Blockade of 12-lipoxygenase expression protects cortical neurons from apoptosis induced by beta-amyloid peptide.

The cyclo-oxygenase (COX) and lipoxygenase (LOX) pathways belong to the eicosanoid synthesis pathway, a major component of the chronic inflammatory process occurring in Alzheimer's disease (AD). Clinical studies reported beneficial effects of COX inhibitors, but little is known about the involvement of LOXs in AD pathogenesis. beta-amyloid peptide (A beta) accumulation contributes to neurodegeneration in AD, but mechanisms underlying A beta toxicity have not been fully elucidated yet. Here, using an antisense oligonucleotide-based strategy, we show that blockade of 12-LOX expression prevents both A beta-induced apoptosis and overexpression of c-Jun, a factor required for the apoptotic process, in cortical neurons. Conversely, the 12-LOX metabolite, 12(S)-HETE (12(S)-hydroxy-(5Z, 8Z, 10E, 14Z)-eicosatetraenoic acid), promoted c-Jun-dependent apoptosis. Specificity of the 12-LOX involvement was further supported by the observed lack of contribution of 5-LOX in this process. These data indicate that blockade of 12-LOX expression disrupts a c-Jun-dependent apoptosis pathway, and suggest that 12-LOX may represent a new target for the treatment of AD.

Effects of dexamethasone and forskolin on neurotensin production in rat hypothalamic cultures.

In the present study, the effects of glucocorticoids and forskolin, an activator of adenylate cyclase, were examined on neurotensin (NT) production from rat hypothalamic neurons in primary culture. Treatment with dexamethasone induced a dose-dependent increase in NT content. The maximum was reached at 1 microM dexamethasone, which induced a 100% increase in NT levels. The effect of dexamethasone was mimicked by the glucocorticoid agonist RU28362 and blocked by the antiglucocorticoid RU38486, suggesting that this effect was mediated through the glucocorticoid receptor. The treatment with dexamethasone also enhanced the number of immunoreactive NTergic cells (92% increase). In contrast to dexamethasone, forskolin affected neither the NT content nor the number of immunoreactive NTergic cells. However, when cells were treated with both dexamethasone and forskolin, a 285% increase in NT content and a 430% increase in the number of immunoreactive NTergic cells were observed, representing 2.8- and 4.7-fold increases, respectively, compared to the effect of dexamethasone alone. Moreover, this combined treatment increased the accumulation of NT in the culture medium (160% increase) as well as the abundance of NT messenger RNA. We conclude from the present findings that dexamethasone and forskolin act synergistically to enhance NT production in hypothalamic neurons.

Subcellular fractionation of pituitary neurointermediate lobes: revelation of various basic proteases.

Homogenates of rat neurointermediate lobes were purified by centrifugation on a Percoll gradient. Lysates of the Percoll gradient fractions were incubated with a synthetic octapeptide and pentapeptide substrate (N-acetyl Lys-Arg-Tyr-Asn-Leu-Thr-Ser-Val-amide and N-acetyl Lys-Arg-Tyr-Asn-Leu-amide), and enzymatic characteristics were compiled. Early assays on nonamidated forms revealed carboxypeptidase activity, whereas with the amide derivatives no carboxypeptidase activity could be detected. These amide substrates were therefore used in all subsequent incubations. High levels of a Leu/Thr and Lys/Arg cleavage were present in fractions almost throughout the Percoll gradient. Cleavages at Tyr/Asn, Thr/Ser, and Arg/Tyr were localized at different regions of the Percoll gradient. Surprisingly, none of the five enzymatic activities appear to be localized in the secretory granule fractions as defined by the presence of immunoreactive beta-endorphin in the gradient. All of the five proteolytic activities have a basic pH optimum (pH 8-9), and four of them seem to be thiol proteases, as categorized by inhibitor studies. The fifth one, namely the Try/Asn cleavage, is more likely to be due to a metalloendopeptidase, since it is activated by Zn2+ and Co2+.

Enzymatic cleavage of pro-opiomelanocortin by anterior pituitary granules. Evidence for a chymotryptic-like enzyme.

This paper presents the results obtained when pig anterior pituitary granule lysates are incubated with rat pro-opiomelanocortin (POMC). The resultant peptides were analyzed by 3 systems of high-performance liquid chromatography. This approach, when coupled with microsequence analysis of the conversion products, allowed the unambiguous identification of a major chymotryptic-like activity (pH optimum around 8) associated with the granule lysates with a specificity directed towards selective Tyr-X and Phe-X bonds within the POMC molecule. Furthermore, these results also demonstrate that although the detected enzyme activity gives rise to products 'resembling' those expected, the characterization of the 'elusive' maturation enzyme responsible for the cleavage at pairs of basic residues remain to be critically evaluated.

Cellular distribution of neurotensin receptors in rat brain: immunohistochemical study using an antipeptide antibody against the cloned high affinity receptor.

Receptors for the neuropeptide, neurotensin, were localized by immunohistochemistry in the rat brain by using an antibody raised against a sequence of the third intracellular loop of the cloned high affinity receptor. Selective receptor immunostaining was observed throughout the brain and brainstem. This immunostaining was totally prevented by preadsorbing the antibody with the immunogenic peptide. The regional distribution of the immunoreactivity conformed for the most part to that of [3H]- or [125I]-neurotensin binding sites previously identified by autoradiography. Thus, the highest levels of immunostaining were observed in the islands of Calleja, diagonal band of Broca, magnocellular preoptic nucleus, pre- and parasubiculum, suprachiasmatic nucleus, anterodorsal nucleus of the thalamus, substantia nigra, ventral tegmental area, pontine nuclei and dorsal motor nucleus of the vagus, all of which had previously been documented to contain high densities of neurotensin binding sites. There were, however, a number of regions reportedly endowed with neurotensin binding sites, including the central amygdaloid nucleus, periaqueductal gray, outer layer of the superior colliculus and dorsal tegmental nucleus, which showed no or divergent patterns of immunostaining, suggesting that they might be expressing a molecularly distinct form of the receptor. At the cellular level, neurotensin receptor immunoreactivity was predominantly associated with perikarya and dendrites in some regions (e.g., in the basal forebrain, ventral midbrain, pons and rostral medulla) and with axons and axon terminals in others (e.g., in the lateral septum, bed nucleus of the stria terminalis, neostriatum, paraventricular nucleus of the thalamus and nucleus of the solitary tract). These data indicate that neurotensin may act both post- and presynaptically in the central nervous system and confirm that some of its effects are exerted on projection neurons. There were also areas, such as the cerebral cortex, nucleus accumbens and para- and periventricular nucleus of the hypothalamus, which contained both immunoreactive perikarya/dendrites and axon terminals, consistent with either a joint association of the receptor with afferent and efferent elements or its presence on interneurons. Taken together, these results also suggest that the neurotensin high affinity receptor protein is associated with a neuronal population that is more extensive than originally surmised from in situ hybridization studies.

High-affinity neurotensin receptors in the rat nucleus accumbens: subcellular targeting and relation to endogenous ligand.

Neurotensin is present in selective mesolimbic dopaminergic projections to the nucleus accumbens (NAc) shell but also is synthesized locally in this region and in the motor-associated NAc core. We examined the electron microscopic immunolabeling of the high-affinity neurotensin receptor (NTR) and neurotensin in these subdivisions of rat NAc to determine the sites for receptor activation and potential regional differences in distribution. Throughout the NAc, NTR immunoreactivity was localized discretely within both neurons and glia. NTR-labeled neuronal profiles were mainly axons and axon terminals with diverse synaptic structures, which resembled dopaminergic and glutamatergic afferents, as well as collaterals of inhibitory projection neurons. These terminals had a significantly higher numerical density in the NAc core than in the shell but were prevalent in both regions, suggesting involvement in both motor and limbic functions. In each region, neurotensin was detected in a few NTR-immunoreactive axon terminals and in terminals that formed symmetric, inhibitory type synapses with NTR-labeled somata and dendrites. The NTR labeling, however, was not seen within these synapses and, instead, was localized to segments of dendritic and glial plasma membranes often near excitatory type synapses. Neuronal NTR immunoreactivity also was associated with cytoplasmic tubulovesicles and nuclear membranes. Our results suggests that, in the NAc shell and core, NTR is targeted mainly to presynaptic sites, playing a role in the regulated secretion and/or retrograde signaling in diverse, neurotransmitter-specific neurons. The findings also support a volume mode of neurotensin actions, specifically affecting excitatory transmission through activation of not only axonal but also dendritic and glial NTR.

Immunologic differentiation of two high-affinity neurotensin receptor isoforms in the developing rat brain.

Earlier studies have demonstrated overexpression of NT1 neurotensin receptors in rat brain during the first 2 weeks of life. To gain insight into this phenomenon, we investigated the identity and distribution of NT1 receptor proteins in the brain of 10-day-old rats by using two different NT1 antibodies: one (Abi3) directed against the third intracellular loop and the other (Abi4) against the C-terminus of the receptor. Immunoblot experiments that used Abi3 revealed the presence of two differentially glycosylated forms of the NT1 receptor in developing rat brain: one migrating at 54 and the other at 52 kDa. Whereas the 54-kDa form was expressed from birth to adulthood, the 52-kDa form was detected only at 10 and 15 days postnatal. Only the 52-kDa isoform was recognized by Abi4. By immunohistochemistry, both forms of the receptor were found to be predominantly expressed in cerebral cortex and dorsal hippocampus, in keeping with earlier radioligand binding and in situ hybridization data. However, whereas Abi4 immunoreactivity was mainly concentrated within nerve cell bodies and extensively colocalized with the Golgi marker alpha-mannosidase II, Abi3 immunoreactivity was predominantly located along neuronal processes. These results suggest that the transitorily expressed 52-kDa protein corresponds to an immature, incompletely glycosylated and largely intracellular form of the NT1 receptor and that the 54-kDa protein corresponds to a mature, fully glycosylated, and largely membrane-associated form. They also indicate that antibodies directed against different sequences of G-protein-coupled receptors may yield isoform-specific immunohistochemical labeling patterns in mammalian brain. Finally, the selective expression of the short form of the NT1 receptor early in development suggests that it may play a specific role in the establishment of neuronal circuitry.

Role of endogenous neurotensin in the behavioral and neuroendocrine effects of cocaine.

The present experiments were designed to assess the role of endogenous neurotensin (NT) in the behavioral response to acute and daily cocaine, after administration of the NT receptor antagonist, SR 48692. Given that glucocorticoids increase the sensitivity to the psychomotor effects of drugs of abuse, we also investigated the effects of SR 48692 on basal and cocaine-induced corticosterone secretion. Acute administration of SR 48692 (1 mg/kg i.p.) reduced the number of rearings induced by cocaine (15 mg/kg i.p.), without modifying horizontal activity. Repeated pretreatment with SR 48692 (1 mg/kg x 5 days) markedly reduced locomotion and rearings after an acute cocaine challenge (day 1), whereas the lower dose of SR 48692 (0.1 mg/kg) had no effect. SR 48692 (1 mg/kg), given daily before cocaine, also decreased cocaine-induced rearing on day 2, but had no effect on the following drug challenges (days 3-10). One week after discontinuing repeated cocaine injections, SR 48692 blocked vertical, but not horizontal, activity induced by an acute cocaine challenge. Rats treated repeatedly with cocaine showed an enhanced behavioral response characterized by the development of stereotypes, which were unaffected by SR 48692. Finally, treatment with SR 48692 did not alter corticosterone circadian secretion nor cocaine-stimulated corticosterone levels, indicating that the attenuation of the behavioral effects of cocaine after NT receptor blockade is not associated with blunted glucocorticoid secretion. These results indicate that administration of SR 48692 attenuates the locomotion and rearing response to cocaine but fails to modify stereotyped behavior, suggesting that SR 48692 modulates the behavioral effects of psychostimulant drugs by acting selectively on the mesolimbic dopaminergic system.

DNA intercalating compounds as potential antitumor agents. 1. Preparation and properties of 7H-pyridocarbazoles.

The DNA intercalating compounds derived from 6H-pyridocarbazole (ellipticines, olivacines) elicit high antitumor properties. In order to get information about the mechanism of action of these agents it is necessary to study structurally related analogues. For this purpose, various derivatives of the four isomeric 7H-pyridocarbazoles were synthesized by a single photochemical process on indolylpyridylethylenes. These derivatives are able to intercalate into DNA. The DNA binding affinities vary in the range of 10(4) to 10(6) M-1, depending mainly on the nature of the substituent, nitrogen quaternization being the most enhancing factor. The position of the pyridinic nitrogen does not markedly affect the DNA binding affinity. Three quaternized compounds elicit a significative but low antileukemic activity on L1210 mice leukemia. The properties of 7H-pyridocarbazoles are discussed and compared to those of 6H-pyridocarbazoles (ellipticines and olivacines).

DNA intercalating compounds as potential antitumor agents. 2. Preparation and properties of 7H-pyridocarbazole dimers.

In order to obtain antitumor agents, various 7H-pyridocarbazole dimers were prepared by quaternization of the pyridinic nitrogens of the different isomeric 7H-pyridocarbazole rings with halogenoamino alicyclic or aliphatic chains. The dimers interact with DNA more markedly than with the corresponding monomers, and the bisintercalation depends upon the nature, the flexibility, and the ionization state of the linking chains. They most often bisintercalate at pH 5 where the chain is protonated and monointercalate at pH 7.4. The apparent binding constants (kap) range from 10(8) to 10(9) M-1 at pH 5 and from 5 X 10(5) to 2 X 10(7) M-1 at pH 7.4. The bisintercalating dimers covered four DNA base pairs, whereas most of the monointercalating dimers covered two bases pairs. The antitumor activity against L1210 murine leukemia is strongly dependent on the position of attachment, the nature of the linking chain, and its rigidity. Three highly active dimers were obtained in the series of 7H-pyrido[4,3-c]carbazole dimers with rigid bis(ethylpiperidinyl) chains. On the other hand, two ellipticine dimers were prepared which were found completely inactive on L1210. These results show that in the series of 7H-pyridocarbazoles the process of dimerization leads to very active antitumor compounds.

Full agonists of CCK8 containing a nonhydrolyzable sulfated tyrosine residue.

The sulfate ester of CCK26-33 or CCK8 (Asp-Tyr(SO3H)-Met-Gly-Trp-Met-Asp-Phe-NH2) borne by the tyrosine residue is a critical determinant of the biological activity of this peptide. In order to increase the stability of this molecule, the sulfated tyrosine has been replaced by a synthetic amino acid (L,D)Phe(p-CH2SO3Na) in which the OSO3H group was replaced by the nonhydrolyzable CH2SO3H group. Both isomers were separated by chromatography and the stereochemistry of the Phe(p-CH2SO3Na) residue in each peptide was established by NMR spectroscopy. The biological activities of the new derivatives Ac[X27, Nle28,Nle31]CCK27-33 were compared with those of Boc[Nle28,Nle31]CCK27-33, an equiactive analogue of CCK8 and Boc[D-Tyr(SO3Na)27,Nle28,Nle31]CCK27-33. Besides their highly enhanced chemical stability, Ac[L-Phe(p-CH2SO3Na)27,Nle28,Nle31]CCK27-33 and Ac[D-Phe(p-CH2SO3Na)27,Nle28,Nle31]CCK27-33 display high affinity for peripheral and central CCK receptors (KI congruent to 10(-9) M) and proved to be full agonists in the stimulation of pancreatic secretion as well as in the in vitro CCK8-induced contractions of the guinea pig ileum.

Synthesis and biological activity of CCK26-33-related analogues modified in position 31.

The role of the amino acid in position 31 of cholecystokinin CCK26-33 in the recognition of central and peripheral receptors was investigated by replacement of methionine-31 by amino acids with side chains of various chemical nature. Thus, phenylalanine, alanine, glutamic acid, and ornithine and its analogue with the epsilon-amino group protected by a benzyloxycarbonyl group were introduced as X residues in Boc(Nle28,X31)-CCK27-33 since the related analogue Boc(Nle28,Nle31)-CCK27-33 was shown to be equipotent to CCK26-33. The binding properties to both mouse brain membranes and guinea pig pancreatic acini and the peripheral activities (amylase secretion and contractile potency on guinea pig ileum) were determined. Whereas the introduction of phenylalanine, alanine, or ornithine residues in position 31 led to compounds that still displayed peripheral agonist properties, the presence of a negative charge in the side chain of the amino acid in position 31 prevented the binding of the peptide to both pancreatic and brain binding sites. Introduction of Phe31 and Ala31 residues increased the specificity of the peptides for the central receptors. Interestingly, when the amine function in the side chain of the ornithine-31 was protected by a benzyloxycarbonyl group, an unusual high affinity for pancreatic binding sites was observed and the related analogue proved to be a new peripheral CCK antagonist.

Pharmacological and molecular characterization of the neurotensin receptor expressed in Sf9 cells.

The rat neurotensin receptor was expressed in Spodoptera frugiperda insect (Sf9) cells using infection with a recombinant baculovirus. Immunoblot experiments performed with an antibody raised against the C-terminus of the receptor showed major bands at 47 (corresponding to the unglycosylated receptor protein) and 50 kDa, and minor bands at 65 and 36 kDa. The expressed receptor bound 125I-neurotensin with high affinity, was coupled to endogenous G-proteins, and agonist-induced inositol phosphate production was observed at early times after infection. These results show that the rat neurotensin receptor retains functional properties when expressed in the heterologous insect cell system.

In vivo regulation of neurotensin receptors following long-term pharmacological blockade with a specific receptor antagonist.

Adaptive changes in brain neurotensin (NT) receptors were investigated in rats after repeated administration of SR 48692, a potent and selective non-peptide NT receptor antagonist. Administration of SR 48692 (1 mg/kg i.p.) for 15 days did not alter NT content in the brain but highly enhanced the expression of NT receptor mRNA as shown by quantitative in situ hybridization. The increase of the signal was observed in numerous areas of the brain, such as the anterior cingulate, perirhinal and retrosplenial cortices, the suprachiasmatic nucleus, the ventral tegmental area, the substantia nigra and the posterior cortical nucleus of the amygdaloid complex. Moreover, the SR 48692 treatment induced the expression of NT receptor mRNA in several nuclei of the diencephalon where it could not be detected in basal conditions. Immunoblot analysis with a specific antibody directed against the rat cloned NT receptor revealed an important increase in NT receptor protein in the brain of SR 48692-treated rats, correlating well with the increase in NT receptor mRNA levels. Surprisingly, the number and the affinity constant of NT binding sites determined on brain membrane homogenates remained unchanged after SR 48692 treatment, even after membrane permeabilization with low concentrations of digitonin. These results suggest that chronic treatment with a specific NT antagonist induces an up-regulation of NT receptors at the level of mRNA and protein. Moreover, they indicate that after a chronic treatment with SR 48692, the number of NT binding sites remains stable in contrast to what is observed after 5-day treatment or with central monoaminergic receptor following their long-term blockade.

Neurotensin receptor down-regulation induced by dexamethasone and forskolin in rat hypothalamic cultures is mediated by endogenous neurotensin.

Neurotensin (NT) has been shown to be involved in neuroendocrine regulation, and the presence of both the peptide and its receptors has been demonstrated in the hypothalamus. In the present study, we show that hypothalamic neurons in primary cultures express the neurotensin receptor (NTR) and we examined a possible regulation of this receptor by glucocorticoids and activators of adenylate cyclase. In the hypothalamic cultures, 125I-NT bound to a single class of binding sites, presenting a selectivity similar to that observed for the high-affinity NTR previously described in the adult rat brain. Radioautographic studies demonstrated that these 125I-NT binding sites were present on 3% of the neurons. A 48-h treatment with forskolin (fsk) decreased 125I-NT binding by 30%. No effect of dexamethasone (dex) alone was found on that parameter. However, a combined treatment with both agents led to a 40% decrease in 125I-NT binding, corresponding to a reduced number of binding sites, and to a 68% decrease in the amount of NTR mRNA. In parallel, the dex plus forsk treatment increased NT release in the incubation medium. Moreover, the decreases in 125I-NT binding and NTR mRNA induced by this treatment were abolished in the presence of an anti-NT antibody or SR 48692, a non-peptidic antagonist of NTR, suggesting that the down-regulation of NTR observed after dex plus fsk treatment was mediated by the release of endogenous NT. Agonist-induced down-regulation of the NTR in this system was confirmed by the application of an exogenous NT analogue, JMV 449. The present findings indicate that, in hypothalamic cultures, dex and fsk indirectly down-regulate NTR expression via the release of endogenous NT.

Autoradiographic localization of cholecystokinin binding sites in human cerebellar system using a [(125)I]CCK8 probe.

Carboxyl-terminal cholecystokinin octapeptide (CCK8) binding sites were studied in the human cerebellar system by autoradiography. High affinity CCK8 binding sites were demonstrated in the main cerebellar afferent nuclei, namely the inferior olivary complex and the pontine nuclei. This localization of CCK8 binding sites was partly correlated with already described CCK containing terminals. In the cerebellar cortex, high affinity CCK8 binding sites were detected with a laminar distribution. Levels were higher in the granular layer (mostly in the superficial part) and lower in the white matter and the Purkinje cell layer. The non-specific binding was homogenous and particularly low (9%) in the cerebellar cortex but a non-specific binding was selectively localized in the deep cerebellar nuclei. Those results illustrate the species variability of CCK binding sites in the cerebellum and are briefly discussed in relation with the low level of CCK immunoreactivity in this structure. The presence of CCK8 binding sites in cerebellar afferent nuclei and cortex suggests a role of CCK in human cerebellar physiology and particularly in the modulation of afferent inputs to the cerebellum.

Autoradiography of CCK receptors in the rat brain using [(3)H]Boc[Nle(28)(31)]CCK(27)-(33) and [(125)I]bolton-hunter CCK(8). Functional significance of subregional distributions.

[(3)H]Boc[Nle(28,31)]CCK(27)-(33) ([(3)H]BDNL-CCK(7)) is a new ligand for cholecystokinin (CCK) receptors, endowed with a high specific activity (100 Ci/mmol). Binding sites for this ligand were visualized in the rat brain by autoradiography [(3)H]BDNL-CCK(7) binds specifically to an apparent single class of CCK receptors on rat striatum sections with a K(d) of 1.76 nM and a B(max) of 57 fmol/mg protein. Unsulfated CCK(8) was two times less potent than sulfated CCK(8) to displace binding of [(3)H]BDNL-CCK(7). Binding sites for [(3)H]BDNL-CCK(7) were present in many brain regions, the highest concentrations occurring in cortex, olfactory bulbs, nucleus accumbens, and medium to high concentrations in striatum, hippocampus, and several nuclei of thalamus, hypothalamus and amygdala. In the same experimental conditions, the binding sites for [(125)I]BH-CCK(8) showed similar specificity and localization. We thus used both ligands to investigate the subregional distributions of CCK receptors in nucleus accumbens and hippocampus, where a highly organized topography of action of CCK has been reported. In nucleus accumbens, the CCK binding sites were concentrated in the anterior portion of the nucleus, whereas very low densities were observed within medial posterior nucleus accumbens, where injection of CCK has been shown to potentiate dopamine-induced hyperlocomotion. p]In hippocampus, CCK receptors were concentrated in the polymorphic zone of the hilus of the dentate gyrus and in stratum lacunosum moleculare of Ammon's horn. Very few receptors were observed in other regions of hippocampus, including stratum pyramidale and stratum moleculare. This is in contrast with the presence of numerous CCK terminals and the potent effect of CCK in these areas. The distributions of CCK receptors reported here in both nucleus accumbens and hippocampus were discussed in correlation with the distribution of CCK neurons and terminals, the related anatomical pathways, and the pharmacological profiles of the effects of CCK in these regions.

Baicalein protects cortical neurons from beta-amyloid (25-35) induced toxicity.

Accumulation of amyloid beta peptide (Abeta) has been suggested to contribute to neurodegeneration in Alzheimer's disease (AD). Since chronic inflammation occurs in AD pathogenesis and lipoxygenases are important mediators of inflammatory processes, we evaluated the effect of lipoxygenase inhibitors on apoptosis induced by Abeta on rat cortical cells. The 12-lipoxygenase inhibitor baicalein attenuated both neuronal apoptosis and c-jun protein over-expression induced by Abeta(25- 35), whereas no protection was found with the broad spectrum lipoxygenase inhibitor nordihydroguaiaretic acid or the 5-lipoxygenase inhibitor caffeic acid. These results suggest that 12-lipoxygenase participates in a c-jun-dependent apoptosis pathway triggered by Abeta(25-35), and that specific 12-lipoxygenase inhibitors might be of interest in AD.

Multiple cleavage sites of cholecystokinin heptapeptide by "enkephalinase".

Degradation of Boc CCK7 (Boc Tyr1 (SO3H)-Met2-Gly3-Trp4-Met5-Asp6-Phe7-NH2), a fully active analog of CCK8, by purified rabbit kidney neutral metalloendopeptidase (enkephalinase) was studied as a basis for the rational design of potent peptidases-resistant analogs of cholecystokinin. Characterization of the metabolites was performed by HPLC using several elution procedures. Three cleavage sites were evidenced: one major at the Asp6-Phe7 bond and two minor at Gly3-Trp4 and Trp4-Met5 bonds. All cleavages were fully inhibited by thiorphan, a potent inhibitor of enkephalinase. The relative importance of the different cleavages was established using several cholecystokinin analogs. At 25 degrees C the half-disappearance time was 18 min for Boc CCK7, Boc[diNle2,5]CCK7 and 70 min for Boc[diNle2,5 D.Asp6]CCK7. Although, half-life of Boc CCK7 and Boc[diNle2,5]CCK7 were identical, the replacement of Met by Nle, a more hydrophobic aminoacid, greatly favoured the cleavage at the Trp4-Nle5 bond which became the major breakdown. This feature was exemplified by the substitution of L.Asp by D.Asp, preventing the Trp4-Nle5 cleavage, which gave rise to the most enkephalinase-resistant analog in this series.

Enzyme-resistant CCK analogs with high affinities for central receptors.

Based on the results of the in vitro metabolism of CCK8 by various peptidases, we have synthesized three CCK analogs: Boc-Tyr(SO3H)-Nle-Gly-Trp-(N- Me)Nle-Asp-Phe-NH2 (compound I), Boc-Tyr(SO3H)-gNle-mGly-Trp-Nle-Asp-Phe-Nh2 (compound II), Boc-Tyr(SO3H)-gNle-mGly-Trp-(N-Me)Nle-Asp-Phe-NH2 (compound III). In in vitro enzymatic degradation studies, these compounds showed a high stability toward either enkephalinase or the enzymes present in crude rat brain membranes preparations. Moreover, in binding studies on guinea pig tissues, these CCK-related peptides were characterized by high apparent affinities for brain CCK receptors and by a broader range of affinities for pancreatic CCK receptors. This broad range of affinities was reflected by their pharmacological potencies in the guinea pig pancreatic amylase release and ileum contraction assays. These enzyme-resistant CCK analogs provide therefore valuable tools to investigate the pharmacology of CCK.