DAG kinase.

Sodium-adenosine cotransport in brush-border membranes from rabbit ileum

To determine the mechanism(s) of transcellular adenosine transport in epithelial tissues that possess an adenosine receptor response, we studied [3H]adenosine uptake using vesicles prepared from isolated brush-border and basolateral membranes of the rabbit ileum. In the presence of the adenosine deaminase inhibitor deoxycoformycin uptake of [3H]adenosine into brush-border membrane vesicles is stimulated fivefold by an inwardly directed Na gradient. Na-dependent [3H]adenosine uptake is enhanced and concentrative under conditions that increase inside negativity of vesicles, thus providing evidence for an electrogenic carrier. Na-dependent adenosine uptake is a saturable function of adenosine concentration with a Michaelis-Menten constant of 17.3 +/- 7.1 microM and maximum transport rate of 216.9 +/- 20.2 pmol.min-1.mg protein-1. Both uridine and inosine inhibit [3H]adenosine uptake, suggesting that the Na-dependent transporter has broad substrate specificity for both purine and pyrimidine ribonucleosides. Na-dependent adenosine uptake is inhibited by dipyridamole but is insensitive to 6-(4-nitrobenzyl)thio-9-beta-D-ribofuranosylpurine. We conclude that adenosine is transported across ileal brush-border membranes by a Na-ribonucleoside cotransport system. In contrast, adenosine uptake in basolateral membranes is not stimulated by a Na gradient. These studies show asymmetry in the distribution of transport systems for adenosine in polarized intestinal epithelia.