Dyn Peptides Research Articles

Brain site-specific regulation of hedonic intake by orexin and DYN peptides: role of the PVN and obesity

ABSTRACT The orexin peptides promote hedonic intake and other reward behaviors through different brain sites. The opioid dynorphin peptides are co-released with orexin peptides but block their effects on reward in the ventral tegmental area (VTA). We previously showed that in the paraventricular hypothalamic nucleus (PVN), dynorphin and not orexin peptides enhance hedonic intake, suggesting they have brain-site-specific effects. Obesity alters the expression of orexin and dynorphin receptors, but whether their expression across different brain sites is important to hedonic intake is unclear. We hypothesized that hedonic intake is regulated by orexin and dynorphin peptides in PVN and that hedonic intake in obesity correlates with expression of their receptors. Here we show that in mice, injection of DYN-A1–13 (an opioid dynorphin peptide) in the PVN enhanced hedonic intake, whereas in the VTA, injection of OXA (orexin-A, an orexin peptide) enhanced hedonic intake. In PVN, OXA blunted the increase in hedonic intake caused by DYN-A1–13. In PVN, injection of norBNI (opioid receptor antagonist) reduced hedonic intake but a subsequent OXA injection failed to increase hedonic intake, suggesting that OXA activity in PVN is not influenced by endogenous opioid activity. In the PVN, DYN-A1–13 increased the intake of the less-preferred food in a two-food choice task. In obese mice fed a cafeteria diet, orexin 1 receptor mRNA across brain sites involved in hedonic intake correlated with fat preference but not caloric intake. Together, these data support that orexin and dynorphin peptides regulate hedonic intake in an opposing manner with brain-site-specific effects.

Molecular identification of the Dyn/Kor system and its potential role in the reproductive axis of goldfish

To ascertain the significance of the dynorphin/kappa-opioid receptor (Dyn/Kor) system in fish reproduction, prodynorphin (pdyn) cDNA was cloned from goldfish. Two Dyn peptides (DynA and DynB) are present in the goldfish prodynorphin precursor. Both DynA and DynB are biologically active as they are able to functionally interact with the goldfish Kor expressed in cultured eukaryotic cells to suppress forskolin-induced CRE promoter activity. RT-PCR analysis showed that pdyn is widely expressed in brain regions, with the highest expression in hypothalamus. During ovarian development, hypothalamic pdyn and kor mRNA levels are lower in the early vitellogenic stage. Then the biological effects of Dyn peptides on salmon gonadotropin releasing hormone (sgnrh), luteinizing hormone beta (lhb) and follicle stimulating hormone beta (fshb) mRNA synthesis were further investigated in goldfish. Intraperitoneal injections of DynA and DynB significantly reduced hypothalamic sgnrh and pituitary lhb and fshb mRNA levels in male goldfish, but these two peptides only down-regulated sgnrh and lhb mRNA expression in female goldfish. In vitro studies revealed that DynA also decreased lhb mRNA levels in primary cultures of pituitary cells, indicating that this peptide can exert its actions at the pituitary level. Our findings suggest that the Dyn/Kor system plays a negative role in regulating the reproductive axis in goldfish.

Comparison with Naloxone of Two Dynorphin a Analogues with K- and δ-Opioid Antagonist Activity

Recently, we have demonstrated that substitution of 1,2,3,4 tetrahyidroisoquinoline-3- carboxylic acid (Tic) in place of Gly2 in dynorphin A-(1-13)-NH2 (DYN) analogue (A) decreased the affinity to the kappa, delta, and micro receptors, and kappa selectivity. The doubly substituted analogue [2',6'-dimethyl-L-tyrosine (Dmt1)-Tic2]DYN (B) exhibited high delta-affinity (Ki=0.39 nM) while micro- and kappa-affinities were only an order of magnitude less (4-5 nM). Bioactivity of [Tic2]DYN peptide (A) on guinea-pig ileum and rabbit jejunum revealed potent delta- and kappa-antagonism thus indicating that the conversion from a kappa-agonist to antagonist occurred with the inclusion of Tic into DYN analogues, similar to the appearance of antagonist properties with delta-opioid agonists containing a Tic2 residue. The present study was undertaken to compare the k- and delta-opioid antagonistic activity of two [Tic2] DYN peptides (A and B) with naloxone a well known non selective opioid receptor antagonist. This comparison was performed by using the model of opioid withdrawal in vitro. Following a 4 min in vitro exposure to U50-488 H (10(-8) M), a selective k opioid receptor agonist, the guinea-pig isolated ileum exhibited a strong contracture after the addition of naloxone (10(-5) M). Also, following a 4 min in vitro exposure to deltorphin II (10(-8) M), a selective delta opioid receptor agonist, the rabbit jejunum exhibited a strong contracture after the addition of naloxone (10(-5) M). Results are expressed as percent of Ach contractions. In our study, we showed that in guinea pig ileum the peptide A (k opioid receptor antagonist) was able to induce a strong contracture at a concentration of 10(-9) M when injected 4 min after U50-488H (10(-8) M). Also, in rabbit jejunum the peptide B (delta-opioid receptor antagonist) was able to induce a strong contracture at a concentration of 10(-10) M when injected 4 min after deltorphin II (10(-8) M). The results of our experiments indicate that both peptide A (k receptor opiod antagonist) and peptide B (alpha receptor opioid antagonist) showed an antagonistic activity higher than naloxone.

ORL1 and opioid receptor preferences of nociceptin and dynorphin A analogues with Dmp substituted for N-terminal aromatic residues

Nociceptin (NOC) and dynorphin A (DYN) analogues containing 2′,6′-dimethylphenylalanine (Dmp) in place of Phe or Tyr in position 1 and/or 4 were synthesized and their metabolic stability and receptor-binding properties were investigated. [Dmp 1]NOC(1–13)-NH 2 ( 1) possessed high ORL1 receptor affinity comparable to that of the parent peptide with substantially improved affinities for κ-, μ-, and δ-opioid receptors. However, Dmp 4 substitution of NOC peptide ( 2) reduced ORL1 receptor affinity. [Dmp 1]DYN(1–13)-NH 2 ( 4) and its Dmp 4 analogue ( 5) possessed a 3-fold greater κ-opioid receptor affinity and improved κ-receptor selectivity compared to the parent peptide. Analogue 4 however exhibited an unexpectedly low in vitro bioactivity (GPI assay), suggesting, the phenolic hydroxyl group at the N-terminal residue in DYN peptide is extremely important for activation of the κ-opioid receptor. Analogue 5 possessed an improved κ-opioid receptor selectivity with an IC 50 ratio of 1(κ)/509(μ)/211598(δ); thus, this peptide may serve as a highly selective κ-receptor agonist for pharmacological study. Dmp 1 substitution in both the NOC and DYN peptides improved metabolic stability toward these peptides, while Dmp 4 substitution provided no additional metabolic stability.

Defective prodynorphin processing in mice lacking prohormone convertase PC2.

Prodynorphin, a multifunctional precursor of several important opioid peptides, is expressed widely in the CNS. It is processed at specific single and paired basic sites to generate various biologically active products. Among the prohormone convertases (PCs), PC1 and PC2 are expressed widely in neuroendocrine tissues and have been proposed to be the major convertases involved in the biosynthesis of hormonal and neural peptides. In this study we have examined the physiological involvement of PC2 in the generation of dynorphin (Dyn) peptides in mice lacking active PC2 as a result of gene disruption. Enzymological and immunological assays were used to confirm the absence of active PC2 in these mice. The processing profiles of Dyn peptides extracted from brains of these mice reveal a complete lack of Dyn A-8 and a substantial reduction in the levels of Dyn A-17 and Dyn B-13. Thus, PC2 appears to be involved in monobasic processing, leading to the generation of Dyn A-8, Dyn A-17, and Dyn B-13 from prodynorphin under physiological conditions. Brains of heterozygous mice exhibit only half the PC2 activity of wild-type mice; however, the levels of Dyn peptides in these mice are similar to those of wild-type mice, suggesting that a 50% reduction in PC2 activity is not sufficient to significantly reduce prodynorphin processing. The disruption of the PC2 gene does not lead to compensatory up-regulation in the levels of other convertases with similar substrate specificity because we find no significant changes in the levels of PC1, PC5/PC6, or furin in these mice as compared with wild-type mice. Taken together, these results support a critical role for PC2 in the generation of Dyn peptides.

Rational design of dynorphin A analogues with δ-receptor selectivity and antagonism for δ- and κ-receptors

Substitution of 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic) in place of Gly 2 in dynorphin A-(1–13)-NH 2 and -(1–11)-NH 2 (DYN) analogues ( 1 and 2) decreased the affinity to the κ, δ, and μ receptors, and κ selectivity. The analogue [ d-Ala 2, des-Gly 3]DYN ( 4), a chimera between deltorphin/dermorphin N-terminal tripeptide and DYN, was virtually inactive for κ-sites while the affinities for δ- and μ-receptors remained essentially unchanged. The doubly substituted analogue [2′,6′-dimethyl- l-tyrosine (Dmt 1)-Tic 2]DYN ( 3) exhibited high δ-affinity ( K i = 0.39 nM) while μ- and κ-affinities were only an order of magnitude less (4–5 nM). Bioactivity of [Tic 2]DYN peptides ( 1–3) on guinea-pig ileum and rabbit jejunum revealed potent δ- and κ-antagonism, while the δ agonist potency of 4 was comparable to DYN. Thus, conversion from a κ-agonist to antagonist occurred with the inclusion of Tic into DYN analogues, similar to the appearance of antagonist properties with δ- and μ-opioid agonists containing a Tic 2 residue.

Antinociceptive effects of dynorphin peptides in a model of inflammatory pain

Dynorphin A (DYN) peptides, administered into the central nervous system, have produced inconsistent analgesic actions in tests using thermal stimuli. This study examined antinociceptive effects of intravenous and intraplantar DYN-(2-17) against noxious pressure in rats with Freund's adjuvant-induced unilateral hindpaw inflammation. The effects of DYN-(2-17) were compared to those of the opioid agonists morphine, ( d-Ala 2, N-Methyl-Phe 4,Gly-ol 5)-enkephalin (DAMGO) and DYN-(1-17). Intravenous DYN-(2-17) (0.188–10 mg/kg) produced dose-dependent elevations of paw pressure thresholds in inflamed and in non-inflamed paws. These effects were similar in magnitude to those of subcutaneous morphine (2 mg/kg), at doses of 0.375–1.5 mg/kg they were significantly greater on the inflamed (right) than on the non-inflamed (left) paw, and they were not reversible by intravenous naloxone (1–10 mg/kg). Intraplantar Dyn-(2-17) (0.001–0.3 mg) was ineffective, whereas both intraplantar DYN-(1-17) (0.15–0.3 mg) and DAMGO (0.008–0.016 mg) produced dose-dependent and naloxone-reversible elevations of paw pressure thresholds. The intraplantar injection of both Dyn peptides produced a transient increase in the volume of non-inflamed paws. These findings suggest that intravenous DYN-(2-17) produces possibly centrally mediated, non-opioid antinociceptive effects against noxious pressure. At certain doses these effects are more potent in inflamed than in non-inflamed paws. In contrast to the opioid peptides DYN-(1-17) and DAMGO, DYN-(2-17) has no peripheral antinociceptive actions.

Molecular basis for dynorphin A selectivity: A chimeric study

The role of Coulombic interactions (1,2,3,4) in the selectivity of DynA fragments toward the ic receptor was investigated by the construction of several rd8 receptor chimeras. The affinity of different Dyn fragments to these chimeric as well as wild type receptors were determined by competition study. Based on the binding profile of DynA fragments at different receptors, it is suggested that while the 8 receptor may contain a high affinity pocket for the Tyr-Gly-Gly-Phe opioid core, the Coulombic interactions between the positive charges in the Dyn peptides and the negative charges in the extracellular (EC) domains of the 1< receptor may play an important role in the selectivity of DynA toward the ic receptor. Restriction sites Aft3 and Bgl2 present in both the rat ~c and 8 receptor cDNA (4, 5) were utilized to produce chimeric 1¢/8 receptors as illustrated in the following figure. Small circles represent the net negative charges in the EC domains.

Dynorphin interaction with the NMDA receptor/channel complex:Correlation with spinal cord injury

Dynorphin A and related peptides have been implicated in the neuropathology of spinal cord injury by a mechanism that may involve an interaction with the NMDA receptor-channel complex. Using radioligand binding and electrophysiological techniques, the present study has investigated the possible post-synaptic interaction between dynorphin (dyn) peptides and the MK-801 related channel site of the NMDA complex in rat brain and spinal cord. Dyn fragments (0.01-10 μM) produced a partial inhibition (45-75%) of [ 3 H]MK-801 binding to cerebral cortex membranes with slope values significantly greater than unity (-1.2 to -2.2)

Evidence for the Corelease of Dynorphin and Glutamate from Rat Hippocampal Mossy Fiber Terminals

Hippocampal mossy fiber (MF) nerve endings may be isolated in a subcellular fraction (P3) that releases both prodynorphin-derived peptides and glutamate (Glu) in a calcium-dependent manner when depolarized. However, this isolation procedure does not yield a pure preparation of MF synaptosomes. The present study evaluates the proportion of dynorphin (Dyn) and Glu that is released from synaptosomes in the P3 fraction that are of MF origin. We have addressed this issue by determining the degree to which a selective lesion of the dentate granule cell/MF system in vivo concomitantly reduces the exocytosis of Dyn and Glu from the P3 subcellular fraction. Unilateral injections of colchicine into the dentate gyrus resulted in a substantial and selective degeneration of the granule cell/MF pathway in the rat hippocampal formation. The overall integrated density of the Timm-stained band, which corresponds to the position of the MF terminal field, was estimated to be reduced by 75%. After this extensive loss of MF boutons, the K(+)-evoked release of Dyn and Glu from the P3 fraction was reduced by 95 and 51%, respectively. The loss of Timm staining and evoked Dyn release indicate that colchicine effectively eliminated MF synaptosomes from the P3 fraction. Those subcellular entities that were not destroyed by colchicine comprised approximately 50% of the protein and evoked Glu release measured by using the P3 fraction. In addition, the present results demonstrate that the inhibitory potency of the kappa opioid agonist U-50,488H was not altered by the elimination of MF boutons from this synaptosomal preparation. This finding indicates that U-50, 488H is capable of suppressing Glu exocytosis from both MF and non-MF synaptosomes. These results are consistent with the hypothesis that Dyn peptides and Glu are co-released from hippocampal MF terminals.