dynorphins and dynorphin-(1-11)

Arodyn (aromatic dynorphin) is a novel analogue of the opioid peptide dynorphin A with a nonbasic N-terminus that exhibits nanomolar affinity (K(i) = 10 nM) and remarkable selectivity for kappa opioid receptors (K(i) ratio (kappa/mu/delta) = 1/174/583). Arodyn completely reverses the agonism of dynorphin A (1-13)NH(2) in a concentration-dependent manner in the adenylyl cyclase assay. Thus arodyn is a novel kappa opioid receptor selective antagonist that will be useful to study these receptors.

Topics: Adenylyl Cyclases; Amino Acid Sequence; Animals; CHO Cells; Cricetinae; Dynorphins; Peptide Fragments; Radioligand Assay; Receptors, Opioid, kappa; Structure-Activity Relationship

We previously have reported four possible binding conformation of dynorphin A (Dyn A) for the central kappa opioid receptors, induced by the address sequence, using a molecular mechanics energy minimization approach. The lowest energy conformation was found to exhibit an alpha-helical conformation in the cyclized address sequence. It was suggested that an alpha-helical conformation in the cyclized address sequence or a helical conformation induced by the conformational characteristics of the message sequence may be important for binding potency and kappa opioid receptor selectivity. Side chain to side chain lactam bridges between the i and i + 4 positions have been shown to stabilize alpha-helical conformation. Thus, a series of cyclic lactam analogues of dynorphin A(1-11)-NH2 have been designed, synthesized and evaluated by the guinea pig brain (GPB) binding assay and guinea pig ileum (GPI) bioassay to evaluate the conformational analysis prediction and, further, to investigate the conformational requirements for high potency and selectivity for kappa opioid receptors. Positions 2-6, 3-7, and 5-9 were chosen as the sites for incorporating cyclic conformational constraints. Cyclization between D-Asp(2) and Lys(6) in c[D-Asp(2),Lys(6)]Dyn A(1-11)-NH2 led to an analogue with pronounced potency and selectivity enhancement for the mu opioid receptor, whereas cyclization between D-Asp(3) and Lys(7) in c[D-Asp(3),Lys(7)]Dyn A(1-11)-NH2 led to a potent ligand (IC(50) 4.9 nM) with kappa receptor selectivity. The other analogues in the series proved to be less selective. The biological results led to the suggestion that the binding conformation for the kappa receptor may have structural requirements that are distinct from those of mu and delta receptors. Interestingly, analogues with a D-Asp at position 2, 3, or 9 were found to be more potent for the kappa receptor than analogues with an L-Asp at the same positions. It is suggested that the incorporation of D-Asp into position 2, 3, or 9 of Dyn A(1-11)-NH2 may have stereochemical and conformational effects on the nearby amino acids which can help discriminate the preference between kappa, mu, and delta receptors.

Topics: Amino Acid Sequence; Animals; Brain; Cell Membrane; Cyclization; Drug Design; Dynorphins; Guinea Pigs; Ileum; Lactams; Male; Molecular Sequence Data; Muscle Contraction; Peptide Fragments; Protein Conformation; Protein Structure, Secondary; Receptors, Opioid, kappa; Structure-Activity Relationship

In sheep, arginine vasopressin (AVP) appears to be a more potent ACTH-releasing factor than ovine corticotrophin-releasing hormone. In order to investigate the neuroendocrine regulation of AVP secretion we have developed a novel system for maintaining fetal ovine hypothalamic neurones in serum-free culture. Hypothalamic neurones derived from fetal sheep at day 70 gestation (term = 145 days) secreted AVP under basal conditions and in response to repeated potassium-induced depolarizations, for up to 35 days in vitro. AVP secretion was time- and calcium-dependent. AVP secreted from ovine hypothalamic cells co-eluted with synthetic AVP on a Sephadex chromatography column and diluted in parallel with AVP standard in the radioimmunoassay. The addition of cortisol (150 nM) to medium bathing ovine hypothalamic cells significantly inhibited basal, and potassium-induced AVP secretion without altering the AVP content of the cell cultures. Furthermore, the opioid peptide [D-Pro10]Dynorphin(1-11) which acts via the kappa opioid receptor, significantly inhibited basal and potassium-stimulated AVP secretion, an effect which was abolished when cells were cultured in the presence of cortisol. These data show that hypothalamic AVP is a site for negative feedback regulation within the ovine hypothalamic-pituitary-adrenal axis. Furthermore, these data suggest that the kappa opioid system inhibits AVP secretion from ovine hypothalamic neurones, a response which is modulated by glucocorticoids.

Topics: Animals; Arginine Vasopressin; Cells, Cultured; Dynorphins; Glucocorticoids; Hypothalamus; Opioid Peptides; Peptide Fragments; Potassium; Radioimmunoassay; Sheep

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Animals; Brain; Dynorphins; Guinea Pigs; In Vitro Techniques; Kinetics; Peptide Fragments; Pyrrolidines; Rats; Receptors, Opioid; Receptors, Opioid, kappa