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hand, oscillations may be part of a scheme in which the cell responds to a Ca 2+ trigger, followed by mechanisms designed to maintain a low intracellular Ca2+ concentration. Recent reviews of this topic are available. ~ 5 24M. J. Berridge, P. H. Cobbold, and IC S. R. Cuthbertson, Philos. Trans. R. Soc. London, B 320, 325 (1988). ,5 M. J. Berridge and A. Galione, FASEB J. 2, 3074 (1988).
[22] I s o l a t i o n o f I n t e s t i n a l E p i t h e l i a l C e l l s a n d Evaluation of Transport Functions B y GEORGE A. KIMMICH
Introduction A great deal of solute "traffic" occurs across the intestinal epithelium in relation to the vital role intestinal tissue plays in nutrient absorption and in' salt and water transfer. These solute fluxes involve transport across two separate plasma membrane boundaries and often involve a metabolic or chemical energy input at one of the two boundaries. The energy-dependent steps allow transfer against a gradient of chemical or electrochemical potential and provide the energetic basis for vectorial transfer of solutes from intestinal lumen to the circulatory system. In addition to its usual absorptive function for salt and water, the intestine also has the capacity to undergo a startling functional transformation and become a secretory organ for salt and water. This capability can be physiologically useful in helping maintain fluidity of the intestinal contents to facilitate digestion in order to allow complete nutrient absorption and to improve peristaltic movement of the lumenal contents through the intestinal tract. In certain disease states, however, including cholera and various diarrheal diseases, intestinal secretion goes beyond the bounds of a regulated activity and can lead to serious clinical problems involving severe dehydration and death due to collapse of the peripheral vasculature. No matter which functional mode is considered, in general, it is the function of the mucosal epithelial cells lining the intestinal villi and the intervillous crypt regions which are responsible for the transport capability of the tissue. A thorough understanding of intestinal transport events therefore implies an understanding of the transport properties of these cells and the regulation of cellular transport events. Many experimental approaches aimed at resolving intestinal transport events have utilized a variety of intact tissue preparations (loops, sacs, METHODS IN ENZYMOLOGY, VOL. 192
Copyright © 1990 by Academic Press, Inc. An rigins of rcproducfion in any form reserved.
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sheets, slices, etc.). These preparations are morphologically complex (villi, crypts, lamina propria, connective and muscle tissue) and contain a variety of cell types which are unrelated in function to those transport events which are characteristic of the tissue. The use of isolated epithelial cells circumvents some of the complexity otherwise associated with interpretation of transport properties observed for the intact tissue preparations. Isolation of Villous Epithelial Cells
General Considerations It is easy to scrape the intestinal mucosal epithelium free from underlying submucosal elements using the edge of a glass slide, ruler, or similar object. 1 The epithelial gemisch produced by this mechanical procedure includes intact cells, cell clumps and considerable cell debris, lamina propria, and connective tissue fragments. Intuitively, one could conclude that this material might be an ideal starting point for subsequent isolation of a functional cell population by differential centrifugation. This ideal cannot be realized, however, due to excessive amounts of mucus released by the scraping procedure which creates a gelatinous mass that prevents easy separation of the smaller tissue elements.2 Extensive proteolysis also occurs, presumably due to release of various proteolytic enzymes from the damaged cells and tissue. For these reasons, scraping or severe abrading of the tissue must be avoided in any procedure for which harvesting of functional epithelial cells is an ultimate aim. On the other hand, a variety of methods can be utilized to release epithelial cells from intestinal tissue without involving a scraping process. These methods include vibration of an intact intestine everted over a metal rod, the end of which is inserted into a Vibra-Mix motor (A.G. Chemap, Zurich, Switzerland). By varying the amplitude and interval of vibration cells can be released from different loci on the intestinal villi. 3 Longer, more vigorous vibration intervals remove cells progressively further down the villus axis toward the crypt region. Crypt cells themselves are not released unless the intestinal wall is distended with air pressure? The vibration procedure is a variation of other approaches involving simple rotation of the everted intestine at varying speeds either with or without mechanical pressure applied to the exposed mucosal surface.5 i F. Dickens and H. Weil-Malherbe, Biochem. J. 35, 7 (1941). 2 j. W. Portcus and B. Clark, Biochem. J. 96, 159 (1965). 3 D. W. Harrison and H. L. Webster, Exp. CellRes. 55, 257 (1969). 4 H. L. Webster and D. D. Harrison, Exp. Cell Res. 56, 245 (1969). 5 F. S. Sjostrand, J. UItrastruct. Res. 22, 424 (1968).
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Additional variations include the use of various chelating agents (EDTA, citrate)6 or proteolytic enzymes (trypsin, collagenase)7 in the medium bathing the tissue while applying mechanical pressure with the fingers. All of the above procedures produce isolated ceils and cell aggregates in differing yields. However, evaluation of metabolic and/or transport properties of the cell populations shows that their functional capability is not maintained for significant intervals.6,8,9 In our own experience, use of chelating agents either alone or in combination with proteolytic enzymes produces a cell population with particularly unstable characteristics. It is not unusual to observe extensive autolysis and loss of functional capability within 20 min when either chelators or proteases have been employed during the preparation. The following procedure for preparation of avian intestinal cells represents an alternative in which the cells exhibit stable transport and metabolism for at least 2 hr following isolation) TM It is important to note that the same procedure when applied to rat intestinal tissue does not produce a useful cell population. Cell yields are low and the functional properties of the isolated cells are very poor. This represents the collective experience of our own laboratory as well as the oral communications from numerous investigators throughout the world. Although we have not evaluated application of the cell isolation procedure to other commonly used laboratory animals extensively, it does appear that cells with acceptable properties can be released from rabbit tissue (G. A. Kimreich, unpublished results).
Isolation of Chicken Intestinal Cells Sacrifice a 4- to 8-week-old chicken by rapid decapitation. Open the abdominal cavity immediately and rapidly excise an intestinal segment beginning at the gizzard and extending for approximately four lengths of the pancreas. Pancreatic tissue occupies the space between the first duodenal loop. The pancreas is a convenient yardstick which varies in length with the age of the chicken in proportion to the length of the developing intestine. For a 6-week-old chicken it will be approximately 2 in. in length so that the intestinal segment removed will approximate 8 in. 6 B. K. Stern, Gastroenterology 51, 855 (1966). 7 B. K. Stern and R. W. Reilly, Nature (London) 205, 563 (1965). s B. K. Stern and W. E. Jensen, Nature (London) 209, 789 (1966). 9 W. G. J. Iemhoff, J. W. O. Van Den Berg, A. M. DePypex, and W. C. Hulsmann, Biochim. Biophys. Acta 215, 229 (1970). lo G. A. Kimmich, Biochemistry 9, 3659 (1970). ,t G. A. Kimmich, in "Methods in Membrane Biology" (E. D. Korn, ed.), Vol. 5, p. 51. Plenum, New York, 1975.
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Rinse out the intestinal contents by flushing 50 ml of 0.9% ice-cold saline through the segment with the aid of a syringe. Slit the intestine lengthwise to make a flat sheet of tissue, and cut the sheet transversely into several segments about 2 in. in length. Incubate the short.tissue segments for 30 rain at 37 ° in 10 ml of isolation medium with gentle oscillation (approximately 100 clam). Isolation medium: 125 m M NaC1, 20 m M HEPES-Tris (pH 7.4), 10 m M mannose, 2.5 m M glutamine, 0.5 m M p-hydroxybutyrate, 3.0 m M K2HPO4, 1.0 m M MgCI2, 1.0 m M CaC12, 1 mg/ml bovine serum albumin (BSA), 1 mg/ml hyaluronidase (Sigma, type I) Glutamine and p-hydroxybutyrate are included in the medium because of work with perfused tissue which indicates that intact intestine metabolizes these two compounds preferentially over other serum constituents, t2 Mannose is rapidly glycolyzed by isolated intestinal cells but is not transported by the Na+-dependent sugar transport system. Together these three fuel molecules allow the isolated cells to establish and maintain more stable ion and sugar gradients than in their absence. After incubation, some cells and cell aggregates may have detached from the tissue. Much more extensive detachment can be induced by mixing the suspension of segments with the tip of a plastic pipet. Sometimes it is necessary to "pin" a segment down on the floor of the incubation beaker with the pipet tip and "sweep" the segment across the floor of the beaker a few times. By this procedure large numbers of cells can be released, as indicated by an increasing degree of opacity of the isolation medium. At this point, pour the contents of the isolation beaker through a section of nylon stocking material in order to filter off the intestinal segments and to remove mucus which will cling to the mesh. Collect the filtrate containing the cell population in a clean plastic beaker. It is important that all procedures be carded out in plasticware in order to avoid fragmentation of the cell population, which can occur when cells are exposed to the microscopically jagged surfaces associated with glass surfaces. Hyaluronidase is removed from the suspension medium by centrifugation of the cells at low speed (100-150 g) and resuspension in isolation medium without hyaluronidase. The cell pellet is very loosely packed by this procedure such that complete removal would require several wash steps. However, one or two washes produces a population with acceptable functional characteristics. Because the cell pellet aggregates readily due to residual mucus it is necessary to resuspend the pellets by gently pulling the 12 H. G. Windmueller and A. E. Spaeth, J. Biol. Chem. 253, 69 (1977).
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cells and medium in and out of a plastic pipet a few times until a visually homogeneous suspension is obtained. If the suspension is examined microscopically at this point, it will be observed to consist of a few isolated cells and cell doublets, but primarily of cell d u m p s containing a few to 10 or 20 cells each. The individual cells will appear rounded morphologically, as should be expected once the restraint represented by neighbor cells has been released. Careful observation will reveal the brush border "mustache" over a portion of the cell membrane representing the original lumenal surface. Despite the lack of fully isolated individual cells, the resuspension can be sampled readily and reproducibly with a narrow-bore micropipet. Replicate samples, carefully taken, wiU contain uniform amounts of total protein. Each chick intestine will yield I to 2 ml of loosely packed cells or about 50 mg of cell protein. Total cell protein can be determined spectrophotometrically by incubating an aliquot of the suspension with Biuret reagent in comparison to BSA standards. 13 For most purposes we usually prepare cell suspensions containing 15-20 nag protein/ml and store the suspensions on ice until they are used experimentally. For best results, the cells should be used within 1 hr of the time of preparation. Because the cells settle and aggregate rapidly, it is always necessary to swirl the suspension vigorously a few times in order to ensure uniform sampling of the stock suspension. It is not advisable to try and achieve further separation of the cell population. This can be accomplished by vigorous stirring or refluxing in and out of a pipet, but only at the expense of the total number of intact cells and concomitant loss of functional properties. Because of the nature of the cell suspension it is difficult to count cells or to determine vital dye staining accurately so that it is better to rely on protein or DNA determinations for standardizing cell numbers. Stability of metabolic and functional capabilities (e.g., ion or organic solute transport) proves to be a more reliable index of viability than do staining techniques. Evaluation of T r a n s p o r t A variety of metabolic parameters can be evaluated readily for the cell populationl°: (1) glucose utilization, (2) lactate production, (3) oxygen consumption, (4) CO2 production from various substrates, and (5) adenine nucleotide ratios or energy charge. The magnitude and stability of each of these parameters individually and collectively convey information relating to biochemical function of the isolated cells. However, they do not convey 13A. G. Gornall, C. S. Bardawill, and M. M. David, J. Biol. Chem. 177, 751 (1949).
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much information regarding the integrity of the plasma membrane for the cell population. Indeed, cell-free extracts of many tissues can catalyze similar metabolic activities. For this reason, it seems likely that the magnitude and stability of solute gradients maintained by the cell population convey more meaningful information about the quality of the cell preparation. For energy-dependent transport events which create transmembrane concentration gradients of solutes, assessment of gradient magnitude and stability provides experimental insight not only to the integrity of the cellular plasma membrane, but also to the set of metabolic events responsible for establishing the energetic forces driving the transport systems and the integration between metabolic and transport events.
Accumulation of ot-Methylglucoside (~-MG) Because of a marked tendency for the cell population to clump and form large aggregates when incubated in a quiescent environment, it is necessary to perform experimental work under conditions in which the suspension is continually shaken at an adequate rate. This provides sufficient homogeneity for reproducible sampling as well as appropriate oxygenation of the medium to sustain the rather high aerobic metabolism of the epithelial cells. In our experience, the surface-to-volume ratio of the incubation vessel exhibits a rather narrow optimum. This relates in part to the necessity for avoiding too great a depth of incubation medium such that the shaking action of the incubator bath can be low enough to allow convenient sampling without being so slow that cells congregate in the lower levels of the suspension, leaving a lower cell density near the surface. Oscillating shaker baths are better than rotary because the latter tend to create higher cell densities in the periphery of the incubation vessel. We have found that a 4-ml total incubation volume in a 50-ml beaker oscillated at 100 cpm provides an ideal set of conditions when the cell density is between 3 to 5 mg protein/ml. In order to conserve isotope and limit the number of cells required we have also used a 2.2- to 2.5-ml volume in 30-ml beakers. An appropriate format for assessing transport capability for several kinds of solutes can be illustrated by evaluating the uptake ofa-[~4C]methylglucoside as follows: Incubation Vessel Preparation. Prepare a set of 30-ml plastic beakers such that each beaker contains 2.2 ml of the same medium used for cell isolation, but without hyaluronidase. In addition, each beaker should contain 100 # M ¢x-methylglucosideand 0.1-0.2 ltCi/ml ofa-[l~]MG. One of these beakers will have no further additions and will be used to assess ot-MG uptake under control conditions. Another will contain 200 # M
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phlorizin, which is a potent inhibitor of Na+-dependent sugar transport in these cells. Other beakers will contain whatever agents one might wish to test in terms of possible action on the sugar transport capability of the cells. For instance, one might wish to test several concentrations of ouabain because of its inhibitory action on Na + transport in order to assess the effect of dissipating the A/ZNa+on the capability of the cells for establishing sugar gradients. In this case, the set of incubation beakers might include the following: All beakers: 2.2ml incubation m e d i u m + 0.25 /zCi a-[14C]MG ( - 0.1/zCi/ml) Beaker l: No addition Beaker 2: + 200 # M phlorizin Beaker 3:+0.5 # M ouabain Beaker 4: + 1.0/all4 ouabain Beaker 5:+2.0/zM ouabain Beaker 6: + 5.0 p M ouabain The beaker set should be incubated at 37 ° with gentle shaking (100 cpm) in order to thermally equilibrate the suspension medium. A stock cell suspension ( - 2 0 mg cell protein/ml) should be thermally equilibrated at the same time. Cellular sugar uptake is initiated by transferring a 0.1-ml sample of cell suspension to each of the experimental beakers at time zero. At intervals after the cell transfer, 100-/zl samples of the incubation medium are taken with the aid of an automatic micropipet. It is important that the shaker continue operating during the sampling procedure in order to achieve uniform samples of the cell suspension. Even a short interruption of the shaking motion can lead to cell aggregation and varying amounts of cell protein from sample to sample with resultant data scatter. In order to study sugar uptake during formation of a steady state sugar gradient, an appropriate sampling schedule might be 1, 5, 10, 20, 40, and 60 rain. For the suggested experimental format (surface-to-volume ratio) it is difficult to take more than six samples per incubation beaker before the medium becomes too shallow for easy sampling. Each 100-/zl sample should be diluted into 4 ml of ice-cold incubation medium (without radioactivity) immediatelyafter being taken. It is convenient in terms of later sample processing to have the diluent for each experimental aliquot in 5-ml polyethylene scintillation minivials. The diluted samples should be centrifuged as soon as possible using a rapidly accelerating tabletop centrifuge. If the six incubations are run in parallel, each set of six samples for a given time point can be processed while waiting to take the sample set for the next time point. After centrifugation
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for 30 sec the supematant is removed by aspiration and 4.0 ml of fresh ice-cold wash medium is added to each tube. After a second centrifugation and supernatant aspiration the cell pellets are ready for extraction and measurement of the amount of isotope accumulated. Using an Autocrit (with modified head) (Clay Adams, Parsippany, NJ) or an MHCT II centrifuge, both wash steps can be completed in less than 3 rain. The carryover of extracellular radioactivity following the two washes can be evaluated by incubating the cells with impermeant [~4C]polyethylene glycol. For the recommended amount of cellular protein this carryover is so small (-0.02 gl or 4 CPM at 0.1 gCi/ml) that it can be ignored when calculating cellular uptake of sugar. Chilling of the diluted cell aliquots is sufficient to prevent subsequent sugar fluxes without the necessity of including any transport inhibitors in the wash medium. This can be evaluated by maintaining diluted samples taken under comparable conditions on ice for varying intervals of time prior to centrifugation and comparing the cellular content of isotope as a function of storage time. For a-MG only 5% of the accumulated sugar is lost to the wash medium even after 20 rain of sample storage time. Because sample processing is typically complete in 2 - 3 min, no correction is required. This should be evaluated for each solute whose transport is examined, however, in order to ascertain if corrections due to solute loss during sample processing are necessary. Once the washed cell pellets are prepared they can be extracted for scintillation counting simply by dissolving each pellet in a 5-ml volume of Liquiscint (National Diagnostics, Manville, N J). If polyethylene minivials are utilized for processing the cell samples, it is only necessary to add the Liquiscint directly to the minivial for a few minutes prior to counting each vial. For the conditions described, the cell pellets do not introduce any quenching or chemiluminescent properties, so no corrections for those events are necessary.
Sample Calculation. If a 10-#1 sample is taken from each incubation beakerprior to adding cells, the count rate determined for each one (CPM) can provide a measure of either the volume specific activity or the picomolar specific activity for the a-[~4C]MG in each experimental run. Volume specific activity (VSA) = (CPM/10) × D, where D is the dilution factor, due to adding cell suspension without isotope to the incubation medium (0.96 for the conditions described earlier). The units for VSA are simply CPM/gl for the final incubation suspension. VSA is useful because it can provide a quick assessment ofintracellular volume/mg of cell protein. For instance, in the experimental case which includes phlorizin, concentrative accumulation of a-MG is inhibited so that the final steady state uptake of sugar simply represents the point at
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which a-MG has equilibrated between the extracellular water and that part of the cellular water to which sugar has access. If the steady state CPM in the presence ofphlorizin is divided by VSA and the result is divided by milligrams cell protein in each experimental aliquot one obtains the microliters of cell water per milligram cellular protein for the cell population. We routinely find a value of between 2.5 and 3 pl/mg cell protein by this procedure. By obtaining a value for the number of cells in each sample this can be converted to a volume per cell basis. However, because of cell aggregation in the suspension, mentioned earlier, it is difficult to determine an accurate value for cell number and we routinely express transport data in terms of uptake per milligram cell protein. Picomolar specific activity (PSA) --- VSA/C, where C is the final concentration ofct-MG in the experimental beakers (in pmol/#l). The units of PSA are CPM per picomole. If CPM for any experimental sample is divided by PSA the result gives a value for the amount (in picomoles) of a-MG accumulated by the cells in that sample over the time interval allowed before sampling. These values can be useful in determining the rate of uptake of a particular solute under different experimental conditions. They are best employed in short-term unidirectional influx experiments in which uptake is linear with time so that initial transport rates can be compared. Such initial rates can provide insight to the magnitude of the driving forces acting on the transport system and allow kinetic analysis of the function of the system for various experimental conditions. Solute Gradients. Assuming that phlorizin-inhibited cells only allow equilibration of sugar between extracellular and intracellular water the steady state counts observed with phlorizin (CPMF0 can also provide an approximation for gradient-forming capacity for any of the other conditions evaluated. It is only necessary to divide CPM for a particular sample by CPM~ to calculate the steady state sugar distribution ratio achieved in that sample. By this procedure we find that chick intestinal cells can routinely establish an apparent steady state sugar gradient of about 70- to 90-fold under control conditions when extracellular ot-MG is 100 gM. Approximately 30 rain is required to achieve the steady state. Stability of the sugar gradient during subsequent incubation provides a measure of the stability of those driving forces acting to create the sugar gradient (Na + gradient and membrane potential). Sugar gradients calculated by the method described above are "apparent" because the procedure assumes that the extracellular sugar concentration has not changed during the experimental interval. For the conditions described where the cellular volume represents about 0.25% of the total incubation volume (2.0 mg cell protein X 3/ll cell water/mg protein in a 2.3-ml volume), a 70-fold accumulation of solute relative to the initial extracellular concentration will decrease the extraceUular concentration by
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TABLE I SUGAR ACCUMULATION RATIOS FOR INTESTINAL EPITHELIAL CELLS INCUBATED AT DIFFERENT OUABAIN CONCENTRATIONS Ouabain concentration (/zM) 0 0.5 1.0 2.0 5.0 0 + 200/.tM Phlorizin
Sugar accumulation ratio* 81 56 36 17 3 1
°Defined after a 40-rain incubation as the ratio of a - p ~ ] M G counts in an aliquot of cells for the indicated ouabain concentration to that observed in the presence of 200/zM phlorizin.
approximately 15- 20%. This depletion of medium solute can be detected by counting the isotope present in an aliquot of the supernatant above the cell pellet following the first centrifugation of the diluted cell samples. When the decrease in extracellular concentration is taken into account the actual accumulation ratio for ~-MG established by the isolated chick cells typically is about 70/(1 - 0 . 1 5 ) or -80-fold. 14 Table I shows the actual accumulation ratios observed for a-MG in the experiment described above for different ouabain concentrations. Urn'directional Influx. As mentioned earlier, short-term experiments similar to those described above can be used to determine the rate of accumulation of solute by the cell suspension. 15 In this situation it is usually necessary to take aliquots of the suspension about every I0-15 sec for a total of 40-60 sec, by which time appreciable backflux begins to occur such that linearity of influx is lost. The data provide information regarding the capacity of the transport system for the particular conditions employed. For any given solute, entry usually occurs by more than one route so that a procedure is needed to identify that part of the total flux which occurs by the route of interest. In the case of a-MG it is convenient to use phlorizin sensitivity as the means of identifying influx via the Na+-dependent sugar carrier. Table II shows the influx of ¢x-MG at an extracellular concentration of 100 # M and at four different phlorizin concentrations in comparison to influx observed without phlorizin or without 14G. A. Kimmich and J. RancHes, Am. J. Physiol. 237, C56 (1978). ~s j. Randles and G. A. Kimmich, Am. J. Physiol. 234, C64 (1978).
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TABLE II UNIDIRECTIONAL INFLUX OF 100 f12~f a-p4C]MG
INTO ISOLATEDCHICKEN INTESTINALCELLS (EFFECT OF PI..ILORIZINAND LACK OF Na +)
Incubation conditions
Unidirectional influxa of/z-MG (pmol/min/mg protein)
Control +0.5/zM Phlorizin + 1.0//.M Phlorizin + 10.0/zM Phlorizin + 200/zM Phlorizin No Na +b
424 358 299 169 25 22
° The influx was determined from five samples taken over a 36-sec interval in each case. Linear uptake is maintained for this interval. Influx ffiCPMt/(PSA.t.mg protein) where t = time of sampling in minutes. NaCI in the incubation medium was replaced by 125 m M tetramethylammonium chloride.
Na +. Note that about 95% of the total a-MG influx is blocked by phlorizin or by lack of sodium.
Sodium Ion-Independent Sugar Transport Intestinal epithelial cells accumulate various sugars via a concentrative Na+-dependent sugar transport system localized in the brush border boundary of the cell. Accumulated sugar escapes from the cell to the circulatory systems via a facilitated diffusion transport system localized in the basolateral boundary. ~-MG satisfies only the Na+-dependent transport system so that it represents the sugar of choice for evaluating function of this system. 16 On the other hand, 2-deoxyglucose (2-DOG) selectively satisfies the Na+-independent carrier so that unidirectional influx of 2-DOG represents a means of studying basolateral transfer of sugar. ~7 Because 2-DOG can be phosphorylated by hexokinase it is not possible to use longer term steady state experiments with 2-DOG in order to determine equilibration time or cellular volumes. Certain nonmetabolized sugars such as 3-O-methylglucose (3-OMG) are transported by both of the intestinal sugar transport systems. At the 16G. A. Kimmich and J. Randles, Am. J. Physiol. 241, C227 (1981). ~7G. A. Kimmich and J. Randles, J. Membr. Biol. 27, 353 (1976).
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ISOLATION OF INTESTINAL EPITHELIAL CELLS TABLE III
EFFECT OF VARIOUS INHImTORSOF INTESTINAL SEROSAL SUGAR TRANSFER(Na+
INDEPENDENT)ON ACCUMULATIONOF 3-[14C]OMG BY ISOLATEDINTESTINALCELLS ( N a + DEPENDENT)
Agent"
Concentration (/tM)
--
--
Dihydroquercitin Theophylline Hesperetin Naringenin Phloretin Apigenin Kaempferol Cytochalasin B
100 5 100 100 100 100 100 100
Steady state 3-OMG accumulation ratiob 10 15 25 28 32 35 43 48 65
"Dihydroquercitin, hesperetin, and naringenin are flavanones. Apigenin and kaempferol are flavones. Phloretin is an analog of the tlavanones in which a heterocyclic ring has been opened. b For 100/zM 3-[t4C]OMG.
steady state, cells transporting 3-OMG accumulate the sugar against a concentration gradient at the brush border and lose it across the basolateral boundary via Na+-independent transfer. The magnitude of the steady state 3-OMG gradient maintained reflects the function of both transport events) + Agents which selectively interfere with the passive facilitated diffusional transfer system will allow the cells to establish better concentration gradients of 3-OMG than control cells can maintain. This can be demonstrated using the same techniques already described, but with 3-[14C]OMG as the test sugar. Phloretin, ts theophylline,~5cytochalasin B, 14 and various flavones or flavanonesIs can all be used to interfere with passive sugar et~ux as shown in Table III. The degree of gradient enhancement is proportional to the degree of inhibition of the Na+-independent transport system for agents which act solely on the facilitated diffusion carrier.
A TP-Depleted Epithelial Cells Isolated intestinal cells can be depleted of more than 90% of their ATP content by a brief incubation with 20 to 80/zM rotenon¢, t9 Because the ATP pool in these cells turns over several times per minute, most of the ~sG. A. Kimmich and J. Randles, Membr. Biochem. 1, 221 (1978). t9 C. Carter-Su and G. A. Kimmich, Am. J. Physiol. 237, C57 (1979).
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depletion occurs in the first minute after rotenone addition. If ouabain is then added to prevent possible slow turnover of the Na+,K+-ATPase due to the residual ATP, it is possible to dissipate the A/zsa+ which is ordinarily established due to monovalent ion transport. The ATP-depleted cells can be utilized experimentally as if they are giant membrane vesicles in order to demonstrate the role for various driving forces acting to energize concentrative transport of sugar or other solutes. They offer significant advantages over conventional plasma membrane vesicles because a more favorable surface-to-volume ratio allows much longer intervals over which influx remains linear with consequent greater ease of flux measurement and better reliability of the experimental values determined. Gradients of chemical potential or electrical potential or both can be experimentally imposed across the plasma membrane of the ATP-depleted cells and the role of each can be determined in terms related to energization of the Na+-dependent transport system? 9~° Unidirectional influx for the solute of interest as well as transient gradient forming capability due to the imposed driving force can each be used for providing insight to the transport mechanism. Imposed Na + Gradients of Defined Magnitude (A~Na+) In order to selectively study effects of a change in Na + gradient, it is necessary to prevent changes in diffusion potentials (AM) due to diffusional flux of the imposed Na + gradient. 2~ This can be accomplished best by the inclusion of high concentrations of highly permeant ions during the ATP depletion procedure and subsequent incubation with Na + in order to "short circuit" the system and "clamp" the potential near zero. For this purpose, we have found that replacing all or part of the NaCl during ATP depletion with tetramethylammonium (TMA +) nitrate is effective. Cells which have been equilibrated with tetramethylammonium (sodium) nitrate can be transferred to an incubation medium with 125 m M sodium nitrate in order to create the experimental Na + gradient. Sometimes in order to ensure that the potential remains constant it is necessary to include potassium nitrate plus valinomycin in both the preincubation and the incubation medium in place of part of the tetramethylammonium nitrate. The high permeability of both NO 3- and K + (with valinomycin) relative to Na + provides an especially stable A¥ when Na + is added hack to the system.22 2oc. Carter-Suand G. A. Kimmich,Am. J. Physiol. 238, C73 (1980). 2~G. A. Kimmichand J. Randles,Biochim. Biophys. Acta 596, 439 (1980). G. A. Kimmich,J. Randles, D. Restrepo,and M. Montrose,Am. J. Physiol. 248, C399 (1985).
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Imposed M e m b r a n e Potentials of Defined Magnitude (A~) If cellsarc A T P depleted in a medium containing 50 m M potassium gluconatc with valinomycin and I00 m M sodium gluconate and then introduced to an incubation medium with 100 m M N a N O 3 and 50 m M tetramethylammonium nitrate,itispossibleto createa diffusionpotential duc to thc imposed gradients of permeant ions but with no imposed Na + gradient.By varying the amount ofextraceHularNO~3 (replacedwith slowly permeant gluconatc) or T M A + (replacedwith K+), itispossibleto manipulate the A ~ over a range of more than 60 m V and to determine the effect on a given Na+-dependent solutetransport system,e3~ Imposed Electrochemical Gradients of Defined Magnitude
(A]]Na+)
By using a combination of the above conditions,gradients of both A ¥ and A~t~+ can be created.A useful approach is to A T P deplete the cellsin potassium gluconate and to transferthem to an incubation medium with NaNO3. A range of A/~+ for differentcellpopulations is constructed by varying the amounts of Na +, K +, and/or NO3- in the two working media. Because the ATP-dcplcted cellscannot establisheitherelement of a A/~N,+ mctabolically, thc imposed gradients will dissipateduc to diffusionalion fluxes.Ncvertbeless,the degree to which an imposed gradientiseffectivein energizing a solute transport system can be assessed by examining the magnitude of "overshoot" or transientgradient-forming capacity the cells exhibit for the solute. Table IV shows the peak accumulation ratio observed for I00 ~ ~ - M G when the various gradientsdescribed above were imposed on isolatedintestinalcells.In each case, the accumulation ratio was defined as the C P M for the peak sample divided by C P M for the sample at eventual steady state(i.e.,fullydissipatedgradients). Measurement of Intracellular Na + and K +
The procedure described for monitoring a-MG accumulation can be modified slightly in order to provide values for intracellular Na + and K + content for different experimental conditions. In this case, instead of dissolving the cell pellets in Liquiscint for scintillation countin~ they are extracted in 3% PCA and an aliquot of the supernatant, after centrifugation of the denatured cellular protein, is used for flame photometry. The G. A. Kimmich, J. Randles, D. Restrepo, and M. Montrose, Ann. N. Y. Acad. Sci. 456, 63 (1985). 24D. Restrepo and G. A. Kimmich, J. Membr. Biol. 87, 159 (1985).
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TABLE IV SUGAR TRANSPORT IN ATP-DEPLETED INTESTINAL CELLS DRIVEN BY AN EXPERIMENTALLY IMPOSEDNa + GRADIENT, MEMBRANE POTENTIAL, OR ELECTROCHEMICAL POTENTIAL GRADIENT FOR Na +
Driving force
AgN~+ A~/ A~N,,+ A~Na+ + phlorizin
Peak sugar gradient 11.0 7.4 19.6 1.0
amount of monovalent ions determined in this manner can be expressed as an intracellular concentration by utilizing a value for cell volume and assuming that each ion is uniformly distributed in the cell water. Concentrations calculated in this manner are not highly reliable, however, because of ion binding to cellular anionic constituents and possible nonuniform distribution among organelles. Both phenomena create an intracellular ion activity which is considerably lower than values determined on the basis of ion amount per volume measurements. This is particularly true for Na +, where the activity measured with ion-selective electrodes is usually about half the calculated concentration. 2s Nevertheless, determination of the amount of intracellular ion under different experimental conditions can give qualitative information regarding changes in the magnitude of ion gradients maintained by the cells. M e a s u r e m e n t of M e m b r a n e Potentials The unidirectional influx or rate of uptake of a lipophilic cation by any cell population should be proportional to the magnitude of the membrane potential maintained by those cells. If the influx is studied as the membrane potential is varied experimentally, one can construct a calibration curve for the relationship between flux and A¥. This calibration curve can be used to determine the membrane potential for situations in which it has not been experimentally created. 22m A useful lipophilic ion to be used for this purpose is [14C]tetraphenylphosphonium (TPP+). Gradients of potassium gluconate imposed on ATP-depleted cells can be used to create potentials of defined magnitude. Valinomycin is used to enhance K + permeability and this, in conjunction 25 j. O'Doherty, J. F. Garcia-Daz, and W. MeD. Armstrong, Science 203, 1349 (1979).
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with the low permeabilityof gluconate, allows formation of a K + equilibrium potential,the magnitude of which can be calculatedfrom the Ncrnst equation22:
A~U= (RT/F) In ([K+lo/[K+]i)
in which T is temperature, F is the Faraday constant, and R is the ideal gas law constant. A useful procedure is to ATP deplete the cells in a medium containing 150 m M potassium gluconate with 20 m M TMA-HEPES buffer and 1 m M CaSO4. No other salt constituents are necessary. In order to initiate the influx measurement the cells are diluted 1 : 10 into 150 m M tetramethylammonium gluconate, 20 raM HEPES, and 1 m M CaSO4 or into media in which various mixtures of tetramethylammonium gluconate and potassium gluconate are used in order to vary the extracellular K + concentration. Valinomycin and 1 # M [~4C]TPP+ are included in the incubation medium and four to five samples are taken during the first minute of incubation in a manner similar to that already described for monitoring unidirectional influx of any solute. All influx values are expressed relative to the value determined when no K + gradient is imposed which is arbitrarily assigned a value of 1.0. This is defined as the flux at a diffusion potential of 0 mV. Relative fluxes for several different K + gradients (i.e., potentials) are given in Table V. The use of relative fluxes instead of absolute values avoids the necessity of having to determine an independent value for the permeability constant for TPP +. For intestinal epithelial cells, the TPP + flux vs A~, relationship is the same as that predicted by the Goldman flux equation. 23m This calibration curve can then be used to determine an unknown potential. Only two flux measurements must be made. In one, the TPP + influx is determined TABLE V [~4C]TPP+ INFLUX INTO INTESTINALCELLS WITH DIFFERENT POTASSIUMGLUCONATEGRADIENTS
[K+].J[K+]o
Aq/ (mV)
Relative TPP + influxa
1 2 5 10
0 --18 --42 --60
1.0 1.4 2.0 2.6
=All flux values are given as a multiple of the flux observed when no K + gradient (A~/) was imposed which was assigned an arbitrary value of 1.0.
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TABLEVI EFFECT OF Na+-DEPENDE~r SUGAR TRANSPORTON" [14C]TPP+ INFLUX IN ISOLATED INTESTINAL CELLS
Incubation conditions
Relative TIP+ influx
A~ (mY)
Control + 10 mMa-MG + Rotenoneand ouabain
2.2 1.9 1.0
-55 -45 0
for the particular experimental conditions for which you wish to determine a A¥. This influx is expressed relative to (i.e., as a multiple or fraction of) the influx measured in cells which are ATP depleted and incubated sufficiently long to dissipate ion gradients. This case is defined as A~ = 0just as in the case described above in which the calibration relationship was defined relative to a situation with no imposed K + gradient. It is now only necessary to pick the A¢/off the calibration curve for the particular flux ratio determined by the two flux measurements (Jv,_ffJv,_o). Table VI shows the TPP + influx ratio and corresponding A~ determined for intestinal cells incubated under standard conditions and with 10 m M a-MG added. Actively accumulated sugars are known to partially depolarize the membrane potential due to electrogenic function of the Na+-dependent sugar transport system. Note that the added sugar causes about a 10-mY depolarization of the membrane potential measured in this manner, just as reported in experiments in which A ¢ was directly monitored with microelectrodes.26 Summary Epithelial cells can be isolated from the small intestine of chickens by a procedure involving hyaluronidase treatment of the intact tissue. The isolated cells retain a high degree of functional activity as assessed by the formation of 70-fold gradients of a-MG. Stability of the sugar gradients reflects maintenance of stable electrochemical Na + gradients across the plasma membrane. The cells can be used to evaluate the properties of Na+-dependent sugar transport, Na+-independent sugar transport, ion transport, metabolism, membrane potentials, and the integration of these events, all of which are important to achieving a stable sugar gradient. 26R. C. Roseand S. O. Schultz, J. Gen. Physiol. 57, 639 (1971).
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hand, oscillations may be part of a scheme in which the cell responds to a Ca 2+ trigger, followed by mechanisms designed to maintain a low intracellular Ca2+ concentration. Recent reviews of this topic are available. ~ 5 24M. J. Berridge, P. H. Cobbold, and IC S. R. Cuthbertson, Philos. Trans. R. Soc. London, B 320, 325 (1988). ,5 M. J. Berridge and A. Galione, FASEB J. 2, 3074 (1988).
[22] I s o l a t i o n o f I n t e s t i n a l E p i t h e l i a l C e l l s a n d Evaluation of Transport Functions B y GEORGE A. KIMMICH
Introduction A great deal of solute "traffic" occurs across the intestinal epithelium in relation to the vital role intestinal tissue plays in nutrient absorption and in' salt and water transfer. These solute fluxes involve transport across two separate plasma membrane boundaries and often involve a metabolic or chemical energy input at one of the two boundaries. The energy-dependent steps allow transfer against a gradient of chemical or electrochemical potential and provide the energetic basis for vectorial transfer of solutes from intestinal lumen to the circulatory system. In addition to its usual absorptive function for salt and water, the intestine also has the capacity to undergo a startling functional transformation and become a secretory organ for salt and water. This capability can be physiologically useful in helping maintain fluidity of the intestinal contents to facilitate digestion in order to allow complete nutrient absorption and to improve peristaltic movement of the lumenal contents through the intestinal tract. In certain disease states, however, including cholera and various diarrheal diseases, intestinal secretion goes beyond the bounds of a regulated activity and can lead to serious clinical problems involving severe dehydration and death due to collapse of the peripheral vasculature. No matter which functional mode is considered, in general, it is the function of the mucosal epithelial cells lining the intestinal villi and the intervillous crypt regions which are responsible for the transport capability of the tissue. A thorough understanding of intestinal transport events therefore implies an understanding of the transport properties of these cells and the regulation of cellular transport events. Many experimental approaches aimed at resolving intestinal transport events have utilized a variety of intact tissue preparations (loops, sacs, METHODS IN ENZYMOLOGY, VOL. 192
Copyright © 1990 by Academic Press, Inc. An rigins of rcproducfion in any form reserved.
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sheets, slices, etc.). These preparations are morphologically complex (villi, crypts, lamina propria, connective and muscle tissue) and contain a variety of cell types which are unrelated in function to those transport events which are characteristic of the tissue. The use of isolated epithelial cells circumvents some of the complexity otherwise associated with interpretation of transport properties observed for the intact tissue preparations. Isolation of Villous Epithelial Cells
General Considerations It is easy to scrape the intestinal mucosal epithelium free from underlying submucosal elements using the edge of a glass slide, ruler, or similar object. 1 The epithelial gemisch produced by this mechanical procedure includes intact cells, cell clumps and considerable cell debris, lamina propria, and connective tissue fragments. Intuitively, one could conclude that this material might be an ideal starting point for subsequent isolation of a functional cell population by differential centrifugation. This ideal cannot be realized, however, due to excessive amounts of mucus released by the scraping procedure which creates a gelatinous mass that prevents easy separation of the smaller tissue elements.2 Extensive proteolysis also occurs, presumably due to release of various proteolytic enzymes from the damaged cells and tissue. For these reasons, scraping or severe abrading of the tissue must be avoided in any procedure for which harvesting of functional epithelial cells is an ultimate aim. On the other hand, a variety of methods can be utilized to release epithelial cells from intestinal tissue without involving a scraping process. These methods include vibration of an intact intestine everted over a metal rod, the end of which is inserted into a Vibra-Mix motor (A.G. Chemap, Zurich, Switzerland). By varying the amplitude and interval of vibration cells can be released from different loci on the intestinal villi. 3 Longer, more vigorous vibration intervals remove cells progressively further down the villus axis toward the crypt region. Crypt cells themselves are not released unless the intestinal wall is distended with air pressure? The vibration procedure is a variation of other approaches involving simple rotation of the everted intestine at varying speeds either with or without mechanical pressure applied to the exposed mucosal surface.5 i F. Dickens and H. Weil-Malherbe, Biochem. J. 35, 7 (1941). 2 j. W. Portcus and B. Clark, Biochem. J. 96, 159 (1965). 3 D. W. Harrison and H. L. Webster, Exp. CellRes. 55, 257 (1969). 4 H. L. Webster and D. D. Harrison, Exp. Cell Res. 56, 245 (1969). 5 F. S. Sjostrand, J. UItrastruct. Res. 22, 424 (1968).
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Additional variations include the use of various chelating agents (EDTA, citrate)6 or proteolytic enzymes (trypsin, collagenase)7 in the medium bathing the tissue while applying mechanical pressure with the fingers. All of the above procedures produce isolated ceils and cell aggregates in differing yields. However, evaluation of metabolic and/or transport properties of the cell populations shows that their functional capability is not maintained for significant intervals.6,8,9 In our own experience, use of chelating agents either alone or in combination with proteolytic enzymes produces a cell population with particularly unstable characteristics. It is not unusual to observe extensive autolysis and loss of functional capability within 20 min when either chelators or proteases have been employed during the preparation. The following procedure for preparation of avian intestinal cells represents an alternative in which the cells exhibit stable transport and metabolism for at least 2 hr following isolation) TM It is important to note that the same procedure when applied to rat intestinal tissue does not produce a useful cell population. Cell yields are low and the functional properties of the isolated cells are very poor. This represents the collective experience of our own laboratory as well as the oral communications from numerous investigators throughout the world. Although we have not evaluated application of the cell isolation procedure to other commonly used laboratory animals extensively, it does appear that cells with acceptable properties can be released from rabbit tissue (G. A. Kimreich, unpublished results).
Isolation of Chicken Intestinal Cells Sacrifice a 4- to 8-week-old chicken by rapid decapitation. Open the abdominal cavity immediately and rapidly excise an intestinal segment beginning at the gizzard and extending for approximately four lengths of the pancreas. Pancreatic tissue occupies the space between the first duodenal loop. The pancreas is a convenient yardstick which varies in length with the age of the chicken in proportion to the length of the developing intestine. For a 6-week-old chicken it will be approximately 2 in. in length so that the intestinal segment removed will approximate 8 in. 6 B. K. Stern, Gastroenterology 51, 855 (1966). 7 B. K. Stern and R. W. Reilly, Nature (London) 205, 563 (1965). s B. K. Stern and W. E. Jensen, Nature (London) 209, 789 (1966). 9 W. G. J. Iemhoff, J. W. O. Van Den Berg, A. M. DePypex, and W. C. Hulsmann, Biochim. Biophys. Acta 215, 229 (1970). lo G. A. Kimmich, Biochemistry 9, 3659 (1970). ,t G. A. Kimmich, in "Methods in Membrane Biology" (E. D. Korn, ed.), Vol. 5, p. 51. Plenum, New York, 1975.
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Rinse out the intestinal contents by flushing 50 ml of 0.9% ice-cold saline through the segment with the aid of a syringe. Slit the intestine lengthwise to make a flat sheet of tissue, and cut the sheet transversely into several segments about 2 in. in length. Incubate the short.tissue segments for 30 rain at 37 ° in 10 ml of isolation medium with gentle oscillation (approximately 100 clam). Isolation medium: 125 m M NaC1, 20 m M HEPES-Tris (pH 7.4), 10 m M mannose, 2.5 m M glutamine, 0.5 m M p-hydroxybutyrate, 3.0 m M K2HPO4, 1.0 m M MgCI2, 1.0 m M CaC12, 1 mg/ml bovine serum albumin (BSA), 1 mg/ml hyaluronidase (Sigma, type I) Glutamine and p-hydroxybutyrate are included in the medium because of work with perfused tissue which indicates that intact intestine metabolizes these two compounds preferentially over other serum constituents, t2 Mannose is rapidly glycolyzed by isolated intestinal cells but is not transported by the Na+-dependent sugar transport system. Together these three fuel molecules allow the isolated cells to establish and maintain more stable ion and sugar gradients than in their absence. After incubation, some cells and cell aggregates may have detached from the tissue. Much more extensive detachment can be induced by mixing the suspension of segments with the tip of a plastic pipet. Sometimes it is necessary to "pin" a segment down on the floor of the incubation beaker with the pipet tip and "sweep" the segment across the floor of the beaker a few times. By this procedure large numbers of cells can be released, as indicated by an increasing degree of opacity of the isolation medium. At this point, pour the contents of the isolation beaker through a section of nylon stocking material in order to filter off the intestinal segments and to remove mucus which will cling to the mesh. Collect the filtrate containing the cell population in a clean plastic beaker. It is important that all procedures be carded out in plasticware in order to avoid fragmentation of the cell population, which can occur when cells are exposed to the microscopically jagged surfaces associated with glass surfaces. Hyaluronidase is removed from the suspension medium by centrifugation of the cells at low speed (100-150 g) and resuspension in isolation medium without hyaluronidase. The cell pellet is very loosely packed by this procedure such that complete removal would require several wash steps. However, one or two washes produces a population with acceptable functional characteristics. Because the cell pellet aggregates readily due to residual mucus it is necessary to resuspend the pellets by gently pulling the 12 H. G. Windmueller and A. E. Spaeth, J. Biol. Chem. 253, 69 (1977).
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cells and medium in and out of a plastic pipet a few times until a visually homogeneous suspension is obtained. If the suspension is examined microscopically at this point, it will be observed to consist of a few isolated cells and cell doublets, but primarily of cell d u m p s containing a few to 10 or 20 cells each. The individual cells will appear rounded morphologically, as should be expected once the restraint represented by neighbor cells has been released. Careful observation will reveal the brush border "mustache" over a portion of the cell membrane representing the original lumenal surface. Despite the lack of fully isolated individual cells, the resuspension can be sampled readily and reproducibly with a narrow-bore micropipet. Replicate samples, carefully taken, wiU contain uniform amounts of total protein. Each chick intestine will yield I to 2 ml of loosely packed cells or about 50 mg of cell protein. Total cell protein can be determined spectrophotometrically by incubating an aliquot of the suspension with Biuret reagent in comparison to BSA standards. 13 For most purposes we usually prepare cell suspensions containing 15-20 nag protein/ml and store the suspensions on ice until they are used experimentally. For best results, the cells should be used within 1 hr of the time of preparation. Because the cells settle and aggregate rapidly, it is always necessary to swirl the suspension vigorously a few times in order to ensure uniform sampling of the stock suspension. It is not advisable to try and achieve further separation of the cell population. This can be accomplished by vigorous stirring or refluxing in and out of a pipet, but only at the expense of the total number of intact cells and concomitant loss of functional properties. Because of the nature of the cell suspension it is difficult to count cells or to determine vital dye staining accurately so that it is better to rely on protein or DNA determinations for standardizing cell numbers. Stability of metabolic and functional capabilities (e.g., ion or organic solute transport) proves to be a more reliable index of viability than do staining techniques. Evaluation of T r a n s p o r t A variety of metabolic parameters can be evaluated readily for the cell populationl°: (1) glucose utilization, (2) lactate production, (3) oxygen consumption, (4) CO2 production from various substrates, and (5) adenine nucleotide ratios or energy charge. The magnitude and stability of each of these parameters individually and collectively convey information relating to biochemical function of the isolated cells. However, they do not convey 13A. G. Gornall, C. S. Bardawill, and M. M. David, J. Biol. Chem. 177, 751 (1949).
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much information regarding the integrity of the plasma membrane for the cell population. Indeed, cell-free extracts of many tissues can catalyze similar metabolic activities. For this reason, it seems likely that the magnitude and stability of solute gradients maintained by the cell population convey more meaningful information about the quality of the cell preparation. For energy-dependent transport events which create transmembrane concentration gradients of solutes, assessment of gradient magnitude and stability provides experimental insight not only to the integrity of the cellular plasma membrane, but also to the set of metabolic events responsible for establishing the energetic forces driving the transport systems and the integration between metabolic and transport events.
Accumulation of ot-Methylglucoside (~-MG) Because of a marked tendency for the cell population to clump and form large aggregates when incubated in a quiescent environment, it is necessary to perform experimental work under conditions in which the suspension is continually shaken at an adequate rate. This provides sufficient homogeneity for reproducible sampling as well as appropriate oxygenation of the medium to sustain the rather high aerobic metabolism of the epithelial cells. In our experience, the surface-to-volume ratio of the incubation vessel exhibits a rather narrow optimum. This relates in part to the necessity for avoiding too great a depth of incubation medium such that the shaking action of the incubator bath can be low enough to allow convenient sampling without being so slow that cells congregate in the lower levels of the suspension, leaving a lower cell density near the surface. Oscillating shaker baths are better than rotary because the latter tend to create higher cell densities in the periphery of the incubation vessel. We have found that a 4-ml total incubation volume in a 50-ml beaker oscillated at 100 cpm provides an ideal set of conditions when the cell density is between 3 to 5 mg protein/ml. In order to conserve isotope and limit the number of cells required we have also used a 2.2- to 2.5-ml volume in 30-ml beakers. An appropriate format for assessing transport capability for several kinds of solutes can be illustrated by evaluating the uptake ofa-[~4C]methylglucoside as follows: Incubation Vessel Preparation. Prepare a set of 30-ml plastic beakers such that each beaker contains 2.2 ml of the same medium used for cell isolation, but without hyaluronidase. In addition, each beaker should contain 100 # M ¢x-methylglucosideand 0.1-0.2 ltCi/ml ofa-[l~]MG. One of these beakers will have no further additions and will be used to assess ot-MG uptake under control conditions. Another will contain 200 # M
330
GASTROINTESTINAL SYSTEM
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phlorizin, which is a potent inhibitor of Na+-dependent sugar transport in these cells. Other beakers will contain whatever agents one might wish to test in terms of possible action on the sugar transport capability of the cells. For instance, one might wish to test several concentrations of ouabain because of its inhibitory action on Na + transport in order to assess the effect of dissipating the A/ZNa+on the capability of the cells for establishing sugar gradients. In this case, the set of incubation beakers might include the following: All beakers: 2.2ml incubation m e d i u m + 0.25 /zCi a-[14C]MG ( - 0.1/zCi/ml) Beaker l: No addition Beaker 2: + 200 # M phlorizin Beaker 3:+0.5 # M ouabain Beaker 4: + 1.0/all4 ouabain Beaker 5:+2.0/zM ouabain Beaker 6: + 5.0 p M ouabain The beaker set should be incubated at 37 ° with gentle shaking (100 cpm) in order to thermally equilibrate the suspension medium. A stock cell suspension ( - 2 0 mg cell protein/ml) should be thermally equilibrated at the same time. Cellular sugar uptake is initiated by transferring a 0.1-ml sample of cell suspension to each of the experimental beakers at time zero. At intervals after the cell transfer, 100-/zl samples of the incubation medium are taken with the aid of an automatic micropipet. It is important that the shaker continue operating during the sampling procedure in order to achieve uniform samples of the cell suspension. Even a short interruption of the shaking motion can lead to cell aggregation and varying amounts of cell protein from sample to sample with resultant data scatter. In order to study sugar uptake during formation of a steady state sugar gradient, an appropriate sampling schedule might be 1, 5, 10, 20, 40, and 60 rain. For the suggested experimental format (surface-to-volume ratio) it is difficult to take more than six samples per incubation beaker before the medium becomes too shallow for easy sampling. Each 100-/zl sample should be diluted into 4 ml of ice-cold incubation medium (without radioactivity) immediatelyafter being taken. It is convenient in terms of later sample processing to have the diluent for each experimental aliquot in 5-ml polyethylene scintillation minivials. The diluted samples should be centrifuged as soon as possible using a rapidly accelerating tabletop centrifuge. If the six incubations are run in parallel, each set of six samples for a given time point can be processed while waiting to take the sample set for the next time point. After centrifugation
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for 30 sec the supematant is removed by aspiration and 4.0 ml of fresh ice-cold wash medium is added to each tube. After a second centrifugation and supernatant aspiration the cell pellets are ready for extraction and measurement of the amount of isotope accumulated. Using an Autocrit (with modified head) (Clay Adams, Parsippany, NJ) or an MHCT II centrifuge, both wash steps can be completed in less than 3 rain. The carryover of extracellular radioactivity following the two washes can be evaluated by incubating the cells with impermeant [~4C]polyethylene glycol. For the recommended amount of cellular protein this carryover is so small (-0.02 gl or 4 CPM at 0.1 gCi/ml) that it can be ignored when calculating cellular uptake of sugar. Chilling of the diluted cell aliquots is sufficient to prevent subsequent sugar fluxes without the necessity of including any transport inhibitors in the wash medium. This can be evaluated by maintaining diluted samples taken under comparable conditions on ice for varying intervals of time prior to centrifugation and comparing the cellular content of isotope as a function of storage time. For a-MG only 5% of the accumulated sugar is lost to the wash medium even after 20 rain of sample storage time. Because sample processing is typically complete in 2 - 3 min, no correction is required. This should be evaluated for each solute whose transport is examined, however, in order to ascertain if corrections due to solute loss during sample processing are necessary. Once the washed cell pellets are prepared they can be extracted for scintillation counting simply by dissolving each pellet in a 5-ml volume of Liquiscint (National Diagnostics, Manville, N J). If polyethylene minivials are utilized for processing the cell samples, it is only necessary to add the Liquiscint directly to the minivial for a few minutes prior to counting each vial. For the conditions described, the cell pellets do not introduce any quenching or chemiluminescent properties, so no corrections for those events are necessary.
Sample Calculation. If a 10-#1 sample is taken from each incubation beakerprior to adding cells, the count rate determined for each one (CPM) can provide a measure of either the volume specific activity or the picomolar specific activity for the a-[~4C]MG in each experimental run. Volume specific activity (VSA) = (CPM/10) × D, where D is the dilution factor, due to adding cell suspension without isotope to the incubation medium (0.96 for the conditions described earlier). The units for VSA are simply CPM/gl for the final incubation suspension. VSA is useful because it can provide a quick assessment ofintracellular volume/mg of cell protein. For instance, in the experimental case which includes phlorizin, concentrative accumulation of a-MG is inhibited so that the final steady state uptake of sugar simply represents the point at
332
GASTROINTESTINAL SYSTEM
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which a-MG has equilibrated between the extracellular water and that part of the cellular water to which sugar has access. If the steady state CPM in the presence ofphlorizin is divided by VSA and the result is divided by milligrams cell protein in each experimental aliquot one obtains the microliters of cell water per milligram cellular protein for the cell population. We routinely find a value of between 2.5 and 3 pl/mg cell protein by this procedure. By obtaining a value for the number of cells in each sample this can be converted to a volume per cell basis. However, because of cell aggregation in the suspension, mentioned earlier, it is difficult to determine an accurate value for cell number and we routinely express transport data in terms of uptake per milligram cell protein. Picomolar specific activity (PSA) --- VSA/C, where C is the final concentration ofct-MG in the experimental beakers (in pmol/#l). The units of PSA are CPM per picomole. If CPM for any experimental sample is divided by PSA the result gives a value for the amount (in picomoles) of a-MG accumulated by the cells in that sample over the time interval allowed before sampling. These values can be useful in determining the rate of uptake of a particular solute under different experimental conditions. They are best employed in short-term unidirectional influx experiments in which uptake is linear with time so that initial transport rates can be compared. Such initial rates can provide insight to the magnitude of the driving forces acting on the transport system and allow kinetic analysis of the function of the system for various experimental conditions. Solute Gradients. Assuming that phlorizin-inhibited cells only allow equilibration of sugar between extracellular and intracellular water the steady state counts observed with phlorizin (CPMF0 can also provide an approximation for gradient-forming capacity for any of the other conditions evaluated. It is only necessary to divide CPM for a particular sample by CPM~ to calculate the steady state sugar distribution ratio achieved in that sample. By this procedure we find that chick intestinal cells can routinely establish an apparent steady state sugar gradient of about 70- to 90-fold under control conditions when extracellular ot-MG is 100 gM. Approximately 30 rain is required to achieve the steady state. Stability of the sugar gradient during subsequent incubation provides a measure of the stability of those driving forces acting to create the sugar gradient (Na + gradient and membrane potential). Sugar gradients calculated by the method described above are "apparent" because the procedure assumes that the extracellular sugar concentration has not changed during the experimental interval. For the conditions described where the cellular volume represents about 0.25% of the total incubation volume (2.0 mg cell protein X 3/ll cell water/mg protein in a 2.3-ml volume), a 70-fold accumulation of solute relative to the initial extracellular concentration will decrease the extraceUular concentration by
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TABLE I SUGAR ACCUMULATION RATIOS FOR INTESTINAL EPITHELIAL CELLS INCUBATED AT DIFFERENT OUABAIN CONCENTRATIONS Ouabain concentration (/zM) 0 0.5 1.0 2.0 5.0 0 + 200/.tM Phlorizin
Sugar accumulation ratio* 81 56 36 17 3 1
°Defined after a 40-rain incubation as the ratio of a - p ~ ] M G counts in an aliquot of cells for the indicated ouabain concentration to that observed in the presence of 200/zM phlorizin.
approximately 15- 20%. This depletion of medium solute can be detected by counting the isotope present in an aliquot of the supernatant above the cell pellet following the first centrifugation of the diluted cell samples. When the decrease in extracellular concentration is taken into account the actual accumulation ratio for ~-MG established by the isolated chick cells typically is about 70/(1 - 0 . 1 5 ) or -80-fold. 14 Table I shows the actual accumulation ratios observed for a-MG in the experiment described above for different ouabain concentrations. Urn'directional Influx. As mentioned earlier, short-term experiments similar to those described above can be used to determine the rate of accumulation of solute by the cell suspension. 15 In this situation it is usually necessary to take aliquots of the suspension about every I0-15 sec for a total of 40-60 sec, by which time appreciable backflux begins to occur such that linearity of influx is lost. The data provide information regarding the capacity of the transport system for the particular conditions employed. For any given solute, entry usually occurs by more than one route so that a procedure is needed to identify that part of the total flux which occurs by the route of interest. In the case of a-MG it is convenient to use phlorizin sensitivity as the means of identifying influx via the Na+-dependent sugar carrier. Table II shows the influx of ¢x-MG at an extracellular concentration of 100 # M and at four different phlorizin concentrations in comparison to influx observed without phlorizin or without 14G. A. Kimmich and J. RancHes, Am. J. Physiol. 237, C56 (1978). ~s j. Randles and G. A. Kimmich, Am. J. Physiol. 234, C64 (1978).
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TABLE II UNIDIRECTIONAL INFLUX OF 100 f12~f a-p4C]MG
INTO ISOLATEDCHICKEN INTESTINALCELLS (EFFECT OF PI..ILORIZINAND LACK OF Na +)
Incubation conditions
Unidirectional influxa of/z-MG (pmol/min/mg protein)
Control +0.5/zM Phlorizin + 1.0//.M Phlorizin + 10.0/zM Phlorizin + 200/zM Phlorizin No Na +b
424 358 299 169 25 22
° The influx was determined from five samples taken over a 36-sec interval in each case. Linear uptake is maintained for this interval. Influx ffiCPMt/(PSA.t.mg protein) where t = time of sampling in minutes. NaCI in the incubation medium was replaced by 125 m M tetramethylammonium chloride.
Na +. Note that about 95% of the total a-MG influx is blocked by phlorizin or by lack of sodium.
Sodium Ion-Independent Sugar Transport Intestinal epithelial cells accumulate various sugars via a concentrative Na+-dependent sugar transport system localized in the brush border boundary of the cell. Accumulated sugar escapes from the cell to the circulatory systems via a facilitated diffusion transport system localized in the basolateral boundary. ~-MG satisfies only the Na+-dependent transport system so that it represents the sugar of choice for evaluating function of this system. 16 On the other hand, 2-deoxyglucose (2-DOG) selectively satisfies the Na+-independent carrier so that unidirectional influx of 2-DOG represents a means of studying basolateral transfer of sugar. ~7 Because 2-DOG can be phosphorylated by hexokinase it is not possible to use longer term steady state experiments with 2-DOG in order to determine equilibration time or cellular volumes. Certain nonmetabolized sugars such as 3-O-methylglucose (3-OMG) are transported by both of the intestinal sugar transport systems. At the 16G. A. Kimmich and J. Randles, Am. J. Physiol. 241, C227 (1981). ~7G. A. Kimmich and J. Randles, J. Membr. Biol. 27, 353 (1976).
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ISOLATION OF INTESTINAL EPITHELIAL CELLS TABLE III
EFFECT OF VARIOUS INHImTORSOF INTESTINAL SEROSAL SUGAR TRANSFER(Na+
INDEPENDENT)ON ACCUMULATIONOF 3-[14C]OMG BY ISOLATEDINTESTINALCELLS ( N a + DEPENDENT)
Agent"
Concentration (/tM)
--
--
Dihydroquercitin Theophylline Hesperetin Naringenin Phloretin Apigenin Kaempferol Cytochalasin B
100 5 100 100 100 100 100 100
Steady state 3-OMG accumulation ratiob 10 15 25 28 32 35 43 48 65
"Dihydroquercitin, hesperetin, and naringenin are flavanones. Apigenin and kaempferol are flavones. Phloretin is an analog of the tlavanones in which a heterocyclic ring has been opened. b For 100/zM 3-[t4C]OMG.
steady state, cells transporting 3-OMG accumulate the sugar against a concentration gradient at the brush border and lose it across the basolateral boundary via Na+-independent transfer. The magnitude of the steady state 3-OMG gradient maintained reflects the function of both transport events) + Agents which selectively interfere with the passive facilitated diffusional transfer system will allow the cells to establish better concentration gradients of 3-OMG than control cells can maintain. This can be demonstrated using the same techniques already described, but with 3-[14C]OMG as the test sugar. Phloretin, ts theophylline,~5cytochalasin B, 14 and various flavones or flavanonesIs can all be used to interfere with passive sugar et~ux as shown in Table III. The degree of gradient enhancement is proportional to the degree of inhibition of the Na+-independent transport system for agents which act solely on the facilitated diffusion carrier.
A TP-Depleted Epithelial Cells Isolated intestinal cells can be depleted of more than 90% of their ATP content by a brief incubation with 20 to 80/zM rotenon¢, t9 Because the ATP pool in these cells turns over several times per minute, most of the ~sG. A. Kimmich and J. Randles, Membr. Biochem. 1, 221 (1978). t9 C. Carter-Su and G. A. Kimmich, Am. J. Physiol. 237, C57 (1979).
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GASTROINTESTINAL SYSTEM
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depletion occurs in the first minute after rotenone addition. If ouabain is then added to prevent possible slow turnover of the Na+,K+-ATPase due to the residual ATP, it is possible to dissipate the A/zsa+ which is ordinarily established due to monovalent ion transport. The ATP-depleted cells can be utilized experimentally as if they are giant membrane vesicles in order to demonstrate the role for various driving forces acting to energize concentrative transport of sugar or other solutes. They offer significant advantages over conventional plasma membrane vesicles because a more favorable surface-to-volume ratio allows much longer intervals over which influx remains linear with consequent greater ease of flux measurement and better reliability of the experimental values determined. Gradients of chemical potential or electrical potential or both can be experimentally imposed across the plasma membrane of the ATP-depleted cells and the role of each can be determined in terms related to energization of the Na+-dependent transport system? 9~° Unidirectional influx for the solute of interest as well as transient gradient forming capability due to the imposed driving force can each be used for providing insight to the transport mechanism. Imposed Na + Gradients of Defined Magnitude (A~Na+) In order to selectively study effects of a change in Na + gradient, it is necessary to prevent changes in diffusion potentials (AM) due to diffusional flux of the imposed Na + gradient. 2~ This can be accomplished best by the inclusion of high concentrations of highly permeant ions during the ATP depletion procedure and subsequent incubation with Na + in order to "short circuit" the system and "clamp" the potential near zero. For this purpose, we have found that replacing all or part of the NaCl during ATP depletion with tetramethylammonium (TMA +) nitrate is effective. Cells which have been equilibrated with tetramethylammonium (sodium) nitrate can be transferred to an incubation medium with 125 m M sodium nitrate in order to create the experimental Na + gradient. Sometimes in order to ensure that the potential remains constant it is necessary to include potassium nitrate plus valinomycin in both the preincubation and the incubation medium in place of part of the tetramethylammonium nitrate. The high permeability of both NO 3- and K + (with valinomycin) relative to Na + provides an especially stable A¥ when Na + is added hack to the system.22 2oc. Carter-Suand G. A. Kimmich,Am. J. Physiol. 238, C73 (1980). 2~G. A. Kimmichand J. Randles,Biochim. Biophys. Acta 596, 439 (1980). G. A. Kimmich,J. Randles, D. Restrepo,and M. Montrose,Am. J. Physiol. 248, C399 (1985).
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ISOLATION OF INTESTINAL EPITHELIAL CELLS
337
Imposed M e m b r a n e Potentials of Defined Magnitude (A~) If cellsarc A T P depleted in a medium containing 50 m M potassium gluconatc with valinomycin and I00 m M sodium gluconate and then introduced to an incubation medium with 100 m M N a N O 3 and 50 m M tetramethylammonium nitrate,itispossibleto createa diffusionpotential duc to thc imposed gradients of permeant ions but with no imposed Na + gradient.By varying the amount ofextraceHularNO~3 (replacedwith slowly permeant gluconatc) or T M A + (replacedwith K+), itispossibleto manipulate the A ~ over a range of more than 60 m V and to determine the effect on a given Na+-dependent solutetransport system,e3~ Imposed Electrochemical Gradients of Defined Magnitude
(A]]Na+)
By using a combination of the above conditions,gradients of both A ¥ and A~t~+ can be created.A useful approach is to A T P deplete the cellsin potassium gluconate and to transferthem to an incubation medium with NaNO3. A range of A/~+ for differentcellpopulations is constructed by varying the amounts of Na +, K +, and/or NO3- in the two working media. Because the ATP-dcplcted cellscannot establisheitherelement of a A/~N,+ mctabolically, thc imposed gradients will dissipateduc to diffusionalion fluxes.Ncvertbeless,the degree to which an imposed gradientiseffectivein energizing a solute transport system can be assessed by examining the magnitude of "overshoot" or transientgradient-forming capacity the cells exhibit for the solute. Table IV shows the peak accumulation ratio observed for I00 ~ ~ - M G when the various gradientsdescribed above were imposed on isolatedintestinalcells.In each case, the accumulation ratio was defined as the C P M for the peak sample divided by C P M for the sample at eventual steady state(i.e.,fullydissipatedgradients). Measurement of Intracellular Na + and K +
The procedure described for monitoring a-MG accumulation can be modified slightly in order to provide values for intracellular Na + and K + content for different experimental conditions. In this case, instead of dissolving the cell pellets in Liquiscint for scintillation countin~ they are extracted in 3% PCA and an aliquot of the supernatant, after centrifugation of the denatured cellular protein, is used for flame photometry. The G. A. Kimmich, J. Randles, D. Restrepo, and M. Montrose, Ann. N. Y. Acad. Sci. 456, 63 (1985). 24D. Restrepo and G. A. Kimmich, J. Membr. Biol. 87, 159 (1985).
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GASTROINTESTINAL SYSTEM
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TABLE IV SUGAR TRANSPORT IN ATP-DEPLETED INTESTINAL CELLS DRIVEN BY AN EXPERIMENTALLY IMPOSEDNa + GRADIENT, MEMBRANE POTENTIAL, OR ELECTROCHEMICAL POTENTIAL GRADIENT FOR Na +
Driving force
AgN~+ A~/ A~N,,+ A~Na+ + phlorizin
Peak sugar gradient 11.0 7.4 19.6 1.0
amount of monovalent ions determined in this manner can be expressed as an intracellular concentration by utilizing a value for cell volume and assuming that each ion is uniformly distributed in the cell water. Concentrations calculated in this manner are not highly reliable, however, because of ion binding to cellular anionic constituents and possible nonuniform distribution among organelles. Both phenomena create an intracellular ion activity which is considerably lower than values determined on the basis of ion amount per volume measurements. This is particularly true for Na +, where the activity measured with ion-selective electrodes is usually about half the calculated concentration. 2s Nevertheless, determination of the amount of intracellular ion under different experimental conditions can give qualitative information regarding changes in the magnitude of ion gradients maintained by the cells. M e a s u r e m e n t of M e m b r a n e Potentials The unidirectional influx or rate of uptake of a lipophilic cation by any cell population should be proportional to the magnitude of the membrane potential maintained by those cells. If the influx is studied as the membrane potential is varied experimentally, one can construct a calibration curve for the relationship between flux and A¥. This calibration curve can be used to determine the membrane potential for situations in which it has not been experimentally created. 22m A useful lipophilic ion to be used for this purpose is [14C]tetraphenylphosphonium (TPP+). Gradients of potassium gluconate imposed on ATP-depleted cells can be used to create potentials of defined magnitude. Valinomycin is used to enhance K + permeability and this, in conjunction 25 j. O'Doherty, J. F. Garcia-Daz, and W. MeD. Armstrong, Science 203, 1349 (1979).
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ISOLATION OF INTESTINAL EPITHELIAL CELLS
339
with the low permeabilityof gluconate, allows formation of a K + equilibrium potential,the magnitude of which can be calculatedfrom the Ncrnst equation22:
A~U= (RT/F) In ([K+lo/[K+]i)
in which T is temperature, F is the Faraday constant, and R is the ideal gas law constant. A useful procedure is to ATP deplete the cells in a medium containing 150 m M potassium gluconate with 20 m M TMA-HEPES buffer and 1 m M CaSO4. No other salt constituents are necessary. In order to initiate the influx measurement the cells are diluted 1 : 10 into 150 m M tetramethylammonium gluconate, 20 raM HEPES, and 1 m M CaSO4 or into media in which various mixtures of tetramethylammonium gluconate and potassium gluconate are used in order to vary the extracellular K + concentration. Valinomycin and 1 # M [~4C]TPP+ are included in the incubation medium and four to five samples are taken during the first minute of incubation in a manner similar to that already described for monitoring unidirectional influx of any solute. All influx values are expressed relative to the value determined when no K + gradient is imposed which is arbitrarily assigned a value of 1.0. This is defined as the flux at a diffusion potential of 0 mV. Relative fluxes for several different K + gradients (i.e., potentials) are given in Table V. The use of relative fluxes instead of absolute values avoids the necessity of having to determine an independent value for the permeability constant for TPP +. For intestinal epithelial cells, the TPP + flux vs A~, relationship is the same as that predicted by the Goldman flux equation. 23m This calibration curve can then be used to determine an unknown potential. Only two flux measurements must be made. In one, the TPP + influx is determined TABLE V [~4C]TPP+ INFLUX INTO INTESTINALCELLS WITH DIFFERENT POTASSIUMGLUCONATEGRADIENTS
[K+].J[K+]o
Aq/ (mV)
Relative TPP + influxa
1 2 5 10
0 --18 --42 --60
1.0 1.4 2.0 2.6
=All flux values are given as a multiple of the flux observed when no K + gradient (A~/) was imposed which was assigned an arbitrary value of 1.0.
340
GASTROINTESTINAL SYSTEM
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TABLEVI EFFECT OF Na+-DEPENDE~r SUGAR TRANSPORTON" [14C]TPP+ INFLUX IN ISOLATED INTESTINAL CELLS
Incubation conditions
Relative TIP+ influx
A~ (mY)
Control + 10 mMa-MG + Rotenoneand ouabain
2.2 1.9 1.0
-55 -45 0
for the particular experimental conditions for which you wish to determine a A¥. This influx is expressed relative to (i.e., as a multiple or fraction of) the influx measured in cells which are ATP depleted and incubated sufficiently long to dissipate ion gradients. This case is defined as A~ = 0just as in the case described above in which the calibration relationship was defined relative to a situation with no imposed K + gradient. It is now only necessary to pick the A¢/off the calibration curve for the particular flux ratio determined by the two flux measurements (Jv,_ffJv,_o). Table VI shows the TPP + influx ratio and corresponding A~ determined for intestinal cells incubated under standard conditions and with 10 m M a-MG added. Actively accumulated sugars are known to partially depolarize the membrane potential due to electrogenic function of the Na+-dependent sugar transport system. Note that the added sugar causes about a 10-mY depolarization of the membrane potential measured in this manner, just as reported in experiments in which A ¢ was directly monitored with microelectrodes.26 Summary Epithelial cells can be isolated from the small intestine of chickens by a procedure involving hyaluronidase treatment of the intact tissue. The isolated cells retain a high degree of functional activity as assessed by the formation of 70-fold gradients of a-MG. Stability of the sugar gradients reflects maintenance of stable electrochemical Na + gradients across the plasma membrane. The cells can be used to evaluate the properties of Na+-dependent sugar transport, Na+-independent sugar transport, ion transport, metabolism, membrane potentials, and the integration of these events, all of which are important to achieving a stable sugar gradient. 26R. C. Roseand S. O. Schultz, J. Gen. Physiol. 57, 639 (1971).
