Deltorphin Transport Across the Blood–Brain Barrier

Proc. Natl. Acad. Sci. USA Vol. 94, pp. 9469–9474, August 1997 Pharmacology

Deltorphin transport across the blood–brain barrier

ANNA FIORI*†,PATRIZIA CARDELLI‡,LUCIA NEGRI§,MARIA ROSARIA SAVI*, ROBERTO STROM‡, AND VITTORIO ERSPAMER§

*Department of Biochemical Sciences ‘‘A. Rossi Fanelli’’, ‡Department of Cellular Biotechnology and Hematology, and §Institute of Medical Pharmacology, University ‘‘La Sapienza’’, 00185 Rome, Italy

Contributed by Vittorio Erspamer, June 30, 1997

ABSTRACT In vivo antinociception studies demonstrate PTS-1, -2, -3, and -4, could accelerate the entrance of opioids that deltorphins are opioid peptides with an unusually high and related peptides into the brain. More recently, the use of blood–brain barrier penetration rate. In vitro, isolated bovine different in vitro models consisting either of fresh isolated brain microvessels can take up deltorphins through a satu- brain microvessels or of cultured brain endothelial cells has rable nonconcentrative permeation system, which is appar- allowed a better investigation of the modality of transport of ently distinct from previously described systems involved in some opioid peptides such as Met-enkephalin and related the transport of neutral amino acids or of enkephalins. analogs (25–27). Deltorphins are naturally occurring peptides Removing Na؉ ions from the incubation medium decreases with high affinity and selectivity for ␦-opioid receptors. These the carrier affinity for deltorphins (؊25%), but does not affect opioids have a D-amino acid residue in position 2 of their the Vmax value of the transport. The nonselective opiate sequence and an amide residue at C terminus that protect them antagonist naloxone inhibits deltorphin uptake by brain mi- from enzymatic hydrolysis (28). In this study, we show that, crovessels, but neither the selective ␦-opioid antagonist nal- when injected intravenously in mice, deltorphins enter BBB to trindole nor a number of opioid peptides with different produce antinociception. Moreover, using isolated bovine affinities for ␦-or␮-opioid receptors compete with deltor- brain microvessels as an in vitro model of the BBB, we phins for the transport. Binding studies demonstrate that ␮-, investigate the presence of transport system(s) for deltorphins ␦-, and ␬-opioid receptors are undetectable in the microvessel in the plasma membranes of the endothelial cells and whether preparation. Preloading of the microvessels with L-glutamine and how such system(s) are susceptible to regulation, in results in a transient stimulation of deltorphin uptake. Glu- particular at the metabolic level. tamine-accelerated deltorphin uptake correlates to the rate of glutamine efflux from the microvessels and is abolished by MATERIALS AND METHODS naloxone. Chemicals. Tyr-ala-Phe-Asp-Val-Val-Gly-NH2 (deltorphin The blood–brain barrier (BBB) accounts for the restricted I; ala, D-Ala), Tyr-ala-Phe-Glu-Val-Val-Gly-NH2 (deltorphin movement of solutes between the vascular and the cerebral II), and Tyr-ala-Gly-NMePhe-glycinol (DAMGO) were ob- compartments. Its anatomical counterpart has been identified tained from Bachem. Tyr-D-Pen-Gly-Phe-D-Pen (DPEDPE), as the endothelial cell membranes of brain microvessels (1–5). Tyr-ala-Phe-Gly-Tyr-Pro-Ser-NH2 (dermorphin), Tyr-Gly- These endothelial cells, joined together by tight junctions (6), Gly-Phe-Leu-OH (Leu-enkephalin), Tyr-Gly-Gly-Phe- form a continuous physical barrier playing a crucial role in Met-OH (Met-enkephalin), Hepes, naloxone, and naltrindole determining the rate at which different compounds can reach, were from Sigma); morphine HCl was from Salars (Como, or in turn leave, the central nervous system (CNS). The Italy); carboxyfluorescein diacetate was from Molecular capacity of any particular molecule to penetrate the BBB Probes. [Lys7]dermorphin was synthesized as indicated by depends essentially on its free diffusion across the cell walls— Negri and coworkers (28). All other chemicals were obtained i.e., on its lipid solubility—or on its specific affinity for a from Merck or Fluka. [Tyrosyl-3,5-3H]Leu-enkephalin (37 carrier-mediated transport system. The use of isolated brain Ci͞mmol; 1 Ci ϭ 37 GBq), [tyrosyl-3,5-3H]deltorphin I (35 microvessels, as an in vitro model of the BBB, has allowed the Ci͞mmol), [tyrosyl-3,5-3H]deltorphin II (54.5 Ci͞mmol), [ty- identification and characterization of some peculiarities of the rosyl-3,5-3H]DAMGO (37 Ci͞mmol), [phenyl-3,4-3H]U-69593 transport systems specific for sugars (7, 8), amino acids (9–13), {(ϩ)-(5␣,7␣,8␤)-N-methyl-N[7-(1-pyrrolidinyl)-1-oxaspiro- electrolytes (14, 15), or transferrin (16). It had been initially (4,5)dec-8-yl]-benzeneacetamide} (47 Ci͞mmol), L-[3,4- assumed that peptides could not enter the CNS via the BBB 3H(N)]glutamine (60 Ci͞mmol), L-[U-14C]leucine (300 mCi͞ (17) and that the entry rate into the CNS of some peptides mmol), L-[U-14C]tyrosine (450 mCi͞mmol), [U-14C]sucrose correlated only with their lipid solubility, suggesting that they (4.63 mCi͞mmol), and Aquasol-2 were obtained from New crossed the BBB by direct diffusion through the phospholipid England Nuclear. bilayer of the endothelial cell membranes. It is now generally Intracerebroventricular (i.c.v.) and Intravenous (i.v.) In- accepted, instead, that different categories of peptides can jections. Male C57BL6 mice weighing 25–28 g (Charles River enter or exit the CNS through the endothelial cell membranes Breeding Laboratories) were housed singly in 20 ϫ 25 cm cages of brain microvessels at a rate higher than that accounted by placed in a thermostatically controlled cabinet at an environ- passive diffusion. In vivo studies have indicated the existence mental temperature of 21°C. Under light diethyl ether anes- of transport systems that allow the selective permeation of the thesia drug solution or vehicle were delivered into the lateral BBB by different peptides, either endogenous or synthetic cerebral ventricle (i.c.v.) by using a modification of the method (18–24). Some years ago, Banks and Kastin (21) have sug- of Haley and McCormick (29). An incision was made in the gested that at least four different transport systems, named Abbreviations: ala, D-Ala; BBB, blood-brain barrier; BBB-PI, BBB The publication costs of this article were defrayed in part by page charge permeability index; DAMGO, Tyr-ala-Gly-NMePhe-glycinol; DPEDPE, Tyr-D-Pen-Gly-Phe-D-Pen; i.c.v., intracerebroventricular; payment. This article must therefore be hereby marked ‘‘advertisement’’ in i.v., intravenous; U-69593, (ϩ)-(5␣,7␣,8␤)-N-methyl-N[7-(1- accordance with 18 U.S.C. §1734 solely to indicate this fact. pyrrolidinyl)-1-oxaspiro(4,5)dec-8-yl]-benzeneacetamide. © 1997 by The National Academy of Sciences 0027-8424͞97͞949469-6$2.00͞0 *To whom reprint requests should be addressed. e-mail: strom@ PNAS is available online at http:͞͞www.pnas.org. dbu.roma1.it.

9469 Downloaded by guest on October 4, 2021 9470 Pharmacology: Fiori et al. Proc. Natl. Acad. Sci. USA 94 (1997)

scalp and the bregma was located. Injections were made Table 1. BBB-PI of deltorphins compared to morphine and other directly through the skull at a point 2 mm caudal and 2 mm opioid peptides lateral to bregma at a depth of 3 mm using a Hamilton i.v. ED50, i.c.v. ED50, BBB-PI microliter syringe with a 26-gauge needle. All i.c.v. injections Compound nmol nmol ϫ 10Ϫ4 were made in a volume of 5 ␮l. For i.v. injections the mouse tail was immersed in moderately hot water (about 40°C) for Deltorphin I 321.0 0.38 12 1–2 min to induce venous dilatation and the injections carried Deltorphin II 364.0 0.27 9 out using a 500 ␮l Hamilton syringe with a 25-gauge needle. DAMGO 315.0 0.04 1 Test of Antinociception. Antinociception responses were Dermorphin 64.0 0.03 3 7 determined by immerging the mouse tail into hot water (55°C). [Lys ]dermorphin 7.4 0.04 28 The latency of the first sign of a rapid tail-flick was taken as Morphine 130.0 18.13 1980 the end point, as indicated by Janssen et al. (30). Before drug For calculation of BB-PI, see text. administration, mice not flicking their tails within 5 sec from hot water immersion were eliminated from the study. After in triplicate by omitting the microvessels, was constantly below drug injection, animals not flicking their tails within 15 sec 100 dpm. were removed from the nociceptive stimulus and assigned a By plotting the initial rate of uptake, v°, as a function of maximal antinociceptive response. The test was repeated every peptide concentration, a typical saturation kinetics pattern was 15 min after i.c.v. or i.v. drug injection and time course found, often superimposed to a diffusional component that antinociception curves were drawn. Peak response (PR), area could, however, be easily subtracted (11, 33). Nonlinear re- under the curve (AUC), and PR͞AUC ratios were calculated gression analysis, performed on the v° vs. v°͞[S] ‘‘Eadie– for the dose producing 50% of the maximum response (ED50). Hofstee plot,’’ which allows the use of simple statistical Five mice were tested for each opioid dose. procedures (34), was used to obtain the optimal estimates of BBB Permeability Index. The values of i.v. and i.c.v. ED50 the kinetic parameters of the transport system(s) and to were multiplied by the respective PR͞AUC ratio to normalize evaluate the standard error impending on them. Because them for degradation and elimination processes, and the BBB labeled sucrose had been shown not to permeate the endo- permeability index (BBB-PI) was calculated as the ratio be- thelial cells (9), the intracellular water space could be evalu- tween i.c.v.͞i.v. normalized ED50 values. Compounds that ated by a procedure similar to that suggested by Betz et al. more easily enter BBB have therefore higher BBB-PI values. (35)—i.e., as the difference between the wet and dry weights Isolation of Microvessels. Fresh bovine brain, obtained from of a microvessels sample collected on a filter minus the sucrose the local slaughterhouse, was transported on ice to the labo- water space. ratory, where meninges were removed. The capillaries were Opioid Binding Sites in Brain Microvessels. [3H]Deltorphin isolated from brain cortex as described by Cangiano et al. (9). I binding experiments were performed on membrane prepa- These microvessels have been shown, by scanning electron rations obtained from homogenates of brain microvessels. microscopy and phase contrast light microscopy, to be prac- Briefly, brain microvessels prepared as described above were tically free from contamination by glial or nerve cells (11). The centrifuged at 40,000 ϫ g and the pellets homogenized for 3 preparations appeared to consist essentially of branching min in Tris⅐HCl buffer (50 mM, pH 7.4) with a Polytron PT microvessel segments enveloped in their basement membrane. 3000 homogenizer (Kinematica, Littau-Luzern, Switzerland). Their ATP content, measured by a luciferin–luciferase assay, The homogenate was centrifuged at 1,000 ϫ g to eliminate was around 643 Ϯ 27 pmol͞mg protein. Passive cell perme- nuclei and debris, and supernatant was collected and centri- ability, tested by measuring at different time intervals the fuged at 40,000 ϫ g. Pellets were washed three times with efflux of carboxyfluorescein from cells previously loaded with buffer and finally resuspended in the buffer at a protein carboxyfluorescein diacetate (31), was negligible; also negli- concentration equal to that of the original microvessel prep- gible was the uptake of labeled sucrose. As compared with the brain gray matter, the microvessels were found to be 10- to 15-fold enriched in some typical enzymatic activities such as ␥-glutamyltranspeptidase and alkaline phosphatase (9). Their contamination with glia, estimated through the immunochemi- cal determination of glial fibrillar acidic protein, was below 3% on a protein basis (11). Uptake Measurements. The initial rate of uptake of labeled peptides by the isolated brain microvessels was measured within the first minute after addition as a function of peptide concentration. Alternatively, the uptake was measured cumu- latively, using a fixed peptide concentration, after increasing time intervals (up to 30 min). For actual measurements, 0.6-ml aliquots of the microvessels suspension, after incubation with the labeled substrate in a phosphate–bicarbonate–Hepes iso- tonic buffer (pH 7.4) (choline being, if required, substituted to Naϩ ions), were poured on a 44-␮m pore nylon sieve on a Millipore vacuum manifold and washed three times with 5 ml of the appropriate cold buffer (9). The sieves with the retained microvessels were then placed in disposable tubes containing 1.8 ml of 1 M NaOH, left overnight at room temperature, and then sonicated for 1 min. Portions were taken for protein determination according to Lowry et al. (32), and 0.5-ml FIG. 1. Effect of temperature on the uptake of [3H]deltorphin I by aliquots were transferred to liquid scintillation vials containing isolated brain microvessels. After standing for 10 min either at 37°C an equal volume of 1 M HCl. After addition of 8 ml of (E)orat4°C(F), the labeled deltorphin I was added and the time Aquasol-2, the vials were counted in a Beckman 9800 liquid course uptake was followed for 30 min. The data in the figure represent scintillation spectrometer. Nonspecific radioactivity, due to the average values Ϯ SD obtained from three different experiments, the binding of labeled peptide to the nylon sieve, when tested each performed in triplicate. Downloaded by guest on October 4, 2021 Pharmacology: Fiori et al. Proc. Natl. Acad. Sci. USA 94 (1997) 9471

Table 2. Effects of glucose and of Naϩ ions on the time course uptake of deltorphins I and II Cumulative uptake, pmol͞mg protein Glucose Naϩ ions (10 mM) (120 mM) 10 sec 5 min 15 min Deltorphin I (50 ␮M) ϩϩ28 Ϯ 4 140 Ϯ 12 164 Ϯ 10 Ϫϩ26 Ϯ 4 120 Ϯ 17 156 Ϯ 16 ϩϪ20 Ϯ 3 104 Ϯ 11 115 Ϯ 9 Deltorphin II (50 ␮M) ϩϩ31 Ϯ 8 171 Ϯ 22 182 Ϯ 12 Ϫϩ27 Ϯ 4 161 Ϯ 20 170 Ϯ 16 ϩϪ30 Ϯ 2 120 Ϯ 15 132 Ϯ 11 After the addition of labeled deltorphin, portions of the microvessels suspension were withdrawn at fixed time intervals and collected as described. Data shown are the average values of three different experiments Ϯ SD.

aration (Х2mg͞ml). The assay mixtures contained, in a final times less than [Lys7]dermorphin, an opioid peptide with an volume of 2 ml, 1 ml of membrane preparation, 0.1 ml of 5 nM unusually high BBB penetration rate (28). tritiated ligand, and 0.9 ml of Tris buffer. The ␮-, ␦-, and Deltorphin Uptake by Isolated Brain Microvessels. Expo- ␬-binding sites were labeled with [3H]DAMGO, [3H]deltor- sure of isolated brain microvessels to either deltorphin I or phin I, and [3H]U-69593, respectively. Nonspecific binding was deltorphin II resulted in a time-dependent increase, within the determined in the presence of 50 ␮M naloxone, 5 ␮M naltrin- microvessels, of 3H-labeled radioactivity. The uptake process dole, or 5 ␮M deltorphin I. After 45 min incubation at 35°C, reached equilibrium within the first 20 min of uptake and the samples were cooled to 4°C and the free ligand was appeared to be temperature-dependent, because the uptake at separated from membrane-bound ligand by filtration under 4°C was less than one-fourth of the total amount transported reduced pressure over Whatman GF͞B filters presoaked with at 37°C, and remained linear for over 30 min (Fig. 1). Similar 0.5% polyethyleneimine. results were also obtained with DAMGO, a ␮-agonist that also has a D-amino acid in position 2 of its sequence. The uptake RESULTS of labeled deltorphin was inhibited by the presence, in the incubation medium, of high concentrations of the same unla- In Vivo Evidence of Deltorphins Permeation Through the beled compound; this self-inhibition indicated that the uptake BBB. Antinociception studies demonstrated that deltorphins I was a saturable process. To clarify whether the isolated mi- and II were the peptides with the highest BBB-PI among the crovessels were indeed able to concentrate deltorphins inside opioid peptides tested, thus most capable of reaching, upon i.v. the endothelial cells, the in͞out ratio was calculated according injection, the brain compartment (Table 1). Deltorphins in- to Betz et al. (35). It could be shown that, under the conditions deed appeared to permeate BBB 4–12 times better than typical of Fig. 1, the in͞out ratio reached a value around 1 after 30 min ␮-opioid receptor agonists such as DAMGO and dermorphin, incubation at 37°C, indicating that the isolated microvessels though 160–220 times less effectively than morphine and 2–3 were unable to concentrate deltorphins. The uptake of del-

FIG. 2. Eadie-Hofstee plot (v° vs. v°͞[S]) of the saturable component of deltorphin II uptake performed in the presence (E) or in the absence (F)ofNaϩions in the incubation medium. Isolated microvessels were assayed for deltorphin II uptake for 1 min at 37°C, between the concentrations ranging from 25 to 1,000 ␮M. After subtraction of the nonsaturable component, the obtained saturation curves (shown in the Inset) or their Eadie–Hofstee transformation were used to estimate the Vmax and the Km values. Data shown are the average values of three different experiments Ϯ SD. Downloaded by guest on October 4, 2021 9472 Pharmacology: Fiori et al. Proc. Natl. Acad. Sci. USA 94 (1997)

Table 3. Effects of some opioid peptides on the time course uptake of [3H]deltorphin II by bovine brain microvessels Cumulative uptake, pmol͞mg protein 10 sec 5 min 15 min 20 min No addition 28 Ϯ 4 110 Ϯ 11 174 Ϯ 20 205 Ϯ 18 ϩ Leu-enkephalin 0.2 mM 45 Ϯ 6 120 Ϯ 9 175 Ϯ 26 229 Ϯ 26 ϩ DPEDPE (0.2 mM) 44 Ϯ 6 175 Ϯ 18 265 Ϯ 16 266 Ϯ 21 ϩ Dermorphin (0.2 mM) 35 Ϯ 6 135 Ϯ 15 145 Ϯ 5 165 Ϯ 21 ϩ DAMGO (0.2 mM) 43 Ϯ 7 125 Ϯ 10 160 Ϯ 13 195 Ϯ 17 The uptake was started by mixing at 37°C, the microvessels suspension with the same buffer but with the addition of the labeled deltorphin II and the peptide indicated. Portions of the microvessels suspension were withdrawn at fixed time intervals and collected as described. Data shown are the average values of three different experiments Ϯ SD.

torphins was markedly decreased in the absence of Naϩ ions, whereas it was not affected by the absence of glucose (Table 2). Evaluation of the Kinetic Parameters of Deltorphin Trans- port. The kinetic analysis of deltorphin transport was per- FIG. 3. Overshoot effect in the time course uptake of [3H]del- formed by measuring, at different substrate concentrations torphin II by Gln-preloaded microvessels. After 20 min preloading and in the presence or absence of Naϩ ions, the uptake of at 37°C with 20 mM Gln (E) or in Gln-free buffer (F), the microvessels were washed and resuspended in prewarmed (37°C) labeled deltorphin in the first minute after its addition to the Gln-free buffer. Immediately after the resuspension, labeled del- microvessels equilibrated at 37°C. The Vmax for deltorphin I torphin II was added and the cumulative uptake followed for 20 min was found to have a value of 251 Ϯ 8 pmol͞mg protein in the (solid lines). Gln efflux is also shown (å, dashed line). The data in presence of Naϩ ions and 245 Ϯ 12 pmol͞mg protein in their the figure represent the mean values Ϯ SD of three different experiments. absence, whereas the apparent Km value was of 114 Ϯ 4 ␮Min the presence of Naϩ ions and 145 Ϯ 10 ␮M in their absence which is a good substrate for both amino acid transport systems (Fig. 2). The kinetic parameters of deltorphin II had similar (Table 4). values. Glutamine Effect on the Deltorphin Transport System(s). Competition with Other Opioid Peptides. To further char- The initial rate of deltorphin uptake was found to be markedly acterize the selectivity of the deltorphin uptake system in increased if the microvessels were preloaded with L-glutamine. bovine brain capillaries, four opioid peptides were assayed for The magnitude of this increase was inversely related to the their capacity to compete for deltorphin transport. At 0.2 mM time interval between glutamine loading and the deltorphin concentration, neither Leu-enkephalin, dermorphin, uptake assay. In the preloaded microvessels, cumulative del- DAMGO, or DPEDPE were capable of modifying the time course uptake of deltorphin by microvessels (Table 3). Competition with Amino Acids. Some of us had shown (36) that, under certain conditions, the A- and L-systems of neutral amino acids transport could cooperate at the BBB level, and that long-chain polar amino acids such as Gln or Met are able to use either system of transport and thus to act as regulatory agents. By checking whether, when added to the incubation medium, given amino acids could compete with deltorphins for the brain capillary transport, we could show that the uptake of deltorphin II was unaffected by the presence of short-chain neutral amino acids such as ␣-methylaminoisobutyric acid, which is specific for the A-system of amino acid transport, as well as by the addition of L-leucine or L-tyrosine, which are selectively transported by the L-system, or of L-glutamine,

Table 4. Effects of some neutral amino acids on the time course uptake of [3H]deltorphin II by bovine brain microvessels Cumulative uptake, pmol͞mg protein 10 sec 5 min 15 min 20 min No addition 28 Ϯ 4 110 Ϯ 11 174 Ϯ 20 205 Ϯ 18 ϩ L-leucine (5 mM) 30 Ϯ 5 140 Ϯ 11 185 Ϯ 14 201 Ϯ 15 ϩ L-tyrosine (1 mM) 40 Ϯ 7 145 Ϯ 10 173 Ϯ 13 192 Ϯ 14 FIG. 4. Effect of the addition of external deltorphin II on Gln ϩ L-Glutamine (5 mM) 30 Ϯ 5 125 Ϯ 6 175 Ϯ 18 200 Ϯ 12 efflux from brain microvessels. Microvessels were preloaded with ϩ MeAIB (5 mM) 29 Ϯ 4 153 Ϯ 10 163 Ϯ 11 199 Ϯ 17 [3H]Gln (final concentration, 20 mM) for 20 min at 37°C, rapidly The uptake was started by mixing, at 37°C, the microvessels sus- washed, and then resuspended in warm Gln-free buffer (E)orin pension with the same buffer but with the addition of the labeled Gln-free buffer containing 0.5 mM L-leucine (ᮀ) or 0.2 mM deltorphin deltorphin II and the desired amino acid. Portions of the microvessels II (F). At fixed time intervals the isolated microvessels were rapidly suspension were withdrawn at fixed time intervals and collected as washed and the internal radioactivity was measured. Data shown are described. Data shown are the average values of three different the mean values Ϯ SD of three different experiments, each performed experiments Ϯ SD. MeAIB, ␣-methylaminoisobutyric acid. in triplicate. Downloaded by guest on October 4, 2021 Pharmacology: Fiori et al. Proc. Natl. Acad. Sci. USA 94 (1997) 9473

shown). Specific binding was absent, and nonspecific binding was less than 1% of the added tritiated ligand.

DISCUSSION In vivo antinociception studies demonstrated that deltorphins are opioid peptides with an unusually high ability of penetration through the mouse BBB. These results confirm those recently obtained by Thomas et al. (37) with deltorphin analogs, using primary endothelium cultures as in vitro BBB model. These authors demonstrated that the transendothelial passage of deltorphins I and II and of some synthetic analogs, which have high BBB permeability coefficients, does not correlate to the molecular weight or to the lipophilicity of the peptide, but critically depends on the N-terminal sequence and on the amino acid residues present in positions 4 and 5. Our present experiments show that the uptake of deltorphins by brain microvessels is mediated, at 37°C, by a saturable nonconcentrative permeation system that appears to be quite distinct from the system(s) that can be utilized by other opioid neuropeptides or which are specific for short- or long-chain neutral amino acids. This deltorphin-specific transport system is insensitive to the presence or absence of glucose, but its FIG. 5. Effect of naloxone and of naltrindole on the uptake of affinity for deltorphins decreases by approximately one-third 3 [ H]deltorphin II by isolated brain microvessels. The microvessels if Naϩ ions are absent from the medium. The efficiency of were preincubated for 10 min at 37°C in the presence of 1.5 mM deltorphin uptake in the presence of Naϩ ions, evaluated at naloxone (F), 1 mM naltrindole (å), or in their absence (E); labeled deltorphin II was then added and the cumulative uptake was peptide concentrations below 100 ␮M (i.e., in terms of the measured at various time intervals. The data in the figure represent Vmax͞Km ratio), was around 2.2 ␮l͞mg of protein per min, the average values Ϯ SD of three different experiments, each whereas at high substrate concentrations the maximal initial performed in triplicate. rate was around 250 pmol͞mg of protein per min; these values are definitely too high to account for any peptide binding not torphin uptake curves had a typical ‘‘overshoot profile’’ (Fig. linked to permeation. 3)—quite similar to that observed for the glutamine- Deltorphin uptake was inhibited by millimolar concentra- stimulated uptake of large hydrophobic neutral amino acids tions of naloxone, in the presence of which only a small (9)—and deltorphin influx was indeed paralleled by an in- diffusional component could be detected. Nevertheless, nal- creased efflux rate of glutamine (Fig. 4). trindole, a selective antagonist to ␦-opioid receptors (36), was Naloxone and Naltrindole Effects on Deltorphin Transport. ineffective. Other opioid receptor ligands, namely Leu- ␦ Addition of 1.5 mM naloxone to the incubation medium failed enkephalin and DPEDPE, which preferentially bind to receptors, or dermorphin or DAMGO, which have a high to affect to any significant extent either the overall perme- selectivity for ␮ receptors, were equally unable to modify the ability of the endothelial cell membranes, as estimated from deltorphin uptake. Finally, binding experiments with highly the rate of carboxyfluorescein efflux, or the transport systems selective ␦ ligands failed to demonstrate the presence of for glucose and for small or large neutral amino acids (data not ␦-opioid receptors in bovine brain endothelial cells. Taken shown). However, naloxone, but not naltrindole, was a strong together, these results indicate that the deltorphin uptake by inhibitor of deltorphin uptake (Fig. 5); the value of the in͞out brain microvessels is not mediated by opioid receptors. ratio was reduced to that of the diffusional component ob- The transient stimulation of deltorphin uptake occurring served at 4°C. Naloxone also abolished the stimulating effect in microvessels preloaded with L-glutamine and the in- exerted by deltorphin on glutamine efflux from glutamine- creased efflux of glutamine upon addition of deltorphin to loaded microvessels (Table 5). the extracellular compartment suggest the possibility of a Absence of Opioid Receptors in Bovine Brain Microvessels. countertransport through some pathway common to glu- In in vitro binding studies, ␮-, ␦-, and ␬-opioid receptors were tamine and to deltorphins. This hypothesis receives further undetectable in our microvessel preparations (data not support from the inhibitory effect exerted by naloxone not only on deltorphin uptake but also on deltorphin-stimulated Table 5. Effect of external deltorphin and͞or of naloxone on Gln glutamine efflux. These results indicate that the coupling efflux from brain microvessels between the two phenomena cannot be ascribed to a mere osmotic effect caused by glutamine efflux. However, it Gln efflux, nmol͞mg protein should be mentioned that a similar hypothesis of counter- 10 sec 5 min 15 min 20 min transport involving glutamine efflux has already been taken Buffer alone 20 Ϯ 116Ϯ115Ϯ214Ϯ2 into consideration (9) as a mechanism capable of increasing ϩDeltorphin II 19 Ϯ 214Ϯ111Ϯ210Ϯ1 the uptake of large hydrophobic amino acids and that it ϩDeltorphin II apparently holds also for Leu-enkephalins (26). These var- ϩ naloxone 22 Ϯ 118Ϯ216Ϯ215Ϯ1 ious permeants undoubtedly utilize different pathways, but ϩNaloxone 19 Ϯ 217Ϯ315Ϯ114Ϯ1 there might be some common pathway capable of acceler- 3 ating the coupled uptake processes. This phenomenon may Microvessels were preloaded with [ H]Gln (external concentration, have some interesting implications at the metabolic level. 20 mM; specific activity, 1.27 mCi͞mmol) for 20 min at 37°C, rapidly washed, and then resuspended in warm Gln-free buffer that contained, For example, high levels of glutamine occur in the cerebral when indicated, 0.2 mM deltorphin II and͞or 1.5 mM naloxone. At compartment as a consequence of hyperammonemia (10), fixed time intervals the isolated microvessels were rapidly washed and and hence some glutamine overload in the brain capillaries the internal radioactivity was measured. Data shown are the average may be expected in patients with hepatic encephalopathy. An values of three different experiments Ϯ SD. increase in the permeability of the BBB to opioids and Downloaded by guest on October 4, 2021 9474 Pharmacology: Fiori et al. Proc. Natl. Acad. Sci. USA 94 (1997)

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