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.

In vitro skin penetration and degradation of peptides and their analysis using a kinetic model.

The main purpose of this study was to estimate the net percutaneous absorption of physiologically active peptides in vitro. The degradation of two peptides, Leu-enkephalin (Enk) and Tyr-Pro-Leu-Gly amide (TPLG), during skin penetration and on the dermal side following penetration, and the prevention of degradation by some protease inhibitors, were investigated using rat skin in vitro. In addition, these permeation and degradation data were analyzed using a kinetic model. These peptides were rapidly degraded in the receptor fluid of a Franz diffusion cell (rate constant: 0.977 h(-1) for Enk and 0.250 h(-1) for TPLG). The addition of phenylmethylsulfonyl fluoride (PMSF) and phenanthroline and the pretreatment of skin with these inhibitors prevented almost completely any degradation in the receptor fluid and skin, respectively. The pretreatment of skin with PMSF and phenanthroline had no effect on the penetration of dextran (1000 Da). The degradation rate constant during skin penetration, calculated from the difference in the penetration rate constants via pretreated and untreated skins, was also high (0.037 h(-1) for Enk and 0.050 h(-1) for TPLG). A kinetic model including an input rate (zero-order), the permeation rate across the viable skin (first-order) and the degradation rate in skin (first-order) was sufficient to describe the apparent steady-state flux of the peptides through skin. We have, thus, established a method for measuring the true flux of peptides across skin in vitro and a kinetic model which simply describes the skin penetration of peptides.

Peptide transport system-1 (PTS-1) for Tyr-MIF-1 and Met-enkephalin differs from the receptors for either.

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.

Regional differences in the metabolism of Tyr-MIF-1 and Tyr-W-MIF-1 by rat brain mitochondria.

Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2) and Tyr-W-MIF-1 (Tyr-Pro-Trp-Gly-NH2) are endogenous neuropeptides with opiate modulating and other CNS effects. After incubation of the tritiated tetrapeptides with fractions of tissue from different areas of rat brain, formation of the metabolites was determined by HPLC. Marked regional differences in degradation were found for both peptides. The metabolism of Tyr-MIF-1, resulting in the formation of the biologically active MIF-1 (Pro-Leu-Gly-NH2), was greater in the mitochondrial than in the synaptosomal fractions. In the mitochondrial fraction, about twice as much MIF-1 was formed in brain cortex than in striatum, diencephalon, or midbrain/pons medulla. These results, showing differential metabolism in various areas of the brain, indicate another means for regulation of the concentrations of neuropeptides.

Perinatal treatment of rats with opiates affects the development of the blood-brain barrier transport system PTS-1.

Previous results have shown that treatment of rats with morphine during the neonatal period can influence development of peptide transport system-1 (PTS-1), the blood-brain barrier transport system for Tyr-MIF-1 and methionine enkephalin. Previous work has suggested that the activity level of PTS-1 correlates with the concentration of methionine enkephalin in the brain. We show here that rats treated peripherally with morphine sulfate (MS) in both the prenatal and neonatal periods have enhanced activity of PTS-1. The degree of enhancement increases with age to reach a 66% increase in comparison with controls at age 9 weeks. The mu agonist MS was more powerful than the kappa agonist ethylketocyclazocine (EKC) or the delta agonist [D-Pen2.5,pCl-Phe4]enkephalin (pCl-DPDPE) in producing this effect. Opiate antagonists had complex effects with methylnaltrexone blocking the action of MS on PTS-1. These results show that the level of PTS-1 activity in adult rats can be modified by perinatal events that affect opiate tone during development.

Periventricular penetration and disappearance of ICV Tyr-MIF-1, DAMGO, tyrosine, and albumin.

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.

Differential metabolism of Tyr-MIF-1 and MIF-1 in rat and human plasma.

The metabolism of the endogenous brain peptides Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2) and MIF-1 (Pro-Leu-Gly-NH2) was determined by HPLC after incubation of the tritiated peptides in human and rat plasma. Degradation of Tyr-MIF-1 was rapid in the plasma from both species, in contrast to the slightly delayed degradation of MIF-1 in rat plasma and the extremely prolonged persistence of MIF-1 in human plasma. In rat plasma, more than half of the intact Tyr-MIF-1 and MIF-1 was degraded within 5 min, in contrast to the 5 days required for 50% degradation of MIF-1 in human plasma at 37 degrees. To slow the rapid rate of metabolism, studies were then performed at 0 degree. Incubation of Tyr-MIF-1 in human plasma at 0 degree for 2 hr resulted in HPLC identification of more Tyr-Pro than Tyr at all times. At 0 degree in rat plasma, however, more Tyr than Tyr-Pro was formed after the first 5 min of incubation of the Tyr-MIF-1 that was labeled on the Tyr. This raised the possibility that the tetrapeptide Tyr-MIF-1 might be serving as a precursor of the tripeptide MIF-1. Incubation of Tyr-MIF-1 tritiated at the Pro under the same conditions with and without Tyr-MIF-1 tritiated at the Tyr showed that Tyr-Pro, not MIF-1, was the predominant degradation product of Tyr-MIF-1. In addition to the metabolism of Tyr-MIF-1 being slower at lower temperatures, it was also slowed by some enzyme inhibitors. After 10 min of incubation at 37 degrees, EDTA appeared to be more effective than bestatin, p-chloromercuribenzoic acid (PCMB), pepstatin, or aprotinin, but after 30 min, bestatin was more effective. Intravenous injection of the tritiated peptides into rats showed short half-time disappearances; again, MIF-1 persisted in blood longer than Tyr-MIF-1. Thus, the results show the rapid metabolism of Tyr-MIF-1 in human and rat plasma, the slightly slower metabolism of MIF-1 in rat plasma, the predominant formation of Tyr-Pro rather than MIF-1 from Tyr-MIF-1, and the markedly delayed metabolism of MIF-1 in human plasma.

Saturable efflux of the peptides RC-160 and Tyr-MIF-1 by different parts of the blood-brain barrier.

Peptides have been shown to be transported in the direction of both blood to brain and brain to blood. Although blood to brain transport is known to occur at both the choroid plexus and the capillary bed of the brain, comprising the two major components of the blood-brain barrier, the location of efflux systems for peptides remains largely unstudied. We adapted established methodologies to study this question for two peptides known to be transported out of the brain after injection into the cerebrospinal fluid (CSF): Tyr-MIF-1, transported by peptide transport system (PTS)-1 and RC-160, a somatostatin analog transported by PTS-5. Radioactive iodide, known to be transported out of the brain primarily by the capillaries, also was studied. We found that after injection into brain tissue, RC-160 and iodide were rapidly transported out of the brain by saturable mechanisms. By contrast, efflux of Tyr-MIF-1 was slow and nonsaturable after injection into brain tissue, but rapid and saturable after injection into the lateral ventricle of the brain. Autoradiography confirmed that peptide injected into brain tissue did not diffuse far from the site of injection during the study period. The results indicate that the efflux system for RC-160 is located at least partly at the capillaries and suggest that the major location for the efflux system of Tyr-MIF-1 is at the choroid plexus.

Opposite direction of transport across the blood-brain barrier for Tyr-MIF-1 and MIF-1: comparison with morphine.

Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2) and MIF-1 (Pro-Leu-Gly-NH2) are endogenous peptides that can exert opiate-related actions on the CNS after peripheral administration. We found that Tyr-MIF-1 radioactively labeled at the tyrosine was transported across the blood-brain barrier (BBB) in the direction of brain to blood by a saturable system. Transport occurred equally well when the tetrapeptide was labeled with 125I or when it was labeled with 3H. [3H]MIF-1 and [3H]morphine were not transported out of the CNS but were retained by the brain after intracerebroventricular injection. Both [3H]MIF-1 and [3H]morphine entered the brain after i.v. injection, with [3H]MIF-1 crossing the BBB by a mechanism that was partially saturable. The entry rate and accumulation of radioactivity by the brain was 50-100 times greater after the i.v. injection of [3H]MIF-1 than after [3H]morphine. The results show that Tyr-MIF-1 labeled with either 3H or 125I can serve equally well for the measurement of transport across the BBB, that MIF-1 has relatively substantial and rapid access from the blood to the CNS by directly crossing the BBB, and that the BBB can differentially regulate the exchange of related substances between the CNS and blood.

Effects of neonatal treatment with Tyr-MIF-1, morphiceptin, and morphine on development, tail flick, and blood-brain barrier transport.

Morphine and endogenous peptides can alter developmental processes, inducing changes that can endure into adulthood. Morphiceptin binds to mu opiate receptors and to non-opiate sites labeled by Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2), an endogenous brain peptide known to modulate opiate effects. Morphine, morphiceptin, Tyr-MIF-1, morphine + Tyr-MIF-1, and morphiceptin+Tyr-MIF-1 (50 micrograms, s.c.) were given to rats during their first week of life. Animals given morphine alone or in combination with Tyr-MIF-1 had significantly lower body weights for the first 3 weeks of life and delayed eye opening on day 16. Rats given morphine had hypersensitive tail flick responses on day 9 while those given morphine + Tyr-MIF-1 were hypersensitive on days 3, 8, and 9. Locomotor, passive avoidance, and rotorod behaviors were not altered by the neonatal treatments. Transport of [125I]Tyr-MIF-1 out of the brain was tested on day 23 and found to be increased by neonatal morphine, an effect that was significantly potentiated by neonatal Tyr-MIF-1. The results indicate that neonatal administration of peptides and opiates can affect later peptide transport across the blood-brain barrier as well as selected developmental characteristics.

Uptake, content, regulation of plasma concentrations, and binding of Tyr-MIF-1 by the adrenals.

The known occurrence of opiates in the adrenals stimulated us to determine whether Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2), an endogenous peptide that can act as an antiopiate, is present in these glands and whether adrenalectomy affects its concentrations in plasma. The presence of Tyr-MIF-1 in the adrenals of the rat was shown by radioimmunoassay (RIA) and high performance liquid chromatography (HPLC). Binding sites for the tetrapeptide also were found in the adrenal. The concentrations and binding of Tyr-MIF-1 were higher in the adrenal medulla than in the adrenal cortex. The adrenals preferentially trapped 125I-Tyr-MIF-1 as compared with 99mTc-albumin after intravenous administration. However, after correction for tissue weight and vascular space, accumulation of 125I-Tyr-MIF-1 by the adrenals was in the general range seen for several other tissues. The concentrations of Tyr-MIF-1-like immunoreactivity determined by RIA in the plasma of adrenalectomized rats were increased in comparison with sham operated or unoperated controls. This approximate doubling was unchanged by the site of sampling: hepatic portal vein, renal vein, inferior vena cava, jugular vein, or truncal blood. Hypophysectomy, removal of the adrenal medulla, or treatment with corticosterone also did not block this increase. Tyr-MIF-1-like immunoreactivity in the plasma of adrenalectomized rats eluted at the same position by HPLC as did the synthetic Tyr-MIF-1 standard. Thus, the adrenal gland contains Tyr-MIF-1 and can affect its concentrations in plasma.

Passage of Tyr-MIF-1 from blood to brain.

Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2) has been shown to be transported from the brain to blood by a saturable system shared with Met-enkephalin and a few other substances. It is not known whether a similar system exists in the opposite direction. Accordingly, the entry rate of 125I-Tyr-MIF-1 from blood to brain was measured by a method involving perfusion of the test substances into the common carotid artery. The rate of entry was obtained from the slope of the line determined by brain to blood ratios at multiple points of time. Penetration of 125I-Tyr-MIF-1 across the blood-brain barrier was found to be 4.444 x 10(-3) ml/g/min, an entry rate significantly higher than that of the vascular marker 125I-albumin. Competition with Tyr-MIF-1 or nonradioactively labeled 127I-Tyr-MIF-1 showed no difference in rate of entry, indicating that the penetration of 125I-tyr-MIF-1 was not saturable. Met-enkephalin and Leu-enkephalin also failed to affect entry of 125I-Tyr-MIF-1. The results indicate that Tyr-MIF-1 can enter the brain from the blood to a greater extent than does albumin, but that this penetration does not involve a saturable system.

Differential effect of aluminum on the blood-brain barrier transport of peptides, technetium and albumin.

Aluminum is a neurotoxin capable of altering membrane structure and function. We investigated whether aluminum also can affect saturable transport across membranes using the blood-brain barrier as our model. Mice were given i.p. or i.v. aluminum (up to 100 mg/kg) as the chloride salt and the disappearance from the brain of several centrally administered substances was measured. We found that aluminum rapidly and profoundly inhibited the saturable system that transports the small, N-tyrosinated peptides Tyr-MIF-1 and the enkephalins from the brain to the blood by acting as a noncompetitive inhibitor. In contrast, the disappearance from the brain of technetium pertechnetate (a substance also transported out of the brain by a different saturable system), albumin or D-Tyr-MIF-1 (a stereoisomer of Tyr-MIF-1 that was confirmed not to be transported by the carrier system) was not affected by aluminum. Aluminum also did not alter either the saturable or nonsaturable component of the uptake of Tyr-MIF-1 by erythrocytes. These findings suggest that one mechanism by which aluminum may induce neurotoxicity is by selective alteration of the transport systems of the blood-brain barrier.