Lysine uptake by human placental microvillous membrane: comparison of system y+ with basal membrane

1995 ◽  
Vol 268 (3) ◽  
pp. C755-C761 ◽  
Author(s):  
T. C. Furesz ◽  
A. J. Moe ◽  
C. H. Smith
Keyword(s):  
Amino Acids ◽  
Maximum Velocity ◽  
Membrane Vesicles ◽  
Basal Membrane ◽  
System Model ◽  
Independent System ◽  
Lysine Concentration ◽  
Menten Constant ◽  
Na Uptake

Transport of lysine by microvillous membranes was investigated by characterization of L-[3H]lysine uptake in membrane vesicles isolated from human placentas. At least one Na(+)-independent system was observed at 22 degrees C and two systems at 37 degrees C. Lysine concentration dependence data were fit by a one- or two-system model with a Michaelis-Menten constant (Km) of 124 +/- 28 microM and a maximum velocity (Vmax) of 33.1 +/- 7.7 pmol.mg protein-1.min-1 at 22 degrees C and with Km values of 1 +/- 0.6 and 245 +/- 51 microM and Vmax values of 0.14 +/- 0.07 and 45.8 +/- 8.7 pmol.mg protein-1.30 s-1 at 37 degrees C. In the presence of N-ethylmaleimide, the uptake (37 degrees C) data were fit by a one-system model with kinetic parameters similar to the lower Km system. Uptake of L-lysine in the absence of Na+ was inhibited completely by L-arginine, L-histidine, and L-homoarginine. In the presence of Na+, uptake was inhibited completely by these same three amino acids and L-leucine but only partially by other neutral amino acids. To compare directly microvillous and basal membrane from the same placenta, we examined the inhibition of 20 microM lysine uptake in the presence of Na+. Inhibition by L-leucine was similar in the two membranes. However, L-homoserine, L-alanine, and L-phenylalanine over a wide concentration range inhibited substantially less in microvillous (at both temperatures) than in basal membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of Na(+)-H+ antiporter activity associated with human cheek epithelial cells

1994 ◽  
Vol 267 (1) ◽  
pp. C84-C93 ◽  
Author(s):  
E. J. McMurchie ◽  
S. L. Burnard ◽  
G. S. Patten ◽  
E. J. Lee ◽  
R. A. King ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Human Subjects ◽  
Maximum Velocity ◽  
Hill Coefficient ◽  
Transport Activity ◽  
Uptake Activity ◽  
Dimethyl Amiloride ◽  
Mouth Wash ◽  
Na Uptake

Na+ transport activity was characterized in human cheek epithelial cells obtained from normotensive adult subjects. The cells were isolated using a mouth-wash procedure and assayed for Na+ uptake using a radioactive (22Na+) rapid filtration assay. Cheek cells displayed proton-dependent Na+ uptake activity that was dependent on the magnitude of the externally directed proton gradient measured using the fluorescent probe 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein to determine intracellular pH. Amiloride, ethylisopropylamiloride (EIPA), 5-(N,N-dimethyl)-amiloride, 5-(N-methyl-N-isobutyl)-amiloride (MIA), and 5-(N,N-hexamethylene)-amiloride (NNHA) all inhibited proton-dependent Na+ uptake, with MIA, EIPA, and NNHA being the most potent. The Michaelis constant (Km) for extracellular Na+ was 5.7 mM, while the maximum velocity for Na(+)-H+ antiporter activity was 4.3 nmol Na+.mg protein-1.30s-1. The Km for intracellular H+ was 0.17 microM, with a Hill coefficient of 0.7. Stimulation by ouabain and inhibition by bumetanide of cheek cell proton-dependent Na+ uptake indicated only relatively low activities of Na(+)-K(+)-ATPase and Na(+)-K(+)-2Cl- cotransport, respectively. These results are consistent with the presence of Na(+)-H+ antiporter activity in cheek cells. Cheek cells therefore provide a convenient, relatively noninvasive source of tissue for examining Na(+)-H+ antiporter activity in human subjects.

Anionic amino acid uptake by microvillous membrane vesicles from human placenta

1989 ◽  
Vol 257 (5) ◽  
pp. C1005-C1011 ◽  
Author(s):  
A. J. Moe ◽  
C. H. Smith
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Glutamate Uptake ◽  
Membrane Vesicles ◽  
Amino Acid Uptake ◽  
Maternal Blood ◽  
Acid Uptake ◽  
Dependent Manner ◽  
Concentration Dependent Manner

The transport mechanisms for anionic amino acids in trophoblast microvillous (maternal facing) membrane were investigated by characterization of L-[3H]aspartate and L-[3H]glutamate uptake in membrane vesicles. Uptake of the anionic amino acids was by a single high-affinity Na+-dependent K+-stimulated cotransporter that is pH sensitive and electrogenic. A second Na+-dependent transporter could not be discriminated, and there was no observable Na+-independent uptake. An outwardly directed K+ gradient (100 mM KCl inside) resulted in a 5- to 10-fold stimulation in glutamate uptake in the presence of Na+. Intravesicular KCl had no effect on transporter affinity but increased transporter velocity in a concentration-dependent manner. Inhibition of Na+-K+-dependent uptake of L-aspartate and L-glutamate (20 mM, 30 s) by 2 mM unlabeled amino acids demonstrated stereoselectivity for L-glutamate but not for L-aspartate. The neutral amino acids (L-alanine, L-threonine, L-serine, L-cysteine, L-phenylalanine) were not effective inhibitors. These data are consistent with an anionic amino acid transporter in the microvillous membrane of the trophoblast, which has characteristics qualitatively similar to the X-AG system found in other epithelia. This system may mediate the concentrative placental uptake of anionic amino acids from maternal blood in utero.

Characterization of human placental activity for transport of amino acids using syncytiotrophoblast brush border membrane vesicles

Placenta ◽  
1992 ◽  
Vol 13 (4) ◽  
pp. A24
Author(s):  
Hideaki Iioka ◽  
Shinobu Akada ◽  
Takako Shimamoto ◽  
Yoshihiko Yamad ◽  
Ikuko S. Moriyama ◽  
...  
Keyword(s):  
Amino Acids ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Membrane Vesicles ◽  
Brush Border Membrane Vesicles

scholarly journals Human placental syncytiotrophoblast expresses two pharmacologically distinguishable types of Na+-H+ exchangers, NHE-1 in the maternal-facing (brush border) membrane and NHE-2 in the fetal-facing (basal) membrane

1992 ◽  
Vol 284 (1) ◽  
pp. 33-38 ◽  
Author(s):  
P Kulanthaivel ◽  
T C Furesz ◽  
A J Moe ◽  
C H Smith ◽  
V B Mahesh ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Membrane Vesicles ◽  
Basal Membrane ◽  
Na Channel ◽  
Normal Term ◽  
Na Uptake ◽  
Pharmacological Profiles

We investigated whether highly purified preparations of basal (fetal-facing) membrane isolated from normal term human placentas possess Na(+)-H+ exchanger activity. Uptake of Na+ into basal membrane vesicles was stimulated many-fold by an outwardly directed H+ gradient. This H(+)-gradient-dependent uptake was inhibitable by amiloride and its analogues. Na+ uptake in these vesicles did not occur via a Na+ channel, as it was not influenced by changes in membrane potential and, in addition, was inhibited by benzamil only at high micromolar concentrations. The results indicate that the human placental basal membrane possesses Na(+)-H+ exchanger activity. We then studied whether this exchanger is similar to or distinct from the Na(+)-H+ exchanger described in brush border (maternal-facing) membrane preparations. For this purpose, we compared the pharmacological characteristics of the basal membrane Na(+)-H+ exchanger with those of the brush border membrane Na(+)-H+ exchanger. The basal membrane exchanger was about 20-fold less sensitive to inhibition by amiloride and about 70-fold less sensitive to inhibition by dimethylamiloride than was the brush border membrane exchanger. The exchanger activity in both membrane preparations was inhibitable by clonidine and cimetidine, but the inhibition patterns with these compounds were markedly different between basal and brush border membrane preparations. These data demonstrate that the basal membrane Na(+)-H+ exchanger is distinct from the brush border membrane Na(+)-H+ exchanger. The pharmacological profiles of these exchangers indicate that the human placental brush border membrane possesses the housekeeping or non-epithelial type Na(+)-H+ exchanger (NHE-1), whereas the basal membrane possesses the epithelial or apical type Na(+)-H+ exchanger (NHE-2).

Localization and functional characterization of Na+/H+ exchanger isoform NHE4 in rat thick ascending limbs

2001 ◽  
Vol 281 (4) ◽  
pp. F707-F717 ◽  
Author(s):  
Régine Chambrey ◽  
Patricia L. St. John ◽  
Dominique Eladari ◽  
Fabienne Quentin ◽  
David G. Warnock ◽  
...  
Keyword(s):  
Rat Kidney ◽  
Basolateral Membrane ◽  
Functional Characterization ◽  
Membrane Vesicles ◽  
Affinity Constant ◽  
Thick Ascending Limb ◽  
Collecting Ducts ◽  
Inhibition Curve ◽  
Na Uptake

The Na+/H+ exchanger NHE4 was cloned from a rat stomach cDNA library and shown to be expressed predominantly in the stomach and less dramatically in the kidney. The role and precise localization of NHE4 in the kidney are still unknown. A polyclonal antibody against a unique NHE4 decapeptide was used for immunohistochemistry in rat kidney. Simultaneous use of antibodies to Tamm-Horsfall glycoprotein and aquaporin-2 or -3 permitted identification of thick ascending limbs and collecting ducts, respectively. The results indicate that NHE4 is highly expressed in basolateral membranes of thick ascending limb and distal convoluted tubule, whereas collecting ducts from cortex to inner medulla and proximal tubules showed weaker basolateral NHE4 expression. Western blot analysis of NHE4 in membrane fractions prepared from the inner stripe of the outer medulla revealed the presence of a 95-kDa protein that was enriched in basolateral membrane vesicles isolated from medullary thick ascending limbs. The inhibition curve of H+-activated 22Na uptake by 5-( N-ethyl- N-isopropyl)amiloride (EIPA) was consistent with the presence, beyond the EIPA high-affinity NHE1 isoform, of an EIPA low-affinity NHE with apparent half-maximal inhibition of 2.5 μM. Kinetic analyses showed that the extracellular Na+ dependence of NHE4 activity followed a simple hyperbolic relationship, with an apparent affinity constant of 12 mM. Intravesicular H+ activated NHE4 by a positive cooperative mechanism. NHE4 had an unusual low affinity for intravesicular H+ with a half-maximal activation value of p K6.21. We conclude that NHE4, like NHE1, is expressed on the basolateral membrane of multiple nephron segments. Nevertheless, these two proteins exhibited dramatically different affinities for intracellular H+, suggesting that they may play distinct physiological roles in the kidney.

Two cationic amino acid transport systems in human placental basal plasma membranes

1991 ◽  
Vol 261 (2) ◽  
pp. C246-C252 ◽  
Author(s):  
T. C. Furesz ◽  
A. J. Moe ◽  
C. H. Smith
Keyword(s):  
Amino Acids ◽  
Time Course ◽  
Plasma Membranes ◽  
Membrane Vesicles ◽  
Basal Membrane ◽  
Transport Systems ◽  
Cationic Amino Acid ◽  
Basal Plasma ◽  
Cationic Amino Acids

Transport of cationic amino acids in basal (fetal facing) plasma membranes was investigated by characterization of L-[3H]lysine and L-[3H]arginine uptake in membrane vesicles isolated from term human placentas. At least two Na(+)-independent systems were present. Lysine concentration dependence data were fit by a two-system model with Km values of 1.0 +/- 0.8 and 223 +/- 57 microM and Vmax values of 0.06 +/- 0.03 and 24.0 +/- 5.8 pmol.mg protein-1.min-1. In the presence of either 10 mM L-leucine or Na+ plus 10 mM L-homoserine, the data were fit by single system models with kinetic parameters similar to the higher and lower Km systems seen in the absence of inhibitors. Uptake of 10 or 20 microM L-lysine in the absence of Na+ showed the higher Km system was inhibited completely by L-arginine, L-homoarginine, and L-histidine. In the presence of Na+, the higher Km system was inhibited completely by L-alanine, L-homoserine, L-leucine, L-phenylalanine, and L-norleucine. The lower Km system was inhibited completely by L-arginine, L-homoarginine, L-histidine, L-leucine, and L-methionine. Time course studies of uptake demonstrated that uptake by either system alone filled the total vesicular space. The basal membrane of human placental syncytiotrophoblast possesses two transport systems for lysine and arginine, resembling the ubiquitous y+ system and the bo,+ system previously described in mouse blastocysts. The higher Vmax of the y+ system suggests that in utero it may mediate transfer of cationic amino acids from the syncytiotrophoblast to the fetus. The role of the high-affinity low-capacity bo,+ system remains to be determined.

Starvation-induced increase of hepatic alanine uptake is related to changes in sensitivity to SH-group reagents

1995 ◽  
Vol 268 (3) ◽  
pp. R598-R604
Author(s):  
A. Felipe ◽  
X. Remesar ◽  
M. Pastor-Anglada
Keyword(s):  
Amino Acids ◽  
Sulfhydryl Group ◽  
Maximum Velocity ◽  
Membrane Vesicles ◽  
Close Correlation ◽  
Affinity Constant ◽  
Plasma Membrane Vesicles ◽  
Adult Rats ◽  
Alanine Transport ◽  
Transport Kinetic

Changes in L-alanine transport in plasma membrane vesicles from livers of control and 24- and 48-h starved adult rats and the sensitivity of alanine uptake to sulfhydryl group reagents [N-ethylmaleimide (NEM) and p-chloromercuribenzenesulfonate (p-CMBS)] were studied. The portal concentration of certain amino acids was measured, and the relationship between L-alanine transport kinetic parameters and amino acid levels was analyzed. Starvation only induced a decrease in portal concentration of these amino acids that are mainly carried by Na(+)-dependent systems (85 and 61% for 24- and 48-h starved rats, respectively). Portal alanine concentration was lower in 24-h starved animals than in control rats (370 vs. 587 microM) and further decreased after 48 h of fasting (228 microM). Starvation induced an increase in maximum velocity (Vmax) values of Na(+)-dependent L-alanine transport (7.19, 8.97, and 12.38 pmol.U 5'-nucleotidase-1.10 s-1 for control and 24- and 48-h starved rats, respectively) with slight, but not significant, changes in the apparent Michaelis constant (Km) values (3.35, 2.63, and 2.20 mM for control and 24- and 48-h starved rats, respectively). Portal alanine showed a directly close correlation with Km values and inverse with Vmax values. The mean affinity constant values for the effects of NEM and p-CMBS on Na(+)-dependent L-alanine transport were lower in 48- (2.57 and 0.13 mM, respectively) and 24-h starved rats (3.59 and 0.32 mM, respectively) than in control rats (8.56 and 0.59 mM, respectively) and showed a directly strong correlation with kinetic characteristics of L-alanine transport and portal alanine concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

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