enkephalin--ala(2)-mephe(4)-gly(5)- and tyrosyl-prolyl-leucyl-glycinamide

Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2) and Met-enkephalin share a saturable transport system (peptide transport system-1, PTS-1) across the blood-brain barrier but do not readily bind to each other's receptors. This information allows the unique opportunity to differentiate the transport protein(s) from the receptors for either peptide in brain endothelial cells. PTS-1 was studied in vitro by allowing radiolabeled Tyr-MIF-1 (125I-Tyr-MIF-1) to bind to the solubilized proteins of isolated murine brain microvessels in the presence or absence of potential inhibitors. Sephadex chromatography separated bound from free labeled peptide. The binding was saturable as shown by inhibition with increasing concentrations of unlabeled Tyr-MIF-1. 125I-Tyr-MIF-1 binding was not inhibited by an unrelated peptide or iodo-tyrosine. D-Tyr-MIF-1 had no effect, demonstrating the stereospecificity of the system. Met-enkephalin decreased the binding of 125I-Tyr-MIF-1 to 84.4% of total, whereas Leu-enkephalin was without effect. Agonists for the mu, delta, and kappa opiate receptors did not change the binding, indicating that the proteins which bound to 125I-Tyr-MIF-1 were not endogenous opiate receptors. The results indicate that, in vitro, Tyr-MIF-1 binds to brain microvessel proteins with characteristics similar to PTS-1.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Analgesics, Non-Narcotic; Analgesics, Opioid; Animals; Binding, Competitive; Blood-Brain Barrier; Capillaries; Carrier Proteins; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalin, Leucine; Enkephalin, Methionine; Iodine Radioisotopes; Male; Mice; Mice, Inbred ICR; MSH Release-Inhibiting Hormone; Receptors, Opioid; Thyrotropin-Releasing Hormone; Tyrosine

The penetration of four radioiodinated materials-Tyr-MIF-1, DAMGO, tyrosine, and albumin-into the periventricular tissue after ICV injection was studied in rats by film autoradiography. Rates of disappearance from the CNS for the injected compounds were also determined by computer-assisted image analysis of the autoradiographic images. The four materials showed distinct patterns of dispersion from the ventricular system, with Tyr-MIF-1 moving farthest into the parenchyma of the brain and albumin primarily restricted to the ventricular space. The other two compounds, tyrosine and DAMGO, had intermediate values. Tyr-MIF-1 also displayed the fastest rate of removal from the brain, which may represent the ability of the peptide to gain access to sites of saturable transport. By contrast, the exit from the brain of DAMGO was minimal, whereas the efflux of albumin and tyrosine was intermediate. These results show the utility of these methods in the simultaneous measurement of both the patterns of distribution within the CNS and the rates of removal from the CNS of compounds injected into the brain.

Topics: Albumins; Analgesics; Animals; Autoradiography; Brain Chemistry; Cerebral Ventricles; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; MSH Release-Inhibiting Hormone; Rats; Rats, Sprague-Dawley; Tyrosine

The relative binding to mu, delta, and kappa opiate receptors was characterized for the brain peptides Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2), Tyr-W-MIF-1 (Tyr-Pro-Trp-Gly-NH2), and two fragments of Tyr-W-MIF-1 (Tyr-Pro-Trp and Tyr-Pro-Trp-Gly) previously shown to have antagonist as well as agonist activity in the guinea pig ileum. Tyr-MIF-1 had relatively low affinity (Ki = 1 microM at the mu site) but high selectivity (400- and 700-fold greater affinity for mu over delta and mu over kappa binding). Tyr-W-MIF-1 (Ki = 71 nM at the mu site) showed higher affinity binding to all three sites than Tyr-MIF-1 while retaining 200-fold selectivity for mu over delta and kappa receptors. The affinity of the fragments of Tyr-W-MIF-1 was lower for mu but higher for delta receptors. We also tested two cyclized analogs of Tyr-W-MIF-1 that were about 200-fold more active than the parent compound in producing analgesia. These analogs showed higher affinity binding to all three opiate receptors. One of the analogs showed binding affinity to mu sites (Ki = 1.3 nM) that was within 3-fold of that of the potent analog of enkephalin, DAMGO. Thus, brain peptides with an N-terminal Tyr-Pro, rather than the Tyr-Gly-Gly-Phe sequence typical of other endogenous opiates, can provide high selectivity for mu opiate receptors. Analogs based on one of them, Tyr-Pro-Trp-Gly-NH2, show high affinity as well as potent analgesic activity.

Topics: Amino Acid Sequence; Animals; Brain; Cerebellum; Endorphins; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Guinea Pigs; Molecular Sequence Data; MSH Release-Inhibiting Hormone; Peptide Fragments; Peptides, Cyclic; Rats; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, kappa; Receptors, Opioid, mu; Structure-Activity Relationship; Synaptic Membranes

The brain peptide Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2) was tested for its effects on electrically stimulated contractions in the guinea pig ileum assay. Tyr-MIF-1 acted as an opiate agonist in reducing these contractions. Its IC50 was about 9 microM, and its effects were reversed by naloxone and CTOP. The ability of Tyr-MIF-1 also to antagonize the inhibitory effects of opiates on electrically stimulated contractions was more evident in the ileum removed from a guinea pig tolerant to morphine or after partial inactivation of opiate receptors with beta-CNA. Similar results were observed with hemorphin. The endogenous peptide Tyr-MIF-1 and the blood-derived peptide hemorphin, therefore, can act as agonists as well as antagonists in the guinea pig ileum. The effects as antagonists are best observed in preparations of ileum with reduced receptor reserve (tolerant or beta-CNA treated) and are consistent with the idea that properties of endogenous peptides as opiate antagonists are enhanced in the tolerant state.

Topics: Amino Acid Sequence; Animals; Dose-Response Relationship, Drug; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalins; Guinea Pigs; Hemoglobins; Ileum; In Vitro Techniques; Male; Molecular Sequence Data; Morphine; MSH Release-Inhibiting Hormone; Muscle Contraction; Muscle, Smooth; Naloxone; Naltrexone; Narcotic Antagonists; Peptide Fragments; Receptors, Opioid; Receptors, Opioid, kappa; Receptors, Opioid, mu

Binding of Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2) to mu and delta opiate receptors was compared with other putative opiate antagonist peptides by displacement of iodinated ligands selective for mu (DAGO, FK33824, and morphiceptin) and delta (DPDPE) receptors. Tyr-MIF-1 and ACTH (1-24 and 1-39) inhibited binding of 125I-DAGO with IC50's of about 1 microM. FMRF-NH2 was about an order of magnitude weaker while CCK-8 and MIF-1 failed to inhibit 50% of binding at concentrations up to 100 microM. Morphiceptin, Tyr-MIF-1, and ACTH were less potent but more efficacious than DAGO, FK33824, morphine, or naloxone in inhibiting the binding of 125I-morphiceptin. Tyr-MIF-1 appeared to have a more selective action at opiate receptors than ACTH; in contrast to their effects at 125I-DAGO-labeled sites, morphiceptin and Tyr-MIF-1 inhibited less than 50% of 125I-DPDPE binding at concentrations up to 10 and 50 microM, while ACTH 1-39 and 1-24 inhibited more than 80% of the binding at 2.5 and 5 microM, respectively. The results indicate that at relatively high concentrations Tyr-MIF-1, like ACTH, can affect binding to the opiate receptor, but unlike ACTH, binding of Tyr-MIF-1 appears relatively selective for the mu site.

Topics: Animals; Binding, Competitive; Brain; Endorphins; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enkephalin, D-Penicillamine (2,5)-; Enkephalins; In Vitro Techniques; Male; MSH Release-Inhibiting Hormone; Naloxone; Rats; Rats, Inbred Strains; Receptors, Opioid; Receptors, Opioid, delta; Receptors, Opioid, mu