Tyr-MIF-1 and Met-enkephalin share a saturable blood-brain barrier transport system

Previous studies have shown that methionine enkephalin and Tyr-MIF-1 are transported from the brain to the blood by a saturable, stereospecific, carrier-mediated process. It was not established by these studies whether Tyr-MIF-1 and methionine enkephalin were transported by the same system or by separate, but overlapping systems. This issue was investigated in anesthetized mice receiving injections containing both 131I-methionine enkephalin and 125I-Tyr-MIF-1 into the lateral ventricle of the brain. Mice were decapitated and the brain to blood transport rate was derived from the residual counts in the brain. It was found that in individual mice, the transport rate for Tyr-MIF-1 correlated highly with the transport rate for methionine enkephalin but not with the transport of iodide. This shows that the transport of Tyr-MIF-1 is closely coupled to the transport of methionine enkephalin but dissociable from the brain to blood transport of iodide. Furthermore, the inability of varying doses of Tyr-MIF-1 or of methionine enkephalin to preferentially self-inhibit is radiolabeled form in comparison with the other peptide shows that, functionally, only a single system exists. Aluminum, a noncompetitive inhibitor of Tyr-MIF-1 transport, was also without preferential inhibition. Thus, under the conditions of these studies, only a single system could be functionally demonstrated for the transport of both Tyr-MIF-1 and methionine enkephalin.     

Regional differences in peptide degradation by rat cerebral microvessels: a potential novel regulatory mechanism for communication between blood and brain

The blood-brain barrier (BBB), composed of the microvessels of cerebral capillary endothelial cells, regulates the passage of peptides into the brain in several ways, mainly by saturable transport or passive diffusion. Here we describe an additional mechanism by which this regulatory function can occur. Cerebral microvessels were isolated from different regions of the brain and incubated with the mu-opiate selective endomorphin-1 (Tyr-Pro-Trp-Phe-NH2) or the opiate-modulating Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2), both tetrapeptides selectively tritiated at the Pro. Degradation was determined by HPLC. For both peptides, the metabolism by microvessels from the cerebral cortex was much greater than that by microvessels from the hypothalamus or pons. For endomorphin-1, the least degradation was in the pons; for Tyr-MIF-1 there was no difference in metabolism by microvessels from the pons or hypothalamus. The results show a novel mechanism at the BBB by which the BBB can selectively regulate the activity of different peptides in different regions of the brain.     

Permeability of the blood-brain barrier to peptides: An approach to the development of therapeutically useful analogs

Peptides have been shown in both in vivo and in vitro systems to cross the blood-brain barrier (BBB) and so affect function on the side contralateral to their origin. Some peptides cross primarily by transmembrane diffusion, a nonsaturable mechanism largely dependent on the lipid solubility of the peptide. Other peptides are transported by saturable systems across the BBB. These transport systems can be in the CNS to blood direction, as in the cases of Tyr-MIF-1 and methionine enkephalin, in the blood to CNS direction, as in the case of peptide T, or bidirectional, as in the case of LHRH. Other factors that also affect the amount of peptide crossing the BBB include binding in blood, volume of distribution, enzymatic resistance, and half-time disappearance from the blood. An in vitro model of the BBB has been characterized and used to confirm that peptides can cross the BBB. Results with the model agree with those obtained in vivo and have been used to study the permeability of the BBB to peptides, the effect of peptides on BBB integrity, the cellular pathway peptides and proteins use to cross the BBB, and the ability of the BBB to degrade peptides. The in vivo and in vitro methods have been used together to develop halogenated enkephalin analogs that are enzymatically resistant, cross the BBB readily to accumulate in areas of the brain rich in opiate receptors, and are powerful analgesics. This shows how the principles elucidated for peptide passage across the BBB can be used to develop therapeutic peptides and how those peptides can be further tested in complementary in vivo and in vitro systems.     

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.     

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.     

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.     

Effect of diphenylhydramine on the Tyr-MIF-1 antinociception in rats.

Tyr-MIF-1 is a representative of the MIF's family of endogenous peptides. It has been isolated from bovine hypothalamus and human parietal cortex that suggests its involvement in nociception. Tyr-MIF-1 can bind to the mu-receptors as well as to its specific non-opiate receptors in the brain. Data in the literature rise the idea that histamine (HA), a well known nociceptive agent, and Tyr-MIF-1 might have a common pathway in their effects on nociception. We tested that possibility by investigation of the combined action of diphenhydramine (DPH, an H (1) -antagonist) and Tyr-MIF-1 on nociception. The changes in the nociceptive effects were examined in the male Wistar rats by the Randall-Sellito paw-pressure (PP) and the tail-flick (TF) tests. Tyr-MIF-1 in a dose of 1 mg/kg exerted strong naloxone-reversible analgesic effects. DPH (100 microg/kg, i.p.) had an antinociceptive action, too. The co-administration of Tyr-MIF-1 and DPH enhanced the antinociceptive effect, as compared to DPH (PP) and to TYR-MIF-1 alone (TF). These effects were reversed when methylene blue (MB, 500 microg/rat) was applied 1h before the combination. However, naloxone (1 mg/kg, i.p.) only slightly affected the antinociceptive effect of DPH and TYR-MIF-1, compared to that of MB. The results obtained confirmed the hypothesis that cyclic nucleotides are involved in the realization of nociceptive effects of both HA and Tyr-MIF-1.     

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 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.     

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.     

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.     

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.     

Tyr-MIF-1 and hemorphin can act as opiate agonists as well as antagonists in the guinea pig ileum.

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.     

Oral administration of Tyr-MIF-1 stimulates gastric emptying and gastrointestinal motility in rodents.

The effects of orally administered Tyr-MIF-1, an agonist of an endogenous antiopiate system, were examined on gastric emptying in mice and gastrointestinal myoelectric activity in rats. Tyr-MIF-1 (5 mg/kg in mice, 20 mg/kg in rats) accelerated gastric emptying of a methylcellulose test meal, increased the frequency of antral spike bursts, and disrupted intestinal migrating myoelectric complexes. These effects were reproduced by a subcutaneous administration of Tyr-MIF-1 at the same dosage. They were blocked by naloxone (1 mg/kg) but not by the kappa receptor subtype antagonist MR 2266 (1 mg/kg). The GABAA antagonist bicuculline (0.5 mg/kg), but not the GABAB antagonist 2-hydroxysaclofen (4 mg/kg), also antagonized the effects of Tyr-MIF-1. These data demonstrate that oral Tyr-MIF-1 stimulates gastric emptying and gastrointestinal motility through a systemic or central action that involves opioid and GABA systems.     

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.     

Effects of neonatal treatment with Tyr-MIF-1 and naloxone on the long-term body weight gain induced by repeated postnatal stressful stimuli

Stressful stimuli repeatedly applied during the first postnatal weeks can induce body weight gain in the mouse during adulthood. This effect can be prevented by injecting naloxone concomitantly with stress. The peptides belonging to the Tyr-MIF-1 family have a great modulating activity on numerous stress-induced phenomena. The aim of the present work was to compare the effect of repeated neonatal injections of Tyr-MIF-1 or naloxone on the long-term body weight gain induced by a stressing procedure applied daily during the first three weeks of life. The results indicate that although naloxone blocked the development of the stress-induced effects, Tyr-MIF-1 potentiated them.     

Transport of nutrients and hormones through the blood-brain barrier

The transport of circulating nutrients (glucose, amino acids, ketone bodies, choline, and purines) through the brain endothelial wall, i.e., the blood-brain barrier (BBB), is an important regulatory step in several substrate-limited pathways of brain metabolism. The in vivo kinetics of nutrient transport has been well characterized in the rat, and the kinetic constants of saturable (Km, Vmax) and nonsaturable (KD) transport through the BBB are now known for more than 30 circulating nutrients. The kinetic constants can be used to gain insight into the important rate-limiting role played by BBB nutrient transport in the regulation of brain metabolism and function. Unlike most nutrients, steroid and thyroid hormones circulate tightly bound to plasma proteins. However, owing to favorable kinetic relationships among brain capillary transit times and rates of hormone dissociation from plasma proteins and hormone diffusion through the brain endothelia, the BBB is able to strip hormones off circulating plasma proteins. With regard to peptide hormone, no specific BBB transport systems for peptides have been identified thus far. However, peptides are able to rapidly distribute into brain interstitial space at the circumventricular organs. In addition, specific receptors for insulin are located on the BBB. The presence of BBB peptide receptors provides a mechanism by which circulating peptides may rapidly influence brain function without the peptide crossing the BBB.     

Peptide transport by germinating barley embryos: Evidence for a single common carrier for di- and oligopeptides

Competition for uptake of a range of amino acids and peptides by germinating barley (Hordeum vulgare L.) embryos was studied. Peptides and amino acids show no competition and are apparently absorbed by independent transport systems. However, peptides of widely different structures do compete and it seems that only a single peptide transport system is present in barley embryos, capable of handling both di- and oligopeptides. The ability of physiological peptides to totally inhibit the uptake of glycylsarcosine indicates they share a common uptake system which previously has been shown to have the properties of an active transport process. The characteristics of the barley peptide transport system are compared with those found in other organisms.     

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.     

Evidence for presence of Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2) in human brain cortex

Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2) was previously isolated from bovine hypothalamus. We have now purified it from the parietal cortex of human brain tissue by gel filtration chromatography and four subsequent high performance liquid chromatographic steps. During isolation, the peptide content was followed by radioimmunoassay and compared with the elution of synthetic Tyr-MIF-1 in identical chromatographic systems. This extends evidence for the presence of Tyr-MIF-1 from bovine to human brain tissue and from hypothalamus to cortex.