Deltorphin transport across the blood–brain barrier

Abstract

In vivo antinociception studies demonstrate that deltorphins are opioid peptides with an unusually high blood–brain barrier penetration rate. In vitro, isolated bovine brain microvessels can take up deltorphins through a saturable nonconcentrative permeation system, which is apparently distinct from previously described systems involved in the transport of neutral amino acids or of enkephalins. Removing Na⁺ ions from the incubation medium decreases the carrier affinity for deltorphins (−25%), but does not affect the V_max value of the transport. The nonselective opiate antagonist naloxone inhibits deltorphin uptake by brain microvessels, but neither the selective δ-opioid antagonist naltrindole nor a number of opioid peptides with different affinities for δ- or μ-opioid receptors compete with deltorphins for the transport. Binding studies demonstrate that μ-, δ-, and κ-opioid receptors are undetectable in the microvessel preparation. Preloading of the microvessels with l-glutamine results in a transient stimulation of deltorphin uptake. Glutamine-accelerated deltorphin uptake correlates to the rate of glutamine efflux from the microvessels and is abolished by naloxone.

The blood–brain barrier (BBB) accounts for the restricted movement of solutes between the vascular and the cerebral compartments. Its anatomical counterpart has been identified as the endothelial cell membranes of brain microvessels (1–5). These endothelial cells, joined together by tight junctions (6), form a continuous physical barrier playing a crucial role in determining the rate at which different compounds can reach, or in turn leave, the central nervous system (CNS). The capacity of any particular molecule to penetrate the BBB depends essentially on its free diffusion across the cell walls—i.e., on its lipid solubility—or on its specific affinity for a carrier-mediated transport system. The use of isolated brain microvessels, as an in vitro model of the BBB, has allowed the identification and characterization of some peculiarities of the transport systems specific for sugars (7, 8), amino acids (9–13), electrolytes (14, 15), or transferrin (16). It had been initially assumed that peptides could not enter the CNS via the BBB (17) and that the entry rate into the CNS of some peptides correlated only with their lipid solubility, suggesting that they crossed the BBB by direct diffusion through the phospholipid bilayer of the endothelial cell membranes. It is now generally accepted, instead, that different categories of peptides can enter or exit the CNS through the endothelial cell membranes of brain microvessels at a rate higher than that accounted by passive diffusion. In vivo studies have indicated the existence of transport systems that allow the selective permeation of the BBB by different peptides, either endogenous or synthetic (18–24). Some years ago, Banks and Kastin (21) have suggested that at least four different transport systems, named PTS-1, -2, -3, and -4, could accelerate the entrance of opioids and related peptides into the brain. More recently, the use of different in vitro models consisting either of fresh isolated brain microvessels or of cultured brain endothelial cells has allowed a better investigation of the modality of transport of some opioid peptides such as Met-enkephalin and related analogs (25–27). Deltorphins are naturally occurring peptides with high affinity and selectivity for δ-opioid receptors. These opioids have a d-amino acid residue in position 2 of their sequence and an amide residue at C terminus that protect them from enzymatic hydrolysis (28). In this study, we show that, when injected intravenously in mice, deltorphins enter BBB to produce antinociception. Moreover, using isolated bovine brain microvessels as an in vitro model of the BBB, we investigate the presence of transport system(s) for deltorphins in the plasma membranes of the endothelial cells and whether and how such system(s) are susceptible to regulation, in particular at the metabolic level.

MATERIALS AND METHODS

Chemicals.

Tyr-ala-Phe-Asp-Val-Val-Gly-NH₂ (deltorphin I; ala, d-Ala), Tyr-ala-Phe-Glu-Val-Val-Gly-NH₂ (deltorphin II), and Tyr-ala-Gly-NMePhe-glycinol (DAMGO) were obtained from Bachem. Tyr-d-Pen-Gly-Phe-d-Pen (DPEDPE), Tyr-ala-Phe-Gly-Tyr-Pro-Ser-NH₂ (dermorphin), Tyr-Gly-Gly-Phe-Leu-OH (Leu-enkephalin), Tyr-Gly-Gly-Phe-Met-OH (Met-enkephalin), Hepes, naloxone, and naltrindole were from Sigma); morphine HCl was from Salars (Como, Italy); carboxyfluorescein diacetate was from Molecular Probes. [Lys⁷]dermorphin was synthesized as indicated by Negri and coworkers (28). All other chemicals were obtained from Merck or Fluka. [Tyrosyl-3,5-³H]Leu-enkephalin (37 Ci/mmol; 1 Ci = 37 GBq), [tyrosyl-3,5-³H]deltorphin I (35 Ci/mmol), [tyrosyl-3,5-³H]deltorphin II (54.5 Ci/mmol), [tyrosyl-3,5-³H]DAMGO (37 Ci/mmol), [phenyl-3,4-³H]U-69593 {(+)-(5α,7α,8β)-N-methyl-N[7-(1-pyrrolidinyl)-1-oxaspiro(4,5)dec-8-yl]-benzeneacetamide} (47 Ci/mmol), l-[3,4-³H(N)]glutamine (60 Ci/mmol), l-[U-¹⁴C]leucine (300 mCi/mmol), l-[U-¹⁴C]tyrosine (450 mCi/mmol), [U-¹⁴C]sucrose (4.63 mCi/mmol), and Aquasol-2 were obtained from New England Nuclear.

Intracerebroventricular (i.c.v.) and Intravenous (i.v.) Injections.

Male C57BL6 mice weighing 25–28 g (Charles River Breeding Laboratories) were housed singly in 20 × 25 cm cages placed in a thermostatically controlled cabinet at an environmental temperature of 21°C. Under light diethyl ether anesthesia drug solution or vehicle were delivered into the lateral cerebral ventricle (i.c.v.) by using a modification of the method of Haley and McCormick (29). An incision was made in the scalp and the bregma was located. Injections were made directly through the skull at a point 2 mm caudal and 2 mm lateral to bregma at a depth of 3 mm using a Hamilton microliter syringe with a 26-gauge needle. All i.c.v. injections were made in a volume of 5 μl. For i.v. injections the mouse tail was immersed in moderately hot water (about 40°C) for 1–2 min to induce venous dilatation and the injections carried out using a 500 μl Hamilton syringe with a 25-gauge needle.

Test of Antinociception.

Antinociception responses were determined by immerging the mouse tail into hot water (55°C). The latency of the first sign of a rapid tail-flick was taken as the end point, as indicated by Janssen et al. (30). Before drug administration, mice not flicking their tails within 5 sec from hot water immersion were eliminated from the study. After drug injection, animals not flicking their tails within 15 sec were removed from the nociceptive stimulus and assigned a maximal antinociceptive response. The test was repeated every 15 min after i.c.v. or i.v. drug injection and time course antinociception curves were drawn. Peak response (PR), area under the curve (AUC), and PR/AUC ratios were calculated for the dose producing 50% of the maximum response (ED₅₀). Five mice were tested for each opioid dose.

BBB Permeability Index.

The values of i.v. and i.c.v. ED₅₀ were multiplied by the respective PR/AUC ratio to normalize them for degradation and elimination processes, and the BBB permeability index (BBB-PI) was calculated as the ratio between i.c.v./i.v. normalized ED₅₀ values. Compounds that more easily enter BBB have therefore higher BBB-PI values.

Isolation of Microvessels.

Fresh bovine brain, obtained from the local slaughterhouse, was transported on ice to the laboratory, where meninges were removed. The capillaries were isolated from brain cortex as described by Cangiano et al. (9). These microvessels have been shown, by scanning electron microscopy and phase contrast light microscopy, to be practically free from contamination by glial or nerve cells (11). The preparations appeared to consist essentially of branching microvessel segments enveloped in their basement membrane. Their ATP content, measured by a luciferin–luciferase assay, was around 643 ± 27 pmol/mg protein. Passive cell permeability, tested by measuring at different time intervals the efflux of carboxyfluorescein from cells previously loaded with carboxyfluorescein diacetate (31), was negligible; also negligible 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 γ-glutamyltranspeptidase and alkaline phosphatase (9). Their contamination with glia, estimated through the immunochemical 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 cumulatively, 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 isotonic buffer (pH 7.4) (choline being, if required, substituted to Na⁺ ions), were poured on a 44-μm 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 aliquots were transferred to liquid scintillation vials containing an equal volume of 1 M HCl. After addition of 8 ml of Aquasol-2, the vials were counted in a Beckman 9800 liquid scintillation spectrometer. Nonspecific radioactivity, due to the binding of labeled peptide to the nylon sieve, when tested in triplicate by omitting the microvessels, was constantly below 100 dpm.

By plotting the initial rate of uptake, v°, as a function of peptide concentration, a typical saturation kinetics pattern was found, often superimposed to a diffusional component that could, however, be easily subtracted (11, 33). Nonlinear regression analysis, performed on the v° vs. v°/[S] “Eadie–Hofstee plot,” which allows the use of simple statistical procedures (34), was used to obtain the optimal estimates of the kinetic parameters of the transport system(s) and to evaluate the standard error impending on them. Because labeled sucrose had been shown not to permeate the endothelial cells (9), the intracellular water space could be evaluated by a procedure similar to that suggested by Betz et al. (35)—i.e., as the difference between the wet and dry weights of a microvessels sample collected on a filter minus the sucrose water space.

Opioid Binding Sites in Brain Microvessels.

[³H]Deltorphin I binding experiments were performed on membrane preparations obtained from homogenates of brain microvessels. Briefly, brain microvessels prepared as described above were centrifuged at 40,000 × g and the pellets homogenized for 3 min in Tris⋅HCl buffer (50 mM, pH 7.4) with a Polytron PT 3000 homogenizer (Kinematica, Littau-Luzern, Switzerland). The homogenate was centrifuged at 1,000 × g to eliminate nuclei and debris, and supernatant was collected and centrifuged at 40,000 × g. Pellets were washed three times with buffer and finally resuspended in the buffer at a protein concentration equal to that of the original microvessel preparation (≅2 mg/ml). The assay mixtures contained, in a final volume of 2 ml, 1 ml of membrane preparation, 0.1 ml of 5 nM tritiated ligand, and 0.9 ml of Tris buffer. The μ-, δ-, and κ-binding sites were labeled with [³H]DAMGO, [³H]deltorphin I, and [³H]U-69593, respectively. Nonspecific binding was determined in the presence of 50 μM naloxone, 5 μM naltrindole, or 5 μM deltorphin I. After 45 min incubation at 35°C, the samples were cooled to 4°C and the free ligand was separated from membrane-bound ligand by filtration under reduced pressure over Whatman GF/B filters presoaked with 0.5% polyethyleneimine.

RESULTS

In Vivo Evidence of Deltorphins Permeation Through the BBB.

Antinociception studies demonstrated that deltorphins I and II were the peptides with the highest BBB-PI among the opioid peptides tested, thus most capable of reaching, upon i.v. injection, the brain compartment (Table 1). Deltorphins indeed appeared to permeate BBB 4–12 times better than typical μ-opioid receptor agonists such as DAMGO and dermorphin, though 160–220 times less effectively than morphine and 2–3 times less than [Lys⁷]dermorphin, an opioid peptide with an unusually high BBB penetration rate (28).

Table 1.

BBB-PI of deltorphins compared to morphine and other opioid peptides

Compound	i.v. ED50, nmol	i.c.v. ED50, nmol	BBB-PI × 10−4
Deltorphin I	321.0	0.38	12
Deltorphin II	364.0	0.27	9
DAMGO	315.0	0.04	1
Dermorphin	64.0	0.03	3
[Lys7]dermorphin	7.4	0.04	28
Morphine	130.0	18.13	1980

For calculation of BB-PI, see text. 

Deltorphin Uptake by Isolated Brain Microvessels.

Exposure of isolated brain microvessels to either deltorphin I or deltorphin II resulted in a time-dependent increase, within the microvessels, of ³H-labeled radioactivity. The uptake process reached equilibrium within the first 20 min of uptake and appeared to be temperature-dependent, because the uptake at 4°C was less than one-fourth of the total amount transported at 37°C, and remained linear for over 30 min (Fig. 1). Similar results were also obtained with DAMGO, a μ-agonist that also has a d-amino acid in position 2 of its sequence. The uptake of labeled deltorphin was inhibited by the presence, in the incubation medium, of high concentrations of the same unlabeled compound; this self-inhibition indicated that the uptake was a saturable process. To clarify whether the isolated microvessels were indeed able to concentrate deltorphins inside the endothelial cells, the in/out ratio was calculated according to Betz et al. (35). It could be shown that, under the conditions of Fig. 1, the in/out ratio reached a value around 1 after 30 min incubation at 37°C, indicating that the isolated microvessels were unable to concentrate deltorphins. The uptake of deltorphins was markedly decreased in the absence of Na⁺ ions, whereas it was not affected by the absence of glucose (Table 2).

Figure 1.

Effect of temperature on the uptake of [³H]deltorphin I by isolated brain microvessels. After standing for 10 min either at 37°C (○) or at 4°C (•), the labeled deltorphin I was added and the time course uptake was followed for 30 min. The data in the figure represent the average values ± SD obtained from three different experiments, each performed in triplicate.

Table 2.

Effects of glucose and of Na⁺ ions on the time course uptake of deltorphins I and II

	Glucose (10 mM)	Na+ ions (120 mM)	Cumulative uptake, pmol/mg protein
			10 sec	5 min	15 min
Deltorphin I (50 μM)	+	+	28  ±  4	140  ±  12	164  ±  10
	−	+	26  ±  4	120  ±  17	156  ±  16
	+	−	20  ±  3	104  ±  11	115  ±  9
Deltorphin II (50 μM)	+	+	31  ±  8	171  ±  22	182  ±  12
	−	+	27  ±  4	161  ±  20	170  ±  16
	+	−	30  ±  2	120  ±  15	132  ±  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 ± SD. 

Evaluation of the Kinetic Parameters of Deltorphin Transport.

The kinetic analysis of deltorphin transport was performed by measuring, at different substrate concentrations and in the presence or absence of Na⁺ ions, the uptake of labeled deltorphin in the first minute after its addition to the microvessels equilibrated at 37°C. The V_max for deltorphin I was found to have a value of 251 ± 8 pmol/mg protein in the presence of Na⁺ ions and 245 ± 12 pmol/mg protein in their absence, whereas the apparent K_m value was of 114 ± 4 μM in the presence of Na⁺ ions and 145 ± 10 μM in their absence (Fig. 2). The kinetic parameters of deltorphin II had similar values.

Figure 2.

Eadie-Hofstee plot (v° vs. v°/[S]) of the saturable component of deltorphin II uptake performed in the presence (○) or in the absence (•) of Na⁺ ions 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 μM. After subtraction of the nonsaturable component, the obtained saturation curves (shown in the Inset) or their Eadie–Hofstee transformation were used to estimate the V_max and the K_m values. Data shown are the average values of three different experiments ± SD.

Competition with Other Opioid Peptides.

To further characterize the selectivity of the deltorphin uptake system in bovine brain capillaries, four opioid peptides were assayed for their capacity to compete for deltorphin transport. At 0.2 mM concentration, neither Leu-enkephalin, dermorphin, DAMGO, or DPEDPE were capable of modifying the time course uptake of deltorphin by microvessels (Table 3).

Table 3.

Effects of some opioid peptides on the time course uptake of [³H]deltorphin II by bovine brain microvessels

	Cumulative uptake, pmol/mg protein
	10 sec	5 min	15 min	20 min
No addition	28  ±  4	110  ±  11	174  ±  20	205  ±  18
+ Leu-enkephalin 0.2 mM	45  ±  6	120  ±  9	175  ±  26	229  ±  26
+ DPEDPE (0.2 mM)	44  ±  6	175  ±  18	265  ±  16	266  ±  21
+ Dermorphin (0.2 mM)	35  ±  6	135  ±  15	145  ±  5	165  ±  21
+ DAMGO (0.2 mM)	43  ±  7	125  ±  10	160  ±  13	195  ±  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 ± SD. 

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 α-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, which is a good substrate for both amino acid transport systems (Table 4).

Table 4.

Effects of some neutral amino acids on the time course uptake of [³H]deltorphin II by bovine brain microvessels

	Cumulative uptake, pmol/mg protein
	10 sec	5 min	15 min	20 min
No addition	28  ±  4	110  ±  11	174  ±  20	205  ±  18
+ l-leucine (5 mM)	30  ±  5	140  ±  11	185  ±  14	201  ±  15
+ l-tyrosine (1 mM)	40  ±  7	145  ±  10	173  ±  13	192  ±  14
+ l-Glutamine (5 mM)	30  ±  5	125  ±  6	175  ±  18	200  ±  12
+ MeAIB (5 mM)	29  ±  4	153  ±  10	163  ±  11	199  ±  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 desired amino acid. 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 ± SD. MeAIB, α-methylaminoisobutyric acid. 

Glutamine Effect on the Deltorphin Transport System(s).

The initial rate of deltorphin uptake was found to be markedly increased if the microvessels were preloaded with l-glutamine. The magnitude of this increase was inversely related to the time interval between glutamine loading and the deltorphin uptake assay. In the preloaded microvessels, cumulative deltorphin uptake curves had a typical “overshoot profile” (Fig. 3)—quite similar to that observed for the glutamine-stimulated uptake of large hydrophobic neutral amino acids (9)—and deltorphin influx was indeed paralleled by an increased efflux rate of glutamine (Fig. 4).

Figure 3.

Overshoot effect in the time course uptake of [³H]deltorphin II by Gln-preloaded microvessels. After 20 min preloading at 37°C with 20 mM Gln (○) or in Gln-free buffer (•), the microvessels were washed and resuspended in prewarmed (37°C) Gln-free buffer. Immediately after the resuspension, labeled deltorphin II was added and the cumulative uptake followed for 20 min (solid lines). Gln efflux is also shown (▴, dashed line). The data in the figure represent the mean values ± SD of three different experiments.

Figure 4.

Effect of the addition of external deltorphin II on Gln efflux from brain microvessels. Microvessels were preloaded with [³H]Gln (final concentration, 20 mM) for 20 min at 37°C, rapidly washed, and then resuspended in warm Gln-free buffer (○) or in Gln-free buffer containing 0.5 mM l-leucine (□) or 0.2 mM deltorphin II (•). At fixed time intervals the isolated microvessels were rapidly washed and the internal radioactivity was measured. Data shown are the mean values ± SD of three different experiments, each performed in triplicate.

Naloxone and Naltrindole Effects on Deltorphin Transport.

Addition of 1.5 mM naloxone to the incubation medium failed to affect to any significant extent either the overall permeability of the endothelial cell membranes, as estimated from the rate of carboxyfluorescein efflux, or the transport systems for glucose and for small or large neutral amino acids (data not shown). However, naloxone, but not naltrindole, was a strong inhibitor of deltorphin uptake (Fig. 5); the value of the in/out ratio was reduced to that of the diffusional component observed at 4°C. Naloxone also abolished the stimulating effect exerted by deltorphin on glutamine efflux from glutamine-loaded microvessels (Table 5).

Figure 5.

Effect of naloxone and of naltrindole on the uptake of [³H]deltorphin II by isolated brain microvessels. The microvessels were preincubated for 10 min at 37°C in the presence of 1.5 mM naloxone (•), 1 mM naltrindole (▴), or in their absence (○); labeled deltorphin II was then added and the cumulative uptake was measured at various time intervals. The data in the figure represent the average values ± SD of three different experiments, each performed in triplicate.

Table 5.

Effect of external deltorphin and/or of naloxone on Gln efflux from brain microvessels

	Gln efflux, nmol/mg protein
	10 sec	5 min	15 min	20 min
Buffer alone	20  ±  1	16  ±  1	15  ±  2	14  ±  2
+ Deltorphin II	19  ±  2	14  ±  1	11  ±  2	10  ±  1
+ Deltorphin II + naloxone	22  ±  1	18  ±  2	16  ±  2	15  ±  1
+ Naloxone	19  ±  2	17  ±  3	15  ±  1	14  ±  1

Microvessels were preloaded with [³H]Gln (external concentration, 20 mM; specific activity, 1.27 mCi/mmol) for 20 min at 37°C, rapidly washed, and then resuspended in warm Gln-free buffer that contained, when indicated, 0.2 mM deltorphin II and/or 1.5 mM naloxone. At fixed time intervals the isolated microvessels were rapidly washed and the internal radioactivity was measured. Data shown are the average values of three different experiments ± SD. 

Absence of Opioid Receptors in Bovine Brain Microvessels.

In in vitro binding studies, μ-, δ-, and κ-opioid receptors were undetectable in our microvessel preparations (data not 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 affinity for deltorphins decreases by approximately one-third if Na⁺ ions are absent from the medium. The efficiency of deltorphin uptake in the presence of Na⁺ ions, evaluated at peptide concentrations below 100 μM (i.e., in terms of the V_max/K_m ratio), was around 2.2 μl/mg of protein per min, whereas at high substrate concentrations the maximal initial rate was around 250 pmol/mg of protein per min; these values are definitely too high to account for any peptide binding not linked to permeation.

Deltorphin uptake was inhibited by millimolar concentrations of naloxone, in the presence of which only a small diffusional component could be detected. Nevertheless, naltrindole, a selective antagonist to δ-opioid receptors (36), was ineffective. Other opioid receptor ligands, namely Leu-enkephalin and DPEDPE, which preferentially bind to δ receptors, or dermorphin or DAMGO, which have a high selectivity for μ receptors, were equally unable to modify the deltorphin uptake. Finally, binding experiments with highly selective δ ligands failed to demonstrate the presence of δ-opioid receptors in bovine brain endothelial cells. Taken together, these results indicate that the deltorphin uptake by brain microvessels is not mediated by opioid receptors.

The transient stimulation of deltorphin uptake occurring in microvessels preloaded with l-glutamine and the increased efflux of glutamine upon addition of deltorphin to the extracellular compartment suggest the possibility of a countertransport through some pathway common to glutamine and to deltorphins. This hypothesis receives further support from the inhibitory effect exerted by naloxone not only on deltorphin uptake but also on deltorphin-stimulated glutamine efflux. These results indicate that the coupling between the two phenomena cannot be ascribed to a mere osmotic effect caused by glutamine efflux. However, it should be mentioned that a similar hypothesis of countertransport involving glutamine efflux has already been taken into consideration (9) as a mechanism capable of increasing the uptake of large hydrophobic amino acids and that it apparently holds also for Leu-enkephalins (26). These various permeants undoubtedly utilize different pathways, but there might be some common pathway capable of accelerating the coupled uptake processes. This phenomenon may have some interesting implications at the metabolic level. For example, high levels of glutamine occur in the cerebral compartment as a consequence of hyperammonemia (10), and hence some glutamine overload in the brain capillaries may be expected in patients with hepatic encephalopathy. An increase in the permeability of the BBB to opioids and related peptides may thus be an additional factor in the pathogenesis of this complex neuropsychiatric syndrome and clearly deserves further attention.

ABBREVIATIONS

ala

d-Ala

BBB

blood-brain barrier

BBB-PI

BBB permeability index

DAMGO

Tyr-ala-Gly-NMePhe-glycinol

DPEDPE

Tyr-d-Pen-Gly-Phe-d-Pen

i.c.v.

intracerebroventricular

i.v.

intravenous

U-69593

(+)-(5α,7α,8β)-N-methyl-N[7-(1-pyrrolidinyl)-1-oxaspiro(4,5)dec-8-yl]-benzeneacetamide