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Peptides crossing the blood–brain barrier: some unusual observations
Brain Research 848 Ž1999. 96–100 www.elsevier.comrlocaterbres
Interactive report
Peptides crossing the blood–brain barrier: some unusual observations 1 Abba J. Kastin a
a, )
, Weihong Pan a , Lawrence M. Maness a , William A. Banks
b
VA Medical Center and Tulane UniÕersity School of Medicine, 1601 Perdido Street, New Orleans, LA 70112-1262 USA b GRECC, VA Medical Center-St. Louis, St. Louis UniÕersity School of Medicine, St. Louis, MO 63106, USA Accepted 12 August 1999
Abstract An interactive blood–brain barrier ŽBBB. helps regulate the passage of peptides from the periphery to the CNS and from the CNS to the periphery. Many peptides cross the BBB by simple diffusion, mainly explained by their lipophilicity and other physicochemical properties. Other peptides cross by saturable transport systems. The systems that transport peptides into or out of the CNS can be highly specific, transporting MIF-1 but not Tyr–MIF-1, PACAP38 but not PACAP27, IL-1 but not IL-2, and leptin but not the smaller ingestive peptides NPY, orexin A, orexin B, CART Ž55–102wMetŽO. 67 x., MCH, or AgRPŽ83–132.. Although the peptides EGF and TGF-a bind to the same receptor, only EGF enters by a rapid saturable transport system, suggesting that receptors and transporters can represent different proteins. Even the polypeptide NGF enters faster than its much smaller subunit b-NGF. The saturable transport of some compounds can be upregulated, like TNF-a in EAE Žan animal model of multiple sclerosis. and after spinal cord injury, emphasizing the regulatory role of the BBB. As has been shown for CRH, saturable transport from brain to blood can exert effects in the periphery. Thus, the BBB plays a dynamic role in the communication of peptides between the periphery and the CNS. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Peptide; Blood–brain barrier; Saturable transport; Diffusion
1. Introduction
2. Size can be relatively unimportant
The millennium is a time for celebration in the neuropeptide field. It has seen the acceptance of several concepts that had long been subject to negative unsubstantiated dogmas. No longer can there be any doubt about the ability of numerous peptides in the periphery to affect the central nervous system ŽCNS. w25,52x or the ability of many of them to cross the blood–brain barrier ŽBBB. w17x so as to be able to exert these effects directly. Similarly, peptides from the CNS can cross the BBB to exert effects in the periphery. The last reviews we published concerning the passage of peptides and polypeptides Žincluding most neurotrophins and cytokines. across the BBB were intended to be more general and comprehensive w5,41x. In the present review, we have selectively chosen a few unusual observations to illustrate some novel concepts in this area.
The aphorism that ‘‘size doesn’t matter’’ may apply to the BBB when the relatively narrow range encompassing peptides and polypeptides is considered. Although an early study found that the molecular weight of peptides does not correlate with their rate of passage across the BBB w1x, the idea still seems somewhat counterintuitive. The relative unimportance of size is most obvious for peptides and polypeptides with saturable transport systems, as the following examples indicate.
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Corresponding author. Tel.: q1-504-568-0811X5884; Fax: q1-504522-8559. 1 Published on the World Wide Web on 24 August 1999.
2.1. Brain-to-blood transport 2.1.1. Tyr–MIF-1 Õs. MIF-1 The same study w1x showing that molecular weight is not a good predictor of the passage of peptides across the BBB also found that lipophilicity is a much better one. An exception to this occurred with low molecular weight peptides with an N-terminal tyrosine ŽTyr. that entered the brain much slower than predicted. Among the possible explanations, a brain-to-blood transport system seemed likely. Using Tyr–MIF-1 ŽTyr–Pro–Leu–Gly–NH 2 . as
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the prototype, we found the existence of saturable systems for the transport of peptides out of the brain. It is easy to understand, therefore, how a brain-to-blood efflux system could obscure determination of blood-to-brain entry when brain uptake after peripheral injection is examined, regardless of whether immunoreactivity or radioactivity is measured. Determination of only immunoreactivity involves the additional problem of separation of endogenous from exogenous material, although this can be resolved with species-specific immunoassays w7x. The transport system for Tyr–MIF-1 is stereospecific. Change of the L-Tyr to its D-form, or the removal of even a single hydroxyl group from the Tyr Žresulting in Phe., prevents saturable transport w14x. Removal of the entire N-terminal Tyr, resulting in the tripeptide MIF-1, also prevents saturable transport out of the brain w4x. MIF-1 was the first endogenous compound shown to exert antiopiate effects w27x. Tyr–MIF-1 shares this property w18x, but in some situations it can act as an opiate agonist w55x. The endogenous opiate Met-enkephalin is a pentapeptide with only the N-terminal Tyr in common with Tyr–MIF-1. Their functional interactions may help explain the unusual observation that the stereospecific system for Tyr–MIF-1 also transports Met-enkephalin w13x, a finding with implications for alcoholism w47x. 2.1.2. CRH Corticotropin-releasing hormone ŽCRH. is more than ten times larger than MIF-1. Although the tripeptide MIF-1 is not transported out of the brain, there is a well characterized saturable efflux system for CRH w32,34x. Moreover, the brain-to-blood transport of CRH is sufficient to exert effects in the periphery w33x. The concept that brain-toblood transport can result in peripheral effects seems more unusual than the concept that blood-to-brain transport can result in central effects. 2.2. Blood-to-brain transport 2.2.1. MIF-1 MIF-1 is saturably transported into the CNS but Tyr– MIF-1 is not w4x. The rate of entry is much faster for MIF-1 than for morphine. Although MIF-1 is remarkably stable in human blood, it is not formed there from Tyr– MIF-1 w26x. Thus, Tyr–MIF-1 but not MIF-1 is saturably transported from brain-to-blood whereas MIF-1 but not Tyr–MIF-1 is saturably transported from blood-to-brain. 2.2.2. Leptin Õs. ingestiÕe peptides Leptin is saturably transported into but not out of the brain w11x. The localization of leptin in the arcuate nucleus after iv administration is not found after icv administration w29x. Since the arcuate appears to be the major site of action of leptin in the control of appetite, it might seem unusual that injection in blood provides a more efficient
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path than is achieved by injection into the cerebrospinal fluid. This directed delivery of leptin from blood to a discrete area of the brain emphasizes the regulatory potential of the BBB. Leptin is a polypeptide Žmore than 100 amino acids.. Many other ingestive substances are peptides Žfewer than 100 amino acids., and most of them also enter the brain. Unlike leptin, however, some do not enter through saturable influx systems even though they apparently interact with leptin. We could not find saturable transport for neuropeptide Y w22x, orexin A, orexin B w23x, cocaine- and amphetamine-regulated transcript ŽCART . Ž55–102 wMetŽO. 67 x. w21x, melanin-concentrating hormone ŽMCH., or agouti-related protein ŽAgRP.Ž83–132. w24x; moreover, none of these feeding-related peptides appear to affect the saturable entry of leptin. 2.2.3. IL-1 Õs. IL-2 Interleukin ŽIL.-1a is transported from blood to brain by a saturable system that also transports IL-1b and IL-1 receptor antagonist ŽIL-1ra. but not tumor necrosis factor-a ŽTNF-a ., IL-2, IL-6, or macrophage inflammatory protein ŽMIP.-1a w15,20x. Of these cytokines, however, only IL-1a has been shown to be saturably concentrated in the posterior division of the septum w28x. IL-1a also is transported into the spinal cord by a saturable system w9x. Although the circumventricular organs ŽCVOs. have a surface area of only 0.02 cm2rg brain as compared with 100–150 cm2rg for the BBB w53x, the misconception persists that most of the penetration of peptides and polypeptides across the BBB occurs through the CVOs. However, less than 5% of the IL-1a injected into blood enters the CVOs w30,46x, and this material is restricted from diffusion into the rest of the brain by the outer layer of cells comprising the CVOs w17,53x. The BBB is not disrupted in such studies w3x. The transport of IL-1a across the BBB is self-inhibited by a dose as low as 2.5 mgrkg w15x. Yet at a dose 20 times higher, there is no self-inhibition Žsaturation. of the passage of IL-2 w54x. As with many of the other unusual observations discussed in this review, it would have been impossible with the present state of knowledge to predict these findings, raising obvious questions about the present criteria for obtaining funding. 2.2.4. IL-6 Õs. other cytokines Essential for studies of the passage of substances across the BBB is determination of the integrity of the material reaching the brain. Most measurements involve homogenization of brain tissue with subsequent release of intracellular enzymes to which the entering substance is not initially exposed. HPLC is the most commonly used procedure, although SDS-PAGE also is suitable, especially for larger polypeptides. Even after correction for processing, apparently much less IL-6 reaches the brain in intact form w9x than has been found for the transport of other cytokines
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like IL-1a w15x, IL-1ra w20x, TNF-a w19x, interferon-g w37x, granulocyte–macrophage colony-stimulating factor ŽGMCSF. w35x, and ciliary neurotrophic factor ŽCNTF. w43x.
3. Saturable transport systems can be upregulated
3.1. EAE 2.2.5. MIP Õs. other cytokines Also essential for studies of the passage of substances across the BBB is determination that the material being measured in the brain actually reaches the parenchyma rather than being attached to the luminal wall of the capillaries comprising the BBB. For almost all the substances studied so far, the capillary depletion method has shown that most of the injected material reaches the brain parenchyma. However, for MIP-1a and MIP-1b, this technique based on differential centrifugation modified with a washout procedure shows that most of the injected MIP is reversibly associated with the endothelia of the BBB w6x. 2.2.6. b-NGF and NGF NGF is composed of five subunits. One of these subunits, b-NGF, is responsible for much of the bioactivity of NGF. Yet the smaller b-NGF crosses the BBB only half as fast as the parent NGF w38x. Using slightly different methods, Poduslo and co-workers also found significant penetration of the BBB by NGF, courageously commenting upon the prevalent ‘‘erroneous conclusions w...x that has plagued brain- and nerve-barrier research w...x that the barrier was impermeable to proteins’’ w48,49x.
Another unusual observation involves experimental autoimmune encephalomyelitis ŽEAE., considered an animal model of multiple sclerosis. In EAE and multiple sclerosis, the BBB is disrupted so that it is more permeable to many substances. Under such circumstances, it might be expected that saturable transport systems also would be disrupted. This was studied with TNF-a, a cytokine implicated in the pathogenesis of EAE, which can exert bimodal actions of either a beneficial or detrimental nature w45x. In the model of EAE used, disruption of the BBB, as reflected by an increased permeability to peripherally administered sucrose and albumin, coincides with the functional deficits of the mice w39x. The previously demonstrated w19x saturable transport of TNF-a, however, is enhanced. Although TNF-a circulates as a trimer of similar size to albumin, the amount of TNF-a entering the CNS is similar to that of the smaller sucrose, both entering twice as fast as albumin w39x. Moreover, the self-inhibition of transport intrinsic to the process of saturation remains intact; this would not be seen if TNF-a crossed the disrupted BBB by diffusion or leakage.
3.2. Spinal cord injury 2.2.7. PACAP38 and PACAP27 The two major members of the pituitary adenylate cyclase activating polypeptide ŽPACAP. family illustrate several unusual findings. First is the observation that there is a saturable transport system into the brain for PACAP38 but not for PACAP27, a peptide with 11 fewer amino acids. Second is the observation that despite the presence of a saturable transport system for PACAP38, a larger percentage of injected PACAP27 enters the brain, probably related to its greater lipophilicity, smaller volume of distribution, and longer half-time disappearance from blood. Third is the observation that there also is a saturable transport system out of the brain that prefers PACAP38 to PACAP27 w12x. The only other peptides for which saturable transport systems w17,51x in both directions have been described are LHRH w16x, arginine vasopressin w56,57x, and leucine enkephalin w10,58x. A few peptides, such as PACAP, can exert neurotrophic properties. The transport of PACAP38 across the BBB Žincluding the blood–spinal cord barrier. from blood to CNS is changed by spinal cord injury ŽSCI. w8x whereas that of the much smaller MSHrACTH analog ebiratide is not affected w36x. Soon after SCI, transport of PACAP38 is decreased, but later there is an upregulation not explained by direct disruption of the vascular barrier w8x.
3.2.1. Complete transection After SCI, permeability of the BBB to sucrose and albumin is increased, as it is in EAE. As also seen in EAE, complete transection of the spinal cord increases the permeability of the BBB to TNF-a w36x. This increase is selective. Even though ebiratide is smaller and has a saturable transport system across the BBB w50x, SCI does not change its rate of entry. By contrast, the saturable entry of TNF-a is enhanced. The increased permeability to TNF-a does not correlate with that to the markers sucrose and albumin w36x.
3.2.2. Partial disruption by compression There is marked dissociation of the permeation of TNFa and the vascular marker albumin during the second phase of BBB opening induced by spinal cord compression w42x. By comparison with albumin, the entry of TNF-a is of much greater magnitude, occurs earlier, lasts for a longer time, involves regions distal as well as proximal to the site of compression, and retains the self-inhibition characteristic of a saturable transport system. The increased entry of TNF-a from blood to the CNS in these conditions could be helpful in restricting secondary tissue damage and in aiding regeneration.
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4. Receptor binding need not reflect transport
Acknowledgements
4.1. EGF Õs. TGF-a
This work was supported by NIH ŽDK54880. and the VA.
Epidermal growth factor ŽEGF. and transforming growth factor a ŽTGF-a . are members of the EGF family of growth factors. As peptides of about 50 amino acids, they are much smaller than the classical neurotrophins. Although EGF and TGF-a bind to the same receptor, they cross the BBB in completely different ways. The rate of entry of EGF into brain is much faster than that for most neurotrophins, cytokines, or even bioactive peptides w40x. As expected for such fast entry, the influx of EGF is inhibited by an excess of EGF, showing the presence of a saturable transport system. It also is inhibited by an excess of TGF-a, but not by a monoclonal antibody against the EGF receptor. Moreover, the transport of EGF remains unchanged in mice with a spontaneous mutation of the EGF receptor w40x. By contrast, very little TGF-a enters the brain, most of it being tightly trapped in the capillary endothelial cells of the cerebral vasculature w44x. Unlike the situation with MIP w6x, the relation of TGF-a with the endothelial cells is not merely that of a loose association removed by washout; most of the TGF-a is not removed by the intracardiac perfusion technique which clears the cerebral vascular space. Thus, even though EGF and TGF-a bind to the same receptor, they do not cross the BBB in the same way. 4.2. Tyr–MIF-1 Õs. Met-enkephalin Tyr–MIF-1 and Met-enkephalin are transported out of the brain by the same efflux system, peptide transport system ŽPTS.-1 w13x. The transporter is down-regulated in alcohol addiction but returns to normal levels of transport relatively quickly after cessation of alcohol ingestion w2x. The sharing of the same transport system by both Tyr– MIF-1 and Met-enkephalin, each with its own receptors, permits determination of whether the transporter is the same as either of the receptors for these peptides. The mere presence of binding of a peptide to a receptor at the endothelial cells of the BBB is certainly not sufficient to establish whether such a receptor provides transport. Recently we found that the saturable transport system for Tyr–MIF-1 and Met-enkephalin differs from the receptors for either peptide w31x.
5. Summary The BBB provides a regulatory system involved in control of communication between the CNS and the rest of the body. It is expected that additional unusual observations will contribute to an eventual comprehensive understanding of the full dynamic role of the BBB.
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Interactive report
Peptides crossing the blood–brain barrier: some unusual observations 1 Abba J. Kastin a
a, )
, Weihong Pan a , Lawrence M. Maness a , William A. Banks
b
VA Medical Center and Tulane UniÕersity School of Medicine, 1601 Perdido Street, New Orleans, LA 70112-1262 USA b GRECC, VA Medical Center-St. Louis, St. Louis UniÕersity School of Medicine, St. Louis, MO 63106, USA Accepted 12 August 1999
Abstract An interactive blood–brain barrier ŽBBB. helps regulate the passage of peptides from the periphery to the CNS and from the CNS to the periphery. Many peptides cross the BBB by simple diffusion, mainly explained by their lipophilicity and other physicochemical properties. Other peptides cross by saturable transport systems. The systems that transport peptides into or out of the CNS can be highly specific, transporting MIF-1 but not Tyr–MIF-1, PACAP38 but not PACAP27, IL-1 but not IL-2, and leptin but not the smaller ingestive peptides NPY, orexin A, orexin B, CART Ž55–102wMetŽO. 67 x., MCH, or AgRPŽ83–132.. Although the peptides EGF and TGF-a bind to the same receptor, only EGF enters by a rapid saturable transport system, suggesting that receptors and transporters can represent different proteins. Even the polypeptide NGF enters faster than its much smaller subunit b-NGF. The saturable transport of some compounds can be upregulated, like TNF-a in EAE Žan animal model of multiple sclerosis. and after spinal cord injury, emphasizing the regulatory role of the BBB. As has been shown for CRH, saturable transport from brain to blood can exert effects in the periphery. Thus, the BBB plays a dynamic role in the communication of peptides between the periphery and the CNS. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Peptide; Blood–brain barrier; Saturable transport; Diffusion
1. Introduction
2. Size can be relatively unimportant
The millennium is a time for celebration in the neuropeptide field. It has seen the acceptance of several concepts that had long been subject to negative unsubstantiated dogmas. No longer can there be any doubt about the ability of numerous peptides in the periphery to affect the central nervous system ŽCNS. w25,52x or the ability of many of them to cross the blood–brain barrier ŽBBB. w17x so as to be able to exert these effects directly. Similarly, peptides from the CNS can cross the BBB to exert effects in the periphery. The last reviews we published concerning the passage of peptides and polypeptides Žincluding most neurotrophins and cytokines. across the BBB were intended to be more general and comprehensive w5,41x. In the present review, we have selectively chosen a few unusual observations to illustrate some novel concepts in this area.
The aphorism that ‘‘size doesn’t matter’’ may apply to the BBB when the relatively narrow range encompassing peptides and polypeptides is considered. Although an early study found that the molecular weight of peptides does not correlate with their rate of passage across the BBB w1x, the idea still seems somewhat counterintuitive. The relative unimportance of size is most obvious for peptides and polypeptides with saturable transport systems, as the following examples indicate.
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Corresponding author. Tel.: q1-504-568-0811X5884; Fax: q1-504522-8559. 1 Published on the World Wide Web on 24 August 1999.
2.1. Brain-to-blood transport 2.1.1. Tyr–MIF-1 Õs. MIF-1 The same study w1x showing that molecular weight is not a good predictor of the passage of peptides across the BBB also found that lipophilicity is a much better one. An exception to this occurred with low molecular weight peptides with an N-terminal tyrosine ŽTyr. that entered the brain much slower than predicted. Among the possible explanations, a brain-to-blood transport system seemed likely. Using Tyr–MIF-1 ŽTyr–Pro–Leu–Gly–NH 2 . as
0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 9 . 0 1 9 6 1 - 7
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the prototype, we found the existence of saturable systems for the transport of peptides out of the brain. It is easy to understand, therefore, how a brain-to-blood efflux system could obscure determination of blood-to-brain entry when brain uptake after peripheral injection is examined, regardless of whether immunoreactivity or radioactivity is measured. Determination of only immunoreactivity involves the additional problem of separation of endogenous from exogenous material, although this can be resolved with species-specific immunoassays w7x. The transport system for Tyr–MIF-1 is stereospecific. Change of the L-Tyr to its D-form, or the removal of even a single hydroxyl group from the Tyr Žresulting in Phe., prevents saturable transport w14x. Removal of the entire N-terminal Tyr, resulting in the tripeptide MIF-1, also prevents saturable transport out of the brain w4x. MIF-1 was the first endogenous compound shown to exert antiopiate effects w27x. Tyr–MIF-1 shares this property w18x, but in some situations it can act as an opiate agonist w55x. The endogenous opiate Met-enkephalin is a pentapeptide with only the N-terminal Tyr in common with Tyr–MIF-1. Their functional interactions may help explain the unusual observation that the stereospecific system for Tyr–MIF-1 also transports Met-enkephalin w13x, a finding with implications for alcoholism w47x. 2.1.2. CRH Corticotropin-releasing hormone ŽCRH. is more than ten times larger than MIF-1. Although the tripeptide MIF-1 is not transported out of the brain, there is a well characterized saturable efflux system for CRH w32,34x. Moreover, the brain-to-blood transport of CRH is sufficient to exert effects in the periphery w33x. The concept that brain-toblood transport can result in peripheral effects seems more unusual than the concept that blood-to-brain transport can result in central effects. 2.2. Blood-to-brain transport 2.2.1. MIF-1 MIF-1 is saturably transported into the CNS but Tyr– MIF-1 is not w4x. The rate of entry is much faster for MIF-1 than for morphine. Although MIF-1 is remarkably stable in human blood, it is not formed there from Tyr– MIF-1 w26x. Thus, Tyr–MIF-1 but not MIF-1 is saturably transported from brain-to-blood whereas MIF-1 but not Tyr–MIF-1 is saturably transported from blood-to-brain. 2.2.2. Leptin Õs. ingestiÕe peptides Leptin is saturably transported into but not out of the brain w11x. The localization of leptin in the arcuate nucleus after iv administration is not found after icv administration w29x. Since the arcuate appears to be the major site of action of leptin in the control of appetite, it might seem unusual that injection in blood provides a more efficient
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path than is achieved by injection into the cerebrospinal fluid. This directed delivery of leptin from blood to a discrete area of the brain emphasizes the regulatory potential of the BBB. Leptin is a polypeptide Žmore than 100 amino acids.. Many other ingestive substances are peptides Žfewer than 100 amino acids., and most of them also enter the brain. Unlike leptin, however, some do not enter through saturable influx systems even though they apparently interact with leptin. We could not find saturable transport for neuropeptide Y w22x, orexin A, orexin B w23x, cocaine- and amphetamine-regulated transcript ŽCART . Ž55–102 wMetŽO. 67 x. w21x, melanin-concentrating hormone ŽMCH., or agouti-related protein ŽAgRP.Ž83–132. w24x; moreover, none of these feeding-related peptides appear to affect the saturable entry of leptin. 2.2.3. IL-1 Õs. IL-2 Interleukin ŽIL.-1a is transported from blood to brain by a saturable system that also transports IL-1b and IL-1 receptor antagonist ŽIL-1ra. but not tumor necrosis factor-a ŽTNF-a ., IL-2, IL-6, or macrophage inflammatory protein ŽMIP.-1a w15,20x. Of these cytokines, however, only IL-1a has been shown to be saturably concentrated in the posterior division of the septum w28x. IL-1a also is transported into the spinal cord by a saturable system w9x. Although the circumventricular organs ŽCVOs. have a surface area of only 0.02 cm2rg brain as compared with 100–150 cm2rg for the BBB w53x, the misconception persists that most of the penetration of peptides and polypeptides across the BBB occurs through the CVOs. However, less than 5% of the IL-1a injected into blood enters the CVOs w30,46x, and this material is restricted from diffusion into the rest of the brain by the outer layer of cells comprising the CVOs w17,53x. The BBB is not disrupted in such studies w3x. The transport of IL-1a across the BBB is self-inhibited by a dose as low as 2.5 mgrkg w15x. Yet at a dose 20 times higher, there is no self-inhibition Žsaturation. of the passage of IL-2 w54x. As with many of the other unusual observations discussed in this review, it would have been impossible with the present state of knowledge to predict these findings, raising obvious questions about the present criteria for obtaining funding. 2.2.4. IL-6 Õs. other cytokines Essential for studies of the passage of substances across the BBB is determination of the integrity of the material reaching the brain. Most measurements involve homogenization of brain tissue with subsequent release of intracellular enzymes to which the entering substance is not initially exposed. HPLC is the most commonly used procedure, although SDS-PAGE also is suitable, especially for larger polypeptides. Even after correction for processing, apparently much less IL-6 reaches the brain in intact form w9x than has been found for the transport of other cytokines
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like IL-1a w15x, IL-1ra w20x, TNF-a w19x, interferon-g w37x, granulocyte–macrophage colony-stimulating factor ŽGMCSF. w35x, and ciliary neurotrophic factor ŽCNTF. w43x.
3. Saturable transport systems can be upregulated
3.1. EAE 2.2.5. MIP Õs. other cytokines Also essential for studies of the passage of substances across the BBB is determination that the material being measured in the brain actually reaches the parenchyma rather than being attached to the luminal wall of the capillaries comprising the BBB. For almost all the substances studied so far, the capillary depletion method has shown that most of the injected material reaches the brain parenchyma. However, for MIP-1a and MIP-1b, this technique based on differential centrifugation modified with a washout procedure shows that most of the injected MIP is reversibly associated with the endothelia of the BBB w6x. 2.2.6. b-NGF and NGF NGF is composed of five subunits. One of these subunits, b-NGF, is responsible for much of the bioactivity of NGF. Yet the smaller b-NGF crosses the BBB only half as fast as the parent NGF w38x. Using slightly different methods, Poduslo and co-workers also found significant penetration of the BBB by NGF, courageously commenting upon the prevalent ‘‘erroneous conclusions w...x that has plagued brain- and nerve-barrier research w...x that the barrier was impermeable to proteins’’ w48,49x.
Another unusual observation involves experimental autoimmune encephalomyelitis ŽEAE., considered an animal model of multiple sclerosis. In EAE and multiple sclerosis, the BBB is disrupted so that it is more permeable to many substances. Under such circumstances, it might be expected that saturable transport systems also would be disrupted. This was studied with TNF-a, a cytokine implicated in the pathogenesis of EAE, which can exert bimodal actions of either a beneficial or detrimental nature w45x. In the model of EAE used, disruption of the BBB, as reflected by an increased permeability to peripherally administered sucrose and albumin, coincides with the functional deficits of the mice w39x. The previously demonstrated w19x saturable transport of TNF-a, however, is enhanced. Although TNF-a circulates as a trimer of similar size to albumin, the amount of TNF-a entering the CNS is similar to that of the smaller sucrose, both entering twice as fast as albumin w39x. Moreover, the self-inhibition of transport intrinsic to the process of saturation remains intact; this would not be seen if TNF-a crossed the disrupted BBB by diffusion or leakage.
3.2. Spinal cord injury 2.2.7. PACAP38 and PACAP27 The two major members of the pituitary adenylate cyclase activating polypeptide ŽPACAP. family illustrate several unusual findings. First is the observation that there is a saturable transport system into the brain for PACAP38 but not for PACAP27, a peptide with 11 fewer amino acids. Second is the observation that despite the presence of a saturable transport system for PACAP38, a larger percentage of injected PACAP27 enters the brain, probably related to its greater lipophilicity, smaller volume of distribution, and longer half-time disappearance from blood. Third is the observation that there also is a saturable transport system out of the brain that prefers PACAP38 to PACAP27 w12x. The only other peptides for which saturable transport systems w17,51x in both directions have been described are LHRH w16x, arginine vasopressin w56,57x, and leucine enkephalin w10,58x. A few peptides, such as PACAP, can exert neurotrophic properties. The transport of PACAP38 across the BBB Žincluding the blood–spinal cord barrier. from blood to CNS is changed by spinal cord injury ŽSCI. w8x whereas that of the much smaller MSHrACTH analog ebiratide is not affected w36x. Soon after SCI, transport of PACAP38 is decreased, but later there is an upregulation not explained by direct disruption of the vascular barrier w8x.
3.2.1. Complete transection After SCI, permeability of the BBB to sucrose and albumin is increased, as it is in EAE. As also seen in EAE, complete transection of the spinal cord increases the permeability of the BBB to TNF-a w36x. This increase is selective. Even though ebiratide is smaller and has a saturable transport system across the BBB w50x, SCI does not change its rate of entry. By contrast, the saturable entry of TNF-a is enhanced. The increased permeability to TNF-a does not correlate with that to the markers sucrose and albumin w36x.
3.2.2. Partial disruption by compression There is marked dissociation of the permeation of TNFa and the vascular marker albumin during the second phase of BBB opening induced by spinal cord compression w42x. By comparison with albumin, the entry of TNF-a is of much greater magnitude, occurs earlier, lasts for a longer time, involves regions distal as well as proximal to the site of compression, and retains the self-inhibition characteristic of a saturable transport system. The increased entry of TNF-a from blood to the CNS in these conditions could be helpful in restricting secondary tissue damage and in aiding regeneration.
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4. Receptor binding need not reflect transport
Acknowledgements
4.1. EGF Õs. TGF-a
This work was supported by NIH ŽDK54880. and the VA.
Epidermal growth factor ŽEGF. and transforming growth factor a ŽTGF-a . are members of the EGF family of growth factors. As peptides of about 50 amino acids, they are much smaller than the classical neurotrophins. Although EGF and TGF-a bind to the same receptor, they cross the BBB in completely different ways. The rate of entry of EGF into brain is much faster than that for most neurotrophins, cytokines, or even bioactive peptides w40x. As expected for such fast entry, the influx of EGF is inhibited by an excess of EGF, showing the presence of a saturable transport system. It also is inhibited by an excess of TGF-a, but not by a monoclonal antibody against the EGF receptor. Moreover, the transport of EGF remains unchanged in mice with a spontaneous mutation of the EGF receptor w40x. By contrast, very little TGF-a enters the brain, most of it being tightly trapped in the capillary endothelial cells of the cerebral vasculature w44x. Unlike the situation with MIP w6x, the relation of TGF-a with the endothelial cells is not merely that of a loose association removed by washout; most of the TGF-a is not removed by the intracardiac perfusion technique which clears the cerebral vascular space. Thus, even though EGF and TGF-a bind to the same receptor, they do not cross the BBB in the same way. 4.2. Tyr–MIF-1 Õs. Met-enkephalin Tyr–MIF-1 and Met-enkephalin are transported out of the brain by the same efflux system, peptide transport system ŽPTS.-1 w13x. The transporter is down-regulated in alcohol addiction but returns to normal levels of transport relatively quickly after cessation of alcohol ingestion w2x. The sharing of the same transport system by both Tyr– MIF-1 and Met-enkephalin, each with its own receptors, permits determination of whether the transporter is the same as either of the receptors for these peptides. The mere presence of binding of a peptide to a receptor at the endothelial cells of the BBB is certainly not sufficient to establish whether such a receptor provides transport. Recently we found that the saturable transport system for Tyr–MIF-1 and Met-enkephalin differs from the receptors for either peptide w31x.
5. Summary The BBB provides a regulatory system involved in control of communication between the CNS and the rest of the body. It is expected that additional unusual observations will contribute to an eventual comprehensive understanding of the full dynamic role of the BBB.
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