Human blood-brain barrier receptors for Alzheimer's amyloid-beta 1- 40. Asymmetrical binding, endocytosis, and transcytosis at the apical side of brain microvascular endothelial cell monolayer.

A soluble monomeric form of Alzheimer's amyloid-beta (1-40) peptide (sAbeta1-40) is present in the circulation and could contribute to neurotoxicity if it crosses the brain capillary endothelium, which comprises the blood-brain barrier (BBB) in vivo. This study characterizes endothelial binding and transcytosis of a synthetic peptide homologous to human sAbeta1-40 using an in vitro model of human BBB. 125I-sAbeta1-40 binding to the brain microvascular endothelial cell monolayer was time dependent, polarized to the apical side, and saturable with high- and low-affinity dissociation constants of 7.8+/-1.2 and 52.8+/-6.2 nM, respectively. Binding of 125I-sAbeta1-40 was inhibited by anti-RAGE (receptor for advanced glycation end products) antibody (63%) and by acetylated low density lipoproteins (33%). Consistent with these data, transfected cultured cells overexpressing RAGE or macrophage scavenger receptor (SR), type A, displayed binding and internalization of 125I-sAbeta1-40. The internalized peptide remains intact > 94%. Transcytosis of 125I-sAbeta1-40 was time and temperature dependent, asymmetrical from the apical to basolateral side, saturable with a Michaelis constant of 45+/-9 nM, and partially sensitive to RAGE blockade (36%) but not to SR blockade. We conclude that RAGE and SR mediate binding of sAbeta1-40 at the apical side of human BBB, and that RAGE is also involved in sAbeta1-40 transcytosis.

Clearance of Alzheimer's amyloid-ss(1-40) peptide from brain by LDL receptor-related protein-1 at the blood-brain barrier.

Elimination of amyloid-ss peptide (Ass) from the brain is poorly understood. After intracerebral microinjections in young mice, (125)I-Ass(1-40) was rapidly removed from the brain (t(1/2)

Evidence for a lactate transport system in the sarcolemmal membrane of the perfused rabbit heart: kinetics of unidirectional influx, carrier specificity and effects of glucagon.

The kinetics and specificity of L-lactate transport into cardiac muscle were studied during a single transit through the isolated perfused rabbit heart using a rapid (15 s) paired-tracer dilution technique. Kinetic experiments revealed that lactate influx was highly stereospecific and saturable with an apparent Kt = 19 +/- 6 mM and a Vmax = 8.4 +/- 1.5 mumol/min per g (mean +/- S.E., n = 14 hearts). At high perfusate concentrations (10 mM), the inhibitors alpha-cyano-4-hydroxycinnamate (Ki = 7.3 mM), pyruvate (Ki = 6.5 mM), acetate (Ki = 19.4 mM) and chloroacetate (Ki = 28 mM) reduced L-lactate influx, and Ki values were estimated assuming a purely competitive interaction of the inhibitors with the monocarboxylate carrier. The monocarboxylic acids [14C]pyruvate and [3H]acetate were themselves transported, and sarcolemmal uptakes of respectively 38 +/- 1% and 70 +/- 8% were measured relative to D-mannitol. Perfusion of hearts for 10-30 min with 0.15 or 1.5 microM glucagon increased myocardial lactate production and simultaneously inhibited tracer uptake of lactate, pyruvate and acetate. It is concluded that a stereospecific lactate transporter exhibiting an affinity for other substituted monocarboxylic acids is operative in the sarcolemmal plasma membrane of the rabbit myocardium.

Kinetics of arginine-vasopressin uptake at the blood-brain barrier.

Uptake of arginine-vasopressin, VP, at the luminal side of the blood-brain barrier (BBB) was studied by means of an in situ brain perfusion technique in the guinea-pig. Kinetic experiments revealed a saturable peptide influx into the parietal cortex, caudate nucleus and hippocampus with Km between 2.1 and 2.7 microM, and Vmax ranging from 4.9 to 5.6 pmol.min-1.g-1. The non-saturable component, Kd, was not significantly different from zero. Influx of VP into the brain was not altered by the presence of the peptide fragments: VP-(1-8), pressinoic acid and [pGlu4,Cyt6]VP-(4-9) at 4.5 microM, nor yet by the aminopeptidase inhibitor, bestatin (0.5 mM) and the L-amino acid transport system substrates, L-tyrosine and L-phenylalanine at 5 mM. At a perfusate concentration of 4.5 microM, the V1-vasopressinergic receptor antagonist, d(CH2)5[Tyr(Me)2]VP, reduced VP influx; regional Ki values, assuming that the observed inhibitions were purely competitive, ranged between 4.7 and 8.5 microM. It is concluded that there is an apparent cerebrovascular permeability to circulating VP due to the presence of a carrier-mediated transport system for the peptide located at the luminal side. The mechanism for VP BBB uptake exhibits no affinity for peptide fragments and large neutral amino acids, but requires reception of the intact molecule, which may be the same initial step for both the BBB VP transporter and the V1-receptor.

Anti-inflammatory, antithrombotic, and neuroprotective effects of activated protein C in a murine model of focal ischemic stroke.

BACKGROUND: Activated protein C (APC) contributes to systemic anticoagulant and anti-inflammatory activities. APC may reduce organ damage by inhibiting thrombin generation and leukocyte activation. Neutrophils and cerebrovascular thrombosis contribute to ischemic neuronal injury, suggesting that APC may be a potential protective agent for stroke. METHODS AND RESULTS: We examined the effects of APC in a murine model of focal ischemia. After middle cerebral artery occlusion/reperfusion, the average survival time in controls was 13.6 hours. Animals that received purified human plasma-derived APC 2 mg/kg IV either 15 minutes before or 10 minutes after stroke induction survived 24 hours and were killed for neuropathological analysis. APC 2 mg/kg given before or after onset of ischemia restored cerebral blood flow, reduced brain infarct volume (59% to 69%; P:<0.003) and brain edema (50% to 61%; P:<0.05), eliminated brain infiltration with neutrophils, and reduced the number of fibrin-positive cerebral vessels by 57% (P:<0.05) and 25% (nonsignificant), respectively. The neuroprotective effect of APC was dose-dependent and associated with significant inhibition of ICAM-1 expression on ischemic cerebral blood vessels (eg, 61% inhibition with 2 mg/kg APC). Intracerebral bleeding was not observed with APC. CONCLUSIONS: APC exerts anti-inflammatory, antithrombotic, and neuroprotective effects in stroke. Central effects of APC are likely to be related to improved maintenance of the blood-brain barrier to neutrophils and to reduced microvascular obstructions and fibrin deposition.

Intravenous RMP-7 increases delivery of ganciclovir into rat brain tumors and enhances the effects of herpes simplex virus thymidine kinase gene therapy.

Herpes simplex virus thymidine kinase (HSV-tk) gene therapy for brain tumors depends on ganciclovir (GCV) and its transport across the blood-brain tumor barrier (BBTB). We examined whether RMP-7, the bradykinin analog and potent BBTB permeabilizer, could enhance the efficacy of GCV treatment of brain tumors by increasing the BBTB delivery of GCV. In vitro, a significant bystander cytocidal effect of GCV was shown in mixed HSV-tk-transduced (HSV-tk+) and control vector-transduced (HSV-tk-) C6 glioma cultures. A dose-dependent cytotoxic effect of GCV on untransformed C6 cells was also shown. In vivo, rats with 100% HSV-tk+ or 100% HSV-tk- intracerebral C6 gliomas were treated for 7 days with intravenous infusions of GCV alone or with GCV and RMP-7 (2.5 microg/kg/day). The growth of HSV-tk+ and HSV-tk- gliomas decreased with increasing doses of GCV. A high dosage (100 mg of GCV/kg/day) eradicated all HSV-tk- and HSV-tk+ tumors. An intermediate dosage (5 mg of GCV/kg/day) reduced the growth of HSV-tk- gliomas by 42% if given alone, and by 88% in combination with RMP-7. A low dosage (0.5 mg of GCV/kg/day) in combination with RMP-7 enhanced the regression of HSV-tk+ gliomas by 87% compared with GCV alone. Low-dose GCV was ineffective in HSV-tk- tumors. RMP-7 increased [3H] GCV tumoral uptake by 2.6- and 1.7-fold in the tumor center and periphery, respectively. We conclude that RMP-7 could be an important adjunctive treatment for suicide gene therapy of brain tumors, while an RMP-7/GCV combination may also have a significant antitumor effect in untransfected gliomas.

Differential regulation of leptin transport by the choroid plexus and blood-brain barrier and high affinity transport systems for entry into hypothalamus and across the blood-cerebrospinal fluid barrier.

Leptin is a circulating hormone that controls food intake and energy homeostasis. Little is known about leptin entry into the central nervous system (CNS). The blood-cerebrospinal fluid (CSF) barrier at the choroid plexus and the blood-brain barrier (BBB) at the cerebral endothelium are two major controlling sites for entry of circulating proteins into the brain. In the present study, we characterized leptin transport across the blood-CSF barrier and the BBB by using a brain perfusion model in lean rats. Rapid, high-affinity transport systems mediated leptin uptake by the hypothalamus (KM = 0.2 ng/ml) and across the blood-CSF barrier (KM = 1.1 ng/ml). High affinity in vivo binding of leptin was also detected in the choroid plexus (KD = 2.6 ng/ml). In contrast, low affinity carriers for leptin (KM = 88 to 345 ng/ml) were found at the BBB in the CNS regions outside the hypothalamus (e.g. cerebral cortex, caudate nucleus, hippocampus). Our findings suggest a key role of high affinity leptin transporters in the hypothalamus and choroid plexus in regulating leptin entry into the CNS and CSF under physiological conditions. Low affinity transporters at the BBB outside the hypothalamus could potentially contribute to overall neuropharmacological effects of exogenous leptin.

Circulating antibody against tumor necrosis factor-alpha protects rat brain from reperfusion injury.

The role of tumor necrosis factor-alpha (TNF alpha) in brain injury is controversial. We studied the effect of anti-TNF-alpha antibody in a rat model of reversible middle cerebral artery occlusion. During focal ischemia and early reperfusion, TNF-alpha was rapidly and transiently released into circulation. Pretreatment with intravenous anti-TNF-alpha antibody reduced cortical (71%, P < 0.015) and subcortical (58%, P < 0.007) injury, enhanced the cerebral blood flow during reperfusion, and improved the neurologic outcome. This further supports the contention that TNF-alpha is a deleterious cytokine in stroke, whereas circulating antibody against TNF-alpha may protect brain from reperfusion injury.

Chronic nicotine treatment enhances focal ischemic brain injury and depletes free pool of brain microvascular tissue plasminogen activator in rats.

Effects of nicotine treatment (4.5 mg/kg of nicotine-free base/day administered s.c. by osmotic minipumps for 14 days) on focal ischemic stroke and expression of tissue plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) in cerebral microvessels were studied in rats in vivo using a reversible (1 h) middle cerebral artery occlusion model. Plasma levels of nicotine and its major metabolite cotinine after 14 days of treatment were 88 and 364 ng/ml, respectively. Nicotine treatment resulted in 35-40% (p < 0.001) decrease in the blood flow in the periphery of the ischemic core during reperfusion, an increase in the neurologic score of 2.6-fold (p < 0.01), and 36% (p < 0.05) and 121% (p < 0.01) increases in the injury and edema volume in the pallium, respectively. A free pool of brain microvascular t-PA antigen was completely depleted by nicotine, while the expression of the PAI-1 antigen and/or PAI-1-t-PA complexes remained unchanged. The relative abundance of cerebromicrovascular t-PA mRNA transcript versus beta-actin mRNA transcript did not change with nicotine. It is concluded that chronic nicotine treatment impairs the restoration of blood flow, worsens the neurologic outcome, and enhances brain injury following an ischemic insult. These nicotine effects are associated with depletion of brain microvascular t-PA antigen.

Brain injury and cerebrovascular fibrin deposition correlate with reduced antithrombotic brain capillary functions in a hypertensive stroke model.

Hemostasis factors may influence the pathophysiology of stroke. The role of brain hemostasis in ischemic hypertensive brain injury is not known. We studied ischemic injury in spontaneously hypertensive rats in relation to cerebrovascular fibrin deposition and activity of different hemostasis factors in brain microcirculation. In spontaneously hypertensive rats subjected to transient middle cerebral artery occlusion versus normotensive Wistar-Kyoto (W-K) rats, infarct and edema volumes were increased by 6.1-fold (P < 0.001) and 5.8-fold (P < 0.001), respectively, the cerebral blood flow (CBF) reduced during middle cerebral artery occlusion (MCAO) by 55% (P < 0.01), motor neurologic score increased by 6.9-fold (P < 0.01), and cerebrovascular fibrin deposition increased by 6.8-fold (P < 0.01). Under basal conditions, brain capillary protein C activation and tissue plasminogen activator activity were reduced in spontaneously hypertensive rats compared with Wistar-Kyoto rats by 11.8-fold (P < 0.001) and 5.1-fold (P < 0.001), respectively, and the plasminogen activator inhibitor-1 antigen and tissue factor activity were increased by 154-fold (P < 0.00001) and 74% (P < 0.01), respectively. We suggest that hypertension reduces antithrombotic mechanisms in brain microcirculation, which may enhance cerebrovascular fibrin deposition and microvascular obstructions during transient focal cerebral ischemia, which results in greater neuronal injury.

Substitution at codon 22 reduces clearance of Alzheimer's amyloid-beta peptide from the cerebrospinal fluid and prevents its transport from the central nervous system into blood.

A point mutation of G to C at codon 693 of the amyloid-beta (Abeta) precursor protein gene results in Glu to Gln substitution at position 22 of the Abeta (AbetaQ22) associated with hereditary cerebrovascular amyloidosis with hemorrhage Dutch type. Factors that regulate AbetaQ22 levels in the central nervous system (CNS) are largely unknown. By using ventriculo-cisternal perfusion technique in guinea pigs, we demonstrated that clearance from the cerebrospinal fluid and transport from the CNS to blood of [(125)I]-AbetaQ22 (1 nM) were reduced by 36% and 52%, respectively, in comparison to the wild type Abeta(1-40) peptide. In contrast to significant uptake and transport of Abeta(1-40) across the brain capillaries and leptomeningeal vessels, AbetaQ22 was not taken up at these CNS vascular transport sites, which was associated with its 53% greater accumulation in the brain. The CNS clearance of Abeta(1-40) was half-saturated at 23.6 nM; AbetaQ22 had about 6.8-fold less affinity for the CNS efflux transporters and its elimination relied mainly on transport across the choroid plexus. Thus, the Dutch mutation impairs elimination of Abeta from brain by reducing its rapid transport across the blood-brain barrier and the vascular drainage pathways, which in turn may result in accumulation of the peptide around the blood vessels and in brain.

Endothelin-1 and monocyte chemoattractant protein-1 modulation in ischemia and human brain-derived endothelial cell cultures.

Brain tissue damage due to ischemia/reperfusion has been shown to be caused, in part, by activated macrophages infiltrating into the post-ischemic brain. Using the Middle Cerebral Artery Occlusion (MCAO) mouse model, this study demonstrated that, in vivo, both endothelin-1 (Et-1), a potent vasoconstrictor, and the macrophage chemokine, monocyte chemoattractant factor-1 (MCP-1) are induced in ischemia. Further studies, using human brain-derived endothelial cells (CNS-EC), showed that in vitro, Et-1 can directly stimulate MCP-1 mRNA expression and MCP-1 protein; and this Et-1-induced MCP-1 production is mediated by the ET(A) receptor. Inflammatory cytokines, tumor necrosis factor alpha and interleukin-1beta, functioned additively and synergistically, respectively, with Et-1 to increase this MCP-1 production. Partial elucidation of the signal transduction pathways involved in Et-1-induced MCP-1 production demonstrated that protein kinase C-, but not cAMP-dependent pathways are involved. These data demonstrate that Et-1, functioning as an inflammatory peptide, increased levels of MCP-1, suggesting a mechanism for chemokine regulation during ischemia/reperfusion injury.

Molecular characterization of a reduced glutathione transporter in the lens.

PURPOSE: To characterize glutathione (GSH) transporter in the lens. METHODS: Poly (A) +RNA isolated from bovine lens was injected into Xenopus laevis oocytes. Oocytes were incubated for 1 hour in either NaCl or sucrose medium containing tracer GSH, and cell-associated radioactivity was determined. Glutathione efflux was determined in lens mRNA injected oocytes preloaded with GSH. Relationship of lens GSH transporter to the two recently cloned sodium-independent hepatic membrane GSH transporters was studied by Northern blot and reverse transcription-polymerase chain reaction (RT-PCR) analyses. Bovine lens mRNA also was probed for gamma glutamyl transpeptidase (GGT) by RT-PCR. RESULTS: Uptake of tracer 35S-GSH could be demonstrated in X. laevis oocytes injected with poly (A) +RNA from bovine lens. Glutathione transport was carrier mediated (Km approximately 1.3 mM) and was sodium independent. High-performance liquid chromatography confirmed that the molecular form of uptake was predominantly (> 98%) as it was for GSH. Poly (A) +RNA-injected oocytes preloaded with 16.5 nmol GSH-oocyte showed GSH efflux at a rate of 2.6 nmol/oocyte per hour. When bovine lens poly (A) +RNA was hybridized with the cDNA probe for the sodium-independent rat canalicular GSH transporter (RcGshT), the transcript for RcGshT was observed. RT-PCR confirmed the presence of RcGshT and showed the absence of rat sinusoidal GSH transporter (RsGshT) and GGT mRNA in rat lens. CONCLUSIONS: The authors have demonstrated for the first time that lens contains mRNA for RcGshT and expresses a low-affinity GSH transporter in oocytes. Glutathione efflux from the apical side of the anterior epithelium and progressive uptake, and inward efflux into cortical fibers, might be explained by expression of RcGshT alone or in combination with as yet unidentified GSH transporters.

RMP-7, a bradykinin agonist, increases permeability of blood-ocular barriers in the guinea pig.

PURPOSE: To determine if the bradykinin agonist, RMP-7, could increase the ocular tissue concentration of agents that normally have limited access across the blood-ocular barriers. The extracellular space marker, sucrose, and the anti-viral drug, ganciclovir, were tested. METHODS: Using the perfused eye method in guinea pigs, RMP-7 (1 microgram/kg over 5 minutes) or saline were administered intraarterially into the ocular circulation before either radiolabeled sucrose or ganciclovir (0.4 to 0.6 microCi/ml per minute). At time intervals ranging from 0.25 minute to 10 minutes, perfused eyes were removed, and the radioactivity within various compartments was measured using liquid scintillography. RESULTS: Pretreatment with RMP-7 significantly increased uptake of both sucrose (up to 4.5-fold) and ganciclovir (up to 2-fold) into the guinea pig retina and lens. Smaller and less consistent effects were observed in other eye compartments. CONCLUSIONS: This report demonstrates that RMP-7 enhances the permeability of blood-ocular barriers, and it provides the first pharmacologic evidence for a means to enhance the concentration of ganciclovir into the retina. Thus, these data support the concept that RMP-7 may prove to be a useful adjunct for enhanced delivery of therapeutics to the eye under conditions in which ocular barriers limit treatment.

Identification of a novel, sodium-dependent, reduced glutathione transporter in the rat lens epithelium.

PURPOSE: To determine whether glutathione (GSH) transporter(s) other than the previously identified rat canalicular GSH transporter (RcGshT) is present in the lens. METHODS: Poly (A) +RNA isolated from rat and guinea pig lens cortex and epithelium was injected into Xenopus laevis oocytes. The effect of sodium removal was determined by measuring cell-associated radioactivity in lenticular epithelium or cortex mRNA injected oocytes (pretreated with acivicin to inhibit gamma glutamyltranspeptidase) after 1 hour of incubation in NaCl medium or choline chloride (Na(+)-free) medium containing tracer GSH (plus unlabeled GSH). The effect of 2 mM bromosulfophthalein-GSH (BSP-GSH) on GSH uptake in the lens epithelium and cortex in NaCl medium at two GSH concentrations also was determined. The molecular form of uptake of GSH in lens epithelial mRNA-injected oocytes was examined by high-performance liquid chromatography. Western blot analysis was performed to study the presence of RcGshT in the cortex and epithelium. RESULTS: Oocytes injected with mRNA from rat and guinea pig lens epithelium and cortex compartments expressed GSH transport. High-performance liquid chromatography confirmed that epithelial uptake was as intact GSH under conditions of inhibition of GSH synthesis with dl-buthionine sulfoximine. The mean GSH uptake (nmol/oocyte per hour) in epithelial mRNA-injected oocytes was significantly reduced (P < 0.01, n = 4 oocyte preparations) under Na(+)-free conditions compared to NaCl medium at 0.05 mM and 2 mM GSH in the medium. Uptake in cortical mRNA-injected oocytes was unaffected by Na+ removal. Lens epithelial uptake exhibited a strong inhibition by BSP-GSH at 0.05 mM (55%) and 2 mM (64%), whereas cortical uptake was unaffected by BSP-GSH. Western blot analysis identified RcGshT in the cortical and epithelial regions. CONCLUSIONS: Results from the current study provide strong evidence for the presence of a hitherto unreported Na(+)-dependent, BSP-GSH inhibitable GSH transporter in the lens epithelium, which may mediate concentrative, basolateral uptake of aqueous GSH consistent with in situ eye perfusion studies. The Na(+)-independent, BSP-GSH insensitive RcGshT may function as an apical GSH effluxer in lens epithelium and in mediating concentration gradient driven inward GSH movement by uptake-efflux in the lens cortex.

Galactose-induced cataract formation in guinea pigs: morphologic changes and accumulation of galactitol.

PURPOSE: To develop and characterize a new model of galactose-induced cataract formation in young, 3- to 4-week-old Hartley guinea pigs. METHODS: Experimental animals were fed 50% galactose in powdered guinea pig chow containing 0.5 g ascorbate/kg diet. Control animals were fed normal powdered guinea pig chow (0.5 g ascorbate/kg diet). Lenses from all animals were subjected to photo-slit-lamp examination, light microscopic analysis, and high-pressure liquid chromatography (HPLC) analysis of polyol content. RESULTS: Photo-slit-lamp examination indicated initial opacities in equatorial subcapsular region between 3 and 5 days in all galactose-fed animals (20/20); opacities progressed toward the anterior pole when diet was extended to 14 days. Histologic analysis of the equatorial changes confirmed progressive cataract formation consisting of small intra-fibrillar vacuoles in the pre-equatorial region (3 days), an increased number of enlarged and coalesced vacuoles (6 days), and progressive tissue swellings with cellular disruption and signs of epithelial multilayering (14 days). The anterior epithelium showed increased cell height and swelling after 3 days of the galactose diet. HPLC analysis of lens tissue indicated progressive accumulation of galactitol, 18 mM after 3 days, which plateaued to about 30 mM between 6 and 14 days. The level of myo-inositol dropped from a control value of 2.8 +/- 0.7 mM to 1.5 +/- 0.7 mM after 3 days, and was nearly undetectable after 14 days of the galactose diet. CONCLUSIONS: The current study suggests that the guinea pig model may serve as a valuable new tool to study sugar-induced cataract formation and to characterize the early morphologic and biochemical events in cataractogenesis.

Passage of delta sleep-inducing peptide (DSIP) across the blood-cerebrospinal fluid barrier.

Unidirectional flux of 125I-labeled DSIP at the blood-tissue interface of the blood-cerebrospinal fluid (CSF) barrier was studied in the perfused in situ choroid plexuses of the lateral ventricles of the sheep. Arterio-venous loss of 125I-radioactivity suggested a low-to-moderate permeability of the choroid epithelium to the intact peptide from the blood side. A saturable mechanism with Michaelis-Menten type kinetics with high affinity and very low capacity (approximate values: Kt = 5.0 +/- 0.4 nM; Vmax = 272 +/- 10 fmol.min-1) was demonstrated at the blood-tissue interface of the choroid plexus. The clearance of DSIP from the ventricles during ventriculo-cisternal perfusion in the rabbit indicated no significant flux of the intact peptide out of the CSF. The results suggest that DSIP crosses the blood-CSF barrier, while the system lacks the specific mechanisms for removal from the CSF found with most, if not all, amino acids and several peptides.

Saturable mechanism for delta sleep-inducing peptide (DSIP) at the blood-brain barrier of the vascularly perfused guinea pig brain.

Cellular uptake of [125I] labelled DSIP at the luminal interface of the blood-brain barrier (BBB) was studied in the ipsilateral perfused in situ guinea pig forebrain. Regional unidirectional transfer constants (Kin) calculated from the multiple-time brain uptake analysis were 0.93, 1.33 and 1.66 microliter.min-1 g-1 for the parietal cortex, caudate nucleus and hippocampus, respectively. In the presence of 7 microM unlabelled DSIP the brain uptake of [125I]-DSIP (0.3 nM) was inhibited, the values of Kin being reduced to 0.23-0.38 microliter.min-1 g-1, values that were comparable with the Kin for mannitol. The rapidly equilibrating space of brain, measured from the intercept of the line describing brain uptake versus time on the brain uptake ordinate, Vi, was greater for [125I]-DSIP than for mannitol; in the presence of unlabelled DSIP this was reduced to that of mannitol, and it was suggested that the larger volume for [125I]-DSIP represented binding at specific sites on the brain capillary membrane. L-tryptophan, the N-terminal residue of DSIP, in concentrations of 7 microM and 1 mM, inhibited Kin without affecting Vi. A moderate inhibition of Kin was obtained by vasopressin ([Arg8]-VP), but only at a concentration as high as 0.2 mM. The results suggest the presence of a high affinity saturable mechanism for transport of DSIP across the blood-brain barrier, with subsequent uptake at brain sites that are highly sensitive to L-tryptophan, and may be modulated by [Arg8]-VP.

Unidirectional uptake of enkephalins at the blood-tissue interface of the blood-cerebrospinal fluid barrier: a saturable mechanism.

The cellular uptake at the blood-tissue interface of the blood-cerebrospinal fluid (CSF) barrier to tyrosyl-3,5-[3H]enkephalin-[5-L-leucine] (abbreviated to Leu-enkephalin) and of its synthetic analogue D-alanine2-tyrosyl-3,5-[3H]enkephalin-[5-D-leucine] (abbreviated to D-Ala2-D-Leu5-enkephalin) was studied in the isolated perfused choroid plexuses from the lateral ventricles of the sheep, using the rapid (less than 30 s), single circulation, paired-tracer dilution technique, in which D-[14C]-mannitol serves as an extracellular marker. Cellular uptake of peptides was estimated by directly comparing venous dilution profiles of [3H] and [14C] radioactivities in the absence and presence of unlabelled peptide, the N-terminal amino acid (L-tyrosine), the typical L-transport system substrate, 2-aminobicyclo(2,2,1)heptane-2-carboxylic acid (BCH) and the inhibitor of aminopeptidase activity, bacitracin. The cellular uptake of both enkephalins was strongly (65-76%) but not completely inhibited by the addition of 5 mM unlabelled peptide to the bolus; the self-inhibition was significantly higher for D-Ala2-D-Leu5-enkephalin than for Leu-enkephalin. The addition to the bolus of L-tyrosine (5 mM), BCH (10 mM) or bacitracin (2 mM) reduced the 3H-radioactivity uptake by the choroid plexus of both enkephalins by 20-40%, the degree of inhibition being greater for [3H]-Leu-enkephalin than for its analogue. It is concluded that during single passage of enkephalins through the choroid plexus circulation, unidirectional uptake at the blood-tissue interface of the blood-CSF barrier consists of two components; a saturable component, which represents uptake of the intact peptide by the choroid epithelium, and a non-saturable component, which reflects enzymatic degradation of peptide in the blood and/or at the barrier, with a liberation of the N-terminal tyrosyl residue. Higher penetration of the blood-CSF barrier by D-Ala2-D-Leu5-enkephalin can be attributed to its greater resistance to hydrolysis.