Biological activity of cholecystokinin (30–33) tetrapeptide analogues

Analogues of the endogenous peptide corresponding to the 30–33 sequence of cholecystokinin (Trp-Met-Asp-Phe-NH₂) were synthesized, and their biological activity was studied. It was shown that, in rats, the N-succinylated Nle² analogue of this tetrapeptide exhibits increased anxiolytic properties in the dark-light chamber test and an enhanced alcohol intake by both the control animals and the alcohol-dependent animals under the conditions of free choice. Introduction of an isopropyl residue into the C-terminal amide of the Nle² analogue resulted in the appearance of anxiogenic and antialcohol activity and the ability to increase the morphine analgesic effect in the tail-flick test on rats. The two synthesized analogues retained an affinity for cholecystokinin receptors.__________Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 2, 2005, pp. 130–139.Original Russian Text Copyright © 2005 by Proskuryakova, Bespalova, Palkeeva, Petrichenko, Pankratova, Shokhonova, Anokhina.     

Correlation between phospholipid breakdown, intracellular calcium mobilization and enzyme secretion in rat pancreatic acini treated with Boc-[Nle28, Nle31]-CCK-7 and JMV180, two cholecystokinin analogues.

In this work in vitro pharmacological profiles of two analogues of the C-terminal heptapeptide of cholecystokinin (CCK) were evaluated. The analogue Boc-[Nle28, Nle31]-CCK-7, a stable analogue of CCK-8, has the same activity profile as CCK-8, and was found to be very potent in stimulating amylase secretion, phospholipid breakdown and [Ca2+]i mobilization from rat pancreatic acini. It can be used as a probe for studying CCK-actions. The CCK-analogue Boc-Tyr(SO3H)-Nle-Gly-Trp-Nle-Asp-2-phenylethylester, (JMV180), which stimulates amylase secretion without inhibition at supramaximal concentrations, has different effects on phospholipid hydrolysis and [Ca2+]i mobilization, compared to CCK-8 and Boc-[Nle28, Nle31]-CCK-7. Compound JMV180 was unable to significantly promote phospholipid breakdown, and was only 50%-60% as efficacious as Boc-[Nle28, Nle31]-CCK-7 in promoting [Ca2+]i mobilization. These findings suggest that low affinity CCK-receptors might be responsible for the supra-maximal inhibition of amylase secretion, and are correlated with phospholipid breakdown and maximal [Ca2+]i mobilization.     

Sulfur-free parathyroid hormone analogues containing D-amino acids: biological properties in vitro and in vivo

Three sulfur-free analogues of bovine parathyroid hormone (bPTH) containing D-amino acids were synthesized by the solid-phase method and their biological properties compared in an in vitro bioassay (rat renal adenylate cyclase assay), a receptor assay for parathyroid hormone (PTH) (canine renal membranes), and an in vivo bioassay (chick hypercalcemia assay). The analogue [Nle8,Nle18,D-Tyr34]-bPTH-(1-34)-amide, which was found to be more than 4 times as potent in vitro as unsubstituted PTH, is the most potent analogue of PTH yet synthesized. The enhanced potency was largely attributable to increased affinity for the PTH receptor. In vivo, however, this analogue was only one-third as potent as bPTH-(1-34). Cumulative evidence suggests that the nearly 15-fold decline in the relative potency when the compound was assayed in vivo is due to the substitution of norleucine for methionine. The other analogues, [D-Val2,Nle8,D-Tyr34]bPTH-(1-34)-amide and [D-Val2,Nle8,Nle18,D=Tyr34]bPTH-(2-34)-amide, were only weakly active in vitro and in vivo, indicating that substitution with D-amino acids at the NH2 terminus of PTH causes markedly diminished receptor affinity. In fact, the placement of a D-amino acid at the NH2 terminus is more deleterious to biological activity than is omission of amino acids at positions 1 and 2.     

Relative stability of alpha-melanotropin and related analogues to rat brain homogenates

alpha-Melanotropin (alpha-MSH) retains less than 1% of its original activity after a 60 min incubation with 10% rat brain homogenate. [Nle4,D-Phe7]-alpha-MSH is nonbiodegradable in rat serum (240 min incubation) and still maintains 10% of its original activity in 10% rat brain homogenate (240 min incubation). The related fragment analogue, Ac-[Nle4,D-Phe7]-alpha-MSH4-10-NH2, retains 50% of its activity after a 240 min incubation in rat brain homogenate, whereas Ac-[Nle4,D-Phe7]-alpha-MSH4-11-NH2 is totally resistant to inactivation by rat brain homogenate. Both [Nle4,D-Phe7]-fragments are resistant to degradation by rat serum, but [Nle4]-alpha-MSH, Ac-[Nle4]-alpha-MSH4-10-NH2 and Ac-[Nle4]-alpha-MSH4-11-NH2 are rapidly inactivated under both conditions. The cyclic melanotropin, [Cys4,Cys10]-alpha-MSH, is inactivated in rat brain homogenate as is the shorter Ac-[Cys4,Cys10]-alpha-MSH4-10-NH2 analogue, but neither cyclic melanotropin is inactivated upon incubation in serum from rats. Ac-[Cys4,D-Phe7,Cys10]-alpha-MSH4-10-NH2 is resistant to inactivation by either rat serum or a brain homogenate. Some of these melanotropin analogues may provide useful probes for the localization and characterization of putative melanotropin receptors in both the central nervous system and peripheral tissues.     

Effects of a Melanotropic Peptide on Melanoma Cell Growth,Metastasis, and Invasion

Melanocyte stimulating hormone (α-MSH, α-melanotropin),Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Ly-Pro-Val-NH₂, regulates melanogenesis within epidermal melanocytes of many animals. An MSH analogue ([Nle4,D-Phe7]α-MSH) that exhibits superpotency and prolonged biological activity has been synthesized, biologically characterized, and is presently in clinical trials to determine its possible clinical use in tanning of the skin. It also has potential for the diagnosis, localization, and chemotherapy of melanoma. The effects of this analogue on the growth, metastatic behavior, and invasive potential of a melanotic variant of Cloudman S-91 murine melanoma are reported here. In an intracutaneous murine model of melanoma cell tumor growth, the analogue did not increase primary tumor growth (size) after the period of administration of the peptide hormone analogue and did not affect spontaneous lung metastases. Survival times for the control and melanotropin-treated groups were similar, suggesting that overall tumor burden was not affected by treatment with the hormone analogue. Last, melanoma cell invasion through a human amniotic basement membrane in vitro was not enhanced compared to untreated cells.     

Establishment of a new short, protease-resistant, affinity labeling reagent for the cholecystokinin receptor

Proteolytic degradation of radioligands is an important source of artifact in affinity labeling of receptor proteins. To complement our previous characterization of the pancreatic acinar cell cholecystokinin (CCK) receptor, we synthesized D-Tyr-Gly[(Nle28,31)CCK-26-33]. The amino terminal D-enantiomer of tyrosine provided a site for oxidative iodination, a free amino group for cross-linking, and rendered the peptide resistant to aminopeptidases. The decapeptide was oxidatively iodinated and purified by reverse-phase HPLC to 2,000 Ci/mmol, to yield a probe which was equal in potency and efficacy to CCK-8, and which bound to rat pancreatic membranes in a rapid, reversible, temperature-dependent, specific, saturable and high affinity manner. This probe was resistant to aminopeptidase degradation, and maintained its ability to bind to receptor after incubation with pancreatic membranes or dispersed cells. Affinity labeling of pancreatic membranes with this analogue identified an Mr = 85,000-95,000 molecule. This analogue offers several advantages over existing probes and should be useful for future studies of this and other CCK receptors.     

Behavioral effect of soymorphin-5-amide in rats

Behavioral effects of YPFVV-NH₂ (an analogue of soymorphin-5, an exorphin derived from a soy protein) in rats were investigated for the first time. Rats of different sex and age were tested. It was shown that the stimulation of locomotion in adult rats (males) was the main effect of the peptide injection. In juvenile rats, in turn, the main effect of YPFVV-NH₂ was related to anxiety. The anxiolytic effect superseded the anxiogenic effect during puberty. Finally, the manifestation of the peptide effects depended on animals’ stress level.     

Cyclic lactam analogues of Ac-[Nle4]alpha-MSH4-11-NH2

Two side-chain cyclic lactam analogues of the 4-11 fragment of alpha-melanocyte-stimulating hormone (alpha-MSH), Ac-[Nle4,D-Orn5,Glu8]alpha-MSH4-11-NH2 and Ac-[Nle4,D-Orn5,D-Phe7,Glu8]alpha-MSH4-11-NH2, were prepared on p-methylbenzhydrylamine resin by using a combination of N alpha-Boc and N alpha-Fmoc synthetic strategies with diphenyl phosphorazidate mediated cyclization. The melanotropin activities of these two analogues were examined and compared relative to those of alpha-MSH, Ac-[Nle4]alpha-MSH4-11-NH2, and Ac-[Nle4,D-Phe7]alpha-MSH4-11-NH2. In the frog (Rana pipiens) skin bioassay, the L-Phe7 17-membered ring cyclic analogue was slightly more potent than the linear Ac-[Nle4]alpha-MSH4-11-NH2 and exhibited prolonged melanotropic bioactivity (greater than or equal to 4 h). In this same assay, the D-Phe7 cyclic analogue was more than 100-fold less potent than the L-Phe cyclic analogue and was 10,000 times less potent than linear Ac-[Nle4,D-Phe7]alpha-MSH4-11-NH2. In the lizard skin (Anolis carolinensis) bioassay, the L-Phe7 cyclic analogue was 100-fold less potent than Ac-[Nle4]alpha-MSH4-11-NH2, while the D-Phe7 cyclic analogue was 10,000-fold less potent than both Ac-[Nle4]alpha-MSH4-11-NH2 and the D-Phe7 linear derivative Ac-[Nle4,D-Phe7]alpha-MSH4-11-NH2. The solution conformation of these two cyclic analogues in dimethyl sulfoxide-d6 was examined by 1D and 2D 500-MHz 1H NMR spectroscopy. Our analysis suggests an H bond stabilized C10 (or C13) turn for the D-Phe7 cyclic structure while the L-Phe7 analogue is more conformationally flexible.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure-function studies of analogues of parathyroid hormone (PTH)-1-34 containing beta-amino acid residues in positions 11-13

The 1-34 N-terminal fragments of human parathyroid hormone (PTH) and PTH-related protein (PTHrP) elicit the full spectrum of bone-relevant activities characteristic of the intact hormones. The structural elements believed to be required for receptor binding and biological activity are two helical segments, one N-terminal and one C-terminal, connected by hinges or flexible points located around positions 12 and 19. To test this hypothesis, we synthesized and characterized the following analogues of PTH-(1-34), each containing single or double substitutions with beta-amino acid residues around the putative hinge located at position 12: I. [Nle(8,18),beta-Ala(11,12),Nal(23),Tyr(34)]bPTH-(1-34)NH(2); II. [Nle(8,18),beta-Ala(12,13),Nal(23),Tyr(34)]bPTH-(1-34)NH(2); III. [Nle(8,18),beta-Ala(11),Nal(23),Tyr(34)]bPTH-(1-34)NH(2); IV. [Nle(8,18),beta-hLeu(11),Nal(23),Tyr(34)]bPTH-(1-34)NH(2); V. [Nle(8,18),beta-Ala(12), Nal(23),Tyr(34)]bPTH-(1-34)NH(2); VI. [Nle(8,18),beta-Ala(13), Nal(23),Tyr(34)]bPTH-(1-34)NH(2) (beta-hLeu = beta-homo-leucine; beta-Ala = beta-alanine; Nal = L-2-naphthyl-alanine; Nle = norleucine). Analogues I and III exhibit very low binding affinity and are devoid of adenylyl cyclase activity. Analogue II, despite its very low binding capacity is an agonist. Biological activity and binding capacity are partially restored in analogue IV, and completely restored in analogues V and VI. The conformational properties of the analogues were investigated in aqueous solution containing dodecylphosphocholine (DPC) micelles as a membrane-mimetic environment using CD, 2D-NMR, and molecular dynamics calculations. All peptides fold partially into the alpha-helical conformation in the presence of DPC micelles, with a maximum helix content in the range of 30-35%. NMR analysis reveals the presence of two helical segments, one N-terminal and one C-terminal, as a common structural motif in all analogues. Incorporation of beta-Ala dyads at positions 11,12 and 12,13 in analogues I and II, respectively, enhances the conformational disorder in this portion of the sequence but also destabilizes the N-terminal helix. This could be one of the possible reasons for the lack of biological activity in these analogues. The partial recovery of binding affinity and biological activity in analogue IV, compared to the structurally similar analogue III, is clearly the consequence of the reintroduction of Leu side-chain of the native sequence. In the fully active analogues V and VI, the helix stability at the N-terminus is further increased. Taken together, these results stress the functional importance of the conformational stability of the helical activation domain in PTH-(1-34). Contrary to expectation, insertion of a single beta-amino acid residue in positions 11, 12, or 13 in analogues III-VI does not favor a disordered structure in this portion of the sequence.     

Synthesis and biological activity of Boc [Nle28, Nle31]CCK27-33, a highly potent CCK8 analogue

A new CCK8 related peptide, Boc[Nle28,Nle31]CCK27-33 (Boc[diNle]CCK7) was synthesized and tested for cholecystokinic activity, at both the peripheral and the central level. This analogue, protected against both chemical oxidation and enzymatic degradation by aminopeptidases, was shown to be equipotent to CCK8 in releasing amylase from rat pancreas fragments. In addition, the EC50 values of Boc[diNle]CCK7 in the guinea pig gallbladder and ileum contraction assays (3.2 nM and 3.0 nM respectively) were similar to those of CCK8 (6.0 nM and 2.0 nM). Moreover both Boc[diNle]CCK7 and CCK8 elicited similar effects on the open field test over the same concentrations range. These results demonstrate the ability of Boc[diNle]CCK7 to be a suitable tool for investigating the physiological role of native CCK8.     

Effects of cholecystokinin octapeptide sulphate ester and unsulphated cholecystokinin octapeptide on active avoidance behaviour in rats

The effects of peripherally administered cholecystokinin octapeptide sulphate ester and its unsulphated form on the active avoidance behaviour of rats were studied. The acquisition of avoidance behaviour was impaired, while extinction was facilitated, following cholecystokinin octapeptide sulphate ester or unsulphated cholecystokinin octapeptide treatment. These peptides had no action on open-field activity. It is concluded that peripherally administered cholecystokinin octapeptide influences acquisition and extinction of active avoidance behaviour and this effect is unrelated to general motor activity of the animals.     

Substitution of norleucine for methionine residues in a crustacean pigment-dispersing hormone

In order to evaluate the structural/functional roles of Met residues in an octadecapeptide pigment-dispersing hormone (PDH: Asn-Ser-Gly-Met-Ile-Asn-Ser-Ile-Leu-Gly-Ile-Pro-Arg-Val-Met-Thr-Glu-Ala- NH2), first described as light-adapting distal retinal pigment hormone (DRPH) from Pandalus, three analogs were synthesized: Nle4-PDH, Nle15-PDH, and Nle4,15-PDH. When tested for melanophore pigment-dispersing activity in destalked Uca, all three Nle-analogs were more potent than unsubstituted PDH. Performic acid oxidation caused a marked loss of potency of PDH, Nle4-PDH, and Nle15-PDH. The analog Nle4,15-PDH was resistant to oxidation and displayed 6-fold higher potency than PDH. Thus Met4 and Met15 are not essential for the PDH activity. The oxidation-induced loss of activity of unsubstituted PDH may result from introduction of oxygen (in methionine sulfone) and a consequent conformational change in the octadecapeptide.     

Evaluation of new base-labile 2-(4-nitrophenylsulfonyl) ethoxycarbonyl (Nsc)-amino acids for solid-phase peptide synthesis.

The 2-(4-nitrophenylsulfonyl)ethoxycarbonyl (Nsc) group is a new base-labile protecting group for solid-phase peptide synthesis, completely interchangeable with the fluorenylmethoxycarbonyl (Fmoc) protecting group, but with certain advantages. In this paper, we report a methodology with Nalpha-Nsc-protected amino acids for the synthesis of some melanotropins important to our research, namely, gamma-melanocyte-stimulating hormone (gamma-MSH), its [Nle3]-analogue, and a cyclic alpha-MSH/beta-MSH hybrid. We developed an efficient protocol for the synthesis of the cyclic MSH analogue that yielded this peptide in >98% purity. The gamma-MSH synthesis, which gave problems with both the Boc and Fmoc strategies, yielded the desired peptide by Nsc-chemistry but was accompanied by side products. Finally, the Nle3-gamma-MSH analogue was synthesized more efficiently using the Fmoc strategy, suggesting that Nsc-chemistry might not be the best methodology for certain sequences.     

Uterine relaxing action of parathyroid hormone: effect of oxidation and methionine substitution

The effects of bPTH-(1-34), oxidized bPTH-(1-34),[Nle8,Nle18, Tyr34] bPTH-(1-34) amide, and oxidized [Nle8,Nle18,Tyr34]bPTH-(1-34)amide were tested in an in vitro rat uterine assay. When bPTH-(1-34) was treated with hydrogen peroxide (H2O2), the ability of this peptide to reduce oxytocin-stimulated uterine contraction in vitro was no longer evident. An analogue of bPTH-(1-34), in which the methionines at positions 8 and 18 were replaced with norleucine ([Nle8,Nle18,Tyr34]bPTH-(1-34)amide), was capable of reducing oxytocin-stimulated uterine contraction. However, when the [Nle8,Nle18,Tyr34]bPTH-(1-34)amide was oxidized, it retained the ability to reduce uterine contraction. Since we have previously shown that H2O2 oxidation affected only the methionines, these results suggest that the methionines are not necessary for the uterine activity of bPTH-(1-34). We have previously shown that oxidation of bPTH-(1-34) also destroys its blood vessel relaxing activity but has no effect in the rat or the Japanese quail hypercalcemic assays. These data combined with the results of the present studies suggest that the uterine and vascular smooth muscle relaxing properties of bPTH-(1-34) may require the same structural conformation and that this conformation is different from that required for the hypercalcemic action of the peptide.     

Structure-function relationship studies of bovine parathyroid hormone [bPTH(1-34)] analogues containing alpha-amino-iso-butyric acid (Aib) residues

The N-terminal 1-34 fragments of the parathyroid hormone (PTH) and parathyroid hormone-related protein (PTHrP) elicit the full spectrum of bone-related biological activities of the intact native sequences. It has been suggested that the structural elements essential for bioactivity are two helical segments located at the N-terminal and C-terminal sequences, connected by hinges or flexible points around positions 12 and 19. In order to assess the relevance of the local conformation around Gly(12) upon biological function, we synthesized and characterized the following PTH(1-34) analogues containing Aib residues: (I) A-V-S-E-I-Q-F-nL-H-N-Aib-G-K-H-L-S-S-nL-E-R-V-E-Nal-L-R-K-K-L-Q-D-V-H-N-Y-NH(2) ([Nle(8,18), Aib(11), Nal(23),Tyr(34)]bPTH(1-34)-NH(2)); (II) A-V-S-E-I-Q-F-nL-H-N-L-Aib-K-H-L-S-S-nL-E-R-V-E-Nal-L-R-K-K-L-Q-D-V-H-N-Y-NH(2) ([Nle(8,18), Aib(12),Nal(23),Tyr(34)]bPTH(1-34)-NH(2)); (III) A-V-S-E-I-Q-F-nL-H-N-L-G-Aib-H-L-S-S-nL-E-R-V-E-Nal-L-R-K-K-L-Q-D-V-H-N-Y-NH(2) ([Nle(8,18), Aib(13), Nal(23),Tyr(34)]bPTH(1-34)-NH(2)); (IV) A-V-S-E-I-Q-F-nL-H-N-Aib-Aib-K-H-L-S-S-nL-E-R-V-E-Nal-L-R-K-K-L-Q-D-V-H-N-YNH(2) ([Nle(8,18), Aib(11,12), Nal(23),Tyr(34)]bPTH(1-34)-NH(2)); (V) A-V-S-E-I-Q-F-nL-H-N-L-Aib-Aib-H-L-S-S-nL-E-R-V-E-Nal-L-R-K-K-L-Q-D-V-H-N-Y-NH(2) ([Nle(8,18), Aib(12,13),Nal(23),Tyr(34)]bPTH(1-34)-NH(2)). (nL= Nle; Nal= L-(2-naphthyl)-alanine; Aib= alpha-amino-isobutyric acid.) The introduction of Aib residues at position 11 in analogue I or at positions 11 and 12 in analogue IV resulted in a 5-20-fold lower efficacy and a substantial loss of binding affinity compared to the parent compound [Nle(8,18), Nal(23),Tyr(34)]bPTH(1-34)-NH(2). Both binding affinity and adenylyl cyclase stimulation activity are largely restored when the Aib residues are introduced at position 12 in analogue II, 13 in analogue III, and 12-13 in analogue V. The conformational properties of the analogues in aqueous solution containing dodecylphosphocholine micelles were studied by CD, two-dimensional (2D) NMR and computer simulations. The results indicated the presence of two helical segments in all analogues, located at the N-terminal and C-terminal sequences. Insertion of Aib residues at positions 12 and 13, or of Aib dyads at positions 11-12 and 12-13, enhances the stability of the N-terminal helix of all analogues. In all analogues the Aib residues are included in the helical segments. These results confirmed the importance of the helical structure in the N-terminal activation domain, as well as of the presence of the Leu(11) hydrophobic side chain in the native sequence, for PTH-like bioactivity.     

The influence of a 2-benzamido-2-(2-oxoindolin-3-iliden) acetic acid derivative on the behavioral activity of rats after traumatic brain injury

We studied the influence of a derivative of 2-benzamido-2-(2-oxoindolin-2-iliden) acetic acid, which is designated as ZNM, on locomotor and orienting–exploratory activity, the state of muscle tonus, and coordination of movements, as well as on the physical stamina of rats after a moderate closed traumatic brain injury. It was revealed that the ZNM derivative promotes a change of the locomotor activity profile with an increase in orienting–exploratory activity and a decrease in the emotionality in animals, an increase in physical stamina in the swimming test with a load, and improved coordination of movements in the rod rotating test. The results of the studies indicate that the pharmacological activity profile of ZNM is similar to that of the reference drug mexidol: cerebroprotective, anxiolytic, and sedative effects.

     

Potent and prolonged melanotropic activities of the alpha-MSH fragment analog, Ac-[Nle4,D-Phe7]-alpha-MSH4-9-NH2

Ac-[Nle4, D-Phe7]-alpha-MSH4-9-NH2 and Ac-[Nle4]-alpha-MSH4-9-NH2, fragment analogs of the tridecapeptide, alpha-melanocyte stimulating hormone (alpha-MSH, alpha-melanotropin), were synthesized. The potency and prolonged activity of the analogs were compared to alpha-MSH in several melanotropin bioassays. The D-Phe-containing hexapeptide was 10 times more active than alpha-MSH in stimulating melanoma tyrosinase activity. This analog was also 10-fold more potent than alpha-MSH in the lizard skin bioassay and about 10-fold less active in the frog skin bioassay. The melanotropic activity of Ac-[Nle4, D-Phe7]-alpha-MSH4-9-NH2 was considerably prolonged compared to alpha-MSH in each of the bioassays. These results demonstrate that the structural requirements for superpotency and prolonged activity of [Nle4, D-Phe7]-substituted analogs reside within this hexapeptide sequence.     

Synthesis of cyclic analogues of cholecystokinin highly selective for central receptors

Cyclic CCK analogues in which positions 28 and 31 have been replaced by lysine residues and whose side chains are bridged by a succinic moiety, were synthesized. They were tested for their ability to inhibit the binding of 125I-BH-CCK-8 to isolated rat pancreatic acini and to guinea pig brain membranes. These cyclic CCK-analogues were compared to the potent CCK analogue Boc-[Nle28,31]-CCK-7 and to Boc-Trp-Leu-Asp-Phe-NH2, analogue of CCK-4. These cyclic compounds appeared to be highly selective for central CCK receptors.     

Electrophysiological studies with new CCK analogs: correlation with binding affinity on B-type receptors

The electrophysiological effects of Boc-D-Asp-Tyr(SO3H)-Nle-D-Lys-Trp-Nle-Asp-Phe-NH2 (compound I) and Boc-gamma-D-Glu-Tyr(SO3H)-Nle-D-Lys-Trp-Nle-Asp-Phe-NH2 (compound II), two cyclic cholecystokinin analogs with high selectivity for CCK-B receptors, as well as the effects of the linear enzyme-resistant analog Boc-[Nle28,Nle31]-CCK7 (BDNL), were compared with those of CCK8 using extracellular recordings in rat hippocampal slices in vitro. Bath applications of the three synthetic compounds resulted in concentration-dependent and reversible increases in single-unit activity. Comparison of equieffective concentrations yielded the following potency rank order: BDNL greater than CCK8 greater than compound II greater than compound I. There was a close correlation (r = .96, slope = 0.98) between the excitatory activities of the analogs and their potencies in displacing radiolabelled CCK8 from CCK-B receptors on rat brain membranes.     

Synthesis of tritium labeled Ac-[Nle4, D-Phe7]-alpha-MSH4-11-NH2: a superpotent melanotropin with prolonged biological activity

Ac-[Nle4, D-Phe7]-alpha-MSH4-11-NH2 an octapeptide, is a melanotropin analogue (Ac-Nle-Glu-His-D-Phe-Arg-Trp-Gly-Lys-NH2), which is a superpotent agonist of frog and lizard skin melanocytes and mouse S 91 (Cloudman) melanoma cells. This melanotropin possesses ultraprolonged activity on melanocytes, both in vitro and in vivo, and the peptide is resistant to inactivation by serum enzymes. The tritium-labeled congener was prepared by direct incorporation of [3H]-labeled norleucine into the peptide. The melanotropic activity of the labeled peptide is identical to the unlabeled analogue. This labeled peptide should be useful for studies on the localization and characterization of melanotropin receptors.