Biological Sciences, North Carolina State University, Raleigh, North Carolina 27695; and 2 Biology Department, Georgia State University, Atlanta, GA 30302-4010
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ABSTRACT |
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In the first of two sets of experiments,
the lumen-to-cell and cell-to-bath transport rates for glycine were
measured in the isolated-perfused medullary pars recta (S3 cells) of
the rabbit proximal tubule at multiple luminal glycine concentrations
(0-2.0 mM). The lumen-to-cell transport of glycine was saturated,
which permitted the calculation of the transport maximum of
disappearance rate of glycine from the lumen
(pmol · min1 · mm tubular
length
1), Km (mM), and
paracellular leak (pmol · min
1 · mm
tubular length
1 · mM
1) values for
this transport mechanism; these values were 4.3, 0.3, and 0.03, respectively. The cell-to-bath transport did not saturate but showed a
linear relationship to cellular glycine concentration, 0.58 pmol · min
1 · mm tubular
length
1 · mM
1. The second set of
experiments characterized the transport rate, cellular accumulation,
and metabolic rate of lumen-to-cell transported [3H]glycine in all segments (cell types) of the proximal
tubule, pars convoluta (S1 cells), cortical pars recta (S2 cells), and medullary pars recta (S3 cells). These proximal tubular segments were
isolated and perfused at a single glycine concentration of 11.2 µM.
From the results of this study and previous work (Barfuss DW and
Schafer JA. Am J Physiol 236: F149-F162, 1979), we
conclude that the axial heterogeneity for glycine lumen-to-cell and
cell-to-bath transport capacity extends to the medullary pars recta (S3
cells; S1 > S2 < S3 for lumen-to-cell transport and S1 > S2 > S3 for cell-to-bath transport). Also, we conclude that
lumen-to-cell transported glycine can be metabolized and its metabolic
rate displays axial heterogeneity (S1 > S2 > S3). The
physiological significances of these transport and metabolic
characteristics of the S3 cell type permits the medullary pars recta to
effectively recover glycine from very low luminal glycine
concentrations and makes glycine available for protective and
maintenance metabolism of the medullary pars recta.
kidney; glutathione; nephron
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INTRODUCTION |
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MUCH IS KNOWN ABOUT THE CHARACTERISTICS of amino acid transport in the epithelial cells of the proximal tubule as determined by several techniques, including whole animal studies, isolated tubule perfusion, microperfusion, cell culture, and membrane vesicle preparations (1, 2, 10, 14, 18, 25). These studies were primarily focused on the transport of the luminal membrane, lumen-to-cell transport. In most of these studies, it is not apparent which of the proximal tubular cell types (S1, S2, or S3) were studied and the metabolic fate of the transported glycine was not determined.
There are two sources of amino acids that are presented to the luminal membrane of the proximal tubule for transport. The first and largest amount is from filtration of blood at the glomerulus, and the second source is the result of digestion of filtered proteins by enzymes located in the brush border. The bulk of the filtered amino acids are removed from the ultrafiltrate by the proximal convoluted tubule (S1 cell type), leaving very little, if any, of the filtered amino acids to be absorbed by the later segments of the proximal tubule, cortical pars recta (S2 cell type), and medullary pars recta (S3 cell type). Free amino acids in the luminal fluid that result from protein digestion are probably absorbed on site or slightly "downstream" of the digestion site. The consequence of the avid lumen-to-cell absorption in the proximal convoluted tubule (S1) is the luminal membrane of the latter segments of the proximal tubule are exposed to very low amino acid concentrations. This implies that these latter segments must acquire the amino acids needed for cellular function from the fluid bathing them, thus the need for transporting amino acids into the tubular epithelia cells at the basolateral membrane. In fact, it has been shown that glycine specifically is avidly transported into the cortical pars recta (S2 cell type) at the basolateral membrane (3).
Because the concentration of amino acids in the luminal fluid of the proximal tubule decreases the farther it flows from the glomerulus, the transport characteristics of the amino acid transport mechanisms need to vary along the proximal tubule to ensure the maximal possible amino acid recovery. Namely, the proximal convoluted tubule (S1) has a much greater transport maximum, lesser affinity, and greater paracellular back-leak (blood-to-lumen) for amino acids than does the cortical proximal straight segment (S2) (3). The latter proximal tubular segments require a much greater affinity and less back-leak to absorb the smaller quantity amino acids from a very low luminal amino acid concentration. This pattern of axial heterogeneity of greater transport capacity and lesser affinity in the proximal convoluted tubule compared with a much lower transport capacity, much greater affinity and lesser back-leak in the proximal straight segments is also found for D-glucose absorption (4).
There are several recent reviews of amino acid transport in the kidney and intestine that indicate that the fractional excretion of glycine varies to a greater extent from species to species than do other amino acids (8, 12, 19, 26, 27). Rats and rabbits have a low urinary excretion coupled with a relatively low absorption rate in the proximal tubule (27). This leads to the conclusion that glycine is absorbed beyond the proximal convoluted tubule. It has also been shown that glycine uptake is Na+ dependent in studies using rabbit renal brush-border membrane vesicles (14) and rat canalicular liver plasma membrane vesicles (18).
The metabolic fate of cellular glycine in the proximal tubular cells is very diversified. In addition to being incorporated into structural and enzymatic proteins and used as an energy source, it is involved in protecting the cell against oxidative and osmotic stress. Glycine participates in protection against oxidative stress by two phenomena. One is the protective effect neutral amino acids, glycine being the most effective, affords renal tissue during anoxic episodes, such as periods of nonperfusion followed by reperfusion of blood (29). The mechanism for this protection has not been definitively determined. The second is that glycine is incorporated into the tri-amino acid GSH (20). GSH is the most abundant antioxidant in cells (5-10 mM). These protective effects of glycine are useful in all segments of the proximal tubule, but the medullary pars recta (S3) is the segment that is exposed to the greatest degree of oxidative and osmotic stress. Because it is located in the outer stripe of the outer medulla, the medullary pars recta (S3) can be subjected to a hypertonic environment during periods of water conservation. Under this condition, one mechanism tubular epithelial cells utilize to maintain cellular volume is to increase their intracellular osmolality by synthesizing organic osmolytes to counteract the extracellular hypertonicity (15). One of these organic osmolytes, betaine (N-trimethylglycine), is synthesized from glycine.
The first part of this study was designed to measure both lumen-to-cell and cell-to-bath transport characteristics for glycine in the medullary pars recta segment (S3), determining the values for transport maximum of the disappearance rate of glycine from the lumen (Jmax), Km, and the nonsaturable coefficient (k; presumably paracellualar leak). In a second series of experiments, we compared the cellular accumulation, extent of metabolism, and lumen-to-cell and cell-to-bath transport rates of [3H]glycine in the S1, S2, and S3 cell types of the proximal tubule.
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MATERIALS AND METHODS |
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Perfusing solutions containing [3H]glycine used in the metabolic experiments of this study were prepared fresh for each experiment. The [3H]glycine had a specific activity of 35.1 Ci/mmol and was stored in ethanol/water (2:98 vol/vol) at 5°C. For perfusion experiments, 20 µl of the [3H]glycine stock solution together with 10 µl of [14C]polyethylene glycol stock solution were dried by a stream nitrogen gas and subsequently reconstituted into 50 µl of artificial perfusion media. The final concentration of [3H]glycine was 11.2 µM. This low glycine concentration was selected to optimize the possibility of measuring any differences in metabolic rate among the three proximal tubule segments (cell types). As determined by HPLC analysis, 96.45 ± 1.27% of the 3H was associated with the glycine peak in the glycine stock solution. [14C]Polyethylene glycol (11.0 mCi/g) was present in the luminal perfusion solution (2 mM) as a volume marker in all perfusion experiments. All isotopes were purchased from New England Nuclear (Boston, MA). All other chemicals were purchased from Sigma (St. Louis, MO).
Perfusion solutions used in experiments to measure the Michaelis-Menten kinetics of the transport of glycine from the luminal fluid in the S3 segment were the same as described above and below but with the addition of nonradiolabeled glycine, resulting in the following final total glycine concentrations: 0.0112, 0.0885, 0.177, 0.354, 1.000, and 2.000 mM. A minimum of five tubules were perfused at each of these concentrations. In these experiments, the volume marker was L-[3H]glucose (14.6 Ci/mmol) and the tracer glycine was [14C]glycine (112.7 mCi/mmol).
The standard artificial perfusion media (3, 4, 20) was
used for perfusion and bathing solutions, which contained (in mM) 145 Na+, 140 Cl, 5.0 K+, 2.5 Ca2+, 1.2 Mg2+, 1.2 SO
Female New Zealand White, specific pathogen-free rabbits were purchased from Myrtle's Rabbitry (Thompson Station, TN). All rabbits were maintained on regular rabbit chow and given water ad libitum. Rabbits were anesthetized with ketamine (Ketastat 50 mg/ml), xylazine (100 mg/ml), and acepromazine maleate (10 mg/ml) purchased from Butler Chemical (Bedford, OH). The dose used was 1 ml/kg body wt of a ketamine-xylazine-acepromazine maleate (1:1:1) stock solution. All experiments were conducted according to the Guide for the Care and Use of Laboratory Animals (Washington, DC: National Academy Press, 1996).
Perfusion methods.
The technique of perfusing isolated segments of the nephron used in
this study was originally described by Burg (6) and subsequently modified in our laboratory (2, 3). Data
collected for this study were from isolated and perfused convoluted
(S1), cortical pars recta (S2), and medullary pars recta (S3) segments of the proximal tubule of the rabbit. Temperature was maintained by a
circulating water bath at 38°C. The lumen-to-bath transport of
glycine was measured as rates of disappearance of
[3H]glycine from the lumen
(JD, Gly;
fmol · min1 · mm tubular
length
1) and appearance of [3H]glycine in
the bathing solution (JA, Gly;
fmol · min
1 · mm tubular
length
1), and its accumulation in the cells of the
tubular segments (µM). Individual segments were dissected
manually from coronal sections (7, 9, 13) and identified
as described in Barfuss et al. (2) while the segments were
bathed in the phosphate-sucrose buffer solution.
Calculations.
All calculations to determine JD, Gly,
JA, Gly, and cell concentration are the same as
calculations previously published (2, 3, 6). Cell volume
(CV) was calculated as in previous studies (3)
from tubular dimensions as shown in the equation
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Transport characteristic studies. The experiments, designed to measure the Michaelis-Menten kinetic parameters (Jmax, Km, and k) of the transport of glycine from the luminal fluid in the medullary pars recta (S3) segment only, were conducted the same as the experiments that measured the metabolic rate of the transported glycine, except the perfusate, collectate, bathing fluid, and cellular extract samples were not subjected to HPLC analysis (see below).
Metabolic studies. Five samples of perfusate, collectate, and bathing fluid were collected from each perfused segment. Two samples were used for HPLC analysis while the remaining three samples were analyzed for total 3H (Beckman 5108 scintillation counter). The single sample of cellular extract per tubular segment was split into halves: one-half was used for HPLC analysis and one-half was used for total 3H analysis.
Chromatography. The dansyl (DNS) derivitization process for [3H]glycine, [3H]GSH, and [3H]metabolites, containing primary amines in each sample subjected to HPLC analysis, was a modification of several protocols (5, 16, 17, 22-24, 28) and was described previously (20). Briefly, samples generated from perfusion experiments were placed in microcentrifuge tubes with 800 µl of artificial perfusion media and 100 µl of a standard solution containing 1.3 mM of GSH, GSSG, glycine, and cysteine-glycine dissolved in 40 mM Li2CO3 at pH 9.5. In addition, 180 µl of 40 mM iodoacetic acid was added to stabilize the free sulfhydryl groups to prevent oxidation of GSH to GSSG. In perfusate, bath, and collectate samples, 8 µl of saturated KOH was added. To the TCA cellular extract of the tubule, 15 µl of saturated KOH was added. Samples were vortexed briefly (30 s), incubated at room temperature for 20 min, and stored at 4°C.
DNS-Cl was added to acetone in a 1.5 mg:1 ml ratio to make a 5.6 mM stock solution. The DNS-Cl solution (500 µl) was added to every sample, shaken gently for 2 min, and allowed to incubate in the dark for 55-60 min. This reaction was pH sensitive and most completely reacted at pH 9.0-9.2. To stop the reaction, 2 ml of chloroform were added and mixed well. This mixture was centrifuged for 5 min to separate the chloroform layer from the aqueous sample. A 1-ml aliquot from the top aqueous layer containing the dansylated products was loaded into the HPLC injector for analysis. The mobile phase was made of eluant A, a 0.05 M sodium acetate solution at pH 4.6, and eluant B, 100% HPLC grade methanol. DNS-glycine, DNS-GSH, DNS-GSSG, and DNS-cysteine-glycine were separated using the following gradient. The gradient program was linear from 0 to 36% eluant B in 6 min. From 6 to 15 min, eluant B continued to run at 36%. From 15 to 21 min, there was a linear gradient from 36 to 60% eluant B that remained constant until 22 min. From 22 to 32 min, the gradient was again linear from 60 to 100% eluant B. Then there was a continuation of 100% eluant B for 5 min to clean the column, followed by a 10-min equilibration period with 100% eluant A to restore initial conditions before a new sample was injected. This procedure generated sufficient fluorescent signals to produce clear chromatographic peaks for GSH, GSSG, glycine, and cysteine-glycine. Purchased dansylated standards of glycine, cysteine, and glutamate eluted ±10 s of the dansylated samples prepared in the laboratory. When tested for yield of dansylated [3H]glycine stock isotope, 96-98% of the 3H was recovered in the chromatographic peak associated with commercially available DNS-glycine. Dansylated standards for GSH, GSSG, and cysteine-glycine were not commercially available and were made and tested in the laboratory. When [3H]GSH was dansylated in the laboratory, 85-90% of the 3H was associated with the DNS-GSH peak and 5-10% was associated with the DNS-GSSG peak. The peak associated with cysteine-glycine was not tested with 3H, but in HPLC runs of collectate, bath, and tubule extracts, no significant 3H was associated with that peak.Confirmation of steady state. After an individual tubule was warmed and perfused, a period of 15 min was allowed for the attainment of steady state. Steady-state conditions were confirmed by samples for 30 min after the warm-up period (15 min) and constant values for all measured parameters. Consequently, for each tubule, samples were collected during the 30-45 min after the 15-min warm-up period.
Mathematical analysis of chromatographic data. For each HPLC analysis of a sample, all individual chromatographic peak and valley effluents were collected separately and counted for the total amount of 3H each contained. The amount of 3H associated with the glycine chromatographic peak was divided by the total amount of 3H in the sample, giving the fraction of the total sample 3H that was associated with glycine. This fraction was used to correct the corresponding samples that were used for calculation of the measured parameters JD, Gly, JA, Gly, and cellular concentration of glycine.
Statistics.
For the determination of the Jmax,
Km, and k for transport of luminal
glycine in the medullary pars recta (S3) only, the data were fitted to
the model
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(1) |
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RESULTS |
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Characterization of glycine transport.
Transport rate of glycine from the luminal fluid and
JA, Gly showed a curvilinear dependency on the
[Gly]ML. This relationship, shown in Fig.
1, appears to saturate and fits a
previously developed model (Eq. 1) (4) for a
combination of active (transcellular) and passive (paracellular)
transepithelial transport of solutes by the proximal tubular
epithelium. Using this model gave estimates for
Jmax, Km, and
k for both JD, Gly and
JA, Gly. There was no significant difference
between the respective values of these three parameters for
JD, Gly and JA, Gly, so
for simplicity only the JD, Gly data are shown
(Table 1) and plotted (Fig. 1, line
plot).
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Glycine metabolism.
The primary data from the glycine metabolism experiments of this study
are summarized in Table 2 and Figs.
4 and
5.
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DISCUSSION |
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Glycine transport characteristics in the medullary pars recta (S3) segment. The transport data of glycine in the medullary pars recta (S3) segment of the proximal tubule measured in this study support the typical two-route model for absorption and transepithelial transport of amino acids, namely, separate paracellular and cellular routes. The cellular route consists of the amino acid being actively transported at the luminal membrane into the cytoplasm and then passively diffusing down its chemical gradient across the basolateral membrane into the peritubular fluid. The paracellular route is the passive diffusion of the amino acid through the tight junction down its concentration gradient. This paracellular transport can be bidirectional from lumen-to-bath or bath-to-lumen, depending on the direction of the chemical gradient of the amino acid. In the isolated perfused tubules in this study, the glycine transport via the paracellular route was from lumen-to-bath because of the outwardly directed concentration gradient, but the in vivo glycine concentration gradient is assumed to be from the peritubular fluid to the lumen. Thus the amino acids back-leak into the luminal fluid from the blood. Too great of a blood-to-lumen paracellular leak (back-leak) would make the absorption of amino acids in the medullary pars recta (S3) segment an inefficient process. This futile cycle is prevented by much less paracellular blood-to-lumen flux, as reflected in the lower k values of the nonsaturable flux in the latter segments of the proximal tubule (Table. 1). In the medullary pars recta (S3) segment, the k value is not statistically different from 0, which greatly prevents glycine from leaking into the luminal fluid in vivo. This optimizes the absorption of glycine in the medullary pars recta (S3) segment. In addition, the paracellular leak for D-glucose is very low in the medullary pars recta (S3) segment compared with the convoluted (S1) and cortical pars recta (S2) segments, again indicating the need to minimize the back-leak of any absorbed substance (D-glucose, glycine, etc.) for its efficient recovery.
The transport of glycine from the luminal fluid at the luminal membrane of the medullary pars recta (S3) appears to have a greater affinity (lower Km) and a slightly greater Jmax than does the cortical pars recta (S2) segment of the proximal tubule (Table 1). This greater affinity of the transport mechanism for glycine permits the S3 cell types of the medullary pars recta to recover glycine against a greater lumen-to-cell concentration gradient than can the S1 or S2 cell types of the convoluted and cortical pars recta segments, respectively, of the proximal tubule. This axial location of the S3 cell types in the medullary pars recta segment is ideal for recovery of filtered glycine or glycine from enzymatic digestion of proteins and peptides occurring in all proximal tubular segments. The fact that Jmax is greater in the medullary pars recta (S3) than in the cortical pars recta (S2) segment may reflect the need for greater transport capacity in the medullary pars recta (S3) segment because of an increased load of glycine from digestion of proteins and peptides "upstream" in the brush border of the cortical pars recta (S2) segment. This same pattern of the medullary pars recta (S3) segment having a greater Jmax and lesser Km than the cortical pars recta (S2) segment is found for the transport of L-arginine (unpublished observations). However, this pattern of a greater Jmax in the medullary pars recta (S3) segment than in the cortical pars recta (S2) segment is not observed for D-glucose (4). This may reflect the very small load of D-glucose normally delivered to the medullary pars recta (S3) segment, because there is no addition of D-glucose to the luminal fluid as a result of upstream digestion of D-glucose-containing compounds and all filtered D-glucose had been absorbed upstream to the medullary pars recta. Finally, of all the proximal tubular segments, the convoluted (S1) segment has the greatest Jmax and Km for glycine and D-glucose (4), reflecting the need to transport large amounts of filtered glycine and D-glucose against a lesser gradient to minimize energy consumption for the recovery of the large filtered loads of these two compounds. This may be the general pattern of transport characteristics for all substances that are filtered and completely absorbed in the proximal tubule: greater transport capacity with lesser affinity in the early (convoluted) proximal tubule with much lesser transport capacity accompanied by greater affinity in the later (cortical and medullary pars recta) segments. To accompany this general pattern of transport characteristic along the proximal tubule with progressively decreasing leakiness of the tight junctions along the proximal tubule results in an effective process for completely recovering filtered substances that have nutritional value. Evidence that the transport of glycine from the luminal fluid to the cytoplasm in the medulla pars recta (S3) segment is an active process is indicated by the cell-to-lumen glycine concentration ratio, which is much greater than unity at all luminal glycine concentrations studied (Fig. 2). As shown in Fig. 2, the cell-to-lumen ratio exponentially increases as [Gly]ML decreases, indicative of an active transport process(es). In addition, the cellular glycine concentrations from the metabolic experiments (Table 2) indicate active luminal transport of glycine in the medulla pars recta (S3) as well as in the convoluted (S1) and cortical pars recta (S2) segments. This is indicated by cellular glycine concentrations significantly greater than [Gly]ML. Cell-to-bathing fluid glycine transport across the basolateral membrane of the medulla pars recta (S3) segment shows no indication of a saturable process over the range of glycine concentrations examined (Fig. 3). The cell-to-bath glycine transport mechanism at the basolateral membrane may have a very low affinity with a relatively limited transport capacity. This is indicated by the greater cellular glycine levels observed in the medullary pars recta (S3) segment than observed in the convoluted (S1) and cortical pars recta (S2) segments, while these latter segments had the same or greater level of transcellular glycine transport compared with the medullary pars recta (S3) segment (Fig. 4 and Table 2). Because the medullary region of the kidney is vulnerable to ischemia and is hypertonic, it could be speculated that the much greater cellular levels of glycine in the medullary pars recta (S3) segment could be important in protection against the toxic effects of hypoxia (29) and/or aiding in the regulation of cell volume. The protective effect of neutral amino acids, glycine being the most effective, against the toxic effects of hypoxia is well documented (29). The medullary region of the kidney is more vulnerable to hypoxia because of lesser blood flow than in the kidney cortex. The much greater cellular levels of glycine in the S3 cell types of the medullary pars recta could be important in proving it protection. This may be more important pathologically that physiologically. The S3 cell types of the medullary pars recta reside in an environment that changes in the osmolality, depending on the state of hydration of the body. In states of dehydration, the medullary region of the kidney becomes hypertonic, requiring the S3 cell types of the medullary pars recta to volume regulate. One of the mechanisms used to prevent cellular crenation in a hypertonic environment is the cellular accumulation of organic osmolytes (15). These cellular organic osmolytes can be acquired from the environment or synthesized. Betaine (N-trimethylglycine) is one of these organic osmolytes and is synthesized from glycine. Thus the greater levels of glycine observed in the S3 cell types of the medullary pars recta could provide a ready source of glycine for betaine synthesis during periods of hypertonicity.Metabolism of transported glycine in the medullary pars recta (S3) segment. HPLC analysis enabled us to determine the percentage of the transported [3H]glycine that remained in the cells and appeared in the bathing solution in the form of [3H]glycine. The analysis of the perfusion samples indicated 95-98% of the 3H was associated with the [3H]glycine chromatographic peak. Therefore, it was assumed that the 3H radiolabel was entering the lumen bound to glycine only. In the cellular samples, the majority (70-85%) of the radiolabel was still present as [3H]glycine, but [3H]metabolites of glycine were registered as well. Most notably, a significant percentage (8-10%) of the 3H radiolabel in the convoluted (S1) segments was recovered under the chromatographic peak corresponding to GSH (20). [3H]GSH was present in the cellular and luminal samples, implying that GSH, and possibly other peptides, could be synthesized and secreted into the lumen (20). The 3H that appeared in the bathing solution samples was predominantly (95-97%) associated with the glycine HPLC peak. This indicates that at the luminal glycine concentration of 11.2 µM, most of the metabolites of the luminally absorbed glycine are not transported across the basolateral membrane in the proximal tubule (cell-to-bathing solution).
In conclusion, we determined that glycine transport from the luminal solution is an active process in the medullary pars recta (S3) segment of the proximal tubule and confirms the presence of axial heterogeneity along the proximal tubule for active glycine transport (S1 >> S2 < S3). In addition, cellular accumulation of glycine also exhibits axial heterogeneity (S1 < S2 << S3). This latter axial heterogeneity of cellular accumulation of glycine could indicate a diminished transport capacity in basolateral membranes of segments more distant from the glomerulus. Certainly, a lesser cell-to-bath transport capacity of the basolateral membrane was found in the medullary pars recta (S3) segment. Also, the back-leak of glycine in the medullary pars recta (S3) is practically nonexistent because of the relative impermeability of the tight junctions to glycine in this segment of the proximal tubule. In addition, it is clear that glycine, which is transported into the cell across the luminal membrane, is available to enter into a variety of cellular metabolic pathways in all segments of the proximal tubule. This glycine metabolic rate exhibited axial heterogeneity, with the segments of the proximal tubule that are closest to the glomerulus having the greatest metabolic rates (S1 > S2 > S3). ![]() |
ACKNOWLEDGEMENTS |
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Angela Westin is acknowledged for perfusing the tubules for the glycine transport studies.
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FOOTNOTES |
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This study was supported by National Institute of Environmental Health Sciences Grant ES-05980 and funds provided by the Biology Department of Georgia State University.
Address for reprint requests and other correspondence: D. W. Barfuss, Biology Dept., PO Box 4010, Georgia State Univ., Atlanta, GA 30302-4010 (E-mail: biodwb{at}langate.gsu.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajprenal.00021.2002
Received 15 January 2002; accepted in final form 24 July 2002.
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