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Characterization of -casozepine, a tryptic peptide from bovine _s1-casein with benzodiazepine-like activity¹

LAURENT MICLO^*2, EMMANUEL PERRIN^*, ALAIN DRIOU^*, VASSILIOS PAPADOPOULOS, NOUREDDINE BOUJRAD^,3, RÉGIS VANDERESSE^¶, JEAN-FRANÇOIS BOUDIER^||, DIDIER DESOR^**, GUY LINDEN^* and JEAN-LUC GAILLARD^*

^* Laboratoire des Biosciences de l’Aliment UA 885 INRA, Faculté des Sciences, Université Henri Poincaré-Nancy 1, Vandoeuvre-lès-Nancy, France;
Departments of Cell Biology, Pharmacology and Neuroscience, Georgetown University Medical Center, Washington, D.C. 20007, USA;
^¶ Laboratoire de Chimie Physique Macromoléculaire UMR CNRS, ENSIC, Nancy, France;
^|| Ingredia, Arras, France; and
^** Laboratoire des Aspects Fonctionnels et du Développement des Comportements URA 1293 CNRS, Faculté des Sciences, Université Henri Poincaré-Nancy 1, Vandoeuvre-lès-Nancy, France

²Correspondence: LBSA, Faculté des Sciences, Université Henri Poincaré-Nancy 1, B.P. 239, 54506 Vandoeuvre-lès-Nancy, France. E-mail: Laurent.Miclo{at}scbiol.uhp-nancy.fr

SPECIFIC AIMS

According to folk wisdom, milk intake improves sleeping or has a calming role, a belief supported by some scientific studies. As caseins contain peptides with physiological roles, we hypothesized that one of their peptides can carry this anxiolytic activity. In this study, _s1-casein tryptic hydrolysate (_s1-CnTH) was tested in vitro on benzodiazepine (BDZ) receptors and checked in vivo for its anticonvulsant and anxiolytic effects in the rat.

PRINCIPAL FINDINGS

1. _s1-CnTH inhibits pentylenetetrazole (PTZ) -induced seizures in Wistar rat
Bovine _s1-CnTH was injected intraperitoneally (i.p.) in a Wistar rat 30 min before an i.p. injection of 60 mg/kg of PTZ to evaluate anticonvulsant activity. Crisis severity parameters, crisis latency, and clonus duration were determined after 45 min observation of behavior. The i.p. injection of 1 mg/kg of _s1-CnTH reduced crisis severity (P<0.02). Other parameters were not significantly different from the control. The i.p. injection of 3 mg/kg of _s1-CnTH increased crisis latency (P<0.005), decreased crisis severity (P<0.002), and decreased clonus duration (P<0.005) of PTZ-induced seizures. The anticonvulsant action of the _s1-CnTH was underestimated because of a sensitization of animals to PTZ during successive experiments.

2. _s1-CnTH displays an anxiolytic activity in the elevated plus maze paradigm
Elevated plus maze test was used to evaluate the anxiolytic effect of the _s1-CnTH. Diazepam (1 mg/kg) and _s1-CnTH (3 mg/kg) enhanced (P<0.02) the percentage of entries in the open arms whereas general activity, represented by entries in closed arms, was not modified (Fig. 1 ).

View larger version (39K): [in this window] [in a new window]   Figure 1. Effect of the s1-CnTH on the performance of Wistar rats in the elevated plus maze paradigm compared with diazepam. 9 NaCl (CTR), 1 mg/kg of diazepam (DZP), or 3 mg/kg of s1-CnTH (TH) were injected by i.p. route 30 min before the experiment. Behavior of animals (n=20, each group) was tape-recorded for 5 min. Values are A) the mean ± SE of the number of entrances into the closed arms of the plus maze; B) the mean ± SE of the percentage of entrances into the open arms (entrances into the open arms to the total entrances). **P < 0.02 by ANOVA.

3. _s1-CnTH displays an anxiolytic activity in the conditioned defensive burying (CDB) experiment
When a rat is shocked once through an electrical probe mounted on a wall of a test cage, it returns to the probe and buries it with bedding material from the floor of the cage. This CDB response is reduced by anxiolytic drugs. Some behavioral sequences such as exploratory approaches to the probe and escape movements from the probe are needed to differentiate agonists from partial inverse agonists. Similar to diazepam, the _s1-CnTH, administrated at 3 mg/kg i.p., decreased the duration of probe burying (P<0.005) (Fig. 2 ). The ratio of the retreats to the approaches decreased in rats treated with _s1-CnTH or diazepam. The CDB paradigm indicates that the _s1-CnTH exerts an anxiolytic activity over rats.

View larger version (28K): [in this window] [in a new window]   Figure 2. Effect of the s1-casein-(f91–100) and s1-CnTH on conditioned defensive burying in the Wistar rat vs. diazepam. 9 NaCl (CTR), 1 mg/kg of diazepam (DZP), 3 mg/kg of s1-CnTH (TH), or 0.4 mg/kg of s1-casein-(f91–100) (PEP) were administered i.p. (n=12, each group) 30 min before the burying test began. Animals’ behavior was tape-recorded for 5 min. Values are A) the mean ± SE of time (seconds) spent to bury the probe; B) the mean ± SE of the percentage of approaches toward the probe followed by retreats (number of retreats to number of approaches). **P < 0.01; ***P < 0.005 by ANOVA.

4. A peptide (-casozepine) of the _s1-CnTH binds on the BDZ site of the GABA_A receptor
Affinity for the BDZ site of the GABA_A receptor from bovine cerebral cortex membranes was measured in competition with [methyl-³H]-flunitrazepam. _s1-CnTH competed with the radioligand for binding on the BDZ site of the GABA_A receptor with an IC₅₀ of 72 µM and a Hill number of 0.8. All peptides of the _s1-CnTH were purified and tested in competition with the radioligand. Only one peptide exhibited affinity for the BDZ site of the GABA_A receptor. The sequence and mass of this peptide corresponded to those of the _s1-casein-(f91–100); it has consequently been named -casozepine. This purified peptide competed against [methyl-³H]-flunitrazepam in binding assays withan IC₅₀ of 88 µM and a Hill number of 0.8 compared to an IC₅₀ value for diazepam of 8.2 nM determined under the same conditions. The synthetic -casozepine also displaced the radioligand from the BDZ site of GABA_A receptor, but was fourfold less potent than the milk-purified peptide. These findings indicate that the _s1-casein-(f91–100) carries the activity of the _s1-CnTH.

5. The -casozepine exhibits an anxiolytic activity in vivo
-Casozepine was tested in the CDB paradigm. This peptide administrated i.p. at 0.4 mg/kg (0.32 µmol/kg) decreased the duration of probe burying compared to saline (P<0.005) (Fig. 2) . Other behavioral parameters show the same modifications as observed with the _s1-CnTH. Effects of -casozepine were comparable to those induced by diazepam injected i.p. at 1 mg/kg (3.5 µmol/kg). The -casozepine seems to be 10-fold more active than diazepam in vivo despite the lower binding affinity of the tryptic fragment for the BDZ site of the GABA_A receptor in vitro.

6. Effects of -casozepine are not mediated by the peripheral-type BDZ receptor (PBR)
PBR are a second class of BDZ binding receptor with predominant mitochondrial localization. The possibility that the peptide would act via the PBR was examined. The binding ability of -casozepine to PBR was tested on MA-10 mouse Leydig cell mitochondrial preparation in competition experiments against two specific ligands of PBR. No displacement of these ligands was observed, indicating that -casozepine did not bind to PBR. Moreover, -casozepine did not significantly affect progesterone production by MA-10 mouse Leydig cells. These results exclude the possibility for PBR to be a target of the -casozepine and suggest that this peptide is specific for the BDZ site of the GABA_A receptor.

CONCLUSIONS

Cow’s milk has long been considered a tranquilizing beverage with sleep-inducing capacity, but the molecular bases of this potential action are not known. Cow or human breast milks contain BDZ-like molecules, even in the absence of BDZ administration. However, the concentrations of these milk BDZ-like compounds are very low—only a few micrograms per liter. This study demonstrated that a peptide stemming from the hydrolysis of _s1-casein by trypsin, the only protease of the gastrointestinal tract that is in equal concentration and activity in the newborn and adult, exhibited a BDZ-like activity. In two behavioral tests, elevated plus maze test and CDB paradigm, _s1-CnTH exhibited the characteristic profile of full BDZ agonists such as diazepam. Moreover, _s1-CnTH suppressed the inhibitory effect of PTZ on the GABAergic system. These results were consistent with the binding of one of the peptides of the _s1-CnTH to the BDZ site of the GABA_A receptor. This peptide, named -casozepine, corresponds to the _s1-casein-(f91–100) (Fig. 3 ). Despite a lower in vitro affinity than diazepam for the BDZ site of the GABA_A receptor, -casozepine might be 10-fold more active in vivo than the BDZ. Although -casozepine displays the characteristic anxiolytic and anticonvulsant effects of BDZ, it does not trigger any side effects. The lack of affinity of -casozepine for PBR and its small effect on steroidogenesis exclude this way of action. Whereas the present results clearly show that -casozepine is acting via the GABA_A receptor, the difference of efficiency between in vivo and in vitro experiments remains unclear. A physiological relevance of modulation of the GABAergic system through the BDZ site of the GABA_A receptor implies the presence of an endogenous ligand.

View larger version (18K): [in this window] [in a new window]   Figure 3. Assessment of biological activity of bovine s1-CnTH and of -casozepine.

Until now, attempts to identify an endogenous agonist ligand of the BDZ site of the GABA_A receptor have failed. A 86 residue endogenous peptide called diazepam binding inhibitor (DBI) inhibits competitively the binding of BDZ to the GABA_A receptor with an IC₅₀ of 4 µM. DBI is the precursor of two peptides, ODN [DBI-(f33–50)] and TTN [DBI-(f17–50)], that carry the BDZ receptor binding activity. However, DBI and its fragments have effects opposite to those of BDZ-like agonists. In behavioral paradigms, DBI and its fragments are anxiogenic, which means they are anxiety factors. Moreover, DBI displays epileptogenic activity. A comparison of the carboxyl-terminal region of DBI (residues 73 to 82) from different species and bovine -casozepine shows a great homology of sequence and structure. This region of DBI does not relate to the ODN or the TTN peptides. The region 73–82 of DBI belongs to the fragment 66–83, which is predicted to form a helix. The fragment 91–100 of the bovine _s1-casein is predicted to insert in a helical region, whereas only 15% of the _s1-casein residues belong to -helix structures. We have shown that the bovine DBI-(f73–82) injected i.p. at 1 mg/kg in Wistar rats displayed an anxiolytic activity in the plus maze experiment. At this time, we cannot conclude whether the DBI-(f73–82) can be generated by in vivo hydrolysis of DBI.

Casein from bovine milk contains a latent peptide with BDZ-like properties that may be released upon proteolytic hydrolysis, which could explain the calming properties attributed to this beverage. This peptide, which displays strong in vivo anxiolytic activity in the rat, is homologous to a peptide of the carboxyl-terminal region of DBI. Our results also raised the possibility that the carboxyl-terminal part of DBI could act as an agonist of physiological importance in regulating anxiety. Moreover, this suggests that bovine -casozepine, or the equivalent in other mammal species, may act as an exogenous ligand for the BDZ site of the GABA_A receptor in newborn brain, thus regulating anxiety in the newborn.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0685fje ; to cite this article, use FASEB J. (June 8, 2001) 10.1096/fj.00-0685fje

³ Present address: Endocrinologie Moléculaire de la Reproduction UMR 6026 CNRS Interactions Cellulaires et Moléculaires, Université de Rennes 1, Campus de Beaulieu, 35042 Rennes Cedex, France.

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