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Regional differences in peptide degradation by rat cerebral microvessels
Life Sciences 69 (2001) 1305–1312
Regional differences in peptide degradation by rat cerebral microvessels A potential novel regulatory mechanism for communication between blood and brain Abba J. Kastin*, Kathy Hahn, James E. Zadina VA Medical Center and Tulane University School of Medicine, New Orleans, LA 70112-1262, USA Received 15 September 2000; accepted 21 March 2001
Abstract 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 (TyrPro-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. © 2001 Elsevier Science Inc. All rights reserved.
Introduction No longer considered static, the blood-brain barrier (BBB) is now known to play an active role in the regulation of the transfer of peptides and polypeptides from blood to brain [1–4]. Some peptides cross the BBB by saturable transport mechanisms while others cross by passive diffusion based on physicochemical properties. For saturable transport the regulatory ability of the BBB is obvious, but for passive diffusion the regulatory ability remains obscure.
* Corresponding author. VA Med Ctr/Tulane Med, 1601 Perdido St., New Orleans, LA 70112-1262. Tel.: 504568-0811, ext. 5884; fax: 504-522-8559. 0024-3205/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 1 )0 1 2 1 1 -5
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The saturable transport of the opiate modulating tetrapeptide Tyr-MIF-1 (Tyr-Pro-LeuGly-NH2) [5] from brain to blood has been extensively studied [6–10]; however, its entry into brain from blood occurs by passive diffusion [11]. Tyr-MIF-1 is rapidly degraded in blood [12] and whole brain homogenates [13], but not in neonatal rat plasma [14] or cerebrospinal fluid [15]. Endomorphin-1 (Tyr-Pro-Trp-Phe-NH2) has the highest affinity and specificity for the mu opiate receptor of any endogenous brain peptide and its analgesic potency is similar to that of morphine [16]. It shares some structural and functional similarities with Tyr-MIF-1, but its metabolism has not been thoroughly investigated. There are regional differences in the metabolism of Tyr-MIF-1 [17] and in the activities of eight peptidases [18] when entire sections of brain tissue are used [17], but no regional differences have been described for the microvessels of the BBB. The enzymatic activities of the capillary endothelial (microvessel) cells have been thoroughly reviewed in general [19] and for some other opiate peptides [20]. Although developmental changes have been observed [18,21], the possibility of regional changes has not been reported. Here, we examine for the first time whether there are regional differences in peptide metabolism in microvessels isolated from different regions of the brain. Since cerebral microvessels constitute the BBB and provide the main site of contact between blood and brain, regional differences in metabolism would provide a unique method of regulation of passage across the BBB, even for substances entering by passive diffusion. Experimental procedures Tyr-MIF-1 was tritiated (specific activity: 40 Ci/mmol) (Amersham, Arlington Heights, IL) on the Pro from the [dehydro-Pro]2 precursor. Endomorphin-1 was tritiated (specific activity: 50 Ci/mmol) (Nycomed Amersham, Buckinghamshire, England) on the Pro by hydrogenation of an unsaturated intermediate. The procedure used for isolation of the capillary microvessels was based on the well-established method of Gerhart et al. [22] as modified for the rodent [23]. Endothelial cells prepared in this way are hardy, grow rapidly, and consist of a single cell type [22]. All plasticware was pre-coated with phosphate-buffered saline and bovine serum albumin. The buffer was MEM (Minimum Essential Medium Eagle, Sigma, St. Louis, MO). Anesthetized male rats (Harlan Sprague-Dawley, Indianapolis, IN) weighing about 300 g were perfused through the heart with 0.9% NaCl by gravity flow. After perfusion, each brain was placed in a petri dish on ice, and the areas of interest removed by careful dissection. Selected regions from 25 rats were combined to obtain sufficient material for analysis. These were homogenized at 4 8C in a Potter-Elvehjem apparatus with a loose fitting Teflon pestle for the first 10 strokes, then with a tight fitting pestle for an additional 5–6 strokes. The homogenate was filtered once through a 300 mm mesh microsieve (Spectrum, Laguna Hills, CA) and twice more through a 100 mm mesh net. An equal volume of 40% dextran was added to the filtrate and the mixture was centrifuged (swinging bucket) at 5000 g for 15 min at 4 8C. The pellets were suspended in MEM, filtered through a 25 mm mesh net (Biodesign, Carmel, NY), and washed three times with MEM. The remaining liquid was removed by pipet and the net was inverted over a buffer-containing petri dish. The agitated microvessel sus-
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pension in the dish was examined by microscopic wet mount to ensure purity and transferred to polypropylene tubes which were centrifuged at 2500 g for 15 min at 4 8C. After aspiration of the supernatant, hibernate A (Life Technologies, Gaithersburg, MD) was added to the pellets which were stored at 4 8C overnight. The next day, the cells were centrifuged at 2500 g for 15 min and resusupended in MEM. Preliminary studies revealed that hibernate A was as least as effective as DMSO after overnight storage, both resulting in somewhat more recovery of intact endomorphin-1 after 2 h incubation than when freshly prepared microvessels without hibernate A were used. Protein content was determined by the Lowry method. 3 H-Tyr-MIF-1 or 3H-endomorphin-1 (3.53106 dpm each) were incubated with 175 mg of microvessel protein in a metabolic shaker (Dubnoff Incu-Shaker, Labline Instruments, Melrose Park, IL). This approximates the standard amount of microvessel protein required for accurate measurement of enzymatic activity [24]. Samples were removed at 30 min, 1 h, 2h, and, for Tyr-MIF-1, 4 h. At these times, 10% cold trifluoroacetic acid (TFA) was added to stop the reaction. After centrifugation, the supernatants were dried in a Speed-Vac (Savant Instruments, Farmingdale, NY) and samples stored in a desiccator at 220 8C until analysis by HPLC. For chromatography, samples were reconstituted in 0.1% TFA/H2O (solvent A), filtered (nylon 66 centrifuge filter; pore size 0.45 mm; Alltech Associates, Deerfield, IL), and applied to a Brownlee RP-18 reversed-phase column (4.6 mm 3 22 cm, width 1.5 cm) with a 1.5 cm RP-18 guard cartridge (Rainen, Woburn, MA). The HPLC system was a Beckman (Fullerton, CA) model 344 with version 5.10 Gold software. The online detector was the INSUS Systems (Tampa, FL) radiochromatogram b-RAM 2B. For Tyr-MIF-1, solvent B was 0.1% TFA in methanol. The gradient consisted of 10% B which was gradually increased to 30% over 35 min, to 33% for the next 10 min, to 35% for the next 5 min, and then to 80% for the final 2 min. With this gradient, Pro eluted at 4 min, Tyr-Pro at 20 min, intact Tyr-MIF-1 at 33–34 min, and the free acid of Tyr-MIF-1 at 37–38 min. As a control for HPLC “shadowing” to ensure that no contamination or carry-over occurred [25], the HPLC column was washed with 80% methanol for 2 h before each analysis, then twice with the same gradient used for the peptides, and a blank sample analyzed . For endomorphin-1, solvent B was 0.1% TFA in acetonitrile. The gradient consisted of 5% solvent B which was gradually increased to 25% over 25 min, to 32% for the next 40 min, and then to 80% for the final 2 min. With this gradient, Pro eluted at 3–4 min, Tyr-Pro at 14– 15 min, and intact endomorphin-1 at 51–53 min. Results A representative HPLC chromatogram for Tyr-MIF-1 before and after 4 h incubation with brain cortical microvessels is shown in figure 1. The inset shows endomorphin-1 before incubation; after 4 h, no endomorphin-1 remained. The percent of intact Tyr-MIF-1 in microvessels from three different regions of the brain at various times up to 4 h after incubation is shown in figure 2. For Tyr-MIF-1, degradation by the cortical microvessels was greater than that by microvessels from the hypothalamus or pons. There was no difference in the rates of degradation between the hypothalamus and pons. Moreover, for microvessels from both hy-
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Fig. 1. HPLC chromatogram of 3H-Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2) before and after incubation for 4 h with the cerebral microvessels that constitute the BBB. P 5 Pro, Y 5 Tyr, YPLG-NH2 5 Tyr-MIF-1, YPLG-OH 5 the free acid of Tyr-MIF-1. The inset shows endomorphin-1 (Tyr-Pro-Trp-Phe-NH2 5 YPWF-NH2) at 0 h; by 4 h none remained. The dotted lines represent the HPLC gradients.
pothalamus and pons, no further degradation of Tyr-MIF-1 occurred after 30 min, the TyrMIF-1 remaining largely (70–76%) intact for the entire 4 h of incubation. Although the accumulation of enough microvessels for measurement of activity is a tedious process requiring a large number of rats, for endomorphin-1 each experiment was repeated twice with microvessels from each brain part at each time. An identical amount of microvessel protein was used for all measurements, although the more highly vascularized areas of the brain may have a smaller proportion of the total number of capillaries. In the repeat experiments performed several months apart, 68.8 – 71.2% endomorphin-1 was intact in the pons after the 2 h incubation , 50.5 – 55.5% in the hypothalamus, and only 29.8 – 32.4% in the cerebral cortex (Fig. 3). The focus of the results was on the hypothesis of regional differences in degradation; no attempt was made to identify the enzymes responsible for the degradation. Discussion Regulation by the BBB can be very selective. Substitution of D-Tyr for L-Tyr, or removal of a hydroxyl group from Tyr (5 Phe), abolishes the saturable brain-to-blood transport of the tetrapeptide Tyr-MIF-1 by Peptide Transport System-1, a system that does not transport the
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Fig. 2. Percentage of Tyr-MIF-1 (YPLG-NH2) remaining in intact form, determined by HPLC, after incubation with the cerebral microvessels from three regions of the brain. HT 5 hypothalamus.
smaller MIF-1 (Pro-Leu-Gly-NH2) [8]. In the opposite direction, Tyr-MIF-1 enters the brain by passive diffusion rather than saturable transport [11]. Passive diffusion is influenced by physicochemical properties such as lipophilicity and hydrogen-bonding [26,27], but does not provide the selective type of feedback control that is characteristic of saturable transport. The self-inhibition of saturable transport provides physiological regulation of transport by varying concentrations of peptide or polypeptide in the blood or brain. Cross-inhibition of saturable transport by a functionally or structurally related substance provides an even broader regulatory role for the BBB. Recently, activation (rather than inhibition) of a saturable transport system by a substance with similar function was described [28]. The present study demonstrates an additional mechanism by which the BBB can exert selective regulatory control: differential degradation of peptides depending on brain region and peptide structure at the BBB itself. For both peptides tested, the largest amount of degradation occurred in microvessels from the cerebral cortex. Metabolism of Tyr-MIF-1 occurred at a similar rate in the hypothalamus and pons while endomorphin-1 was metabolized faster in the hypothalamus than in pons. Thus, the rate of degradation was specific to the region and peptide. The cortex shows relatively sparse endomorphin-1-like immunoreactivity in studies involving immunocytochemistry [29] and radioimmunoassay [16]. Although fewer axonal projections to this region is likely to be the major reason, the present study indicates that rapid
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Fig. 3. Percentage of endomorphin-1 (YPWF-NH2) remaining in intact form, determined by HPLC, after incubation with the cerebral microvessels from three regions of the brain in two large experiments (1 and 2) repeated several months apart. HT 5 hypothalamus.
metabolism by microvessels could contribute to the paucity of endomorphin in cortical regions. Perhaps the biological effects of endomorphin in regions like the cortex are potentially so great that special mechanisms are required to reduce its concentrations. By comparison, the hypothalamus contains cell bodies and dense immunoreactive endomorphin-1 fibers that are positioned to be involved in modulating nociceptive and autonomic stimuli as well as other homeostatic functions such as ingestive, neuroendocrine, and thermoregulatory processes [29]. Similarly, the parabrachial nuclei in the pons are critical regions for autonomic function including cardiovascular, respiratory, and neuroendocrine function as well as pain processing. The dense staining for the endomorphins together with the high mu opiate receptor density in these nuclei implicate the endomorphins as regulators of these functions. Microvessels in the hypothalamus and pons metabolized Tyr-MIF-1 and endomorphin-1 more slowly than did those in the cortex. It is unlikely that the regional differences in metabolism are the result of different concentrations of residual endogenous peptide because extensive washing of the microvessels in salt-containing media would reduce the endogenous peptides to concentrations that are negligible with regard to enzyme saturation. A more likely explanation is that metabolic processes that are more proximal to the signaling pathway, such as enzymes in the synaptic plasma membranes, play a greater regulatory role than microvessels in
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these regions of relatively high endomorphin concentrations. In areas such as the cortex and hippocampus, degradation by microvesssels may play a more prominent role. Thus, the results establish that cerebral microvessels can provide a novel mechanism for regulation of the activity of peptides crossing the BBB. This involves differential metabolism at the BBB that is dependent on the brain region and the structure of the peptide attempting to cross there. Together with the well-established process of saturable transport, this novel mechanism demonstrates a multi-faceted role for the BBB in peptide regulation. Acknowledgements Supported by the VA, NIH, and the US Army Medical Research Acquisition Activity (DAMD17-00-1-0113). References 1. Kastin AJ, Pan W, Maness LM, Banks WA. Peptides crossing the blood-brain barrier: some unusual observations. Brain Research 1999;848:96–100. 2. Strand FL. Neuropeptides: Regulators of Physiological Processes. Cambridge, MA: MIT Press, 1999. 3. Egleton RD, Davis TP. Bioavailability and transport of peptides and peptide drugs into the brain. Peptides 1997;18:1431–9. 4. Davson H, Segal MB. Physiology of the CSF and Blood-Brain Barriers. New York: CRC Press, 1995. 5. Horvath A, Kastin AJ. Isolation of tyrosine-melanocyte-stimulating hormone release-inhibiting factor 1 from bovine brain tissue. Journal of Biological Chemistry 1989;264:2175–9. 6. Banks WA, Kastin AJ, Fischman AJ, Coy DH, Strauss SL. Carrier-mediated transport of enkephalins and N-Tyr-MIF-1 across blood-brain barrier. American Journal of Physiology 1986;251:E477–E482. 7. Banks WA, Kastin AJ, Michals EA. Tyr-MIF-1 and met-enkephalin share a saturable blood-brain barrier transport system. Peptides 1987;8:899–903. 8. Banks WA, Kastin AJ, Michals EA, Barrera CM. Stereospecific transport of Tyr-MIF-1 across the bloodbrain barrier by peptide transport system-1. Brain Research Bulletin 1990;25:589–92. 9. Banks WA, Kastin AJ. Opposite direction of transport across the blood-brain barrier for Tyr-MIF-1 and MIF-1: comparison with morphine. Peptides 1994;15:23–9. 10. Banks WA, Kastin AJ. New Concepts of a Blood-Brain Barrier. In: Greenwood J, Begley DJ, Segal MB, Lightman S editors. Plenum Press, 1995. p.p.111–117. 11. Barrera CM, Banks WA, Kastin AJ. Passage of Tyr-MIF-1 from blood to brain. Brain Research Bulletin 1989;23:439–42. 12. Kastin AJ, Hahn K, Erchegyi J, Zadina JE, Hackler L, Palmgren M, Banks WA. Differential metabolism of Tyr-MIF-1 and MIF-1 in rat and human plasma. Biochemical Pharmacology 1994;47:699–709. 13. Kastin AJ, Hahn K, Zadina JE, Banks WA, Hackler L. Melanocyte-stimulating hormone release-inhibiting factor-1 (MIF-1) can be formed from Tyr-MIF-1 in brain mitochondria but not in brain homogenate. Journal of Neurochemistry 1995;64:1855–9. 14. Kastin AJ, Hahn K, Banks WA, Zadina JE. Delayed degradation of Tyr-MIF-1 in neonatal rat plasma. Peptides 1994;15:1561–3. 15. Kastin AJ, Banks WA, Hahn K, Zadina JE. Extreme stability of Tyr-MIF-1 in CSF. Neuroscience Letters 1994;174:26–8. 16. Zadina JE, Hackler L, Ge L-J, Kastin AJ. A potent and selective endogenous agonist for the -opiate receptor. Nature 1997;386:499–502. 17. Kastin AJ, Hahn K, Banks WA, Zadina JE. Regional differences in the metabolism of Tyr-MIF-1 and Tyr-WMIF-1 by rat brain mitochondria. Biochemical Pharmacology 1998;55:33–6. 18. Dauch P, Masuo Y, Vincent JP, Checler F. A survey of the cerebral regionalization and ontogeny of eight exoand endopeptidases in murines. Peptides 1993;14:593–9.
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19. Brownlees J, Williams CH. Peptidases, peptides, and the mammalian blood-brain barrier. Journal of Neurochemistry 1993;60:793–803. 20. Brownson EA, Abbruscato TJ, Gillespie TJ, Hruby V, Davis TP. Effect of peptidases at the blood-brain barrier on the permeability of enkephalin. Journal of Pharmacology and Experimental Therapeutics 1994;270:675–80. 21. Brust P, Bech A, Kretzchmar R, Bergmann R. Developmental changes of enzymes involved in peptide degradation in isolated rat brain microvessels. Peptides 1994;15:1085–8. 22. Gerhart DZ, Broderius MA, Drews LR. Cultured human and canine endothelial cells from brain microvessels. Brain Research Bulletin 1988;21:785–93. 23. Banks WA, Akerstrom V, Kastin AJ. Adsorptive endocytosis mediates the passage of HIV-1 across the BBB: evidence for a post-internalization coreceptor. Journal of Cell Science 1998;111:533–40. 24. Pardidge WM, Eisenberg J, Yamada T. Rapid sequestration and degradation of somatostatin analogues by isolated brain microvessels. Journal of Neurochemistry 1985;44:1178–84. 25. Fischman AJ, Kastin AJ, Graf MV. HPLC shadowing: artifacts in peptide characterization monitored by RIA. Peptides 1984;5:1007–10. 26. Banks WA, Kastin AJ. Peptides and the blood-brain barrier: lipophilicity as a predictor of permeability. Brain Research Bulletin 1985;15:287–92. 27. Chikhale EG, Ng KY, Burton PS, Borchardt RT. Hydrogen bonding potential as a determinant of the in vitro and in situ blood-brain barrier permeability of peptides. Pharmaceutical Research 1994;11:412–9. 28. Kastin AJ, Akerstrom V, Pan W. Activation of urocortin transport into brain by leptin. Peptides 2000;21:1 811–8. 29. Martin-Schild S, Gerall AE, Kastin AJ, Zadina JE. Differential distribution of endomorphin 1- and endomorphin 2-like immunoreactivities in the CNS of the rodent. Journal of Comparative Neurology 1999;405:450–71.
Regional differences in peptide degradation by rat cerebral microvessels A potential novel regulatory mechanism for communication between blood and brain Abba J. Kastin*, Kathy Hahn, James E. Zadina VA Medical Center and Tulane University School of Medicine, New Orleans, LA 70112-1262, USA Received 15 September 2000; accepted 21 March 2001
Abstract 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 (TyrPro-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. © 2001 Elsevier Science Inc. All rights reserved.
Introduction No longer considered static, the blood-brain barrier (BBB) is now known to play an active role in the regulation of the transfer of peptides and polypeptides from blood to brain [1–4]. Some peptides cross the BBB by saturable transport mechanisms while others cross by passive diffusion based on physicochemical properties. For saturable transport the regulatory ability of the BBB is obvious, but for passive diffusion the regulatory ability remains obscure.
* Corresponding author. VA Med Ctr/Tulane Med, 1601 Perdido St., New Orleans, LA 70112-1262. Tel.: 504568-0811, ext. 5884; fax: 504-522-8559. 0024-3205/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 1 )0 1 2 1 1 -5
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The saturable transport of the opiate modulating tetrapeptide Tyr-MIF-1 (Tyr-Pro-LeuGly-NH2) [5] from brain to blood has been extensively studied [6–10]; however, its entry into brain from blood occurs by passive diffusion [11]. Tyr-MIF-1 is rapidly degraded in blood [12] and whole brain homogenates [13], but not in neonatal rat plasma [14] or cerebrospinal fluid [15]. Endomorphin-1 (Tyr-Pro-Trp-Phe-NH2) has the highest affinity and specificity for the mu opiate receptor of any endogenous brain peptide and its analgesic potency is similar to that of morphine [16]. It shares some structural and functional similarities with Tyr-MIF-1, but its metabolism has not been thoroughly investigated. There are regional differences in the metabolism of Tyr-MIF-1 [17] and in the activities of eight peptidases [18] when entire sections of brain tissue are used [17], but no regional differences have been described for the microvessels of the BBB. The enzymatic activities of the capillary endothelial (microvessel) cells have been thoroughly reviewed in general [19] and for some other opiate peptides [20]. Although developmental changes have been observed [18,21], the possibility of regional changes has not been reported. Here, we examine for the first time whether there are regional differences in peptide metabolism in microvessels isolated from different regions of the brain. Since cerebral microvessels constitute the BBB and provide the main site of contact between blood and brain, regional differences in metabolism would provide a unique method of regulation of passage across the BBB, even for substances entering by passive diffusion. Experimental procedures Tyr-MIF-1 was tritiated (specific activity: 40 Ci/mmol) (Amersham, Arlington Heights, IL) on the Pro from the [dehydro-Pro]2 precursor. Endomorphin-1 was tritiated (specific activity: 50 Ci/mmol) (Nycomed Amersham, Buckinghamshire, England) on the Pro by hydrogenation of an unsaturated intermediate. The procedure used for isolation of the capillary microvessels was based on the well-established method of Gerhart et al. [22] as modified for the rodent [23]. Endothelial cells prepared in this way are hardy, grow rapidly, and consist of a single cell type [22]. All plasticware was pre-coated with phosphate-buffered saline and bovine serum albumin. The buffer was MEM (Minimum Essential Medium Eagle, Sigma, St. Louis, MO). Anesthetized male rats (Harlan Sprague-Dawley, Indianapolis, IN) weighing about 300 g were perfused through the heart with 0.9% NaCl by gravity flow. After perfusion, each brain was placed in a petri dish on ice, and the areas of interest removed by careful dissection. Selected regions from 25 rats were combined to obtain sufficient material for analysis. These were homogenized at 4 8C in a Potter-Elvehjem apparatus with a loose fitting Teflon pestle for the first 10 strokes, then with a tight fitting pestle for an additional 5–6 strokes. The homogenate was filtered once through a 300 mm mesh microsieve (Spectrum, Laguna Hills, CA) and twice more through a 100 mm mesh net. An equal volume of 40% dextran was added to the filtrate and the mixture was centrifuged (swinging bucket) at 5000 g for 15 min at 4 8C. The pellets were suspended in MEM, filtered through a 25 mm mesh net (Biodesign, Carmel, NY), and washed three times with MEM. The remaining liquid was removed by pipet and the net was inverted over a buffer-containing petri dish. The agitated microvessel sus-
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pension in the dish was examined by microscopic wet mount to ensure purity and transferred to polypropylene tubes which were centrifuged at 2500 g for 15 min at 4 8C. After aspiration of the supernatant, hibernate A (Life Technologies, Gaithersburg, MD) was added to the pellets which were stored at 4 8C overnight. The next day, the cells were centrifuged at 2500 g for 15 min and resusupended in MEM. Preliminary studies revealed that hibernate A was as least as effective as DMSO after overnight storage, both resulting in somewhat more recovery of intact endomorphin-1 after 2 h incubation than when freshly prepared microvessels without hibernate A were used. Protein content was determined by the Lowry method. 3 H-Tyr-MIF-1 or 3H-endomorphin-1 (3.53106 dpm each) were incubated with 175 mg of microvessel protein in a metabolic shaker (Dubnoff Incu-Shaker, Labline Instruments, Melrose Park, IL). This approximates the standard amount of microvessel protein required for accurate measurement of enzymatic activity [24]. Samples were removed at 30 min, 1 h, 2h, and, for Tyr-MIF-1, 4 h. At these times, 10% cold trifluoroacetic acid (TFA) was added to stop the reaction. After centrifugation, the supernatants were dried in a Speed-Vac (Savant Instruments, Farmingdale, NY) and samples stored in a desiccator at 220 8C until analysis by HPLC. For chromatography, samples were reconstituted in 0.1% TFA/H2O (solvent A), filtered (nylon 66 centrifuge filter; pore size 0.45 mm; Alltech Associates, Deerfield, IL), and applied to a Brownlee RP-18 reversed-phase column (4.6 mm 3 22 cm, width 1.5 cm) with a 1.5 cm RP-18 guard cartridge (Rainen, Woburn, MA). The HPLC system was a Beckman (Fullerton, CA) model 344 with version 5.10 Gold software. The online detector was the INSUS Systems (Tampa, FL) radiochromatogram b-RAM 2B. For Tyr-MIF-1, solvent B was 0.1% TFA in methanol. The gradient consisted of 10% B which was gradually increased to 30% over 35 min, to 33% for the next 10 min, to 35% for the next 5 min, and then to 80% for the final 2 min. With this gradient, Pro eluted at 4 min, Tyr-Pro at 20 min, intact Tyr-MIF-1 at 33–34 min, and the free acid of Tyr-MIF-1 at 37–38 min. As a control for HPLC “shadowing” to ensure that no contamination or carry-over occurred [25], the HPLC column was washed with 80% methanol for 2 h before each analysis, then twice with the same gradient used for the peptides, and a blank sample analyzed . For endomorphin-1, solvent B was 0.1% TFA in acetonitrile. The gradient consisted of 5% solvent B which was gradually increased to 25% over 25 min, to 32% for the next 40 min, and then to 80% for the final 2 min. With this gradient, Pro eluted at 3–4 min, Tyr-Pro at 14– 15 min, and intact endomorphin-1 at 51–53 min. Results A representative HPLC chromatogram for Tyr-MIF-1 before and after 4 h incubation with brain cortical microvessels is shown in figure 1. The inset shows endomorphin-1 before incubation; after 4 h, no endomorphin-1 remained. The percent of intact Tyr-MIF-1 in microvessels from three different regions of the brain at various times up to 4 h after incubation is shown in figure 2. For Tyr-MIF-1, degradation by the cortical microvessels was greater than that by microvessels from the hypothalamus or pons. There was no difference in the rates of degradation between the hypothalamus and pons. Moreover, for microvessels from both hy-
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Fig. 1. HPLC chromatogram of 3H-Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2) before and after incubation for 4 h with the cerebral microvessels that constitute the BBB. P 5 Pro, Y 5 Tyr, YPLG-NH2 5 Tyr-MIF-1, YPLG-OH 5 the free acid of Tyr-MIF-1. The inset shows endomorphin-1 (Tyr-Pro-Trp-Phe-NH2 5 YPWF-NH2) at 0 h; by 4 h none remained. The dotted lines represent the HPLC gradients.
pothalamus and pons, no further degradation of Tyr-MIF-1 occurred after 30 min, the TyrMIF-1 remaining largely (70–76%) intact for the entire 4 h of incubation. Although the accumulation of enough microvessels for measurement of activity is a tedious process requiring a large number of rats, for endomorphin-1 each experiment was repeated twice with microvessels from each brain part at each time. An identical amount of microvessel protein was used for all measurements, although the more highly vascularized areas of the brain may have a smaller proportion of the total number of capillaries. In the repeat experiments performed several months apart, 68.8 – 71.2% endomorphin-1 was intact in the pons after the 2 h incubation , 50.5 – 55.5% in the hypothalamus, and only 29.8 – 32.4% in the cerebral cortex (Fig. 3). The focus of the results was on the hypothesis of regional differences in degradation; no attempt was made to identify the enzymes responsible for the degradation. Discussion Regulation by the BBB can be very selective. Substitution of D-Tyr for L-Tyr, or removal of a hydroxyl group from Tyr (5 Phe), abolishes the saturable brain-to-blood transport of the tetrapeptide Tyr-MIF-1 by Peptide Transport System-1, a system that does not transport the
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Fig. 2. Percentage of Tyr-MIF-1 (YPLG-NH2) remaining in intact form, determined by HPLC, after incubation with the cerebral microvessels from three regions of the brain. HT 5 hypothalamus.
smaller MIF-1 (Pro-Leu-Gly-NH2) [8]. In the opposite direction, Tyr-MIF-1 enters the brain by passive diffusion rather than saturable transport [11]. Passive diffusion is influenced by physicochemical properties such as lipophilicity and hydrogen-bonding [26,27], but does not provide the selective type of feedback control that is characteristic of saturable transport. The self-inhibition of saturable transport provides physiological regulation of transport by varying concentrations of peptide or polypeptide in the blood or brain. Cross-inhibition of saturable transport by a functionally or structurally related substance provides an even broader regulatory role for the BBB. Recently, activation (rather than inhibition) of a saturable transport system by a substance with similar function was described [28]. The present study demonstrates an additional mechanism by which the BBB can exert selective regulatory control: differential degradation of peptides depending on brain region and peptide structure at the BBB itself. For both peptides tested, the largest amount of degradation occurred in microvessels from the cerebral cortex. Metabolism of Tyr-MIF-1 occurred at a similar rate in the hypothalamus and pons while endomorphin-1 was metabolized faster in the hypothalamus than in pons. Thus, the rate of degradation was specific to the region and peptide. The cortex shows relatively sparse endomorphin-1-like immunoreactivity in studies involving immunocytochemistry [29] and radioimmunoassay [16]. Although fewer axonal projections to this region is likely to be the major reason, the present study indicates that rapid
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Fig. 3. Percentage of endomorphin-1 (YPWF-NH2) remaining in intact form, determined by HPLC, after incubation with the cerebral microvessels from three regions of the brain in two large experiments (1 and 2) repeated several months apart. HT 5 hypothalamus.
metabolism by microvessels could contribute to the paucity of endomorphin in cortical regions. Perhaps the biological effects of endomorphin in regions like the cortex are potentially so great that special mechanisms are required to reduce its concentrations. By comparison, the hypothalamus contains cell bodies and dense immunoreactive endomorphin-1 fibers that are positioned to be involved in modulating nociceptive and autonomic stimuli as well as other homeostatic functions such as ingestive, neuroendocrine, and thermoregulatory processes [29]. Similarly, the parabrachial nuclei in the pons are critical regions for autonomic function including cardiovascular, respiratory, and neuroendocrine function as well as pain processing. The dense staining for the endomorphins together with the high mu opiate receptor density in these nuclei implicate the endomorphins as regulators of these functions. Microvessels in the hypothalamus and pons metabolized Tyr-MIF-1 and endomorphin-1 more slowly than did those in the cortex. It is unlikely that the regional differences in metabolism are the result of different concentrations of residual endogenous peptide because extensive washing of the microvessels in salt-containing media would reduce the endogenous peptides to concentrations that are negligible with regard to enzyme saturation. A more likely explanation is that metabolic processes that are more proximal to the signaling pathway, such as enzymes in the synaptic plasma membranes, play a greater regulatory role than microvessels in
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these regions of relatively high endomorphin concentrations. In areas such as the cortex and hippocampus, degradation by microvesssels may play a more prominent role. Thus, the results establish that cerebral microvessels can provide a novel mechanism for regulation of the activity of peptides crossing the BBB. This involves differential metabolism at the BBB that is dependent on the brain region and the structure of the peptide attempting to cross there. Together with the well-established process of saturable transport, this novel mechanism demonstrates a multi-faceted role for the BBB in peptide regulation. Acknowledgements Supported by the VA, NIH, and the US Army Medical Research Acquisition Activity (DAMD17-00-1-0113). References 1. Kastin AJ, Pan W, Maness LM, Banks WA. Peptides crossing the blood-brain barrier: some unusual observations. Brain Research 1999;848:96–100. 2. Strand FL. Neuropeptides: Regulators of Physiological Processes. Cambridge, MA: MIT Press, 1999. 3. Egleton RD, Davis TP. Bioavailability and transport of peptides and peptide drugs into the brain. Peptides 1997;18:1431–9. 4. Davson H, Segal MB. Physiology of the CSF and Blood-Brain Barriers. New York: CRC Press, 1995. 5. Horvath A, Kastin AJ. Isolation of tyrosine-melanocyte-stimulating hormone release-inhibiting factor 1 from bovine brain tissue. Journal of Biological Chemistry 1989;264:2175–9. 6. Banks WA, Kastin AJ, Fischman AJ, Coy DH, Strauss SL. Carrier-mediated transport of enkephalins and N-Tyr-MIF-1 across blood-brain barrier. American Journal of Physiology 1986;251:E477–E482. 7. Banks WA, Kastin AJ, Michals EA. Tyr-MIF-1 and met-enkephalin share a saturable blood-brain barrier transport system. Peptides 1987;8:899–903. 8. Banks WA, Kastin AJ, Michals EA, Barrera CM. Stereospecific transport of Tyr-MIF-1 across the bloodbrain barrier by peptide transport system-1. Brain Research Bulletin 1990;25:589–92. 9. Banks WA, Kastin AJ. Opposite direction of transport across the blood-brain barrier for Tyr-MIF-1 and MIF-1: comparison with morphine. Peptides 1994;15:23–9. 10. Banks WA, Kastin AJ. New Concepts of a Blood-Brain Barrier. In: Greenwood J, Begley DJ, Segal MB, Lightman S editors. Plenum Press, 1995. p.p.111–117. 11. Barrera CM, Banks WA, Kastin AJ. Passage of Tyr-MIF-1 from blood to brain. Brain Research Bulletin 1989;23:439–42. 12. Kastin AJ, Hahn K, Erchegyi J, Zadina JE, Hackler L, Palmgren M, Banks WA. Differential metabolism of Tyr-MIF-1 and MIF-1 in rat and human plasma. Biochemical Pharmacology 1994;47:699–709. 13. Kastin AJ, Hahn K, Zadina JE, Banks WA, Hackler L. Melanocyte-stimulating hormone release-inhibiting factor-1 (MIF-1) can be formed from Tyr-MIF-1 in brain mitochondria but not in brain homogenate. Journal of Neurochemistry 1995;64:1855–9. 14. Kastin AJ, Hahn K, Banks WA, Zadina JE. Delayed degradation of Tyr-MIF-1 in neonatal rat plasma. Peptides 1994;15:1561–3. 15. Kastin AJ, Banks WA, Hahn K, Zadina JE. Extreme stability of Tyr-MIF-1 in CSF. Neuroscience Letters 1994;174:26–8. 16. Zadina JE, Hackler L, Ge L-J, Kastin AJ. A potent and selective endogenous agonist for the -opiate receptor. Nature 1997;386:499–502. 17. Kastin AJ, Hahn K, Banks WA, Zadina JE. Regional differences in the metabolism of Tyr-MIF-1 and Tyr-WMIF-1 by rat brain mitochondria. Biochemical Pharmacology 1998;55:33–6. 18. Dauch P, Masuo Y, Vincent JP, Checler F. A survey of the cerebral regionalization and ontogeny of eight exoand endopeptidases in murines. Peptides 1993;14:593–9.
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19. Brownlees J, Williams CH. Peptidases, peptides, and the mammalian blood-brain barrier. Journal of Neurochemistry 1993;60:793–803. 20. Brownson EA, Abbruscato TJ, Gillespie TJ, Hruby V, Davis TP. Effect of peptidases at the blood-brain barrier on the permeability of enkephalin. Journal of Pharmacology and Experimental Therapeutics 1994;270:675–80. 21. Brust P, Bech A, Kretzchmar R, Bergmann R. Developmental changes of enzymes involved in peptide degradation in isolated rat brain microvessels. Peptides 1994;15:1085–8. 22. Gerhart DZ, Broderius MA, Drews LR. Cultured human and canine endothelial cells from brain microvessels. Brain Research Bulletin 1988;21:785–93. 23. Banks WA, Akerstrom V, Kastin AJ. Adsorptive endocytosis mediates the passage of HIV-1 across the BBB: evidence for a post-internalization coreceptor. Journal of Cell Science 1998;111:533–40. 24. Pardidge WM, Eisenberg J, Yamada T. Rapid sequestration and degradation of somatostatin analogues by isolated brain microvessels. Journal of Neurochemistry 1985;44:1178–84. 25. Fischman AJ, Kastin AJ, Graf MV. HPLC shadowing: artifacts in peptide characterization monitored by RIA. Peptides 1984;5:1007–10. 26. Banks WA, Kastin AJ. Peptides and the blood-brain barrier: lipophilicity as a predictor of permeability. Brain Research Bulletin 1985;15:287–92. 27. Chikhale EG, Ng KY, Burton PS, Borchardt RT. Hydrogen bonding potential as a determinant of the in vitro and in situ blood-brain barrier permeability of peptides. Pharmaceutical Research 1994;11:412–9. 28. Kastin AJ, Akerstrom V, Pan W. Activation of urocortin transport into brain by leptin. Peptides 2000;21:1 811–8. 29. Martin-Schild S, Gerall AE, Kastin AJ, Zadina JE. Differential distribution of endomorphin 1- and endomorphin 2-like immunoreactivities in the CNS of the rodent. Journal of Comparative Neurology 1999;405:450–71.