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Inhibitory effect of neurotensin on proline
Regulatory Peptides, 46 (1993) 543-547 © 1993 Elsevier Science Publishers B.V. All rights reserved 0167-0115/93/$06.00
543
REGPEP 01377
Inhibitory effect of neurotensin on proline Hani A.H. Abd-U1-Salam and Camille F. Nassar Department of Physiology, Faculty of Medicine, American Universityof Beirut, Beirut (Lebanon) (Received 29 October 1992; revised version received 1 February 1993; accepted 8 February 1993)
Key words: Neurotensin; Intestinal absorption
Summary The effect of neurotensin (NT) on proline absorption across rat jejunum was investigated using the singlepass perfusion technique. This study showed that intravenous administration of NT produced a dose-dependent inhibition of proline absorption. Thus, NT at a 0.16 pmol/kg/min concentration gave 10~o decrease in proline absorption while 0.32 and 1.6 pmol/kg/min concentration gave 317o and 45?0 decrease, respectively. In the absence of Na, proline absorption decreased to 77~o from control values. No change in proline absorption was noticed when NT at a concentration of 0.32 pmol/kg/min was intravenously injected in the absence of sodium from the perfusion solution. Water absorption did not show significant changes (P> 0.05) in presence or absence of NT. Moreover, NT did not produce a significant change (P> 0.2) in intracellular proline accumulation. NT inhibited proline absorption through an indirect mechanism that is Na-dependent and independent of changes in water absorption.
Introduction Neurotensin (NT), a tridecapeptide [1] isolated originally from bovine hypothalamus during isolation of substance P [2] is found to be distributed in the central and peripheral nervous system, and the peripheral endocrine system [3]. The gastrointestinal tract is considered as both, the major source of NT Correspondence to: C.F. Nassar, Department of Physiology, Faculty of Medicine, American University of Beirut, Beirut, Lebanon.
(about 85 ~o of total NT in the rat) and an important target organ for this peptide [4]. Immunochemical studies show that gut NT is largely concentrated in apparently secretory cells in intestinal villi and crypts [5]. Dense core vesicles that can be stained histochemically for NT were found in the basolateral portion of the cell [6]. In a recent study, Armstrong et al. [7] noticed that intravenous infusion of NT increased oleic acid translocation across the rat small intestine. In addition, perfusing fatty acids in the intestine caused an
544
increase in plasma concentration of N T [8]. This demonstrated a direct effect of N T on solute absorption across the small intestine. So far, there are no reports on the effect of N T on translocation of amino acids across the rat intestine. This study is an attempt to determine the effect of N T on amino acid absorption and the mechanism involved in this process.
Materials and Methods
In vivo study Animalpreparation. Adult female Sprague-Dawley rats weighing 150-250 g fed ad libitum and fasted one day before the day of experiment were used. The animals were anaesthetized with an intraperitoneal injection of 35 mg of sodium pentobarbital (Nembutal) per kg body weight. A tracheotomy was done on all animals to allow for adequate ventilation. The abdominal cavity was opened by a midline incision and the jejunum was exposed with its vascular supply kept intact. A segment of the jejunum was cannulated by an inlet 5 cm caudal to the ligament of Treitz and an outlet 15-20 cm distal to it. The segment was replaced in the abdomen after removal of the luminal contents by perfusing 50 ml of modified Ringer solution (NaC1 140 raM, K H C O 3 10 mM, K2HPO 4 1.7 raM, KHzPO 4 0.2 mM, MgCI 2 1.2 mM). During all experiments, the perfusate was kept at constant temperature of 37 °C and the jejunal segment was perfused by a peristaltic pump at a constant rate of 0.67 ml/min for a period of 120 min. N T effect on proline absorption was investigated by infusing the rats intravenously with different concentrations of NT ranging between 0.16 and 3.2 pmol/ kg/min. N T was dissolved in 0.9~o NaC1. The effect of N T on proline absorption in the absence of Na was determined by replacing sodium chloride with choline chloride in the perfusion solution. Control rats received 0.9~o NaC1. Measurement of intestinal absorption. The perfusate consisted of modified Ringer solution contain-
ing phenol red (15 rag/l), 1 mM L-proline and trace quantities of its labelled isotope. The effluent solution was collected every 10 min for 120 rain. Proline absorption was calculated from the isotope content of the initial and effluent solutions that was determined by liquid scintillation. Water absorption was determined by measuring phenol red concentration by spectrophotometry at 560 nm. The intestinal segment was resected, blotted on Whatman No. 1 filter paper, its length measured and weighed on a Sartorius balance. Absorption was calculated from the rate of disappearance of the labelled amino acid from the perfusion solution, after correction for water transport.
In vitro study Tissue preparation. Female Sprague-Dawley rats weighing 150-250 g were utilized in all experiments. The rats were anaesthetized by an intraperitoneal injection of 35 mg sodium pentobarbital per kg body weight. The abdomen was opened by a midline incision and the jejunum was removed and placed immediately in oxygenated ice-cold phosphate buffered saline (PBS) solution. The jejunum was freed from the mesenteric and fat attachments and cut longitudinally into strips 1 cm in length. Measurement of intracellular proline concentration. Jejunal strips, each weighing between 50-80 mg, were incubated in oxygenated PB S solution (pH 7.4) containing [laC]proline, and shaked at 60-65 cycles/ rain in water bath at 37°C for 1 h. After the incubation period, the strips were removed from the incubation solution and immediately dipped in icecold isotonic mannitol solution. Each strip was blotted with Whatman No. 1 filter paper, its wet weight determined by a Sartorius balance, and then extracted in 2 ml of 0.1 M H N O 3 for more than 4 h. Aliquots of the tissue extracts and incubation media were counted for their [ lac]proline content in a liquid scintillation counter. The effect of N T on proline uptake across the rat small intestine was determined by preincubating the jejunal strips for 30 min with 3.2 pmol/kg NT in a
545
water bath at 37 ° C. The tissues were then processed in the same manner as described before. ~
LO~
4.
o= 8E3-
Results
In vivo study The effect of N T on intestinal proline absorption: concentration study. The effect of N T was studied by infusing N T in the jugular vein with different concentrations of this peptide ranging between 0.16 pmol/kg/min to 3.2 pmol/kg/min and measuring the absorption of proline at 10 min intervals. The results (Fig. 1) clearly indicate an inhibitory effect of N T which is dose-dependent. The inhibitory effect of N T is significant at concentrations ranging between 0.32 pmol/kg/min and 3.2 pmol/kg/min (P<0.001). The maximal inhibitory effect of N T is noticed at 1.6 pmol/kg/min (P<0.001) and higher doses did not show an increase in this effect ( P > 0.9). Intestinal absorption of proline: combined effect of sodium (Na) and NT. The effect of the absence of sodium in the perfusion solution was also deter-
4:4444
E~ 4
5~
o ~ 2&
5'0
6'0
7(D
80 90 100 Time (min)
110
120
130
Fig. 2. The effect of N T intravenous infusion on proline absorption when the jejunum was perfused with Na-free perfusion solution. Proline absorption was determined every 10 min. until the end of the experiment. Bars represent _+ S.E.M. Symbols: m , control; + , Na-free medium; ×, Na-free medium + 3.2 pmol/kg/min N T concentration.
mined. Proline absorption in a Na-free Ringer solution was determined as described previously, and the results are shown in Fig. 2. It can be noticed that the absence of Na from the perfusion solution caused a 77% decrease in proline absorption. However, intravenous administration of N T at a concentration of 3.2 pmol/kg/min did not alter the level of proline absorption ( P > 0.9) when sodium was removed from the perfusion solution (Fig. 2).
~o 3
aE
a-Control b-Na-free c-Controi~N] d-Na-free÷NT
E u E
5 4
o
50
6'0
70
80 90 100 Time (mm)
110
120
130
L
o 3 ~3
Fig. 1. The effect of N T infusion on proline absorption in the rat jejunum. Proline absorption was determined every 10 rain. until the end of the experiment. Bars indicate + S.E.M. Symbols: • control; + , control+ 0.16 pmol/kg/min N T concentration; O , control + 0.32 pmol/kg/min N T concentration; [~, control + 1.60 pmol/kg/min N T concentration; x , Control + 3.20 pmol/kg/min N T concentration.
L 2
O
-
-
Fig. 3. Effect of 3.2 pmol/kg/min N T on water absorption in the presence and absence of Na ÷ from the perfusion medium.
546
Effect of N T on water absorption. Water absorption was measured in the presence and absence of Na. The effect of N T was determined in both sets of experiments. N T showed no effect on water transport neither in the presence (P>0.05) nor in the absence of Na ( P > 0.05) (Fig. 3). Accordingly, the change in proline absorption noticed after NT administration was not secondary to changes in water transport but most probably was due to a direct effect on proline absorption. In vitro study Measurement of intracellular proline accumulation. The effect of N T on proline uptake was investigated in vitro. Jejunal strips were preincubated in 3.2 pmol/kg N T for 30 min, then incubated in 1 mM proline for 1 h. The incubation solution was oxygenated and kept at 37 °C in a water bath. The results indicate that there is no significant difference ( P > 0.2) between proline uptake by the intestinal strips in the presence and absence of 3.2 pmol/kg NT. The fact that the In/Out ratio measured in the intestinal cells preincubated with N T reached 5.90 which is not significantly different ( P > 0.2) from the control value (5.85) suggests that the active component of the transport process was not affected by preincubating the tissues with NT. Discussion Intestinal epithelial ceils have the ability to concentrate amino acids against an electrochemical gradient. Previous studies indicate that proline absorption follows three pathways; an imino carrier specific for proline responsible for 60~o of total transport, a non-specific neutral system believed to be the ASC system responsible for 35 ~o of the total uptake, and the remaining 5~o are due to diffusional processes [ 9]. Hence, the active component which accounts for 95 ~o of total transport is Na-dependent. Our results show an inhibition of proline uptake in the absence of Na equivalent to around 7 7 ~ , which would corroborate previous reports.
N T is a peptide that has several effects at various sites in the body. It causes an increase in glucagon level as well as a decrease in insulin level in serum [ 10]. In the gastrointestinal tract, N T has been shown to increase contractility of longitudinal muscle of isolated guinea pig ileum [ 11 ] as well as to decrease gastric motility [ 12] and lower esophageal sphincter pressure [13]. In addition, N T increases vascular permeability in feline ileum [14]. In vitro, binding studies show high affinity of NT binding to membranes from guinea pig ileum [ 11 ]. This work is an attempt to elucidate the mechanism by which NT regulates proline absorption across the small intestine. Our data indicate that NT inhibits proline uptake at a concentration of 1.6 pmol/kg/min. Higher doses did not have a greater effect. The concentrations used in this study are within the range of N T concentration in human plasma. The fasting concentration of NT in plasma was reported as 1 pM and it increased to 7.2 pM after a lunch meal [15]. N T at its maximal effect inhibits up to 46~o of total proline uptake in vivo. This suggests that the N T effect could be largely on the Na-dependent imino carrier which is resposible for 60~o of the total uptake [9]. However, N T has no effect on proline absorption in the absence of Na. This could suggest that N T effect is a Na-mediated process. There exists around 75 ~o inhibition of proline uptake when Na is removed from our perfusion solution. This may be due to the inactivation of the Na-dependent imino carrier as well as the Nadependent non-specific proline uptake mechanisms. Our results in vivo study showed no effect of NT when it was incubated with the intestinal strips. However, N T is a large polypeptide molecule that makes it impermeant to the intestinal cell membrane. Hence, it could be suggested - on theoretical basis - that N T did not cross the brush border membrane and did not cause any conformational changes in the imino acid carrier, and therefore no effect was noticed. However, when N T was administered intravenously, a definite inhibitory effect on proline absorption was noticed. This inhibition is produced
547
due probably to an effect of NT or one or more of its metabolites on the basolateral membrane. In vitro binding studies show higher affinity specific binding to membranes of the guinea pig ileum [ 11]. Data indicate that the NT modulatory effect is a saturable process suggesting saturation of binding sites at the basolateral membrane inducing intracellular mechanisms that alter the imino carrier activity. This implies that binding of NT on the basolateral membrane may have caused functional and/or structural changes that produced proline inhibition. Moreover, since the absence of Na alleviated this effect of NT, it could be suggested also that NT is causing this inhibition through a Na-dependent regulatory mechanism. The effect of NT on water transport was determined. Our results show that NT had no effect on water transport across the rat jejunum. This implies that the observed changes in proline uptake were not secondary to changes in water transport. Accordingly, it could be suggested that proline absorption occurs via a nonsolvent drag mechanism, and that the effect of NT on proline uptake is not translated through a change in water transport. Moreover, since NT causes an increase of glucagon level and a decrease of insulin in blood, it could imply that the mechanism of inhibition is mediated through the release of a hormone or any other mediator that has a modulatory effect on the imino carrier. In conclusion, NT inhibits proline absorption through an indirect mechanism that is Na-dependent and independent of change in water absorption.
References 1 Carraway, R. and Leeman, S.E., The synthesis ofneurotensin, J. Biol. Chem., 250 (1975) 1912-1918.
2 Carraway, R. and Leemen, S.E., The isolation of a new hypotensive peptide, neurotensin, from bovine hypothalami, J. Biol. Chem., 248 (1973) 6854-6861. 3 Rokaeus, A., Studies on NT as a hormone, Acta. Physiol. Scand. (Suppl.), 501 (1981) 1-62. 4 Carraway, R. and Leeman, S.E., Characterization of radioimmunoassyable NT in the rat: its differential distribution in the CNS, small intestine and stomach, J. Biol. Chem., 251 (1976) 7045-7052. 5 Sundler, F., H~kanson, R., Hammer, R.A., Alumets, J., Carraway, R., Leeman, S.E. and Zimrnerman, E.A., Immunohistochemical localization of N T in endocrine cells of the gut, Cell Tissue Res., 178 (1977) 313-321. 6 Helmsteideter, V., Feurle, G.E. and Forssman, W.G., Ultrastructural identification of new cell type-the N-cell as a source of neurotensin in the gut mucosa, Cell Tissue Res., 184 (1977) 445-448. 7 Armstrong, M., Parker, M., Ferris, C. and Leeman, S.E., N T stimulates oleic acid translocation across rat small intestine, Am. J. Physiol., 251 (1986) G823-829. 8 Ferris, C.F., Armstrong, M.J., George, J.K., Stevens, C.A., Carraway, R.E. and Leeman, S.E., Alcohol and fatty acid stimulation of N T release from rat small intestine, Endocrinology, 116 (1985) 1133-1138. 9 Stevens, B.R. and Wright, E.M., Substrate specificity of the intestinal brush border proline/sodium transporter, J. Membr. Biol., 87 (1985) 27-34. 10 Brown, M., Villareal, J. and Vale, W., NT and substance P: Effects on plasma insulin and glucagon levels, Metabolism, 25 (1976) 1459-1461. 11 Kitabgi, P. and Freychet, F., Neurotensin: contractile activity, specific binding, and lack of cyclic nucleotide in intestinal smooth muscle, Eur. J. Pharmacol., 55 (1979) 34-42. 12 Andersson, S., Resell, S., Vjelmquist, V., Chang, D. and Folkers, K., Inhibition of gastric acid and intestinal motor activity in dogs by (Gln4)-NT, Acta. Physiol. Scand., 100 (1977) 231235. 13 Corruzi, G. and Bertacinni, G., Effects of some vasoactive peptides on the lower esophageal sphincter, Pharmacol. Res. Commun., 12 (1980) 965-973. 14 Harper, S.L., Barrowman, J.A., Kvietys, P.R. and Granger, D.N., Effect of neurotensin on intestinal capillary permeability and blood flow, Am. J. Physiol., 247 (1984) G161-166. 15 Flaten, O. and Hanssen, L.E., Concentration of neurotensin in human plasma after glucose, meals and lipids, Acta Physiol. Stand., 114(2) (1982) 311-313.
543
REGPEP 01377
Inhibitory effect of neurotensin on proline Hani A.H. Abd-U1-Salam and Camille F. Nassar Department of Physiology, Faculty of Medicine, American Universityof Beirut, Beirut (Lebanon) (Received 29 October 1992; revised version received 1 February 1993; accepted 8 February 1993)
Key words: Neurotensin; Intestinal absorption
Summary The effect of neurotensin (NT) on proline absorption across rat jejunum was investigated using the singlepass perfusion technique. This study showed that intravenous administration of NT produced a dose-dependent inhibition of proline absorption. Thus, NT at a 0.16 pmol/kg/min concentration gave 10~o decrease in proline absorption while 0.32 and 1.6 pmol/kg/min concentration gave 317o and 45?0 decrease, respectively. In the absence of Na, proline absorption decreased to 77~o from control values. No change in proline absorption was noticed when NT at a concentration of 0.32 pmol/kg/min was intravenously injected in the absence of sodium from the perfusion solution. Water absorption did not show significant changes (P> 0.05) in presence or absence of NT. Moreover, NT did not produce a significant change (P> 0.2) in intracellular proline accumulation. NT inhibited proline absorption through an indirect mechanism that is Na-dependent and independent of changes in water absorption.
Introduction Neurotensin (NT), a tridecapeptide [1] isolated originally from bovine hypothalamus during isolation of substance P [2] is found to be distributed in the central and peripheral nervous system, and the peripheral endocrine system [3]. The gastrointestinal tract is considered as both, the major source of NT Correspondence to: C.F. Nassar, Department of Physiology, Faculty of Medicine, American University of Beirut, Beirut, Lebanon.
(about 85 ~o of total NT in the rat) and an important target organ for this peptide [4]. Immunochemical studies show that gut NT is largely concentrated in apparently secretory cells in intestinal villi and crypts [5]. Dense core vesicles that can be stained histochemically for NT were found in the basolateral portion of the cell [6]. In a recent study, Armstrong et al. [7] noticed that intravenous infusion of NT increased oleic acid translocation across the rat small intestine. In addition, perfusing fatty acids in the intestine caused an
544
increase in plasma concentration of N T [8]. This demonstrated a direct effect of N T on solute absorption across the small intestine. So far, there are no reports on the effect of N T on translocation of amino acids across the rat intestine. This study is an attempt to determine the effect of N T on amino acid absorption and the mechanism involved in this process.
Materials and Methods
In vivo study Animalpreparation. Adult female Sprague-Dawley rats weighing 150-250 g fed ad libitum and fasted one day before the day of experiment were used. The animals were anaesthetized with an intraperitoneal injection of 35 mg of sodium pentobarbital (Nembutal) per kg body weight. A tracheotomy was done on all animals to allow for adequate ventilation. The abdominal cavity was opened by a midline incision and the jejunum was exposed with its vascular supply kept intact. A segment of the jejunum was cannulated by an inlet 5 cm caudal to the ligament of Treitz and an outlet 15-20 cm distal to it. The segment was replaced in the abdomen after removal of the luminal contents by perfusing 50 ml of modified Ringer solution (NaC1 140 raM, K H C O 3 10 mM, K2HPO 4 1.7 raM, KHzPO 4 0.2 mM, MgCI 2 1.2 mM). During all experiments, the perfusate was kept at constant temperature of 37 °C and the jejunal segment was perfused by a peristaltic pump at a constant rate of 0.67 ml/min for a period of 120 min. N T effect on proline absorption was investigated by infusing the rats intravenously with different concentrations of NT ranging between 0.16 and 3.2 pmol/ kg/min. N T was dissolved in 0.9~o NaC1. The effect of N T on proline absorption in the absence of Na was determined by replacing sodium chloride with choline chloride in the perfusion solution. Control rats received 0.9~o NaC1. Measurement of intestinal absorption. The perfusate consisted of modified Ringer solution contain-
ing phenol red (15 rag/l), 1 mM L-proline and trace quantities of its labelled isotope. The effluent solution was collected every 10 min for 120 rain. Proline absorption was calculated from the isotope content of the initial and effluent solutions that was determined by liquid scintillation. Water absorption was determined by measuring phenol red concentration by spectrophotometry at 560 nm. The intestinal segment was resected, blotted on Whatman No. 1 filter paper, its length measured and weighed on a Sartorius balance. Absorption was calculated from the rate of disappearance of the labelled amino acid from the perfusion solution, after correction for water transport.
In vitro study Tissue preparation. Female Sprague-Dawley rats weighing 150-250 g were utilized in all experiments. The rats were anaesthetized by an intraperitoneal injection of 35 mg sodium pentobarbital per kg body weight. The abdomen was opened by a midline incision and the jejunum was removed and placed immediately in oxygenated ice-cold phosphate buffered saline (PBS) solution. The jejunum was freed from the mesenteric and fat attachments and cut longitudinally into strips 1 cm in length. Measurement of intracellular proline concentration. Jejunal strips, each weighing between 50-80 mg, were incubated in oxygenated PB S solution (pH 7.4) containing [laC]proline, and shaked at 60-65 cycles/ rain in water bath at 37°C for 1 h. After the incubation period, the strips were removed from the incubation solution and immediately dipped in icecold isotonic mannitol solution. Each strip was blotted with Whatman No. 1 filter paper, its wet weight determined by a Sartorius balance, and then extracted in 2 ml of 0.1 M H N O 3 for more than 4 h. Aliquots of the tissue extracts and incubation media were counted for their [ lac]proline content in a liquid scintillation counter. The effect of N T on proline uptake across the rat small intestine was determined by preincubating the jejunal strips for 30 min with 3.2 pmol/kg NT in a
545
water bath at 37 ° C. The tissues were then processed in the same manner as described before. ~
LO~
4.
o= 8E3-
Results
In vivo study The effect of N T on intestinal proline absorption: concentration study. The effect of N T was studied by infusing N T in the jugular vein with different concentrations of this peptide ranging between 0.16 pmol/kg/min to 3.2 pmol/kg/min and measuring the absorption of proline at 10 min intervals. The results (Fig. 1) clearly indicate an inhibitory effect of N T which is dose-dependent. The inhibitory effect of N T is significant at concentrations ranging between 0.32 pmol/kg/min and 3.2 pmol/kg/min (P<0.001). The maximal inhibitory effect of N T is noticed at 1.6 pmol/kg/min (P<0.001) and higher doses did not show an increase in this effect ( P > 0.9). Intestinal absorption of proline: combined effect of sodium (Na) and NT. The effect of the absence of sodium in the perfusion solution was also deter-
4:4444
E~ 4
5~
o ~ 2&
5'0
6'0
7(D
80 90 100 Time (min)
110
120
130
Fig. 2. The effect of N T intravenous infusion on proline absorption when the jejunum was perfused with Na-free perfusion solution. Proline absorption was determined every 10 min. until the end of the experiment. Bars represent _+ S.E.M. Symbols: m , control; + , Na-free medium; ×, Na-free medium + 3.2 pmol/kg/min N T concentration.
mined. Proline absorption in a Na-free Ringer solution was determined as described previously, and the results are shown in Fig. 2. It can be noticed that the absence of Na from the perfusion solution caused a 77% decrease in proline absorption. However, intravenous administration of N T at a concentration of 3.2 pmol/kg/min did not alter the level of proline absorption ( P > 0.9) when sodium was removed from the perfusion solution (Fig. 2).
~o 3
aE
a-Control b-Na-free c-Controi~N] d-Na-free÷NT
E u E
5 4
o
50
6'0
70
80 90 100 Time (mm)
110
120
130
L
o 3 ~3
Fig. 1. The effect of N T infusion on proline absorption in the rat jejunum. Proline absorption was determined every 10 rain. until the end of the experiment. Bars indicate + S.E.M. Symbols: • control; + , control+ 0.16 pmol/kg/min N T concentration; O , control + 0.32 pmol/kg/min N T concentration; [~, control + 1.60 pmol/kg/min N T concentration; x , Control + 3.20 pmol/kg/min N T concentration.
L 2
O
-
-
Fig. 3. Effect of 3.2 pmol/kg/min N T on water absorption in the presence and absence of Na ÷ from the perfusion medium.
546
Effect of N T on water absorption. Water absorption was measured in the presence and absence of Na. The effect of N T was determined in both sets of experiments. N T showed no effect on water transport neither in the presence (P>0.05) nor in the absence of Na ( P > 0.05) (Fig. 3). Accordingly, the change in proline absorption noticed after NT administration was not secondary to changes in water transport but most probably was due to a direct effect on proline absorption. In vitro study Measurement of intracellular proline accumulation. The effect of N T on proline uptake was investigated in vitro. Jejunal strips were preincubated in 3.2 pmol/kg N T for 30 min, then incubated in 1 mM proline for 1 h. The incubation solution was oxygenated and kept at 37 °C in a water bath. The results indicate that there is no significant difference ( P > 0.2) between proline uptake by the intestinal strips in the presence and absence of 3.2 pmol/kg NT. The fact that the In/Out ratio measured in the intestinal cells preincubated with N T reached 5.90 which is not significantly different ( P > 0.2) from the control value (5.85) suggests that the active component of the transport process was not affected by preincubating the tissues with NT. Discussion Intestinal epithelial ceils have the ability to concentrate amino acids against an electrochemical gradient. Previous studies indicate that proline absorption follows three pathways; an imino carrier specific for proline responsible for 60~o of total transport, a non-specific neutral system believed to be the ASC system responsible for 35 ~o of the total uptake, and the remaining 5~o are due to diffusional processes [ 9]. Hence, the active component which accounts for 95 ~o of total transport is Na-dependent. Our results show an inhibition of proline uptake in the absence of Na equivalent to around 7 7 ~ , which would corroborate previous reports.
N T is a peptide that has several effects at various sites in the body. It causes an increase in glucagon level as well as a decrease in insulin level in serum [ 10]. In the gastrointestinal tract, N T has been shown to increase contractility of longitudinal muscle of isolated guinea pig ileum [ 11 ] as well as to decrease gastric motility [ 12] and lower esophageal sphincter pressure [13]. In addition, N T increases vascular permeability in feline ileum [14]. In vitro, binding studies show high affinity of NT binding to membranes from guinea pig ileum [ 11 ]. This work is an attempt to elucidate the mechanism by which NT regulates proline absorption across the small intestine. Our data indicate that NT inhibits proline uptake at a concentration of 1.6 pmol/kg/min. Higher doses did not have a greater effect. The concentrations used in this study are within the range of N T concentration in human plasma. The fasting concentration of NT in plasma was reported as 1 pM and it increased to 7.2 pM after a lunch meal [15]. N T at its maximal effect inhibits up to 46~o of total proline uptake in vivo. This suggests that the N T effect could be largely on the Na-dependent imino carrier which is resposible for 60~o of the total uptake [9]. However, N T has no effect on proline absorption in the absence of Na. This could suggest that N T effect is a Na-mediated process. There exists around 75 ~o inhibition of proline uptake when Na is removed from our perfusion solution. This may be due to the inactivation of the Na-dependent imino carrier as well as the Nadependent non-specific proline uptake mechanisms. Our results in vivo study showed no effect of NT when it was incubated with the intestinal strips. However, N T is a large polypeptide molecule that makes it impermeant to the intestinal cell membrane. Hence, it could be suggested - on theoretical basis - that N T did not cross the brush border membrane and did not cause any conformational changes in the imino acid carrier, and therefore no effect was noticed. However, when N T was administered intravenously, a definite inhibitory effect on proline absorption was noticed. This inhibition is produced
547
due probably to an effect of NT or one or more of its metabolites on the basolateral membrane. In vitro binding studies show higher affinity specific binding to membranes of the guinea pig ileum [ 11]. Data indicate that the NT modulatory effect is a saturable process suggesting saturation of binding sites at the basolateral membrane inducing intracellular mechanisms that alter the imino carrier activity. This implies that binding of NT on the basolateral membrane may have caused functional and/or structural changes that produced proline inhibition. Moreover, since the absence of Na alleviated this effect of NT, it could be suggested also that NT is causing this inhibition through a Na-dependent regulatory mechanism. The effect of NT on water transport was determined. Our results show that NT had no effect on water transport across the rat jejunum. This implies that the observed changes in proline uptake were not secondary to changes in water transport. Accordingly, it could be suggested that proline absorption occurs via a nonsolvent drag mechanism, and that the effect of NT on proline uptake is not translated through a change in water transport. Moreover, since NT causes an increase of glucagon level and a decrease of insulin in blood, it could imply that the mechanism of inhibition is mediated through the release of a hormone or any other mediator that has a modulatory effect on the imino carrier. In conclusion, NT inhibits proline absorption through an indirect mechanism that is Na-dependent and independent of change in water absorption.
References 1 Carraway, R. and Leeman, S.E., The synthesis ofneurotensin, J. Biol. Chem., 250 (1975) 1912-1918.
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