GASTROENTEROLOGY
1992;103:1817-1822
Sodium Transport in Human Intestinal Basolateral Membrane Vesicles ZAFAR
ZAMIR,
JEANNE
A. BARRY,
and KRISHNAMURTHY
Department of Medicine, Zablocki Veterans Administration Wisconsin, Milwaukee, Wisconsin
The current investigation was aimed at characterizing transport pathways for Na+ in basolateral membrane vesicles (BLMV) isolated from organ donor jejunum and ileum. An outward proton gradient [pH inside, 5.5; pH outside, 7.51 led to a 4-Sfold stimulation of transport rates compared with the absence of proton-gradient conditions in both human jejunal and ileal BLMV. Voltage-clamping the vesicles (K+ inside = K+ outside + valinomycin) reduced the uptake of 22Na by 2O%, indicating a minor conductive component of Na+ transport. Uptake of 22Na (1mmol/L) in voltage-clamped BLMV was inhibited 70% by 2 mmol/L amiloride. Li+ and NH,+ inhibited transport of 22Na into voltageclamped BLMV. Transport of Na+ exhibited saturation kinetics, and the Michaelis constant (I&) and V mex values for jejunum and ileum were similar [I&,, 27 f 3 mmol/L (jejunum) and 18 f 2 mmol/L (ileum); V,,,, 19 + 2 nmol . mg protein-’ +min-’ (jejunum) and 16 + 1 nmol- mg protein-’ - min-’ (ileum)]. V,,, values were ~15% of those reported for brush border membrane, whereas K,,, values were comparable. The results show that Na+ transport in human jejunal and ileal BLMV occurs via an Na+/H+ exchanger and a minor conductive pathway. he amiloride-sensitive Na+-H+ antiporter appears to be present in most if not all cell types.’ This exchanger present at the gastrointestinal and renal epithelial brush border membrane (BBM) plays a key role in Na+ and Cl- absorption, whereas the antiport at basolateral membrane (BLM) has been implicated in a variety of other cell functions, including regulation of cytoplasmic pH and initiation of cell growth and proliferation.‘4 This exchanger has been characterized using BBM vesicles (BBMV) from rat, rabbit, and human ileum,5-‘o human jejunum,l’ and rat co1on.‘2 Recently it was also investigated with different techniques in BLM from rabbit enterocytes,13 frog skin,14 oxyntic cells,15 rabbit cortical collecting tubule,16 proximal tubule of tiger salamander,17 and rat co10n.‘8*‘g Studies of Naf-H+ exchanger on the apical and basolateral surface of
T
RAMASWAMY
Medical Center, and the Medical College of
cultured porcine kidney cells (LLC-PK,) by Haggerty et a1.2oshowed that the apical system had a 50% inhibitory concentration (IC,,) for ethylisopropylamiloride of 13 pmol/L, whereas the basolateral system had an (I&,) of 44 nmol/L. These results led to suggest that there may be two distinct forms of Na+-H+ antiporter with separate genetic control. Similarly, Knickelbein et a1.21 described two distinct antiporters in rabbit ileal villus membrane vesicles on the basis of different kinetic characteristics and amiloride sensitivity. In addition to Na+-H+ exchange, a sodium bicarbonate cotransport has been shown in BLM vesicles (BLMV) isolated from rat kidney cortical cells,22,23 rat liver,24,25 rabbit renal cortex,‘” and rat colon.” However, very little information is available regarding Na+-H+ exchange and the related pathways at the human BLM. The current studies were aimed to investigate sodium transport in the human jejunal and ileal BLMV and to compare the pathways for Na+ transport at brush border membrane (BBM) and BLM. Our results show the presence of a distinct Na+-H+ exchanger in human intestinal BLMV. Materials and Methods Preparation
of Human
BLMV
Human small intestine from ligament of Treitz to the end of ileum was obtained at the time of organ donation after removal of all transplantable organs. It was opened along its antimesenteric border, rinsed with icecold 0.9% NaCl, and divided into four equal segments. The first segment was used to obtain jejunal mucosa and the last for ileal mucosa. The mucosal scrapings were kept frozen at -70°C in 5-10-g quantities and was used within 6 months. A total of five donor intestines was used for this study. On the morning of each experiment, BLMV were prepared by differential Percoll (Pharmacia, Piscataway, NJ) gradient centrifugation as described by Kikuchi and Ghishan.27 Briefly, the mucosal scraping was homogenized in Polytron (model PT 10/35; Brinkmann, Westbury, NY) for 30 seconds at full speed in 200 mL of buffer containing This is a U.S. government
work. There are no restrictions
its use. 0016-5085/92/$0.00
on
1818
ZAMIR
GASTROENTEROLOGY Vol. 103, No. 6
ET AL.
250 mmol/L sucrose, 10 mmol/L Tris-HEPES buffer (pH 7.5), 0.5 mmol/L Na, ethylenediaminetetraacetic acid, and 0.15 mmol/L phenylmethylsulfonyl fluoride (PMSF). The homogenate was centrifuged for 20 minutes at 2500g (model J2-21 Rotor JA-20; Beckman, Fullerton, CA) at 4°C. Supernatant was collected and centrifuged at 22,OOOg for 25 minutes. At the end of this spin, the supernatant was discarded; the resulting fluffy layer of pellet was resuspended in 200 mL of buffer containing 250 mmol/L sucrose, 10 mmol/L Tris-HEPES buffer (pH 7.5), 0.1 mmol/L PMSF, and 0.1 mmol/L MgCl, and homogenized in a glass Teflon homogenizer (20 strokes). The resulting homogenate was mixed with Percoll at a concentration of 15% and then centrifuged at 48,000g for 40 minutes. A distinct band of BLMV was seen at the upper one fourth of the Percoll gradient. This band was aspirated by a fine glass pipette and resuspended in the appropriate transport buffer followed by centrifugation at 48,000g for 15 minutes. The pellet was once again resuspended in the transport buffer and centrifuged at 48,000g for 15 minutes. The final pellet was resuspended in the desired transport buffer to a protein concentration of approximately 10 + 3 mg/mL, and the vesicles were used for transport studies after at least 1 hour at room temperature.
Transport
Studies
Transport studies were performed by the rapid filtration method of Hopfer et al.ZBAll experiments were performed at room temperature. Uptake was initiated by diluting the vesicle suspension lo-fold into the incubation medium containing ‘*Na+. At the desired time points, 50 pL (50-100 pg of protein)-aliquots of the reaction mix were transferred to 1 mL of ice-cold stop solution. Transport measurements, washing of filters, and counting were performed as previously described.g*” To correct for binding to filter, 45 pL of incubation medium was transferred to 1 mL of ice-cold stop solution and followed by addition of 5 pL of vesicles, and the mixture was immediately pipetted out on a prewetted filter. Radioactivity remaining on the filters after washing was used in calculations to correct for nonspecific binding. The composition of loading and incubation buffers is described in figure legends. Unless indicated otherwise, the stop solutions and the washing solutions consisted of 100 mmol/L potassium gluconate and 50 mmol/L Tris-2-[N-morpholinolethanesulfonic acid (pH 5.5) 50 mmol/L Tris-HEPES (pH 7.5), or 50 mmol/L TrisHEPES (pH 8) with appropriate concentrations of mannitol to maintain iso-osmolarity with the loading and the incubation buffers. Results are expressed as picomoles of Na+ uptake per milligrams of protein. Uptake studies were always performed in triplicate. The standard errors of the triplicate were less than +5% of the mean value, and therefore are not shown. Each experiment was repeated with at least three different membrane preparations. By use of different membrane preparations, qualitatively similar results were obtained, but in view of variation in intravesicular space between preparations, only results of typical experiments are shown in the figures. Protein content was assessed by the method of Lowry et a1.2g using bovine serum albumin as standard.
Materials 22Na+ (44.0 MBq/mL) was obtained from Amersham (Arlington Heights, IL). Amiloride was a gift from Merck, Sharpe & Dohme (West Point, PA). Membrane filters (0.45~pm pore size) were obtained from Sartorius Filters (Bohemia, NY). DIDS (4, 4’-di-isothiocyanato-2, 2’stilbenedisulfonic acid) was obtained from Aldrich Chemical Co., Milwaukee, WI). All other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO) and were of the highest purity available. Valinomycin was used as ethanolic stock, but final ethanol concentration in incubation medium never exceeded 0.7%.
Results Purity of Membrane
Vesicles
The purity of membrane vesicles was determined by measuring K+-stimulated p-nitrophenyl phosphatase activity. 3o The vesicles showed g-10fold enrichment compared with the homogenate. The stimulation of D-glucose transport by Na+ was not observed, indicating the absence of contamination with BBM. Studies of inhibition of D-glucose transport by phloretin and cytochalasin-B further confirmed the basolateral origin of the D-glucose transport system. Effect of pH Gradient
on Na+ Uptake
Figure 1 depicts the effects of the presence and absence of a pH gradient on Na+ uptake in human jejunal BLMV. Compared with the absence of pH
800
600
”
1
0
\\
;
;
;
9;
TIME (minutes)
Figure 1. Effect of pH gradient on Na+ uptake in human jejunal BLMV. Vesicles were preloaded with 100mmol/L potassium gluconate, 115 mmol/L mannitol, and 50 mmol Tris-Mes buffer (pH 5.5). Uptake was determined after diluting vesicles 10 times into incubation medium with same composition as the loading buffer or with 100 mmol/L potassium gluconate, 100 mmol/L mannitol, 50 mmol/L Tris-HEPES buffer (pH 7.5), and 1 mmol/L “Na+ as sodium gluconate. (0) pH,, 5.5; p&, 7.5. (m) pH,, 5.5; PH., 5.5.
December 19%
SODIUM TRANSPORT IN BLMV
Efiect of Amiloride
1819
on Na+ Transport
Amiloride has been established to be a specific inhibitor of Na+-H+ exchange process.4 To confirm that pH stimulation of Na+ uptake under voltage-clamped conditions was caused by a sodium proton antiporter, the effect of amiloride was examined by determining the effects of varying concentrations of amiloride. Figure 5 illustrates that amiloride inhibited Na+ uptake in jejunal BLMV in a dose-dependent fashion; analysis of the results using a Dixon plot (insert) yielded an inhibitor constant, Ki of 30 umol/L. 0
1
5
2 TIME
90
(minutes)
Figure 2. Effect of pH gradient on Na+ uptake in human ileal BLMV. Vesicles were preloaded with 100mmol/L potassium gluconate, 115 mmol/L mannitol, and 50 mmol/L Tris-Mes buffer (pH 5.5). Uptake was determined after diluting vesicles 10 times into incubation medium with same composition as the loading buffer or with 100 mmol/L potassium gluconate, 100 mmol/L mannitol, 50 mmol/L Tris-HEPES buffer (pH 7.5), and 1 mmol/L “Na+ as sodium gluconate. (0) pH,, 5.5; pH, 7.5. (B) pH,, 5.5; pH, 5.5.
gradient (pHi = pH, 5.5), an outwardly directed proton gradient (pHi, 5.5; pH,, 7.5) stimulated sodium uptake with an overshoot of l&fold over the equilibrium value. These results indicate that sodium uptake is coupled to proton efflux from the vesicles. Qualitatively similar results were obtained when ileal BLMV were studied (Figure 2).
Effect of Membrane
Kinetic Characteristics
of Na+-H+ Exchanger
Figure 6 depicts the effect of varying sodium concentrations (l-100 mmol/L) on the initial rates of pH-stimulated Na+ uptake in ileal BLMV. Uptake of Na+ occurred by a saturable process conforming to Michaelis-Menten kinetics. Kinetic analysis of Na+ uptake yielded a Michaelis constant (K,) of 18 f 2 prommol/L and a V,,, of 16 f 1 nmol.mg tein-’ - min-‘. Similar results showing saturation kinetics were obtained when jejunal BLMV were studied, with a K, of 27 ? 3 mmol/L and a V,,, of 19 rt 2 nmol - mg protein-’ - min-* (data not shown). Effect of Monovalent
Cations on Na+ Uptake
To determine the specificity of exchange process for cations, the ability of various monovalent
60 1
Potential
Because sodium and proton conductances could be responsible for apparent coupling of transport, the effect of a membrane potential in the absence of a pH gradient on “Na+ transport by jejunal BLMV was studied. An interior negative membrane potential was induced by preincubating the vesicles in 100 mmol/L KC1 and then diluting in incubation medium with 21 pmol/L valinomycin but without potassium. Figure 3 shows that sodium uptake rates at 0.1,0.2, and 0.3 minute were enhanced by an interior negative membrane potential; however, this increase in uptake was not inhibited by 1 mmol/L amiloride. When Na+ uptake studies were repeated in jejunal vesicles voltage-clamped with equal internal and external concentration of potassium and valinomycin and under pH gradient conditions (Figure 4), the reduction in uptake was only 2O%, showing that conductance is only a minor component of Na+ transport in BLMV. Similar results were obtained with ileal BLMV (results not shown).
, I / I I , , I / / , / I
L
K+, = K+. + vat
K+i > K+o + VA
Figure 3. Effect of membrane potential on Na+ uptake in human jejunal BLMV. Vesicles were preloaded with 100 mmol/L potassium gluconate, 100 mmof/L mannitol, and 50 mmol/L TrisHEPES buffer (pH 7.5). Uptake was determined at 0.1 @I),0.2 (Cl), and 0.3 (0) minutes after diluting 5 pL of vesicles in 45 pL of incubation medium containing either 100 mmol/L tetramethylammonium (TMA) gluconate or 100 mmol/L potassium gluconate, 50 mmol/L Tris-HEPES buffer (pH 7.5), 100 mmol/L mannitol, and 21 Pmol/L valinomycin, and 1 mmol/L %a+ as sodium gluconate. pH,, 7.5; pH,, 7.5.
1820
ZAMIR
ET AL.
GASTROENTEROLOGYVol. 103,No.6
4 TIME
(minutes)
Figure 4. Effect of voltage clamping on “Na+ uptake into human jejunal BLMV. Vesicles were preloaded with 20 mmol/L potassium gluconate, 80 mmol/L TMA, 115 mmol/L mannitol, and 50 mmol/L Tris-MES buffer (pH 5.5) and incubated in a medium containing 20 mmol/L potassium gluconate, 80 mmol/L TMA, 100 mmol/L mannitol, 50 mmol/L Tris-HEPES buffer (pH 7.5), 21 pmol/L valinomycin, and 1 mmol/L **Na+ as sodium gluconate (m) (pH,, 5.5; pHO, 7.5 + valinomycin). Vesicles were preloaded with 100 mmol/L potassium gluconate, 115mmol/L mannitol, and 50 mmol/L Tris-MES buffer (pH 5.5) and incubated in a medium containing 100 mmol/L potassium gluconate, 100 mmol/L mannitol, 50 mmol/L Tris-HEPES buffer (pH 7.5), and 1 mmol/L “Na+ as sodium gluconate (0) (pHi, 5.5; pHO, 7.5).
change process between BBM and BLM shows that there are significant differences in kinetic characteristics of Na+-H+ exchanger in the BBMV and BLMV. The K, values for Na+ uptake are similar for jejunal (29 f 6 mmol/L) and ileal (27 f 1 mmol/L) BBMV’,” and jejunal(27 + 3 mmol/L) and ileal(18 + 2 mmol/ L) BLMV. However, the V,,, values for jejunal(190 + 18 nmol - mg protein-’ - min-‘) and ileal (946 f 20 nmol - mg protein-l - min-‘) BBMV”” are severalfold higher than the values calculated for BLMV (19 f 2 and 16 + 1 nmol - mg protein-* - min-’ for jejunum and ileum, respectively). Knickelbein et alzl recently reported somewhat different results on the kinetic properties of the Na+-H+ exchanger in BBMV and BLMV from rabbit villus cells. They reported that the K,,, for Na+ transport was twofold greater on the BLM than on the BBM (46.3 f 3.4 vs. 28.8 + 2.3 mmol/L). They also found that the V,,, for Na+ transport was higher on the BLM than on the BBM pro(91.8 + 3.3 vs. 42.2 k 12.0 nmol-mg tein? . min-I). It is not known whether this variation in results could be ascribed to the species difference. Similar to what has been reported by other investigators,20~2*~31 we found that in human jejunal BLMV this exchanger is more sensitive to amiloride than the Na+-H+ exchanger in BBM. The Ki for amiloride was 30 umol/L, which is 3-4-fold lower than the K, for Na+-H+ exchanger (jejunal BBMV, 99 umol/L;
cations to compete with Na+ uptake was studied. Table 1 shows the percentage inhibition of 1 mmol/L “Na+ uptake by 20 mmol/L concentration of various cations under voltage-clamped conditions in jejunal BLMV. Uptake was inhibited by lithium, unlabeled sodium, and ammonium ions but not by other cations such as potassium, rubidium, and cesium. Discussion
Our previous investigations focused on the Na+-H+ exchange process in BBMV prepared from human ileum and jejunumgr” and showed that it plays a role in absorption of sodium chloride. Recent studies with various animal species showing evidence that this exchanger serves different functions and has distinct characteristics at BLM prompted us to characterize it in human small intestinal BLMV. Our results show that an outwardly directed proton gradient stimulated Na+ uptake in BLMV in the presence and absence of voltage-clamping. Amiloride inhibited this uptake under voltage-clamped conditions. Sodium uptake under voltage-clamped conditions was a saturable process, conforming to Michaelis-Menten kinetics. Lithium, ammonium, and unlabeled sodium ions inhibited this uptake. These results confirm the presence of Na+-H+ exchange mechanism in human intestinal BLMV. A comparison of the characteristics of the Naf ex-
0
I
I
I
0
50
100
I
150
I
200
AMILORIDE CONC. (PM)
Figure 5. Effect of amiloride on “Na+ uptake in human jejunal BLMV. Vesicles were preloaded with 100mmol/L potassium gluconate, 95 mmol/L mannitol, and 50 mmol/L Tris-MES buffer (pH 5.5) and incubated in a medium containing 100 mmol/L potassium gluconate, 50 mmol/L mannitol, 50 mmol/L TrisHEPES buffer (pH 8), 21 pmol/L valinomycin, 1 mmol/L “Na+ as gluconate, and various concentrations of amiloride. =Na+ uptake determined at 6 seconds is expressed as a percentage of that for vesicles without amiloride. The insert shows a Dixon plot of the absolute values of Na+ uptake with various amiloride concentrations.
December
1992
SODIUM TRANSPORT
ileal BBMV, 140 pmol/L) in human BBM.‘,” The specificity of human jejunal BLMV Na+-H+ exchanger for monovalent cations is the same as reported for renal cortex32,33 and human jejunum and ileum.gs’1 We did not find any significant inhibition by external K+, which is similar to the results reported by Knickelbein et al.‘l for rabbit villus epithelial cells and by Orsenigo et al. for rat jejunum.34 Besides the Na+-H+ antiport mechanism, the BLM has a conductive pathway for Na+ as evidenced by the following: (a) Voltage clamping reduced uptake by -20% under pH gradient conditions and (b) negative membrane potential increased uptake, which was not inhibited by amiloride. It should be mentioned that recent studies of Acra et aL31 showed the absence of any conductive pathway for Na+. The reason for this difference in results is not readily apparent. The basolateral Na+-H+ exchanger has been implicated in a variety of “house-keeping” cell functions such as regulation of cell pH and initiation of cell growth and proliferation. l4 The additional contribution of Na+/HCO,- symport process to homeostasis 1500
1000
500
Y
;r 5
0
20
40
60
80
100
SODIUM CONC. (mM)
Figure 6. Effect of external Na+ concentration on initial rates of “Na+ uptake in human ileal BLMV. Vesicles were preloaded with 130 mmol/L TMA gluconate, 20 mmol/L potassium gluconate, 15 mmol/L mannitol, and 50 mmol/L Tris-MES buffer (pH 5.5) and diluted into medium with an equivalent K+ concentration with 21 pmol/L valinomycin in which total TMA and sodium salt concentration was maintained at 130 mmol/L with appropriate addition of TMA gluconate. The stop and washing solutions consisted of 150 mmol/L potassium gluconate and 50 mmol/L Tris-HEPES (pH 7.5) or 150 mmol/L potassium gluconate, 15 mmol/L mannitol, and 60 mmol/L Tris-MES (pH 5.5). The pH gradient (~$5.5; pH, 7.5) induced uptake of **Na+ at 6 seconds was determined in the presence of increasing concentrations of sodium gluconate ranging from 1 to 100 mmol/L (the uptake was determined to be linear at 6 seconds). The graph is plotted after correcting the actual uptake values by subtracting the uptake values in the absence of a pH gradient. A Lineweaver-Burk plot of the data is shown in the insert.
Table 1. Effect of Cations on Na+ Uptake
IN BLMV
1821
in Human
Jejunai BLMV Cation Li+ NH,+ Na’ K+ Rb+ CS+
(20 mmol/L)
% Inhibition 48 + 2 53 * 2 31 f 2 2f5 9+6 2+7
NOTE. Values are mean + SEM (n = 3). Vesicles are preloaded with 20 mmol/L potassium gluconate, 80 mmol/L TMA gluconate, 115 mmol/L mannitol, and 50 mmol/L Tris-MES buffer (pH 5.5) and diluted into an external medium containing 20 mmol/L potassium gluconate, 60 mmol/L TMA gluconate, 100 mmoI/L mannitol, 50 mmol/L Tris-HEPES buffer (pH 7.5), 21 pmol/L valinomycin, 1 mmol/L “Na+, and 20 mmol/L of the Cl- salts of indicated cations. Uptake was measured at 6 seconds.
of cell pH has been suggested by the studies of Grass1 et a1.23,26in rat and rabbit renal cortex. Further studies by Soleimani et a1.35showed a stoichiometry of 3 HCO,:l Na+ using a thermodynamic approach. However, studies performed by Hagenbuch and MureP in rat intestinal BLMV and by Knickelbein et al.‘* in rabbit BLMV did not show any stimulation of Na+ uptake by HCO,- and hence the absence of a Na+/ HCO,- symport process. The presence of Na+/ HCO,- symport system has been shown in rat coionic BLMV by Rajendran et a1.l’ and by others for rat liver.24~25Our preliminary experiments showed that Naf uptake in human jejunal BLMV was stimulated 1.5-Z-fold when an inward HCO,- gradient was imposed in addition to an outward proton gradient. DIDS, which is a Na/HCO, cotransport inhibitor,Z6 decreased this uptake by 50%. This is suggestive of the existence of a Na/HCO, symport process in addition to Na+-H+ exchange. However, this stimulation was inhibited by amiloride (1 mmol/L). Similar results have been reported for rat colonic BLMV and for rat liver.1g,24*25However, this interesting aspect of Na+ transport needs to be further evaluated by exhaustive experiments before a definite conclusion can be drawn; this is the focus of ongoing experiments in our laboratory. In summary, we have shown that a kinetically distinct Na+-H+ exchanger exists in human small intestinal BLMV. This exchanger also differs from BBM Na+-H+ antiporter in amiloride sensitivity. Our preliminary studies are suggestive of a DIDS-sensitive Na+/HCO,- symport process. Further experiments need to be performed to fully characterize the Na+/ HCO,- symport pathway and its stoichiometry in human BLMV. These studies may shed further light on the role of transport pathways for Na+ in pH homeostasis of enterocytes.
1822 ZAMIR ET AL.
References 1. Grinstein
S, Rothstein A. Mechanisms of regulation of the Na’/H+ exchanger. J Membr Biol 1986;90:1-12. 2. Mahnensmith RL, Aronson P.S. The plasma membrane sodium-hydrogen exchanger and its role in physiological and pathophysiological processes. Circ Res 1985;56:773-788. 3. Pouyssegur J, Franchi A, Kohno M, L’Allemain G, Paris S. Na+-H+ exchange and growth control in fibroblasts: a genetic approach. Curr Top Membr Transport 1986;26:202-218. 4. Aronson PS. Kinetic properties of the plasma membrane Na+H+ exchanger. Annu Rev Physiol 1985;47:545-560. 5. Liedtke CM, Hopfer II. Mechanism of Cl- translocation across small intestinal brush-border membrane. I. Absence of Na+Cl- cotransport. Am J Physiol 1982;242:G263-G271. 6. Liedtke CM, Hopfer U. Mechanism of Cl- translocation across small intestinal brush-border membrane. II. Absence of Na+Cl- cotransport. Am J Physiol 1982;242:G272-G280. 7. Knickelbein R, Aronson PS, Atherton W, Dobbins JW. Sodium and chloride transport across rabbit ileal brush border. I. Evidence for Na-H exchange. Am J Physiol1983;245:G504-G510. a. Knickelbein R, Aronson PS, Schron CM, Seifter J, Dobbins JW. Sodium and chloride transport across rabbit ileal brush border. II. Evidence for Cl-HCO, exchange and mechanism of coupling. Am J Physiol 1985;249:G236-G245. 9. Ramaswamy K, Harig JM, Kleinman JG, Harris MS, Barry JA. Sodium-proton exchange in human ileal brush border membrane vesicles. Biochim Biophys Acta 1989;981:193-199. 10. Kikuchi K, Abumrad NN, Ghishan FK. Na+/H+ exchange by brush border membrane vesicles of human ileal small intestine. Gastroenterology 1988;95:388-393. 11. Kleinman JG, Harig JM, Barry JA, Ramaswamy K. Na+ and H+ transport in human jejunal brush-border membrane vesicles. Am J Physiol1988;255:G206-G211. 12. Foster ES, Dudeja PK, Brasitus TA. Na+-H+ exchange in rat colonic brush-border membrane vesicles. Am J Physiol 1986;250:G781-G787. 13. Barros F, Dominguez P, Velasco G, Lazo PS. Na+/H+ exchange is present in basolateral membranes from rabbit small intestine. Biochem Biophys Res Commun 1986;134:827-834. 14. Ehrenfeld J, Cragoe EJ Jr, Harvey BJ. Evidence for a Na+/H+ exchanger at the basolateral membranes of the isolated frog skin epithelium: effect of amiloride analogues. Pflugers Arch 1987;409:200-207. 15. Forte JG, Wolosin JM. HCl secretion by the gastric oxyntic cell. In: Johnson LR, ed. Physiology of the gastrointestinal tract. Volume 1. 2nd ed. New York: Raven, 1987:853-863. 16. Chaillet JR, Lopes AG, Boron WF. Basolateral Na-H exchange in the rabbit cortical collecting tubule. J Gen Physiol 1985;86:795-812. 17. Boron WF, Boulpaep EL. Intracellular pH regulation in the renal proximal tubule of the salamander. Na-H exchange. J Gen Physiol 1983;81:29-52. 18. Dudeja PK, Foster ES, Brasitus TA. Na+-H+ antiporter of rat colonic basolateral membrane vesicles. Am J Physiol 1989;257:G624-G632. 19. Rajendran VM, Oesterlin M, Binder HJ. Sodium uptake across basolateral membrane of rat distal colon. J Clin Invest 1991;88:1379-1385. 20. Haggerty JG, Agarwal N, Reilly RF, Adelberg EA, Slayman CW. Pharmacologically different Na/H antiporters on the apical and basolateral surfaces of cultured porcine kidney cells (LLC-PKi). Proc Nat1 Acad Sci USA 1988;85:6797-6801.
GASTROENTEROLOGY Vol. 103, No. 6
21. Knickelbein RG, Aronson PS, Dobbins JW. Characterization of Na+/H+ exchangers on villus cells in rabbit ileum. Am J Physiol 1990;259:G802-G806. 22. Hagenbuch B, Stange G, Murer H. Sodium-bicarbonate cotransport occurs in rat kidney cortical membranes but not in rat small intestinal basolateral membranes. Biochem J 1987;246:543-545. 23. Grass1 SM, Holohan PD, Ross CR. HCO,- transport in basolatera1 membrane vesicles isolated from rat renal cortex. J Biol Chem 1987;262:2682-2687. 24. Felipe A, Moule SK, McGivan JD. Bicarbonate stimulation of Na+ transport in liver basolateral plasma membrane vesicles requires the presence of a transmembrane pH gradient. Biochim Biophys Acta 1990;1029:61-66. 25. Renner EL, Lake JR, Scharschmidt BF, Zimmerli B, Meier PJ, Rat hepatocytes exhibit basolateral Na+/HCO,- cotransport. J Clin Invest 1989;83:1225-1235. 26. Grass1 SM, Aronson PS. Na+/HCO,- co-transport in basolatera1 membrane vesicles isolated from rabbit renal cortex. J Biol Chem 1986;261:8778-8783. 27. Kikuchi K, Ghishan FK. Phosphate transport by basolateral plasma membranes of human small intestine. Gastroenterology 1987;93:106-113. 28. Hopfer U, Nelson K, Perrotto J, Isselbacher KJ. Glucose transport in isolated brush border membrane from rat small intestine. J Biol Chem 1973;248:25-32. 29. Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265-275. 30. Murer H, Ammann E, Bibber J, Hopfer II. The surface membrane of the small intestinal epithelial cell. I. Localization of adenyl cyclase. Biochim Biophys Acta 1976;433:509-519, 31. Acra SA, Dykes W, Nylander W, Ghishan FK. Characterization of a distinct Na+/H* exchanger in the basolateral membranes of human jejunum (abstr). Gastroenterology 1991;100:A678. 32. Kinsella JL, Aronson P.S. Interaction of NH,+ and Li+ with the renal microvillus membrane Na+-H+ exchanger. Am J Physiol 1981;241:C220-C226. 33. Aronson PS. Mechanisms of active H+ secretion in the proximal tubule. Am J Physiol 1983;245:F647-F659. 34. Orsenigo MN, Rosco M, Zoppi S, Faelli A. Characterization of basolateral membrane Na/H antiport in rat jejunum. Biochim Biophys Acta 1990;1026:64-68. 35. Soleimani M, Grass1 SM, Aronson PS. Stoichiometry of Na+/ HCO,- cotransport in basolateral membrane vesicles isolated from rabbit renal cortex. J Clin Invest 1987;79:1276-1280.
Received December 27,1991.Accepted July 7,1992. Address requests for reprints to: Krishnamurthy Ramaswamy, Ph.D., Section of Digestive and Liver Diseases, University of Illinois at Chicago, 840 South Wood Street (M/C 787), Chicago, Illinois 60612. Supported by Grant DK33349 from the National Institute of Diabetes and Digestive and Kidney Diseases and by the Department of Veterans Affairs. This work was presented in part at the annual meeting of the American Gastroenterological Association in New Orleans, Louisiana, 1991, and was published in abstract form (Gastroenterology 1991;1oo:A710). The authors thank Anita Tredeau for her secretarial assistance.
1992;103:1817-1822
Sodium Transport in Human Intestinal Basolateral Membrane Vesicles ZAFAR
ZAMIR,
JEANNE
A. BARRY,
and KRISHNAMURTHY
Department of Medicine, Zablocki Veterans Administration Wisconsin, Milwaukee, Wisconsin
The current investigation was aimed at characterizing transport pathways for Na+ in basolateral membrane vesicles (BLMV) isolated from organ donor jejunum and ileum. An outward proton gradient [pH inside, 5.5; pH outside, 7.51 led to a 4-Sfold stimulation of transport rates compared with the absence of proton-gradient conditions in both human jejunal and ileal BLMV. Voltage-clamping the vesicles (K+ inside = K+ outside + valinomycin) reduced the uptake of 22Na by 2O%, indicating a minor conductive component of Na+ transport. Uptake of 22Na (1mmol/L) in voltage-clamped BLMV was inhibited 70% by 2 mmol/L amiloride. Li+ and NH,+ inhibited transport of 22Na into voltageclamped BLMV. Transport of Na+ exhibited saturation kinetics, and the Michaelis constant (I&) and V mex values for jejunum and ileum were similar [I&,, 27 f 3 mmol/L (jejunum) and 18 f 2 mmol/L (ileum); V,,,, 19 + 2 nmol . mg protein-’ +min-’ (jejunum) and 16 + 1 nmol- mg protein-’ - min-’ (ileum)]. V,,, values were ~15% of those reported for brush border membrane, whereas K,,, values were comparable. The results show that Na+ transport in human jejunal and ileal BLMV occurs via an Na+/H+ exchanger and a minor conductive pathway. he amiloride-sensitive Na+-H+ antiporter appears to be present in most if not all cell types.’ This exchanger present at the gastrointestinal and renal epithelial brush border membrane (BBM) plays a key role in Na+ and Cl- absorption, whereas the antiport at basolateral membrane (BLM) has been implicated in a variety of other cell functions, including regulation of cytoplasmic pH and initiation of cell growth and proliferation.‘4 This exchanger has been characterized using BBM vesicles (BBMV) from rat, rabbit, and human ileum,5-‘o human jejunum,l’ and rat co1on.‘2 Recently it was also investigated with different techniques in BLM from rabbit enterocytes,13 frog skin,14 oxyntic cells,15 rabbit cortical collecting tubule,16 proximal tubule of tiger salamander,17 and rat co10n.‘8*‘g Studies of Naf-H+ exchanger on the apical and basolateral surface of
T
RAMASWAMY
Medical Center, and the Medical College of
cultured porcine kidney cells (LLC-PK,) by Haggerty et a1.2oshowed that the apical system had a 50% inhibitory concentration (IC,,) for ethylisopropylamiloride of 13 pmol/L, whereas the basolateral system had an (I&,) of 44 nmol/L. These results led to suggest that there may be two distinct forms of Na+-H+ antiporter with separate genetic control. Similarly, Knickelbein et a1.21 described two distinct antiporters in rabbit ileal villus membrane vesicles on the basis of different kinetic characteristics and amiloride sensitivity. In addition to Na+-H+ exchange, a sodium bicarbonate cotransport has been shown in BLM vesicles (BLMV) isolated from rat kidney cortical cells,22,23 rat liver,24,25 rabbit renal cortex,‘” and rat colon.” However, very little information is available regarding Na+-H+ exchange and the related pathways at the human BLM. The current studies were aimed to investigate sodium transport in the human jejunal and ileal BLMV and to compare the pathways for Na+ transport at brush border membrane (BBM) and BLM. Our results show the presence of a distinct Na+-H+ exchanger in human intestinal BLMV. Materials and Methods Preparation
of Human
BLMV
Human small intestine from ligament of Treitz to the end of ileum was obtained at the time of organ donation after removal of all transplantable organs. It was opened along its antimesenteric border, rinsed with icecold 0.9% NaCl, and divided into four equal segments. The first segment was used to obtain jejunal mucosa and the last for ileal mucosa. The mucosal scrapings were kept frozen at -70°C in 5-10-g quantities and was used within 6 months. A total of five donor intestines was used for this study. On the morning of each experiment, BLMV were prepared by differential Percoll (Pharmacia, Piscataway, NJ) gradient centrifugation as described by Kikuchi and Ghishan.27 Briefly, the mucosal scraping was homogenized in Polytron (model PT 10/35; Brinkmann, Westbury, NY) for 30 seconds at full speed in 200 mL of buffer containing This is a U.S. government
work. There are no restrictions
its use. 0016-5085/92/$0.00
on
1818
ZAMIR
GASTROENTEROLOGY Vol. 103, No. 6
ET AL.
250 mmol/L sucrose, 10 mmol/L Tris-HEPES buffer (pH 7.5), 0.5 mmol/L Na, ethylenediaminetetraacetic acid, and 0.15 mmol/L phenylmethylsulfonyl fluoride (PMSF). The homogenate was centrifuged for 20 minutes at 2500g (model J2-21 Rotor JA-20; Beckman, Fullerton, CA) at 4°C. Supernatant was collected and centrifuged at 22,OOOg for 25 minutes. At the end of this spin, the supernatant was discarded; the resulting fluffy layer of pellet was resuspended in 200 mL of buffer containing 250 mmol/L sucrose, 10 mmol/L Tris-HEPES buffer (pH 7.5), 0.1 mmol/L PMSF, and 0.1 mmol/L MgCl, and homogenized in a glass Teflon homogenizer (20 strokes). The resulting homogenate was mixed with Percoll at a concentration of 15% and then centrifuged at 48,000g for 40 minutes. A distinct band of BLMV was seen at the upper one fourth of the Percoll gradient. This band was aspirated by a fine glass pipette and resuspended in the appropriate transport buffer followed by centrifugation at 48,000g for 15 minutes. The pellet was once again resuspended in the transport buffer and centrifuged at 48,000g for 15 minutes. The final pellet was resuspended in the desired transport buffer to a protein concentration of approximately 10 + 3 mg/mL, and the vesicles were used for transport studies after at least 1 hour at room temperature.
Transport
Studies
Transport studies were performed by the rapid filtration method of Hopfer et al.ZBAll experiments were performed at room temperature. Uptake was initiated by diluting the vesicle suspension lo-fold into the incubation medium containing ‘*Na+. At the desired time points, 50 pL (50-100 pg of protein)-aliquots of the reaction mix were transferred to 1 mL of ice-cold stop solution. Transport measurements, washing of filters, and counting were performed as previously described.g*” To correct for binding to filter, 45 pL of incubation medium was transferred to 1 mL of ice-cold stop solution and followed by addition of 5 pL of vesicles, and the mixture was immediately pipetted out on a prewetted filter. Radioactivity remaining on the filters after washing was used in calculations to correct for nonspecific binding. The composition of loading and incubation buffers is described in figure legends. Unless indicated otherwise, the stop solutions and the washing solutions consisted of 100 mmol/L potassium gluconate and 50 mmol/L Tris-2-[N-morpholinolethanesulfonic acid (pH 5.5) 50 mmol/L Tris-HEPES (pH 7.5), or 50 mmol/L TrisHEPES (pH 8) with appropriate concentrations of mannitol to maintain iso-osmolarity with the loading and the incubation buffers. Results are expressed as picomoles of Na+ uptake per milligrams of protein. Uptake studies were always performed in triplicate. The standard errors of the triplicate were less than +5% of the mean value, and therefore are not shown. Each experiment was repeated with at least three different membrane preparations. By use of different membrane preparations, qualitatively similar results were obtained, but in view of variation in intravesicular space between preparations, only results of typical experiments are shown in the figures. Protein content was assessed by the method of Lowry et a1.2g using bovine serum albumin as standard.
Materials 22Na+ (44.0 MBq/mL) was obtained from Amersham (Arlington Heights, IL). Amiloride was a gift from Merck, Sharpe & Dohme (West Point, PA). Membrane filters (0.45~pm pore size) were obtained from Sartorius Filters (Bohemia, NY). DIDS (4, 4’-di-isothiocyanato-2, 2’stilbenedisulfonic acid) was obtained from Aldrich Chemical Co., Milwaukee, WI). All other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO) and were of the highest purity available. Valinomycin was used as ethanolic stock, but final ethanol concentration in incubation medium never exceeded 0.7%.
Results Purity of Membrane
Vesicles
The purity of membrane vesicles was determined by measuring K+-stimulated p-nitrophenyl phosphatase activity. 3o The vesicles showed g-10fold enrichment compared with the homogenate. The stimulation of D-glucose transport by Na+ was not observed, indicating the absence of contamination with BBM. Studies of inhibition of D-glucose transport by phloretin and cytochalasin-B further confirmed the basolateral origin of the D-glucose transport system. Effect of pH Gradient
on Na+ Uptake
Figure 1 depicts the effects of the presence and absence of a pH gradient on Na+ uptake in human jejunal BLMV. Compared with the absence of pH
800
600
”
1
0
\\
;
;
;
9;
TIME (minutes)
Figure 1. Effect of pH gradient on Na+ uptake in human jejunal BLMV. Vesicles were preloaded with 100mmol/L potassium gluconate, 115 mmol/L mannitol, and 50 mmol Tris-Mes buffer (pH 5.5). Uptake was determined after diluting vesicles 10 times into incubation medium with same composition as the loading buffer or with 100 mmol/L potassium gluconate, 100 mmol/L mannitol, 50 mmol/L Tris-HEPES buffer (pH 7.5), and 1 mmol/L “Na+ as sodium gluconate. (0) pH,, 5.5; p&, 7.5. (m) pH,, 5.5; PH., 5.5.
December 19%
SODIUM TRANSPORT IN BLMV
Efiect of Amiloride
1819
on Na+ Transport
Amiloride has been established to be a specific inhibitor of Na+-H+ exchange process.4 To confirm that pH stimulation of Na+ uptake under voltage-clamped conditions was caused by a sodium proton antiporter, the effect of amiloride was examined by determining the effects of varying concentrations of amiloride. Figure 5 illustrates that amiloride inhibited Na+ uptake in jejunal BLMV in a dose-dependent fashion; analysis of the results using a Dixon plot (insert) yielded an inhibitor constant, Ki of 30 umol/L. 0
1
5
2 TIME
90
(minutes)
Figure 2. Effect of pH gradient on Na+ uptake in human ileal BLMV. Vesicles were preloaded with 100mmol/L potassium gluconate, 115 mmol/L mannitol, and 50 mmol/L Tris-Mes buffer (pH 5.5). Uptake was determined after diluting vesicles 10 times into incubation medium with same composition as the loading buffer or with 100 mmol/L potassium gluconate, 100 mmol/L mannitol, 50 mmol/L Tris-HEPES buffer (pH 7.5), and 1 mmol/L “Na+ as sodium gluconate. (0) pH,, 5.5; pH, 7.5. (B) pH,, 5.5; pH, 5.5.
gradient (pHi = pH, 5.5), an outwardly directed proton gradient (pHi, 5.5; pH,, 7.5) stimulated sodium uptake with an overshoot of l&fold over the equilibrium value. These results indicate that sodium uptake is coupled to proton efflux from the vesicles. Qualitatively similar results were obtained when ileal BLMV were studied (Figure 2).
Effect of Membrane
Kinetic Characteristics
of Na+-H+ Exchanger
Figure 6 depicts the effect of varying sodium concentrations (l-100 mmol/L) on the initial rates of pH-stimulated Na+ uptake in ileal BLMV. Uptake of Na+ occurred by a saturable process conforming to Michaelis-Menten kinetics. Kinetic analysis of Na+ uptake yielded a Michaelis constant (K,) of 18 f 2 prommol/L and a V,,, of 16 f 1 nmol.mg tein-’ - min-‘. Similar results showing saturation kinetics were obtained when jejunal BLMV were studied, with a K, of 27 ? 3 mmol/L and a V,,, of 19 rt 2 nmol - mg protein-’ - min-* (data not shown). Effect of Monovalent
Cations on Na+ Uptake
To determine the specificity of exchange process for cations, the ability of various monovalent
60 1
Potential
Because sodium and proton conductances could be responsible for apparent coupling of transport, the effect of a membrane potential in the absence of a pH gradient on “Na+ transport by jejunal BLMV was studied. An interior negative membrane potential was induced by preincubating the vesicles in 100 mmol/L KC1 and then diluting in incubation medium with 21 pmol/L valinomycin but without potassium. Figure 3 shows that sodium uptake rates at 0.1,0.2, and 0.3 minute were enhanced by an interior negative membrane potential; however, this increase in uptake was not inhibited by 1 mmol/L amiloride. When Na+ uptake studies were repeated in jejunal vesicles voltage-clamped with equal internal and external concentration of potassium and valinomycin and under pH gradient conditions (Figure 4), the reduction in uptake was only 2O%, showing that conductance is only a minor component of Na+ transport in BLMV. Similar results were obtained with ileal BLMV (results not shown).
, I / I I , , I / / , / I
L
K+, = K+. + vat
K+i > K+o + VA
Figure 3. Effect of membrane potential on Na+ uptake in human jejunal BLMV. Vesicles were preloaded with 100 mmol/L potassium gluconate, 100 mmof/L mannitol, and 50 mmol/L TrisHEPES buffer (pH 7.5). Uptake was determined at 0.1 @I),0.2 (Cl), and 0.3 (0) minutes after diluting 5 pL of vesicles in 45 pL of incubation medium containing either 100 mmol/L tetramethylammonium (TMA) gluconate or 100 mmol/L potassium gluconate, 50 mmol/L Tris-HEPES buffer (pH 7.5), 100 mmol/L mannitol, and 21 Pmol/L valinomycin, and 1 mmol/L %a+ as sodium gluconate. pH,, 7.5; pH,, 7.5.
1820
ZAMIR
ET AL.
GASTROENTEROLOGYVol. 103,No.6
4 TIME
(minutes)
Figure 4. Effect of voltage clamping on “Na+ uptake into human jejunal BLMV. Vesicles were preloaded with 20 mmol/L potassium gluconate, 80 mmol/L TMA, 115 mmol/L mannitol, and 50 mmol/L Tris-MES buffer (pH 5.5) and incubated in a medium containing 20 mmol/L potassium gluconate, 80 mmol/L TMA, 100 mmol/L mannitol, 50 mmol/L Tris-HEPES buffer (pH 7.5), 21 pmol/L valinomycin, and 1 mmol/L **Na+ as sodium gluconate (m) (pH,, 5.5; pHO, 7.5 + valinomycin). Vesicles were preloaded with 100 mmol/L potassium gluconate, 115mmol/L mannitol, and 50 mmol/L Tris-MES buffer (pH 5.5) and incubated in a medium containing 100 mmol/L potassium gluconate, 100 mmol/L mannitol, 50 mmol/L Tris-HEPES buffer (pH 7.5), and 1 mmol/L “Na+ as sodium gluconate (0) (pHi, 5.5; pHO, 7.5).
change process between BBM and BLM shows that there are significant differences in kinetic characteristics of Na+-H+ exchanger in the BBMV and BLMV. The K, values for Na+ uptake are similar for jejunal (29 f 6 mmol/L) and ileal (27 f 1 mmol/L) BBMV’,” and jejunal(27 + 3 mmol/L) and ileal(18 + 2 mmol/ L) BLMV. However, the V,,, values for jejunal(190 + 18 nmol - mg protein-’ - min-‘) and ileal (946 f 20 nmol - mg protein-l - min-‘) BBMV”” are severalfold higher than the values calculated for BLMV (19 f 2 and 16 + 1 nmol - mg protein-* - min-’ for jejunum and ileum, respectively). Knickelbein et alzl recently reported somewhat different results on the kinetic properties of the Na+-H+ exchanger in BBMV and BLMV from rabbit villus cells. They reported that the K,,, for Na+ transport was twofold greater on the BLM than on the BBM (46.3 f 3.4 vs. 28.8 + 2.3 mmol/L). They also found that the V,,, for Na+ transport was higher on the BLM than on the BBM pro(91.8 + 3.3 vs. 42.2 k 12.0 nmol-mg tein? . min-I). It is not known whether this variation in results could be ascribed to the species difference. Similar to what has been reported by other investigators,20~2*~31 we found that in human jejunal BLMV this exchanger is more sensitive to amiloride than the Na+-H+ exchanger in BBM. The Ki for amiloride was 30 umol/L, which is 3-4-fold lower than the K, for Na+-H+ exchanger (jejunal BBMV, 99 umol/L;
cations to compete with Na+ uptake was studied. Table 1 shows the percentage inhibition of 1 mmol/L “Na+ uptake by 20 mmol/L concentration of various cations under voltage-clamped conditions in jejunal BLMV. Uptake was inhibited by lithium, unlabeled sodium, and ammonium ions but not by other cations such as potassium, rubidium, and cesium. Discussion
Our previous investigations focused on the Na+-H+ exchange process in BBMV prepared from human ileum and jejunumgr” and showed that it plays a role in absorption of sodium chloride. Recent studies with various animal species showing evidence that this exchanger serves different functions and has distinct characteristics at BLM prompted us to characterize it in human small intestinal BLMV. Our results show that an outwardly directed proton gradient stimulated Na+ uptake in BLMV in the presence and absence of voltage-clamping. Amiloride inhibited this uptake under voltage-clamped conditions. Sodium uptake under voltage-clamped conditions was a saturable process, conforming to Michaelis-Menten kinetics. Lithium, ammonium, and unlabeled sodium ions inhibited this uptake. These results confirm the presence of Na+-H+ exchange mechanism in human intestinal BLMV. A comparison of the characteristics of the Naf ex-
0
I
I
I
0
50
100
I
150
I
200
AMILORIDE CONC. (PM)
Figure 5. Effect of amiloride on “Na+ uptake in human jejunal BLMV. Vesicles were preloaded with 100mmol/L potassium gluconate, 95 mmol/L mannitol, and 50 mmol/L Tris-MES buffer (pH 5.5) and incubated in a medium containing 100 mmol/L potassium gluconate, 50 mmol/L mannitol, 50 mmol/L TrisHEPES buffer (pH 8), 21 pmol/L valinomycin, 1 mmol/L “Na+ as gluconate, and various concentrations of amiloride. =Na+ uptake determined at 6 seconds is expressed as a percentage of that for vesicles without amiloride. The insert shows a Dixon plot of the absolute values of Na+ uptake with various amiloride concentrations.
December
1992
SODIUM TRANSPORT
ileal BBMV, 140 pmol/L) in human BBM.‘,” The specificity of human jejunal BLMV Na+-H+ exchanger for monovalent cations is the same as reported for renal cortex32,33 and human jejunum and ileum.gs’1 We did not find any significant inhibition by external K+, which is similar to the results reported by Knickelbein et al.‘l for rabbit villus epithelial cells and by Orsenigo et al. for rat jejunum.34 Besides the Na+-H+ antiport mechanism, the BLM has a conductive pathway for Na+ as evidenced by the following: (a) Voltage clamping reduced uptake by -20% under pH gradient conditions and (b) negative membrane potential increased uptake, which was not inhibited by amiloride. It should be mentioned that recent studies of Acra et aL31 showed the absence of any conductive pathway for Na+. The reason for this difference in results is not readily apparent. The basolateral Na+-H+ exchanger has been implicated in a variety of “house-keeping” cell functions such as regulation of cell pH and initiation of cell growth and proliferation. l4 The additional contribution of Na+/HCO,- symport process to homeostasis 1500
1000
500
Y
;r 5
0
20
40
60
80
100
SODIUM CONC. (mM)
Figure 6. Effect of external Na+ concentration on initial rates of “Na+ uptake in human ileal BLMV. Vesicles were preloaded with 130 mmol/L TMA gluconate, 20 mmol/L potassium gluconate, 15 mmol/L mannitol, and 50 mmol/L Tris-MES buffer (pH 5.5) and diluted into medium with an equivalent K+ concentration with 21 pmol/L valinomycin in which total TMA and sodium salt concentration was maintained at 130 mmol/L with appropriate addition of TMA gluconate. The stop and washing solutions consisted of 150 mmol/L potassium gluconate and 50 mmol/L Tris-HEPES (pH 7.5) or 150 mmol/L potassium gluconate, 15 mmol/L mannitol, and 60 mmol/L Tris-MES (pH 5.5). The pH gradient (~$5.5; pH, 7.5) induced uptake of **Na+ at 6 seconds was determined in the presence of increasing concentrations of sodium gluconate ranging from 1 to 100 mmol/L (the uptake was determined to be linear at 6 seconds). The graph is plotted after correcting the actual uptake values by subtracting the uptake values in the absence of a pH gradient. A Lineweaver-Burk plot of the data is shown in the insert.
Table 1. Effect of Cations on Na+ Uptake
IN BLMV
1821
in Human
Jejunai BLMV Cation Li+ NH,+ Na’ K+ Rb+ CS+
(20 mmol/L)
% Inhibition 48 + 2 53 * 2 31 f 2 2f5 9+6 2+7
NOTE. Values are mean + SEM (n = 3). Vesicles are preloaded with 20 mmol/L potassium gluconate, 80 mmol/L TMA gluconate, 115 mmol/L mannitol, and 50 mmol/L Tris-MES buffer (pH 5.5) and diluted into an external medium containing 20 mmol/L potassium gluconate, 60 mmol/L TMA gluconate, 100 mmoI/L mannitol, 50 mmol/L Tris-HEPES buffer (pH 7.5), 21 pmol/L valinomycin, 1 mmol/L “Na+, and 20 mmol/L of the Cl- salts of indicated cations. Uptake was measured at 6 seconds.
of cell pH has been suggested by the studies of Grass1 et a1.23,26in rat and rabbit renal cortex. Further studies by Soleimani et a1.35showed a stoichiometry of 3 HCO,:l Na+ using a thermodynamic approach. However, studies performed by Hagenbuch and MureP in rat intestinal BLMV and by Knickelbein et al.‘* in rabbit BLMV did not show any stimulation of Na+ uptake by HCO,- and hence the absence of a Na+/ HCO,- symport process. The presence of Na+/ HCO,- symport system has been shown in rat coionic BLMV by Rajendran et a1.l’ and by others for rat liver.24~25Our preliminary experiments showed that Naf uptake in human jejunal BLMV was stimulated 1.5-Z-fold when an inward HCO,- gradient was imposed in addition to an outward proton gradient. DIDS, which is a Na/HCO, cotransport inhibitor,Z6 decreased this uptake by 50%. This is suggestive of the existence of a Na/HCO, symport process in addition to Na+-H+ exchange. However, this stimulation was inhibited by amiloride (1 mmol/L). Similar results have been reported for rat colonic BLMV and for rat liver.1g,24*25However, this interesting aspect of Na+ transport needs to be further evaluated by exhaustive experiments before a definite conclusion can be drawn; this is the focus of ongoing experiments in our laboratory. In summary, we have shown that a kinetically distinct Na+-H+ exchanger exists in human small intestinal BLMV. This exchanger also differs from BBM Na+-H+ antiporter in amiloride sensitivity. Our preliminary studies are suggestive of a DIDS-sensitive Na+/HCO,- symport process. Further experiments need to be performed to fully characterize the Na+/ HCO,- symport pathway and its stoichiometry in human BLMV. These studies may shed further light on the role of transport pathways for Na+ in pH homeostasis of enterocytes.
1822 ZAMIR ET AL.
References 1. Grinstein
S, Rothstein A. Mechanisms of regulation of the Na’/H+ exchanger. J Membr Biol 1986;90:1-12. 2. Mahnensmith RL, Aronson P.S. The plasma membrane sodium-hydrogen exchanger and its role in physiological and pathophysiological processes. Circ Res 1985;56:773-788. 3. Pouyssegur J, Franchi A, Kohno M, L’Allemain G, Paris S. Na+-H+ exchange and growth control in fibroblasts: a genetic approach. Curr Top Membr Transport 1986;26:202-218. 4. Aronson PS. Kinetic properties of the plasma membrane Na+H+ exchanger. Annu Rev Physiol 1985;47:545-560. 5. Liedtke CM, Hopfer II. Mechanism of Cl- translocation across small intestinal brush-border membrane. I. Absence of Na+Cl- cotransport. Am J Physiol 1982;242:G263-G271. 6. Liedtke CM, Hopfer U. Mechanism of Cl- translocation across small intestinal brush-border membrane. II. Absence of Na+Cl- cotransport. Am J Physiol 1982;242:G272-G280. 7. Knickelbein R, Aronson PS, Atherton W, Dobbins JW. Sodium and chloride transport across rabbit ileal brush border. I. Evidence for Na-H exchange. Am J Physiol1983;245:G504-G510. a. Knickelbein R, Aronson PS, Schron CM, Seifter J, Dobbins JW. Sodium and chloride transport across rabbit ileal brush border. II. Evidence for Cl-HCO, exchange and mechanism of coupling. Am J Physiol 1985;249:G236-G245. 9. Ramaswamy K, Harig JM, Kleinman JG, Harris MS, Barry JA. Sodium-proton exchange in human ileal brush border membrane vesicles. Biochim Biophys Acta 1989;981:193-199. 10. Kikuchi K, Abumrad NN, Ghishan FK. Na+/H+ exchange by brush border membrane vesicles of human ileal small intestine. Gastroenterology 1988;95:388-393. 11. Kleinman JG, Harig JM, Barry JA, Ramaswamy K. Na+ and H+ transport in human jejunal brush-border membrane vesicles. Am J Physiol1988;255:G206-G211. 12. Foster ES, Dudeja PK, Brasitus TA. Na+-H+ exchange in rat colonic brush-border membrane vesicles. Am J Physiol 1986;250:G781-G787. 13. Barros F, Dominguez P, Velasco G, Lazo PS. Na+/H+ exchange is present in basolateral membranes from rabbit small intestine. Biochem Biophys Res Commun 1986;134:827-834. 14. Ehrenfeld J, Cragoe EJ Jr, Harvey BJ. Evidence for a Na+/H+ exchanger at the basolateral membranes of the isolated frog skin epithelium: effect of amiloride analogues. Pflugers Arch 1987;409:200-207. 15. Forte JG, Wolosin JM. HCl secretion by the gastric oxyntic cell. In: Johnson LR, ed. Physiology of the gastrointestinal tract. Volume 1. 2nd ed. New York: Raven, 1987:853-863. 16. Chaillet JR, Lopes AG, Boron WF. Basolateral Na-H exchange in the rabbit cortical collecting tubule. J Gen Physiol 1985;86:795-812. 17. Boron WF, Boulpaep EL. Intracellular pH regulation in the renal proximal tubule of the salamander. Na-H exchange. J Gen Physiol 1983;81:29-52. 18. Dudeja PK, Foster ES, Brasitus TA. Na+-H+ antiporter of rat colonic basolateral membrane vesicles. Am J Physiol 1989;257:G624-G632. 19. Rajendran VM, Oesterlin M, Binder HJ. Sodium uptake across basolateral membrane of rat distal colon. J Clin Invest 1991;88:1379-1385. 20. Haggerty JG, Agarwal N, Reilly RF, Adelberg EA, Slayman CW. Pharmacologically different Na/H antiporters on the apical and basolateral surfaces of cultured porcine kidney cells (LLC-PKi). Proc Nat1 Acad Sci USA 1988;85:6797-6801.
GASTROENTEROLOGY Vol. 103, No. 6
21. Knickelbein RG, Aronson PS, Dobbins JW. Characterization of Na+/H+ exchangers on villus cells in rabbit ileum. Am J Physiol 1990;259:G802-G806. 22. Hagenbuch B, Stange G, Murer H. Sodium-bicarbonate cotransport occurs in rat kidney cortical membranes but not in rat small intestinal basolateral membranes. Biochem J 1987;246:543-545. 23. Grass1 SM, Holohan PD, Ross CR. HCO,- transport in basolatera1 membrane vesicles isolated from rat renal cortex. J Biol Chem 1987;262:2682-2687. 24. Felipe A, Moule SK, McGivan JD. Bicarbonate stimulation of Na+ transport in liver basolateral plasma membrane vesicles requires the presence of a transmembrane pH gradient. Biochim Biophys Acta 1990;1029:61-66. 25. Renner EL, Lake JR, Scharschmidt BF, Zimmerli B, Meier PJ, Rat hepatocytes exhibit basolateral Na+/HCO,- cotransport. J Clin Invest 1989;83:1225-1235. 26. Grass1 SM, Aronson PS. Na+/HCO,- co-transport in basolatera1 membrane vesicles isolated from rabbit renal cortex. J Biol Chem 1986;261:8778-8783. 27. Kikuchi K, Ghishan FK. Phosphate transport by basolateral plasma membranes of human small intestine. Gastroenterology 1987;93:106-113. 28. Hopfer U, Nelson K, Perrotto J, Isselbacher KJ. Glucose transport in isolated brush border membrane from rat small intestine. J Biol Chem 1973;248:25-32. 29. Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951;193:265-275. 30. Murer H, Ammann E, Bibber J, Hopfer II. The surface membrane of the small intestinal epithelial cell. I. Localization of adenyl cyclase. Biochim Biophys Acta 1976;433:509-519, 31. Acra SA, Dykes W, Nylander W, Ghishan FK. Characterization of a distinct Na+/H* exchanger in the basolateral membranes of human jejunum (abstr). Gastroenterology 1991;100:A678. 32. Kinsella JL, Aronson P.S. Interaction of NH,+ and Li+ with the renal microvillus membrane Na+-H+ exchanger. Am J Physiol 1981;241:C220-C226. 33. Aronson PS. Mechanisms of active H+ secretion in the proximal tubule. Am J Physiol 1983;245:F647-F659. 34. Orsenigo MN, Rosco M, Zoppi S, Faelli A. Characterization of basolateral membrane Na/H antiport in rat jejunum. Biochim Biophys Acta 1990;1026:64-68. 35. Soleimani M, Grass1 SM, Aronson PS. Stoichiometry of Na+/ HCO,- cotransport in basolateral membrane vesicles isolated from rabbit renal cortex. J Clin Invest 1987;79:1276-1280.
Received December 27,1991.Accepted July 7,1992. Address requests for reprints to: Krishnamurthy Ramaswamy, Ph.D., Section of Digestive and Liver Diseases, University of Illinois at Chicago, 840 South Wood Street (M/C 787), Chicago, Illinois 60612. Supported by Grant DK33349 from the National Institute of Diabetes and Digestive and Kidney Diseases and by the Department of Veterans Affairs. This work was presented in part at the annual meeting of the American Gastroenterological Association in New Orleans, Louisiana, 1991, and was published in abstract form (Gastroenterology 1991;1oo:A710). The authors thank Anita Tredeau for her secretarial assistance.
