Our experiments were designed to investigate the effects of zerumbone pretreatment on cholecystokinin octapeptide (CCK-8)-induced acute pancreatitis in rats.

Male Wistar rats weighing 240 to 280 g were divided into a control group, a group treated with CCK-8, a group receiving 20 mg/kg zerumbone before CCK-8 administration, and a group treated with zerumbone only.

The serum amylase and lipase activities and the pancreatic weight-body weight ratio were significantly reduced by zerumbone pretreatment, but the drug failed to influence the histological parameters of pancreatitis. The anti-inflammatory effects of the drug were manifested in decreases in the cytosolic interleukin 6 and tumor necrosis factor alpha concentrations and an elevation in the I-kappaB concentration, whereas the antioxidant ability of zerumbone was demonstrated by reductions in inducible nitric oxide synthase, Mn- and Cu/Zn-superoxide dismutase activities in the zerumbone-treated rats.

Zerumbone ameliorated the changes of several parameters of acute pancreatitis probably by interfering with I-kappaB degradation, but in the applied dose, it failed to influence the histology of the disease.

Tyrosine sulfation is one of the most common post-translational modifications in secreted and transmembrane proteins and a key modulator of extracellular protein-protein interactions. Several proteins known to be tyrosine sulfated play important roles in physiological processes, and in some cases a direct link between protein function and tyrosine sulfation has been established. In blood coagulation, tyrosine sulfation of factor VIII is required for efficient binding of von Willebrand factor; in leukocyte adhesion, tyrosine sulfation of the P-selectin glycoprotein ligand-1 mediates high-affinity binding to P-selectin; and in leukocyte chemotaxis, tyrosine sulfation of chemokine receptors is required for optimal interaction with chemokine ligands. Furthermore, tyrosine sulfation has been implicated in several infectious diseases. In particular, tyrosine sulfation of the HIV-1 co-receptor CCR5 is required for viral entry into host cells and tyrosine sulfation of the Duffy antigen/receptor for chemokines is crucial for erythrocyte invasion by the malaria parasite plasmodium vivax. Despite increasing interest in tyrosine sulfation in recent years, the sulfoproteome still remains largely unexplored. To date, only a relatively small number of sulfotyrosine-containing peptides and proteins have been identified, and a specific role for tyrosine sulfation has not been established for most of these. Here, we provide an overview of the biology and enzymology of tyrosine sulfation and discuss recent developments in preparative and analytical methods that are central to sulfoproteome research.

Penetratin is a cationic cell-penetrating peptide that has been frequently used for the intracellular delivery of polar bioactive compounds. Recent studies have just revealed the major role of polyanionic membrane proteoglycans and cholesterol-enriched lipid rafts in the uptake of the peptide. Both proteoglycans and lipid-rafts influence inflammatory processes by binding a wide array of proinflammatory mediators; thus, we decided to analyze the effect of penetratin on in vitro and in vivo inflammatory responses. Our in vitro luciferase gene assays demonstrated that penetratin decreased transcriptional activity of nuclear factor-kappaB (NF-kappaB) in tumor necrosis factor (TNF)-stimulated L929 fibroblasts and lipopolysaccharide-activated RAW 264.7 macrophages. Penetratin also inhibited TNF-induced intercellular adhesion molecule-1 expression in human endothelial HMEC-1 cells. Exogenous heparan sulfate abolished the in vitro NF-kappaB inhibitory effects of the peptide. Uptake experiments showed that penetratin was internalized by all of the above-mentioned cell lines in vitro and rapidly entered the cells of the lung and pancreas in vivo. In an in vivo rat model of acute pancreatitis, a disease induced by elevated activities of stress-responsive transcription factors like NF-kappaB, pretreatment with only 2 mg/kg penetratin attenuated the severity of pancreatic inflammation by interfering with IkappaB degradation and subsequent nuclear import of NF-kappaB, inhibiting the expression of proinflammatory genes and improving the monitored laboratory and histological parameters of pancreatitis and associated oxidative stress.

The cell-permeant MG132 tripeptide (Z-Leu-Leu-Leu-aldehyde) is a peptide aldehyde proteasome inhibitor that also inhibits other proteases, including calpains and cathepsins. By blocking the proteasome, this tripeptide has been shown to induce the expression of cell-protective heat shock proteins (HSPs) in vitro. Effects of MG132 were studied in an in vivo model of acute pancreatitis. Pancreatitis was induced in male Wistar rats by injecting 2 x 100 microug/kg cholecystokinin octapeptide intraperitoneally (ip) at an interval of 1 h. Pretreating the animals with 10 mg/kg MG132 ip before the induction of pancreatitis significantly inhibited IkappaB degradation and subsequent activation of nuclear factor-kappaB (NF-kappaB). MG132 also increased HSP72 expression. Induction of HSP72 and inhibition of NF-kappaB improved parameters of acute pancreatitis. Thus MG132 significantly decreased serum amylase, pancreatic weight/body weight ratio, pancreatic myeloperoxidase activity, proinflammatory cytokine concentrations, and the expression of pancreatitis-associated protein. Parameters of oxidative stress (GSH, MDA, SOD, etc.) were improved in both the serum and the pancreas. Histopathological examinations revealed that pancreatic specimens of animals pretreated with the peptide demonstrated milder edema, cellular damage, and inflammatory activity. Our findings show that simultaneous inhibition of calpains, cathepsins, and the proteasome with MG132 prevents the onset of acute pancreatitis.

To assess the effect of our novel cell-permeable nuclear factor-kappaB (NF-kappaB) inhibitor peptide PN50 in an experimental model of acute pancreatitis. PN50 was produced by conjugating the cell-penetrating penetratin peptide with the nuclear localization signal of the NF-kappaB p50 subunit.

Pancreatitis was induced in male Wistar rats by administering 2X100 mug/kg body weight of cholecystokinin-octapeptide (CCK) intraperitoneally (IP) at an interval of 1 h. PN50-treated animals received 1 mg/kg of PN50 IP 30 min before or after the CCK injections. The animals were sacrificed 4 h after the first injection of CCK.

All the examined laboratory (the pancreatic weight/body weight ratio, serum amylase activity, pancreatic levels of TNF-alpha and IL-6, degree of lipid peroxidation, reduced glutathione levels, NF-kappaB binding activity, pancreatic and lung myeloperoxidase activity) and morphological parameters of the disease were improved before and after treatment with the PN50 peptide. According to the histological findings, PN50 protected the animals against acute pancreatitis by favoring the induction of apoptotic, as opposed to necrotic acinar cell death associated with severe acute pancreatitis.

Our study implies that reversible inhibitors of stress-responsive transcription factors like NF-kappaB might be clinically useful for the suppression of the severity of acute pancreatitis.

The effect of a recombinant RGD (arginine-glycine-aspartic acid)-containing fusion protein, cellulose-binding domain (CBD)-RGD, on the cellular adhesion to a biomedical polyurethane (PU) was evaluated. A series of different cell lines, as well as freshly harvested animal cells, were grown on the PU surfaces with or without CBD-RGD, in serum or serum-free media. The results showed that the enhancement of cellular attachment by CBD-RGD varied with cell types. This is believed to be a result of the unique integrin receptors on each type of cell surface. The existence of certain divalent ions (Mg2+ and Mn2+) may increase the efficacy of the CBD-RGD, in a cell type-dependent manner. The fusion protein was also found to inhibit the platelet activation. The effect of CBD-RGD was further examined in two other substrate materials, poly(L-lactide) (PLLA) and poly(lactide-co-glycolide) (PLGA). The effect on cellular adhesion correlated with the amount of CBD-RGD physically adsorbed on the material surface.

Vasorelaxation caused by some antifibrinogen RGD (Arg-Gly-Asp-containing) peptides and their basic mechanism of action was studied on rabbit isolated thoracic aortic rings preconstricted with 0.25 microM phenylephrine. GRGDS (Gly-Arg-Gly-Asp-Ser-OH) and RGDV (Arg-Gly-Asp-Val-OH) caused dose-dependent relaxation. RGDS (Arg-Gly-Asp-Ser-OH) had a biphasic effect (a transient relaxation followed by a contraction) while GRGDS-[SE] (Gly-Arg-Gly-Asp-Ser(SO3)-OH) did not change the isometric tone of precontracted aortic preparations. GRGDS and RGDV exerted no relaxing effect on endothelium-denuded blood vessels suggesting that the vascular action of these peptides was entirely dependent on the presence of functionally intact endothelium. L-NG-Nitro-arginine (30 microM) attenuated the relaxation induced by GRGDS and abolished that induced by RGDV. All of the four RGD congeners inhibited ADP-induced aggregation of human platelets. These findings indicate that the relaxant effect of RGDV is mediated exclusively by the nitric oxide pathway, but GRGDS could cause, besides nitric oxide release, the release of another substance which is different from nitric oxide. Because the rank order of the vasorelaxant potencies of RGD peptides differed from that found for their anti-aggregatory activities, a vascular effector mechanism mediated by an RGD-recognizing structure other than the known glycoprotein IIb/IIIa-like RGD-binding site is suggested.

Peptides 31D and VF13, corresponding to the rabies virus nucleo- and glycoproteins, respectively, vigorously stimulate T helper cells of the appropriate specificity. Earlier we showed how internal and external glycosylation affects the major histocompatibility complex molecule (MHC)-binding ability and conformation of these T-cell epitopes (Otvos et al. (1994) Biochim. Biophys. Acta 1224, 68-76; Otvos et al. (1995) Biochim. Biophys. Acta 1267, 55-64). In the current report, we examined the T-helper cell stimulatory ability after introduction of a new set of post-translational modifications. To obtain general information concerning the effects of amino acid side-chain modifications on other biochemical properties of protein fragments, we studied the serum stability and the conformation of the 31D and VF13 peptides. We found that the extent of the reduction of the T-cell stimulatory activity depends upon the location in the sequence of the host amino acid residue. Generally, beta-linked sugars in mid-chain positions had a greater inhibitory effect than alpha-linked sugars attached to identical amino acids. In a case where mid-chain glycosylation just marginally reduced the T-cell stimulatory activity, the beta-linked glycopeptide was significantly more resistant to serum proteases. This finding suggests that addition of beta-linked carbohydrates might be superior to the addition of alpha-linked sugars for vaccine development, and generally for peptide agonist drug design. In addition, data presented here provide the first documentation that phosphorylation and sulfation of tyrosine residues may retain the MHC-binding ability and T-cell stimulatory activity of class II epitopes. The sulfated and the phosphorylated 31D peptides exhibited considerably increased serum stability compared to the unmodified parent peptide. Finally, all post-translational modifications destabilized the dominant alpha-helical or turn structures of the peptides presented in aqueous trifluoroethanol mixtures. While the circular dichroism spectra of the alpha- and beta-linked VF13 glycopeptides with monosaccharides were almost indistinguishable, the structure of the glycopeptides depended upon the length of the sugar moiety. Significantly, incorporation of sulfate or phosphate groups resulted in identical peptide conformations.

In conscious female Wistar rats with chronic lateral cerebroventricular cannula, the thermoregulatory effects of CCK-8, ceruletide and prostaglandin E1 (PGE1) were studied. In addition, the possible involvement of type A or type B receptors of CCK-8 in thermoregulatory effects of PGE1 and CCK-8 was also investigated. In the normothermic rat an intracerebroventricular (i.c.v.) injection of CCK-8 or ceruletide induced a thermogenic response with tail-skin vasoconstriction and a resulting rise in colonic temperature (Tc). There was a significant negative correlation between the starting level of Tc and the extent of rise in Tc following an i.c.v. administration of PGE1, CCK-8 or ceruletide. Subcutaneously injected CCK-8 caused decreases in Tc in a cool ambient temperature as also described by others. The fever-like response to i.c.v. injected CCK-8 was attenuated by a CCK type B receptor blocker, but not by a CCK type A receptor blocker. Conversely, the hypothermic response to peripherally administered CCK-8 was attenuated by a type A receptor blocker, but not by a type B receptor blocker. Neither of these CCK-receptor blockers influenced the fever caused by an i.c.v. injection of PGE1. It is concluded that in normothermic rats the thermogenic response observed after i.c.v. injection of CCK-8 and ceruletide is the most likely central thermoregulatory change mediated by CCK type B receptors, while the well-known hypothermic response observed after peripheral injection of these peptides might also be explained by their direct effect on variables influencing some of the thermoregulatory effector mechanisms at the periphery.

Previous work indicates that both the C-terminal phenylalanine amide and the tryptophan moieties of cholecystokinin (CCK) are critical pharmacophores for interaction with either the A or B receptor subtypes. We have examined a series of analogs of Ac-CCK-7 [Ac-Tyr(SO3H)-Met-Gly-Trp-Met-Asp-Phe33-NH2] (2) in which the phenyl ring of the C-terminal Phe-NH2 has been modified. Compounds were assessed in binding assays using homogenated rat pancreatic membranes and bovine striatum as the source of CCK-A and CCK-B receptors respectively and for anorectic activity after intraperitoneal administration to rats. Substitution of a number of cycloalkyl or bicyclic aryl moieties for the phenyl ring of phenylalanine33 including cyclopentyl (20), cyclohexyl (21), cyclooctyl (23), 2-(5,6,7,8-tetrahydro)naphthyl (26), 2-naphthyl (27), and 1-naphthyl (29) led to analogs with 10-70 times the anorectic potency of 2. The anorectic activity of 21 was blocked by the specific CCK-A receptor antagonist MK-329. Other bulky aliphatic groups in place of the phenylalanine33 aromatic ring such as isopropyl, 2-adamantyl and cyclohexylmethyl gave derivatives similar to 2 in potency. While most of the new compounds were comparable to CCK in binding assays, 23, 26, 27 and 29 were exceptionally potent with IC50s 10(-11)-10(-14) M in the pancreas. Compounds 23 and 29 were further evaluated for their ability to stimulate amylase secretion and found to have potencies similar to that of CCK. The dissociation between potency in the binding and amylase secretion assays suggests that they may interact with a high affinity binding site which is not coupled to amylase secretion. We conclude that CCK receptors possess a generous hydrophobic pocket capable of accommodating large alkyl groups in place of the side chain of phenylalanine33 and that the pharmacological profile of CCK analogs can be tailored by appropriate exploitation of this finding.

Cholecystokinin represents a family of gut hormones which among other activities, have been proposed to participate in satiety signaling. Ac-CCK-7[Ac-Tyr(SO3H)-Met-Gly-Trp30-Met-Asp-Phe-NH2 (2)] possesses the full spectrum of activity and potency of the intact hormone; thus analogs of 2 may be useful as anorectic agents. A series of derivatives has been prepared in which the tryptophan indole moiety of 2 has been modified. The new compounds were assayed in CCK binding assays using homogenated rat pancreatic membranes and bovine striatum as a source of CCK-A and CCK-B receptors respectively and in vivo in rats for anorectic activity. Although previous studies have concluded that the indole ring of Trp30 is a critical pharmacophore for the interaction of CCK with both its A and B type receptors, we find 2-Nal30-Ac-CCK-7 (20) to be nearly equipotent to 2 in both CCK binding and as an anorectic agent sensitive to blockade by the Merck CCK-A receptor antagonist MK-329. The extreme structural sensitivity of this anorectic activity is illustrated by the 1-naphthylalanine30 (19) and (benzo[b]thien-2-yl)alanine30 (21) analogs which are 30 and 100 times less potent than 2 respectively. Other mono- and bicyclic Trp30 replacements, including substituted phenylalanines, 3-quinolinylalanine, and 2-(5,6,7,8-tetrahydro)naphthylalanine, gave inactive compounds.

Substitution of the -OSO3H group in the sulfated-tyrosine by the non-hydrolyzable-CH2SO3H group was the first described modification of the sulfate ester that does not affect CCK8 activity. In addition to its capacity to mimic the sulfated tyrosine residue, the amino acid Phe(p-CH2SO3Na) was shown to be stable in acidic media, including HF containing mixtures. The synthesis of Boc-Phe(p-CH2SO3Na)-OH in racemic and resolved forms and its introduction into the sequence of CCK8 by solid phase using standard Boc/benzyl synthesis conditions and BOP as coupling reagent is now reported. The two CCK8 analogues containing the L- or the D-Phe(p-CH2SO3Na) residue, obtained in satisfactory yields, were separated by HPLC and the stereochemistry of Phe(p-CH2SO3Na) residue in each peptide was established by NMR spectroscopy and confirmed by a separate solid phase synthesis in which the pure L isomer was used. Both CCK8 analogues displayed high affinities for peripheral and central receptors (KI approximately 1 nM) and proved to be full agonists in the stimulation of pancreatic amylase secretion. The "stabilized-CCK8 peptide", easily prepared by solid phase, could replace the native peptide in biochemical and pharmacological studies. Moreover the modified amino acid Phe (p-CH2SO3Na) could also be used in solid phase synthesis to prepare a wide variety of CCK analogues and more generally, peptides analogues containing the acid-labile O-sulfated tyrosine.

The model peptide TRH was successfully synthesized using benzotriazol-1-yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP reagent). The coupling reactions were carried out in N,N-dimethylformamide or N-methylpyrrolidone. These solvents allowed the incorporation of the N-terminal pyroglutamic acid residue into the peptide chain, without using the derivative bearing the N-benzyloxycarbonyl group, which acts as a solubility promoter. A comparative racemization study showed that Boc-His(Tos) can be coupled by means of BOP reagent with less racemization than with DCC when the amount of diisopropylethylamine (DIEA) is kept minimal (same ratio of equivalents as for Boc-His(Tos), i.e. 3 equiv.). However, with the use of a larger amount of DIEA in the coupling mixture (9 equiv.), approximately 3% of epimer was found in the crude product. Our study showed that even under low DIEA conditions, the rate of coupling of the residues with BOP remained comparable to that observed with DCC.

Intraperitoneal (ip) injection of cholecystokinin octapeptide sulfate ester (CCK; 5, 10 and 50 micrograms/kg) reduced the hypothermic response to 8 mg/kg and 32 mg/kg systemic morphine in restrained and freely moving rats, respectively. The hyperthermia elicited by a subcutaneous (sc) injection of 8 mg/kg morphine to freely moving rats was not diminished by CCK pretreatment. CCK (5 micrograms/kp ip) completely prevented the restraint stress-induced hyperthermia. Naloxone (1 mg/gk sc) was effective in antagonizing both the hyperthermic and the hypothermic effects of morphine and the stress-induced emotional hyperthermia. These results support the hypothesis that CCK may contribute to regulation of the endogenous opioid system.

Three new analogues of N alpha-hydroxysulfonyl-[Nle28,31]CCK26-33 are reported in which the C-terminal L-Phe33 residue has been replaced by L-Leu, D-Phe or N-methyl-L-Phe. Biological evaluation in a series of binding and bioassays demonstrates that both L-stereochemistry and an aromatic side chain at position-33 are essential for full agonist activity. While the L-Leu33 and D-Phe33 analogues had reduced potencies in stimulating contraction of the guinea pig ileum or gall bladder, the D-Phe33 analogue was fourfold selective for the ileum. This latter analogue also exhibited apparent partial agonism in the rat pancreatic amylase release assay. The N-methyl-L-Phe33 analogue was almost equipotent to the parent analogue in all bioassays, suggesting that this modification might be useful for introducing enzymatic stability in CCK analogues.

Rats received an i.p. injection of cholecystokinin-octapeptide sulfate ester (CCK; 4, 10 or 50 micrograms/kg) or physiological saline at dark onset, and the 24-h sleep-wake cycle (12-h-dark and 12-h-light phases), spontaneous motor activity and brain temperature (Tbr) were recorded. EEG activity was studied through spectral analysis for 2.5 h, and food intake was measured at the end of postinjection hour 1. In response to CCK, non-REM sleep increased at the expense of wakefulness, and the sleep-promoting effect was substantiated by an increase in EEG slow-wave activity. Motor activity, Tbr and food intake decreased. The effects vanished in postinjection hour 2; the diurnal rhythms were not modified. The changes varied as a function of the dose: the effects were significant following 10 micrograms/kg, and even higher in response to 50 micrograms/kg CCK. The results indicate that i.p. CCK definitely promotes non-REM sleep. This effect may belong to the behavioral sequence elicited by the peptide, which is often attributed to satiety. As evidenced by the reduction of Tbr, CCK also exerts strong autonomic actions, which might interfere with the behavioral responses.

Subcutaneous injection of cholecystokinin octapeptide (CCK-8) (0.005-1.25 mg/kg) elicited dose-dependent hypothermia in rats. The threshold of the response was between 0.01 and 0.05 mg/kg and the dose-response curve levelled off at doses larger than 0.2-0.5 mg/kg. Warm and cold ambient temperatures decreased and increased the response, respectively. Pretreatment with capsaicin, morphine, naloxone, atropine, haloperidol or propranolol did not affect the response to CCK-8, whereas pretreatment with phenoxybenzamine and a large dose of proglumide, an antagonist for CCK-receptors, attenuated the hypothermia. It seems that neither capsaicin-sensitive thermal and non-thermal afferents, nor opiate mechanisms are involved in the response, but alpha-adrenoceptors might be of some importance in the hypothermia. Non-sulphated-CCK-8, the C-terminal tetrapeptide and hexapeptide, [D-Ala4]-CCK-8 and [D-Met6]-CCK-8 were ineffective. Chronic treatment with CCK-8 resulted in the development of tolerance to the thermoregulatory effect, while the hypothermic responses to apomorphine and capsaicin were not affected. It seems that the tolerance cannot be attributed to conditioned homeostatic reactions.

The dose-response curves for the anticonvulsive activity of sulfated and nonsulfated cholecystokinin octapeptide (CCK-8-SE and CCK-8-NS) against picrotoxin-induced (6 mg/kg SC) seizures were assessed either following or without pretreatment with a single high dose of CCK-8-SE or CCK-8-NS, to examine acute tolerance to the effect after IP injections in mice. As CCK-8-SE or CCK-8-NS pretreatment, a 1.6 mumole/kg dose was injected 2 hr prior to the second injection. No acute tolerance to the anticonvulsive activity was demonstrated, and CCK-8-NS pretreatment significantly potentiated its own anticonvulsive activity. Chronic (8-day) daily treatment with a 0.16 mumole/kg dose of CCK-8-SE or CCK-8-NS antagonized seizures by picrotoxin, presumably in a cumulative manner. To investigate the interactions of CCK octapeptides with other anticonvulsive agents, picrotoxin-induced seizures were antagonized with several doses of diazepam following or without acute, high-dose pretreatment with CCK-8-SE or CCK-8-NS. The two octapeptides only slightly modified the activity of diazepam: CCK-8-SE pretreatment displayed a tendency to antagonize it, while CCK-8-NS pretreatment to potentiate it. The results suggest that multiple treatment with CCK-8 induces sensitization of CCK receptors mediating anticonvulsive activity.

The usefulness of a strong acid, such as MSA or TFMSA/TFA, as a deprotecting reagent in peptide synthesis was examined. By synthesizing several structurally related brain-gut-skin triangle peptides, a number of advantageous features of the thioanisole-mediated deprotecting procedure were demonstrated. New amino acid derivatives, Arg(Mts), Trp(Mts) and Asp(OChp), were introduced to improve the synthetic methodology of complex peptides and the superior properties of Cys(Ad) were evaluated.

In most laboratories CCK-8(s) has been found to be the biologically active form of CCK-8 in the CNS. The role of CCK-8(ns) has scarcely been investigated and is poorly understood. CCK-8(s) exerts a transmitter and/or modulator role in this projection. CCK-8(ns), on the other hand, profoundly affects DA-ergic neurotransmission in the nigrostriatal DA-ergic projection. The octapeptide modulates the turnover and release of DA from this neuron population. DA-mediated behavioral reactions are also modulated by CCK-8(ns). We should emphasize that the biological importance of CCK-8(ns) in the CNS has hitherto generally been neglected. Our results point to the equivalence of CCK-8(s) and CCK-8(ns) in the CNS in most biological tests. In some cases the latter compound is the more potent one. In most of these tests the C-terminal fragment (tetragastrin = CCK-4) also proved to be active. It is most likely that a brain receptor population exists which can bind both forms of CCK-8 and even CCK-4. Nevertheless, the CNS could contain binding sites which bind only CCK-8(s) as a ligand. We have found that an unidentified sulfotransferase of the brain can sulfate CCK-8(ns) and thereby provide a ligand for the special receptors of CCK-8(s). It is likely that CCK modulates the turnover and release of DA, and vice versa. Theoretically, different biochemical mechanisms could exist for interactions between CCK octapeptides and DA. We have focused our investigations on the enzymic sulfation-desulfation processes of both CCK-8 and DA and have devised a hypothetical model for the possible interactions. Both CCK-8(ns) and DA could be sulfated in vivo, this enzymic reaction generally requiring active sulfate (PAPS). These two compounds could compete for the limited pool of PAPS, and thus CCK-8 and DA could mutually regulate their levels in the same cell by influencing one of the metabolic (DA) or synthetic (CCK-8(s)) pathways. CCK-8(s) also might provide the O-sulfate group for DA by enzymic transformation, and, conversely, DA-O-sulfate may sulfate CCK-8(ns) in a similar way. These trans-sulfation processes could also mutually determine the concentrations of DA and CCK-8 co-existing in one cell. Experiments to prove these models are planned.

Based on recent findings identifying the initial degradative cleavage of CCK-8 at the Met3-Gly4 bond by a metalloendopeptidase, two analogues of CCK-8 with D-Ala and D-Trp substitutions at the Gly4 position were synthesized as stable analogues. Their stability to proteolysis by brain membranes and their binding potency at central CCK receptors were quantified. Both peptides are stable to degradation by peptidases in cortical synaptic membrane preparations. The analogues are nearly equipotent to CCK-8 in their affinities for inhibition of 125I-CCK-33 binding to guinea pig cortical membranes. L-Ala and L-Trp substituted peptides were synthesized for comparison. Both these peptides are degraded by synaptic membranes and the L-Trp substituted peptide possesses a greatly reduced affinity for central CCK receptors. Therefore, the structure of CCK due to the D conformation of Gly is more capable of interacting with brain CCK receptors. Further conformational analysis will establish whether the stabilized structure is a beta-bend or a beta-turn. Since these peptides are highly potent and stable to brain proteolysis they may be useful as stable CCK analogues for in vivo application.