Melanocyte-Stimulating Hormone Release-Inhibiting Factor-1 (MIF-1) Can Be Formed from Tyr-MIF-1 in Brain Mitochondria but Not in Brain Homogenate

Abstract: Two samples of the peptide tyrosine-melanocyte-stimulating hormone release-inhibiting factor-1 (Tyr-MIF-1; Tyr-Pro-Leu-Gly-NH₂) were tritiated on different amino acids (Tyr or Pro) and incubated together at 37°C with fractions of rat brain. The amount of intact tetrapeptide remaining was determined by HPLC. By 3 min, most of the Tyr-MIF-1 was degraded. Because similar amounts of [³H]Pro and [³H]Tyr appeared after incubation of the Tyr-MIF-1 peptides in brain homogenate, even as early as 30 s, examination of only this crude preparation would misleadingly indicate that Tyr-MIF-1 is not a precursor of melanocyte-stimulating hormone release-inhibiting factor-1 (MIF-1; Pro-Leu-Gly-NH₂) in brain tissue. However, incubation of the mitochondrial fractions of brain under the same conditions resulted in more than three times as much [³H]Tyr being formed as [³H]Pro, with accompanying accumulation of MIF-1. Addition of excess MIF-1 to the mitochondrial fraction completely suppressed the formation of MIF-1 and more than doubled the amount of Tyr-MIF-1 remaining intact. When Tyr-MIF-1 tritiated only on the Tyr was added to the mitochondrial fraction, the main peaks of radioactivity appeared only at the positions of Tyr and Tyr-MIF-1, not at the position of Tyr-Pro. The results indicate that Tyr-MIF-1 can serve as a precursor of MIF-1 in brain mitochondria, an effect not evident when crude brain homogenate is used.     

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-NH₂) 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-NH₂) not only shows antiopiate activity, but also considerable selectivity for the mu-opiate binding site. Tyr-W-MIF-1 (Tyr-Pro-Trp-Gly-NH₂) 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-NH₂ and endomorphin-2 is Tyr-Pro-Phe-Phe-NH₂. 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.     

MIF-1 and Tyr-MIF-1 augment GABA-stimulated benzodiazepine receptor binding

Behavioral evidence in laboratory animals and human beings indicates possible links between the endogenous opiate and gamma-aminobutyric acid (GABA)-benzodiazepine receptor systems, especially with regard to antagonistic properties. To assess possible interactions between endogenous opiate antagonists and benzodiazepine receptor binding, we evaluated the effects of the peptides MIF-1 and Tyr-MIF-1 on benzodiazepine receptor binding in mouse brain membranes. Neither peptide affected receptor binding in cortex over a broad dose range, but both peptides significantly augmented GABA-stimulated benzodiazepine receptor binding at GABA concentrations of 10(-8) and 10(-7) M. Rosenthal-Scatchard analysis indicated that the increase in binding was largely due to increased apparent affinity. Both peptides augmented GABA-enhanced binding at low doses (MIF-1 10(-11) M, Tyr-MIF-1 10(-13) M) with decreased effects at higher doses. In cerebellum and brainstem, MIF-1 tended to enhance GABA-stimulated binding but Tyr-MIF-1 was inactive. These results indicate benzodiazepine-opiate and benzodiazepine-peptide interactions.     

Inhibitory influences of MIF-1 (PLG) and Tyr-MIF-1 (YPLG) on aggression and defeat-induced analgesia in mice

The effects of prolyl-leucyl-glycinamide (MIF-1, PLG), tyrosine-prolyl-leucyl-glycinamide (Tyr-MIF-1, YPLG) and the exogenous opiate antagonist, naloxone, on aggressive interactions and defeat-induced analgesia were examined in male mice. All three substances reduced the number of bites required to obtain defeat in subordinate mice during aggressive encounters, as well as blocking subsequent defeat-induced analgesia. Tyr-MIF-1 had significantly greater inhibitory effects than MIF. These results suggest that both MIF and Tyr-MIF-1 may function as endogenous opioid antagonists and have inhibitory influences on aggression, with the antagonistic effects of Tyr-MIF-1 being more potent than those of MIF-1.     

Differential inhibitory effects of MIF-1, Tyr-MIF-1, naloxone and beta-funaltrexamine on body rotation-induced analgesia in the meadow vole, Microtus pennsylvanicus

The effects of body rotation in a horizontal plane and various opiate antagonists on the nociceptive responses of a day-active microtine rodent, the meadow vole, Microtus pennsylvanicus, were examined. Intermittent rotation (70 rpm, schedule of 30 sec on, 30 sec off) for 30 min induced significant analgesic responses in the voles for 15 min after rotation. These increases in thermal response latency were blocked by intraperitoneal pretreatment with either naloxone or the irreversible mu opiate receptor antagonist beta-funaltrexamine (beta-FNA; 10 mg/kg; 24 hr pretreatment). This antagonistic effect of beta-FNA indicates mu opioid involvement in the mediation of rotation-induced analgesia. The antiopiate peptides MIF-1 (Pro-Leu-Gly-NH2) and Tyr-MIF-1 also significantly reduced, though did not completely block, body rotation-induced opiate analgesia. This suggests that Tyr-MIF-1 and MIF-1 have significant antagonistic effects on mu opioid systems that are involved in the mediation of stress (rotation)-induced analgesia.     

Existence of antiopiate systems as illustrated by MIF-1/Tyr-MIF-1

Evidence is presented that the small peptides MIF-1/Tyr-MIF-1 are part of an endogenous antiopiate system that may function to balance the opiate system. We review the biological activity, behavioral activity, and functional effects of this proposed opiate antagonist system. In addition, we suggest, based on antinociceptive mechanisms, that the individual components of the antiopiate system might function differently from naloxone.     

Delayed degradation of Tyr-MIF-1 in neonatal rat plasma

Peptides like Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH₂) that are administered during the neonatal period can result in biological effects persisting into the adult period. The possibility that Tyr-MIF-1 might have a prolonged half-life in neonatal blood was investigated by HPLC of plasma obtained from 4-day-old rat pups. More than half (65%) of the tritiated Tyr-MIF-1 incubated with neonatal rat plasma at 37°C remained in intact form at 30 min compared with less than a quarter of the Tyr-MIF-1 incubated with adult rat plasma. The calculated half-life of the tetrapeptide incubated in neonatal plasma was 50.2 min, compared with 13.8 min for adult plasma (p<0.01). The simultaneous addition of Tyr-MIF-1 tritiated on the Tyr and Tyr-MIF-1 tritiated on the Pro showed the formation of equal amounts of the free amino acids Tyr and Pro; this indicates that Tyr-MIF-1 is not a precursor of MIF-1 in neonatal rat plasma. The results show that the degradation of Tyr-MIF-1 is significantly delayed in plasma from neonatal rats, suggesting the possibility that the metabolism of other peptides and different types of compounds also may be delayed during the perinatal period.     

Isolation of tyrosine-melanocyte-stimulating hormone release-inhibiting factor 1 from bovine brain tissue

Although Tyr-melanocyte-stimulating hormone release-inhibiting factor 1 (MIF-1) (Tyr-Pro-Leu-Gly-NH2) can exert a number of biological actions in the brain and elsewhere, it has never been isolated from any tissue. Accordingly, we attempted to purify it from acetic acid extracts of bovine brain tissue by gel filtration chromatography and several different high performance liquid chromatographic systems. Peptide content was followed by a specific and sensitive radioimmunoassay with an antibody that was generated against synthetic Tyr-MIF-1. In each of the five applied high performance liquid chromatographic systems, the immunoreactive fractions coincided exactly with the elution time of synthetic Tyr-MIF-1 in the control runnings. The structure of the isolated peptide was identified by microsequence analysis as the tetrapeptide Tyr-Pro-Leu-Gly-NH2 and shown to be biologically active.     

MIF-1 and Tyr-MIF-1 antagonize morphine and opioid but not non-opioid stress-induced analgesia in the snail, Cepaea nemoralis

The effects of prolyl-leucyl-glycinamide (MIF-1, PLG), tyrosine-prolyl-leucyl-glycinamide (Tyr-MIF-1, YPLG) and naloxone on morphine and warm and cold stress-induced increases in the latency of the thermal (40 degrees C hot plate) avoidance behaviors of the terrestrial snail, Cepaea nemoralis, were examined. All three substances blocked the morphine- and warm stress-induced opioid analgesia, while having no effects on non-opioid cold stress-induced analgesia. Tyr-MIF-1 had a significantly greater inhibitory effect than MIF-1. These results indicate that MIF-1 and Tyr-MIF-1 antagonize the antinociceptive effects of exogenous opiates and opioid-mediated analgesia in snails in a manner analogous to that described for mammals. This raises the possibility of an evolutionary conservation of functional opioid antagonists.     

Chronic, but not acute, administration of morphine alters antiopiate (Tyr-MIF-1) binding sites in rat brain

Opiate addiction could involve a change in the binding of endogenous antiopiates. A candidate for such a role is Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2), a brain peptide that can antagonize exogenous and endogenous opiates and bind to opiate receptors. Its primary action, however, may be through its own binding site in brain, which we now report is altered by chronic administration of morphine. Rats given morphine pellets had reduced binding of both iodinated and tritiated Tyr-MIF-1 on day 5, when substantial tolerance is evident. In contrast, mu and delta opiate receptors were increased. Acute injection of an analgesic dose of morphine did not reduce Tyr-MIF-1 binding, indicating that chronic administration is required for the change. These findings open new approaches to the study of addiction by focusing on antiopiate activity.     

Hemorphins, cytochrophins, and human beta-casomorphins bind to antiopiate (TYR-MIE-1) as well as opiate binding sites in rat brain

Novel peptides with opiate activity, derived from endogenous sources (human and bovine casomorphins from milk, hemorphins from hemoglobin, and cytochrophins from mitochondrial cytochrome b), were tested for their ability to inhibit binding of the brain peptide Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2) to its high affinity sites in rat brain. The order of potency in inhibiting binding of 125I-Tyr-MIF-1 was: hemorphin and bovine casomorphins greater than Tyr-MIF-1 greater than cytochrophins greater than human casomorphins. Naloxone and DAMGO were ineffective at inhibiting Tyr-MIF-1 binding. The results provide evidence that, in addition to their ability to bind to mu opiate receptors, these novel endogenous peptides with opiate activity and a peptide (Tyr-MIF-1) with antiopiate properties also bind to a non-opiate site labeled by Tyr-MIF-1. These sites could be involved in a balance between opiate and antiopiate peptides.     

Interactions between the antiopiate Tyr-MIF-1 and the mu opiate morphiceptin at their respective binding sites in brain

The interactions between Tyr-MIF-1, a brain peptide with antiopiate activity, and the beta-casomorphins, a family of peptides derived from milk protein with opiate activity, were investigated by in vitro binding assays. Specific binding of 125I-Tyr-MIF-1 to rat brain membranes was displaced with high potency by beta-casomorphin, morphiceptin, and the morphiceptin analog PL017 but not by the analgesically inactive analog D-Pro2-morphiceptin or by several other ligands for classical delta, kappa, or sigma opiate receptors. In addition, Tyr-MIF-1 displaced 125I-morphiceptin from its binding sites in brain with affinities similar to those of unlabeled morphiceptin and PL017. These results, which include the first demonstration of a binding site in brain for labeled morphiceptin, indicate that brain antiopiate Tyr-MIF-1 and the beta-casomorphin derived peptides with opiate activity may share a common binding site or cross-react at each other's site. This suggests a possible mechanism of action for endogenous antiopiate-opiate interactions.     

Synthesis and biological activity of a lysine-containing cyclic analog of [Leu5]enkephalin

The cyclic analogue of [Leu5]enkephalin--cyclo (Lys-Tyr-Gly-Gly-Phe-Leu) and two corresponding linear hexapeptides with lysine residue attached to the N- or C-terminus of the molecule have been synthesized by classical methods of peptide chemistry. The addition of lysine residue to the N-terminus of cyclization of the molecule reduce the interaction of these analogues with both central and peripheral opiate receptors. The addition of lysine residue to the C-terminus of the molecule through the epsilon-amino group does not affect the interaction of the analogue with mu-receptors but reduces approximately tenfold its affinity for delta-receptors. All three analogues have analgesic potency similar to that of [Leu5]enkephalin as assayed after intracisternal administration to mice.     

MIF-1, Tyr-MIF-1, and MSH: Control of MSH Release and Extra-pigmentary Effects

New brain peptides are being discovered with increasing frequency.The discovery of multiple forms of MSH and evidence for a novelpeptide structurally related to MIF-1 may provide avenues fornew insights into the mechanisms controlling MSH release Demonstrationthat levels of the new tetrapeptide Tyr-MIF-1 are higher insome parts of the brain than in others and can be altered byneuroendocrine manipulations raises the possibility that thispeptide may affect CNS functions regardless of any effects onMSH release. Early studies on the extra-pigmentary effects ofMSH and MIF-1, which ushered in the field of brain peptides,provide models for the exploration of these possibilities. Explanationsfor unusual dose-response relationships, chronic effects afterneonatal injections, and the mechanisms by which peripherallyadministered peptides reach the brain to exert behavioral andEEG actions remain to be found     

MIF-1 antagonizes warm-, but not cold-water stress-induced analgesia: Dissociation from immobility

Forced swimming in warm or cold water can lead to immobility and analgesia in mice. In this report we demonstrate that the peptide MIF-1 (Pro-Leu-Gly-NH₂) was able to attenuate the analgesia induced by swimming in warm water, but not that induced by swimming in cold water. In addition, we show that the analgesia and the immobility may be differentially mediated since MIF-1 was able to reduce the immobility at doses different from those necessary to reduce the analgesia. These results confirm previous research indicating that MIF-1 may act as an anti-opiate and further demonstrate that MIF-1 affects analgesia induced by stress.     

New analogue of cyclolinopeptide B modified by amphiphilic residue of alpha-hydroxymethylmethionine

In order to evaluate the role and influence of the methionine residue on the biological activity of cyclolinopeptide B, an analogue with methionine residue in position 7 replaced by the amphiphilic (S)-alpha-hydroxymethylmethionine residue was synthesized. This peptide exhibits high immunosuppressive activity in the cellular, and to a lesser degree in the humoral immune response, comparable to that of CsA. In addition, the peptide was devoid of toxicity, even at high doses.     

Cysteic- and homocysteic acid-S-(aminoiminomethyl) amides: a new class of modified arginines useful in peptide synthesis.

A novel, practical synthesis of the title compounds and their derivatives are described. The protecting groups for amino, guanidino and carboxylic functions of substituted amides of cysteic and homocysteic acid were selected with the aim of making the amino acid derivatives synthons for peptide synthesis both in solution and by the solid phase method. Studying the structure-activity relationship some new kyotorphin, [Leu] kyotorphin and MIF-1 analogues, containing the unusual amino acid cysteic acid-S-(aminoiminomethyl) amide (sArg) in position two, have been prepared. It is a very promising compound, a structural analogue of arginine and an efficient antagonist in its metabolism.     

Isolation of a novel tetrapeptide with opiate and antiopiate activity from human brain cortex: Tyr-Pro-Trp-Gly-NH2 (Tyr-W-MIF-1).

A novel tetrapeptide, Tyr-Pro-Trp-Gly-NH2 (Tyr-W-MIF-1), was purified from extracts of frontal cortex of human brain tissue by several consecutive reversed-phase high performance liquid chromatographic steps followed by a radioimmunoassay originally developed for Tyr-Pro-Leu-Gly-NH2 (Tyr-MIF-1). Sequencing, mass spectrometric analysis, and comparison of its chromatographic behavior with that of the synthetic peptide confirmed the structure. Like Tyr-MIF-1, which was previously isolated from human brain tissue, Tyr-W-MIF-1 can inhibit the binding of 3H-DAMGO (selective for mu opiate receptors) to rat brain and can act as an opiate agonist as well as antagonist. Tyr-W-MIF-1 was a more potent opiate agonist than Tyr-MIF-1, the free acid of Tyr-W-MIF-1, and the structurally related hemoglobin-derived opiate peptide hemorphin-4 (Tyr-Pro-Trp-Thr) in the guinea pig ileum. Each of these peptides acted as opiate antagonists on the ileum from morphine-tolerant guinea pigs; the free acid of Tyr-W-MIF-1 was the most potent antagonist in inhibiting the activity of DAMGO. The results demonstrate the presence in human brain of a new member of the Tyr-MIF-1 family of biologically active peptides.     

Structural comparison between retro-inverso and parent peptides: Molecular basis for the biological activity of a retro-inverso analogue of the immunodominant fragment of VP1 coat protein from foot-and-mouth disease virus

Antibodies induced against intact foot-and-mouth disease Virus (FMDV) particles bind to the retro-inverso analogue of fragment 141–159 of the viral coat protein VP1 of FMDV, variant A, equally well as to the parent peptide. A conformational investigation of this retro-inverso peptide was carried out by nmr spectroscopy and restrained molecular modeling in order to identify the structural basis for the antigenic mimicry between these retro-inverso and parent peptides. In 100% trifluoroethanol a well-defined left-handed α-helical region exists from residue 150 to residue 159, which is consistently present in all conformational families obtained from restrained modelling. A less-defined left-handed helical region is present in the tract 144–148, which is also consistent for all structures. Conformational flexibility exists about Gly149, which leads to two types of structures, either bent or linear. In the bent structures, a three-residue inverse tight turn is found, which can be classified as an inverse γ-turn centered at Gly149. The overall structural features of the retro-inverso peptide are shown to be similar to those of the parent L-peptide. The two molecules, however, are roughly mirror images because they share inherently chiral secondary structure elements. By comparing these conformational conclusions with the x-ray structure of the Fab complex of a corresponding VP1 antigenic fragment, a rationale is proposed to account for the topological requirements of specific recognition that are implied by the equivalent antigenic activity of the natural and retro-inverso compounds. © 1997 John Wiley & Sons, Inc. Biopoly 41: 569–590, 1997.     

Functional characterisation of the 1-18 fragment of esculentin-1b, an antimicrobial peptide from Rana esculenta

Esculentin-1 is a 46-amino acid residue peptide isolated from skin secretions of Rana esculenta, displaying the most potent antimicrobial activity among the bioactive molecules found in the secretion, with negligible effects on eukaryotic cell membranes. From skin secretions, the 19-46 fragment of esculentin-1, devoid of antibacterial activity, was also isolated. We studied in detail the activity of the N-terminal fragment (1-18) of esculentin-1 using a synthetic amidated analogue. The results show that this fragment is highly active against most bacterial and fungal species, although at a lower extent than the full-length peptide, being four-fold more potent against Phytophthora nicotianae. It has a reduced activity against human erythrocytes with respect to the full-length peptide. The killing curves in liquid medium are similar for the two molecules and the shorter peptide is able to increase the bacterial outer and inner membrane permeability. Overall these data indicate that the antimicrobial properties of esculentin-1 are exerted by its N-terminal 1-18 region and that the positively charged residue distribution as well as peptide length represent important determinants for cell selectivity.