448
GASTROINTESTINAL SYSTEM
[28]
[28] C a l c i u m T r a n s p o r t b y I n t e s t i n a l E p i t h e l i a l C e l l Basolateral Membrane B y JULIAN R. F. WALTERS a n d MILTON M . WEISER
Introduction The absorption of calcium ion (Ca2+) by the intestine involves several steps as Ca 2+ moves through the epithelial cell. The nature of these separate steps, and the role of the vitamin D metabolite 1,25-dihydroxycholecalciferol [1,25(OH)2D3] in their control, have recently been reviewed, l To be absorbed, Ca 2+ must traverse the apical brush border membrane, the intracellular cytoplasm, and eventually the basolateral membrane. Within the cell, certain subcellular organeiles have been shown to be capable of sequestering Ca2+; these include the endoplasmic reticulum, the Golgi apparatus, mitochondria, and lysosomes. Additionally, epithelial cells have a specific Ca2+-binding protein (CaBP, calbindin-Dgu~ in mammals), which is thought to increase the amount of Cae+ diffusing across the cell by responding either directly to the action of 1,25(OH)2D 3 or to increased intracellular Ca 2+. As the concentrations of free Ca 2+ within the cell are submicromolar, whereas those in the lumen of the intestine and in the extracellular fluid are millimolar, the entry of Ca 2+ is downhill and probably a passive process not requiting energy, though transport systems have been described at the lumenal membrane. However, Ca 2+ extrusion at the basolateral membrane must be an energy-requiring process since it must move Ca 2+ against a 10,000-fold concentration gradient. Some of this energy appears to come from the Na + gradient created by the Na + pump which then drives Na+/Ca 2+ exchange, but plasma membranes also possess an ATP-dependent Ca 2+ pump which extrudes Ca 2+ from the cell linked to the hydrolysis of ATP. This chapter will describe the methodology for studies of the Ca 2+ pump in basolateral-enriched membrane vesicles of rat intestinal epithelial cells. Calcium Ion Uptake by Intestinal M e m b r a n e Vesicles The basic methods adopted in our laboratory for the study of vesicular Ca 2+ transport will be described first. 2 Factors important in the preparation of membranes and methods which have defined the properties of transport t F. Bronner, D. Pansu, and W. D. Stein, Am. J. Physiol. 250, G561 (1986). 2 j. R. F. Waiters and M. M. Weiser, Am. J. Physiol. 252, G170 (1987).
METHODS IN ENZYMOLOGY, VOL 192
Copyright © 1990 by Academic Pr'-~, Inc. All rights of reproduction in any form reserved.
[28]
INTESTINAL BASOLATERAL MEMBRANE C a 2+ TRANSPORT
449
will be described subsequently. The methodology used in the intestine is similar to that described in other chapters of this series for inside-out vesicles from red blood cells. 3 Materials. The typical reagents employed in the assay are as follows. Dilution buffer. 135 m M KC1, 5 m M MgC12, 10 m M imidazole-acetate, pH 7.5 Calcium buffer: Made up at 2 X final concentration in dilution buffer. This 2X buffer contains 1 m M EGTA, 45CAC12 to give about 100,000 cpm/25/~l (from stock supplied at 10-40 mCi/Mg Ca2+), and additional nonradioactive CaCI 2 to give the appropriate total Ca 2+ which will result in the desired submicromolar free Ca 2+. The composition of the Ca 2+ EGTA buffer is determined by a computer program employing published association constants and methods for the interactions between Ca 2+, Mg2+, H +, EGTA, and ATP,4 and includes corrections for temperature and ionic strength. Three important considerations are (1) the critical dependence on pH of the free Ca+, (2) the purity of the EGTA used (typically 95-97%), and (3) the contaminating levels of Ca 2+ inevitably found in any laboratory water. These additional amounts of contaminating Ca 2+, typically l0 aM, can be measured with a Ca2+-sensitive electrode and included in the calculation of the Ca 2+ to be added to the buffer to result in the correct total Ca 2+. For example, the solution which will give 0.5 a M free Ca2+ can be calculated to require approximately 460 lzMtotal Ca 2+ with 500 #MEGTA, 5 m M Mg2+, 3 m M A T P at pH 7.5. With laboratory water containing 10 a M Ca 2+, an additional 450 IzM Ca2+ needs to be present, so 900 IzM Ca 2+ is added to the 2 X concentrated buffer. This Ca 2+ is obtained from both the 45Ca2+ and nonradioactive CaCI2 stock ATP solution: 30 m M Tris-ATP in dilution buffer adjusted to pH 7.5 Additional compounds, such as inhibitors or stimulatory proteins, are dissolved in dilution buffer and the pH checked Membrane filters: Nitrocellulose filters, 0.45-am pore size, 25-mm diameter, soaked in 10 m M CaC12 for a few minutes before use Wash solution: Dilution buffer containing l0 m M CaC12 These reagents may be stored frozen for several months.
Procedure. Transport studies are performed in a final volume of 100/zl. Aliquots of membranes (20 #1) containing 10 to 30/zg of membrane protein in dilution buffer are distributed to disposable glass or plastic tubes 3 T. R. Hinds and F. F. Vincenzi, this series, Vol. 102, p. 47. 40. Scharff, Anal Chim. Acta 109, 291 (1979).
450
GASTROINTESTINAL SYSTEM
[28]
on ice. Additional compounds may be added in another 20/~l dilution buffei, and then 50/~l of the 2X concentrated calcium buffer is added. These tubes are mixed and kept on ice until the individual uptakes are determined. After a 10-min preincubation at the final temperature (usually 25 o), l0/~1 of the ATP solution is added and the tube mixed by a brief vortexing. Following a 1-min incubation, a 90-#1 sample is pipetted onto a nitrocellulose disk in a vacuum suction apparatus and washed twice with 2.5 ml of ice-cold wash solution. Filtration is completed in about 15 sec after which the nitrocellulose filter is dissolved in aqueous scintillation fluid and the radioactivity counted in a liquid scintillation counter. Calculation of Results. In order to determine ATP-independent uptake, ATP is replaced with dilution buffer. Calcium ion uptake in ATP-free solutions is near maximal at 1 rain in submicromolar Ca 2+ buffers and is thought to represent Ca 2+ binding. ATP-independent Ca 2+ uptake is subtracted from uptake in the presence of ATP to give ATP-dependent Ca 2+ transport. Membrane-free blanks are also filtered and give values for binding of Ca :+ directly to the filters. An aliquot of the uptake solution is used as an internal standard to convert counts per minute to nanomoles of Ca 2+ using the total Ca 2+ present in the Ca :+ buffer. Results are usually expressed as 1-min rates, though uptake is linear for at least 2 min. Experiments are performed with at least triplicate determinations. This procedure is not the only one which will produce satisfactory results. Reagents may be added in different orders and volumes may be altered. For instance, an alternative method has been employed in which the uptake reaction is started by adding the 2 X Ca 2+ buffer. In this case, though, the membranes are not preincubated at the final submicromolar Ca 2+ concentration, which may be important in determining the actions of stimulatory proteins. Similar values, however, are found for both ATP-dependent and -independent Ca 2+ uptake. Effects on Ca 2+ U p t a k e of Different Preparations of Basolateral M e m b r a n e Vesicles
Membrane Preparation. Basolateral-enriched membrane fractions have been prepared by several similar methods from intestinal cells. The method we use is modified from the one described in 1978. 5 We have found that one must start with isolated cells to demonstrate satisfactorily the ATP-dependent Ca2+ pump. This is discussed below. Homogenates of isolated intestinal epithelial cells are prepared using a Polytron (Brinkmann, Westbury, NY), setting 8, for 1 rain, and then centrifuged at 1500 g M. M. Weiser, M. M. Neumeier, A. Quaroni, and K. Kirsch, J. CellBiol. 77, 722 (1978).
[28]
INTESTINAL BASOLATERAL MEMBRANE C a 2+ TRANSPORT
451
for 15 min to remove unbroken cells, nuclei, and brush border membranes. The supernatant is centrifuged at 105,000 g for 15 min to collect a crude membrane fraction. Basolateral membranes are further purified using sorbitol density-gradient centrifugation for 2 h r at 200,000 g and the fractions washed and resuspended in dilution buffer. The fractions with densities similar to, or less than, 40 g/dl sorbitol have been shown to be enriched for the basolateral membrane marker, Na+,K+-ATPase activity. Calcium ion transport has been found to be associated with these membranes rather than with the more dense membranes enriched for sucrase activity, a brush border membrane marker (Fig. 1). Essentially similar membrane preparations have been employed by other groups of workers who have studied basolateral membrane Ca 2+ transport. 6-8 Enrichment of Na+,K+-ATPase activity in excess of 10-fold have been difficult to achieve with intestinal homogenates, and though separation from brush border membranes and mitochondria is relatively easy, contamination with endoplasmic reticulum markers has been more difficult to overcome. Other properties of the vesicles that may affect Ca2+ transport include vesicle size, degree of sealing and orientation. Size may be estimated from electron micrographs, and quantified more easily from the equilibration volume of molecules such as glucose or mannitol. These give estimates ranging from 1 to 8 gl/mg protein. 2,6 Orientation can be estimated from enzyme latencies, though these determinations may be affected through stimulation of activities by the detergents employed. Most estimates for basolateral membranes give figures of roughly 50% in inside-out and rightside-out orientations.7 It is assumed that only inside-out vesicles will be in the correct orientation for measuring the function of the basolateral membrane Ca 2+ pump since substrates are added to the external vesicle surface which, if activated by ATP, would represent the cytoplasmic domain of the membranes in the intact ,cells. We have used these vesicles immediately after preparation for Ca 2+ transport studies. Although Ca 2+ uptake rates remain stable for several hours when the membranes are kept on ice, substantial loss of activity occurs overnight. Freezing also results in a large and variable reduction of the transport rate, though other workers appear to be able to accept -50% loss of activity as there was no change in the affinity of the pump for Ca2+.9 6 H. N. Nellans and J. E. Popovitch, J. Biol. Chem. 256, 9932 (1981). 7 W. E. J. M. Ghijsen, M. D. de Jong, and C. H. van Os, Biochim. Biophys. Acta 689, 327 (1982). s B. Hildmann, A. Schmidt, and H. Muter, J. Membr. Biol. 65, 55 (1982). 9 W. E. J. M. Ghijsen, C. H. van Os, C. W. Heizmann, and H. Murer, Am. J. Physiol. 251, G223 (1986).
452
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Fro. I. Distributionof vesicularCa 2+ transportand marker enzymes on a sorbitol density gradient. Crude membrane fractionswere prepared from rat duodena and separated on a discontinuous sorbitoldensity gradient.2 Fractions were coUected from the 30 to the 50-60 g/dl interfaceand washed. ATP-dependent Ca 2+ was measured at 0.5/tM free Ca 2+ (seetext), and Na+,K+-ATPase and sucrasc were measured as previouslydescribed.5
Cell Preparation Methods. The intestine, particularly when compared with other cell types, is sensitive to variations in the methods used to prepare the cells which are homogenized to give subcellular fractions, t° It is important not to overlook the effects of these factors on the properties of Ca2+ uptake in basolateral membrane vesicles. Also important are the differences in epithelial cell function that are found in the various regions of the small intestine and along the crypt-villus axis of differentiation. We have been able to demonstrate ATP-dependent Ca2+ uptake only in vesicles prepared from isolated intestinal cells and not from scrapings of intestinal epithelium, n One explanation appears to be the high levels of nonesterified fatty acids found in membranes prepared from scrapings, t2 These fatty acids probably result from lipolysis of other lipids during the homogenization of the serapings; much higher phospholipase A activity m M. M. Weiser, J. R. F. Wa]ters, and J. R. Wilson, Int. Rev. CytoL 101, 1 (1986). ii j. R. F. Waiters, P. J. Horvath, and M. M. Weiser, in "Epithelial Calcium and Phosphate Transport: Molecular and Cellular Aspects" (F. Bronner and M. Peterlik, eds.), p. 187. Alan R. Liss, New York, 1984. ,2 j. R. F. Walters, P. J. Horvath, and M. M. Weiser, Gastroenterology 91, 34 (1986).
[28]
INTESTINAL BASOLATERAL MEMBRANE C a 2÷ TRANSPORT
453
can be shown in homogenates of intestinal scrapings than in isolated cells. In addition to binding C a 2+ and so increasing the ATP-independent Ca2+ uptake, there may be direct effects of lipids on the plasma membrane Ca 2+ pump. Isolated cell preparations may be better in this regard as they are more extensively washed prior to homogenization. This washing may be removing a greater proportion of adherent pancreatic enzymes or inhibiting or removing epithelial cell phospholipases. Another related problem is the effect of proteolytic enzymatic activity on the Ca 2+ pump. It has been shown that there are differences in basolateral membrane Ca2+ transport from two isolated cell preparations ~3and it was postulated that this was because of differences in amounts of tryptic or other proteolytic activity. Ileal and jejunal membranes seemed more susceptible to this than duodenal; unfortunately this effect could not be prevented by commonly used trypsin inhibitors but only by modifications to the physical methods used for cell isolation. A third problem in preparing basolateral membranes, particularly related to isolated cell methods, is that redistribution of intracellular and cell surface markers may occur. Consequently, the relative purification factors for basolateral, brush border, and endoplasmic reticulum membranes become lower with prolonged preparation and washing procedures. ~°,~4Despite this concern, preparations of "basolateral" membranes show coincident enrichment for Ca2+ pump and Na+,K+-ATPase activities. Thus, either both activities have redistributed themselves at the same rate and to the same location or the redistribution is selective for the enzyme studied, e.g., alkaline phosphatase. These concerns must be considered in studies of C a 2+ transport by intestinal membrane vesicles; unfortunately there is no clearly superior method for cell isolation at present. Investigation of the Properties of Ca 2+ Uptake To confirm that Ca2+ uptake by membrane vesicles is transport by an ATP-dependent Ca 2+ pump, certain basic properties should be investigated to help differentiate ATP-dependent Ca 2+ transport from Ca 2+ binding. An absolute dependency on ATP should be shown. The time course of Ca2+ uptake by basolateral-enriched membrane vesicles, in the presence and absence of ATP, is shown in Fig. 2. Also shown is that ADP will not substitute for ATP; other nucleotide triphosphates such as GTP or CTP were also ineffective in supporting transport. Estimates of the affinity of the intestinal pump for ATP gave a Km of under 50/~M. 13E. J. J. M. van Corven, M. D. de Jong, and C. H. van Os, Cell Calcium 7, 89 (1986). 14C. A. Ziomek, S. Schulman, and M. Edidin, J. Cell Biol. 86, 849 (1980).
454
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FI~. 2. ATP dependence. Basolateral-enriched membranes were prepared from pooled duodenal villus and crypt cells. Calcium ion uptake was measured at 25" with 0.5/zM free Ca 2+ in the presence of 3 m M ATP (O) 3 m M ADP (1), and in the absence of ATP or ADP (O). Results are means _ SEM of four determinations. (Reprinted from Ref. 2.)
Calcium ion transport by the Ca 2+ pump can be most conveniently measured at 25*; uptake is greater at more physiologic temperatures though the rate is linear for a shorter period of time (Fig. 3). However, when experiments are performed on ice, Ca 2+ uptake is reduced to levels similar to those seen in the absence of ATP. As binding is much less temperature dependent than trans-membrane transport, this result also indicates that Ca 2+ uptake is by the Ca 2+ pump. The at~nity of the epithelial cell pump for Ca 2+ should be determined. This has been studied by several groups. We have found a Km of 0.3/zM, 2 which is similar to that found by Ghijsen et al. 7 Another group has found a much lower figure6 but used a different Ca 2+ buffer system. The free Ca 2+ and hence the Km values depend greatly upon the association constants and calculations used in the determination of the Ca2+-buffering solutions. There is the additional problem in that the Ca2+/EGTA complex has been postulated to be recognized by the Ca2+ site on the pump, giving an apparent stimulatory effect of EGTA independent of the effect on free Ca 2+ concentrations? 5 This has not yet been fully resolved for the intestinal basolateral membrane Ca 2+ pump, but appears to be an explanation for the discrepancy in measured KIn. Ca 2+ ionophores, such as A23187, should be used to show that the accumulation of Ca 2+ by the membranes is in fact intravesicular (Fig. 4). A23187 is dissolved in ethanol or dimethyl sulfoxide (DMSO) and added in micromolar concentrations such that the concentration of the solvent does not exceed 1%. This concentration of ethanol has been shown to not 15N. Kotagal, J. R. Colca, and M. L. McDaniel, J. Biol Chem. 258, 4808 (1983).
[28]
INTESTINAL BASOLATERAL MEMBRANE C a 2+ TRANSPORT
455
= 2O
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, TIME
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FIG. 3. Temperature dependence. Calcium ion uptake was determined in duodenal basolateral-enriched membranes in the presence of 0.5 pM free Ca 2+ and 3 m M ATP at 35 ° (&) 25 ° (0) and on ice ([3). Results are means + SEM of four determinations. (Reprinted from Ref. 2.)
affect Ca 2+ uptake. When the ionophore is added to the membranes before ATP, the amount of Ca 2+ associated with the vesicles is reduced to a level similar to those usually seen in the absence of ATP or when uptake is performed on ice. Further, when A23187 is added after Ca 2+ has been taken up by the vesicles, the concentrated intravesicular Ca 2+ will be rapidly released through the membrane pores created by the ionophore.
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FIO. 4. Effects of A23187 on Ca 2+ uptake by duodenal basolateral-enriched membrane vesicles. Membranes were incubated at 25 ° with 3 m M A T P and 0.5 g M free Ca 2+. Values are the means + SE of three or four determinations. Uptake was determined for 30 min with A23187 (9 # M ) added before ATP and Ca 2+ ((3). Uptake in the absence ofA23187 (0); at 30 min (9 gM) A23187 was added and rapidly reduced Ca 2+ uptake. (Reprinted from Ref. 2.)
456
GASTROINTESTINAL SYSTEM
[28]
Prevention of ATP-dependent Ca 2+ uptake and release of intravesicular accumulations by A23817 confirms that the Ca 2+ is concentrated in a soluble form within the vesicles. From determinations of the volume of the vesicles, it can be calculated that the concentration of Ca z+ inside the vesicles at equilibrium exceeds 5 mM, so a 10,000-fold gradient can be maintained by the pump. The subcellular origin of the CaZ+-uptake activity in these membrane fractions may be defined by the actions of established inhibitors of Ca 2+ pumps. Orthovanadate is a powerful inhibitor of most ATPases and will inhibit membrane Ca 2+ uptake with a Ki of less than 1/tM, a figure similar to those of other plasma membrane pumps. The mitochondrial Ca 2+ pump inhibitors such as oligomycin or azide should have no effect. In sodiumfree conditions, ouabain or other Na + pump inhibitors should have no action on Ca :+ accumulation. The effects of Na + on Ca 2+ uptake through the Na+/Ca 2+ exchanger found in plasma membranes have been studiedS,~6; the effects of this system on Ca2+ uptake can best be avoided by omitting Na + from solutions used in the study of the ATP-dependent pump. Calmodulin, a ubiquitous M~ 16,800 protein, has been shown to stimulate various plasma membrane Ca 2+ pumps in a direct manner independent of the action of the specific calmodulin-independent protein kinase. As calmodulin binds up to four Ca 2+ atoms with affinities appropriate for intracellular levels, this is thought to provide a means of control for the Ca 2+ pump. In the intestine, basolateral membrane Ca 2+ uptake is also stimulated by calmodulin under appropriate conditions, but the effect is not as great as that described for the erythrocyte pump. The problem appears to be the retained, tightly bound endogenous calmodulin which obscures an effect of added stimulator. Stimulations of 20-50% at 0.5-1 /~M free Ca 2+ can be found with 10/~g/ml calmodulin (about 0.6/~M) 6 though even this relatively small effect is only seen with inclusion of 5 m M EGTA in the homogenization buffer7 or preincubation with submicromolar Ca 2+.~7The effect of calmodulin appears predominantly to be on the Km of the pump; in the intestine even in the absence of exogenous calmodulin, the Km for Ca 2+ is under 1/~M, similar to the calmodulin-stimulated pump of other tissues. Calmodulin can be detected in basolateral membranes, 9 including those that have been treated with procedures that in other tissues would have removed it. The effects of drugs with calmodulin antagonistic properties have also been investigated; again the results are 16 W. E. J. M. Ghijsen, M. D. de Jong, and C. H. van Os, Biochim. Biophys. Acta 730, 85 (1983). ~7j. R. F. Waiters, Am. J. Physiol. 256, G124 (1989).
[28]
INTESTINAL BASOLATERAL MEMBRANE C a 2+ TRANSPORT
457
not definitive. It may be that the interaction of calmodulin with the intestinal basolateral Ca2+ pump is different from that in the red cell or other tissues, an interaction that may more specifically define this pump. The intestinal vitamin D-dependent Ca2+-binding protein (CaBP, calbindin-D9 u~) affects Ca2+ absorption and might also be expected to influence the basolateral Ca2+ pump. Our studies suggest that this may be the case~7although others have not found such an action.9 Concentrations of CaBP are included in the uptake medium at submicromolar Ca2+; with appropriate C a 2+ buffering this does not significantly affect the free Ca2+ concentrations or specific activity of 45Ca2+. The effects of CaBP may be dependent on the saturation of the protein with Ca2+; the methodology required to confirm or disprove such an effect of CaBP on the pump remains to be determined. The rate of Ca2+ transport in basolateral membrane vesicles has been shown to be affected by the vitamin D status of the animal3 ,~s Along with CaBP and other actions at the brush border membrane, an increase in basolateral Ca2+ transport may represent a method of controlling the overall rate of Ca2+ absorption. Rats can be made vitamin D deficient by a combination of a synthetic diet without vitamin D supplementation, a Ca2+-free diet for 2 weeks to deplete stores of vitamin D through conversion to 1,25(OH)2D3, and an environment lit only by incandescent light to prevent synthesis in the skin of vitamin D by the action of UV light. ~9 These animals have reduced serum Ca2+ concentrations and bone histology compatible with vitamin D deficiency. Intestinal cells and basolateral membrane vesicles have been prepared from these animals by methods detailed above. Purification of marker enzymes, and other studies to show nonspecific effects on vesicles, have been performed with only minor differences found. Vesicular Ca2+ transport rates, however, are reduced by almost 50% in the duodenum, an effect which appears to be on the V ~ and is particularly apparent in membranes of cells at the villus tip. When vitamin D-deficient animals were given repleting doses of 1,25(OH)2D3, 125 ng in ethanol by intravenous injection, the increase after 6 hr in vesicular Ca2+ transport was predominantly in the developing cells lower down the villus.2 Thus, any studies characterizing aspects of Ca2+ absorption should investigate the effects of vitamin D status and consider differences related to intestinal regions and to the crypt-villus axis of differentiation. Ca2+-ATPase enzymatic activity, i.e., the hydrolysis of inorganic phosis W. E. J. M. Ghijsen, and C. H. van Os, Biochim. Biophys. Acta 689, 170 (1982). ~9j. H. Bloor, A. Dasmahapatra, M. M. Weiser, and W. D. Klohs, Biochem. J. 208, 567
(1982).
458
GASTROINTESTINAL SYSTEM
[28]
phate from ATP, is a function of the Ca 2+ pump and should be related to the ATP-dependent transport of Ca 2+. In the intestine, difficulties have been encountered in making this correlation, partly because of the additional hydrolysis of ATP which, once attributed to alkaline phosphatase activity, 2° appears to be a function of ectonucleotide phosphatase activity. 2~ Using similar Mg 2+ and Ca 2+ concentrations as those described for uptake measurements, we found that the high level of this Ca 2+- and ATP-independent Mg2+-ATPase activity prevented accurate assessment of the small increment of any Ca2+-dependent ATP hydrolysis. Consequently, Ca2+-dependent ATPase activity was best measured at reduced free Mg 2+ concentrations of 1 #M. This was achieved with a total Mg + concentration of 47/zM in the presence of 500/zM EGTA, 3 m M ATP, and 509/~M total Ca 2÷ at pH 7.5. Under these conditions, basolateral Ca2+-ATPase activity was similar to values reported by others, but the properties were not the same as those described for vesicular Ca 2+ transport. 2! Thus, calcium ion-dependent ATP hydrolysis in these membranes is not solely a function of the Ca 2+ pump, but represents other enzymatic activities, as has been described in other tissues, including the liver. Conditions for the determination of that portion of ATP hydrolysis due only to the Ca 2+ pump remain to be determined. Basolateral Membrane Ca 2+ Transport in Other Species Most of the details of the intestinal basolateral membrane Ca 2+ pump have been determined with experiments in the rat. Some studies have been performed with chick basolateral membranes; these are particularly relevant as this species has been well studied regarding the action of vitamin D. These findings are broadly similar to those with the rat, including a dependence on the vitamin D status of the animal. 22 Vesicular Transport of Ca 2+ in Other Intestinal Subcellular M e m b r a n e s This chapter has concentrated on the vesicular transport of Ca 2+ by the basolateral membrane of the intestine. We have not described methods or 2oW. E. J. M. Ghijsen, M. D. de Jong, and C. H. van Os, Biochim. Biophys. Acta 599, 538 (1980). 21T. C. Moy,J. R. F. Waiters, and M. M. Weisex,Biochim. Biophys. Res. Commun. 141, 979 (1987). 22j. S. Chandler, S. A. Meyer,and R. H. Wasserman, in "Vitamin D: Chemical, Biochemical and Clinical Update" (A. W. Norman, K. Schaefer, H.-G. G-rigoleit, and D. v. Herrath, eds.), p. 408. de Gruyter, Berlin, 1985.
[29]
ELECTRICAL MEASUREMENTS IN LARGE INTESTINE
459
results of studies conducted by others attempting to measure Ca2+ transport at the brush border membrane.23,u Only one paper deals with transport by the endoplasmic reficulum in any detail.25 Though it appears that the Gelgi may also have a C a 2+ transport system, u earlier results were complicated by high binding of Ca2+ to nonesterified.fatty adds in membranes prepared from intestinal serapings. 12In these studies with the intestine, it seems that the major methodological problem remains the preparation of enriched membranes in sufficient quantities, and unaltered so that they reasonably reflect the in vivo condition of the membrane domain. This has been largely true with duodenal basolateral-enfiched membranes where vesicular Ca 2+ transport has most of the properties expected from studies of C a 2+ a b s o r p t i o n . More recently a transcript for an intestinal plasma membrane C a 2+ pump has been detected which responded to vitamin D similarly to that shown for Ca 2+ uptake by basolateral membranes. 26 Acknowledgments This work is supported by NII-I Grants AM-32336 and AM-35015, and by the Troup Fund of Buffalo General Hospital, Buffalo, New York. 23A. Miller [ ] and F. Bronner, Biochem. J. 196, 391 (1981). 24 H. Rasmussen, O. Fontaine, E. E. Max, and D. B. P. Goodman, J. Biol. Chem. 254, 2993 (1979). 25 M. J. Rubinoffand H. N. Nellans, J. Biol. Chem. 260, 7824 (1985). 2~j. Zelinski, D. E. Sykes, and M. M. Weiser, Gastroenterology 98, A560 (1990).
[29] E l e c t r i c a l M e a s u r e m e n t s
in L a r g e I n t e s t i n e (Including Caecum, Colon, Rectum) B y ULRICH HEGEL a n d MICHAEL FROMM
Introduction The main function of the large intestine is the conservation of water and ions. Electrophysiological methods are therefore of predominant importance in the analysis of physiological as well as of pathological functional states of this organ. Apart from this more applied aspect large intestinal mucosa has increasingly been used as a model to study basic mechanisms of epithelial ion transport which are of relevance also for other tubular organs such as the nephron or excretory ducts of exocrine METHODS 1N ENZYMOLOGY, VOL. 192
Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
GASTROINTESTINAL SYSTEM
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[28] C a l c i u m T r a n s p o r t b y I n t e s t i n a l E p i t h e l i a l C e l l Basolateral Membrane B y JULIAN R. F. WALTERS a n d MILTON M . WEISER
Introduction The absorption of calcium ion (Ca2+) by the intestine involves several steps as Ca 2+ moves through the epithelial cell. The nature of these separate steps, and the role of the vitamin D metabolite 1,25-dihydroxycholecalciferol [1,25(OH)2D3] in their control, have recently been reviewed, l To be absorbed, Ca 2+ must traverse the apical brush border membrane, the intracellular cytoplasm, and eventually the basolateral membrane. Within the cell, certain subcellular organeiles have been shown to be capable of sequestering Ca2+; these include the endoplasmic reticulum, the Golgi apparatus, mitochondria, and lysosomes. Additionally, epithelial cells have a specific Ca2+-binding protein (CaBP, calbindin-Dgu~ in mammals), which is thought to increase the amount of Cae+ diffusing across the cell by responding either directly to the action of 1,25(OH)2D 3 or to increased intracellular Ca 2+. As the concentrations of free Ca 2+ within the cell are submicromolar, whereas those in the lumen of the intestine and in the extracellular fluid are millimolar, the entry of Ca 2+ is downhill and probably a passive process not requiting energy, though transport systems have been described at the lumenal membrane. However, Ca 2+ extrusion at the basolateral membrane must be an energy-requiring process since it must move Ca 2+ against a 10,000-fold concentration gradient. Some of this energy appears to come from the Na + gradient created by the Na + pump which then drives Na+/Ca 2+ exchange, but plasma membranes also possess an ATP-dependent Ca 2+ pump which extrudes Ca 2+ from the cell linked to the hydrolysis of ATP. This chapter will describe the methodology for studies of the Ca 2+ pump in basolateral-enriched membrane vesicles of rat intestinal epithelial cells. Calcium Ion Uptake by Intestinal M e m b r a n e Vesicles The basic methods adopted in our laboratory for the study of vesicular Ca 2+ transport will be described first. 2 Factors important in the preparation of membranes and methods which have defined the properties of transport t F. Bronner, D. Pansu, and W. D. Stein, Am. J. Physiol. 250, G561 (1986). 2 j. R. F. Waiters and M. M. Weiser, Am. J. Physiol. 252, G170 (1987).
METHODS IN ENZYMOLOGY, VOL 192
Copyright © 1990 by Academic Pr'-~, Inc. All rights of reproduction in any form reserved.
[28]
INTESTINAL BASOLATERAL MEMBRANE C a 2+ TRANSPORT
449
will be described subsequently. The methodology used in the intestine is similar to that described in other chapters of this series for inside-out vesicles from red blood cells. 3 Materials. The typical reagents employed in the assay are as follows. Dilution buffer. 135 m M KC1, 5 m M MgC12, 10 m M imidazole-acetate, pH 7.5 Calcium buffer: Made up at 2 X final concentration in dilution buffer. This 2X buffer contains 1 m M EGTA, 45CAC12 to give about 100,000 cpm/25/~l (from stock supplied at 10-40 mCi/Mg Ca2+), and additional nonradioactive CaCI 2 to give the appropriate total Ca 2+ which will result in the desired submicromolar free Ca 2+. The composition of the Ca 2+ EGTA buffer is determined by a computer program employing published association constants and methods for the interactions between Ca 2+, Mg2+, H +, EGTA, and ATP,4 and includes corrections for temperature and ionic strength. Three important considerations are (1) the critical dependence on pH of the free Ca+, (2) the purity of the EGTA used (typically 95-97%), and (3) the contaminating levels of Ca 2+ inevitably found in any laboratory water. These additional amounts of contaminating Ca 2+, typically l0 aM, can be measured with a Ca2+-sensitive electrode and included in the calculation of the Ca 2+ to be added to the buffer to result in the correct total Ca 2+. For example, the solution which will give 0.5 a M free Ca2+ can be calculated to require approximately 460 lzMtotal Ca 2+ with 500 #MEGTA, 5 m M Mg2+, 3 m M A T P at pH 7.5. With laboratory water containing 10 a M Ca 2+, an additional 450 IzM Ca2+ needs to be present, so 900 IzM Ca 2+ is added to the 2 X concentrated buffer. This Ca 2+ is obtained from both the 45Ca2+ and nonradioactive CaCI2 stock ATP solution: 30 m M Tris-ATP in dilution buffer adjusted to pH 7.5 Additional compounds, such as inhibitors or stimulatory proteins, are dissolved in dilution buffer and the pH checked Membrane filters: Nitrocellulose filters, 0.45-am pore size, 25-mm diameter, soaked in 10 m M CaC12 for a few minutes before use Wash solution: Dilution buffer containing l0 m M CaC12 These reagents may be stored frozen for several months.
Procedure. Transport studies are performed in a final volume of 100/zl. Aliquots of membranes (20 #1) containing 10 to 30/zg of membrane protein in dilution buffer are distributed to disposable glass or plastic tubes 3 T. R. Hinds and F. F. Vincenzi, this series, Vol. 102, p. 47. 40. Scharff, Anal Chim. Acta 109, 291 (1979).
450
GASTROINTESTINAL SYSTEM
[28]
on ice. Additional compounds may be added in another 20/~l dilution buffei, and then 50/~l of the 2X concentrated calcium buffer is added. These tubes are mixed and kept on ice until the individual uptakes are determined. After a 10-min preincubation at the final temperature (usually 25 o), l0/~1 of the ATP solution is added and the tube mixed by a brief vortexing. Following a 1-min incubation, a 90-#1 sample is pipetted onto a nitrocellulose disk in a vacuum suction apparatus and washed twice with 2.5 ml of ice-cold wash solution. Filtration is completed in about 15 sec after which the nitrocellulose filter is dissolved in aqueous scintillation fluid and the radioactivity counted in a liquid scintillation counter. Calculation of Results. In order to determine ATP-independent uptake, ATP is replaced with dilution buffer. Calcium ion uptake in ATP-free solutions is near maximal at 1 rain in submicromolar Ca 2+ buffers and is thought to represent Ca 2+ binding. ATP-independent Ca 2+ uptake is subtracted from uptake in the presence of ATP to give ATP-dependent Ca 2+ transport. Membrane-free blanks are also filtered and give values for binding of Ca :+ directly to the filters. An aliquot of the uptake solution is used as an internal standard to convert counts per minute to nanomoles of Ca 2+ using the total Ca 2+ present in the Ca :+ buffer. Results are usually expressed as 1-min rates, though uptake is linear for at least 2 min. Experiments are performed with at least triplicate determinations. This procedure is not the only one which will produce satisfactory results. Reagents may be added in different orders and volumes may be altered. For instance, an alternative method has been employed in which the uptake reaction is started by adding the 2 X Ca 2+ buffer. In this case, though, the membranes are not preincubated at the final submicromolar Ca 2+ concentration, which may be important in determining the actions of stimulatory proteins. Similar values, however, are found for both ATP-dependent and -independent Ca 2+ uptake. Effects on Ca 2+ U p t a k e of Different Preparations of Basolateral M e m b r a n e Vesicles
Membrane Preparation. Basolateral-enriched membrane fractions have been prepared by several similar methods from intestinal cells. The method we use is modified from the one described in 1978. 5 We have found that one must start with isolated cells to demonstrate satisfactorily the ATP-dependent Ca2+ pump. This is discussed below. Homogenates of isolated intestinal epithelial cells are prepared using a Polytron (Brinkmann, Westbury, NY), setting 8, for 1 rain, and then centrifuged at 1500 g M. M. Weiser, M. M. Neumeier, A. Quaroni, and K. Kirsch, J. CellBiol. 77, 722 (1978).
[28]
INTESTINAL BASOLATERAL MEMBRANE C a 2+ TRANSPORT
451
for 15 min to remove unbroken cells, nuclei, and brush border membranes. The supernatant is centrifuged at 105,000 g for 15 min to collect a crude membrane fraction. Basolateral membranes are further purified using sorbitol density-gradient centrifugation for 2 h r at 200,000 g and the fractions washed and resuspended in dilution buffer. The fractions with densities similar to, or less than, 40 g/dl sorbitol have been shown to be enriched for the basolateral membrane marker, Na+,K+-ATPase activity. Calcium ion transport has been found to be associated with these membranes rather than with the more dense membranes enriched for sucrase activity, a brush border membrane marker (Fig. 1). Essentially similar membrane preparations have been employed by other groups of workers who have studied basolateral membrane Ca 2+ transport. 6-8 Enrichment of Na+,K+-ATPase activity in excess of 10-fold have been difficult to achieve with intestinal homogenates, and though separation from brush border membranes and mitochondria is relatively easy, contamination with endoplasmic reticulum markers has been more difficult to overcome. Other properties of the vesicles that may affect Ca2+ transport include vesicle size, degree of sealing and orientation. Size may be estimated from electron micrographs, and quantified more easily from the equilibration volume of molecules such as glucose or mannitol. These give estimates ranging from 1 to 8 gl/mg protein. 2,6 Orientation can be estimated from enzyme latencies, though these determinations may be affected through stimulation of activities by the detergents employed. Most estimates for basolateral membranes give figures of roughly 50% in inside-out and rightside-out orientations.7 It is assumed that only inside-out vesicles will be in the correct orientation for measuring the function of the basolateral membrane Ca 2+ pump since substrates are added to the external vesicle surface which, if activated by ATP, would represent the cytoplasmic domain of the membranes in the intact ,cells. We have used these vesicles immediately after preparation for Ca 2+ transport studies. Although Ca 2+ uptake rates remain stable for several hours when the membranes are kept on ice, substantial loss of activity occurs overnight. Freezing also results in a large and variable reduction of the transport rate, though other workers appear to be able to accept -50% loss of activity as there was no change in the affinity of the pump for Ca2+.9 6 H. N. Nellans and J. E. Popovitch, J. Biol. Chem. 256, 9932 (1981). 7 W. E. J. M. Ghijsen, M. D. de Jong, and C. H. van Os, Biochim. Biophys. Acta 689, 327 (1982). s B. Hildmann, A. Schmidt, and H. Muter, J. Membr. Biol. 65, 55 (1982). 9 W. E. J. M. Ghijsen, C. H. van Os, C. W. Heizmann, and H. Murer, Am. J. Physiol. 251, G223 (1986).
452
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Fro. I. Distributionof vesicularCa 2+ transportand marker enzymes on a sorbitol density gradient. Crude membrane fractionswere prepared from rat duodena and separated on a discontinuous sorbitoldensity gradient.2 Fractions were coUected from the 30 to the 50-60 g/dl interfaceand washed. ATP-dependent Ca 2+ was measured at 0.5/tM free Ca 2+ (seetext), and Na+,K+-ATPase and sucrasc were measured as previouslydescribed.5
Cell Preparation Methods. The intestine, particularly when compared with other cell types, is sensitive to variations in the methods used to prepare the cells which are homogenized to give subcellular fractions, t° It is important not to overlook the effects of these factors on the properties of Ca2+ uptake in basolateral membrane vesicles. Also important are the differences in epithelial cell function that are found in the various regions of the small intestine and along the crypt-villus axis of differentiation. We have been able to demonstrate ATP-dependent Ca2+ uptake only in vesicles prepared from isolated intestinal cells and not from scrapings of intestinal epithelium, n One explanation appears to be the high levels of nonesterified fatty acids found in membranes prepared from scrapings, t2 These fatty acids probably result from lipolysis of other lipids during the homogenization of the serapings; much higher phospholipase A activity m M. M. Weiser, J. R. F. Wa]ters, and J. R. Wilson, Int. Rev. CytoL 101, 1 (1986). ii j. R. F. Waiters, P. J. Horvath, and M. M. Weiser, in "Epithelial Calcium and Phosphate Transport: Molecular and Cellular Aspects" (F. Bronner and M. Peterlik, eds.), p. 187. Alan R. Liss, New York, 1984. ,2 j. R. F. Walters, P. J. Horvath, and M. M. Weiser, Gastroenterology 91, 34 (1986).
[28]
INTESTINAL BASOLATERAL MEMBRANE C a 2÷ TRANSPORT
453
can be shown in homogenates of intestinal scrapings than in isolated cells. In addition to binding C a 2+ and so increasing the ATP-independent Ca2+ uptake, there may be direct effects of lipids on the plasma membrane Ca 2+ pump. Isolated cell preparations may be better in this regard as they are more extensively washed prior to homogenization. This washing may be removing a greater proportion of adherent pancreatic enzymes or inhibiting or removing epithelial cell phospholipases. Another related problem is the effect of proteolytic enzymatic activity on the Ca 2+ pump. It has been shown that there are differences in basolateral membrane Ca2+ transport from two isolated cell preparations ~3and it was postulated that this was because of differences in amounts of tryptic or other proteolytic activity. Ileal and jejunal membranes seemed more susceptible to this than duodenal; unfortunately this effect could not be prevented by commonly used trypsin inhibitors but only by modifications to the physical methods used for cell isolation. A third problem in preparing basolateral membranes, particularly related to isolated cell methods, is that redistribution of intracellular and cell surface markers may occur. Consequently, the relative purification factors for basolateral, brush border, and endoplasmic reticulum membranes become lower with prolonged preparation and washing procedures. ~°,~4Despite this concern, preparations of "basolateral" membranes show coincident enrichment for Ca2+ pump and Na+,K+-ATPase activities. Thus, either both activities have redistributed themselves at the same rate and to the same location or the redistribution is selective for the enzyme studied, e.g., alkaline phosphatase. These concerns must be considered in studies of C a 2+ transport by intestinal membrane vesicles; unfortunately there is no clearly superior method for cell isolation at present. Investigation of the Properties of Ca 2+ Uptake To confirm that Ca2+ uptake by membrane vesicles is transport by an ATP-dependent Ca 2+ pump, certain basic properties should be investigated to help differentiate ATP-dependent Ca 2+ transport from Ca 2+ binding. An absolute dependency on ATP should be shown. The time course of Ca2+ uptake by basolateral-enriched membrane vesicles, in the presence and absence of ATP, is shown in Fig. 2. Also shown is that ADP will not substitute for ATP; other nucleotide triphosphates such as GTP or CTP were also ineffective in supporting transport. Estimates of the affinity of the intestinal pump for ATP gave a Km of under 50/~M. 13E. J. J. M. van Corven, M. D. de Jong, and C. H. van Os, Cell Calcium 7, 89 (1986). 14C. A. Ziomek, S. Schulman, and M. Edidin, J. Cell Biol. 86, 849 (1980).
454
GASTROINTESTINAL SYSTEM
I28]
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FI~. 2. ATP dependence. Basolateral-enriched membranes were prepared from pooled duodenal villus and crypt cells. Calcium ion uptake was measured at 25" with 0.5/zM free Ca 2+ in the presence of 3 m M ATP (O) 3 m M ADP (1), and in the absence of ATP or ADP (O). Results are means _ SEM of four determinations. (Reprinted from Ref. 2.)
Calcium ion transport by the Ca 2+ pump can be most conveniently measured at 25*; uptake is greater at more physiologic temperatures though the rate is linear for a shorter period of time (Fig. 3). However, when experiments are performed on ice, Ca 2+ uptake is reduced to levels similar to those seen in the absence of ATP. As binding is much less temperature dependent than trans-membrane transport, this result also indicates that Ca 2+ uptake is by the Ca 2+ pump. The at~nity of the epithelial cell pump for Ca 2+ should be determined. This has been studied by several groups. We have found a Km of 0.3/zM, 2 which is similar to that found by Ghijsen et al. 7 Another group has found a much lower figure6 but used a different Ca 2+ buffer system. The free Ca 2+ and hence the Km values depend greatly upon the association constants and calculations used in the determination of the Ca2+-buffering solutions. There is the additional problem in that the Ca2+/EGTA complex has been postulated to be recognized by the Ca2+ site on the pump, giving an apparent stimulatory effect of EGTA independent of the effect on free Ca 2+ concentrations? 5 This has not yet been fully resolved for the intestinal basolateral membrane Ca 2+ pump, but appears to be an explanation for the discrepancy in measured KIn. Ca 2+ ionophores, such as A23187, should be used to show that the accumulation of Ca 2+ by the membranes is in fact intravesicular (Fig. 4). A23187 is dissolved in ethanol or dimethyl sulfoxide (DMSO) and added in micromolar concentrations such that the concentration of the solvent does not exceed 1%. This concentration of ethanol has been shown to not 15N. Kotagal, J. R. Colca, and M. L. McDaniel, J. Biol Chem. 258, 4808 (1983).
[28]
INTESTINAL BASOLATERAL MEMBRANE C a 2+ TRANSPORT
455
= 2O
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FIG. 3. Temperature dependence. Calcium ion uptake was determined in duodenal basolateral-enriched membranes in the presence of 0.5 pM free Ca 2+ and 3 m M ATP at 35 ° (&) 25 ° (0) and on ice ([3). Results are means + SEM of four determinations. (Reprinted from Ref. 2.)
affect Ca 2+ uptake. When the ionophore is added to the membranes before ATP, the amount of Ca 2+ associated with the vesicles is reduced to a level similar to those usually seen in the absence of ATP or when uptake is performed on ice. Further, when A23187 is added after Ca 2+ has been taken up by the vesicles, the concentrated intravesicular Ca 2+ will be rapidly released through the membrane pores created by the ionophore.
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FIO. 4. Effects of A23187 on Ca 2+ uptake by duodenal basolateral-enriched membrane vesicles. Membranes were incubated at 25 ° with 3 m M A T P and 0.5 g M free Ca 2+. Values are the means + SE of three or four determinations. Uptake was determined for 30 min with A23187 (9 # M ) added before ATP and Ca 2+ ((3). Uptake in the absence ofA23187 (0); at 30 min (9 gM) A23187 was added and rapidly reduced Ca 2+ uptake. (Reprinted from Ref. 2.)
456
GASTROINTESTINAL SYSTEM
[28]
Prevention of ATP-dependent Ca 2+ uptake and release of intravesicular accumulations by A23817 confirms that the Ca 2+ is concentrated in a soluble form within the vesicles. From determinations of the volume of the vesicles, it can be calculated that the concentration of Ca z+ inside the vesicles at equilibrium exceeds 5 mM, so a 10,000-fold gradient can be maintained by the pump. The subcellular origin of the CaZ+-uptake activity in these membrane fractions may be defined by the actions of established inhibitors of Ca 2+ pumps. Orthovanadate is a powerful inhibitor of most ATPases and will inhibit membrane Ca 2+ uptake with a Ki of less than 1/tM, a figure similar to those of other plasma membrane pumps. The mitochondrial Ca 2+ pump inhibitors such as oligomycin or azide should have no effect. In sodiumfree conditions, ouabain or other Na + pump inhibitors should have no action on Ca :+ accumulation. The effects of Na + on Ca 2+ uptake through the Na+/Ca 2+ exchanger found in plasma membranes have been studiedS,~6; the effects of this system on Ca2+ uptake can best be avoided by omitting Na + from solutions used in the study of the ATP-dependent pump. Calmodulin, a ubiquitous M~ 16,800 protein, has been shown to stimulate various plasma membrane Ca 2+ pumps in a direct manner independent of the action of the specific calmodulin-independent protein kinase. As calmodulin binds up to four Ca 2+ atoms with affinities appropriate for intracellular levels, this is thought to provide a means of control for the Ca 2+ pump. In the intestine, basolateral membrane Ca 2+ uptake is also stimulated by calmodulin under appropriate conditions, but the effect is not as great as that described for the erythrocyte pump. The problem appears to be the retained, tightly bound endogenous calmodulin which obscures an effect of added stimulator. Stimulations of 20-50% at 0.5-1 /~M free Ca 2+ can be found with 10/~g/ml calmodulin (about 0.6/~M) 6 though even this relatively small effect is only seen with inclusion of 5 m M EGTA in the homogenization buffer7 or preincubation with submicromolar Ca 2+.~7The effect of calmodulin appears predominantly to be on the Km of the pump; in the intestine even in the absence of exogenous calmodulin, the Km for Ca 2+ is under 1/~M, similar to the calmodulin-stimulated pump of other tissues. Calmodulin can be detected in basolateral membranes, 9 including those that have been treated with procedures that in other tissues would have removed it. The effects of drugs with calmodulin antagonistic properties have also been investigated; again the results are 16 W. E. J. M. Ghijsen, M. D. de Jong, and C. H. van Os, Biochim. Biophys. Acta 730, 85 (1983). ~7j. R. F. Waiters, Am. J. Physiol. 256, G124 (1989).
[28]
INTESTINAL BASOLATERAL MEMBRANE C a 2+ TRANSPORT
457
not definitive. It may be that the interaction of calmodulin with the intestinal basolateral Ca2+ pump is different from that in the red cell or other tissues, an interaction that may more specifically define this pump. The intestinal vitamin D-dependent Ca2+-binding protein (CaBP, calbindin-D9 u~) affects Ca2+ absorption and might also be expected to influence the basolateral Ca2+ pump. Our studies suggest that this may be the case~7although others have not found such an action.9 Concentrations of CaBP are included in the uptake medium at submicromolar Ca2+; with appropriate C a 2+ buffering this does not significantly affect the free Ca2+ concentrations or specific activity of 45Ca2+. The effects of CaBP may be dependent on the saturation of the protein with Ca2+; the methodology required to confirm or disprove such an effect of CaBP on the pump remains to be determined. The rate of Ca2+ transport in basolateral membrane vesicles has been shown to be affected by the vitamin D status of the animal3 ,~s Along with CaBP and other actions at the brush border membrane, an increase in basolateral Ca2+ transport may represent a method of controlling the overall rate of Ca2+ absorption. Rats can be made vitamin D deficient by a combination of a synthetic diet without vitamin D supplementation, a Ca2+-free diet for 2 weeks to deplete stores of vitamin D through conversion to 1,25(OH)2D3, and an environment lit only by incandescent light to prevent synthesis in the skin of vitamin D by the action of UV light. ~9 These animals have reduced serum Ca2+ concentrations and bone histology compatible with vitamin D deficiency. Intestinal cells and basolateral membrane vesicles have been prepared from these animals by methods detailed above. Purification of marker enzymes, and other studies to show nonspecific effects on vesicles, have been performed with only minor differences found. Vesicular Ca2+ transport rates, however, are reduced by almost 50% in the duodenum, an effect which appears to be on the V ~ and is particularly apparent in membranes of cells at the villus tip. When vitamin D-deficient animals were given repleting doses of 1,25(OH)2D3, 125 ng in ethanol by intravenous injection, the increase after 6 hr in vesicular Ca2+ transport was predominantly in the developing cells lower down the villus.2 Thus, any studies characterizing aspects of Ca2+ absorption should investigate the effects of vitamin D status and consider differences related to intestinal regions and to the crypt-villus axis of differentiation. Ca2+-ATPase enzymatic activity, i.e., the hydrolysis of inorganic phosis W. E. J. M. Ghijsen, and C. H. van Os, Biochim. Biophys. Acta 689, 170 (1982). ~9j. H. Bloor, A. Dasmahapatra, M. M. Weiser, and W. D. Klohs, Biochem. J. 208, 567
(1982).
458
GASTROINTESTINAL SYSTEM
[28]
phate from ATP, is a function of the Ca 2+ pump and should be related to the ATP-dependent transport of Ca 2+. In the intestine, difficulties have been encountered in making this correlation, partly because of the additional hydrolysis of ATP which, once attributed to alkaline phosphatase activity, 2° appears to be a function of ectonucleotide phosphatase activity. 2~ Using similar Mg 2+ and Ca 2+ concentrations as those described for uptake measurements, we found that the high level of this Ca 2+- and ATP-independent Mg2+-ATPase activity prevented accurate assessment of the small increment of any Ca2+-dependent ATP hydrolysis. Consequently, Ca2+-dependent ATPase activity was best measured at reduced free Mg 2+ concentrations of 1 #M. This was achieved with a total Mg + concentration of 47/zM in the presence of 500/zM EGTA, 3 m M ATP, and 509/~M total Ca 2÷ at pH 7.5. Under these conditions, basolateral Ca2+-ATPase activity was similar to values reported by others, but the properties were not the same as those described for vesicular Ca 2+ transport. 2! Thus, calcium ion-dependent ATP hydrolysis in these membranes is not solely a function of the Ca 2+ pump, but represents other enzymatic activities, as has been described in other tissues, including the liver. Conditions for the determination of that portion of ATP hydrolysis due only to the Ca 2+ pump remain to be determined. Basolateral Membrane Ca 2+ Transport in Other Species Most of the details of the intestinal basolateral membrane Ca 2+ pump have been determined with experiments in the rat. Some studies have been performed with chick basolateral membranes; these are particularly relevant as this species has been well studied regarding the action of vitamin D. These findings are broadly similar to those with the rat, including a dependence on the vitamin D status of the animal. 22 Vesicular Transport of Ca 2+ in Other Intestinal Subcellular M e m b r a n e s This chapter has concentrated on the vesicular transport of Ca 2+ by the basolateral membrane of the intestine. We have not described methods or 2oW. E. J. M. Ghijsen, M. D. de Jong, and C. H. van Os, Biochim. Biophys. Acta 599, 538 (1980). 21T. C. Moy,J. R. F. Waiters, and M. M. Weisex,Biochim. Biophys. Res. Commun. 141, 979 (1987). 22j. S. Chandler, S. A. Meyer,and R. H. Wasserman, in "Vitamin D: Chemical, Biochemical and Clinical Update" (A. W. Norman, K. Schaefer, H.-G. G-rigoleit, and D. v. Herrath, eds.), p. 408. de Gruyter, Berlin, 1985.
[29]
ELECTRICAL MEASUREMENTS IN LARGE INTESTINE
459
results of studies conducted by others attempting to measure Ca2+ transport at the brush border membrane.23,u Only one paper deals with transport by the endoplasmic reficulum in any detail.25 Though it appears that the Gelgi may also have a C a 2+ transport system, u earlier results were complicated by high binding of Ca2+ to nonesterified.fatty adds in membranes prepared from intestinal serapings. 12In these studies with the intestine, it seems that the major methodological problem remains the preparation of enriched membranes in sufficient quantities, and unaltered so that they reasonably reflect the in vivo condition of the membrane domain. This has been largely true with duodenal basolateral-enfiched membranes where vesicular Ca 2+ transport has most of the properties expected from studies of C a 2+ a b s o r p t i o n . More recently a transcript for an intestinal plasma membrane C a 2+ pump has been detected which responded to vitamin D similarly to that shown for Ca 2+ uptake by basolateral membranes. 26 Acknowledgments This work is supported by NII-I Grants AM-32336 and AM-35015, and by the Troup Fund of Buffalo General Hospital, Buffalo, New York. 23A. Miller [ ] and F. Bronner, Biochem. J. 196, 391 (1981). 24 H. Rasmussen, O. Fontaine, E. E. Max, and D. B. P. Goodman, J. Biol. Chem. 254, 2993 (1979). 25 M. J. Rubinoffand H. N. Nellans, J. Biol. Chem. 260, 7824 (1985). 2~j. Zelinski, D. E. Sykes, and M. M. Weiser, Gastroenterology 98, A560 (1990).
[29] E l e c t r i c a l M e a s u r e m e n t s
in L a r g e I n t e s t i n e (Including Caecum, Colon, Rectum) B y ULRICH HEGEL a n d MICHAEL FROMM
Introduction The main function of the large intestine is the conservation of water and ions. Electrophysiological methods are therefore of predominant importance in the analysis of physiological as well as of pathological functional states of this organ. Apart from this more applied aspect large intestinal mucosa has increasingly been used as a model to study basic mechanisms of epithelial ion transport which are of relevance also for other tubular organs such as the nephron or excretory ducts of exocrine METHODS 1N ENZYMOLOGY, VOL. 192
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