[25]

INTESTINAL NaC1 TRANSPORT IN VESICLES

389

DMSO. Cells are frozen at a density of 5-10 million/ml/tube. We freeze the cells using a BF-5 biological freezer from Union Carbide. The freezing protocol is 30 min at 4 ° at level C, 30 min at level C over the liquid N2 tank, and finally 90 min at level A over the liquid N 2. The tubes are then attached to the canes and immersed in liquid nitrogen. Viability upon thawing rapidly at 37 ° should be >50%. Acknowledgments The T u cell line was originally established by Dr. Hideo Masui and colleagues at the University of California, San Diego, California. Most of the methods described are modifications of commonly used methods established by Dr. Gordon Sato, Dr. Hans Us.sing Dr. Joseph Handler, Dr. Marcelino Cereijido, and Dr. Milton Saier. Dr. Kenneth Mandel and Dr. James McRoberts are the two key associates who designed and carried out most of the experiments. Mr. Greg Beuerlein provided technical assitance. Ms. Bambi Beuerlein and Ms. Carol Gaul typed and edited the chapter. This work was supported by Grants R01 AM28305 and R01 AM35932 from the National Institues of Health, a grant from the University of California Cancer Research Coordinating Committee, a grant from The Burroughs Wellcome Fund and a grant from the National Foundation for Ileitis and Colitis, Inc. Dr. K. Dharmsathaphorn is a recipient of a Research Career Development Aware, AM01146, from the National Institues of Health and an American Gastroenterological Association/Glaxo Research Scholar Award. Dr. James Madam is a recipient of an American Gastroenterological/Ross Research Scholar Award.

[25] S o d i u m C h l o r i d e T r a n s p o r t P a t h w a y s Membrane Vesicles

in I n t e s t i n a l

B y ULRICH HOPFER

Overview of Models of Intestinal NaCl Transport The classical tools for investigating epithelial electrolyte transport consist mainly of measurements of radioactive tracer and electrical fluxes across intact epithelia or of short-term tracer uptake or release by epithelial cells. Major, new biochemical and cell biological approaches were developed in the 1970s and include localization of key transport enzymes, i.e., transport ATPases, and cell ffactionation with separate isolation of membrane vesicles from the lumenal and basolateral regions of epithelial cells. The membrane vesicle systems provide opportunities to investigate molecular events associated with the transport of Na + and C1- across these membranes. U. Hopfer, K. Nelson, J. Perrotto, and K. J. Isselbacher, £ BioL Chem. 248, 25 (1973).

METHODS IN ENZYMOLOGY, VOL. 192

Copyright © 1990 by Academic Press, Inc. All fights of reproduction in any form reserved.

390

GASTROINTESTINAL SYSTEM

[25]

Several different modes of Na ÷ and C1- transport can be distinguished in the intestine. 2,3 One major criterion is the overall flux of electrical charges associated with transepithelial NaC1 flow which can be easily measured in terms of the short-circuit current; the transport is termed electrogenic if under short-circuited conditions the "active" net flow of either Na + or C1- across the epithelial cell layer is associated with a corresponding electrical flux. Other criteria are obligatory involvement of other solutes and the direction of NaC1 transport. The following modes have been defined: (1) Sodium-nutrient cotransport (e.g., sodium-glucose cotransport) which, under physiological conditions, results in nutrient-dependent Na + absorption, (2) electrically neutral NaCI absorption, which is typically found in the small intestine of mammals and birds, but is also present in the colon of Na+-replete rats, (3) electrogenic Na + absorption, which is typically present in the most distal portion of the gut and regulated by mineralocorticoids, (4) K+-dependent, electrogenic NaC1 absorption, which is typically found in the intestine of carnivorous fish, and (5) NaC1 secretion. This chapter focuses on modes 2 and 5, i.e., electrically neutral NaCI absorption and NaC1 secretion. Figure 1 describes the current model for electrically neutral NaC1 absorption and Fig. 2 the one for NaC1 secretion. Although the overall picture of salt movements across the epithelium appears well supported by experimental findings, molecular information about transporters involved and regulation of their activity is still missing or grossly incomplete. Therefore, well-established methods that exist for isolation of intestinal plasma membranes as well as assays for ATP-independent Na + and C1- transport across intestinal plasma membranes will be summarized, with particular emphasis on methods in use in the author's laboratory. Figure 1 illustrates that NaC1 absorption comes about by the presence of (1) C1-/HCO3- and (2) Na+/H + exchangers in the lumenal plasma membrane in tandem with primary active Na + transport via the (3) Na+,K+-ATPase in the basolateral plasma membrane. The (4) CI- exit pathway in the basolateral plasma membrane is not yet well investigated in mammalian enterocytes. In the steady state, the cell must recycle the K 4 that is pumped into the cell by the Na+,K+-ATPase in exchange for Na +. This extra K + leaves the cell through (5) K + channels. While the existence of Na+/H + exchange,4-~4 Na+,K+-ATPase, ~5-~7 as well as K + channels 18,~9 2U. Hopferand C. M. Liedtke,Annu. Rev. Physiol. 49, 51 (1987). 3M. Field, M. C. Rao, and E. B. Chang,N. Engl. J. Med. 321, 800, 879 (1989). 4H. Murer,U. Hopfer,and R. Kinne,Biochem. J. 154, 597 (1976). 5R. Knickelbein,P. S. Aronson,W. Atherton, and J. W. Dobbins, Am. J. Physiol. 245, G504 (1983). 6H. J. Binder,G. Stange,H. Murer, B. Stieger,and H. P. Hauri,Am. Z Physiol. 251,G382 (1986).

[25]

I N T E S T I N ANaCI L TRANSPORT IN VESICLES BLOOD

~

LUMEN

CI-

ul

HCO

k,L_..., []

~ 3 N a 2 K+

391

,

COz

+ >

=

t O - Na +

FIG. 1. Model of intestinal NaC1 absorption from the lumenal to the blood-side compartment. Overall, active transepithelial absorption is a result of(l) CI-/HCO3- and (2) Na+/H + exchanges at the brush border membrane; primary active movement of Na+out of the cell by (3) Na+,K+-ATPase and passive exit of (4) C1- and (5) K + at the basolateral plasma membrane. The nature of (4) C1- exit is not well understood. Carbonic anhydrase (CA) serves to accelerate equilibration of CO2 with HCO3- and protons.

is generally accepted, the nature of the C1- transporters at both plasma membranes and the types of transport reactions are still debated. For example, at the brush border membrane electrically neutral C1- transport has been demonstrated; however, it is experimentally difficult to distinguish between C1-/HCO3- exchange, C1-/OH- exchange, and HC1 cotransport. Na+/H + exchange activity is prominent in brush border membranes,

7 I. W. Booth, G. Strange, H. Murer, T. R. Fenton, and P. J. Milla, Lancet 1, 1066 (1985). 8 E. S. Foster, P. K. Dudeja, and T. A. Brasitus, Am. J. Physiol. 250, G781 (1986). 9 R. Fuchs, J. Graft, and M. Peterlik, Biochem. J. 230, 441 (1985). to j. N. Howard and G. A. Ahearn, J. Exp. Biol. 135, 65 (1988). H j. G. Kleinman, J. M. Harig, J. A. Barry, and K. Ramaswamy, Am. J. Physiol. 255, G206 (1988). 12K. Kikuchi, N. N. Abumrad, and F. K. Ghishan, Gastroenterology 95, 388 (1988). t3 y. Miyamoto, D. F. Balkovetz, V. Ganapathy, T. Iwatsubo, M. Hanano, and F. Leibach, J. Pharmacol. Exp. Ther. 245, 823 (1988). 14K. Ramaswamy, J. M. Harig, J. G. Kleinman, M. S. Harris, and J. A. Barry, Biochim. Biophys. Acta 981, 193 (1989). ~5C. Stifling, J. CellBiol. 53, 704 (1972). i~ V. Harms and E. Wright, i Membr. Biol. 53, 119 (1980). ~7P. B. Vengesa and U. Hopfer, J. Histochem. Cytochem. 27, 1231 (1979). ~s E. Grasset, P. Gunter-Smith, and S. G. Sehultz, J. Membr. Biol. 71, 89 (1983). 19j. Costantin, S. Alealen, A. de Souza Otero, W. P. Dubinsky, and S. G. Sehultz, Proc. Natl. Acad. Sci. U.S.A. 86, 5212 (1989).

392

GASTROINTESTINAL SYSTEM

[9.5]

but has also been described in basolateral plasma membrane vesicles. 20-22 The physiological role of basolateral Na+/H + exchange is assumed to be a proton pump transporting protons out of the cell into blood. This proton pumping at the basolateral side accompanies HCO3- secretion at the lumenal pole, indicating the importance of Na+/H + exchange in basolateral membranes for overall HCO3- secretion from blood to lumen. An alternative view regards this Na+/H + exchange as a compensating reaction for HCO3- secretion at the lumenal pole, thus maintaining cellular pH homeostasis. In some species, e.g., rodents, basolateral Na+/H + exchange may also play a role for concentrative C1- uptake (see next paragraph). Figure 2 illustrates the steps involved in NaCl secretion: Secondary active Cl- uptake (6) into the cell at the basolateral pole, recycling of Na + through the Na+,K+-ATPase (3), and o f K + through K + channels (5) at the basolateral pole, and exit of C1- via C1- channels (7) in the lumenal plasma membrane. The secondary active C1- uptake across the basolateral plasma membrane probably occurs by loop diuretic-sensitive Na+/K+/2C1 - cotransport in humans and rabbits and the combination of Na+/H + and C1-/HCO3- exchangers in rats. However, little information about these basolateral transporters has been obtained through flux studies in isolated plasma membrane vesicles. The lumenal Cl- channel is thought to reside mainly in crypt cells3; however, it can also be detected in fully differentiated enterocytes and brush border membrane vesicles. 23-2~ Based on intestinal physiology and pathology, the transporter distribution is probably not homogeneous along the longitudinal axis. The same consideration applies to the crypt-villus axis. Knickelbein et a[. 21 have actually demonstrated that Na+/H + exchange exists in both brush border and basolateral membranes of villus cells of rabbit ileum, but only in basolateral membranes of crypt cells.

Isolation of Brush Border Membranes Highly purified membrane vesicles from the brush border region can be isolated with relative ease. Two major types of preparations are commonly 20 F. Barros, P. Dominguez, G. Velasco, and P. S. Lazo, Biochem. Biophys. Res. Commun. 134, 827 (1986). 21 R. G. Knickelbein, P. S. Aronson, and J. W. Dobbins, J. Clin. Invest. 82, 2158 (1988). 22 A. J. Moe, J. A. Hollywood, and M. J. Jackson, Comp. Biochem. Physiol. 93, 845 (1989). 23 F. Giraldez, F. V. Sepulveda, and D. N. Sheppard, J. Physiol. (London) 395, 597 (1988). 23"F. Giraldez, K. Y. Murray, F. V. Sepulveda, and D. N. Sheppard, J. Physiol. 416, 517 (1989). 24 C. P. Stewart and L. A. Turnber~ Am. J. Physiol. 257, G334 (1989). 25 G. W. Forsyth and S. E. Gabriel, J. Membr. Biol. 107, 137 (1989). 26 U. Hopfer, Int. Congr. Physiol. Sci. 31st, S1023 (abstract) (1989).

[25]

INTESTINAL N a C I TRANSPORT IN VESICLES BLOOD

2 C1K+

393

LUMEN

~f tl ~ ~ 11 -

C1

[(6)-

-

~

.

~

(7)

l~'T~a) 3 Na+ 2K+

if}

;

5)

K+ Na +

FIG. 2. Model of intestinal NaC1 secretion from the blood side to the lumen. Overall, transepithelial active secretion is achieved by concentrative C1- uptake at the basolateral plasma membrane via (6) Na+/K+/2CI- cotransport. This uptake is driven by the physiological Na+ gradient (extraceHular > intracelhilar). In the steady state, Na+ is extruded back across the basolateral plasma membrane by (3) Na+,K+-ATPase and K + leaves through specific (5) K + channels. Chloride ion is secreted through (7) Cl- channels in the brush border membrane. Charge compensation for the C1- movement into the lumen is mainly by Na+ as the major extracellular cation moving through intercellular space and junction.

employed. The first one involves thorough homogenization of mucosal scrapings, aggregation of nonbrush border membranes and particulates by divalent cations, and subsequent differential centrifugation (see the section, Aggregation Method). 2~s The other method is based on an initial isolation of intact brush borders29,s° and subsequent depolymedzation of the cytoskeletal material associated with the brush border membrane (see the section, Depolymerization Method). al The quantity and quality of isolated membrane preparations are usually assessed on the basis of yield, enrichment, and specific activity of marker enzymes, such as sucrase or alkaline phosphatase for the brush border membrane and Na+,K+-ATPase for the basolateral membrane. With the aggregation method, brush border membranes can be prepared with 25- to 30-fold enrichments in sucrase relative to a homogenate of mucosal scrapings at a yield of 30 to 50%. The depolymerization method 27j. Schmitz, H. Preiser, D. Maestracci, B. K. Ghosh, J. J. Cerda, and R. K. Crane, Biochim. Biophys. Acta 323, 98 (1973). 2s H. Hauser, K. Howell, R. M. C. Dawson, and D. E. Boyer, Biochim. Biophys. Acta. 602, 567 (1980). G. G. Forstncr, S. M. Sabesin, and K. J. Is~lbacher, Biochem. J. 106, 381 (1968). so A. Eichholz and R. IC Crane, this series, Vol. 31 p. 123 (1974). 3t U. Hopfer, T. D. Crowe, and B. Tandler, AnaL Biochem. 131, 447 (1983).

394

GASTROINTESTINAL SYSTEM

[9-5]

can yield enrichments of 2 to 4 times that of the aggregation method (i.e., 60- to 120-fold enrichments of brush border marker enzymes) at a yield of 40 to 70%. The membranes prepared by the two methods are not identical, e.g., because of differences in associated cytoskeletal material. In addition, more cytosolic contaminants are trapped by the aggregation method when vesicles are formed during the initial homogenization. While the vesicle orientation with both preparations is predominantly right side out based on electron microscopy and detergent activation of marker enzymes, the depolymerization method provides access to the cytosolic side at the intermediate stage of isolated brush borders because the cytoskeleton prevents resealing of the membrane. Aggregation Method This methodology is based on the report by Schmitz et a/. 27 with many modifications by others. 32 The following protocol provides a brief outline of the essential steps in this method. All procedures are carried out on ice or with ice-cold solutions. 1. The small intestine is removed as intact tube from the animal, rinsed with cold buffered saline, and inverted over a thin rod. Excess mucus is removed by wiping with paper tissues. 2. The mucosa is scraped with glass slides, and the scrapings are suspended in about 20X their volume in a low ionic strength buffer containing 0.1 M mannitol, 10 m M Tris-HEPES, pH 7.4 The scrapings are then thoroughly homogenized with a high-speed blender (e.g., a Sorvall Omnimixer (Dupont Instruments, Wilmington, DE) at full speed for 5 rain). 3. Either l0 m M (final concentration) MgC12 o r CaC12 is added to the homogenate to aggregate nonbrush border particulates during a period of 10-15 min. 4. The aggregated material is removed by low-speed centrifugation at 6000 g for 10 rain. 5. The brush border membranes are collected by medium-speed centrifugation at 45,000 g for 30 min. Steps 3 - 5 of this protocol can be repeated to increase the purification of the brush border membrane. The efficiency of the aggregation is dependent not only on the divalent cation, but also on the protein concentration. The aggregation method has been successfully used with many animal species. Although both Ca 2+ and Mg2+ aggregate nonbrush border material, the results with these two different cations are not identical.2s,33 32H. Muter, P. Gmaj, B. Stieger, and B. Hagenbuch, this series Vol. 172, p. 346 (1989). 33H. Aubry, A. R. Merrill, and P. A. Proulx, Biochim. Biophys. Acta 856, 610 (1986).

[25]

INTESTINAL NaC1 TRANSPORT IN VESICLES

395

Depolymerization Method This method is based on an initial isolation of brush borders as intact "'organelles" from small intestinal scrapings and subsequent separation of cytoskeletal material from the membrane. Procedures for the preparation of brush borders of many different species have previously been described.3° A modification for rat small intestine is provided below. 1. Small intestinal scrapings are obtained as described above for the aggregation method. 2. The intestinal serapings from 1 rat (about 0.5 g) are suspended in 0.5 liter of homogenization buffer (5 m M EGTA, 1 m M HEPES adjusted to pH 7.4 with Tris hydroxide) and homogenized in a blender for about 10 sec. The time must be optimized for each blender and volume, based on an optimal recovery of intact brush borders: if shearing is insufficient, brush borders are not liberated from the cells; if sheafing is too much, the brush borders are broken into small pieces that cannot be isolated by low-speed centrifugation. The effectiveness of the homogenization can be quantitated by the yield of marker enzyme(s) in the purified brush border fraction obtained in step 5. 3. The homogenate is passed through a Nitex (Tetko Inc., Elmsford, NY) (30 gin) filter and subsequently a thin layer of Pyrex glass wool (Coming Glass Works, Coming, NY) to remove debris and nuclei. Both filter and glass wool are prewashed with homogenization buffer. 4. The filtered homogenate is centrifuged at 1000 g for 15 min to collect the intact brush borders. 5. The 1000 g, low-speed pellet is resuspended in the homogenization buffer by careful trituration and centrifuged again at 1000 g for 10 min. The new low-speed pellet is resuspended in a small volume of homogenization buffer and an aliquot examined by light microscopy for purity. At least 90% of visible particles should be brush borders. If necessary, the fourth step can be repeated to achieve greater purity. The resulting brush borders are stable for several hours, particularly if Ca 2+ concentrations are kept low and the ionic strength is above 0.03 at pH 7.4. The cytoskeleton can be separated from the membranes by a cycle of depolymerization and repolymerization of the microfilaments that form the core of brush borders. Shearing of the brush borders after depolymerization of the microfilaments results in vesicularion of the membrane, preventing reassembly of the original filaments. The de- and repolymerization can be manipulated using the conditions for G and F actin transformarion. The depolymerization can be achieved by high concentrations of chaotropic ions. 31 The method described below is based on the lability of

396

GASTROINTESTINAL SYSTEM

[25]

microtilaments to high pressure. ~ The following steps have proved useful for high yields of membranes. 6. The purified brush border pellet is suspended in the membrane preparation buffer (0.3 M mannitol, l0 m M HEPES, adjusted to pH 7.4 with Tris, 1 m M EGTA) at about 0.5 to 1.0 mg/ml and is exposed to pressures of more than 1600 psi of an inert gas (e.g., N2) in a Parr cell disruption bomb (Parr Instrument Co., Moline, IL) for 15 min. 7. The suspension is then slowly released from the bomb, resulting in homogenization of the brush border membranes due to sheafing and cavitation. 8. The microfilaments are reaggregated by adjusting the MgC12 concentration to l0 mM. The aggregated material is subsequently removed by centrifugation at 1500 g for 5 min. 9. The brush border membranes remain in the supernatant and can be collected by centdfugation at 45,000 g for 20 min. The depolymerization of the cytoskeleton in the bomb can be enhanced by inclusion of 0.1 m M ATP and/or micromolar concentrations of free Ca 2+ in the membrane preparation buffer. Isolation of Basolateral Plasma Membranes

In contrast to the brush border region, the basolateral plasma membrane of enterocytes lacks unique features that distinguish it from those of other cells. Therefore, the method for isolating this membrane resembles that of the plasma membrane from other, more generic mammalian cells, consisting of a combination of differential and density centrifugations. Actually, to ensure that the isolated plasma membrane originates from enterocytes, it is important to first isolate a relatively pure enterocyte fraction. The procedure by Bjerknes and Cheng35 works well for small rodents. It entails intraarterial or intracardiac injection of a solution containing 30 m M EDTA to break the attachments of epithelial cells to the basement lamina. The epithelial cells can subsequently be removed from the gut mucosa by either shaking or very light scrapings. The following protocol is an adaptation of the method by Scalera et al. 36 It consists of an initial isolation of entire sheets of epithelial cells and L. G. Tilney and R. R. CardeH, Jr., J. CellBiol. 47, 408 (1970). 3s M. Bjerknes and H. Cheng, Anta. Rec. 199, 565 (1981). 36 V. Scalera, C. Storelli, C. Storelli-Joss, W. Haase, and H. Muter, Biochem. J. 186, 177 (1980).

[25]

INTESTINAL NaC1 TRANSPORT IN VESICLES

397

subsequent preparation of basolateral plasma membrane from the isolated cells. The actual membrane preparation can, in turn, be subdivided into (1) isolation of a crude, basolateral plasma membrane fraction that is collected as a "fluffy" layer on top of the mitochondrial pellet after centrifugation, and (2) isolation of the final, purified membrane by density centrifugation in a Percoll gradient. Na+,K+-ATPase or K+-stimulated, neutral phosphatase serves as marker enzyme to assess purification and yield of basolateral plasma membrane. The crude plasma membrane fraction typically is threefold enriched in the marker enzyme at a yield of about 50%. The Percoll gradient provides an additional fourfold purification at a yield of about 25% relative to the homogenate.

Isolation of Enterocytes 1. Rats are anesthetized with sodium pentobarbital ip (about 60 mg/kg body wt). 2. The abdomen is opened by a midline abdominal incision and the small intestine rinsed with 37", phosphate-buffered 0.9% saline through two small incisions in the gut wall opposite the mesenteries. 3. After the portal vein is severed, about 150 ml of warm (37°), phosphate-buffered 0.9% saline containing additionally 30 m M EDTA, pH 7.4, is injected into the left ventricle. The effectiveness of the intestinal perfusion can be judged by "blanching" of the intestinal wall. 4. The small intestine is removed and inverted over a glass rod. Cells are released by shaking or light scraping of the glass rod, which is suspended in a cold sucrose buffer (250 mMsucrose, 10 mMHEPES adjusted to pH 7.4 with Tris, 0.5 m M Na2EDTA, and 0.1 m M phenylmethylsulfonyl fluoride as serine protease inhibitor). The isolated cells serve as starting material for the subsequent isolation of plasma membranes. 5. Cells are collected by low-speed centrifugation, resuspended in about 30 ml of the sucrose buffer/animal, and homogenized in a glassTeflon homogenizer at 600-1200 rpm for 10-25 strokes. The speed and number of strokes must be optimized for each homogenizer with respect to recovery of basolateral plasma membrane marker in the "fluffy layer" (see step 7 below). 6. The heavy particulate material is removed by centrifugation at 2300 g for 15 min and the supernatant retained. 7. A crude basolateral plasma membrane fraction is collected from this

398

GASTROINTESTINAL SYSTEM

[25]

supernatant by centrifugation at 15,000 g for 20 min. The basolateral plasma membrane is enriched in a "fluffy," white layer above the denser, yellow mitochondrial pellet. For greater yield, the heavy, 2300 g particulate material from the previous step can be rehomogenized with a glass- Teflon homogenizer and spun again at 2300 g for 15 min. In this case, the two supernatants are combined before the 15,000 g centrifugation. 8. The white, fluffy layer from step 7 is resuspended in the sucrose buffer, but with 0.1 m M MgC12 instead of EDTA, and Percoll added to make 12 to 14%. 9. The membrane suspension is centrifuged in a vertical SorvaU SS90 rotor at 27,000 g for 15 min. The basolateral membranes collect in a band about one-fourth down from the top. 10. The band containing most of the marker enzyme is removed and the membranes collected by high-speed centrifugation (>200,000 g for 45 min) on top of a dense, glassy Percoll pellet. Transport Measurements:

General Considerations

The general design principles applicable to transport experiments with membrane vesicles have been discussed prcviouslyY Therefore, only specific issues of importance for electrolyte transport arc included in this chapter. One of the problems in electrolyte transport studies is that fixed charges on the inside of vesicles give rise to the so-caUed Gibbs-Donnan potential which influences the equilibrium distribution of ions across the vesicle membrane. This effect is expressed as apparent binding or exclusion of ions. The magnitude of the potential can be calculated from ion concentration gradients at equilibrium: A ~F = - ( R T / z F ) In([ iz]'m/[izlo,t)

where R = gas constant, T = absolute temperature, F = Faraday constant, z -- charge of ion, [ij -- concentration of ion i of charge z. The presence of a Gibbs-Donnan potential can be inferred if several different ions give the same value for A~P. The apparent ratio of ion concentrations between intra- and extravesicular medium, which converts to A~P,is equal to "equilibrium uptake of the ion" divided by "the intravesicular space," divided again by "its medium concentration." The space, in turn, can be calculated from the equilibrium uptake of a neutral, nonbinding solute, such as glucose. At equilibrium, the intravesicular concentration of such a neutral solute corresponds to that of the medium so that the 37U. Hopfer, this series, Vol. 172, p. 313 (1989).

[25]

INTESTINAL NaC1 TRANSPORT IN VESICLES

399

"intravesicular space" is equal to "uptake" divided by "medium concentration" (e.g., uptake in moles per milligram protein divided by solute concentration in moles per liter to yield liter per nag protein). The intravesicular space of intestinal plasma membrane vesicles typically ranges from 1 to 5 #l/mg protein. For rat small intestinal brush border membranes, an inside-negative Gibbs-Donnan potential of about 10 mV has been inferred from the findings of equilibrium uptakes that are higher for Na + and lower for the anions CI- and sulfate relative to uncharged ~glucose. 38 Sodium Ion Transport Na+/H+ Exchange The existence of Na+/H + exchange as the major form of nutrient-independent Na + transport across the brush border membrane of the small intestine is supported by four major types of experimental results. 1. pH gradients can drive concentrative uptake of Na ÷, independent from proton diffusion potentials. 2. Sodium ion gradients can drive concentrative proton movements in the absence of diffusion potentials. 3. Na+/Na + exchange rates across the brush border membrane are not dependent on the nature of the anion, excluding Na+/anion cotransport models. 4. The diuretic amiloride and some of its derivatives, which are inhibitors of Na +/H + exchange in other systems, inhibit Na + transport in isolated membrane vesicles. pH Gradient-Driven Na + Uptake The Na+/H + exchange mechanism implies that a proton gradient can drive uphill Na + transport. This prediction can be tested by measuring 22Na+ uptake by membrane vesicles in the presence of a pH gradient, whereby the lower pH is on the inside. For such experiments, the following considerations are important. 1. Membrane vesicles must be preloaded with a relatively impermeant buffer such as HEPES or 2-(N-morpholino)ethane sulfonic acid (MES) at a low pH (e.g., 5.5). The preloading can be accomplished by prolonged incubations and homogenizations in such a low-pH buffer. Alternatively, 3s C. M. Liedtke and U. Hopfer, Am. £ Physiol. 242, G263, G272 (1982).

400

G A S T R O I N T E S T I NSYSTEM AL

[25]

the membrane vesicles are prepared in a solution containing impermeant buffer components that by themselves have a low pH, but which are adjusted to the higher pH (e.g., 7.4) of the usual membrane preparation buffer with permeant base (e.g., imidazole or ethanolamine). Once the membrane vesicles are isolated, their intravesicular pH can easily be lowered by removing this premeant component of the buffer (e.g., by gel filtration or washing of membranes in a buffer of appropriate low pH and without the permeant base). 2. A pH gradient is established between the intravesicular compartment and the incubation medium at the beginning of the incubation with labeled Na +. 3. The establishment of proton diffusion potentials may have to be prevented by inclusion of high and equal concentrations of K + in both the intravesicular and extravesicular medium as well as valinomycin to ensure that the K + conductance is high. 4. Sodium ion transport must be effectively quenched at the termination of the incubation period and vesicles separated from the medium containing the labeled Na +. The quenching can be accomplished by icecold solutions containing 0.05 to 0.1 M MgSO4, an impermeant buffer of the same pH as the incubation medium, and sufficient mannitol to bring the osmolarity of the quench solution to that of the incubation medium. Vesicles can be conveniently collected on nitrocellulose filters (0.45 to 0.6 #m). A Na+/H + exchanger can utilize the energy of a pH gradient into Na + uptake into membrane vesicles against a concentration gradient. Since the pH gradient usually dissipates with prolonged incubation periods, the Na + gradient also collapses with it and Na + uptake as a function of time presents the picture of an overshoot. The overshooting portion, but not the equilibrium uptake, should be dependent on the pH gradient. Observations of such overshooting uptake are generally considered sufficient to justify the conclusion that the membranes possess functional Na+/H + exchangers provided the overshoot persists in the absence of a diffusion potential and is not due to time-dependent changes in intravesicular space. Because of the presence of dissipative proton fluxes (not coupled to Na + uptake), the buffer capacity of the intravesicular medium must be kept relatively high and the Na + concentration in the uptake medium relatively low (typical ratio, 50: 1). Figure 3 shows overshooting Na + uptake by brush border membranes driven by a pH gradient. The initial uptake can be inhibited about 90% by 1 m M amiloride, which is thought to inhibit the transporter. However, this drug also dissipates the pH gradient and this effect may contribute to the

[25]

INTESTINAL N a C I TRANSPORT IN VESICLES

401

,Ig

o B

E

D

1:: ._= O O~

0

0

I

2

3

4

5

6

T i m e (rain)

Fro. 3. pH gradient-driven concentrative uptake ofNa + by intestinal brush border membrane vesicles. The membranes were preloaded with 50 mM MES/Tris buffer, pH 5.5, followed by incubation in a medium containing 0.5 mM Na2SO4 and (0) 50 mM MES/Tris, pH 5.5; (13) 50 mM HEPES/Tris, pH 7.5; (&) 50 m M HEPES/Tris, pH 7.5 plus 5 pg of carbonyl cyanide p-trifluoromethoxyphenylhydrazone to increase the proton conductance and set up a proton diffusion potential. Overshooting Na+ uptake is seen only in the presence of a pH gradient. In addition, the overshoot is not influenced very much by a diffusion potential. (Reproduced with permission from Ref. 4.)

inhibition of uptake. 39 Other modulators ofNa+/H + exchange appear to be lipid fluidity in general and free fatty adds. Interestingly, while lipid fluidity correlates positively4°-43 with Na+/H + exchange activity, free fatty acids by themselves were shown to inhibit. 44

Sodium Ion Gradient-Driven Proton Transport Experimental conditions can also be rigged so that Na + gradients can drive H + transport through the Na÷/H + exchanger. The proton move39 W. P. Dubinsky, Jn, and R. A. Frizzell, Am. J. Physiol. 245, C157 (1983). 4o T. A. Brasitus, P. K. Dudeja, and E. S. Foster, Biochim. Biophys. Acta 938, 483 (1988). 4~ p. K. Dudcja, E. S. Foster, R. Dahiya, and T. A. Brasitus, Biochim. Biophys. Acta899, 222 (1987). 42 p. K. Dudcja, E. S. Foster, and T. A. Brasitus, Biochim. Biophys. Acta 905, 485 (1987). 43 p. K. Dudcja, E. S. Foster, and T. A. Brasitus, Biochim. Biophys. Acta 859, 61 (1986). 44 C. Tiruppathi, Y. Miyamoto, V. Ganapathy, and F. H. Leibach, Biochem. Pharmacol. 37, 1399 (1988).

402

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ments can be conveniently measured by pH changes of the medium with pH electrodes. 4 Experimental design considerations are similar to those for pH gradient-driven Na+ uptake, except that the roles of Na + and protons are reversed. In other words, large Na+ gradients and Na + fluxes as well as low pH buffering capacities are essential to achieve appropriate sensitivities to detect Na+-driven proton movements.

Na+/Na+ Exchange Na+/Na+ exchange experiments are useful under two different types of conditions: (1) tracer ~Na + uptake into vesicles that have been preloaded with high concentrations of nonradioactive Na + and (2) 22Na+ uptake at chemical equilibrium. The advantage of preloading with high concentrations of unlabeled Na+ is that tracer 22Na+ is accumulated to high levels, i.e., the transport assay with vesicles is very sensitive. This method was employed with crude membrane vesicles from rat colon to study amiloride-sensitive Na+ channels.+5,46 The equilibrium condition has the advantage of well-defined concentrations in both intra- and extravesicular compartments, which is particularly well suited for kinetic experiments.37 At equilibrium, changes in rates of 22Na+ movement across the membrane are clearly interpretable in terms of activation or inhibition of transporter(s) without interference from dissipation of gradients across the membrane. The condition was important for studies investigating coupling of transport of Na+ to that of other solutes. Early models of electrically neutral NaC1 absorption included NaC1 cotransport,47 analogous to Na+-glucose cotransport. Such cotransport models can be tested because they predict that the obligatory cosubstrate appears as activator of the labeled substrate whose transport is measured. This prediction applies to net flux as well as isotope exchange conditions.4s NaC1 cotransport could be excluded in rat and rabbit brush border membranes on the basis of independence of the rate of Na+/Na+ exchange from the magnitude of the C1- concentration between 0 and 0.15 M (Fig. 4).3s This finding provides strong support for parallel Na+/H + and CI-/HCO3exchangers, which appear coupled in intact tissue only under steady state conditions of salt transport measurements. 4s R. J. Bridges, E. J. Cragoe, Jr., R. A. Frizzell, and D. J. Benos, Am. J. Physiol. 256, C67 (1989). 4~R. J. Bridges, H. Garty, D. J. Benos, and W. Rummel, Am. J. Physiol. 254, C484 (1988). 47 R. A. Frizzell, M. Field, and S. G. Schultz, Am. J. Physiol. 236, F1 (1979). 48 U. Hopfer and C. M. Liedtke, Membr. Biochem. 4, 11 ( 1981) [plus Errata in issue 4 of Vol. 4 (1982)].

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Na+/Na + exchange experiments are carried out by preincubating membrane vesicles under the desired conditions of pH and medium composition, including 22Na+. The preincubation must be sufficiently long so that any pH or ion gradients are abolished. The rate of Na÷/Na + exchange can then be measured during the incubation period by replacing 22Na+ in the medium with unlabeled Na + and following release of 22Na+ from the vesicles. The effect of an experimental variable, such as the C1- concentration in the experiment in Fig. 4, is tested in parallel aliquots of membrane vesicles. The aliquots are treated identically, including measurements of the Na+/Na + exchange rates, except for preineubation and incubation with different CI- concentrations. Although rates of Na+/Na + exchange can be measured both as 22Na+ uptake or release, 22Na+ release is usually employed because it requires less radiolabel.

pH Gradient-Dissipation Measured with Acridine Orange One of the methods for measuring pH gradients between medium and the interior of vesicles takes advantage of fluorescence quenching of heterocyclic amines when they are accumulated in vesicles.49 Suitable dyes are 9-aminoaeridine and acridine orange. These amines are accumulated by membrane vesicles preloaded with a pH that is more acid than the medium. The basis of the fluorescent quench of 9-aminoaeridine by pH gradients was investigated only recently.5°,51 These studies suggest that the dye method is probably not reliable for quantitative measurements of the kinetics of pH changes since fluorescence decay and pH changes after a pH jump did not coincide. The method has been widely used, however, to probe for existence of intestinal Na+/H + exchange.5,8,4°,42,43,52In light of the discovery that the kinetics of fluorescence quench are not identical with intravesicular pH changes, some of the conclusions from the intestinal vesicle studies may have to be reevaluated. The method consists of preloading the membrane vesicles with a lowpH buffer of an impermeant acid-base pair, subsequent suspension of the vesicles in a medium of higher pH, and equilibration with about 5-10 # M acridine orange (excitation, 493 nm; emission. 527-535 rim). This manipulation results in quenching of acridine orange fluorescence due to dye uptake by the vesicles. With time, the fluorescence recovers. The recovery can be accelerated by Na + influx from the medium, which is interpreted as Na+/H + exchange. 49 H. Rottenberg, this series, Vol. 54, p. 547 (1979). so S. Grzesiek and N. A. Dencher, Biochim. Biophys. Acta 938, 411 (1988). 51 S. Grzesiek, H. Otto, and N. A. Dencher, Biophys. J. 55, 1101 (1989). s2 C. B. Cassano, B. Stieger, and H. Murer, Pfl~gers Arch. 400, 309 (1984).

404

[25]

GASTROINTESTINAL SYSTEM

.0B

I

!

t,to

o

z 04

-

r,

I

Z [ N o ' ] - 1 5 0 mM

~ o2 ~0 3,

0

frOm M C I -

7s

5"OreM S0~- 2"5

100

1~0

O

FIG. 4. Independence of Na+/Na + exchange from C1-. Aliquots of brush border membranes were preincubated at 25°for 75 rain containing different concentrations of NaCI or Na2SO4 as indicated. The rate of Na+/Na + exchange was subsequently measured from the 22Na+ uptake from the medium. The lack of stimulation of Na+/Na+ exchange by elindicates that it is not a cosubstrate for Na+ transport. (Reproduced with permission from Ref. 38.)

Electrophysiology of Isolated Plasma Membrane Vesicles The conductive permeabilities of intestinal brush border membranes to ions have been evaluated with potential-sensitive, fluorescent indicators, such as cyanine dyes.s3-ss The studies have been most informative when they were concerned with Na+-nutrient cotransporters, but the methodology is generally useful to evaluate ion movements associated with electrical currents. However, patch clamping can usually provide more direct information.

Regulation of Na+/H+ Exchange Cons'~erable evidence from intact tissue studies suggests that electroneutral NaC1 transport is acutely regulated: Classical secretagogues, such as vasoactive intestinal peptide and cholinergic agonists, decrease this transport, while ¢x-adrenergic agonists increase it. The molecular mechanisms by which the transporter activity is regulated are not known definitively, although some mechanisms appear to persist in isolated brush border membranes. Brasitus' group has demonstrated a positive correlation between Vm~ of Na+/H + exchange and lipid fluidity of the brush border membrane, as measured by anisotropy of fluorescent probes (diphenyl 83R. D. Gunther and E. M. Wright, J. Membr. Biol. 74, 85 (1983). R. D. Gunther, R. E. Sch¢ll, and E. M. Wright, J. Membr. Biol. 78, l l 9 (1984). ss E. M. Wright, Am. J. Physiol. 246, F363 (1984).

[25]

INTESTINAL NaC1 TRANSPORT IN VESICLES

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hexatdene and anthroylstearic acid).4°,42,43Donowitz and colleagues have provided evidence that the Na+/H + exchanger can be down-regulated by Ca2+/calmodulin-stimulated protein kinase.56,57 Chloride Ion Transport There is considerable evidence from measurements in intact tissues, isolated cells, as well as isolated membrane vesicles that the intestinal brush border membrane contains both C1- conductance and electroneutral C1-/ anion exchange (or HC1 cotransport) pathways.23-26,5s.59The C1- conductance, but possibly also C1-/anion exchanger(s), are highly regulated in intact cells. The C1- conductance is activated by agents that stimulate salt secretion (e.g., cholinergic agents, vasoactive intestinal peptide, histamine, prostaglandins). The simultaneous presence of both electrogenic and electrically silent transport systems in the same membrane can produce ditficulties in terms of accurately measuring C1- flux through either system or ascribing the observed properties of CI- uptake or flux to a particular type of transporter. While there is no evidence for primary ~active C1- transport in vertebrate intestine, Gerencser has obtained data supporting ATP-dependent, concentrative C1- uptake by membrane vesicles from Aplysia intestinal brush borders? 9-6' Attempts to distinguish between electrically neutral and electrogenic C1- transport pathways have exploited the following features of intestinal electrolyte transport: 1. C1-/HCO3- exchange activity appears to be more prevalent in the ileum than the jejunum. 2. Stilbene sulfonates, such as 4-acetamido-4'-isothiocyanostilbene2,2'-disulfonate (SITS) or 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS), appear to inhibit CI-/HCO3- exchange from the external side, although relatively high concentrations of 1-5 m M arc required.2s,62-~ On 56R. P. Rood, E. Emmer, J. Wesolek, J. McCuUen, Z. Husain, M. E. Cohen, R. S. Braithwaite, H. Muter, G. W. G. Sharp, and M. Donowitz, J. Clin. Invest. 82, 1091 (1988). 57E. Emmer, R. P. Rood, J. H. Wesolek, M. E. Cohen, R. S. Braithwaite, G. W. G. Sharp, H. Muter, and M. Donowitz, J. Membr. Biol. 108, 207 (1989). 5s M. Montrose, J. Randles, and G. A. Kimmieh, Am. J. Physiol. 253, C693 (1987). 59G. A. Gerencser, Am. J. Physiol. 254, R127 (1988). 60G. A. Gerencser, Comp. Biochem. Physiol. 90, 621 (1988). 61 G. A. Gerencser, J. F. White, D. Gradmann, and S. L. Bonting, Am. J. Physiol. 255, R677

(1988). 62R. G. Knickelbcin, P. S. Aronson, and J. W. Dobbins, J. Membr. Biol. 88, 199 (1985). 63C. D. Brown, C. R. Dunk, and L. A. Turnberg, Am. J. Physiol. 257, G661 (1989). 64M. Vasseur, M. Cauzac, and F. Alvarados, Biochem. J. 263, 775 (1989).

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the other hand, micromolar concentrations of SITS inhibit C1- conductance from the cytosolic side in vesicles (however, Bridges et al. concluded from bilayer studies that colonic O - conductance can be inhibited by SITS and DIDS from the external side65). 3. Pretreatment with secretagogues enhances C1- conductance present in isolated brush border membranes. There is evidence for at least two different C1-/anion exchangers in the brush border membrane based on substrate specificity and sensitivity to stilbene disulfonate inhibitors (SITS, DIDS): (1) C1-/HCO3- exchange and (2) C1-/oxalate exchange.T M In addition, a C1-/SO4 exchanger is present in basolateral plasma membranes.69 The molecular nature of C1-/ HCO3- exchange is still debated since it is difficult to experimentally distinguish between CI-/HCO3- exchange, C1-/OH- exchange, and HC1 cotransport. In analogy with the red cell exchanger, C1-/HCO3- exchange is usually assumed for the intestine. The major experimental evidence for this interpretation is that preloading of vesicles with HCO3- stimulates greater overshooting C1- uptake in rabbit brush border membranes. 62 However, the interpretation of this result as supporting a CI-/HCO3exchange mechanism has been criticized because HCO3- preloading could contribute to a pH gradient more favorable for concentrative C1- uptake, regardless of the exact mechanism responsible for electrically neutral C1movement.64 Chloride ion transport studies in isolated, intestinal membrane vesicles have been carried out in ways analogous to those of Na+ studies. The same methodologies are useful. Chloride ion uptake by membrane vesicles can be studied with 360-. Although the specific activity of radiolabeled ~sC1-is low, it is sufficient for studies down to 1 m M C1-. Alternatively, other ions, such as 125I-, have been used as alternate substrates for the transporter, e7 Chloride ion transport across brush border membranes is effectively quenched by ice-cold solutions containing 0.05-0.1 M magnesium glucohate, an impermeant buffer such as HEPES or MES, adjusted to the pH of the incubation medium, 1 m M diphenylamine-2-carboxylate, and mannitol to bring the osmolarity to that of the incubation medium. Diphenyl65 R. J. Bridges, R. T. Worrell, R. A. Frizzell, and D. J. Benos, Am. J. Physiol. 256, C902 (1989). C. M. Liedtke and U. Hopfer, Biochem. Biophys. Res. Commun. 76, 579 (1977). 67 A. B. Vaandrager and H. R. De Jonge, Biochim. Biophys. Acta 939, 305 (1988). 68 R. G. Knickelbein, P. S. Aronson, and J. W. Dobbins, J. Clin. Invest. 77, 170 (1986). 69 C. M. Schron, R. G. Knickelbein, P. S. Aronson, and J. W. Dobbins, Am. J. Physiol. 253, G404 (1987).

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INTESTINAL N a C I T R A N S P O R T

IN VESICLES

407

amine-2-carboxylate appears to be an inhibitor of both electrogenic and electrically silent C1- transport. 7°

CI-/HCO 3- Exchange/pH Gradient-Driven CI- Uptake The activity of C1-/OH- exchange (or HCI cotransport) is measured as rate of 360- uptake in response to a pH gradient which is more alkaline inside the vesicles. Membrane vesicles are preloaded with an impermeant buffer (e.g., HEPES at pH 7.5-7.8) and then exposed to medium with a low pH (e.g., pH 5.0-6.0) in the presence of 1-4 m M 360-. If C1-/OHexchange is sutficiently active, this protocol results in an overshooting 36C1- uptake, even in the absence of a proton diffusion potential (short circuited by high intra- and extravesicular K + and valinomycin). The involvement of HCO3- as one of the physiological substrates for C1-/anion exchange is experimentaUy difficult to evaluate. Because of the conversion of CO2 into HCO3- and vice versa, the presence of CO2 is required to establish and maintain defined HCO 3- concentrations. Although the conversion is often slow in bulk solutions, it may be relatively fast in the small volumes (/tl) used for membrane incubations. In addition, brush border membranes contain membrane-bound carbonic anhydrase71 that speeds up the conversion. To test for C1-/HCO3- exchange, 3~C1uptake is measured after inclusion of COTJHCO3- in the preincubation period and establishment of an HCO 3- gradient (intravesicular > medium). 71 HCO3- clearly interacts with one of the C1-/anion exchangers since ~C1- uptake and C1-/C1- exchange can be inhibited by the presence of HCOa-. 67

Potential-Driven Cl- Uptake To probe for the presence of C1- channels in vesicles, 36C1- uptake is measured in response to an inside-positive diffusion potential. Thus, membrane vesicles that are incubated with potassium gluconate (K+ gradient, outside > inside) and valinomycin produce overshooting 36C1- uptake. The major arguments for suggesting that this uptake is transporter mediated are inhibition by diphenylamine-2-carboxylic acid and stilbene disulfonates, such as SITS, from the cytosolie side. The ratio of potassium giuconate to C1- is usually kept at a ratio of about 50:1, resulting in overshooting C1- uptake of three- to fourfold above equilibrium. ToL. Reuss, J. L. Constantin, and J. E. Bazile, Am. J. Physiol. 253, C79 (1987). 7~R. Knickelbein, P. S. Aronson, C. M. Schron, J. Seifter, and J. W. Dobbins, Am. J. Physiol. 249, G236 (1985).

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One of the interesting recent observations is that this C1- conductance in isolated brush border membrane vesicles depends on the treatment of the membranes72 as well as exposure of the intact tissue to secretagogues.26

CI-/CI- Exchange The transporters that catalyze Cl-/anion exchange and electrogenic CImovement presumably also mediate CI-/C1- exchange at equilibrium. This experimental condition has been used to demonstrate that CI- is not transported by a putative NaCI cotransporter across the brush border membrane. The C1-/CI- exchange rate was shown to be saturable and independent from the Na + concentration) 8 These types of experiments are designed similarly as described above for Na+/Na + exchange at equilibrium: Membrane vesicles are preincubated with about 40 m M labeled ~C1- and varying Na + concentrations (and other conditions as desired) and the rate of ~CI- efflux is measured during the subsequent incubation period after replacement of 36C1- in the medium with nonradioactive C1-. The C1- concentration in the exchange experiments must be higher than in pH and HCO 3- gradient-driven experiments to provide sufficient radioactive counts that are remaining in the membranes and therewith sufficient experimental accuracy. Acknowledgements The work presented from this laboratory was supported by grants from the National Institutes of Health (DK-25170) and the Cystic Fibrosis Foundation.

72A. B. Vaandrager, M. C. Ploemacher, and H. R. de Jonge, Biochim. Biophys. Acta. 856, 325 (1986).