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Enzymatic inhibition of lysine transport across the small intestine In vivo
Camp. Biodem. Physioi. Vol.
@A, No. 1, pp. 61-64, 1988
03~-9629/88
$3.00 + 0.00
0 1988 Pergamon Journals Ltd
Printed in Great Britain
ENZYMATIC INHIBITION OF LYSINE TRANSPORT ACROSS THE SMALL INTESTINE IN VIVO CAMILLE F. NASSAR, GABI M. WAZZ, MICHEL G. NASSER and ZOHEIR M. HABBAL Departments
of Physiology and Laboratory Medicine, Faculty of Medicine, American University of Beirut, Beirut, Lebanon (Received 22 April 1987)
Abstract-l. Trypsin, at different concentrations, significantly inhibited lysine absorption (P < 0.05) in a dose-dependent pattern. 2. Maximum inhibition equivalent to 35% below control value was reached with lOpg/ml (100 BAEE units) trypsin with a non-reversible inhibitory effect. 3. Chymotrypsin at 10 pg/ml produced a significant decrease (P < 0.05) of lysine absorption although it did not exceed 5%. Perfusion of both enzymes did not show an additive inhibitory effect. 4. Lysine absorption showed a 39% decrease with 10 pg/ml trypsin and 1 x lo-‘M ouabain, whereas ouabain alone produced 34% inhibition. 5. Lysine absorption showed a 71% decrease with 10 &ml trypsin in a sodium-free medium, and 70% inhibition with Na-free medium alone. 6. The inhibition of lysine absorption after trypsin treatment could be due to’inhibition of the active ~rn~nent of lysine transport.
INTRODUCTJON
Ringer solution (NaCl 140 mM, KHCOs 10mM, KrHPQ 1.2mM, KH,PO, 0.2 mM, MgCl, 1.2 mM, CaCl, 1.2mM) and 100 ml air and then replaced in the abdomen. During all experiments the perfusate was kept at a pH 7.4, a temperature of 37”C, and oxygenated continuously. The jejunal segment was perfused by a peristaltic pump, at a constant rate of 0.67 ml/min for a minimum time interval of 120 min.
Previous reports (Nassar et af., 1983, 1986) on the effect of enzymatic treatment on amino acid absorp tion across the small intestine showed that trypsin and chymotrypsin exhibited specific inhibition of amino acid accumulation in intestinal cells and influx across the mucosal membrane in a dose-dependent pattern. It was suggested also that since lysine is activety transpose into the intestinal cells (McLeod
~e~arern~nt of ~ntest~~f absorpti~
and Tyor, 1967) and the entry process across the brush-border membrane is primarily responsible for the overall active transport of this amino acid (Munck and Schultz, 1969), then the inhibitory effect of trypsin on lysine transport could be due to alterations in the membrane structure and/or to an inhibition of the active step in the absorption mechanism. These observations are not supported by studies to characterize the mechanism of action of trypsin on lysine transport. The present investigation examines the effect and mechanism of action of trypsin and chymotrypsin on lysine transport across the small intestine in vivo.
The perfusate consisted of Ringer solution containing phenol red (15 mg/l), 1 mM lysine and trace quantities of its labelled isotope. The effluent solution was collected every 10min. The labelled isotope content of the initial and effluent solutions was determined by liquid scintillation, and its Phenol Red concentration by spcctrophotometry. The intestinal segment was then resected and dried in an oven for at least 18 hr to determine its dry weight. Absorption was calculated from the rate of disappearance of the labelled amino acid from the perfusate solution taking into account water transport as measured by the change of Phenol Red concentration as described previously (Hajjar et al., 1979). Effect of trypsin and ~~yrnotrypsi~ on iysine transport The effect of trypsin and chymotrypsin on lysine absorption was investigated separately and in combination by perfusing the intestinal segments with a Ringer solution containing the enzymes at different concentrations ranging from 0.5 to 20pg/ml. The intestine was then perfused with the amino acid as described earlier, and the net absorption of lysine was determined.
MATERIALS AND METHODS Animal Preparation Sprague-Dawley rats of both sexes, weighing 130-170 g, were maintained on normal food and water intake, until the day of the experiment when they were anaes~et~~ with intraperitoneal injection of 50 mg of sodium pcntobarbital per kg body weight. In all experiments a tracheostomy was performed to allow for artificial respiration when needed. The abdominal cavity was opened by a midline incision, An inlet cannula in a jejunal segment was placed 4cm caudal to the ligament of Treitz and an outlet cannula 25-30cm distal to it. The segment was flushed with SOml isotonic
of trypsin and ouabain on iysine absorption Two sets of experiments were conducted. In the first set, lysine absorption was determined after perfusing the intestinal segment with Ringer solution containing 1 x lo-‘M ouabain. In the second set the intestinal segment was perfused with 1 x lo-‘M ouabain and lOgg/ml trypsin Eflect
61
CAMILLEF. NASSARefal.
62
TRYPSIN
~NCENTRA~ION
o&m0
Fig. 1. The effect of increasing concentration of trypsin on lysine absorption. Each point is the mean of five determinations. Bars indicate + SEM.
i
1
E +/ . before perfusing it with lysine. Amino acid absorption was calculated in both sets. E#ect of trypin on lysine transport in a Na “-free ceded This set is conducted in a similar procedure to the previous one; however, the effect of a Na+-free Ringer solution was determined by replacing NaCl with choline chloride.
60
100
140
180
TIME tminl
Fig. 3. The time course of lysine absorption in the jejunum of the rat showing no recovery from the effect of 10 fig/ml trypsin after an 80min perfusion. Closed squares: the perfusion of intestinal segment with trypsin. The 8GlOO min period is a washing period. Open squares: the perfusion of the intestinal segment with the Ringer solution.
RESULTS Intestinal absorption of lysine: time study Preliminary experiments to determine lysine absorption across the intestine as a function of time were conducted. t_.-Lysine absorption was determined every 1Omin and the results of these experiments show that lysine uptake was variable during the first 4O-min time interval following which it reached steady state. This steady state was maintained until the end of the 2-hr period of perfusion. Effect of enzymatic
treatment
The effect of trypsin was studied by me-perfusing the jejunal segment with different con~ntrations of this enzyme ranging between 0.5 and 20 pg/ml, and then measuring the absorption of lysine. The results
(Fig. 1) indicate that the inhibitory
effect of trypsin
is dose-dependent, with the maximal effect noticed at 10 fig/ml (100 BAEE units). Figure 2 shows that when the jejunal segment was pre-perfused with 10 pg/ml trypsin a 35% decrease in lysine absorption is noticed. Similarly, the intestinal se@rtent was perfused with chymotrypsin and lysine absorption was determined. The maxims effect of chymotrypsin was noticed at a concentration of lO,~g/ml which produced a significant decrease in lysine transport although it did not exceed 5% (Fig. 2). The combined effect of trypsin and chymotrypsin on lysine absorption was also determined. The treatment of the jejunal segment with lO~g/ml trypsin and lO~g~m1 chymotr~sin produced a 33.5% decrease in the amino acid absorption (Fig. 2) which is not significantly different (P > 0.3) from the inhibitory effect of trypsin alone. Thus, there was no additive effect of both enzymes. The reversibility of the trypsin effect was also investigated. After an 80-min perfusion with 10 pg/mi trypsin, the intestine was washed for 10min by perfusing the jejunal segment with isotonic saline; then the intestine was perfused for an additional 90 min with the Ringer solution containing lysine. The results (Fig. 3) show that there was no significant increase (P > 0.05) in lysine absorption after washing the intestine from trypsin and removing the enzyme from the perfusate. Thus, it could be suggested that trypsin has a non-reversible effect on lysine absorption. Effect of trypsin and ouabain
L
50
loo TIME (r-n‘” 1
Fig. 2. The effects of trypsin (m-m), chymotrypsin (0-Q) and trypsin +chymotrypsin (0-O) on lysine absorption (~-~). Trypsin decreased lysine transport by 35%, chymotrypsin by 5% and their combination by 34%. Each point is the mean + SEM of five determinations.
In order to study the effect of trypsin on the active components of lysine absorption mechanism, ouabain was used to inhibit the active step of lysine transport before treating the intestinal segment with the enzyme. The jejunal segment was perfused intraluminally with Ringer solution containing 1 x low4 M ouabain. Lysine absorption showed a 33.9% decrease from the steady-state value, whereas perfusion with Ringer solution containing 1x
Enzymatic inhibition of Lysine transport
63
G
t
E
‘I
50
1Ol TIME
Cm(n)
Fig. 4. Effect of lOpg/ml trypsin in the presence of 1 x 10e4 M ouabain. Closed circles: control steady-state. Open circles: the effect of IO pg/ml trypsin. Open triangles: the effect of 1 x 10m4M ouabain. Closed triangles: the combined effect of 10 pg/ml trypsin and 1 x 10m4M ouabain. No significant difference was noticed between the effect of trypsin and ouabain (P > 0.5). Vertical bars indicate + SEM.
10e4 M ouabain and 10 pg/ml trypsin showed a 39% decrease in lysine absorption (Fig. 4). Although no statistical difference (P > 0.5) is observed between the effect of 10 pg/ml trypsin and that of 1 x 10m4M ouabain, yet the difference between 1 x low4 M ouabain and 1 x 10m4M ouabain + 10 pg/ml trypsin is statistically significant (P < 0.05). Effect
of trypsin in a Na +-free medium
The effect of the absence of sodium from the perfusing solution, and its effect in the presence of trypsin on lysine absorption was also determined. The absence of Na+ caused a 70% decrease in lysine absorption, whereas the addition of 10 pg/ml trypsin in the Na+-free Ringer solution decreased lysine absorption by 71% only (Fig. 5). DISCUSSION
In this study, lysine transport across the rat jejunum was investigated by evaluating the effect of trypsin and chymotrypsin on steady-state lysine absorption. Preliminary studies showed that the absorption of lysine reached a steady-state after 40min of perfusion with the amino acid. Similar findings on other amino acids were reported by Hajjar et al. (1981). Nassar et al. (1982) demonstrated that the concentration of lysine reached an intracellular/ extracellar ratio of nearly 2.0, which would reflect an accumulation of lysine against a concentration gradient within the cell. A careful examination of the effect of trypsin digestion, using different concentrations of the enzyme, on intestinal segments indicates that the diminished ability of trypsin-treated intestinal segments to absorb lysine correlates with the increase in the concentration of trypsin perfused within these segments. Hence trypsinization of the intestinal segment at 10 pg/ml in the perfusate resulted in a 35% decrease in lysine absorption com-
50
100 TIME (m(n)
Fig. 5. Effect of 10 pg/ml trypsin in a Na-free solution on lysine absorption. Closed circles: control state. Closed squares: the absorption of lysine in a Ringer medium. Open squares: the effect of 10 pg/ml in Na-free medium.
Ringer steadyNa-free trypsin
pared to 5 1% decrease in intracellular lysine concentration reported for in vitro study. Moreover, chymotrypsin inhibited lysine absorption by only 5%, whereas it did not show any significant effect on the intracellular lysine concentration, which indicates that the in oiuo findings are compatible with those reported on lysine absorption in oitro (Nassar et al., 1982). In another study on the effect of trypsin on renal brush-border vesicles, Hsu et al. (1982) showed that not only the uptake of several groups of amino acids, including lysine, across renal brush border membrane decreased after trypsin treatment, but that also the uptake of n-glucose and a-methyl-Dglucoside was inhibited. These data indicate that there is a global depression of transport systems in trypsinized membranes in renal brush-border vesicles which might be extrapolated to the intestinal brushborder membrane. Chymotrypsin did not show such a prominent effect. Other types of cells showed a similar differential pattern with respect to enzymatic digestion. The chymotrypsin fragmentation pattern of glycophorin A in intact red blood cells was different from that generated by trypsin treatment (Dzandu et al., 1985). To characterize the nature of trypsin inhibitory mechanism on lysine absorption, the effect of treating the intestinal segment with the enzyme in the presence of ouabain and absence of sodium was evaluated. Our findings may suggest that the effect of trypsin could be due to specific alteration in the membrane structure and/or to an inhibition of the active step in the transport system. Our results demonstrated that perfusing the intestine with 1 x 10e4M ouabain inhibited lysine absorption to a level that is very close to the trypsin-treated value. However, when both were infused, lysine inhibition was increased by only 2%, although it was statistically significant from trypsin treatment alone. This might suggest that structural changes in the membrane contribute minimally to the inhibitory effect of trypsin, the major factor probably being the inhibition of the active step in the lysine transport mechanism. However, trypsin
64
CAMILLEF. NASSARet al
treatment did not inhibit further lysine absorption caused by the absence of sodium in the Ringer solution. Thus, it could be suggested that the effect of trypsin is more likely to be on the lysine active component. Previous evidence has shown that (Na+-K+ )ATPase in animal cell membranes consists of two polypeptides, a large mol. wt polypeptide and a sialoglycoprotein (Kyte, 1971; Jorgensen, 1974). Trypsin treatment resulted in the cleavage of the large polypeptide into two fragments and the rapid loss of (Na+-K+)ATPase activity (Giotta, 1975; Lo and Titus, 1978). Other experiments (Giotta, 1975) using red cell membranes indicate that trypsin is attacking (Na+-K+)ATPase from the cytoplasmic side of the membrane. This finding supports the report of Hodges et al. (1973) that trypsin could be internalized and thus its inhibitory effect could be translated through inhibiting the active component of lysine transport rather than through alterations in the membrane structure. REFERENCES Dzandu J. K., Deh M. E. and Wise G. E. (1985) A re-examination of the effects of chymotrypsin and trypsin on the erythrocyte membrane surface topology. Biochem. Res. Comm. 126, 5&58. Giotta G. J. (1975) Native (Na+-K+)-dependent adenosine triphosphatase has two trypsin-sensitive sites. .I. biol. Chem. 250, 5159-5164.
Hajjar J. J., Murphy D. M. and Scheig R. L. (1979) Mechanism of inhibition of alanine absorption by Na ricinoleate. Am. J. Physiol. 236, E534E558. Hajjar J. J. and Tomicic T. (1981) Effect of chronic ethanol consumption on leucine absorption in the rat small intestine. Digestion 22, 17&l 76. Hodges G. M., Livingstone D. C. and Franks L. M. (1973) The localization of trypsin in cultured mammalian cells. J. Cell Sci. 12, 887-902. Hsu B. Y., Corcoran S. M. and Segal S. (1982) The effect on AA transport of trypsin treatment of rat renal brush border membranes. Biochem. biophys. Acta 689, 181-183. Jorgensen P. L. (1974) Purification and characteristics of (Na+-K+)-ATPase. Biochem. biophys. Acta 356, 3652. Kyte J. (1971) Purification of the sodium and potassium dependent adenosine triphosphate from renal medulla. J. biol. Chem. 246, 415714165. Lo T. N. and Titus E. 0. (1978) Effects of ligands on conformationallv dependent trypsinolysis of (Na+-K+) activated ATP. 3. bi‘ol. Chem. %3, 4432-4438: McLeod M. E. and Tyor M. P. (1967) Transport of basic amino acids bv hamster intestine. Am. J. Phvsiol. 213, 163-168. _ Munck B. G. and Schultz S. G. (1969) Lysine transport across isolated rabbit ileum. J. gen. Physiol. 53, 157-182. Nassar C. F. and Haddad M. E (198i) Enzymatic inhibition of lysine transport across rat and turtle small intestine. Comp. Biochem. Physiol. 76, 153-156. Nassar C. F., Semrani P., Nasser M. and Habbal Z. M. (1986) The effect of enzymatic treatment on phenylalanine transport mechanisms across rat small intestine. Comp. Biochem. Physiol. 83, 271-274.
@A, No. 1, pp. 61-64, 1988
03~-9629/88
$3.00 + 0.00
0 1988 Pergamon Journals Ltd
Printed in Great Britain
ENZYMATIC INHIBITION OF LYSINE TRANSPORT ACROSS THE SMALL INTESTINE IN VIVO CAMILLE F. NASSAR, GABI M. WAZZ, MICHEL G. NASSER and ZOHEIR M. HABBAL Departments
of Physiology and Laboratory Medicine, Faculty of Medicine, American University of Beirut, Beirut, Lebanon (Received 22 April 1987)
Abstract-l. Trypsin, at different concentrations, significantly inhibited lysine absorption (P < 0.05) in a dose-dependent pattern. 2. Maximum inhibition equivalent to 35% below control value was reached with lOpg/ml (100 BAEE units) trypsin with a non-reversible inhibitory effect. 3. Chymotrypsin at 10 pg/ml produced a significant decrease (P < 0.05) of lysine absorption although it did not exceed 5%. Perfusion of both enzymes did not show an additive inhibitory effect. 4. Lysine absorption showed a 39% decrease with 10 pg/ml trypsin and 1 x lo-‘M ouabain, whereas ouabain alone produced 34% inhibition. 5. Lysine absorption showed a 71% decrease with 10 &ml trypsin in a sodium-free medium, and 70% inhibition with Na-free medium alone. 6. The inhibition of lysine absorption after trypsin treatment could be due to’inhibition of the active ~rn~nent of lysine transport.
INTRODUCTJON
Ringer solution (NaCl 140 mM, KHCOs 10mM, KrHPQ 1.2mM, KH,PO, 0.2 mM, MgCl, 1.2 mM, CaCl, 1.2mM) and 100 ml air and then replaced in the abdomen. During all experiments the perfusate was kept at a pH 7.4, a temperature of 37”C, and oxygenated continuously. The jejunal segment was perfused by a peristaltic pump, at a constant rate of 0.67 ml/min for a minimum time interval of 120 min.
Previous reports (Nassar et af., 1983, 1986) on the effect of enzymatic treatment on amino acid absorp tion across the small intestine showed that trypsin and chymotrypsin exhibited specific inhibition of amino acid accumulation in intestinal cells and influx across the mucosal membrane in a dose-dependent pattern. It was suggested also that since lysine is activety transpose into the intestinal cells (McLeod
~e~arern~nt of ~ntest~~f absorpti~
and Tyor, 1967) and the entry process across the brush-border membrane is primarily responsible for the overall active transport of this amino acid (Munck and Schultz, 1969), then the inhibitory effect of trypsin on lysine transport could be due to alterations in the membrane structure and/or to an inhibition of the active step in the absorption mechanism. These observations are not supported by studies to characterize the mechanism of action of trypsin on lysine transport. The present investigation examines the effect and mechanism of action of trypsin and chymotrypsin on lysine transport across the small intestine in vivo.
The perfusate consisted of Ringer solution containing phenol red (15 mg/l), 1 mM lysine and trace quantities of its labelled isotope. The effluent solution was collected every 10min. The labelled isotope content of the initial and effluent solutions was determined by liquid scintillation, and its Phenol Red concentration by spcctrophotometry. The intestinal segment was then resected and dried in an oven for at least 18 hr to determine its dry weight. Absorption was calculated from the rate of disappearance of the labelled amino acid from the perfusate solution taking into account water transport as measured by the change of Phenol Red concentration as described previously (Hajjar et al., 1979). Effect of trypsin and ~~yrnotrypsi~ on iysine transport The effect of trypsin and chymotrypsin on lysine absorption was investigated separately and in combination by perfusing the intestinal segments with a Ringer solution containing the enzymes at different concentrations ranging from 0.5 to 20pg/ml. The intestine was then perfused with the amino acid as described earlier, and the net absorption of lysine was determined.
MATERIALS AND METHODS Animal Preparation Sprague-Dawley rats of both sexes, weighing 130-170 g, were maintained on normal food and water intake, until the day of the experiment when they were anaes~et~~ with intraperitoneal injection of 50 mg of sodium pcntobarbital per kg body weight. In all experiments a tracheostomy was performed to allow for artificial respiration when needed. The abdominal cavity was opened by a midline incision, An inlet cannula in a jejunal segment was placed 4cm caudal to the ligament of Treitz and an outlet cannula 25-30cm distal to it. The segment was flushed with SOml isotonic
of trypsin and ouabain on iysine absorption Two sets of experiments were conducted. In the first set, lysine absorption was determined after perfusing the intestinal segment with Ringer solution containing 1 x lo-‘M ouabain. In the second set the intestinal segment was perfused with 1 x lo-‘M ouabain and lOgg/ml trypsin Eflect
61
CAMILLEF. NASSARefal.
62
TRYPSIN
~NCENTRA~ION
o&m0
Fig. 1. The effect of increasing concentration of trypsin on lysine absorption. Each point is the mean of five determinations. Bars indicate + SEM.
i
1
E +/ . before perfusing it with lysine. Amino acid absorption was calculated in both sets. E#ect of trypin on lysine transport in a Na “-free ceded This set is conducted in a similar procedure to the previous one; however, the effect of a Na+-free Ringer solution was determined by replacing NaCl with choline chloride.
60
100
140
180
TIME tminl
Fig. 3. The time course of lysine absorption in the jejunum of the rat showing no recovery from the effect of 10 fig/ml trypsin after an 80min perfusion. Closed squares: the perfusion of intestinal segment with trypsin. The 8GlOO min period is a washing period. Open squares: the perfusion of the intestinal segment with the Ringer solution.
RESULTS Intestinal absorption of lysine: time study Preliminary experiments to determine lysine absorption across the intestine as a function of time were conducted. t_.-Lysine absorption was determined every 1Omin and the results of these experiments show that lysine uptake was variable during the first 4O-min time interval following which it reached steady state. This steady state was maintained until the end of the 2-hr period of perfusion. Effect of enzymatic
treatment
The effect of trypsin was studied by me-perfusing the jejunal segment with different con~ntrations of this enzyme ranging between 0.5 and 20 pg/ml, and then measuring the absorption of lysine. The results
(Fig. 1) indicate that the inhibitory
effect of trypsin
is dose-dependent, with the maximal effect noticed at 10 fig/ml (100 BAEE units). Figure 2 shows that when the jejunal segment was pre-perfused with 10 pg/ml trypsin a 35% decrease in lysine absorption is noticed. Similarly, the intestinal se@rtent was perfused with chymotrypsin and lysine absorption was determined. The maxims effect of chymotrypsin was noticed at a concentration of lO,~g/ml which produced a significant decrease in lysine transport although it did not exceed 5% (Fig. 2). The combined effect of trypsin and chymotrypsin on lysine absorption was also determined. The treatment of the jejunal segment with lO~g/ml trypsin and lO~g~m1 chymotr~sin produced a 33.5% decrease in the amino acid absorption (Fig. 2) which is not significantly different (P > 0.3) from the inhibitory effect of trypsin alone. Thus, there was no additive effect of both enzymes. The reversibility of the trypsin effect was also investigated. After an 80-min perfusion with 10 pg/mi trypsin, the intestine was washed for 10min by perfusing the jejunal segment with isotonic saline; then the intestine was perfused for an additional 90 min with the Ringer solution containing lysine. The results (Fig. 3) show that there was no significant increase (P > 0.05) in lysine absorption after washing the intestine from trypsin and removing the enzyme from the perfusate. Thus, it could be suggested that trypsin has a non-reversible effect on lysine absorption. Effect of trypsin and ouabain
L
50
loo TIME (r-n‘” 1
Fig. 2. The effects of trypsin (m-m), chymotrypsin (0-Q) and trypsin +chymotrypsin (0-O) on lysine absorption (~-~). Trypsin decreased lysine transport by 35%, chymotrypsin by 5% and their combination by 34%. Each point is the mean + SEM of five determinations.
In order to study the effect of trypsin on the active components of lysine absorption mechanism, ouabain was used to inhibit the active step of lysine transport before treating the intestinal segment with the enzyme. The jejunal segment was perfused intraluminally with Ringer solution containing 1 x low4 M ouabain. Lysine absorption showed a 33.9% decrease from the steady-state value, whereas perfusion with Ringer solution containing 1x
Enzymatic inhibition of Lysine transport
63
G
t
E
‘I
50
1Ol TIME
Cm(n)
Fig. 4. Effect of lOpg/ml trypsin in the presence of 1 x 10e4 M ouabain. Closed circles: control steady-state. Open circles: the effect of IO pg/ml trypsin. Open triangles: the effect of 1 x 10m4M ouabain. Closed triangles: the combined effect of 10 pg/ml trypsin and 1 x 10m4M ouabain. No significant difference was noticed between the effect of trypsin and ouabain (P > 0.5). Vertical bars indicate + SEM.
10e4 M ouabain and 10 pg/ml trypsin showed a 39% decrease in lysine absorption (Fig. 4). Although no statistical difference (P > 0.5) is observed between the effect of 10 pg/ml trypsin and that of 1 x 10m4M ouabain, yet the difference between 1 x low4 M ouabain and 1 x 10m4M ouabain + 10 pg/ml trypsin is statistically significant (P < 0.05). Effect
of trypsin in a Na +-free medium
The effect of the absence of sodium from the perfusing solution, and its effect in the presence of trypsin on lysine absorption was also determined. The absence of Na+ caused a 70% decrease in lysine absorption, whereas the addition of 10 pg/ml trypsin in the Na+-free Ringer solution decreased lysine absorption by 71% only (Fig. 5). DISCUSSION
In this study, lysine transport across the rat jejunum was investigated by evaluating the effect of trypsin and chymotrypsin on steady-state lysine absorption. Preliminary studies showed that the absorption of lysine reached a steady-state after 40min of perfusion with the amino acid. Similar findings on other amino acids were reported by Hajjar et al. (1981). Nassar et al. (1982) demonstrated that the concentration of lysine reached an intracellular/ extracellar ratio of nearly 2.0, which would reflect an accumulation of lysine against a concentration gradient within the cell. A careful examination of the effect of trypsin digestion, using different concentrations of the enzyme, on intestinal segments indicates that the diminished ability of trypsin-treated intestinal segments to absorb lysine correlates with the increase in the concentration of trypsin perfused within these segments. Hence trypsinization of the intestinal segment at 10 pg/ml in the perfusate resulted in a 35% decrease in lysine absorption com-
50
100 TIME (m(n)
Fig. 5. Effect of 10 pg/ml trypsin in a Na-free solution on lysine absorption. Closed circles: control state. Closed squares: the absorption of lysine in a Ringer medium. Open squares: the effect of 10 pg/ml in Na-free medium.
Ringer steadyNa-free trypsin
pared to 5 1% decrease in intracellular lysine concentration reported for in vitro study. Moreover, chymotrypsin inhibited lysine absorption by only 5%, whereas it did not show any significant effect on the intracellular lysine concentration, which indicates that the in oiuo findings are compatible with those reported on lysine absorption in oitro (Nassar et al., 1982). In another study on the effect of trypsin on renal brush-border vesicles, Hsu et al. (1982) showed that not only the uptake of several groups of amino acids, including lysine, across renal brush border membrane decreased after trypsin treatment, but that also the uptake of n-glucose and a-methyl-Dglucoside was inhibited. These data indicate that there is a global depression of transport systems in trypsinized membranes in renal brush-border vesicles which might be extrapolated to the intestinal brushborder membrane. Chymotrypsin did not show such a prominent effect. Other types of cells showed a similar differential pattern with respect to enzymatic digestion. The chymotrypsin fragmentation pattern of glycophorin A in intact red blood cells was different from that generated by trypsin treatment (Dzandu et al., 1985). To characterize the nature of trypsin inhibitory mechanism on lysine absorption, the effect of treating the intestinal segment with the enzyme in the presence of ouabain and absence of sodium was evaluated. Our findings may suggest that the effect of trypsin could be due to specific alteration in the membrane structure and/or to an inhibition of the active step in the transport system. Our results demonstrated that perfusing the intestine with 1 x 10e4M ouabain inhibited lysine absorption to a level that is very close to the trypsin-treated value. However, when both were infused, lysine inhibition was increased by only 2%, although it was statistically significant from trypsin treatment alone. This might suggest that structural changes in the membrane contribute minimally to the inhibitory effect of trypsin, the major factor probably being the inhibition of the active step in the lysine transport mechanism. However, trypsin
64
CAMILLEF. NASSARet al
treatment did not inhibit further lysine absorption caused by the absence of sodium in the Ringer solution. Thus, it could be suggested that the effect of trypsin is more likely to be on the lysine active component. Previous evidence has shown that (Na+-K+ )ATPase in animal cell membranes consists of two polypeptides, a large mol. wt polypeptide and a sialoglycoprotein (Kyte, 1971; Jorgensen, 1974). Trypsin treatment resulted in the cleavage of the large polypeptide into two fragments and the rapid loss of (Na+-K+)ATPase activity (Giotta, 1975; Lo and Titus, 1978). Other experiments (Giotta, 1975) using red cell membranes indicate that trypsin is attacking (Na+-K+)ATPase from the cytoplasmic side of the membrane. This finding supports the report of Hodges et al. (1973) that trypsin could be internalized and thus its inhibitory effect could be translated through inhibiting the active component of lysine transport rather than through alterations in the membrane structure. REFERENCES Dzandu J. K., Deh M. E. and Wise G. E. (1985) A re-examination of the effects of chymotrypsin and trypsin on the erythrocyte membrane surface topology. Biochem. Res. Comm. 126, 5&58. Giotta G. J. (1975) Native (Na+-K+)-dependent adenosine triphosphatase has two trypsin-sensitive sites. .I. biol. Chem. 250, 5159-5164.
Hajjar J. J., Murphy D. M. and Scheig R. L. (1979) Mechanism of inhibition of alanine absorption by Na ricinoleate. Am. J. Physiol. 236, E534E558. Hajjar J. J. and Tomicic T. (1981) Effect of chronic ethanol consumption on leucine absorption in the rat small intestine. Digestion 22, 17&l 76. Hodges G. M., Livingstone D. C. and Franks L. M. (1973) The localization of trypsin in cultured mammalian cells. J. Cell Sci. 12, 887-902. Hsu B. Y., Corcoran S. M. and Segal S. (1982) The effect on AA transport of trypsin treatment of rat renal brush border membranes. Biochem. biophys. Acta 689, 181-183. Jorgensen P. L. (1974) Purification and characteristics of (Na+-K+)-ATPase. Biochem. biophys. Acta 356, 3652. Kyte J. (1971) Purification of the sodium and potassium dependent adenosine triphosphate from renal medulla. J. biol. Chem. 246, 415714165. Lo T. N. and Titus E. 0. (1978) Effects of ligands on conformationallv dependent trypsinolysis of (Na+-K+) activated ATP. 3. bi‘ol. Chem. %3, 4432-4438: McLeod M. E. and Tyor M. P. (1967) Transport of basic amino acids bv hamster intestine. Am. J. Phvsiol. 213, 163-168. _ Munck B. G. and Schultz S. G. (1969) Lysine transport across isolated rabbit ileum. J. gen. Physiol. 53, 157-182. Nassar C. F. and Haddad M. E (198i) Enzymatic inhibition of lysine transport across rat and turtle small intestine. Comp. Biochem. Physiol. 76, 153-156. Nassar C. F., Semrani P., Nasser M. and Habbal Z. M. (1986) The effect of enzymatic treatment on phenylalanine transport mechanisms across rat small intestine. Comp. Biochem. Physiol. 83, 271-274.