JOURNAL OF CELLULAR PHYSIOLOGY 142533-538 (1990)
Effect of K + Channel-Blockers on Sugar Uptake by Isolated Chicken Enterocges M.C. MONTERO, M.L. CALONCE, I. BOLUFER, AND A. ILUNDAlN* Department of Physiology, P h a r m x y Faculty of the University of Sevllle, 4 I0 I2 Sevilie, Spain The effects of Ba2+, quinine, verapamil, and CaLi - free saline solutions on sugar active transport have been investigated in isolated chicken enterocytes. Ba' +, quinine, and verapamil, which have been 5hown to inhibit &'+-activated K+ channels, decreased basal and theophylline-dependent 3-0-methylglucose (30-MG) accumulation. CaL+-free conditions reduced 3-0-MG uptake in theophylline-treated enterocytes, but it had no effect in control cells. O n the other hand, the uptake of a non-actively transported sugar, 2-deoxyglucose @DOG), by control or theophylline-treated cells wa5 not modified by the presence of verapamil or by Ca' +-removal. 3-0-MG increased ouabain-sensitive Na+-efflux, hut had no effect on either K + efflux or K + uptake. However, in the presence of Ba'+, K' uptake wa5 stimulated by 3-0-MG, and this increase was prevented by ouabain. All these findings are discussed in terms of the role that K + permeability may play in cellular homeostasis during sugar active transport.
We have recently reported (Montero e t al., 1988) that K + efflux from reloaded (86Rb)chicken enterocytes is inhibited by Bag+, quinine, and verapamil and unaffected by Ca2' removal from the incubation buffer. Previous studies (Ilundain et al., 1985) on the effects of Ca2+-free conditions or verapamil on sugar transport in intact rat small intestine suggested that intracellular Ca2+ might decrease sugar permeability across the basolateral cell boundary. Ba' has also been shown to affect intestinal transport function; thus, Ba2 induced intestinal secretion (Hardcastle et al., 1983), decreased transepithelial sugar transport (Alcalde and Ilundain, 1988) in intact rat ileum, and decreased sugar uptake by isolated rabbit enterocytes (Brown and Sepulveda, 1985b). In the current work, we present evidence suggesting that the effects of Ca2+-freesolutions, verapamil, and Ba2' on sugar transport could be explained in terms of changes in K' conductance and t h a t K + permeability may play a role in the energization of sugar accumulation in chicken enterocytes. +
+
MATERIALS AND METHODS Cell isolation and solutions Four-to-six week old Hubbard chickens have been used in the current study. Unless otherwise stated, the composition of the incubation buffer was (in mM): NaC1, 80; CaC12, 1; mannitol, 100; K2HP04,3; MgCl,, 1; Tris-HC1 (pH = 7.41, 20; and 1 mg/ml bovine serum albumin. The Ca"-free buffer was prepared by omitting calcium from the standard buffer. When the concentration of a modifier was greater than 10 mM, isotonicity was maintained by reducing the mannitol concentration. The isolation buffer contained standard buffer plus 1 mg/ml hyaluronidase. Cells were isolated following the method described 0 1990 WILEY-LISS, INC
by Kimmich (1975). Briefly, the animals were decapitated and a section of the mid-intestine of about 25 cm was removed. The tissue was rinsed clean with ice-cold Tris-HC1 buffered solution, then everted and opened longitudinally. Pieces of 3-4 cm were then transferred to isolation buffer and incubated a t 37°C in a shaking water bath (70 cyclesimin) for 30 min. After the incubation, the cells were separated by gentle shaking and washed twice with standard buffer without hyaluronidase, and resuspended in standard buffer. Cell viability, determined by the trypan blue technique (Girardi et al., 19561, was 65%. All the modifiers were present in the incubation buffer from the start of the incubation period.
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K + uptake measurements K' uptake was measured a t 37"C, using cells a t a final concentration of 15-25 mg cell proteiniml. Incubations were started by adding 1 ml of cell suspension to 4 ml of buffer containing 0.25 pCiiml "Rb, as a tracer for K + (Brown and Sepulveda, 1985a; Petersen, 1986) and 0.1 pCiiml [14C]PEG4,000 a s a n extracellular space marker. The cell suspension and incubation buffer (both prewarmed at 37°C) were mixed in a 20 ml polyethylene beaker held in a thermostated bath and shaken at 70 cyclesimin to maintain adequate stirring and uniform unity of the cell suspension. Uptake was terminated by diluting 200 p1 cell suspension in 500 pl ice-cold buffer and the cells separated by centrifugation (lO,OOOg, 20 s) through a 250 p1 layer of the oil mixture di-n-butyl phtha1ate:dinonyl phthalate, 3:2 vlv. The cell pellets were lysed in 1 ml distilled water, and the Received March 6, 1989; accepted October 25, 1989.
"To whom reprint requestsicorrespondence should be addressed.
534
MONTERO ET AL
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Fig. 1. Effects of verapamil and Ca2’-free conditions on 3-0-MG uptake by untreated and theophylline-treated chicken enterocytes. 3-0-MG uptake was measured in the absence ( 0 , 0 ) and in the presence (A,0) of 7 mM theophylline. A Absence (filled symbols) or pres-
Time ( m i n ) ence (open symbols) of 0.2 mM verapamil. B: In the presence (filled symbols) or absence (open symbols) of 1 mM Ca’ ’ . Results are the means ? S.E. of five experiments.
Na efflux measurements The isolated cells were preincubated in a medium containing 3 pCi 22Na+during 30 min in the presence of ouabain (200 pM) in order to improve the Na’ loading. Then, the cells were washed twice in ice-cold buffer without isotope and ouabain and resuspended in buffer to a final concentration of 20-30 mg cell protein/ml. The rate of 22Na+ loss from the cells was then meaSugar uptake measurements For these experiments, the incubation buffer con- sured by diluting 0.5 ml of the preloaded cell suspentained either 3-0-MG (1 mM and 0.2 pCiiml [l4C]3- sion into 19.5 ml of tracer free-buffer (37°C). Aliquots 0-MG) or 2-DOG (1mM and 0.02 pCiiml [14C]2-DOG) (500 pl) were taken a t the start of the incubation, for and 0.1 pCi/ml 13H]PEG 4,000 as a n extracellular the estimation of total initial radiolabeled sodium, and space marker. After separating the cells by centrifuga- a t 2, 4, 8, and 12 min. The cells were pelleted through tion through the oil layer a s indicated above, the cell the oil mixture layer a s indicated above, and the isopellets were lysed in 200 p.1 perchloric acid (3%)and tope in the supernatant and in the pellet was counted counted by liquid scintillation. 3-0-MG was also used by liquid scintillation. Na efflux was calculated as to determine cell volume. This was done by measuring indicated for “Rb efflux. the equilibrium uptake (30 min) of 3-0-MG in the presStatistics ence of 0.1 mM phlorizin. Results are expressed as mean ? S.E. Statistical significance was evaluated by the two-tailed Student’s tK + efflux measurements test for unpaired variates. Cell suspension (40-60 mg cell proteiniml) was first Materials preloaded by incubation at 37°C in a shaking water VeraPamil, quinine, 3-0-MG, 2-DOG, and theophylbath for 30 min in the presence of“Rb (12-14 pCi/ml), The cells were then washed twice in radiosotope-free, line were Obtained fromSigma, St. Louis, MO; hyaluronice-co]d buffer and resuspended in buffer to a final con- idase from Merck, Darmstadt, FRG; and “Rb, ‘%a+, centration of 20-30 mg cell proteiniml. The rate of [‘4C13-0-MG, 14C-PEG, and [l4C12-DOG from Amer86Rb loss from the cells was then measured by diluting sham International PLC. All reagents were of analytic 0.5 ml of the preloaded cell suspension into 4.5 ml of grade. radioisotope-free buffer and kept at 37°C. Aliquots (200 RESULTS pl) were taken a t 0 , 2 , 4 , 8 , and 12 min, and the “Rb in Effect of Ca2+-free conditions, verapamil, Ba2+, the cell pellet was estimated as indicated above. K + and quinine on sugar accumulation efflux was calculated as percent of “Rb remaining in The time course of 1 mM 3-0-MG uptake by isolated the cells and expressed a s a n apparent efflux rate coefficient. Throughout this paper, this efflux rate is re- chicken enterocytes, under different experimental conferred as “K efflux” and has the units of min-I. ditions, is shown in Figures 1 and 2. Since cell volume content of @Rbin the incubation media and solubilized pellets was estimated b measuring its Cerenkov radiation. The amount of 8Rb uptake was calculated taking into account the trapped extracellular volume estimated from the amount of [14C]PEG4,000 associated with the pellet.
+
+
+
535 0
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f
a
60.
L
A.
L 0
n
r) c
0
E
Time ( m i d Fig. 2. Effects of Ba2+ and quinine on 3-0-MC uptake by untreated and theophylline-treated chicken enterocytes. 3-0-MG uptake was measured in the absence ( 0 , 0)and in the presence (A,A)of 7 mM
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represents 3 t 0.11 (n = 25 experiments) ~ 1 3 - 0 - M G spaceimg cell protein, a t the steady-state, the intrato-extracellular sugar concentration gradient established by control cells was 11.6 nmol/pl. Theophylline, by inhibiting the basolateral, Na -independent sugar transport system (Randles and Kimmich, 19781, prevents the accumulated sugar to leave the cell, and, hence, it will increase steady-state cell sugar accumulation. As can be seen in Figures 1 and 2, theophylline increases the sugar concentration gradient by a factor of 2.3, which agrees with previous reports (Randles and Kimmich, 1978; Moreto et al., 1984). VerapamilO.2 mM, Ba2+ 5 mM, and quinine 125 +M reduced the accumulation of 3-0-MG to about 74, 68, and 53% of the control value, respectively, whereas Ca2+ removal from the bathing solutions had no significant effect on sugar accumulation. All these modifiers decreased, although they did not abolish, the theophylline-dependent increase in sugar accumulation. The inhibition induced by verapamil (41%), Ba2+ (35%),or quinine (55%)was greater than that of Ca2+free conditions (32%). +
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Effect of Ca2+-freeconditions and verapamil on 2-DOG uptake It has been reported (Randles and Kimmich, 1978; Kimmich and Randles, 1979) that 2-DOG is a specific substrate for the Na +-independent,non-concentrative, facilitated sugar transport system and that its initial rate of uptake by chicken enterocytes is inhibited by theophylline (Randles and Kimmich, 1978; Moreto et al., 1984). Figure 3 shows that theophylline inhibited the uptake of 2-DOG by chicken enterocytes. However, neither basal 2-DOG uptake nor the effect of theophylline on 2-DOG uptake were modified by Ca2+-freeconditions or verapamil. Effect of theophylline on K + efflux As previously reported (Montero et al., 1988) the rate constant of K + efflux from preloaded ($'Rb)
Time (min) theophylline, and in the absence (filled symbols) or presence (open symbols) of 5 mM Ba2' (A), or 125 p , ~quinine (B). Results are the means i S.E. of four experiments.
chicken enterocytes has a mean value of 0.073 t 0.004 min-' (n = 5). The presence of 7 mM theophylline (see Fig. 4) in the bathing solution increased "Rb efflux. The rate constant of K + efflux in the presence oftheophylline was 0.115 k 0.003imin.
Effect of 3-0-MG on Na+ and K + efflux N a + efflux from preloaded (22Na+ chicken enterocytes was monitored in the absence and presence of 3-0-MG and in the absence or presence of 1 mM ouabain. Figure 5 shows that 20 mM 3-0-MG increases the rate constant of N a + efflux from its basal level of 0.127 ? 0.004 to 0.224 ? O.O07/min. In the presence of 1 mM ouabain, the rate constant of N a t efflux was 0.078 t 0.002/min, and it was not significantly modified by 30-MG. Thus, ouabain prevented the sugar-induced increase in N a + efflux. Figure 4 shows that K ' efflux was not modified by the presence of 3-0-MG in the incubation buffer. Effect of 3-0-MG on K + uptake The uptake of K f ("Rb) by isolated chicken enterocytes in the presence and absence of modifiers is shown in Figure 6. As can be seen, 20 mM 3-0-MG or 5 mM Ba2+ has no si nificant effect on K + uptake. However, when both Bag+ and 3-0-MG were present in the incubation buffer, the steady-state K uptake was significantly increased. On the other hand, this increase in K f uptake was not observed in the presence of 1mM ouabain. +
DISCUSSION Two separated systems operating in series are involved in the active absorption of sugars across the intestinal epithelia. Sugars are first accumulated in the enterocytes by a Na -I -dependent, concentrative transport system localized a t the apical membrane. This transport system, which has been shown to be rheogenic, is driven by the cellular membrane poten-
536
MONTERO ET AL.
A
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2b Time
(s)
Fig. 3. Effects of verapamil and CaZ -free conditions on the initial rate of 2-DOG uptake by isolated chicken enterocytes. 2-DOG uptake was measured a t 20,40, and 60 min incubation periods, in the absence ( 0 , 0 ) and in the presence (A,A) of 7 mM theophylline. A Absence +
Y
.-
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60
Time (s) (filled symbols) or presence (open symbols) of 0.2 mM verapamil. B: In the presence (filled symbols) or absence (open symbols) of 1 mM Ca2 ' . Results are the means t- S.E. of four experiments.
ao
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4
Time
(Ittiti)
Fig. 4. Effect of theophylline or 3-0-MG on K ' efflux from preloaded "Rb chicken enterocytes. The percentage of "Rb content remaining in the cells is plotted on a logarithmic scale against time; 1009 is the initial radioactivity present in the samples a t 0 min. ( 0 ) 20 mM mannitol, (A)20 mM 3-0-MG, ( W ) 7 mM theophylline. Each point represents the mean value of four independent determinations.
tial as well as by the N a + chemical gradient (Kimmich, 1981). As a result, sugar active transport depolarizes the apical membrane potential and increases Na+ pump activity. K t permeability is involved in the generation and maintenance of membrane potential, and the current view is that K' permeability may serve both for energization of organic solute transport and for protecting the cells from marked changes in K content during the transport of organic and inorganic solutes (Schultz, 1981). Sugar efflux across the basolateral boundary of en-
1
: a 2
4
l i m e (min) Fig. 5 . Effect of 3-0-MG on Na' efflux from preloaded (""a '1 chicken enterocytes. The percentage of "Na ' content remaining in the cells is plotted on a logarithmic scale against time. ( 0 ) 20 mM mannitol, (0) 20 mM 3-0-MG, (A)1mM ouabain, (A)1 mM ouabain + 20 mM 3-0-MG.
terocytes has been shown to take place via a Na+-independent, facilitated diffusion system that is inhibited by theophylline. Therefore, in the presence of theophylline, the cells are allowed to establish much greater sugar concentration gradients (Randles and Kimmich, 1978; Kimmich and Randles, 1979; Moreto et al., 1984). Previous work (Ilundain e t al., 1985) with intact rat
SUGAR UPTAKE AND K ' PERMEABILITY IN ENTEROCYTES
537
line reduced the initial rate of 2-DOG uptake and both Ca2+-free conditions and verapamil were without effect on 2-DOG uptake, both in the resence and absence of theophylline. Therefore, Cap' -free conditions amil were not interfering with the inhibitory theophylline on the passive sugar transport
140-
100-
ges in K + permeability could modify steadyar accumulation by modifying the electrical membrane potential and, hence, the driving force for active sugar uptake. The following observations suggest that Ca2'-free conditions and verapamil might affect steady-state sugar accumulation via a decrease in K' permeability:
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20
T i m e (min) Fig. 6. K ' uptake by isolated chicken enterocytes in the presence of 20 mM mannitol ( 0 , 0)or 20 mM 3-0-MG (A,A,and in the absence (filled symbols) or presence (open symbols) of 5 mM Ba"' . The lower part of the graph represents the uptake of K' in the presence of 1 mM ouabain: 20 mM mannitol, .( 0), 20 mM 3-0-MG (O), in the absence (filled symbols) or presence (open symbols) of 5 mM Ba'+.
1. We have previously found (Montero e t al., 1988) that verapamil, but not Ca2+-free conditions, decreased the rate constant of K' efflux from preloaded ("Rb) chicken enterocytes. Therefore, under basal conditions, only those modifiers that affect K 'permeability were capable of affecting sugar transport, i.e., verapamil. 2. Ca2+-freeconditions impaired sugar uptake only in the presence of theoDhvlline. and theoDhvlline raises the rate constant of Ki efflux. Since theoGhylline has been shown to increase mucosal Ca" -permeability (11undain and Naftalin, 1979a) and intracellular free Ca2+ concentration (Ilundain and Naftalin, 197913) in intact intestine, Ca2 -free conditions might affect sugar transport by preventing the theophylline-dependent increase in K permeability. These findings also suggest that theophylline may increase steady-state sugar accumulation by two mechanisms: i) by inhibiting sugar efflux, a s already reported (Randles and Kimmich, 1978; Moreto et al., 1983), and ii) by increasing K' efflux and, hence, the electrical driving force for active sugar uptake. +
+
ileum showed that theophylline increased tissue sugar concentration and reduced mucosal to serosal sugar fluxes. Ca2'-free solutions and the Ca" -channel blocker verapamil (Rosenberger and Triggle, 1978; Guerrero and Martin, 1984) decreased tissue sugar accumulation and also significantly reduced the theophylline effects on sugar transport. These findings led to the suggestion that theophylline may act by increasing intracellular free Ca2+ concentration, which, in turn, might reduce sugar permeability a t the basolateral border of the enterocytes. In the present work, we present evidence consistent with the view that the effects of the Ca2+-free buffer and verapamil on intestinal sugar transport may be due to changes in K' permeability and that as shown for rabbit enterocytes (Browm and Sepulveda, 1985b) K permeability might play a role in the energization of continued active sugar transport in chicken enterocytes. As previously reported (Randles and Kimmich, 1978; Moreto et al., 1984), theophylline increased the steadystate 3-0-MG accumulation. Removal of Ca2+ from the bathing buffer or the presence of verapamil significantly reduced the theophylline-dependent increase in sugar accumulation. Verapamil, but not Ca2'-free conditions, also decreased sugar uptake in the absence of theophylline. These findings could suggest that the inhibitory action of theophylline on sugar transport is mediated by a n increase in Ca2' entry into the cells. However, the results obtained when 2-DOG uptake (a specific substrate for the Na+ -independent, facilitateddiffusion, sugar transport system) was studied did not support this view. Thus, as previously reported (Randles and Kimmich, 1978; Moreto et al., 1984),theophyl+
It has been reported that in rat ileum Ba2+ increases tissue sugar accumulation (Alcalde and Ilundain, 1988). However, as previously found in rabbit enterocytes (Brown and Sepulveda, 1985b), Ba2 ' decreases sugar accumulation in chicken enterocytes (see Fig. 2). Since Ba2' has been shown to decrease K + efflux in these cells (Montero et al., 19881, its effect on sugar uptake could be also explained in terms of a decreased driving force for sugar accumulation. The differences in results between intact intestine and isolated enterocytes could he due to the presence of muscularis mucosae in the intact tissue preparation. Thus, Ba2 causes smooth muscle contraction (Antonio et al., 1973), and a n increase in smooth muscle tone, which would lead to decreased permeability of the non-epithelial barriers, could explain the effects of Ba2+ on sugar transport observed in intact tissue. All the findings discussed so far suggest that K + permeability might play a role in the energization of continued active sugar accumulation in chicken enterocytes. Further evidence for this role of K' permeability is provided by the results obtained in the presence of quinine. Quinine decreased K t efflux from chicken enterocytes (Montero et al., 1988) and decreased sugar uptake by these cells, both in the presence and absence of theophylline. The current work also shows that although ouabain+
538
MONTERO ET AL.
sensitive Na’ efflux was increased by 3-0-MG, indicating that the Na+ pump has been stimulated, K’ efflux was not modified. However, when K + uptake was monitored, the results provided indirect evidence for a n increase in K c efflux associated with a n increase in pump activity. Thus, when K’ exit was inhibited by Ba2+,a sugar-dependent increase in K accumulation was observed, and this increase was prevented by 1 mM ouabain. The lack of effect of 30-MG on net K’ uptake in the absence of Ba2+ suggests that the extra K + entry, induced by the sugardependent activation of the pump, may be compensated by a n increase in K’ efflux. Brown and Sepulveda (1985b) have shown that actively transported organic solutes increased K t efflux from rabbit enterocytes. The reason for the differences between our results and those obtained in rabbit enterocytes on K + efflux could be that the changes in K t efflux, induced by the uptake of organic solutes, in our preparation are masked by the greater basal rates of K + efflux in chicken enterocytes (0.07 vs. 0.03imin). In summary, the present findings suggest that K’ permeability might play a role in energization and dissipation of the extra pump-mediated K + entry during active accumulation of sugar in isolated chicken enterocytes. They also suggest that it may not be correct to identify the intestine’s response to modifiers as exclusive to the enterocytes. They could also affect nonepithelial tissue, such as smooth muscle. An additional observation is that theophylline, besides inhibiting sugar efflux (Randles and Kimmich, 1978; Moreto et al., 19831, may increase sugar accumulation by increasing the electrical driving force for active sugar uptake, via a n increase in K’ efflux. +
ACKNOWLEDGMENTS The work was supported by a grant from the Spanish “Comision Interministerial de Ciencia y Tecnologia” PB86-0513.
LITERATURE CITED Alcalde, A.I., and Ilundain, A. (1988) The effect of BaCI, on intestinal sugar transport in the rat. Rev. Esp. Fisiol., 44(2):25-28. Antonio, A., Rocha, E., Silva, M., and Yashuda, Y. (1973) The tachy-
phylactic effect of barium on intestinal smooth muscle. Arch. Int. Pharmacodyn. Ther., 204.260-267. Brown, P.D., and Sepulveda, F.V. (1985a) A rabbit jejunal isolated enterocyte preparation suitable for transport studies. J. Physiol. (Land.),363:257-270. Brown, P.D., and Sepdlveda, F.V. (1985b) Potassium movements associated with amino acids and sugar transport in enterocytes isolated from rabbit jejunum. J. Physiol. (Land.),36.3.271-285. Girardi, A.J., McMichael, H., and Henle, W. (1956) The use of HeLa cells in suspension for the quantitative study of virus propagation. Virology, 2:532-544. Guerrero, J.R., and Martin, S.A. (1984) Verapamil: Full spectrum calcium channel blocking agent: An overview. Med. Res. Rev., 4: 87-109. Hardcastle, J., Hardcastle, P.T., and Noble, J.M. (1983) The effect of barium chloride on intestinal secretion in the rat. J. Physiol. (Lond.),344:69-80. Ilundain, A., and Naftalin, R.J. (1979a) The role of calcium in theophylline-induced intestinal secretion. J. Physiol, (Land.),296:101102p. Ilundain, A., and Naftalin, R.J. (1979b) Role of calcium-dependent regulator in intestinal secretion. Nature, 279.446-448. Ilundain, A., Alcalde, A.I., Barcina, Y., and Larralde, J. (1985) Calcium-dependence of sugar transport in rat small intestine. Biochim. Biophys. Acta, 818.67-72. Kimmich, G.A. (1975) Preparation and characterization of isolated intestinal epithelial cells and their use in studying intestinal transport. In: Methods in Membrane Biology. E. Korth, ed. Plenum Press, New York, vol. IV, pp. 51-115. Kimmich, G.A. (1981) Intestinal absorption of sugars. In: Physiology of the Gastrointestinal Tract.” L.R. Johnson, ed. Raven Press, New York, chap. 41, pp. 1035-1061. Kimmich, G.A., and Randles, J. (1979) Energetics of sugar transport by isolated intestinal epithelial cells: effects of cytochalasin B. Am. J . Physiol., 6(1):C56-C63. Montero, M.C., Bolufer, J., and Ilundain, A. (1988) Potassium transport in epithelial cells isolated from small intestine of the chicken. Pflugers Arch., 412t422-426. Moreto, M., Planas, J.M., De Gabriel, C., and Santos, F.J. (1984) Involvement of cellular cyclic AMP in theophylline-induced sugar accumulation in chicken intestinal epithelial cells. Biochim. Biophys. Acta, 771.68-73. Petersen, O.H. (1986) Calcium-activated potassium channels and fluid secretion in exocrine glands. Am. J. Physiol., 251 :Gl-G13. Randles, J., and Kimmich, G.A. (1978) Effects of phloretin and theophylline on 3-0-methylglucose transport by intestinal epithelial cells. Am. J . Physiol., 3(2):C64-C72. Rosenberger, L., and niggle, D.J. (1978) Calcium, calcium translocation, and specific calcium antagonists. In: Calcium in Drug Action. G.B. Weiss, ed. Plenum Press, New York, chap. 1, pp, 3-31. Schultz, S.G. (1981) Homocellular regulatory mechamisms in sodiumtransporting epithelia: Avoidance of extinction by “flush-through,” Am. J. Physiol., 241:F579-F590.
Effect of K + Channel-Blockers on Sugar Uptake by Isolated Chicken Enterocges M.C. MONTERO, M.L. CALONCE, I. BOLUFER, AND A. ILUNDAlN* Department of Physiology, P h a r m x y Faculty of the University of Sevllle, 4 I0 I2 Sevilie, Spain The effects of Ba2+, quinine, verapamil, and CaLi - free saline solutions on sugar active transport have been investigated in isolated chicken enterocytes. Ba' +, quinine, and verapamil, which have been 5hown to inhibit &'+-activated K+ channels, decreased basal and theophylline-dependent 3-0-methylglucose (30-MG) accumulation. CaL+-free conditions reduced 3-0-MG uptake in theophylline-treated enterocytes, but it had no effect in control cells. O n the other hand, the uptake of a non-actively transported sugar, 2-deoxyglucose @DOG), by control or theophylline-treated cells wa5 not modified by the presence of verapamil or by Ca' +-removal. 3-0-MG increased ouabain-sensitive Na+-efflux, hut had no effect on either K + efflux or K + uptake. However, in the presence of Ba'+, K' uptake wa5 stimulated by 3-0-MG, and this increase was prevented by ouabain. All these findings are discussed in terms of the role that K + permeability may play in cellular homeostasis during sugar active transport.
We have recently reported (Montero e t al., 1988) that K + efflux from reloaded (86Rb)chicken enterocytes is inhibited by Bag+, quinine, and verapamil and unaffected by Ca2' removal from the incubation buffer. Previous studies (Ilundain et al., 1985) on the effects of Ca2+-free conditions or verapamil on sugar transport in intact rat small intestine suggested that intracellular Ca2+ might decrease sugar permeability across the basolateral cell boundary. Ba' has also been shown to affect intestinal transport function; thus, Ba2 induced intestinal secretion (Hardcastle et al., 1983), decreased transepithelial sugar transport (Alcalde and Ilundain, 1988) in intact rat ileum, and decreased sugar uptake by isolated rabbit enterocytes (Brown and Sepulveda, 1985b). In the current work, we present evidence suggesting that the effects of Ca2+-freesolutions, verapamil, and Ba2' on sugar transport could be explained in terms of changes in K' conductance and t h a t K + permeability may play a role in the energization of sugar accumulation in chicken enterocytes. +
+
MATERIALS AND METHODS Cell isolation and solutions Four-to-six week old Hubbard chickens have been used in the current study. Unless otherwise stated, the composition of the incubation buffer was (in mM): NaC1, 80; CaC12, 1; mannitol, 100; K2HP04,3; MgCl,, 1; Tris-HC1 (pH = 7.41, 20; and 1 mg/ml bovine serum albumin. The Ca"-free buffer was prepared by omitting calcium from the standard buffer. When the concentration of a modifier was greater than 10 mM, isotonicity was maintained by reducing the mannitol concentration. The isolation buffer contained standard buffer plus 1 mg/ml hyaluronidase. Cells were isolated following the method described 0 1990 WILEY-LISS, INC
by Kimmich (1975). Briefly, the animals were decapitated and a section of the mid-intestine of about 25 cm was removed. The tissue was rinsed clean with ice-cold Tris-HC1 buffered solution, then everted and opened longitudinally. Pieces of 3-4 cm were then transferred to isolation buffer and incubated a t 37°C in a shaking water bath (70 cyclesimin) for 30 min. After the incubation, the cells were separated by gentle shaking and washed twice with standard buffer without hyaluronidase, and resuspended in standard buffer. Cell viability, determined by the trypan blue technique (Girardi et al., 19561, was 65%. All the modifiers were present in the incubation buffer from the start of the incubation period.
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K + uptake measurements K' uptake was measured a t 37"C, using cells a t a final concentration of 15-25 mg cell proteiniml. Incubations were started by adding 1 ml of cell suspension to 4 ml of buffer containing 0.25 pCiiml "Rb, as a tracer for K + (Brown and Sepulveda, 1985a; Petersen, 1986) and 0.1 pCiiml [14C]PEG4,000 a s a n extracellular space marker. The cell suspension and incubation buffer (both prewarmed at 37°C) were mixed in a 20 ml polyethylene beaker held in a thermostated bath and shaken at 70 cyclesimin to maintain adequate stirring and uniform unity of the cell suspension. Uptake was terminated by diluting 200 p1 cell suspension in 500 pl ice-cold buffer and the cells separated by centrifugation (lO,OOOg, 20 s) through a 250 p1 layer of the oil mixture di-n-butyl phtha1ate:dinonyl phthalate, 3:2 vlv. The cell pellets were lysed in 1 ml distilled water, and the Received March 6, 1989; accepted October 25, 1989.
"To whom reprint requestsicorrespondence should be addressed.
534
MONTERO ET AL
I
I
A
0
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0
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15
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Fig. 1. Effects of verapamil and Ca2’-free conditions on 3-0-MG uptake by untreated and theophylline-treated chicken enterocytes. 3-0-MG uptake was measured in the absence ( 0 , 0 ) and in the presence (A,0) of 7 mM theophylline. A Absence (filled symbols) or pres-
Time ( m i n ) ence (open symbols) of 0.2 mM verapamil. B: In the presence (filled symbols) or absence (open symbols) of 1 mM Ca’ ’ . Results are the means ? S.E. of five experiments.
Na efflux measurements The isolated cells were preincubated in a medium containing 3 pCi 22Na+during 30 min in the presence of ouabain (200 pM) in order to improve the Na’ loading. Then, the cells were washed twice in ice-cold buffer without isotope and ouabain and resuspended in buffer to a final concentration of 20-30 mg cell protein/ml. The rate of 22Na+ loss from the cells was then meaSugar uptake measurements For these experiments, the incubation buffer con- sured by diluting 0.5 ml of the preloaded cell suspentained either 3-0-MG (1 mM and 0.2 pCiiml [l4C]3- sion into 19.5 ml of tracer free-buffer (37°C). Aliquots 0-MG) or 2-DOG (1mM and 0.02 pCiiml [14C]2-DOG) (500 pl) were taken a t the start of the incubation, for and 0.1 pCi/ml 13H]PEG 4,000 as a n extracellular the estimation of total initial radiolabeled sodium, and space marker. After separating the cells by centrifuga- a t 2, 4, 8, and 12 min. The cells were pelleted through tion through the oil layer a s indicated above, the cell the oil mixture layer a s indicated above, and the isopellets were lysed in 200 p.1 perchloric acid (3%)and tope in the supernatant and in the pellet was counted counted by liquid scintillation. 3-0-MG was also used by liquid scintillation. Na efflux was calculated as to determine cell volume. This was done by measuring indicated for “Rb efflux. the equilibrium uptake (30 min) of 3-0-MG in the presStatistics ence of 0.1 mM phlorizin. Results are expressed as mean ? S.E. Statistical significance was evaluated by the two-tailed Student’s tK + efflux measurements test for unpaired variates. Cell suspension (40-60 mg cell proteiniml) was first Materials preloaded by incubation at 37°C in a shaking water VeraPamil, quinine, 3-0-MG, 2-DOG, and theophylbath for 30 min in the presence of“Rb (12-14 pCi/ml), The cells were then washed twice in radiosotope-free, line were Obtained fromSigma, St. Louis, MO; hyaluronice-co]d buffer and resuspended in buffer to a final con- idase from Merck, Darmstadt, FRG; and “Rb, ‘%a+, centration of 20-30 mg cell proteiniml. The rate of [‘4C13-0-MG, 14C-PEG, and [l4C12-DOG from Amer86Rb loss from the cells was then measured by diluting sham International PLC. All reagents were of analytic 0.5 ml of the preloaded cell suspension into 4.5 ml of grade. radioisotope-free buffer and kept at 37°C. Aliquots (200 RESULTS pl) were taken a t 0 , 2 , 4 , 8 , and 12 min, and the “Rb in Effect of Ca2+-free conditions, verapamil, Ba2+, the cell pellet was estimated as indicated above. K + and quinine on sugar accumulation efflux was calculated as percent of “Rb remaining in The time course of 1 mM 3-0-MG uptake by isolated the cells and expressed a s a n apparent efflux rate coefficient. Throughout this paper, this efflux rate is re- chicken enterocytes, under different experimental conferred as “K efflux” and has the units of min-I. ditions, is shown in Figures 1 and 2. Since cell volume content of @Rbin the incubation media and solubilized pellets was estimated b measuring its Cerenkov radiation. The amount of 8Rb uptake was calculated taking into account the trapped extracellular volume estimated from the amount of [14C]PEG4,000 associated with the pellet.
+
+
+
535 0
A 80.
70-
f
a
60.
L
A.
L 0
n
r) c
0
E
Time ( m i d Fig. 2. Effects of Ba2+ and quinine on 3-0-MC uptake by untreated and theophylline-treated chicken enterocytes. 3-0-MG uptake was measured in the absence ( 0 , 0)and in the presence (A,A)of 7 mM
-
represents 3 t 0.11 (n = 25 experiments) ~ 1 3 - 0 - M G spaceimg cell protein, a t the steady-state, the intrato-extracellular sugar concentration gradient established by control cells was 11.6 nmol/pl. Theophylline, by inhibiting the basolateral, Na -independent sugar transport system (Randles and Kimmich, 19781, prevents the accumulated sugar to leave the cell, and, hence, it will increase steady-state cell sugar accumulation. As can be seen in Figures 1 and 2, theophylline increases the sugar concentration gradient by a factor of 2.3, which agrees with previous reports (Randles and Kimmich, 1978; Moreto et al., 1984). VerapamilO.2 mM, Ba2+ 5 mM, and quinine 125 +M reduced the accumulation of 3-0-MG to about 74, 68, and 53% of the control value, respectively, whereas Ca2+ removal from the bathing solutions had no significant effect on sugar accumulation. All these modifiers decreased, although they did not abolish, the theophylline-dependent increase in sugar accumulation. The inhibition induced by verapamil (41%), Ba2+ (35%),or quinine (55%)was greater than that of Ca2+free conditions (32%). +
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Effect of Ca2+-freeconditions and verapamil on 2-DOG uptake It has been reported (Randles and Kimmich, 1978; Kimmich and Randles, 1979) that 2-DOG is a specific substrate for the Na +-independent,non-concentrative, facilitated sugar transport system and that its initial rate of uptake by chicken enterocytes is inhibited by theophylline (Randles and Kimmich, 1978; Moreto et al., 1984). Figure 3 shows that theophylline inhibited the uptake of 2-DOG by chicken enterocytes. However, neither basal 2-DOG uptake nor the effect of theophylline on 2-DOG uptake were modified by Ca2+-freeconditions or verapamil. Effect of theophylline on K + efflux As previously reported (Montero et al., 1988) the rate constant of K + efflux from preloaded ($'Rb)
Time (min) theophylline, and in the absence (filled symbols) or presence (open symbols) of 5 mM Ba2' (A), or 125 p , ~quinine (B). Results are the means i S.E. of four experiments.
chicken enterocytes has a mean value of 0.073 t 0.004 min-' (n = 5). The presence of 7 mM theophylline (see Fig. 4) in the bathing solution increased "Rb efflux. The rate constant of K + efflux in the presence oftheophylline was 0.115 k 0.003imin.
Effect of 3-0-MG on Na+ and K + efflux N a + efflux from preloaded (22Na+ chicken enterocytes was monitored in the absence and presence of 3-0-MG and in the absence or presence of 1 mM ouabain. Figure 5 shows that 20 mM 3-0-MG increases the rate constant of N a + efflux from its basal level of 0.127 ? 0.004 to 0.224 ? O.O07/min. In the presence of 1 mM ouabain, the rate constant of N a t efflux was 0.078 t 0.002/min, and it was not significantly modified by 30-MG. Thus, ouabain prevented the sugar-induced increase in N a + efflux. Figure 4 shows that K ' efflux was not modified by the presence of 3-0-MG in the incubation buffer. Effect of 3-0-MG on K + uptake The uptake of K f ("Rb) by isolated chicken enterocytes in the presence and absence of modifiers is shown in Figure 6. As can be seen, 20 mM 3-0-MG or 5 mM Ba2+ has no si nificant effect on K + uptake. However, when both Bag+ and 3-0-MG were present in the incubation buffer, the steady-state K uptake was significantly increased. On the other hand, this increase in K f uptake was not observed in the presence of 1mM ouabain. +
DISCUSSION Two separated systems operating in series are involved in the active absorption of sugars across the intestinal epithelia. Sugars are first accumulated in the enterocytes by a Na -I -dependent, concentrative transport system localized a t the apical membrane. This transport system, which has been shown to be rheogenic, is driven by the cellular membrane poten-
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MONTERO ET AL.
A
€3
2b Time
(s)
Fig. 3. Effects of verapamil and CaZ -free conditions on the initial rate of 2-DOG uptake by isolated chicken enterocytes. 2-DOG uptake was measured a t 20,40, and 60 min incubation periods, in the absence ( 0 , 0 ) and in the presence (A,A) of 7 mM theophylline. A Absence +
Y
.-
40
60
Time (s) (filled symbols) or presence (open symbols) of 0.2 mM verapamil. B: In the presence (filled symbols) or absence (open symbols) of 1 mM Ca2 ' . Results are the means t- S.E. of four experiments.
ao
\
I
\
2
8
4
Time
(Ittiti)
Fig. 4. Effect of theophylline or 3-0-MG on K ' efflux from preloaded "Rb chicken enterocytes. The percentage of "Rb content remaining in the cells is plotted on a logarithmic scale against time; 1009 is the initial radioactivity present in the samples a t 0 min. ( 0 ) 20 mM mannitol, (A)20 mM 3-0-MG, ( W ) 7 mM theophylline. Each point represents the mean value of four independent determinations.
tial as well as by the N a + chemical gradient (Kimmich, 1981). As a result, sugar active transport depolarizes the apical membrane potential and increases Na+ pump activity. K t permeability is involved in the generation and maintenance of membrane potential, and the current view is that K' permeability may serve both for energization of organic solute transport and for protecting the cells from marked changes in K content during the transport of organic and inorganic solutes (Schultz, 1981). Sugar efflux across the basolateral boundary of en-
1
: a 2
4
l i m e (min) Fig. 5 . Effect of 3-0-MG on Na' efflux from preloaded (""a '1 chicken enterocytes. The percentage of "Na ' content remaining in the cells is plotted on a logarithmic scale against time. ( 0 ) 20 mM mannitol, (0) 20 mM 3-0-MG, (A)1mM ouabain, (A)1 mM ouabain + 20 mM 3-0-MG.
terocytes has been shown to take place via a Na+-independent, facilitated diffusion system that is inhibited by theophylline. Therefore, in the presence of theophylline, the cells are allowed to establish much greater sugar concentration gradients (Randles and Kimmich, 1978; Kimmich and Randles, 1979; Moreto et al., 1984). Previous work (Ilundain e t al., 1985) with intact rat
SUGAR UPTAKE AND K ' PERMEABILITY IN ENTEROCYTES
537
line reduced the initial rate of 2-DOG uptake and both Ca2+-free conditions and verapamil were without effect on 2-DOG uptake, both in the resence and absence of theophylline. Therefore, Cap' -free conditions amil were not interfering with the inhibitory theophylline on the passive sugar transport
140-
100-
ges in K + permeability could modify steadyar accumulation by modifying the electrical membrane potential and, hence, the driving force for active sugar uptake. The following observations suggest that Ca2'-free conditions and verapamil might affect steady-state sugar accumulation via a decrease in K' permeability:
60-
1 10
3o
20
T i m e (min) Fig. 6. K ' uptake by isolated chicken enterocytes in the presence of 20 mM mannitol ( 0 , 0)or 20 mM 3-0-MG (A,A,and in the absence (filled symbols) or presence (open symbols) of 5 mM Ba"' . The lower part of the graph represents the uptake of K' in the presence of 1 mM ouabain: 20 mM mannitol, .( 0), 20 mM 3-0-MG (O), in the absence (filled symbols) or presence (open symbols) of 5 mM Ba'+.
1. We have previously found (Montero e t al., 1988) that verapamil, but not Ca2+-free conditions, decreased the rate constant of K' efflux from preloaded ("Rb) chicken enterocytes. Therefore, under basal conditions, only those modifiers that affect K 'permeability were capable of affecting sugar transport, i.e., verapamil. 2. Ca2+-freeconditions impaired sugar uptake only in the presence of theoDhvlline. and theoDhvlline raises the rate constant of Ki efflux. Since theoGhylline has been shown to increase mucosal Ca" -permeability (11undain and Naftalin, 1979a) and intracellular free Ca2+ concentration (Ilundain and Naftalin, 197913) in intact intestine, Ca2 -free conditions might affect sugar transport by preventing the theophylline-dependent increase in K permeability. These findings also suggest that theophylline may increase steady-state sugar accumulation by two mechanisms: i) by inhibiting sugar efflux, a s already reported (Randles and Kimmich, 1978; Moreto et al., 1983), and ii) by increasing K' efflux and, hence, the electrical driving force for active sugar uptake. +
+
ileum showed that theophylline increased tissue sugar concentration and reduced mucosal to serosal sugar fluxes. Ca2'-free solutions and the Ca" -channel blocker verapamil (Rosenberger and Triggle, 1978; Guerrero and Martin, 1984) decreased tissue sugar accumulation and also significantly reduced the theophylline effects on sugar transport. These findings led to the suggestion that theophylline may act by increasing intracellular free Ca2+ concentration, which, in turn, might reduce sugar permeability a t the basolateral border of the enterocytes. In the present work, we present evidence consistent with the view that the effects of the Ca2+-free buffer and verapamil on intestinal sugar transport may be due to changes in K' permeability and that as shown for rabbit enterocytes (Browm and Sepulveda, 1985b) K permeability might play a role in the energization of continued active sugar transport in chicken enterocytes. As previously reported (Randles and Kimmich, 1978; Moreto et al., 1984), theophylline increased the steadystate 3-0-MG accumulation. Removal of Ca2+ from the bathing buffer or the presence of verapamil significantly reduced the theophylline-dependent increase in sugar accumulation. Verapamil, but not Ca2'-free conditions, also decreased sugar uptake in the absence of theophylline. These findings could suggest that the inhibitory action of theophylline on sugar transport is mediated by a n increase in Ca2' entry into the cells. However, the results obtained when 2-DOG uptake (a specific substrate for the Na+ -independent, facilitateddiffusion, sugar transport system) was studied did not support this view. Thus, as previously reported (Randles and Kimmich, 1978; Moreto et al., 1984),theophyl+
It has been reported that in rat ileum Ba2+ increases tissue sugar accumulation (Alcalde and Ilundain, 1988). However, as previously found in rabbit enterocytes (Brown and Sepulveda, 1985b), Ba2 ' decreases sugar accumulation in chicken enterocytes (see Fig. 2). Since Ba2' has been shown to decrease K + efflux in these cells (Montero et al., 19881, its effect on sugar uptake could be also explained in terms of a decreased driving force for sugar accumulation. The differences in results between intact intestine and isolated enterocytes could he due to the presence of muscularis mucosae in the intact tissue preparation. Thus, Ba2 causes smooth muscle contraction (Antonio et al., 1973), and a n increase in smooth muscle tone, which would lead to decreased permeability of the non-epithelial barriers, could explain the effects of Ba2+ on sugar transport observed in intact tissue. All the findings discussed so far suggest that K + permeability might play a role in the energization of continued active sugar accumulation in chicken enterocytes. Further evidence for this role of K' permeability is provided by the results obtained in the presence of quinine. Quinine decreased K t efflux from chicken enterocytes (Montero et al., 1988) and decreased sugar uptake by these cells, both in the presence and absence of theophylline. The current work also shows that although ouabain+
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MONTERO ET AL.
sensitive Na’ efflux was increased by 3-0-MG, indicating that the Na+ pump has been stimulated, K’ efflux was not modified. However, when K + uptake was monitored, the results provided indirect evidence for a n increase in K c efflux associated with a n increase in pump activity. Thus, when K’ exit was inhibited by Ba2+,a sugar-dependent increase in K accumulation was observed, and this increase was prevented by 1 mM ouabain. The lack of effect of 30-MG on net K’ uptake in the absence of Ba2+ suggests that the extra K + entry, induced by the sugardependent activation of the pump, may be compensated by a n increase in K’ efflux. Brown and Sepulveda (1985b) have shown that actively transported organic solutes increased K t efflux from rabbit enterocytes. The reason for the differences between our results and those obtained in rabbit enterocytes on K + efflux could be that the changes in K t efflux, induced by the uptake of organic solutes, in our preparation are masked by the greater basal rates of K + efflux in chicken enterocytes (0.07 vs. 0.03imin). In summary, the present findings suggest that K’ permeability might play a role in energization and dissipation of the extra pump-mediated K + entry during active accumulation of sugar in isolated chicken enterocytes. They also suggest that it may not be correct to identify the intestine’s response to modifiers as exclusive to the enterocytes. They could also affect nonepithelial tissue, such as smooth muscle. An additional observation is that theophylline, besides inhibiting sugar efflux (Randles and Kimmich, 1978; Moreto et al., 19831, may increase sugar accumulation by increasing the electrical driving force for active sugar uptake, via a n increase in K’ efflux. +
ACKNOWLEDGMENTS The work was supported by a grant from the Spanish “Comision Interministerial de Ciencia y Tecnologia” PB86-0513.
LITERATURE CITED Alcalde, A.I., and Ilundain, A. (1988) The effect of BaCI, on intestinal sugar transport in the rat. Rev. Esp. Fisiol., 44(2):25-28. Antonio, A., Rocha, E., Silva, M., and Yashuda, Y. (1973) The tachy-
phylactic effect of barium on intestinal smooth muscle. Arch. Int. Pharmacodyn. Ther., 204.260-267. Brown, P.D., and Sepulveda, F.V. (1985a) A rabbit jejunal isolated enterocyte preparation suitable for transport studies. J. Physiol. (Land.),363:257-270. Brown, P.D., and Sepdlveda, F.V. (1985b) Potassium movements associated with amino acids and sugar transport in enterocytes isolated from rabbit jejunum. J. Physiol. (Land.),36.3.271-285. Girardi, A.J., McMichael, H., and Henle, W. (1956) The use of HeLa cells in suspension for the quantitative study of virus propagation. Virology, 2:532-544. Guerrero, J.R., and Martin, S.A. (1984) Verapamil: Full spectrum calcium channel blocking agent: An overview. Med. Res. Rev., 4: 87-109. Hardcastle, J., Hardcastle, P.T., and Noble, J.M. (1983) The effect of barium chloride on intestinal secretion in the rat. J. Physiol. (Lond.),344:69-80. Ilundain, A., and Naftalin, R.J. (1979a) The role of calcium in theophylline-induced intestinal secretion. J. Physiol, (Land.),296:101102p. Ilundain, A., and Naftalin, R.J. (1979b) Role of calcium-dependent regulator in intestinal secretion. Nature, 279.446-448. Ilundain, A., Alcalde, A.I., Barcina, Y., and Larralde, J. (1985) Calcium-dependence of sugar transport in rat small intestine. Biochim. Biophys. Acta, 818.67-72. Kimmich, G.A. (1975) Preparation and characterization of isolated intestinal epithelial cells and their use in studying intestinal transport. In: Methods in Membrane Biology. E. Korth, ed. Plenum Press, New York, vol. IV, pp. 51-115. Kimmich, G.A. (1981) Intestinal absorption of sugars. In: Physiology of the Gastrointestinal Tract.” L.R. Johnson, ed. Raven Press, New York, chap. 41, pp. 1035-1061. Kimmich, G.A., and Randles, J. (1979) Energetics of sugar transport by isolated intestinal epithelial cells: effects of cytochalasin B. Am. J . Physiol., 6(1):C56-C63. Montero, M.C., Bolufer, J., and Ilundain, A. (1988) Potassium transport in epithelial cells isolated from small intestine of the chicken. Pflugers Arch., 412t422-426. Moreto, M., Planas, J.M., De Gabriel, C., and Santos, F.J. (1984) Involvement of cellular cyclic AMP in theophylline-induced sugar accumulation in chicken intestinal epithelial cells. Biochim. Biophys. Acta, 771.68-73. Petersen, O.H. (1986) Calcium-activated potassium channels and fluid secretion in exocrine glands. Am. J. Physiol., 251 :Gl-G13. Randles, J., and Kimmich, G.A. (1978) Effects of phloretin and theophylline on 3-0-methylglucose transport by intestinal epithelial cells. Am. J . Physiol., 3(2):C64-C72. Rosenberger, L., and niggle, D.J. (1978) Calcium, calcium translocation, and specific calcium antagonists. In: Calcium in Drug Action. G.B. Weiss, ed. Plenum Press, New York, chap. 1, pp, 3-31. Schultz, S.G. (1981) Homocellular regulatory mechamisms in sodiumtransporting epithelia: Avoidance of extinction by “flush-through,” Am. J. Physiol., 241:F579-F590.
