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Membrane potential changes associated with differentiation of enterocytes in the rat intestinal villi in culture
DEVELOPMENTAL
BIOLOGY
94, 284-290 (1982)
Membrane Potential Changes Associated with Differentiation of Enterocytes in the Rat Intestinal Villi in Culture WAKOH TSUCHIYA’ Department
of Physiology,
Received February
AND
Faculty of Medicine,
YASUNOBU
OKADA
Kyoto University,
Kyoto 606, Japan
3, 1982; accepted in revised form June 28, 1982
Fragmented villi of the small intestine isolated from newborn rats were maintained in culture for periods of up to 4 weeks. In culture, floating globular villi and adherent villi were distinguished. Intracellular recordings were made from both types of villi. The membrane potential was highest (about -70 mV) in the cells at the tip of the villus and was lowest (about -18 mV) in the cells near the crypt region, showing a continuous gradation according to the cell location. The membrane potential in cultured mature enterocytes was due to the contribution of the electrogenic Na+ pump as well as to the Ca*+-activated K+ conductance. Both the components were temperature and metabolic energy dependent. Based on these data, it is suggested that the Ca*’ transport mechanism and the electrogenic Na’ pump are involved in the process of cell differentiation or maturation of the intestinal epithelia. INTRODUCTION
The villi of the small intestine consist of a constantly renewed population of epithelial cells. The crypt region contains the mitotically active cells which migrate up to the villus and become progressively differentiated into the mature absorptive cells after the end of cell division (Leblond and Messier, 1958). The different,iation of the mitotically active crypt cells to absorptive villous cells is accompanied by changes in cell morphology (Palay and Karlin, 1959; van Dongen et al., 1976) and in various enzyme activities (Dahlqvist and Nordstrtim, 1966; Moog and Grey, 1966; NordstrGm et al., 1968, Webster and Harrison, 1969; Weiser 1973; Charney et al., 1974; de Both and Plaisier, 1974; Quill and Weiser, 1975; Gall et al., 1977; Raul et al., 1977; Pothier and Hugon, 1980). However, little is known about the membrane potential changes during cell differentiation along the villus-crypt axis. We have examined membrane potential changes associated with the cell maturation and the underlying ionic mechanisms in cultured villi. Some of these data have been published in a short report (Tsuchiya et al, 1980). MATERIALS
AND
METHODS
Tissue culture. Morphological maturation of the rat small intestine is known to be accomplished by 19 to 22 days of gestation (DeRitis et al., 1975; Madara et al., 1981). In the present study, newborn small intestines were employed for tissue culture. Wistar rats were decapitated immediately after birth before receiving co1 Present address: Department of Dermatology, Kyoto University, Kyoto 606, Japan.
Faculty of Medicine,
284 0012-1606/82/120284-07$02.00/O Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.
lostrum. The entire small intestine was then quickly resected. The luminal surface of the intestine was washed with a culture medium (Medium 199, Nissui) with a syringe, and the tissue (Fig. 1A) was minced into small pieces. The fragmented villi (Fig. 1B) thus obtained were placed in Falcon plastic dishes (60 X 15 mm), containing 5 ml of the culture medium supplemented with 20% bovine serum, streptomycin (100 pg/ ml), and penicillin (63 pg/ml). The dishes were then kept in a humidified incubator equilibrated with 5% CO2 in air at 37°C. The culture medium was replaced every other day. Electrophysiology. The procedures used for intracellular recordings have been described in a previous paper (Okada et al., 1977a). KC1 (3 M)-filled glass microelectrodes, with a resistance of 15-30 MO were used. Their tip potentials were less than 5 mV. The electrophysiological experiments were performed under an inverted phase-contrast microscope (Chiyoda T-2). Unless otherwise noted, the experiments were performed in phosphate-buffered saline (PBS) which contains 5.4 mM Kt , 129.3 mM Na+, 1.8 mM Ca2+, 0.8 mM Mg’+, and 83 mM Cl- (pH = 7.3 f 0.1). Membrane potentials were measured through a high-input impedance preamplifier (WPI M701). Intracellular injection of Ca2’ ions was performed by a method described elsewhere (Okada et al., 1979). A suction micropipet was employed to stabilize a floating villus during the electrode penetration. Data were accepted only when a stable membrane potential was maintained for at least 1 min and when the extracellular potential level observed upon withdrawal of the electrode agreed with the initial zero level. The experiments were done usually at 37”C, but the bath
TSUCHIYA
AND
OKADA
Enterocyte
75
FIG. 1. Small intestinal epithelia of a newborn rat, Day 1, before culture. (A) Whole epithelium. Hematoxylin-eosin (X170). (B) Fragmented villi immediately aft.er mincing. Phase contrast (X340).
Membrane
Differentiation
285
were covered with a single layer of columnar epithelial cells and contained lamina propria (Fig. 2B). As the culture period increased, the volume of contents as well as the diameter of the floating villi gradually increased, suggesting transepithelial solute accumulation. While the floating villi swelled, the epithelial cells became flattened. No changes in the cell number occurred during 2 weeks of culture. No sign of cell mitosis was ever noticed in any histological sections of the floating villi. Thus, the cells constituting floating villi appear to be nonmitotic mature enterocytes. In contrast, from the villi adhered to the plastic substrate, an increasing number of epithelial cells migrated and formed a growing monolayer (Fig. 3). A few mitotic cells were often found in the monolayer, but never in the upper region (free end) of adherent villi. Thus, the cells in the monolayer appear to correspond to undifferentiated crypt enterocytes, since cell proliferation is known to be confined exclusively to the crypt zone in small intestinal epithelia (Leblond and Messier, 1958). Both types of fragmented villi were viable in culture for 4 weeks, but morphological studies revealed some sign of deterioration as evidenced by cell desquamation after 2 weeks of culture. Some epithelial cells in the monolayer of the adherent villi were viable for over 1 month, but were progressively overwhelmed by increasing fibroblastic cells. Stable membrane potentials were recorded from epithelial cells in the two types of villi. All the cells examined were assumed to be enterocytes, since the epi-
temperature was reduced to 56°C by circulating icecold water, if necessary. Ethanol was used as vehicle for verapamil (a gift from Eisai Co., Tokyo). The addition of ethanol did not affect membrane potentials at least in the doses used. After applying any drugs, the cells were washed with the control PBS, and the recovery of membrane potentials .to the original level was always confirmed. Morphology. Studies with a phase-contrast microscope (Nikon Optiphoto NT) were made, in a living state, on the tissue culture dishes. Tissues fixed with Bouin’s fluid were studied after hematoxylin-eosin staining. RESULTS
Two types of cultured villi. After 2 days in culture, two distinct types of the fragmented villi were discernible. The first was the globule-type floating villi (Fig. 2A), 50 to 500 pm in diameter, which were formed as a result of the open-end closure. The second was the adherent villi (Fig. 3), which adhered to the bottom of the culture dish with a central mounded region. The floating villi
FIG. 2. Floating
contrast
globule-type villi cultured for 2 days. (A) Phase (X425). (B) Hematoxylin-eosin (X300).
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DEVELOPMENTAL BIOLOGY
thelium of the newborn rat small intestine contains only a few goblet cells (Fig. 1A). The magnitude of the membrane potentials measured in the adherent villi after 2 to 9 days of culture varied, depending upon the cell location, as shown in Fig. 4. The membrane potentials were highest (approximately -70 mV) at the top of the adherent villi, while the cells located near the base showed lower membrane potentials; the monolayer epithelial cells directly attached to the substrate were characterized by the lowest membrane potential (-15 to -20 mV). The addition of glycine (20 r&I) rapidly induced a significant depolarization (about 20 mV) in the cells at the villous tip, but this response was never observed in the monolayer cells (data not shown). Such a glycine-evoked depolarization has been reported to be associated with an active transport of the amino acid by mature absorbing enterocytes in the rat small intestine (Okada et al., 1977b; Okada, 1979). Thus, it can be concluded that the absorbing villus cells have higher membrane potentials than the undifferentiated crypt cells in the adherent villi. The magnitude of the mem-
VOLUME94,1982 -80 c
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FIG. 4. Membrane potential profiles recorded in an adherent villus after 4 days of culture (0) and in a floating villus after 2 days of culture (A). Both types of cultured villi are schematically drawn. Arrows show the direction of successive recordings. The same profiles were obtained even when the direction of punctures was reversed.
FIG. 3. Adherent villi. Phase contrast. (X140). (B) Cultured for 7 days (X280).
(A) Cultured
for 3 days
brane potentials measured in the floating vilii after 2 days of culture was also dependent on the cell location as seen in Fig. 4. The membrane potential profiles in the floating villi exhibited a continuous gradation, but the gradient was less marked than that in the adherent villi. The smaller the diameter of floating villi, the less deviated the membrane potentials from about -70 mV. This fact suggests that the floating villi mainly consist of mature absorbing enterocytes in the crest of the villi. Supporting this inference, glycine-evoked depolarization was found in almost all the cells in floating villi. Furthermore, the potentials of all the cells in the floating villi were similar (about -70 mV), regardless of cell location, when the culture period was prolonged (up to 2 weeks). Thus, the magnitude of the membrane potential apparently reflects the degree of cell differentiation in the cultured villi. Membrane properties of uillous cells in culture. The electrical membrane properties of mature villous enterocytes were examined, using the floating villi cultured for 3 to 6 days. The diameter of these villi was less than 150 pm. Figure 5A illustrates the effects of the external K+ concentration ([K+],) on the membrane potential of the villous enterocyte in floating villi. As [K+], was increased from 5.4 mM, the membrane became depolarized. These changes were approximately linear against
TSUCHIYA AND OKADA
Enterocyte
Membrane
0'
3
287
Differentiation
I 10
I loo ~n+lo30 (m-M)
([K+] ,,) on the membrane potentials. (A) Membrane potentials obtained from the villous FIG. 5. Effects of the external K’ concentration cells in a floating villus cultured for 3 to 6 days. To cancel out the physical effect of the temperature, the data are presented as the reduced membrane potentials (-F/RT X membrane potentials). (0) Measured under the control condition (with 1.8 mM at 37°C). (a) Measured under the external Ca’+-free condition at 37°C. (0) Measured at 6°C with 1.8 mM Ca’+. (B) Membrane potentials obtained in adherent villi cultured for 3 to 5 days under the control condition. (0) Obtained from the villous tip cells. (0) Obtained from the monolayer crypt cells. To change the IK+L. . __.NaCl was replaced with equimolar amounts of KCl, or vice versa, in a PBS bath. Each point represents the mean value of 26 - 115 observations with the standard deviation of less than +6.9 mV
log [K’],, with a slope of about 50 mV/decade. However, a decrease of [K’10 from 5.4 to 1.4 r&L4 also caused a slight depolarization by several millivolts (Fig. 5A, open circles). Upon rapid cooling of the villi from 37 to 6”C, the cell was immediately depolarized. This change was readily reversible after rewarming, being accompanied by a slight transient hyperpolarization (up to about -75 to -80 mV). At low temperatures, the [K+lo dependency was markedly depressed (Fig. 5A, squares). Several metabolic inhibitors were applied to test whether the temperature-dependent component of the membrane potential is generated by an energy-consuming process. The application of NaF or iodoacetic acids (IAA), which are glycolysis inhibitors, depolarized the membrane within 15 min to almost the same level (Fig. 6) as that under the cooli.ng condition (Fig. 5A, squares). In contrast, KCN, one of the respiratory inhibitors, did not significantly affect the membrane potential (Fig. 6). Thus, the temperature dependence of the membrane potential appeared to be mediated by a metabolic energy mainly derived from glycolysis. The membrane potential at 37°C was also dependent on the extracellular Ca2+. The deprivation of external calcium depolarized the membrane by about 20 mV at normal [K+],, and the [K’10 dependency of the membrane potential was considerably suppressed (Fig. 5A, triangles). The application of EGTA to the external Ca’+-free solution did not cause a further depolarization (Fig. 6). Addition of calcium to the Ca2+-free solution readily hyperpolarized the membrane to the control
value. Sr2+ or Mn2+ (but not Mg2+) ions can be substituted for Ca2+ ions. Even in the absence of external Ca2+, the intracellular injection of Ca2+ restored the membrane potential (Fig. 6). The addition of Ba2+ ions, an inhibitor of Ca2+-activated K+ conductance (Porzig, 1977: Gorman and Hermann, 1979), rapidly abolished (60) I
ing Villi FIG. 6. Membrane potentials under a variety of con dit from the villous cells in floating villi cultured for 3 to 6 days. Data were collected within 30 min after adding Ba*+, verapamil, or EGTA, within 10 min after adding NaF or IAA, and within 20 min after adding KCN. Numbers in parentheses indicate the number of cells impaled. Vertical bars represent standard deviations. *, Significantly different from the control values at P < 0.05.
288
DEVELOPMENTAL BIOLOGY
the Ca2+-dependent component of the membrane potential (Fig. 6). Verapamil, a Ca2+ channel blocker (Kohlhardt et al., 1972), also abolished the Ca2+-dependent component (Fig. 6). The calcium deprivation did not affect the membrane potential at 6°C. Thus, it is concluded that the temperature-dependent component of the membrane potential of villous mature enterocytes is partly due to the Ca2+-activated K+ conductance (Lew and Ferreira, 1978; Meech, 1978) and that the intracellular Ca2+ ions responsible for the activation of K+ conductance are mainly transported from the outside of the cell, probably through Ca2+ channels. The membrane was transiently hyperpolarized after rewarming from 6 to 37°C (see above). The transient (for about 2-3 min) hyperpolarization became more pronounced (from -80 to -100 mV) upon rewarming after the cells were exposed to a K+-free PBS at 2°C. Under the latter condition, the cells would have been loaded with Na+ ions. Thus, the prominent hyperpolarization suggests the presence of an electrogenic Na+ pump in mature villous enterocytes in culture. Supporting this hypothesis, the application of ouabain depolarized the membrane down to about -50 mV within 10 min (Fig. 6); yet, ouabain did not affect the membrane potential at 6°C. Therefore, the electrogenic Na+ pump appears to be an additional component involved in the temperature-dependent membrane potential in villous cells. The properties of membrane potentials in mature villous cells were also examined in the adherent villi cultured for 2 to 9 days. The data obtained from the cells at the tip region of villi (Fig. 5B, open circles, and Fig. 7A) were essentially the same as those obtained from the mature cells in the floating villi (Figs. 5A and 6). Membrane potentials in crypt cells in culture. The membrane potentials of the monolayer epithelial cells in the adherent villi after 2 to 9 days of culture were about -18 mV (Fig. 7B). The potentials were less affected by the [K+], changes than those obtained in the villous cells, as shown in Fig. 5B (solid circles). Cooling the cells (at 5°C) depolarized the membrane only slightly (to about -14 mV), as did the addition of ouabain to the same level within 10 min (Fig. 7B). The degree of depolarization produced by cooling or ouabain (Fig. 7B) was, however, very scanty compared to that in the villous cells (Fig. 7A). In addition, the application of Ba2+ never affected the membrane potential profiles in the monolayer enterocytes in the adherent villi. Thus, it seems reasonable to conclude that the membrane potential in undifferentiated crypt cells lacks the Ca’+activated K+ conductance component and has a very small component of the electrogenic Na+ pump. Other undetermined factors might be involved in the difference of the ionic mechanisms underlying the membrane
VOLUME 94, 1982
O- A). Villous
Cells
B).Crypt
in Adherent
Cells
Villi
FIG. 7. Membrane potentials under a variety of conditions obtained from the villous cells (A) and the crypt cells (B) in adherent villi cultured for 2 to 9 days. Numbers in parentheses, vertical bars, and stars are the same as in Fig. 6.
potentials between the villous and the crypt cells because the membrane potential in villous cells at 5-6°C was still greater than that in crypt cells at 37°C (Figs. 5 and 7). DISCUSSION
There have been several attempts to establish cell lines derived from the small intestinal epithelial cells (Henle and Deinhardt, 1957; Lichtenberger et al., 1973; Quaroni et al., 1979) or to culture the isolated enterocytes (Raul et aE., 1978). Induction of differentiation of these cells to the mature absorbing cells seems difficult in vitro (Raul et al., 1978; Inui et al., 1980). It is possible that the intestinal cell differentiation in uiuo depends upon a complex set of different factors (Quaroni and May, 1980), including epithelial-mesenchymal interactions (Haffen et al., 1981). Thus, the organ culture of explanted intestinal epithelia may provide a favorable approach for the electrophysiological studies with respect to the intestinal differentiation along the villuscrypt axis. However, in the previous organ culture system (Browning and Trier, 1969), the explants could be maintained only for short periods (up to 24 to 48 hr) and were mobile, interfering with a stable penetration of microelectrodes, when the whole villi were exposed to electrolyte solutions. These difficulties could be avoided in the present study by culturing fragmented villi of newborn (Day 1) rat small intestine (see Materials and Methods). The cultured fragmented villi were classified into the adherent villi and globule-type floating villi. Cell mitosis
TSUCHIYA AND OKADA
Enterocyte
was found in the monolayer of the adherent villi, but it was never observed in the upper region of the adherent villi or in the floating villi. Glycine-evoked depolarization was found in the cells in the floating villi or the top region of the adherent villi, but the monolayer cells of the adherent villi lack’ed this response. Based on these results, it is concluded that the floating villi and the free end of the adherent villi consist of mature absorbing enterocytes, whereas the attached end of the adherent villi consist of undifferentiated crypt cells. The rat coionic crypt cells have been reported to be much more adherent to the substrate than the villous cells (Patnaik et al., 1981). Therefore, it is likely that the adherent villi are formed by the attachment of crypt cells around the cut end of the fragmented villi. On the other hand, if the cut end of the fragmented villi consists of villous cells, the villi would be incapable of attaching, thereby forming the floating villi. The membrane potential was highest in the enterocytes at the crest of a villus and lower in those closer to the crypt region in cultured villi (Fig. 4). This finding is in agreement with the observations on the isolated rabbit ileum (Hirschhorn and Frazier, 1973). Since amino acid-dependent membrane depolarizations, presumably associated with amino acid absorption (Okada, 1979), were observed in the villous cells with a high membrane potential, the potential gradient observed along a villus may reflect some events related to the differentiation of enterocytes to absorbing cells. The difference in membran’e potential was finely graded, depending upon cell location along the cultured villus. This coincides with gra.dual changes in morphological (van Dongen et al., 19:76) and biochemical properties (Nordstrom et al., 1968; Weiser, 1973; Raul et al., 1977; Pothier and Hugon, 1980) according to the cell location along a villus. The low membrane potential observed in the crypt cells cannot be attributed to the low K+ concentration gradient since the intracellular K+ concentration measured by the electron-probe microanalysis is higher in the crypt cells than in the villous cells (Cameron et al., 1979). The present study has shown that the membrane potentials of crypt enterocytes are less sensitive to temperature or cellular metabolic energy than those of villous cells. The temperatureor energy-dependent component of the membrane potential in mature villous cells in culture was found to be regulated by the Ca’+-activated K+ conductance as well as by the electrogenic Na+ pump. Since the Ca’+-activated K+ conductance in the villous cells depends on the operation of verapa.mil-sensitive Ca2+ channels, it is possible that the Ca2+-dependent component of the membrane potential might be related to the activity of Ca2+ transport in villous mature cells. In this connection, it is noteworthy that the distribution of calcium-
Membrane
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binding proteins appears to have a gradient increasing from the crypt to the tip of the villus (Marche et al., 1980). Also, Na+-K+-ATPase activity (Charney et al., 1974) and ouabain-sensitive Na+ efflux (Gall et al., 1977) are known to be higher in the villus tip than in the crypt cells in rat small intestine. Thus, the electrogenic Na+ pump found in cultured villous cells may reflect the activity of Na+-K+-ATPase. A significant contribution of the electrogenic Nat pump to the membrane potential has been found in mature enterocytes in isolated rat small intestine (Okada et al., 1978). It is suggested that the development of the electrogenic Na+ pump activity and the appearance of the verapamil-sensitive Ca” channel or the Ca2+-activated K+ channel are related to cell differentiation or maturation in intestinal epithelia. The authors are indebted to Professor A. Inouye for his valuable advice and financial support of this study. We are grateful to Professor M. Kuno for critical reading of the manuscript. Thanks are also due to Dr. S. Ueda for his collaboration in a part of this culture work.
REFERENCES BROWNING, T. H., and TRIER, J. S. (1969). Organ culture of mucosal biopsies of human small intestine. J. Clin. Znuest. 48, 1423-1432. CAMERON, I. L., SMITH, N. K. R., and POOL, T. B. (1979). Element concentration changes in mitotically active and postmitotic enterocytes. An X-ray microanalysis study. J. Cell Biol. 80, 444-450. CHARNEY, A. N., GOTS, R. E., and GIANNELLA, R. A. (1974). (Na+Kf)-stimulated adenosinetriphosphatase in isolated intestinal villus tip and crypt cells. Biochim. Biophys. Acta 367, 265-270. DAHLQVIST, A., and NORDSTROM, C. (1966). The distribution of disaccharidase activities in the villi and crypts of the small-intestinal mucosa. Biochim. Biophys. Acta 113, 624-626. DE BOTH, N. J., and PLAISIER, H. (1974). The influence of changing cell kinetics on functional differentiation in the small intestine of the rat. A study of enzymes involved in carbohydrate metabolism. J. H&o&em. Cytochem. 22, 352-360. DERITIS, G., FALCHUK, Z. M., and TRIER, J. S. (1975). Differentiation and maturation of cultured fetal rat jejunum. Dew Biol. 45, 304317. GALL, D. G., CHAPMAN, D., KELLY, M., and HAMILTON, J. R. (1977). Nat transport in jejunal crypt cells. Gastroenterology 72, 452-456. GORMAN, A. L. F., and HERMANN, A. (1979). Internal effects of divalent cations on potassium permeability in molluscan neurones. J. Physiol. 296, 393-410. HAFFEN, K., KEDINGER, M., SIMON, P. M., and RAUL, F. (1981). Organogenetic potentialities of rat intestinal epithelioid cell culture. Differentiation 18, 97-103. HENLE, G., and DEINHARDT, F. (1957). The establishment of strains of human cells in tissue culture. J. Zmmunol. 79, 54-59. HIRSCHHORN, N., and FRAZIER, H. S. (1973). The electrical profile of stripped, isolated rabbit ileum. Hopkins Med. J. 132, 271-281. INUI, K., QUARONI, A., TILLOTSON, L. G., and ISSELBACHER, K. J. (1980). Amino acid and hexose transport by cultured crypt cells from rat small intestine. Amer. J. Physiol 239, C190-C196. KOHLHARDT, M., BAUER, B., KRAUSE, H., and FLECKENSTEIN, A. (1972). Differentiation of the transmembrane Na and Ca channels in mammalian cardiac fibres by the use of specific inhibitors. PfEiigers Arch. 335, 309-322.
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LEBLOND, C. P., and MESSIER, B. (1958). Renewal of chief cells and goblet cells in the small intestine as shown by radioautography after injection of thymidine-H3 into mice. Anat. Rec. 132, 247-259. LEW, V. L., and FERREIRA, H. G. (1978). Calcium transport and the properties of a calcium-activated potassium channel in red cell membranes. Curr. Top. Membr. Transp. 10, 217-277. LICHTENBERGER, L., MILLER, L. R., ERWIN, D. N., and JOHNSON, L. R. (1973). Effect of pentagastrin on adult rat duodenal cells in culture. Gastroenterology. 65, 242-251. MADARA, J. L., NEUTRA, M. R., and TRIER, J. S. (1981). Junctional complexes in fetal rat small intestine during morphogenesis. Deu. Biol. 86, 170-178. MARCHE, P., CASSIER, P., and MATHIEU, H. (1980). Intestinal calciumbinding protein. A protein indicator of enterocyte maturation associated with the terminal web. Cell Tiss. Res. 212, 63-72. MEECH, R. W. (1978). Calcium-dependent potassium activation in nervous tissues. Annu. Reu. Biophys. Bioeng. 7, 1-18. Moot, F., and GREY, R. D. (1966). Spatial and temporal differentiation of alkaline phosphatase on the intestinal villi of the mouse. J. Cell Biol. 32, Cl-C6. NORDSTROM, C., DAHLQVIST, A., and JOSEFSSON, J. (1968). Quantitative determination of enzymes in different parts of the villi and crypts of rat small intestine. Comparison of alkaline phosphatase, disaccharidases and dipeptidase. J. H&o&em. Cytochem. 15, 713721.
OKADA, Y. (1979). Solute transport process in intestinal epithelial cells. Membr. Biochem. 2, 339-365. OKADA, Y., DOIDA, Y., ROY, G., TSUCHIYA, W., INOUYE, K., and INOUYE, A. (1977a). Oscillations of membrane potential in L cells. I. Basic Characteristics. J. Membr. Biol. 35, 319-335. OKADA, Y., IRIMAJIRI, A., TSUCHIYA, W., and INOUYE, A. (1978). Contribution of an electrogenic sodium pump to the membrane potential in the intestinal epithelial cell. Jpn. J. Physiol. 28, 511-525. OKADA, Y., TSUCHIYA, W., and INOUYE, A. (1979). Oscillations of membrane potential in L cells. IV. Role of intracellular Ca2+ in hyperpolarizing excitability. J. Membr. Biol. 47, 357-376. OKADA, Y., TSUCHIYA, W., IRIMAJIRI, A., and INOUYE, A. (1977b). Electrical properties and active solute transport in rat small intestine. I. Potential profile changes associated with sugar and amino acid transports. J. Membr. Biol. 31, 205-219.
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PALAY, S. L., and KARLIN, L. J. (1959). An electron microscopic study of the intestinal villus. I. The fasting animal. J. Biophys. Biochem. Cytol. 5, 363-371. PATNAIK. R., MAZUMDER, A., BHAGAVAN, B. S., and NAIR, P. P. (1981). Characterization of rat colonic epithelial cell populations. Cell Differen. 10, 147-156. PORZIG, H. (1977). Studies on the cation permeability of human red cell ghosts. J. Membr. Biol. 31, 317-349. POTHIER, P., and HUGON, J. S. (1980). Characterization of isolated villus and crypt cells from the small intestine of the adult mouse. Cell Tissue Res. 2 11, 405-418. QUARONI, A., and MAY, R. J. (1980). Establishment and characterization of intestinal epithelial cell cultures. Methods Cell Biol. 21B, 403-427.
QUARONI, A., WANDS, J., TRELSTAD, R. L., and ISSELBACHER, K. J. (1979). Epithelioid cell cultures from rat small intestine. Characterization by morphologic and immunologic criteria. J. Cell Biol. 80, 248-265.
QUILL, H., and WEISER, M. M. (1975). Adenylate and guanylate cyclase activities and cellular differentiation in rat small intestine. Gastroenterology 69, 470-478. RAUL, F., KEDINGER, M., SIMON, P., GRENIER, J., and HAFFEN, K. (1978). Behaviour of isolated rat intestinal cells maintained in suspension or monolayer cultures. Biol. Cell 33, 163-168. RAUL, F., SIMON, P., KEDINGER, M., and HAFFEN, K. (1977). Intestinal enzymes activities in isolated villus and crypt cells during postnatal development of the rat. Cell Tiss. Res. 176, 167-178. TSUCHIYA, W., OKADA, Y., and INOUYE, A. (1980). Membrane potential measurements in cultured intestinal villi. Membr. Biochem. 3, 147-153. VAN DONGEN, J. M., VISSER, W. J., DAEMS, W. T., and GOLJAARD, H. (1976). The relation between cell proliferation, differentiation and ultrastructural development in rat intestinal epithelium. Cell Tiss. Res. 174, 183-199. WEBSTER, H. L., and HARRISON, D. D. (1969). Enzymic activities during the transformation of crypt to columnar intestinal cells. Exp. Cell Res. 56, 245-253. WEISER, M. M. (1973). Intestinal epithelial cell surface membrane glycoprotein synthesis. I. An indicator of cellular differentiation. J. Biol. Chem. 248, 2536-2541.
BIOLOGY
94, 284-290 (1982)
Membrane Potential Changes Associated with Differentiation of Enterocytes in the Rat Intestinal Villi in Culture WAKOH TSUCHIYA’ Department
of Physiology,
Received February
AND
Faculty of Medicine,
YASUNOBU
OKADA
Kyoto University,
Kyoto 606, Japan
3, 1982; accepted in revised form June 28, 1982
Fragmented villi of the small intestine isolated from newborn rats were maintained in culture for periods of up to 4 weeks. In culture, floating globular villi and adherent villi were distinguished. Intracellular recordings were made from both types of villi. The membrane potential was highest (about -70 mV) in the cells at the tip of the villus and was lowest (about -18 mV) in the cells near the crypt region, showing a continuous gradation according to the cell location. The membrane potential in cultured mature enterocytes was due to the contribution of the electrogenic Na+ pump as well as to the Ca*+-activated K+ conductance. Both the components were temperature and metabolic energy dependent. Based on these data, it is suggested that the Ca*’ transport mechanism and the electrogenic Na’ pump are involved in the process of cell differentiation or maturation of the intestinal epithelia. INTRODUCTION
The villi of the small intestine consist of a constantly renewed population of epithelial cells. The crypt region contains the mitotically active cells which migrate up to the villus and become progressively differentiated into the mature absorptive cells after the end of cell division (Leblond and Messier, 1958). The different,iation of the mitotically active crypt cells to absorptive villous cells is accompanied by changes in cell morphology (Palay and Karlin, 1959; van Dongen et al., 1976) and in various enzyme activities (Dahlqvist and Nordstrtim, 1966; Moog and Grey, 1966; NordstrGm et al., 1968, Webster and Harrison, 1969; Weiser 1973; Charney et al., 1974; de Both and Plaisier, 1974; Quill and Weiser, 1975; Gall et al., 1977; Raul et al., 1977; Pothier and Hugon, 1980). However, little is known about the membrane potential changes during cell differentiation along the villus-crypt axis. We have examined membrane potential changes associated with the cell maturation and the underlying ionic mechanisms in cultured villi. Some of these data have been published in a short report (Tsuchiya et al, 1980). MATERIALS
AND
METHODS
Tissue culture. Morphological maturation of the rat small intestine is known to be accomplished by 19 to 22 days of gestation (DeRitis et al., 1975; Madara et al., 1981). In the present study, newborn small intestines were employed for tissue culture. Wistar rats were decapitated immediately after birth before receiving co1 Present address: Department of Dermatology, Kyoto University, Kyoto 606, Japan.
Faculty of Medicine,
284 0012-1606/82/120284-07$02.00/O Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.
lostrum. The entire small intestine was then quickly resected. The luminal surface of the intestine was washed with a culture medium (Medium 199, Nissui) with a syringe, and the tissue (Fig. 1A) was minced into small pieces. The fragmented villi (Fig. 1B) thus obtained were placed in Falcon plastic dishes (60 X 15 mm), containing 5 ml of the culture medium supplemented with 20% bovine serum, streptomycin (100 pg/ ml), and penicillin (63 pg/ml). The dishes were then kept in a humidified incubator equilibrated with 5% CO2 in air at 37°C. The culture medium was replaced every other day. Electrophysiology. The procedures used for intracellular recordings have been described in a previous paper (Okada et al., 1977a). KC1 (3 M)-filled glass microelectrodes, with a resistance of 15-30 MO were used. Their tip potentials were less than 5 mV. The electrophysiological experiments were performed under an inverted phase-contrast microscope (Chiyoda T-2). Unless otherwise noted, the experiments were performed in phosphate-buffered saline (PBS) which contains 5.4 mM Kt , 129.3 mM Na+, 1.8 mM Ca2+, 0.8 mM Mg’+, and 83 mM Cl- (pH = 7.3 f 0.1). Membrane potentials were measured through a high-input impedance preamplifier (WPI M701). Intracellular injection of Ca2’ ions was performed by a method described elsewhere (Okada et al., 1979). A suction micropipet was employed to stabilize a floating villus during the electrode penetration. Data were accepted only when a stable membrane potential was maintained for at least 1 min and when the extracellular potential level observed upon withdrawal of the electrode agreed with the initial zero level. The experiments were done usually at 37”C, but the bath
TSUCHIYA
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FIG. 1. Small intestinal epithelia of a newborn rat, Day 1, before culture. (A) Whole epithelium. Hematoxylin-eosin (X170). (B) Fragmented villi immediately aft.er mincing. Phase contrast (X340).
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were covered with a single layer of columnar epithelial cells and contained lamina propria (Fig. 2B). As the culture period increased, the volume of contents as well as the diameter of the floating villi gradually increased, suggesting transepithelial solute accumulation. While the floating villi swelled, the epithelial cells became flattened. No changes in the cell number occurred during 2 weeks of culture. No sign of cell mitosis was ever noticed in any histological sections of the floating villi. Thus, the cells constituting floating villi appear to be nonmitotic mature enterocytes. In contrast, from the villi adhered to the plastic substrate, an increasing number of epithelial cells migrated and formed a growing monolayer (Fig. 3). A few mitotic cells were often found in the monolayer, but never in the upper region (free end) of adherent villi. Thus, the cells in the monolayer appear to correspond to undifferentiated crypt enterocytes, since cell proliferation is known to be confined exclusively to the crypt zone in small intestinal epithelia (Leblond and Messier, 1958). Both types of fragmented villi were viable in culture for 4 weeks, but morphological studies revealed some sign of deterioration as evidenced by cell desquamation after 2 weeks of culture. Some epithelial cells in the monolayer of the adherent villi were viable for over 1 month, but were progressively overwhelmed by increasing fibroblastic cells. Stable membrane potentials were recorded from epithelial cells in the two types of villi. All the cells examined were assumed to be enterocytes, since the epi-
temperature was reduced to 56°C by circulating icecold water, if necessary. Ethanol was used as vehicle for verapamil (a gift from Eisai Co., Tokyo). The addition of ethanol did not affect membrane potentials at least in the doses used. After applying any drugs, the cells were washed with the control PBS, and the recovery of membrane potentials .to the original level was always confirmed. Morphology. Studies with a phase-contrast microscope (Nikon Optiphoto NT) were made, in a living state, on the tissue culture dishes. Tissues fixed with Bouin’s fluid were studied after hematoxylin-eosin staining. RESULTS
Two types of cultured villi. After 2 days in culture, two distinct types of the fragmented villi were discernible. The first was the globule-type floating villi (Fig. 2A), 50 to 500 pm in diameter, which were formed as a result of the open-end closure. The second was the adherent villi (Fig. 3), which adhered to the bottom of the culture dish with a central mounded region. The floating villi
FIG. 2. Floating
contrast
globule-type villi cultured for 2 days. (A) Phase (X425). (B) Hematoxylin-eosin (X300).
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thelium of the newborn rat small intestine contains only a few goblet cells (Fig. 1A). The magnitude of the membrane potentials measured in the adherent villi after 2 to 9 days of culture varied, depending upon the cell location, as shown in Fig. 4. The membrane potentials were highest (approximately -70 mV) at the top of the adherent villi, while the cells located near the base showed lower membrane potentials; the monolayer epithelial cells directly attached to the substrate were characterized by the lowest membrane potential (-15 to -20 mV). The addition of glycine (20 r&I) rapidly induced a significant depolarization (about 20 mV) in the cells at the villous tip, but this response was never observed in the monolayer cells (data not shown). Such a glycine-evoked depolarization has been reported to be associated with an active transport of the amino acid by mature absorbing enterocytes in the rat small intestine (Okada et al., 1977b; Okada, 1979). Thus, it can be concluded that the absorbing villus cells have higher membrane potentials than the undifferentiated crypt cells in the adherent villi. The magnitude of the mem-
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FIG. 4. Membrane potential profiles recorded in an adherent villus after 4 days of culture (0) and in a floating villus after 2 days of culture (A). Both types of cultured villi are schematically drawn. Arrows show the direction of successive recordings. The same profiles were obtained even when the direction of punctures was reversed.
FIG. 3. Adherent villi. Phase contrast. (X140). (B) Cultured for 7 days (X280).
(A) Cultured
for 3 days
brane potentials measured in the floating vilii after 2 days of culture was also dependent on the cell location as seen in Fig. 4. The membrane potential profiles in the floating villi exhibited a continuous gradation, but the gradient was less marked than that in the adherent villi. The smaller the diameter of floating villi, the less deviated the membrane potentials from about -70 mV. This fact suggests that the floating villi mainly consist of mature absorbing enterocytes in the crest of the villi. Supporting this inference, glycine-evoked depolarization was found in almost all the cells in floating villi. Furthermore, the potentials of all the cells in the floating villi were similar (about -70 mV), regardless of cell location, when the culture period was prolonged (up to 2 weeks). Thus, the magnitude of the membrane potential apparently reflects the degree of cell differentiation in the cultured villi. Membrane properties of uillous cells in culture. The electrical membrane properties of mature villous enterocytes were examined, using the floating villi cultured for 3 to 6 days. The diameter of these villi was less than 150 pm. Figure 5A illustrates the effects of the external K+ concentration ([K+],) on the membrane potential of the villous enterocyte in floating villi. As [K+], was increased from 5.4 mM, the membrane became depolarized. These changes were approximately linear against
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I loo ~n+lo30 (m-M)
([K+] ,,) on the membrane potentials. (A) Membrane potentials obtained from the villous FIG. 5. Effects of the external K’ concentration cells in a floating villus cultured for 3 to 6 days. To cancel out the physical effect of the temperature, the data are presented as the reduced membrane potentials (-F/RT X membrane potentials). (0) Measured under the control condition (with 1.8 mM at 37°C). (a) Measured under the external Ca’+-free condition at 37°C. (0) Measured at 6°C with 1.8 mM Ca’+. (B) Membrane potentials obtained in adherent villi cultured for 3 to 5 days under the control condition. (0) Obtained from the villous tip cells. (0) Obtained from the monolayer crypt cells. To change the IK+L. . __.NaCl was replaced with equimolar amounts of KCl, or vice versa, in a PBS bath. Each point represents the mean value of 26 - 115 observations with the standard deviation of less than +6.9 mV
log [K’],, with a slope of about 50 mV/decade. However, a decrease of [K’10 from 5.4 to 1.4 r&L4 also caused a slight depolarization by several millivolts (Fig. 5A, open circles). Upon rapid cooling of the villi from 37 to 6”C, the cell was immediately depolarized. This change was readily reversible after rewarming, being accompanied by a slight transient hyperpolarization (up to about -75 to -80 mV). At low temperatures, the [K+lo dependency was markedly depressed (Fig. 5A, squares). Several metabolic inhibitors were applied to test whether the temperature-dependent component of the membrane potential is generated by an energy-consuming process. The application of NaF or iodoacetic acids (IAA), which are glycolysis inhibitors, depolarized the membrane within 15 min to almost the same level (Fig. 6) as that under the cooli.ng condition (Fig. 5A, squares). In contrast, KCN, one of the respiratory inhibitors, did not significantly affect the membrane potential (Fig. 6). Thus, the temperature dependence of the membrane potential appeared to be mediated by a metabolic energy mainly derived from glycolysis. The membrane potential at 37°C was also dependent on the extracellular Ca2+. The deprivation of external calcium depolarized the membrane by about 20 mV at normal [K+],, and the [K’10 dependency of the membrane potential was considerably suppressed (Fig. 5A, triangles). The application of EGTA to the external Ca’+-free solution did not cause a further depolarization (Fig. 6). Addition of calcium to the Ca2+-free solution readily hyperpolarized the membrane to the control
value. Sr2+ or Mn2+ (but not Mg2+) ions can be substituted for Ca2+ ions. Even in the absence of external Ca2+, the intracellular injection of Ca2+ restored the membrane potential (Fig. 6). The addition of Ba2+ ions, an inhibitor of Ca2+-activated K+ conductance (Porzig, 1977: Gorman and Hermann, 1979), rapidly abolished (60) I
ing Villi FIG. 6. Membrane potentials under a variety of con dit from the villous cells in floating villi cultured for 3 to 6 days. Data were collected within 30 min after adding Ba*+, verapamil, or EGTA, within 10 min after adding NaF or IAA, and within 20 min after adding KCN. Numbers in parentheses indicate the number of cells impaled. Vertical bars represent standard deviations. *, Significantly different from the control values at P < 0.05.
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the Ca2+-dependent component of the membrane potential (Fig. 6). Verapamil, a Ca2+ channel blocker (Kohlhardt et al., 1972), also abolished the Ca2+-dependent component (Fig. 6). The calcium deprivation did not affect the membrane potential at 6°C. Thus, it is concluded that the temperature-dependent component of the membrane potential of villous mature enterocytes is partly due to the Ca2+-activated K+ conductance (Lew and Ferreira, 1978; Meech, 1978) and that the intracellular Ca2+ ions responsible for the activation of K+ conductance are mainly transported from the outside of the cell, probably through Ca2+ channels. The membrane was transiently hyperpolarized after rewarming from 6 to 37°C (see above). The transient (for about 2-3 min) hyperpolarization became more pronounced (from -80 to -100 mV) upon rewarming after the cells were exposed to a K+-free PBS at 2°C. Under the latter condition, the cells would have been loaded with Na+ ions. Thus, the prominent hyperpolarization suggests the presence of an electrogenic Na+ pump in mature villous enterocytes in culture. Supporting this hypothesis, the application of ouabain depolarized the membrane down to about -50 mV within 10 min (Fig. 6); yet, ouabain did not affect the membrane potential at 6°C. Therefore, the electrogenic Na+ pump appears to be an additional component involved in the temperature-dependent membrane potential in villous cells. The properties of membrane potentials in mature villous cells were also examined in the adherent villi cultured for 2 to 9 days. The data obtained from the cells at the tip region of villi (Fig. 5B, open circles, and Fig. 7A) were essentially the same as those obtained from the mature cells in the floating villi (Figs. 5A and 6). Membrane potentials in crypt cells in culture. The membrane potentials of the monolayer epithelial cells in the adherent villi after 2 to 9 days of culture were about -18 mV (Fig. 7B). The potentials were less affected by the [K+], changes than those obtained in the villous cells, as shown in Fig. 5B (solid circles). Cooling the cells (at 5°C) depolarized the membrane only slightly (to about -14 mV), as did the addition of ouabain to the same level within 10 min (Fig. 7B). The degree of depolarization produced by cooling or ouabain (Fig. 7B) was, however, very scanty compared to that in the villous cells (Fig. 7A). In addition, the application of Ba2+ never affected the membrane potential profiles in the monolayer enterocytes in the adherent villi. Thus, it seems reasonable to conclude that the membrane potential in undifferentiated crypt cells lacks the Ca’+activated K+ conductance component and has a very small component of the electrogenic Na+ pump. Other undetermined factors might be involved in the difference of the ionic mechanisms underlying the membrane
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FIG. 7. Membrane potentials under a variety of conditions obtained from the villous cells (A) and the crypt cells (B) in adherent villi cultured for 2 to 9 days. Numbers in parentheses, vertical bars, and stars are the same as in Fig. 6.
potentials between the villous and the crypt cells because the membrane potential in villous cells at 5-6°C was still greater than that in crypt cells at 37°C (Figs. 5 and 7). DISCUSSION
There have been several attempts to establish cell lines derived from the small intestinal epithelial cells (Henle and Deinhardt, 1957; Lichtenberger et al., 1973; Quaroni et al., 1979) or to culture the isolated enterocytes (Raul et aE., 1978). Induction of differentiation of these cells to the mature absorbing cells seems difficult in vitro (Raul et al., 1978; Inui et al., 1980). It is possible that the intestinal cell differentiation in uiuo depends upon a complex set of different factors (Quaroni and May, 1980), including epithelial-mesenchymal interactions (Haffen et al., 1981). Thus, the organ culture of explanted intestinal epithelia may provide a favorable approach for the electrophysiological studies with respect to the intestinal differentiation along the villuscrypt axis. However, in the previous organ culture system (Browning and Trier, 1969), the explants could be maintained only for short periods (up to 24 to 48 hr) and were mobile, interfering with a stable penetration of microelectrodes, when the whole villi were exposed to electrolyte solutions. These difficulties could be avoided in the present study by culturing fragmented villi of newborn (Day 1) rat small intestine (see Materials and Methods). The cultured fragmented villi were classified into the adherent villi and globule-type floating villi. Cell mitosis
TSUCHIYA AND OKADA
Enterocyte
was found in the monolayer of the adherent villi, but it was never observed in the upper region of the adherent villi or in the floating villi. Glycine-evoked depolarization was found in the cells in the floating villi or the top region of the adherent villi, but the monolayer cells of the adherent villi lack’ed this response. Based on these results, it is concluded that the floating villi and the free end of the adherent villi consist of mature absorbing enterocytes, whereas the attached end of the adherent villi consist of undifferentiated crypt cells. The rat coionic crypt cells have been reported to be much more adherent to the substrate than the villous cells (Patnaik et al., 1981). Therefore, it is likely that the adherent villi are formed by the attachment of crypt cells around the cut end of the fragmented villi. On the other hand, if the cut end of the fragmented villi consists of villous cells, the villi would be incapable of attaching, thereby forming the floating villi. The membrane potential was highest in the enterocytes at the crest of a villus and lower in those closer to the crypt region in cultured villi (Fig. 4). This finding is in agreement with the observations on the isolated rabbit ileum (Hirschhorn and Frazier, 1973). Since amino acid-dependent membrane depolarizations, presumably associated with amino acid absorption (Okada, 1979), were observed in the villous cells with a high membrane potential, the potential gradient observed along a villus may reflect some events related to the differentiation of enterocytes to absorbing cells. The difference in membran’e potential was finely graded, depending upon cell location along the cultured villus. This coincides with gra.dual changes in morphological (van Dongen et al., 19:76) and biochemical properties (Nordstrom et al., 1968; Weiser, 1973; Raul et al., 1977; Pothier and Hugon, 1980) according to the cell location along a villus. The low membrane potential observed in the crypt cells cannot be attributed to the low K+ concentration gradient since the intracellular K+ concentration measured by the electron-probe microanalysis is higher in the crypt cells than in the villous cells (Cameron et al., 1979). The present study has shown that the membrane potentials of crypt enterocytes are less sensitive to temperature or cellular metabolic energy than those of villous cells. The temperatureor energy-dependent component of the membrane potential in mature villous cells in culture was found to be regulated by the Ca’+-activated K+ conductance as well as by the electrogenic Na+ pump. Since the Ca’+-activated K+ conductance in the villous cells depends on the operation of verapa.mil-sensitive Ca2+ channels, it is possible that the Ca2+-dependent component of the membrane potential might be related to the activity of Ca2+ transport in villous mature cells. In this connection, it is noteworthy that the distribution of calcium-
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binding proteins appears to have a gradient increasing from the crypt to the tip of the villus (Marche et al., 1980). Also, Na+-K+-ATPase activity (Charney et al., 1974) and ouabain-sensitive Na+ efflux (Gall et al., 1977) are known to be higher in the villus tip than in the crypt cells in rat small intestine. Thus, the electrogenic Na+ pump found in cultured villous cells may reflect the activity of Na+-K+-ATPase. A significant contribution of the electrogenic Nat pump to the membrane potential has been found in mature enterocytes in isolated rat small intestine (Okada et al., 1978). It is suggested that the development of the electrogenic Na+ pump activity and the appearance of the verapamil-sensitive Ca” channel or the Ca2+-activated K+ channel are related to cell differentiation or maturation in intestinal epithelia. The authors are indebted to Professor A. Inouye for his valuable advice and financial support of this study. We are grateful to Professor M. Kuno for critical reading of the manuscript. Thanks are also due to Dr. S. Ueda for his collaboration in a part of this culture work.
REFERENCES BROWNING, T. H., and TRIER, J. S. (1969). Organ culture of mucosal biopsies of human small intestine. J. Clin. Znuest. 48, 1423-1432. CAMERON, I. L., SMITH, N. K. R., and POOL, T. B. (1979). Element concentration changes in mitotically active and postmitotic enterocytes. An X-ray microanalysis study. J. Cell Biol. 80, 444-450. CHARNEY, A. N., GOTS, R. E., and GIANNELLA, R. A. (1974). (Na+Kf)-stimulated adenosinetriphosphatase in isolated intestinal villus tip and crypt cells. Biochim. Biophys. Acta 367, 265-270. DAHLQVIST, A., and NORDSTROM, C. (1966). The distribution of disaccharidase activities in the villi and crypts of the small-intestinal mucosa. Biochim. Biophys. Acta 113, 624-626. DE BOTH, N. J., and PLAISIER, H. (1974). The influence of changing cell kinetics on functional differentiation in the small intestine of the rat. A study of enzymes involved in carbohydrate metabolism. J. H&o&em. Cytochem. 22, 352-360. DERITIS, G., FALCHUK, Z. M., and TRIER, J. S. (1975). Differentiation and maturation of cultured fetal rat jejunum. Dew Biol. 45, 304317. GALL, D. G., CHAPMAN, D., KELLY, M., and HAMILTON, J. R. (1977). Nat transport in jejunal crypt cells. Gastroenterology 72, 452-456. GORMAN, A. L. F., and HERMANN, A. (1979). Internal effects of divalent cations on potassium permeability in molluscan neurones. J. Physiol. 296, 393-410. HAFFEN, K., KEDINGER, M., SIMON, P. M., and RAUL, F. (1981). Organogenetic potentialities of rat intestinal epithelioid cell culture. Differentiation 18, 97-103. HENLE, G., and DEINHARDT, F. (1957). The establishment of strains of human cells in tissue culture. J. Zmmunol. 79, 54-59. HIRSCHHORN, N., and FRAZIER, H. S. (1973). The electrical profile of stripped, isolated rabbit ileum. Hopkins Med. J. 132, 271-281. INUI, K., QUARONI, A., TILLOTSON, L. G., and ISSELBACHER, K. J. (1980). Amino acid and hexose transport by cultured crypt cells from rat small intestine. Amer. J. Physiol 239, C190-C196. KOHLHARDT, M., BAUER, B., KRAUSE, H., and FLECKENSTEIN, A. (1972). Differentiation of the transmembrane Na and Ca channels in mammalian cardiac fibres by the use of specific inhibitors. PfEiigers Arch. 335, 309-322.
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LEBLOND, C. P., and MESSIER, B. (1958). Renewal of chief cells and goblet cells in the small intestine as shown by radioautography after injection of thymidine-H3 into mice. Anat. Rec. 132, 247-259. LEW, V. L., and FERREIRA, H. G. (1978). Calcium transport and the properties of a calcium-activated potassium channel in red cell membranes. Curr. Top. Membr. Transp. 10, 217-277. LICHTENBERGER, L., MILLER, L. R., ERWIN, D. N., and JOHNSON, L. R. (1973). Effect of pentagastrin on adult rat duodenal cells in culture. Gastroenterology. 65, 242-251. MADARA, J. L., NEUTRA, M. R., and TRIER, J. S. (1981). Junctional complexes in fetal rat small intestine during morphogenesis. Deu. Biol. 86, 170-178. MARCHE, P., CASSIER, P., and MATHIEU, H. (1980). Intestinal calciumbinding protein. A protein indicator of enterocyte maturation associated with the terminal web. Cell Tiss. Res. 212, 63-72. MEECH, R. W. (1978). Calcium-dependent potassium activation in nervous tissues. Annu. Reu. Biophys. Bioeng. 7, 1-18. Moot, F., and GREY, R. D. (1966). Spatial and temporal differentiation of alkaline phosphatase on the intestinal villi of the mouse. J. Cell Biol. 32, Cl-C6. NORDSTROM, C., DAHLQVIST, A., and JOSEFSSON, J. (1968). Quantitative determination of enzymes in different parts of the villi and crypts of rat small intestine. Comparison of alkaline phosphatase, disaccharidases and dipeptidase. J. H&o&em. Cytochem. 15, 713721.
OKADA, Y. (1979). Solute transport process in intestinal epithelial cells. Membr. Biochem. 2, 339-365. OKADA, Y., DOIDA, Y., ROY, G., TSUCHIYA, W., INOUYE, K., and INOUYE, A. (1977a). Oscillations of membrane potential in L cells. I. Basic Characteristics. J. Membr. Biol. 35, 319-335. OKADA, Y., IRIMAJIRI, A., TSUCHIYA, W., and INOUYE, A. (1978). Contribution of an electrogenic sodium pump to the membrane potential in the intestinal epithelial cell. Jpn. J. Physiol. 28, 511-525. OKADA, Y., TSUCHIYA, W., and INOUYE, A. (1979). Oscillations of membrane potential in L cells. IV. Role of intracellular Ca2+ in hyperpolarizing excitability. J. Membr. Biol. 47, 357-376. OKADA, Y., TSUCHIYA, W., IRIMAJIRI, A., and INOUYE, A. (1977b). Electrical properties and active solute transport in rat small intestine. I. Potential profile changes associated with sugar and amino acid transports. J. Membr. Biol. 31, 205-219.
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PALAY, S. L., and KARLIN, L. J. (1959). An electron microscopic study of the intestinal villus. I. The fasting animal. J. Biophys. Biochem. Cytol. 5, 363-371. PATNAIK. R., MAZUMDER, A., BHAGAVAN, B. S., and NAIR, P. P. (1981). Characterization of rat colonic epithelial cell populations. Cell Differen. 10, 147-156. PORZIG, H. (1977). Studies on the cation permeability of human red cell ghosts. J. Membr. Biol. 31, 317-349. POTHIER, P., and HUGON, J. S. (1980). Characterization of isolated villus and crypt cells from the small intestine of the adult mouse. Cell Tissue Res. 2 11, 405-418. QUARONI, A., and MAY, R. J. (1980). Establishment and characterization of intestinal epithelial cell cultures. Methods Cell Biol. 21B, 403-427.
QUARONI, A., WANDS, J., TRELSTAD, R. L., and ISSELBACHER, K. J. (1979). Epithelioid cell cultures from rat small intestine. Characterization by morphologic and immunologic criteria. J. Cell Biol. 80, 248-265.
QUILL, H., and WEISER, M. M. (1975). Adenylate and guanylate cyclase activities and cellular differentiation in rat small intestine. Gastroenterology 69, 470-478. RAUL, F., KEDINGER, M., SIMON, P., GRENIER, J., and HAFFEN, K. (1978). Behaviour of isolated rat intestinal cells maintained in suspension or monolayer cultures. Biol. Cell 33, 163-168. RAUL, F., SIMON, P., KEDINGER, M., and HAFFEN, K. (1977). Intestinal enzymes activities in isolated villus and crypt cells during postnatal development of the rat. Cell Tiss. Res. 176, 167-178. TSUCHIYA, W., OKADA, Y., and INOUYE, A. (1980). Membrane potential measurements in cultured intestinal villi. Membr. Biochem. 3, 147-153. VAN DONGEN, J. M., VISSER, W. J., DAEMS, W. T., and GOLJAARD, H. (1976). The relation between cell proliferation, differentiation and ultrastructural development in rat intestinal epithelium. Cell Tiss. Res. 174, 183-199. WEBSTER, H. L., and HARRISON, D. D. (1969). Enzymic activities during the transformation of crypt to columnar intestinal cells. Exp. Cell Res. 56, 245-253. WEISER, M. M. (1973). Intestinal epithelial cell surface membrane glycoprotein synthesis. I. An indicator of cellular differentiation. J. Biol. Chem. 248, 2536-2541.