Developmental changes in glutamine transport by rat jejunal basolateral membrane vesicles

The ontogeny of glutamine uptake by jejunal basolateral membrane vesicles (BLMV) was studied in suckling and weanling rats and the results were compared with adult rats. Glutamine uptake was found to represent a transport into an osmotically active space and not mere binding to the membrane surface. Temperature dependency indicated a carrier-mediated process with optimal pH of 7.0. Transport of glutamine was Na+ (out greater than in) gradient dependent with a distinct "overshoot" phenomenon. The magnitude of the overshoot was higher in suckling compared with weanling rats. The uptake kinetics and inhibition profile indicated the existence of two major transport pathways. A Na(+)-dependent system correlated with System A showed tolerance to System N and System ASC substrates, and a Na(+)-independent system similar to the classical L system that favors leucine and BCH. The Vmax for the Na(+)-dependent system was higher in suckling compared with weanling and adult rats. The Vmax for the Na(+)-dependent system was 0.86 +/- 0.17, 0.64 +/- 0.8, and 0.41 +/- 0.9 nmol.mg protein-1.10 sec-1 for suckling, weanling, and adult rats, respectively. The Vmax for the Na(+)-independent system was 0.68 +/- 0.08, 0.50 +/- 0.03, and 0.24 +/- 0.03 nmol.mg protein-1.10 sec-1 for suckling, weanling, and adult rats, respectively. We conclude that glutamine uptake undergoes developmental changes consistent with more activity and/or number of glutamine transporters during periods of active cellular proliferation and differentiation.     

Biotin transport in rat intestinal brush-border membrane vesicles

Transport of biotin across rat intestinal brush-border membrane was examined using the brush-border membrane vesicle (BBMV) technique. Uptake of biotin by BBMV is the result of transport of the substrate into the intravesicular space with negligible binding to membrane surfaces. In the presence of a Na+ gradient (out greater than in), transport of biotin was higher with a transient 'overshoot' phenomenon. In comparison, transport of biotin in the presence of a choline gradient (out greater than in) was lower with no 'overshoot' phenomenon. In both jejunal and ileal BBMV, the transport of biotin as a function of concentration was saturable in the presence of a Na+ gradient (out greater than in) but was linear in the presence of a choline gradient (out greater than in). Vmax of the Na+-dependent transport system was 0.88 and 0.37 pmol/mg protein per s and apparent Kt was 7.57 and 7.85 microM in jejunal and ileal BBMV, respectively. Structural analogues inhibited the transport process of biotin. Unlike the electrogenic transport of D-glucose, the transport of the anionic biotin was not affected by imposing a relatively positive intravesicular potential with the use of valinomycin and an inwardly-directed K+ gradient, suggesting that biotin transport is most probably an electroneutral process. This suggestion was further supported by studies on biotin transport in the presence of anions of different lipid permeability. The results of this study demonstrate that biotin transport across rat intestinal brush-border membrane is by a carrier-mediated, Na+-dependent and electroneutral process. Furthermore, transport of biotin is higher in the jejunum than the ileum.     

Transport of carnosine by mouse intestinal brush-border membrane vesicles

The characteristics of carnosine (beta-alanyl-L-histidine) transport have been studied using purified brush-border membrane vesicles from mouse small intestine. Uptake curves did not exhibit any overshoot phenomena, and were similar under Na+, K+ or choline+ gradient conditions (extravesicular greater than intravesicular). However, uptake of histidine showed an overshoot phenomenon in the presence of a Na+-gradient. There was no detectable hydrolysis of carnosine during 15 min of incubation with membrane vesicles under conditions used for transport experiments. Analysis of intravesicular contents further showed the complete absence of the constituent free amino acids of carnosine, and indicates that intact carnosine is transported. Studies on the effect of concentration on peptide uptake revealed that transport occurred by a saturable process conforming to Michaelis-Menten kinetics with a Km of 9.6 +/- 1.4 mM and a Vmax of 2.9 +/- 0.2 nmol/mg protein per 0.4 min. Uptake of carnosine was inhibited by both di- and tripeptides with a maximum inhibition of 68% by glycyl-L-leucyltyrosine. These results clearly demonstrate that carnosine is transported intact by a carrier-mediated, Na+-independent process.     

Calcium uptake kinetics into brush-border membrane vesicles: higher Vmax in the spontaneously hypertensive rat than in normotensive control

Arterial hypertension in the spontaneously hypertensive rat (SHR) is associated with an abnormal Ca2+ homeostasis, compared with its normotensive control, the Wistar Kyoto rat (WKY). In particular, epithelial Ca2+ transport is perturbed, with intestinal absorption and renal tubular reabsorption being decreased in the adolescent and adult SHR. In the present study we examined Ca2+ uptake into isolated duodenal brush-border membrane vesicles (BBMV) in 12-14 week-old male rats. This uptake can be separated in a nonsaturable and a saturable component. The latter follows Michaelis-Menten kinetics. Vmax of this component was found to be significantly higher (p less than 0.05) in SHR than in WKY (0.58 +/- 0.19 versus 0.35 +/- 0.06 nmol/mg protein x 10 sec, mean +/- SD) whereas Km did not differ. Thus, the defect in the intestinal Ca2+ absorption previously identified in the SHR of this age is not due to a decrease in Ca uptake at the level of the duodenal brush-border membrane, but is most likely located in the baso-lateral membrane.     

Transport of methotrexate in basolateral membrane vesicles from rat liver.

Transport of the antifolate cancer drug methotrexate was studied in vesicles isolated from the basolateral membrane of rat liver. Transport of methotrexate by basolateral membrane vesicles (BLMVs) was mostly via uptake into an osmotically active intravesicular space, with some binding (approximately 9%), as shown by initial uptake studies and by varying medium osmolarity with increasing concentrations of sucrose. Methotrexate transport was linear for the first 20 s of incubation. Transport was not affected by imposition of a Na+ gradient across the vesicular membrane. Transport of methotrexate displayed a broad pH optimum: at an intravesicular pH of 7.5, the initial rate of uptake was not significantly different at extravesicular pH values ranging from 5.5 to 7.5, but uptake was less at extravesicular pH of 5.0 or 8.0. Methotrexate transport was saturable: Km = 0.15 +/- 0.05 microM and Vmax = 11.4 +/- 1.1 pmol 10 s-1 mg-1 protein. Methotrexate uptake into BLMVs was not inhibited by 5-methyltetrahydrofolate nor by 5-formyltetrahydrofolate but was weakly inhibited by folic acid in a concentration-dependent manner. Uptake was also inhibited by anion-exchange inhibitor 4,4'-diisothio-cyanostilbene-2,2'-disulfonic acid (DIDS), and by the structurally unrelated anions ATP, ADP, Cl-, SO4(2-), and oxalate2-. Adenosine (no negative charge) had no effect on transport. When vesicles were preloaded with anions (ADP, SO4(2-), oxalate2-) such that an anion gradient existed from the intra- to the extravesicular compartment, and methotrexate uptake was measured, no stimulation of uptake was seen. Methotrexate uptake into rat liver BLMVs was electrogenic as shown by stimulation of the initial rate of uptake by a valinomycin-imposed K+ diffusion potential across the vesicular membrane. These results suggest that methotrexate is transported into the hepatocyte across the basolateral membrane by an electrogenic, multispecific anion carrier system.     

Ontogeny of glutamine transport by rat liver plasma membrane vesicles.

Glutamine metabolism in the liver is essential for gluconeogenesis and ureagenesis. During the suckling period there is high hepatic protein accretion and the portal vein glutamine concentration is twice that in the adult, whereas hepatic vein glutamine concentration is similar between adult and suckling rats. Therefore, we hypothesized that glutamine uptake by the liver could be greater in the suckling period compared to the adult period. The present studies were, therefore, designed to investigate the transport of glutamine by plasma membranes of rat liver during maturation (suckling--2-week old, weanling--3-week old and adult--12-week old). Glutamine uptake by the plasma membranes of the liver represented transport into an osmotically sensitive space in all age groups. Inwardly directed Na+ gradient resulted in an "overshoot" phenomenon compared to K+ gradient. The magnitude of the overshoot was greater in suckling rats plasma membranes compared to adult membranes. Glutamine uptake under Na+ gradient was electrogenic and maximal at pH 7.5, whereas uptake under K+ gradient was electroneutral. Glutamine uptake with various concentrations of glutamine under Na+ gradient was saturable in all age groups with a Vmax of 1.5 +/- 0.1, 0.7 +/- 0.1 and 0.5 +/- 0.06 nmoles/mg protein/10 seconds in suckling, weanling and adult rats, respectively (P < 0.01). Km values were 0.6 +/- 0.1, 0.5 +/- 0.1 and 0.5 +/- 0.1 mM respectively. Vmax for Na(+)-independent glutamine uptake were 0.6 +/- 0.1, 0.55 +/- 0.07 and 0.54 +/- 0.06 nmoles/mg protein with Km values of 0.54 +/- 0.2, 0. +/- 0.1 and 0.5 +/- 0.2 mM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

A carrier-mediated transport for folate in basolateral membrane vesicles of rat small intestine.

The mechanism of exit of folate from the enterocyte, i.e. transport across the basolateral membrane, is not known. In this study we examined, using basolateral membrane vesicles, the transport of folic acid across the basolateral membrane of rat intestine. Uptake of folic acid by these vesicles represents transport of the substrate into the intravesicular compartment and not binding to the membrane surface. The rate of folic acid transport was linear for the first 1 min of incubation but decreased thereafter, reaching equilibrium after 5 min of incubation. The transport of folic acid was: (1) saturable as a function of concentration with an apparent Km of 0.6 +/- 0.17 microM and Vmax. of 1.01 +/- 0.11 pmol/30 s per mg of protein; (2) inhibited in a competitive manner by the structural analogues 5-methyltetrahydrofolate and methotrexate (Ki = 2 and 1.4 microM, respectively); (4) electroneutral; (5) Na+-independent; (6) sensitive to the effect of the anion exchange inhibitor 4,4'-di-isothiocyanatostilbene-2,2'-disulphonic acid (DIDS). These data indicate the existence of a carrier-mediated transport system for folic acid in rat intestinal basolateral membrane and demonstrate that the transport process is electroneutral, Na+-independent and sensitive to the effect of anion exchange inhibition.     

Ca2+ transport and Ca2+-dependent ATP hydrolysis by Golgi vesicles from lactating rat mammary glands.

Ca2+ transport across mammary-gland Golgi membranes was measured after centrifugation of the membrane vesicles through silicone oil. In the presence of 2.3 microM free Ca2+ the vesicles accumulated 5.8 nmol of Ca2+/mg of protein without added ATP, and this uptake was complete within 0.5 min. In the presence of 1 mM-ATP, Ca2+ was accumulated at a linear rate for 10 min after the precipitation of intravesicular Ca2+ with 10 mM-potassium oxalate. ATP-dependent Ca2+ uptake exhibited a Km of 0.14 microM for Ca2+ and a Vmax. of 3.1 nmol of Ca2+/min per mg of protein. Ca2+-dependent ATP hydrolysis exhibited a Km of 0.16 microM for Ca2+ and a Vmax. of 10.1 nmol of Pi/min per mg of protein. The stoichiometry between ATP-dependent Ca2+ uptake and Ca2+-stimulated ATPase varied between 0.3 and 0.7 over the range 0.03-8.6 microM-Ca2+. Both Ca2+ uptake and Ca2+-stimulated ATPase were strongly inhibited by orthovanadate, which suggests that the major mechanism by which Golgi vesicles accumulate Ca2+ is through the action of the Ca2+-stimulated ATPase. However, Ca2+ uptake was also decreased by the protonophore CCCP (carbonyl cyanide m-chlorophenylhydrazone), indicating that it may occur by other mechanisms too. The effect of CCCP may be related to the existence of transmembrane pH gradients (delta pH) in these vesicles: the addition of 30 microM-CCCP reduced delta pH from a control value of 1.06 to 0.73 pH unit. Golgi vesicles also possess a Ca2+-efflux pathway which operated at an initial rate of 0.5-0.57 nmol/min per mg of protein.     

Isolation and characterization of brush border membrane vesicles from pig small intestine

1. Brush border membrane vesicles (BBMV) were isolated from swine mid-intestine by a MgCl2 precipitation and sucrose density gradient centrifugation. 2. Transport of D-glucose and L-alanine were Na+-stimulated and into an osmotically sensitive space. 3. Estimates of kinetic parameters for Na+-dependent D-glucose transport were: apparent Kt = 1.8 mM and Jmax = 16.8 nmol/mg protein/min. 4. Results of experiments with the delta pH sensitive fluorescent probe 9-aminoacridine indicated independent mechanisms for Na+-dependent glucose transport and Na+/H+ exchange. 5. This study demonstrates that pig BBMV provide a useful model for investigating intestinal membrane transport.     

Limited Blood-Brain Barrier Transport of Polyamines

Transport of polyamines across the blood-brain barrier of adult rats was examined by measuring the amount of radioactivity that reached the forebrain 5 s after a "bolus" intracarotid injection. The values were expressed by the brain uptake index (BUI), which is the percentage of material transported in relation to freely diffusible water in a single passage through the brain. Transport was restricted as indicated by the respective BUI values, presented as means +/- SD (number of animals): putrescine, 5.3 +/- 0.8 (11); spermidine, 6.1 +/- 1.3 (7); and spermine, 5.8 +/- 0.5 (4). A kinetic study of the transport of [14C]putrescine showed that transport due to passive diffusion accounted for the majority of the observed influx (66% at 1 mM putrescine). However, a small saturable component exists with a Km value of 4-5 mM and a Vmax of 30 nmol X min-1 X g-1. This Km value is considerably higher than the circulating levels of the polyamine in the normal mature animal, and thus is unlikely to be of physiological significance. Competition studies indicated that putrescine does not interact with carriers for adenosine, arginine, choline, or leucine.     

Role of N-linked oligosaccharides in the transport activity of the Na+/H+ antiporter in rat renal brush-border membrane

The role of N-linked oligosaccharide side chains in the biogenesis and function of Na+-coupled transporters in renal luminal brush-border membrane (BBM) is not known. We examined the question of how in vivo inhibition by alkaloid swainsonine of alpha-mannosidase, a key enzyme in processing of glycoproteins in the Golgi apparatus, affects Na+/H+ antiport and Na+/Pi symport as well as activities of other transporters and enzymes in rat renal BBM. Administration of swainsonine to thyroparathyroidectomized rats, control or treated with 3,5,3'-triiodothyronine, markedly decreased the rate of Na+/H+ antiport, but had no effect on the rate of Na+/Pi symport across renal BBM vesicles (BBMV). Moreover, administration of swainsonine did not change activities of Na+ gradient, ([extravesicular Na+] greater than [intravesicular Na+])-dependent transport of D-glucose, L-proline, or the amiloride-insensitive 22Na+ uptake by BBMV; the activities of the BBM enzymes alkaline phosphatase, gamma-glutamyltransferase, or leucine aminopeptidase in BBMV were also not changed. The in vitro enzymatic deglycosylation of BBM by incubating freshly isolated BBMV with bacterial endoglycosidase F also resulted in a decreased rate of Na+/H+ antiport, but not Na+-coupled symports of Pi, L-proline, and D-glucose, or the activities of the BBM enzymes were not significantly affected. Similar incubation with endoglycosidase H was without effect on any of these parameters. Both the modification of BBMV glycoproteins by administration fo swainsonine in vivo as well as the in vitro incubation of BBMV with endoglycosidase F resulted in a decrease of the apparent Vmax of Na+/H+ antiport, but did not change the apparent Km of this antiporter for extravesicular Na+ and did not increase H+ conductance of BBM. Taken together, our findings suggest that intact N-linked oligosaccharide chains of the biantennary complex type in renal BBM glycoproteins are required, directly or indirectly, for the transport function of the Na+/H+ antiporter inserted into BBM of renal proximal tubules.     

Creatine Transport in Cultured Cells of Rat and Mouse Brain

Astroglia-rich cultures derived from brains of newborn rats or mice use a transport system for the uptake of creatine. The uptake system is saturable, Na+-dependent, and highly specific for creatine and Na+. Kinetic studies on rat cells revealed a Km value for creatine of 45 microM, a Vmax of 17 nmol x h-1 x (mg of protein)-1, and a Km value of 55 mM for Na+. The carrier is competitively inhibited by guanidinopropionate (Ki = 15 microM). No such transport system was found in neuron-rich primary cultures from embryonic rat brain. It is hypothesized that creatine transport is an astroglial rather than a neuronal function.     

Dicarboxylate transport in renal basolateral and brush-border membrane vesicles.

Characteristics of succinate transport were determined in basolateral and brush-border membrane vesicles (BLMV and BBMV, respectively) isolated in parallel from rabbit renal cortex. The uptake of succinate was markedly stimulated by the imposition of an inwardly directed Na+ gradient, showing an "overshoot" phenomenon in both membrane preparations. The stimulation of succinate uptake by an inwardly directed Na+ gradient was not significantly affected by pH clamp or inhibition of Na(+)-H+ exchange. The Na(+)-dependent and -independent succinate uptakes were not stimulated by an outwardly directed pH gradient. The Na dependence of succinate uptake exhibited sigmoidal kinetics, with Hill coefficients of 2.17 and 2.38 in BLMV and BBMV, respectively. The Na(+)-dependent succinate uptake by BLMV and BBMV was stimulated by a valinomycin-induced inside-negative potential. The Na(+)-dependent succinate uptake by BLMV and BBMV followed a simple Michaelis-Menten kinetics, with an apparent Km of 22.20 +/- 4.08 and 71.52 +/- 0.14 microM and a Vmax of 39.0 +/- 3.72 and 70.20 +/- 0.96 nmol/(mg.min), respectively. The substrate specificity and the inhibitor sensitivity of the succinate transport system appeared to be very similar in both membranes. These results indicate that both the renal brush-border and basolateral membranes possess the Na(+)-dependent dicarboxylate transport system with very similar properties but with different substrate affinity and transport capacity.     

Na+-H+ exchange in cardiac sarcolemmal vesicles

The transport of Na+ by a purified sarcolemmal vesicular preparation from canine ventricular tissue was studied as a function of both internal and external pH. The uptake of Na+ into sarcolemmal vesicles increased upon raising the extravesicular pH of the reaction medium. Half-maximal uptake of Na+ was observed at a pHo of about 8.1 and maximal uptake occurred at pH 8.6. The uptake of Na+ by sarcolemma was also dependent upon the intravesicular pH. Na+ uptake into sarcolemmal vesicles was greatly attenuated in the absence of a H+ gradient across the membrane. Transport of Na+ was potently inhibited by amiloride, a known blocker of Na+-H+ exchange. LiCl was also an effective inhibitor of Na+ transport. In the presence of optimal H+ gradients, Na+ uptake was linear for the first 5 seconds of the reaction and exhibited a Vmax of 290 nmol Na+/mg per min and a KNa of 3.5 mM. These experiments strongly indicate the presence of a Na+-H+ exchange system in cardiac sarcolemma. This activity appeared to be relatively specific for this membrane fraction. The identification of Na+-H+ exchange activity in a sarcolemmal vesicular fraction from the heart will permit extensive characterization of the regulation and kinetics of this antiporter in future investigations.     

Activity of membranous dopamine beta-monooxygenase within chromaffin granule ghosts. Interaction with ascorbate

The role of intra- and extravesicular ascorbate has been investigated in dopamine beta-monooxygenase (D beta M) turnover using adrenal medulla chromaffin granule ghosts. Resealing of vesicle ghosts with high levels of intravesicular ascorbate leads to viable vesicles, as evidenced from the high rates of the ATP-dependent accumulation of tyramine, Vmax = 14 +/- 1 nmol/min.mg and Km = 20 +/- 6 microM. However, the D beta M-catalyzed conversion of tyramine to octopamine occurs slowly, Vmax = 0.50 +/- 0.13 nmol/min.mg and Km = 29 +/- 18 mM. When ascorbate is present instead in the external buffer, the D beta M rate increases 3.6-fold for a final Vmax = 1.8 +/- 0.2 and Km = 1.2 +/- 0.3 mM. This relatively high rate of enzyme turnover is retained in ghosts resealed with a large excess of ascorbate oxidase, ruling out contamination by intravesicular ascorbate as the source of enzyme activity. The synergistic effect of intravesicular ascorbate was examined under conditions of 2 mM external ascorbate, showing that the enzymatic rate increases 2.7-fold, from 1.2 (0 internal ascorbate) to 3.2 +/- 0.4 nmol/min.mg (saturating internal ascorbate). This result confirms that high levels of internal ascorbate are not damaging to intravesicular D beta M. These studies demonstrate very clearly that external ascorbate is the preferred reductant for the membranous form of D beta M in chromaffin granule ghosts.     

Characterization of the mitochondrial Na+-H+ exchange. The effect of amiloride analogues

The kinetic properties and inhibitor sensitivity of the Na+-H+ exchange activity present in the inner membrane of rat heart and liver mitochondria were studied. (1) Na+-induced H+ efflux from mitochondria followed Michaelis-Menten kinetics. In heart mitochondria, the Km for Na+ was 24 +/- 4 mM and the Vmax was 4.5 +/- 1.4 nmol H+/mg protein per s (n = 6). Basically similar values were obtained in liver mitochondria (Km = 31 +/- 2 mM, Vmax = 5.3 +/- 0.2 nmol H+/mg protein per s, n = 4). (2) Li+ proved to be a substrate (Km = 5.9 mM, Vmax = 2.3 nmol H+/mg protein per s) and a potent competitive inhibitor with respect to Na+ (Ki approximately 0.7 mM). (3) External H+ inhibited the mitochondrial Na+-H+ exchange competitively. (4) Two benzamil derivatives of amiloride, 5-(N-4-chlorobenzyl)-N-(2',4'-dimethyl)benzamil and 3',5'-bis(trifluoromethyl)benzamil were effective inhibitors of the mitochondrial Na+-H+ exchange (50% inhibition was attained by approx. 60 microM in the presence of 15 mM Na+). (5) Three 5-amino analogues of amiloride, which are very strong Na+-H+ exchange blockers on the plasma membrane, exerted only weak inhibitory activity on the mitochondrial Na+-H+ exchange. (6) The results indicate that the mitochondrial and the plasma membrane antiporters represent distinct molecular entities.     

Ontogeny of the Na(+)-H+ exchanger in rat ileal basolateral membrane vesicles.

This study was designed to examine the activity of the Na(+)-H+ exchanger across the basolateral membranes of the ileal enterocyte and its developmental pattern. The function of the Na(+)-H+ exchanger was studied using a well validated basolateral membrane vesicle technique. Na+ uptake represented transport into the vesicle rather than binding as validated by initial rate studies. Na+ uptake represented an electroneutral process as shown by studies in which negative membrane potential was induced by the ionophore valinomycin. Various outwardly directed pH gradients significantly stimulated Na+ uptake compared with no pH gradient conditions at all age groups. However, the magnitude of stimulation was significantly different between the age groups with more marked stimulation of amiloride-sensitive Na+ uptake occurring in adolescent rats as compared to weanling or suckling rats. The amiloride sensitivity of the pH stimulated Na+ uptake was investigated using [Amiloride] = 10(-2)-10(-5) M at pHi/pHo = 5.2/7.5. At 10(-2) M amiloride concentration, Na+ uptake was inhibited by 80%, 70%, 77%, in the basolateral membranes of adolescent, weanling and suckling rats, respectively. Dixon plot analysis in both adolescent and weanling rats was consistent with two amiloride binding sites, a low affinity system and a high affinity system. In the suckling rat, on the other hand, the data supported a single high affinity binding site. Kinetic studies revealed a Km for amiloride-sensitive Na+ uptake of 12.6 +/- 6.6, 10.2 +/- 1.77, 9.46 and Vmax of 4.83 +/- 1.22, 4.47 +/- 0.36 and 8.08 +/- 1.92 n.mol.mg.protein-1.7 s-1 in suckling, weanling and adolescent rats, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ontogeny of mitochondrial calcium transport in spontaneously hypertensive (SHR) and WKY rats

The current studies were designed to investigate calcium uptake by intestinal jejunal sacs as well as in intestinal mitochondria of spontaneously hypertensive rats and their genetically matched WKY control rats. Kinetics of jejunal calcium uptake by jejunal sacs of adult SHR and WKY rats showed a significant decrease in Vmax of calcium uptake in SHR (227 +/- 24 versus 423 +/- 22 nmol.g tissue-1.3 min-1) compared to WKY rats P less than 0.001. To explore the intracellular handling of calcium by the intestinal mitochondria, calcium uptake was characterized by intestinal mitochondria before (suckling and weanling periods) and after (adult period) development of hypertension. Calcium uptake by intestinal mitochondria was driven by ATP in the presence of succinate as a respiratory substrate. Calcium uptake was stimulated several fold by the presence of ATP compared to no ATP conditions. Maximal calcium uptake occurred between 15-30 min and was significantly greater in adult SHR and WKY rats compared to corresponding values in weanling and suckling rats. Maximal ATP dependent calcium uptake in adult, weanling and suckling WKY rats was significantly greater compared to corresponding mean values in each age group in SHR (P less than 0.001). Oligomycin (10 micrograms/mg protein) inhibited calcium uptake partially. Ruthenium red (0.25 microM), 1 mM sodium azide and 0.5 mM dinitrophenol inhibited calcium uptake by more than 80% in both SHR and WKY rats. Kinetic parameters for ATP stimulated calcium uptake at 10 s revealed a Vmax of 0.56 +/- 0.6, 3.46 +/- 0.23 and 3.95 +/- 0.52 nmol/mg protein/10 s in suckling, weanling and adult WKY rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of sodium-dependent and sodium-independent nucleoside transport systems in rabbit brush-border and basolateral plasma-membrane vesicles from the renal outer cortex.

The transport of uridine into rabbit renal outer-cortical brush-border and basolateral membrane vesicles was compared at 22 degrees C. Uridine was taken up into an osmotically active space in the absence of metabolism for both types of membrane vesicles. Uridine influx by brush-border membrane vesicles was stimulated by Na+, and in the presence of inwardly directed gradients of Na+ a transient overshoot phenomenon was observed, indicating active transport. Kinetic analysis of the saturable Na+-dependent component of uridine flux indicated that it was consistent with Michaelis-Menten kinetics (Km 12 +/- 3 microM, Vmax. 3.9 +/- 0.9 pmol/s per mg of protein). The sodium:uridine coupling stoichiometry was found to be consistent with 1:1 and involved the net transfer of positive charge. In contrast, uridine influx by basolateral membrane vesicles was not dependent on the cation present and was inhibited by nitrobenzylthioinosine (NBMPR). NBMPR-sensitive uridine transport was saturable (Km 137 +/- 20 microM, Vmax. 5.2 +/- 0.6 pmol/s per mg of protein). Inhibition of uridine flux by NBMPR was associated with high-affinity binding of NBMPR to the basolateral membrane (Kd 0.74 +/- 0.46 nM). Binding of NBMPR to these sites was competitively blocked by adenosine and uridine. These results indicate that uridine crosses the brush-border surface of rabbit proximal renal tubule cells by Na+-dependent pathways, but permeates the basolateral surface by NBMPR-sensitive facilitated-diffusion carriers.