The influence of diphenylhydantoin on intestinal glucose absorption in the rat

European Journal o f Pharmacology 28 (1974) 310- 315 © North-Holland Publishing Company

THE INFLUENCE ABSORPTION

OF DIPHENYLHYDANTOIN

ON INTESTINAL

GLUCOSE

IN THE RAT

Hubertus VAN REES, Frederik A. DE W O L F F and Erik L. NOACH Department of Pharmacology, University o f Leiden, Wassenaarseweg62, Leiden, The Netherlands

Received 1 March 1974, accepted 28 May 1974 H. VAN REES, F.A. DE WOLFF and E.L. NOACH, The influence o f diphenylhydantoin on intestinal glucose absorption in the rat, European J. Pharmacol. 28 (1974) 310-315. The influence of diphenylhydantoin (DPH) on intestinal glucose absorption was studied by perfusing an isolated jejunal loop of a rat under urethane anaesthesia. DPH produced dose dependent enhancement of glucose absorption. Since DPH also stimulates the absorption of 3-O-methylglucose, but not fructose, the stimulatory effect was considered to be primarily on facilitated transport. The highest concentration of DPH in the perfusate (10-4 M) stimulated the appearance of sodium in an originally sodium-free perfusate, while ouabain (1.25 mg/kg body weight, i.v.) diminished this process. On the basis of these results an additional sodium pump, directed towards the lumen, is postulated in the brush border of the intestinal epithelial cell. The discrepancy between the results of in vivo and in vitro experiments concerning the influence of sodium ions on intestinal glucose absorption, and the possible physiological role of the proposed apical sodium pump are discussed. Glucose absorption

Diphenylhydantoin

Sodium pump

1. Introduction Glucose absorption from the intestine is thought to depend on a concentration gradient o f sodium ions over the brush border of intestinal epithelial cells (Crane, 1965; K o t y k and Jana~ek, 1970; Schultz and Curran, 1970a; Goldner, 1973). According to the sodium gradient hypothesis, it is essential that the sodium concentration inside the cell is lower than in the lumen. The low intracellular sodium concentration is maintained by a sodium pump, which is most probably situated in the latero-basal plasma membrane of the intestinal epithelial cell (Schulz and Curran, 1970b; Goldner, 1973). Thus facilitated transport through the brush border membrane is thought to be connected with the presence of a mobile carrier protein, which moves from the luminal to the intracellular side of the membrane only when the sodium as well as the glucose sites are occupied. The anti-epileptic activity of diphenylhydantoin (DPH) has been attributed to its ability to stimulate

the sodium pump in brain cells (Woodbury, 1955), which phenomenon has been confirmed in studies on muscle in vitro (Bihler and Shaw, 1971). If the sodium pump of the intestinal epithelial cells is also stimulated by DPH, the sodium gradient over the brush border will be augmented, or at least better maintained. In view of the Crane-model this would mean that the active glucose absorption must be stimulated by DPH. The present investigation is aimed at testing this hypothesis.

2. Material and methods Male rats, 200 g, were used from our Wistar-derived laboratory strain. They were maintained on a standard laboratory diet (Hope Farms). Food was withheld during the night before the experiments, but water was given ad libitum. The experimental technique is similar to that used by Barr and Riegelman (1970) and De Bruijne et al. (1971), and was suitable

H. Van Rees et al., Diphenylhydantoin and glucose absorption

for simultaneous perfusion of the jejunal loops of 2 rats. Under urethane anaesthesia (3.2 g/kg body weight, i.p.), a dose adequate for the whole period of perfusion, the abdomen was opened by a midline incision. A plastic cannula (4 5 mm) was inserted in the jejunum in a distal direction, directly adjacent to the plica duodeno-jejunalis, and tied firmly to keep it in place and to prevent leakage. 20 vascular arcades distal from the first cannula, a second cannula was inserted in a proximal direction and tied likewise. The proximal cannula was connected with the perfusion system by the inner tube of a double-walled tube. Through the outer tube water of 37°C was circulated to keep the perfusate in the inner tube on the desired temperature. The distal cannula emptied itself in a glass receptacle from which the perfusate was pumped by a finger pump (Biihler MP2) into the inner one of the double-walled tube, and thence into the proximal cannula. The temperature of the rats was monitored by an electrical thermometer (Electrolaboriet, Kopenhagen), and was kept at 37°C with a heating lamp. At the beginning of each experiment, the circulation system was rinsed with 25 ml of the control perfusate without sugar, which was discarded after one circulation. The perfusate was circulated at a rate of 10 ml/min, usually for 65 rain. At certain time intervals samples of 0.1 or 0.2 ml were taken from the end of the distal cannula for chemical assays. At the conclusion of the experiment the final volume of the perfusate was estimated with a calibrated cylinder. The rats were killed by cutting the abdominal aorta, and the jejunal loop between the two cannulae was removed from the mesentery. The isolated gut was gently pressed on filter paper to remove adhering fluid and mucus, and the wet weight was established. After drying to constant weight at IO0°C, the dry weight was determined. The perfusates were made isotonic with either sodium sulphate (100mM) or choline chloride (150 mM) and adjusted at 37°C to pH 7.4 with a saturated Tris solution. The initial concentration of the sugars (glucose, fructose or 3-O-methylglucose) was 5.6 mM. The concentration of the drugs in the perfusates is shown in the results. In the samples of the perfusate the glucose concentration was determined by a glucose oxidase-peroxi-

31 l

dase method, using o-dianisidine as the chromogen. The procedure was equal to the second step in the disaccharidase assay of Dahlquist (1968). The sodium concentration in the samples was determined after appropriate dilution by means of an Eppendorf Flame Photometer with a propane flame. The radioactivity of the samples containing D-fructose -~ 4C or 3-O-methyl-14C-D-glucose (both from New England Nuclear) was counted in a Packard Tri-Carb liquid scintillation spectrometer. To each sample of 0.1 ml of perfusate 10 ml of a scintillation fluid was added in a glass counting vial. This fluid consisted of tolue n e : T r i t o n X-100:water = 8 : 6 : 1, with 2,5-diphenyloxazole (PPO, 7 g/l) as the scintillating agent. The first five minutes of perfusion were considered to be an equilibrium period. All following determinations of the amount of sugar present in the perfusate were expressed as a percentage of the 5 min's value and calculated per 100 mg dry weight of the relevant intestinal loop, assuming that the sugar disappearance is a first order process. The sodium appearance is expressed as the total amount (in/~moles) present at each point of time, also on the basis of 100 mg dry weight of the relevant intestinal loop. In both calculations the small changes in volume owing to sampling and fluid absorption or secretion were taken into account. All chemicals were of analytical grade, generally purchased from either E. Merck, Darmstadt, K o c h Light Laboratories, or BDH Chemicals. Ouabain was obtained from Merck; diphenylhydantoin was a generous gift from Chemische Industrie 'Katwijk' N.V. For statistical significance an analysis of variance was applied; in the figures the mean value plus or minus the standard error of the mean value are shown.

3. Results 3.1. Disappearance o f sugars from the perfusate

Glucose disappearance from a perfusate made isotonic with 100 mM sodium sulphate is shown in table 1. DPH, 1.8 × 10-4 M, enhanced glucose disappearance (p < 0.05). Fig. 1 shows the disappearance of glucose from an originally sodium-free perfusate made isotonic with

312

H. Van Rees et al., Diphenylhydantoin and glucose absorption

Table 1 The influence of DPH on the glucose content of a sodium sulphate perfusate. "A" denotes the glucose concentration (~g/ml) in the perfusate, while "B" denotes the values expressed as percentage of the 5 min's value, calculated per 100 mg dry weight of the intestinal loop. DPH concentration

Perfusion time (min) 5

15

30

45

60

None (controls)

A B

802 +- 30* 100

588 -+ 38 91.8-+ 0.9

324 ± 14 78.5 -+ 1.9

175 -+41 66.4 -+ 3.3

95 -+ 27 57.4-+ 3.6

1.8 ×

At B'~

790 -+ 26 100

505 ± 33 88.5 -+ 2.1

268 +- 33 77.5 +- 1.8

131 +-25 63.9 -+ 2.0

49 -+ 16 51.1 -+ 2.2

10 - 4

M

* Means +-S.E.M. based on 6 determinations in each group. t Statistically significant difference when compared with the control series (analysis of variance: p < 0.05). 150 mM choline chloride in the presence of DPH in concentrations of 10-4, 10-s and 10-6 M. Here too, DPH enhanced glucose disappearance in a dose dependent manner. In a similar perfusate, digitoxin at about the maximal solubility (2/ag/ml) diminished glucose disappearance slightly but not significantly, although the total dose of digitoxin was increased by doubling the volume of the perfusate from 25 to 50 ml. Ouabain (1.25 mg/kg body weight, given i.v. 30 rain before the experiment did not affect rate of glucose disappearance. Increasing the dose of ouabain resulted in death. Probably low sensitivity of the rat to cardiac glycosides (Robinson, 1970), precluded an % GLUCOSE

•

-

"~'~-'~

....

OPH

H

(7)

10- 4 H (7)

effect like that reported by C ~ k y and Hara (1965) and Kimmich (1970) with in vitro systems of frog and chicken, respectively. To exclude the possibility that DPH enhances glucose disappearance by a stimulatory effect on glucose metabolism inside the epithelial cell, similar experiments were performed using fructose or 3-O-methylglucose (3-OMEG) instead of glucose. Fructose is metabolized like glucose, but it is not actively transported by the same mechanism as glucose (Gracey et al., 1972) and 3-OMEG is absorbed in the same way as glucose, but it is not metabolized (Schulz and Curran, 1970b). The stimulatory effect of DPH on the 3OMEG disappearance from the perfusate is shown in fig. 2. DPH had no effect on the fructose disappearance in similar experiments. % 3 - OHEO 100-

~

o - - o CONTROL (9)

" ~ . " " ' - - L ~ ~

..... oP.

0ol

"-.~ \ \ ~ \

f', - ? \

!t

,1 \

gS-

min.

Fig. 1. The dose dependent influence of DPH on the glucose content of a choline chloride perfusate. The glucose values are expressed as a percentage of the amount present at 5 min and calculated per 100 g dry weight of the intestinal loop. Mean values +- S.E.M. are plotted semilogarithmically against time, with number of experiments between parentheses.

5

15

25

35

45

55

S5 min.

Fig. 2. The influence of DPH (10 -4 M) on the 3-OMEG content of a choline chloride perfusate (of. legend to fig. 1) (p < 0.01).

H. Van Rees et aL, Diphenylhydantoin and glucose absorption ju motes Na+

YJT .,"A1/;i~"~iJ+o CONTROL (6) ~.---'~.DPH

/////,~/

50

313

stimulated, compared to controls (fig. 3), while lower concentrations of DPH (10 -s and 10-6 M) lacked effect. Ouabain (1.25 mg/kg body weight, given i.v. 30 min before the experiment) diminished sodium appearance (fig. 4). Thi; effect could also be demonstrated by using a perfusate-containing digitoxin at about the maximal solubility (2/ag/ml), although it was not reproducible in all experiments.

(6)

4. Discussion

/ 5

15

2S

35

45

56

65 rain.

Fig. 3. The influence o f DPH (10 -4 M) on the sodium appearance in a choline chloride perfusate (p < 0.001). The mean

values -+ S.E.M. denote the total amounts of sodium ions present in the perfusate, per 100 mg of dry weight of the intestinal loop. The number of experiments are given between parentheses.

3.2. Appearance o f sodium ions in the perfusate

Perfusing an intestinal loop in vivo with an originally sodium-free medium containing 5.6 mM glucose, resulted in sodium accumulation in the perfusate, the source of which is not known (Saltzman et al., 1972). If DPH was present in the perfusate in a concentration of 10-4 M, the sodium appearance was ,u motes Na+

150 150

~ /. , ~

-- OVABAIN(6)

rain..

Fig. 4. The influence of ouabain (1.25 mg/kg body weight, i.v., 30 min before the experiment) on the sodium appear-

ance in a choline chloride perfusate (ef. legend to fig. 3) (p < 0.01).

Although sodium-facilitated glucose transfer across the brush border of mucosal epithelial cells has been well documented (Crane, 1965; Schulz and Curran, 1970a; Goldner, 1973), most experiments in the literature have been performed in vitro. However, Levine et al. (1970) demonstrated that after only 5 min's incubation of everted sacs of rat intestine at 37°C the epithelium begins to be disrupted from the lamina propria, which process is progressive. Plattner et al. (1970) showed that changes in the ultrastructure of rat intestinal epithelium occurred during in vitro incubations. Therefore, for our experiments we chose an in vivo method, in which no morphological alterations had occurred in the intestine after one hour of perfusion (De Bruijne et al., 1971). There are only a few reports on glucose absorption in vivo (Cs~ky and Zollicoffer, 1960; Annegers, 1964, in rat and dog, respectively), demonstrating a clear inhibition of sugar uptake when sodium is omitted from the perfusate. Olsen and Ingelfinger (1968) noted only a slight inhibition of glucose disappearance when the ileum and jejunum of human subjects were perfused with a sodium-free solution containing glucose in a concentration of 3.4 mM or less. No effect of omitting sodium from the perfusate was noted when the glucose concentration was 6 mM or more. Also Saltzman et al. (1972) could not demonstrate the influence of sodium on glucose transfer across the brush border in perfusion experiments in human, rat and dog ileum. They concluded, however, that their results did not disprove the sodium gradient theory, because of the possibility of a 'microclimate' having a high sodium concentration adjacent to the brush border. Our results show that glucose may disappear at a rather fast rate even in an originally sodium-free perfusate (fig. 1, controls).

314

H. Van R ees et al., Diphenylhydantoin and glucose absorption

Under such conditions DPH is capable of accelerating glucose disappearance in a dose dependent fashion, as well as enhancing the appearance of sodium in the perfusate. For the following reasons the effect of DPH on glucose absorption cannot be solely dependent on the appearance of sodium in the lumen. First, the effect of DPH on glucose disappearance also occurs with a perfusate with a high sodium concentration, and second at lower DPH concentrations (10 -s and 10-6 M), the glucose effect is present while the sodium effect is not. We therefore conclude that the stimulatory effect o f DPH on glucose disappearance from the perfusate is principally the result of an influence on facilitated transfer. Probably (cf. Woodbury, 1955 and Bihler and Shaw, 1971) this effect results from stimulation of the sodium pump located at the latero-basal plasma membrane of the mucosal epithelial cell. However, no decreased glucose absorption was found, when cardiac glycosides were given and the sodium pump was expected to be inhited. A possible effect of DPH on intracellular glucose metabolism is excluded by the experiments using fructose and 3-OMEG. This is confirmed by experiments with freeze-clamped jejunum, in which the levels of energy-rich phosphates in the intestinal wall were shown to be unaffected after perfusion with or without DPH (De Wolff, 1973). The sodium appearance in the perfusate is considered to be at least partly an active process. For, if this phenomenon was passive, then inhibition of the latero-basal sodium pump by ouabain would lead to an augmented appearance of sodium in the lumen because of the resulting higher intrecellular sodium concentration and not to a decrease. Similarly a stimulatory effect of DPH on the sodium pump in the latero-basal plasma membranes would be expected to decrease the appearance of sodium in the lumen whereas an increase was found. We conclude, therefore, that in addition to the well-known sodium pump in the plasma membrane (the 'latero-basal' pump), an 'apical' pump directed towards the lumen, is present in intestinal epithelium. The existence of two different sodium pumps in jejunal epithelial cells is consistent with the findings of Quigley and Gotterer (1972), who found different kinetic parameters for brush border and plasma membrane Na'-K+-ATPase. Furthermore we found different threshold concentrations of DPH and of cardiac

glycosides for the effect on glucose disappearance from the perfusate and on sodium appearance therein. The physiological role of the proposed 'apical' pump may be a regulatory one. If the intraluminal sodium concentration is too low to result in a sodium gradient over the brush border, this pump may furnish sodium ions from an intracellular site to the outside of the brush border, after which facilitated transfer can take place. This hypothesis is consistent with the existence of a 'microclimate' with a high sodium concentration adjacent to the brush border, as proposed by Saltzman et al. (1972). Part of the extruded sodium ions will diffuse into the perfusate, leading to the observed sodium accumulation. In any case our hypothesis may supply a bridge between the conflicting results from in vitro and in vivo experiments. In vitro the medium at the mucosal side of an everted sac preparation can be kept sodium-free, while the 'apical' pump may be damaged owing to the deleterious effects on the intestinal epithelium, as described by Levine et al. (1970). In vivo the 'apical' pump may serve as a means to warrant glucose absorption from the gut content, even when the sodium ion concentration is very low.

Acknowledgements The technical assistance of Mrs. M. de Jongh-de Bois and Miss G.A.H. Duijts is gratefully acknowledged. Diphenylhydantoin was generously supplied by Chemische Industrie 'Katwijk' N.V. The investigations were financially supported by the Dutch Organization for Purely Scientific Research (Fungo-ZWO).

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Plattner, H., J. Klima, A. Mehnert and H. Berger, 1970, Quantitative and qualitative changes of structure and ultrastructure of intestinal epithlia during incubation in vitro, Virchows Arch. B Zellpath. 6, 337. Quigley, J.P. and G.S. Gotterer, 1972, A comparison of the NaK-ATPase activities found in isolatedbrush border and plasma membrane of the rat intestinal mucosa, Biochim. Biophys. Acta 255,107. Robinson, J.W.L., 1970, The difference in sensitivity to cardiac steroids of NaK-ATPase and amino acid transport in the intestinal mucosa of the rat and other species, J. Physiol. 206, 41. Saltzman, D.A., F.C. Rector and J.S. Fordtran, 1972, The role of intraluminal sodium in glucose absorption in vivo, J. Clin. Invest. 51,876. Schultz, S.G. and P.F. Curran, 1970a, Coupled transport of sodium and organic solutes, Physiol. Rev. 50, 637. Schultz, S.G. and P.F. Curran, 1970b, Stimulation of intestinal sodium absorption by sugars, Amer. J. Clin. Nutr. 23, 437. Wolff, F.A. de, 1973, Drug effects on intestinal epithelium, Thesis, Leiden University, p. 74. Woodbury, D.M., 1955, Effect of diphenylhydantoin on electrolytes and radiosodium turnover in brain and other tissues of normal, hyponatremic and postictal rats, J. Pharmacol. Exptl. Therap. 115, 74.