Synthesis of an MIF-1 analogue containing enantiopure (S)-α-trifluoromethyl-proline and biological evaluation on nociception.

The synthesis and the effect of a novel MIF-1 analogue on nociception during acute pain in rat model are reported. The synthesis of this enantiopure trifluoromethyl group containing tripeptide was performed through a peptide coupling reaction between the HCl. Leu-Gly-NH2 and the (S)-α-Tfm-proline. The analgesic effect of the CF3-(MIF-1) 2 has been evaluated in vivo on rat model by paw pressure (PP) and hot plate (HP) tests and compared to the native peptide MIF-1. Highest analgesic effect was observed with CF3-(MIF-1) 2 only in PP test. In order to study the mechanisms of nociception induced by the studied peptides, the involvement of the opioid and the nitric oxideergic systems was investigated. The results are in favor of a participation of both system since pretreatment, 20 min before injection of the CF3-(MIF-1) 2, with the non-competitive antagonist of opiate receptors naloxone, the nitric oxide synthase (NOS) inhibitor l-N(G)-nitroarginine ester (l-NAME) or the nitric oxide (NO) donor l-arginine (l-Arg) significantly decreased the pain perception in PP and HP tests.

PAOPA, a potent analogue of Pro-Leu-glycinamide and allosteric modulator of the dopamine D2 receptor, prevents NMDA receptor antagonist (MK-801)-induced deficits in social interaction in the rat: implications for the treatment of negative symptoms in schizophrenia.

The aim of this study was to investigate whether a potent analogue of the endogenous brain peptide l-prolyl-l-leucyl-glycinamide (PLG), (3(R)-[(2(S)-pyrrolidinylcarbonyl)amino]-2-oxo-1-pyrrolidineacetamide (PAOPA), can prevent the induction of social withdrawal caused by sub-chronic treatment with the non-competitive NMDA (N-methyl-l-aspartate) receptor antagonist, MK-801. Results indicate that MK-801 (0.5 mg/kg) significantly decreased social interaction following sub-chronic treatment (7 days). Treatment with PAOPA (1 mg/kg) blocked the effects of MK-801, and increased the amount of time spent in social interaction in comparison to control animals. These results provide evidence for the development of peptidomimetic compounds for the treatment of social withdrawal and related negative symptoms associated with schizophrenia.

Behavioral effects of neuropeptides in rodent models of depression and anxiety.

In recent years, studies have advocated neuropeptide systems as modulators for the behavioral states found in mood disorders such as depression and anxiety disorders. Neuropeptides have been tested in traditional animal models and screening procedures that have been validated by known antidepressants and anxiolytics. However, it has become clear that although these tests are very useful, neuropeptides have distinct behavioral effects and dose-dependent characteristics, and therefore, use of these tests with neuropeptides must be done with an understanding of their unique characteristics. This review will focus on the behavioral actions of neuropeptides and their synthetic analogs, particularly in studies utilizing various preclinical tests of depression and anxiety. Specifically, the following neuropeptide systems will be reviewed: corticotropin-releasing factor (CRF), urocortin (Ucn), teneurin C-terminal associated peptide (TCAP), neuropeptide Y (NPY), arginine vasopressin (AVP), oxytocin, the Tyr-MIF-1 family, cholecystokinin (CCK), galanin, and substance P. These neuropeptide systems each have a unique role in the regulation of stress-like behavior, and therefore provide intriguing therapeutic targets for mood disorder treatment.

Effect of Tyr-MIF-1 peptides on blood ACTH and corticosterone concentration induced by three experimental models of stress.

1. Studies, using a wide variety of stressors, have clearly indicated that the pattern of neuroendocrine response is dependent upon the stress stimulus applied. 2. The Tyr-MIF-1 family of peptides (Tyr-MIF-1s) includes MIF-1, Tyr-MIF-1, Tyr-W-MIF-1 and Tyr-K-MIF-1. These neuropeptides, neuromodulators are able to inhibit the expression of some forms of stress-induced analgesia. 3. The aim of this study was to compare changes in ACTH and corticosterone (CORT) concentration after various stressors (immobilization, cold and heat), as well as after injection of investigated Tyr-MIF-1s peptides. 4. According to our results, hypothalamic-pituitary-adrenal (HPA) system was activated by all the stressors applied. Heat and immobilization are stronger stressors, as the exposure of animals to a high ambient temperature and immobilization resulted in the highest rise of plasma ACTH and CORT concentration when compared with cold stress. Moreover, all the investigated peptides from Tyr-MIF-1 family, administered after application of stressors, inhibited the elevations in adrenocorticotropic hormone (ACTH) and corticosterone (CORT) plasma concentrations significantly. 5. In conclusion, the various stressors applied seem to induce a different response of the HPA system as judged by quantitative changes in ACTH and CORT release. We suggest that Tyr-MIF-1 peptides may possess anti-stressor effects, as they inhibited stress-induced rising in two hormones that were investigated.

From MIF-1 to endomorphin: the Tyr-MIF-1 family of peptides.

The Tyr-MIF-1 family of small peptides has served a prototypic role in the introduction of several novel concepts into the peptide field of research. MIF-1 (Pro-Leu-Gly-NH(2)) was the first hypothalamic peptide shown to act "up" on the brain, not just "down" on the pituitary. In several situations, including clinical depression, MIF-1 exhibits an inverted U-shaped dose-response relationship in which increasing doses can result in decreasing effects. This tripeptide also can antagonize opiate actions, and the first report of such activity also correctly predicted the discovery of other endogenous antiopiate peptides. The tetrapeptide Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH(2)) not only shows antiopiate activity, but also considerable selectivity for the mu-opiate binding site. Tyr-W-MIF-1 (Tyr-Pro-Trp-Gly-NH(2)) is an even more selective ligand for the mu receptor, leading to the discovery of two more Tyr-Pro tetrapeptides that have the highest specificity and affinity for this site. These are the endomorphins: endomorphin-1 is Tyr-Pro-Trp-Phe-NH(2) and endomorphin-2 is Tyr-Pro-Phe-Phe-NH(2). Tyr-MIF-1 proved, contrary to the then prevailing dogma, that peptides can be saturably transported across the blood-brain barrier by a quantifiable transport system. Unexpectedly, the Tyr-MIF-1 transporter is shared with Met-enkephalin. In the era in which it was doubtful whether a peripheral peptide could exert CNS effects, the Tyr-MIF-1 family of peptides also explicitly showed that they can exert more than one central action that persists longer than their half-lives in blood. These peptides clearly illustrate that the name of a peptide restricts neither its actions nor its conceptual implications.

A Tyr-W-MIF-1 analog containing D-Pro2 discriminates among antinociception in mice mediated by different classes of mu-opioid receptors.

The antagonism by Tyr-D-Pro-Trp-Gly-NH2 (D-Pro2-Tyr-W-MIF-1), a Tyr-Pro-Trp-Gly-NH2 (Tyr-W-MIF-1) analog, of the antinociception induced by the mu-opioid receptor agonists Tyr-W-MIF-1, [D-Ala2,NMePhe4,Gly(ol)5]-enkephalin (DAMGO), Tyr-Pro-Trp-Phe-NH2 (endomorphin-1), and Tyr-Pro-Phe-Phe-NH2 (endomorphin-2) was studied with the mouse tail-flick test. D-Pro2-Tyr-W-MIF-1 (0.5-3 nmol) given intracerebroventricularly (i.c.v.) had no effect on the thermal nociceptive threshold. High doses of D-Pro2-Tyr-W-MIF-1 (4-16 nmol) administered i.c.v. produced antinociception with a low intrinsic activity of about 30% of the maximal possible effect. D-Pro2-Tyr-W-MIF-1 (0.25-2 nmol) co-administered i.c.v. showed a dose-dependent attenuation of the antinociception induced by Tyr-W-MIF-1 or DAMGO without affecting endomorphin-2-induced antinociception. A 0.5 nmol dose of D-Pro2-Tyr-W-MIF-1 significantly attenuated Tyr-W-MIF-1-induced antinociception but not DAMGO- or endomorphin-1-induced antinociception. The highest dose (2 nmol) of D-Pro2-Tyr-W-MIF-1 almost completely attenuated Tyr-W-MIF-1-induced antinociception. However, that dose of D-Pro2-Tyr-W-MIF-1 significantly but not completely attenuated endomorphin-1 or DAMGO-induced antinociception, whereas the antinociception induced by endomorphin-2 was still not affected by D-Pro2-Tyr-W-MIF-1. Pretreatment i.c.v. with various doses of naloxonazine, a mu1-opioid receptor antagonist, attenuated the antinociception induced by Tyr-W-MIF-1, endomorphin-1, endomorphin-2, or DAMGO. Judging from the ID50 values for naloxonazine against the antinociception induced by the mu-opioid receptor agonists, the antinociceptive effect of Tyr-W-MIF-1 is extremely less sensitive to naloxonazine than that of endomorphin-1 or DAMGO. In contrast, endomorphin-2-induced antinociception is extremely sensitive to naloxonazine. The present results clearly suggest that D-Pro2-Tyr-W-MIF-1 is a selective antagonist for the mu2-opioid receptor in the mouse brain. D-Pro2-Tyr-W-MIF-1 may also discriminate between Tyr-W-MIF-1-induced antinociception and the antinociception induced by endomorphin-1 or DAMGO, which both show a preference for the mu2-opioid receptor in the brain.

Study on the antinociceptive action of Tyr-K-MIF-1, a peptide from the MIF family.

1. Tyr-K-MIF-1 is a melanocyte inhibiting factor (MIF) neuropeptide, isolated from the brain. Opposite to other MIFs (Tyr-MIF-1, Tyr-W-MIF-1), it has a very low affinity for opiate mu-receptors, but interacts with Tyr-MIF-1 specific binding sites. Tyr-MIF-1 and Tyr-W-MIF-1 evoke antinociception mainly by activating opioid receptors. We investigated the possible antinociceptive effect of Tyr-K-MIF-1 and the involvement of histaminergic system in its mechanism of action. 2. Tested on rats by paw-pressure test, Tyr-K-MIF-1 (0.5, 1 and 2 mg kg(-1)) was associated with short-lasting analgesia, which was abolished by naloxone (1 mg kg(-1)). 3. Injected intraperitoneally (i.p.) 15 min before Tyr-K-MIF-1, antagonists of H(1) (diphenhydramine, 100 mg kg(-1)) or H(2) (famotidine, 0.3 and 0.6 mg kg(-1)) histamine receptors diminished peptide antinociceptive effect. Simultaneous H(1)- and H(2) blockade, as well as pretreatment with 5 mg kg(-1) dimaprit (H(2) agonist) abolished Tyr-K-MIF-1-induced analgesia. Tyr-K-MIF-1-induced analgesia was also abolished by treatment with R-(alpha)-methylhistamine (10 mg kg(-1), i.p.), an H(3) histamine receptor agonist that acts to inhibit histamine release. 4. Our results together with data reported in the literature support the conclusion that activation of the histaminergic system is involved in the mechanism of Tyr-K-MIF-1-induced antinociception.

Synthesis of enantiopure pyrrolidine-derived peptidomimetics and oligo-beta-peptides via nucleophilic ring-opening of beta-lactams.

The synthesis of the two enantiomers of pyrrolidine-derived spiro beta-lactams by resolution with D- and L-Boc phenylalanine is described. The potential of these optically active spiro beta-lactams on the synthesis of peptidomimetics as analogues of melanostatin is evaluated. Theoretical studies of several models, at the Becke3LYP/6-31+G* level of theory, together with previous experimental evidences from our group, gathered by NMR, allow us to design structures that can efficiently mimic some biologically active peptide-type molecules. On the other hand, the spiro beta-lactams have shown their utility in the preparation of beta-peptides. As an example, a homo-tetra-beta-peptide was synthesized. This research will continue in the future in order to obtain higher peptides with potential biological activity.

Involvement of spinal mu1-opioid receptors on the Tyr-d-Arg-Phe-sarcosine-induced antinociception.

The involvement of spinal mu-opioid receptor subtypes on the antinociception induced by i.t.-administered Tyr-D-Arg-Phe-sarcosine (TAPS), a N-terminal tetrapeptide analog of dermorphin, was determined in mice tail-flick test. Intrathecal administration of TAPS produced the marked inhibition of the tail-flick response in a dose-dependent manner. The antinociception induced by TAPS was completely eliminated by i.t.-co-administration of Tyr-D-Pro-Phe-Phe-NH2 (D-Pro2-endomorphin-2), the mu1-opioid receptor antagonist, whereas i.t. co-treatment with Tyr-D-Pro-Trp-Phe-NH2 (D-Pro2-endomorphin-1) or Tyr-D-Pro-Trp-Gly-NH2 (D-Pro2-Tyr-W-MIF-1), the mu2-opioid receptor antagonists, did not affect the TAPS-induced antinociception. In contrast, the antinociception induced by i.t.-administered [D-Ala2,N-MePhe4,Gly-ol5]enkephalin was significantly attenuated by i.t.-co-administration of D-Pro2-endomorphin-1 or D-Pro2-Tyr-W-MIF-1, but not D-Pro2-endomorphin-2. These results suggest that TAPS may stimulate spinal mu1-opioid receptors to produce the antinociception.

Design and synthesis of photoaffinity-labeling ligands of the L-prolyl-L-leucylglycinamide binding site involved in the allosteric modulation of the dopamine receptor.

Pro-Leu-Gly-NH(2) (PLG), in addition to its endocrine effects, possesses the ability to modulate dopamine D(2) receptors within the central nervous system. However, the precise binding site of PLG is unknown. Potential photoaffinity-labeling ligands of the PLG binding site were designed as tools to be used in the identification of the macromolecule that possesses this binding site. Six different photoaffinity-labeling ligands were designed and synthesized on the basis of the gamma-lactam PLG peptidomimetic 1. The 4-azidobenzoyl and 4-azido-2-hydroxybenzoyl photoaffinity-labeling moieties were placed at opposite ends of PLG peptidomimetic 1 to generate a series of ligands that potentially could be used to map the PLG binding site. All of the compounds that were synthesized possessed activity comparable to or better than PLG in enhancing [(3)H]-N-propylnorapomorphine agonist binding to dopamine receptors. Photoaffinity ligands that were cross-linked to the receptor preparation produced a modulatory effect that was either comparable to or greater than the increase in agonist binding produced by the respective ligands that were not cross-linked to the dopamine receptor. The results indicate that these photoaffinity-labeling agents are binding at the same allosteric site as PLG and PLG peptidomimetic 1.

Synthesis and utilization of 13C and 15N backbone-labeled proline: NMR study of synthesized oxytocin with backbone-labeled C-terminal tripeptide amide.

The 13C and 15N backbone-labeled proline was prepared using Oppolzer's method based on application of a sultam as chiral auxiliary. This isotopomer was used in the synthesis of the 13C, 15N backbone-labeled C-terminal tripeptide amide fragment of neurohypophyseal hormone oxytocin. Finally, this tripeptide amide was coupled by segment condensation with N-Boc- or N-Fmoc-tocinoic acid, followed by N-deprotection with TFA or piperidine. The labeled oxytocin exhibited biological activity identical with that of natural oxytocin. A detailed 1H, 13C and 15N NMR study confirmed the assigned oxytocin conformation containing a beta-turn in the cyclic part of the molecule, stabilized by H-bond(s) that can be perturbed by the C-terminal tripeptide amide moiety as indicated by comparison of NMR data for both the tocine ring in oxytocin and tocinoic acid.

A Tyr-W-MIF-1 analog containing D-Pro2 acts as a selective mu2-opioid receptor antagonist in the mouse.

The antagonistic properties of Tyr-d-Pro-Trp-Gly-NH(2) (d-Pro(2)-Tyr-W-MIF-1), a Tyr-Pro-Trp-Gly-NH(2)(Tyr-W-MIF-1) analog, on the antinociception induced by the mu-opioid receptor agonists Tyr-W-MIF-1, [d-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO), Tyr-Pro-Trp-Phe-NH(2) (endomorphin-1), and Tyr-Pro-Phe-Phe-NH(2) (endomorphin-2) were studied in the mouse paw-withdrawal test. d-Pro(2)-Tyr-W-MIF-1 injected intrathecally (i.t.) had no apparent effect on the thermal nociceptive threshold. d-Pro(2)-Tyr-W-MIF-1 (0.1-0.4 nmol) coadministered i.t. showed a dose-dependent attenuation of the antinociception induced by Tyr-W-MIF-1 without affecting endomorphin- or DAMGO-induced antinociception. However, higher doses of d-Pro(2)-Tyr-W-MIF-1 (0.8-1.2 nmol) significantly attenuated endomorphin-1- or DAMGO-induced antinociception, whereas the antinociception induced by endomorphin-2 was still not affected by d-Pro(2)-Tyr-W-MIF-1. Pretreatment i.t. with various doses of naloxonazine, a mu(1)-opioid receptor antagonist, attenuated the antinociception induced by Tyr-W-MIF-1, endomorphin-1, endomorphin-2, or DAMGO. Judging from the ID(50) values for naloxonazine against the antinociception induced by the mu-opioid receptor agonists, the antinociceptive effect of Tyr-W-MIF-1 is extremely less sensitive to naloxonazine than those of endomorphin-1 or DAMGO. In contrast, endomorphin-2-induced antinociception is extremely sensitive to naloxonazine. The present results clearly suggest that d-Pro(2)-Tyr-W-MIF-1 is the selective antagonist to be identified for the mu(2)-opioid receptor in the mouse spinal cord. d-Pro(2)-Tyr-W-MIF-1 may also discriminate between Tyr-W-MIF-1-induced antinociception and the antinociception induced by endomorphin-1 or DAMGO, all of which show a preference for the mu(2)-opioid receptor in the spinal cord.

Effects of Tyr-MIF's family of peptides on immobilization stress-induced antinociception in rats.

UNLABELLED: The Tyr-MIF-1 family of peptides includes MIF-1, Tyr-MIF-1, Tyr-W-MIF-1 and Tyr-K-MIF-1, which have been isolated from bovine hypothalamus and cortex of human brain. Data in the literature suggest that peptides of Tyr-MIF-1's family have opioid-like and anti-opioid actions. All these peptides interact with opioid receptors and in addition bind to non-opiate sites specific for each of the peptides and able to inhibit the expression of some forms of stress-induced analgesia (SIA) in various species. AIM: To examined the effects of the Tyr-MIF-1's peptides on immobilization stress-induced antinociception. METHODS: Tyr-MIF-1's peptides were given to male Wistar rats intraperitoneally before or after 1 hour of restraint. The changes in the mechanical nociceptive threshold of the animals were measured by the Randall-Selitto paw pressure test. RESULTS: Immobilization of the rats increased the pain threshold at least 1 h. Tyr-MIF-1's peptides have contrasting effects on immobilization stress-induced antinociception in paw-pressure test in rats. When administered before immobilization procedure they potentiated the immobilization stress-induced antinociception, while if given after immobilization, they reduced it. Antinociceptive effects of Tyr-MIF-1, Tyr-W-MIF-1 and Tyr-K-MIF-1 were reduced in condition of stress. CONCLUSION: Tyr-MIF-1's peptides exerted antiopioide effects under condition of stress in paw-pressure test. These antiopioide effects were more pronounced when peptides were injected after stress exposure.

Antiopioid properties of the TYR-MIF-1 family.

The Tyr-MIF-1 family of peptides includes MIF-1, Tyr-MIF-1, Tyr-W-MIF-1 and Tyr-K-MIF-1, which have been isolated from bovine hypothalamus and human brain cortex. All these peptides interact with opioid receptors and in addition bind to non-opiate sites specific for each of the peptides. Data in the literature suggest that peptides of the Tyr-MIF-1 family (Tyr-MIF-1s) have antiopioid and opioid- like effects. It is known that some anti-opioid peptides (AOP) could reverse morphine-induced analgesia in rodents and men and able to inhibit the expression of some forms of stress-induced analgesia (SIA) in various species. We examined the effects of the Tyr-MIF-1 peptides (all in dose 1 mg/kg i.p.) in the male Wistar rats on morphine-induced analgesia in acute pain using the paw-pressure (PP) and the tail-flick (TF) tests and on immobilization stress-induced antinociception using the PP test. Our results showed that the Tyr-MIF-1 peptides significantly decreased the analgesic effect of morphine (1 mg/kg i.p.) in both tests used. Immobilization of the rats increased the pain threshold for at least 1 h. The Tyr-MIF-1 peptides reduced stress-induced antinociception in PP test. In conclusion, our findings indicate that Tyr-MIF-1s modulate the analgesic effects of morphine and SIA, which corresponds with the hypothesis about AOP mentioned above.

Development of a new family of conformationally restricted peptides as potent nucleators of beta-turns. Design, synthesis, structure, and biological evaluation of a beta-lactam peptide analogue of melanostatin.

Novel enantiopure (i)-(beta-lactam)-(Gly)-(i+3) peptide models, defined by the presence of a central alpha-alkyl-alpha-amino-beta-lactam ring placed as the (i+1) residue, have been synthesized in a totally stereocontrolled way by alpha-alkylation of suitable N-[bis(trimethylsilyl)methyl]-beta-lactams. The structural properties of these beta-lactam pseudopeptides have been studied by X-ray crystallography, Molecular Dynamics simulation, and NOESY-restrained NMR simulated annealing techniques, showing a strong tendency to form stable type II or type II' beta-turns either in the solid state or in highly coordinating DMSO solutions. Tetrapeptide models containing syn- or anti-alpha,beta-dialkyl-alpha-amino-beta-lactam rings have also been synthesized and their conformations analyzed, revealing that alpha-alkyl substitution is essential for beta-turn stabilization. A beta-lactam analogue of melanostatin (PLG amide) has also been prepared, characterized as a type-II beta-turn in DMSO-d6 solution, and tested by competitive binding assay as a dopaminergic D2 modulator in rat neuron cultured cells, displaying moderate agonist activity in the micromolar concentration range. On the basis of these results, a novel peptidomimetic design concept, based on the separation of constraint and recognition elements, is proposed.

Involvement of nitric oxide in the nociception of kyotorphin, TYR-CAV and MIF-s analogues in the rat spinal cord.

The spinal analgesic effects of Kyotorphin (Kyo) and Melanocyte-inhibiting factor (MIF-l) were studied during acute pain in rats chronically implanted with intrathecal (i.t.) cannulas. Kyo (5 micrograms), t-Cav (5 micrograms), Tyr-Cav (5 micrograms), L-NAME (1500 micrograms), MIF-Cav (200-400 micrograms) and MIF-sLeu (200 micrograms) exerted antinociceptive effects in both tests. The coadministration of Kyo + L-NAME enhanced the nociceptive effect compared with L-NAME (PP) or Kyo alone (PP, TF). The combination of Tyr-Cav + L-NAME enhanced the antinociceptive effect compared with L-NAME (PP) or Tyr-Cav alone (TF, PP). MIF-l (200 micrograms) had a weak antinociceptive effect in both tests. The coadministration of MIF-Cav + L-NAME enhanced the nociceptive effect compared with L-NAME (TF) or MIF-Cav alone (TF). The combination of MIF-sLeu + L-NAME enhanced the antinociceptive effect compared with L-NAME (TF) or MIF-sLeu alone (TF, PP). The results suggest that nitric oxide (NO) is involved in the antinociceptive effects of neuropeptides in the rat spinal cord.

Involvement of H2-receptors in the mechanism of analgesic action of Tyr-MIF-1.

The antinociceptive effects of H2-agents cimetidine (CIM) and dimaprit (DMP) as well as their effects on the Tyr-MIF-1-evoked analgesia have been studied after intraperitoneal (i.p.) administration in rats. In the paw-pressure (PP) test Tyr-MIF-1 (1 mg/kg), CIM (50 and 100mg/kg) and DMP (5 and 10mg/kg) induced analgesia. Injected before DMP, naloxone (NAL) and CIM diminished or completely prevented the pain-relieving effect of H2-agonist DMP. The antinociceptive effect of Tyr-MIF-1 has been potentiated by DMP dose-dependently. CIM (50mg/kg) decreased the antinociceptive action of the combination Tyr-MIF-1 + DMP, while CIM (100mg/kg) expressed a weaker inhibitory effect on it. The data obtained clearly show that H2-receptor activation is involved in the mechanism of the Tyr-MIF-1 antinociceptive action.

Inhibition of hematopoietic progenitor cell growth by Tyr-MIF, an endogenous opiate modulator, and its degradation products.

There is increasing evidence that neuronal factors can affect hematopoietic cell proliferation. Endogenous opioids with specificity for several opioid receptor classes were tested for their ability to inhibit murine and human hematopoietic progenitor cell proliferation. Tyr-MIF, an opioid tetrapeptide (H-Tyr-Pro-Leu-Gly-NH2), demonstrated a dose-dependent inhibition of colony formation at concentrations < 10 uM, inhibiting M-CSF and G-CSF-responsive progenitor cells equally. Tyr-MIF did not inhibit the number of colonies responsive to recombinant interleukin 3 (rmIL-3) or recombinant murine granulocyte-macrophage colony stimulating factor (rmGM-CSF), but significantly reduced colony size of GM-CSF responsive colonies. Colony formation by human low density and CD34+ marrow cells in response to G-CSF was also inhibited by Tyr-MIF and was more sensitive to inhibition than murine progenitor cells. Colony formation by single CD34+ cells was also inhibited by Tyr-MIF, indicating an effect directly on progenitor cells. Incubation of marrow cells in liquid culture and removal of Tyr-MIF prior to quantitating progenitor cell proliferation demonstrated that opioid-induced inhibition was reversible. The inhibitory effect of Tyr-MIF was not blocked by naloxone, a mu receptor specific antagonist, or diminished in mu opioid receptor deficient mice. HPLC analysis of cell-free culture medium containing Tyr-MIF showed no presence of the parent peptide after 24 h while progenitor cell inhibitory activity was retained. Analysis of potential degradation products of Tyr-MIF indicated that only H-Gly-NH9 or H-Gly-NH2 containing peptides inhibited colony forming unit (CFU) proliferation. These results indicate that Tyr-MIF is a reversible inhibitor of mature hematopoietic progenitor cell proliferation, and that this effect is most likely mediated by the degradation product H-Gly-NH2. Potential applications including protection of myeloid cells after cytosuppresive therapy are discussed.

Endomorphins, Met-enkephalin, Tyr-MIF-1, and the P-glycoprotein efflux system.

The P-glycoprotein (P-gp) transport system, responsible for the efflux of many therapeutic drugs out of the brain, recently has been shown to transport the endogenous brain opiate endorphin. We used P-gp knockout mice (Mdr1a) and their controls to determine where P-gp is involved in the saturable efflux systems of four other endogenous opiate-modulating peptides across the blood-brain barrier (BBB). After injection of endomorphin-1 (Tyr-Pro-Trp-Phe-NH(2)), endomorphin-2 (Tyr-Pro-Phe-Phe-NH(2)), Met-enkephalin (Tyr-Gly-Gly-Phe-Met-OH), and Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH(2)) into the lateral ventricle of the mouse brain, residual radioactivity was measured at 0, 2, 5, 10, and 20 min later. The results showed no difference in the disappearance of any of these peptides from the brains of knockout mice compared with their controls. This demonstrates that unlike endorphin and morphine, P-gp does not seem to be required for the brain-to-blood transport of the endomorphins, Met-enkephalin, or Tyr-MIF-1 across the BBB.

Saturable brain-to-blood transport of endomorphins.

Opiate-modulating tetrapeptides such as tyrosine-melanocyte-stimulating hormone-release inhibiting factor-1 (Tyr-MIF-1; Tyr-Pro-Leu-Gly-NH2) and Tyr-W-MIF-1 (Tyr-Pro-Trp-Gly-NH2) are saturably transported from brain to blood. We examined whether two recently described endogenous opiate tetrapeptides with similar structures, the mu-specific endomorphins, also are transported across the blood-brain barrier (BBB). We found that the efflux rates of endomorphin-1 (Tyr-Pro-Trp-Phe-NH2) and endomorphin-2 (Tyr-Pro-Phe-Phe-NH2) were each self-inhibited by an excess of the respective endomorphin, thereby defining saturable transport. Cross-inhibition of the transport of each endomorphin by the other indicated shared transport. By contrast, no inhibition of the efflux of either endomorphin resulted from coadministration of Tyr-MIF-1, indicating that peptide transport system-1 (PTS-1) was not involved. Tyr-W-MIF-1, which is partially transported by PTS-1, significantly (P<0.01) decreased the transport of endomorphin-1 and tended (P=0.051) to decrease the transport of endomorphin-2, consistent with its role as both an opiate and antiopiate. Although involved in modulation of pain, coinjection of calcitonin gene-related peptide or constriction of the sciatic nerve did not appear to inhibit endomorphin efflux. Thus, the results demonstrate the existence of a new efflux system across the BBB which saturably transports endomorphins from brain to blood.