The fat-induced satiety factor oleoylethanolamide suppresses feeding through central release of oxytocin.

Oleoylethanolamide (OEA) is a biologically active lipid amide that is released by small-intestinal enterocytes during the absorption of dietary fat and inhibits feeding by engaging the nuclear receptor, peroxisome proliferator-activated receptor-alpha (PPAR-alpha). Previous studies have shown that the anorexic effects of systemically administered OEA require the activation of sensory afferents of the vagus nerve. The central circuits involved in mediating OEA-induced hypophagia remain unknown. In the present study, we report the results of in situ hybridization and immunohistochemistry experiments in rats and mice, which show that systemic injections of OEA (5-10 mg kg(-1), intraperitoneal) enhance expression of the neuropeptide oxytocin in magnocellular neurons of the paraventricular nucleus (PVN) and supraoptic nucleus (SON) of the hypothalamus. No such effect is observed with other hypothalamic neuropeptides, including vasopressin, thyrotropin-releasing hormone and pro-opiomelanocortin. The increase in oxytocin expression elicited by OEA was absent in mutant PPAR-alpha-null mice. Pharmacological blockade of oxytocin receptors in the brain by intracerebroventricular infusion of the selective oxytocin antagonist, L-368,899, prevented the anorexic effects of OEA. The results suggest that OEA suppresses feeding by activating central oxytocin transmission.

The chemokine Bv8/prokineticin 2 is up-regulated in inflammatory granulocytes and modulates inflammatory pain.

Neutrophil migration into injured tissues is invariably accompanied by pain. Bv8/prokineticin 2 (PK2), a chemokine characterized by a unique structural motif comprising five disulfide bonds, is highly expressed in inflamed tissues associated to infiltrating cells. Here, we demonstrate the fundamental role of granulocyte-derived PK2 (GrPK2) in initiating inflammatory pain and driving peripheral sensitization. In animal models of complete Freund's adjuvant-induced paw inflammation the development and duration of pain temporally correlated with the expression levels of PK2 in the inflamed sites. Such an increase in PK2 mRNA depends mainly on a marked up-regulation of PK2 gene transcription in granulocytes. A substantially lower up-regulation was also detected in macrophages. From a pool of peritoneal granulocytes, elicited in rats by oyster glycogen, we purified the GrPK2 protein, which displayed high affinity for the prokineticin receptors (PKRs) and, when injected into the rat paw, induced hypersensitivity to noxious stimuli as the amphibian prokineticin Bv8 did. Mice lacking PKR1 or PKR2 developed significantly less inflammation-induced hyperalgesia in comparison with WT mice, confirming the involvement of both PKRs in inflammatory pain. The inflammation-induced up-regulation of PK2 was significantly less in pkr1 null mice than in WT and pkr2 null mice, demonstrating a role of PKR1 in setting PK2 levels during inflammation. Pretreatment with a nonpeptide PKR antagonist, which preferentially binds PKR1, dose-dependently reduced and eventually abolished both prokineticin-induced hypernociception and inflammatory hyperalgesia. Inhibiting PK2 formation or antagonizing PKRs may represent another therapeutic approach for controlling inflammatory pain.

Bv8/Prokineticins and their Receptors A New Pronociceptive System.

Bv8 is a small protein secreted by frog skin. Mammalian homologues of Bv8, the prokineticins PK1 and PK2, and their G-protein coupled receptors prokineticin receptor 1 (PKR1) and prokineticin receptor 2 (PKR2) have been identified and linked to several biological effects as gut motility, neurogenesis, angiogenesis, circadian rhythms, hematopoiesis, and nociception. Emerging evidences indicated that prokineticins are also associated with pathologies of the reproductive and nervous system, myocardial infarction, and tumorigenesis. Bv8 elicits a dose-dependent reduction in nociceptive threshold to thermal, mechanical, and chemical stimuli. The prokineticin receptors are present in a fraction of C- and Adelta-fiber neurons also expressing the vanilloid receptors, TRPV1 and TRPA1. Mice lacking PKR genes exhibit impaired Bv8-induced hyperalgesia, develop deficient responses to noxious heat, capsaicin, and protons and show reduced thermal and mechanical hypersensitivity to paw inflammation, indicating a requirement for PKR signaling in activation and sensitization of primary afferent fibers. Bv8/PK2 is highly expressed by neutrophils and other inflammatory cells and must be considered as new pronociceptive mediators in inflamed tissues. Bv8-like hyperalgesic activity was demonstrated in extracts of rat inflammatory granulocytes. Bv8 stimulates macrophage and T lymphocyte to differentiate towards an inflammatory and Th1 profile indicating that Bv8/PK2 plays a role in immunoinflammatory responses. Blockade of PKRs may represent a novel therapeutic strategy in acute and inflammatory pain conditions.

Triazine compounds as antagonists at Bv8-prokineticin receptors.

On the basis of a Janssen's patent, we approached a new synthesis of some 1,3,5-triazin-4,6-diones as potential non peptidic prokineticin receptor antagonists, containing the following substitutions: (N(1) and N(5) link a 4-methoxybenzyl and a 4-ethylbenzyl, respectively; C(2) can link an amino-ethyl-guanidine (reference compound 1) or an ethylendiamine (2) or an amino-ethyl-amino-2-imidazoline (3). New compounds were assessed for PKR1 and PKR2 affinity. Antagonist properties were evaluated as inhibition of 1 nM Bv8-induced intracellular Ca2+ mobilization.

The prokineticin receptor agonist Bv8 decreases IL-10 and IL-4 production in mice splenocytes by activating prokineticin receptor-1.

BACKGROUND: Bv8, prokineticin-1, or endocrine gland-vascular endothelial growth factor, and prokineticin-2 are recently isolated peptide agonists of two G protein-coupled receptors, prokineticin receptor-1 (PROKR 1) and PROKR 2, and have been described as affecting a number of myeloid cell functions. We evaluated the impact of Bv8 on lymphoid cells by investigating its ability to modulate T cell cytokine balance in mouse. RESULTS: The production of T-helper1 cytokines (IL-2, IFN-gamma and IL-1beta), the T-helper 2 cytokine IL-4, and the anti-inflammatory cytokine IL-10 by mouse splenocytes was evaluated after polyclonal stimulation or immunisation with the keyhole limpet hemocyanin protein antigen by measuring cytokine levels. When added in vitro to Con-A-stimulated splenocytes, Bv8 significantly increased IL-1beta and decreased IL-4 and IL-10; IL-2 and IFN-gamma were not affected. Similar results were obtained when Bv8 was administered in vivo. In KLH-immunised mice, splenocytes restimulated in vitro with KLH and Bv8 produced significantly smaller amounts of IL-4 and IL-10. KLH-induced IL-10 and IL-4 production was also significantly blunted in animals administered Bv8 in vivo at the time of KLH immunisation or two weeks later. The Bv8-induced effects were lost in mice lacking the PROKR 1 gene, thus indicating that PROKR 1 is the receptor involved in the modulation of cytokines. CONCLUSION: These findings indicate that Bv8/prokineticin-1 is a novel modulator of lymphoid functions, and may be a suitable target for new immunopharmacological strategies.

The prokineticin receptor agonist Bv8 increases GABA release in the periaqueductal grey and modifies RVM cell activities and thermoceptive reflexes in the rat.

The prokineticin Bv8, a small protein secreted by the skin of the Bombina variegata frog, is a potent agonist of both the identified prokineticin receptors, the G-protein-coupled PK-R1 and PK-R2. We found in this study that intraperiaqueductal grey (PAG) Bv8, 100 and 200 pmol per rat, exerted a pronociceptive action and caused opposite effects on the ongoing rostral ventromedial medulla (RVM) On- and Off-cell activities in rats. Bv8 increased and decreased the ongoing activity of RVM On and Off cells, respectively. Bv8 decreased tail flick latency and increased the pause and shortened the onset of the Off-cell pause. Bv8 did not change either the tail flick-induced On-cell burst of activity or the onset of On-cell peak firing. Microdialysis analysis, applied in combination with the plantar test, showed that intra-PAG perfusion with Bv8 (0.25 and 0.5 pm) increased GABA, but not glutamate, extracellular levels, and decreased thermoceptive thresholds. These findings show that stimulation of PAG PK-Rs might worsen pain perception and this effect is consistent with both RVM On- and Off-cell ongoing and tail flick-related activities, as well as with the increase induced by Bv8 in PAG GABA levels.

Bv8/Prokineticin proteins and their receptors.

The Bv8/Prokineticins (PKs) are a new family of peptides identified in frog, fish, reptiles and mammals that signal through two highly homologous G-protein coupled receptors, PKR1 and PKR2. Bv8/PK proteins possess a unique structural motif comprising five disulfide bonds and a completely conserved N-terminal hexapeptide sequence that is essential for the peptide's biological activities. Over the past few years, several biological functions of Bv8/PK proteins have been elucidated. This review considers all the published data on the action and physiological role of this new biological system implicated in angiogenesis and neurogenesis, in reproduction and cancer and in regulating physiological functions that underly circadian rhythms, such as the sleep/wake cycle, hormone secretion and ingestive behaviors. The high expression level of human Bv8/PK2 in bone marrow, lymphoid organs and leukocytes suggested an involvement of these peptides in hematopoiesis and in inflammatory and immunomodulatory processes. Our review highlights the role of the Bv8/PK and their receptor system in setting the pain threshold under normal and pathological conditions.

Novel glycosylated [Lys(7)]-dermorphin analogues: synthesis, biological activity and conformational investigations.

Syntheses of the [Lys(7)]- and [Hyp(6),Lys(7)]-dermorphin analogues in which either Tyr(5) or Hyp(6) are O-glucosylated are described. For comparison, the carbohydrate-free peptides have also been prepared. Structural investigations by FT-IR and CD measurements were carried out on the synthetic analogues and some preliminary pharmacological experiments were also performed. The biological potency of the glucosylated analogues was compared with that of the micro-opioid receptor agonist dermorphin in GPI preparations. Glucosylation of either Tyr(5) or Hyp(6) reduces the potency of both [Lys(7)]-dermorphin and [Hyp(6),Lys(7)]-dermorphin. The effect induced by the Tyr(5) glucosylation is quite strong and the potency of both peptides is reduced by about 150 times. A similar but less dramatic effect is induced by the glucosylation of the Hyp(6) residue, and the potency of the parent peptide is reduced by about 15 times. The presence of acetyl groups on the sugar hydroxyl functions further reduces the agonistic potency of the glucosylated analogues. The analgesic potency of [Hyp(6),Lys(7)]-, [Hyp(betaGlc)(6),Lys(7)]- and [Tyr(betaGlc)(5),Lys(7)]-dermorphin were also tested in vivo by the tail-flick test. The glucosylated hydroxyproline-containing analogue is 8-10 times less active than the parent peptide, but its analgesic effect lasts significantly longer.

Impaired nociception and inflammatory pain sensation in mice lacking the prokineticin receptor PKR1: focus on interaction between PKR1 and the capsaicin receptor TRPV1 in pain behavior.

Bv8, prokineticin-1 or EG-VEGF (endocrine gland-derived vascular endothelial growth factor), and prokineticin-2, are naturally occurring peptide agonists of two G-protein-coupled receptors (GPCRs), prokineticin receptor 1 (PKR1) and PKR2. PKRs are expressed in neurons in the CNS and peripheral nervous system and many dorsal root ganglion (DRG) cells expressing PKRs also express transient receptor potential vanilloid receptor-1 (TRPV1). Mice lacking the pkr1 gene were generated to explore the role of the PKR1 receptor in nociceptive signaling and in nociceptor sensitization. When compared with wild-type littermates, mice lacking the pkr1 gene showed impaired responsiveness to noxious heat, mechanical stimuli, capsaicin, and protons. In wild-type mice, activation of PKRs by the PKR agonist Bv8 caused hyperalgesia and sensitized to the actions of capsaicin. pkr1-null mice exhibited impaired responses to Bv8 but showed normal hyperalgesic responses to bradykinin and PGE2 (prostaglandin E2). Conversely, trpv1-null mice showed a reduced pronociceptive response to Bv8. Additionally, pkr1-null mice showed diminished thermal hyperalgesia after acute inflammation elicited by mustard oil and reduced pain behavior after chronic inflammation produced by complete Freund's adjuvant. The number of neurons that responded with a [Ca2+]i increase to Bv8 exposure was five times lower in pkr1-null DRG cultures than in wild-type cultures. Furthermore, Bv8-responsive neurons from pkr1-null mice showed a significant reduction in the [Ca2+]i response to capsaicin. These findings indicate a modulatory role of PKR1 in acute nociception and inflammatory pain and disclose a pharmacological interaction between PKR1 and TRPV1 in nociceptor activation and sensitization.

New 2',6'-dimethyl-L-tyrosine (Dmt) opioid peptidomimetics based on the Aba-Gly scaffold. Development of unique mu-opioid receptor ligands.

The Aba-Gly scaffold, incorporated into Dmt-Tic ligands (H-Dmt-Tic-Gly-NH-CH2-Ph, H-Dmt-Tic-Gly-NH-Ph, H-Dmt-Tic-NH-CH2-Bid), exhibited mixed micro/delta or delta opioid receptor activities with micro agonism. Substitution of Tic by Aba-Gly coupled to -NH-CH2-Ph (1), -NH-Ph (2), or -Bid (Bid=1H-benzimidazole-2-yl) (3) shifted affinity (Ki(micro)=0.46, 1.48, and 19.9 nM, respectively), selectivity, and bioactivity to micro-opioid receptors. These compounds represent templates for a new class of lead opioid agonists that are easily synthesized and suitable for therapeutic pain relief.

6-N,N-dimethylamino-2,3-naphthalimide: a new environment-sensitive fluorescent probe in delta- and mu-selective opioid peptides.

A new environment-sensitive fluorophore, 6-N,N-(dimethylamino)-2,3-naphthalimide (6DMN) was introduced in the delta-selective opioid peptide agonist H-Dmt-Tic-Glu-NH(2) and in the mu-selective opioid peptide agonist endomorphin-2 (H-Tyr-Pro-Phe-Phe-NH(2)). Environment-sensitive fluorophores are a special class of chromophores that generally exhibit a low quantum yield in aqueous solution but become highly fluorescent in nonpolar solvents or when bound to hydrophobic sites in proteins or membranes. New fluorescent delta-selective irreversible antagonists (H-Dmt-Tic-Glu-NH-(CH(2))(5)-CO-Dap(6DMN)-NH(2) (1) and H-Dmt-Tic-Glu-Dap(6DMN)-NH(2) (2)) were identified as potential fluorescent probes showing good properties for use in studies of distribution and internalization of delta receptors by confocal laser scanning microscopy.

Sensitization of transient receptor potential vanilloid 1 by the prokineticin receptor agonist Bv8.

Small mammalian proteins called the prokineticins [prokineticin 1 (PK1) and PK2] and two corresponding G-protein-coupled receptors [prokineticin receptor 1 (PKR1) and PKR2] have been identified recently, but the physiological role of the PK/PKR system remains mostly unexplored. Bv8, a protein extracted from frog skin, is a convenient and potent agonist for both PKR1 and PKR2, and injection of Bv8 in vivo causes a potent and long-lasting hyperalgesia. Here, we investigate the cellular basis of hyperalgesia caused by activation of PKRs. Bv8 caused increases in [Ca]i in a population of isolated dorsal root ganglion (DRG) neurons, which we identified as nociceptors, or sensors for painful stimuli, from their responses to capsaicin, bradykinin, mustard oil, or proteases. Bv8 enhanced the inward current carried by the heat and capsaicin receptor, transient receptor potential vanilloid 1 (TRPV1) via a pathway involving activation of protein kinase Cepsilon (PKCepsilon), because Bv8 caused translocation of PKCepsilon to the neuronal membrane and because PKC antagonists reduced both the enhancement of current carried by TRPV1 and behavioral hyperalgesia in rodents. The neuronal population expressing PKRs consisted partly of small peptidergic neurons and partly of neurons expressing the N52 marker for myelinated fibers. Using single-cell reverse transcriptase-PCR, we found that mRNA for PKR1 was mainly expressed in small DRG neurons. Exposure to GDNF (glial cell line-derived neurotrophic factor) induced de novo expression of functional receptors for Bv8 in a nonpeptidergic population of neurons. These results show that prokineticin receptors are expressed in nociceptors and cause heat hyperalgesia by sensitizing TRPV1 through activation of PKCepsilon. The results suggest a role for prokineticins in physiological inflammation and hyperalgesia.

Bv8, the amphibian homologue of the mammalian prokineticins, induces a proinflammatory phenotype of mouse macrophages.

1.--The small protein Bv8, isolated from the amphibian skin, belongs to a novel family of secreted proteins linked to several biological effects. We describe the expression of Bv8/prokineticins and their receptors in mouse macrophages, and characterize their proinflammatory activities. 2.--The rodent analogue of Bv8, prokineticin-2, is expressed by macrophages, as well as its G-protein-coupled receptor prokineticin receptor (PKR-1 and PKR-2). PKR-1 is expressed more abundantly. 3.-- Bv8 induces potent chemotaxis of macrophages at concentrations as low as 10(-12) M. 4.-- It stimulates lipopolysaccharide-induced production of the proinflammatory cytokines IL-1 and IL-12, reducing that of the anti-inflammatory cytokine IL-10. The effects are observed starting at the very low concentration of 10(-11) M. 5.--Effects on chemotaxis and cytokine are not pertussis-toxin sensitive, but are completely prevented by addition of the phospholipase inhibitor U73122, suggesting a G(q) protein is involved in the Bv8-induced effects. 6.--Studies in PKR-1 knockout mice indicate that all the activities exerted by Bv8 on macrophages are mediated by the PKR-1 receptor. 7.--In conclusion, Bv8 appears to be able to induce the macrophage to migrate and to acquire a proinflammatory phenotype.

Modulators of pain: Bv8 and prokineticins.

Bv8 is a small protein secreted by frog skin. Mammalian homologues of Bv8, the prokineticins PK1 and PK2, and their G-protein coupled receptors PKR1 and PKR2 have been identified and linked to several biological effects. Bv8 elicits a dose-dependent reduction in nociceptive threshold to thermal and mechanical stimuli applied to the skin of tail and paw of rats and mice and increases the sensitivity to nociceptive mediators as capsaicin and prostaglandins. The receptors for Bv8/PKs are present in a fraction of peptidergic population of C-fibre neurons, and in a fraction of A myelinated-fibre neurons. In mouse and rat dorsal root ganglia, PKR-expressing neurons also express TRPV1 and the activation of PKRs sensitises TPRV1 to the action of capsaicin. Mice lacking PKR1 gene exhibit impaired Bv8-induced hyperalgesia, develop deficient responses to noxious heat, capsaicin and protons and show reduced thermal and mechanical hypersensitivity to paw inflammation, indicating a requirement for PKR1 signalling associated with activation and sensitisation of primary afferent fibres. PKs are highly expressed by neutrophils and other inflammatory cells and must be considered as new pronociceptive mediators in inflammatory tissues. Bv8-like hyperalgesic activity was demonstrated in extracts of rat inflammatory granulocytes. Bv8 stimulate macrophage and T lymphocyte to differentiate between an inflammatory and Th1 profile indicating that Bv8/PK proteins play a role in immuno-inflammatory responses. Blockade of PKRs may represent a novel therapeutic strategy in acute and inflammatory pain conditions.

Conversion of the potent delta-opioid agonist H-Dmt-Tic-NH-CH(2)-bid into delta-opioid antagonists by N(1)-benzimidazole alkylation(1).

N(1)-Alkylation of 1H-benzimidizole of the delta agonist H-Dmt-Tic-NH-CH(2)-Bid with hydrophobic, aromatic, olefinic, acid, ethyl ester, or amide (1-6) became delta antagonists (pA(2)=8.52-10.14). delta- and micro-Opioid receptor affinities were high (K(i)delta=0.12-0.36 nM and K(i)micro=0.44-1.42 nM). Only delta antagonism (pA(2)=8.52-10.14) was observed; micro agonism (IC(50)=30-450 nM) was not correlated with changes in alkylating agent or delta antagonism, and some compounds yielded mixed delta antagonism/micro agonism.

Biological activities of Bv8 analogues.

1 The small protein Bv8, secreted by the skin of the frog Bombina variegata, belongs to a novel family of secreted proteins whose orthologues have been identified in snakes (MIT) and in mammals (prokineticins (PKs)). A characteristic feature of this protein family is the same N-terminal sequence, AVITGA, and the presence of 10 cysteines with identical spacing in the C-terminal domain. Two closely related G protein-coupled receptors that mediate signal transduction of Bv8/PKs have been cloned (PK-R1 and PK-R2). In mammals, the Bv8/PK protein family is involved in a number of biological activities such as ingestive behaviours, circadian rhythms, angiogenesis and pain sensitization. 2 In an attempt to identify the structural determinants required for the pronociceptive activity of Bv8, we prepared Bv8 derivatives lacking one (des-Ala-Bv8) or two (des-Ala-Val-Bv8) residues from the N-terminus. 3 des-Ala-Bv8 displayed a receptor affinity five times lower than that of Bv8, it was five times less potent in inducing [Ca(2+)](i) transients and in causing p42/p44 MAPK phosphorylation in CHO-cells expressing PK-R1 and PK-R2. Moreover, dA-Bv8 was about 20 times less potent than Bv8 in inducing hyperalgesia in rats. 4 The deletion of the first two amino acids of Bv8 abolished any biological activity both 'in vitro' and 'in vivo'; however, des-AlaVal-Bv8 is able to antagonize the Bv8-induced hyperalgesia, binding the PK-Rs on peripheral and central projections of the primary sensitive neurons.

[D-Ala2]-Deltorphin I peptoid and retropeptoid analogues: synthesis, biological activity and conformational investigations.

The synthesis is described of a [D-Ala2]-deltorphin I peptoid analogue in which all amino acid residues have been substituted by the corresponding N-alkylglycine residues. The [D-Ala2]-deltorphin I retropeptoid was also prepared as well as [Ala1 ,D-Ala2]-deltorphin 1 and the corresponding peptoid. Structural investigations by FT-IR and fluorescence measurements were carried out on the synthetic analogues and on some [D-Ala2]-deltorphin 1 peptide-peptoid hybrids previously prepared. According to the fluorescence measurements the distance between the aromatic residues in the deltorphin I peptoid and retropeptoid is similar to that suggested for the delta- and micro-opioids, respectively. Measurements of CD in the presence of beta-cyclodextrin, and some preliminary pharmacological experiments were also performed. No dichroic bands are present in the spectrum of the [Ntyr1,D-Ala2]-deltorphin I, but an increasing dichroic effect appears in the spectra of both the deltorphin I peptoid and retropeptoid. Activity tests on isolated organ preparations showed that the modifications made produced a dramatic decrease in the agonistic activity of the synthetic derivatives.

Direct influence of C-terminally substituted amino acids in the Dmt-Tic pharmacophore on delta-opioid receptor selectivity and antagonism.

A series of 17 analogues were developed on the basis of the general formula H-Dmt-Tic-NH-CH(R)-R' (denotes chirality; R = charged, neutral, or aromatic functional group; R' = -OH or -NH(2)). These compounds were designed to test the following hypothesis: the physicochemical properties of third-residue substitutions C-terminal to Tic in the Dmt-Tic pharmacophore modify delta-opioid receptor selectivity and delta-opioid receptor antagonism through enhanced interactions with the mu-opioid receptor. The data substantiate the following conclusions: (i) all compounds had high receptor affinity [K(i)(delta) = 0.034-1.1 nM], while that for the mu-opioid receptor fluctuated by orders of magnitude [K(i)(mu) = 15.1-3966 nM]; (ii) delta-opioid receptor selectivity [K(i)(mu)/K(i)(delta)] declined 1000-fold from 22,600 to 21; (iii) a C-terminal carboxyl group enhanced selectivity but only as a consequence of the specific residue; (iv) amidated, positive charged residues [Lys-NH(2) (6), Arg-NH(2) (7)], and a negatively charged aromatic residue [Trp-OH (11)] enhanced mu-opioid affinity [K(i)(mu) = 17.0, 15.1, and 15.7 nM, respectively], while Gly-NH(2) (8), Ser-NH(2) (10), and His-OH (12) were nearly one-tenth as active; and (v) D-isomers exhibited mixed effects on mu-opioid receptor affinity (2' << 3' < 4' < 1' < 5') and decreased delta-selectivity in D-Asp-NH(2) (1') and D-Lys(Ac)-OH (5'). The analogues exhibited delta-opioid receptor antagonism (pA(2) = 6.9-10.07) and weak mu-opioid receptor agonism (IC(50) > 1 microM) except H-Dmt-Tic-Glu-NH(2) (3), which was a partial delta-opioid receptor agonist (IC(50) = 2.5 nM). Thus, these C-terminally extended analogues indicated that an amino acid residue containing a single charge, amino or guanidino functionality, or aromatic group substantially altered the delta-opioid receptor activity profile (selectivity and antagonism) of the Dmt-Tic pharmacophore, which suggests that the C-terminal constituent plays a major role in determining opioid receptor activity as an "address domain".

Bv8, the amphibian homologue of the mammalian prokineticins, modulates ingestive behaviour in rats.

1. The small protein Bv8, secreted by the skin of the frog Bombina variegata, belongs to a novel family of secreted proteins whose mammalian orthologues have been identified and named prokineticins (PK-1 and PK-2). 2. Bv8 (from 2.5 to 60 pmol) injected into the lateral ventricles of rat brain suppressed diurnal, nocturnal, deprivation-induced and neuropeptide Y-stimulated feeding and stimulated diurnal drinking. Nocturnal drinking was increased only in fasted rats. 3. PK-2 mRNA is expressed in discrete areas of the rat brain, including the suprachiasmatic nucleus (SCN), medial preoptic area (MPA) and nucleus of the solitary tract (NTS). In the SCN neurons, PK-2 mRNA is highest during the light phase of the circadian cycle and undetectable during the dark phase. 4. The G-protein-coupled receptor prokineticin receptor 2 (PKR-2), which binds Bv8 and PK-2 with high affinity, is mainly expressed in the piriform cortex, paraventricular thalamic nucleus, parataenial nucleus (PT), SCN, hypothalamic paraventricular (PVH) and dorsomedial (DMH) nuclei, arcuate nucleus (ARC) and subfornical organ (SFO) of the rat brain. 5. Bv8 microinjected into the ARC, at doses from 0.02 to 2.0 pmol during night-time or from 0.2 to 5 pmol in 24-h-fasted rats, selectively suppressed feeding without affecting drinking. When injected into the SFO, Bv8 (from 0.2 to 2 pmol) stimulated drinking but did not affect feeding. Bv8 injections into other brain areas left rat ingestive behaviours unchanged. 6. We hypothesize that PK-2-rich projections from SCN neurons to PKR-expressing ARC neurons could transmit the circadian rhythm of feeding, whereas inputs from the PK-2-expressing NTS neurons to the PKR-2-expressing SFO neurons could transmit visceral information on the water-electrolyte balance and osmotic regulation.

Synthesis and opioid activity of N,N-dimethyl-Dmt-Tic-NH-CH(R)-R' analogues: acquisition of potent delta antagonism.

N,N-Dimethylation of the H-Dmt-Tic-NH-CH(R)-R' series of compounds produced no significant affect on the high delta-opioid receptor affinity (K(i)=0.035-0.454 nM), but dramatically decreased that for the micro-opioid receptor. The effect of N-methylation was independent of the length of the linker (R); however, the bioactivities were affected by the chemical composition of the third aromatic group (R'): phenyl (Ph) (5'-8') elicited a greater reduction in micro-affinity (40-70-fold) compared to analogues containing 1H-benzimidazole-2-yl (Bid) (9-fold). The major consequences of N,N-dimethylation on in vitro bioactivity were: (i). a loss of delta-agonism coupled with the appearance of potent delta antagonism (4'-7') (pA(2)=8.14-9.47), while 1 exhibited only a 160-fold decreased delta agonism (1') and the delta antagonism of 8 enhanced >10-fold (pA(2)=10.62, 8'); and (ii). a consistent loss of micro-affinity resulted in enhanced delta-opioid receptor selectivity. With the exception of compound 1', the change in the hydrophobic environment at the N-terminus and formation of a tertiary amine by N,N-dimethylation in analogues of the Dmt-Tic pharmacophore produced potent delta-selective antagonists.