THE JOURNAL (c’ 1984 by The American Society of Biological Chemists, Inc.

OF BIOLOGICAL CHEMISTRY Vol. 259, No. 8, Issue of April 25. pp. 5295-5300, 1984 Printed in U. S. A

Effect of Ammonia on Amino Acid Uptake by Brain Microvessels* (Received for publication, July 20, 1983)

Patrizia Cardelli-Cangiano, Carlo Cangianoz, James Howard Jamese, Fabrizio CeciS, Josef E. Fischere, and Roberto Stromll From the Departments of Human Biopathology and the $Third Department of Internal Medicine, University of Rome “La Sapienza,” Rome, Italy, the §Department of Surgery, Cincinnati General Hospital and University of Cincinnati Medical Center, Cincinnati, Ohio 45267, and Consiglio Nazionnle delle Richerche, Center for Molecular Biology, Rome, Italy

NH: ions, at a concentration (0.25 mM) similar to that found in the plasma of patients with hepatic en- cephalopathy, cause, in vitro, a significant stimulation of the uptake by brain microvessels of large neutral amino acids, without any effect on the uptake of a- methylaminoisobutyric acid, glutamic acid, or lysine. Such a stimulation occurs essentially through an in- crease of the maximal transport capacity (Vm=) of the saturable component. It is apparently mediated by the intracellular formation of glutamine, which is then exchanged, through the L-system of transport, for large neutral amino acids such as leucine, phenylala- nine, or tyrosine. At higher concentrations (r0.5 mM), NHt ions cause also a decrease of carrier affinity for neutral amino acids, which counteracts the stimulatory effect on their uptake.

Severe hepatic failure and portal-systemic shunting are usually associated, both in patients and in experimental ani- mals, with hyperammonemia as well as with high brain aro- matic amino acid levels (1-8). These findings have been recently linked together by suggesting that high brain concen- trations of ammonia may produce, through a rise of brain Gln, an increase of neutral amino acid transport across the blood-brain barrier (4).

Previous studies have shown that brain microvessels (which represent the anatomical counterpart of the blood-brain bar- rier) isolated from rats with portacaval anastomosis take up large neutral amino acids more rapidly than controls (9). These results were correlated to high intracellular Gln levels. We have also recently reported that Gln is able, in uitro, to exert a direct stimulatory effect on the L-system-mediated uptake of large neutral amino acids by isolated brain micro- vessels (10).

The present study was carried out to clarify the in vitro effect of NH: ions on amino acid uptake by isolated brain microvessels. Our results indicate that NH: ions act by in- creasing the endothelial cell content of Gln, which in turn is responsible for a higher L-system-mediated uptake of large

~ _ _ _ ~ _ _ _ _ _ ~ _ _ _ _

* This work was supported by Grant AM252638 from the National Institutes of Health and a grant from the University of Rome (Re- search Project “Membrane Phenomena”). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

P To whom correspondence should be addressed at, Istituto di Chimica Biologica, Universita degli Studi “La Sapienza” di Roma, Piazzale Aldo Mor0 5, 00185 Rome, Italy.

neutral amino acids. The rise in Gln levels can tentatively be attributed to the presence of a Gln synthetase activity inside the endothelial cells of the microvessels.

MATERIALS AND METHODS

Chemicals-The following materials were obtained from New England Nuclear: L-[“C(U)]L~U, ~-[‘~c(U)]Phe, ~-[‘~c(U)]Tyr , L- [‘4C(U)]Glu, ~-[‘~c(U)]Lys, [carbo~yl-~~C]aminocyclopentane-l-car- boxylic acid (cycloleucine), [carbo~yl-’~C]a-methylamin~i~~butyric acid, [carboxyl-’4C]~~-~-aminobicyclo-2,2,1-heptanecarhoxylic acid, [‘4C(U)]sucrose, and Aquasol 2. Collagenase from Clostridium histo- lyticum, methionine sulfoximine, and N-2-hydroxyethylpiperazine- N’-2-ethanesulfonic acid were obtained from Sigma. Carboxyfluores- cein diacetate was obtained from Molecular Probes Inc. (Junction City, OR). Luciferase, purified from the firefly Photinus pyralis, and D - ( - ) - 2 - (6’ - hydroxybenzothiazoly) - A’ - thiazoline - 4 -carbonic acid were from LKB-Wallac (Turku, Finland). All other chemicals were from Merck (Darmstadt, W. Germany) or from Fluka (Buchs, Swit- zerland).

Isolation of Microuessels-Microvessels were isolated from gray matter of fresh bovine brain essentially as described by Hjelle et al. (11) with minor modifications (9-10). Briefly, gray matter was ho- mogenized by hand in buffer (l:l, w : ~ ) containing 122 mM NaCI, 15 mM NaHC03, 10 mM glucose, 3 mM KCl, 1.4 mM CaCl,, 1.2 mM MgS04, 0.4 mM KZHPO~, and 10 mM HEPES,’ pH 7.4, equilibrated with 95% 0 2 + 5% CO,. The homogenate was poured on a nylon sieve (86 pm pore size) and washed with a spray of ice-cold buffer. The material retained on the sieve was rehomogenized and washed again with a spray of cold buffer. The isolated vessels were then resuspended in buffer and kept, until use, in a plastic tube at 0 “C. For some experiments, after the isolation step, the microvessels were resus- pended in a sodium-free buffer, obtained by substituting NaCl and NaHC03, respectively, with choline-HC1 and KHCO,. When indi- cated, the microvessels were digested with 0.75 mg/ml of crude collagenase for 20 min at 37 “C. In another series of experiments the microvessels were exposed to 5 mM MSO, a durable inhibitor of the glutamine synthetase activity (12, 13) for 20 min at 37 “C. In both cases, collagenase or MSO were subsequently removed by pouring microvessels suspensions through a 86 pm pore size nylon sieve; the retained material was then extensively washed and resuspended, until use, in ice-cold buffer.

Incubation and Transport Assay-The uptake of labeled amino acids by isolated brain microvessels was measured either cumulatively a t given time intervals (up to 30 min) using fixed amino acid concen- trations or within the first 2 min as a function of amino acid concentration. When indicated, the microvessels were suspended in a buffer containing fixed concentrations of NH:HCO; and/or 100 FM 2,4-dinitrophenol at a final pH of 7.4. After the addition of the labeled test substance, 600-p1 portions of the microvessel suspension were withdrawn at various times, poured on a 44-pm pore nylon sieve on a vacuum manifold, and washed 3 times with 5 ml of cold buffer. The sieves with the retained microvessels were then placed in dispos- able tubes containing 1.8 ml of 1 N NaOH, left overnight at room temperature, and then subjected to sonication for 1 min. Portions

‘ The abbreviations used are: HEPES, N-2-hydroxyethylpipera- zine-N’-2-ethanesulfonic acid; MSO, methionine sulfoximine; MeAIB, a-methylaminoisobutyric acid; BCH, DL-fl-aminobicyclo- Z,Z,l-heptanecarboxylic acid.

5295

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5296 Effect of Ammonia on Amino Acid Uptake by Brain Microvessels were withdrawn for protein determination (14) using bovine serum albumin as standard, and 0.5 ml were transferred to liquid scintilla- tion counting vials containing an equal volume of 1 N HCI. After addition of 10 ml of Aquasol 2, the vials were counted in a Packard Tri-Carb liquid scintillation spectrometer. Nonspecific radioactivity due to the binding of the labeled amino acid to the nylon sieve was also tested in triplicate by omitting the microvessels; the radioactivity remaining on the sieve was less than 70 dpm.

Kinetic Analysis-When plotted as a function of amino acid con- centration, the initial (2 min) rate of uptake showed the presence of a saturable component superimposed on a nonsaturable one. As previously described (9, 10, 15), the latter was evaluated and then subtracted from the overall experimental curve. Following this pro- cedure, the values of the kinetic parameters of the saturable compo- nent were then calculated using the S/u uersus S "Hanes plot" which allows straightforward statistical analysis, according to Wilkinson (16). The data were subjected to nonlinear regression analysis to obtain the optimal estimate of the kinetic parameters (the nonsatur- able free diffusional component, Kd; the maximal transport velocity, Vm.,,, and the carrier affinity, K,) and to evaluate the standard error impending on them.

Enzyme Assays-The microvessels were subjected to homogeniza- tio in a Potter-Elvehjem motor-driven apparatus in the buffer appro- priate to the subsequent assay. Alkaline phosphatase was measured using 5 mM p-nitrophenyl phosphate as substrate in a reaction mixture containing 50 mM MgC12,5 mM CaCI2, 100 mM KCI, and 100 mM Tris-HCI, pH 9. The reaction was initiated by addition of 0.1 ml of the microvessels homogenate in a final volume of 1 ml. The mixture was then incubated at 37 "C for 20 min and the reaction stopped by addition of 2 ml of 1 N NaOH and by cooling in a ice bath. The insoluble material was then removed by centrifugation for 10 min a t 3000 X g, and the absorbance at 420 nm, determined for each sample, converted in micromoles.min" using a p-nitrophenol standard (17). y-Glutamyltranspeptidase activity was determined using l-y-gluta- myl-p-nitroanilide as substrate, according to Orlowski and Meister (18). 5'-Nucleotidase activity was determined in a reaction mixture containing 40 mM Verona1 buffer, pH 7.5, 20 mM MnSO,, and 1 mM 5'-AMP in a final volume of 2 ml. The mixture was incubated 30 min at 37 "C in a shaking bath and the reaction stopped by addition of 2 ml of 20% trichloroacetic acid. Insoluble material was removed by centrifugation for 5 min at 3000 X g and aliquots of the supernatants were used for inorganic phosphate determination (19). The assay was carried out in the presence or in the absence of Ni2+ ions (0.1 mM NiCI2) which selectively inhibit 5'-nucleotidase activity without af- fecting nonspecific phosphatases. Glutamine synthetase activity was measured according to Wellner and Meister (20).

Determination of Intracellular Gln-Of suspended microvessels, 10 ml (corresponding to -20 mg of protein) were centrifuged for 5 min at 4 "C at 1000 X g, the supernatant decanted, and the pellet homog- enized in 1 ml (total volume) of H20; 0.1 ml was then used for protein determination (12), and 100 pl of 40% sulfosalicylic acid were added to the remaining homogenate. After centrifugation, the supernatant was subjected to amino acid analysis in a Beckman 121 MB autolaintic amino acid analyzer using lithium citrate buffers, which allow the separate determination of Glu, Gln, and Asn.

Integrity and Viability of Microvessel Preparations-Phase-con- trast light microscopy and scanning electron microscopy showed that the isolated brain microvessels were free from contamination by nerve or glial cells (Fig. 1). Our microvessel preparations were found to be impermeable to trypan blue, a t variance with the findings reported by Williams et al. (21). Cell permeability was also tested by measuring at different time intervals the efflux of carboxyfluorescein from cells previously loaded with carboxyfluorescein diacetate (22). As shown in Table I, the release of carboxyfluorescein within the first 30 min was less than 20% of the total intracellular concentration. The microvessel suspensions were found to be enriched, with respect to the gray matter, with some enzymatic activities such as y-glutamyl- transpeptidase and alkaline phosphatase (10). Moreover, 5'-nucleo- tidase and Gln synthetase activities were still detectable after colla- genase treatment, suggestinga very close association of these enzymes with the endothelial cells (10).

Determination of Intracellular ATP Content-After a 20-min in- cubation at 37 "C in different experimental conditions, 0.5-ml aliquots of microvessels suspension were mixed with an equal volume of ice- cold 10% trichloroacetic acid containing 2 mM EDTA. After 15 min

FIG. 1. Scanning electron microscopy of isolated brain mi- crovessels. The sample was suspended in a modified Karnovsky's fixative. containing 2.5% glutaraldehyde, a t 4 "C overnight. The fix- ative was removed from the microvessels by filtration through a Nucleopore membrane of 5 pm pore size. The membrane was then dried with CO, using amyl acetate as the transitional solvent. The dried sample was then coated with gold and examined in a JEOL JSM-35U scanning electron microscope at 25 kV.

TABLE I Carboxyflwrescein efflux from isolated brain microvessels

In the presence of various concentrations of NH: ions the isolated microvessels, suspended in buffer a t pH 7.4 at 150 pg/protein/ml, were loaded with carboxyfluorescein diacetate (50 pglml). The car- boxyfluorescein efflux was followed at 37 "C within the first 30 min. The values are expressed as percentage of exit with respect to the total intracellular content of carboxyfluorescein.

NH: ions

"

Carboxyfluorescein efflux

5 min 30 rnin mM 9%

6.04 k 0.01 12.31 f 0.13 0.05 7.05 f 0.03 14.12 f 0.14 0.25 6.10 f 0.04 14.01 f 0.12 1.00 8.07 f 0.04 20.10 f 0.10

at 4 "C the samples were centrifuged 5 min at 4000 X g. The pellet was washed three times with distilled water and finally resuspended in 0.5 ml of 1 N NaOH, aliquots being then withdrawn for protein determination. In order to remove the trichloroacetic acid from the samples, the supernatants were extracted three times with water- saturated diethyl ether. The ATP levels were measured in the neu- tralized extracts by the luciferine-luciferase procedure (23) using a LKB luminometer model 1251.

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Effect of Ammonia on Amino Acid Uptake by Brain Microvessels 5297

RESULTS

Effect of NHI Ions on Amino Acid Uptake-The time course of the uptake of different amino acids in the presence or absence of 0.25 mM NH: ions is shown in Figs. 2 and 3. When ammonia was present in the medium, there was a statistically significant increase (p 5 0.05) of the uptake of Phe, Tyr, Leu, and cycloleucine (a nonmetabolizable synthetic amino acid), as well as of BCH, specifically transported by the L-system (24). At low amino acid concentrations, the effect of ammonia was dose-dependent, reaching a maximal stimulation at 0.25 mM (Fig. 4) and declining at higher concentrations. This stimulation was, in the presence of 0.25 mM NH: ions, exerted

0 BUFFER BUFFER+ NH~HCOJ

2 1 2-1 T Y R

e .- c

0

u)

c 2 2 j GLU Ac 2 4 L Y S

1 0 s 5 15 30 1 0 s 5 15 30 MIN MIN

TIME FIG. 2. A 30-min uptake of different "C-amino-acids ob-

served in the presence or absence of 0.25 mM NH: ions. Data shown are the means from three different experiments (+S.D.). * indicates p I 0.05, by Student's t test for unpaired data.

0 5 15 TlME(mm)

FIG. 3. A 15-min uptake of BCH observed in the presence (0) or absence (0) of 0.25 mM NHI ions. Data shown are the means of three different experiments +S.D.

1: 0 5 1 i

N+MI

FIG. 4. Effect of different concentrations of NH: ions on [I4C]Leu uptake. After 20-min incubation in the presence of various concentration of NH: ions, the microvessels were left 5 min in the presence of the labeled amino acid and the internal radioactivity was then measured. Data shown are the mean of three different experi- ments +S.D.

TABLE I1 Kinetic parameters of amino acid transport by isolated brain

microvessels in the presence or absence of two ammonium bicarbonate concentrations

Initial rates of uptake, measured at various amino acid concentra- tions, were subjected to statistical analysis to obtain the values of the nonsaturable free diffusional component (Kd), of the maximal velocity (Vmm), and of the carrier affinity (K,,,). The values are expressed as the mean (+S.D.) of three different determinations.

mM pmol. min" . mg protein" @f

Leucine 0.76 300+ 15 145 f 10 0.25 0.82 418 f 10 146 f 8 1.00 0.74 599 f 16 190 + 12

Phenylalanine 0.58 214f 14 1 4 0 f 9 0.25 0.66 314 f 11 135 + 15

Lysine 1.50 242 f 13 103 + 8 0.25 1.40 1 8 7 f 16 95f 6

essentially on the V,,, values, which, for Leu and Phe, were 1.4 times higher than those of controls (Table 11); the K,,, and the Kd values were unaffected.

Exposure of the microvessels to 1 mM NH: ions resulted in a further increase of the Vmax value for neutral amino acids, but there was a parallel increase of the K , values (Table 11) which explained why, in a standard assay, the uptake of a labeled neutral amino acid (present in low concentrations) was not different from controls (Fig. 4). In these experimental conditions (ie. in the presence of 1 mM NH: ions) the Kd values were unaffected also, although a slight increase of the carboxyfluorescein efflux from microvessels could be detected (Table I).

No significant modifications were apparently exerted by 0.25 mM NH: ions on the uptake of MeAIB, Glu, or Lys (Figs. 2 and 5; Table 11); the ammonia-related effects appear there- fore to be restricted to the L-system-mediated uptake of large neutral amino acids. This hypothesis is apparently confirmed by experiments in which the microvessels were digested with collagenase; such a treatment caused a 2-fold increase of the Vmax for Leu uptake, with a severe reduction of the V,,, for MeAIB uptake and without any significant modification of the K,,, values for either Leu or MeAIB. In these collagenase- treated microvessels, addition of 0.25 mM NH: ions caused a relevant increase of the V,,, for Leu uptake (Table 111).

Pretreatment of capillaries with MSO (Table IV) or with 2,4-dinitrophenol (Fig. 6), as well as omission of Na' ions from the incubation medium, completely counteracted the effects of NH: on large neutral amino acids uptake.

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5298 Effect of Ammonia on Amino Acid Uptake by Brain Microvessels

I 150,

0 10 20 30

TIME (min)

FIG. 5. [14C]MeAIB uptake observed in the presence (0) or absence (0) of 0.25 mM NH: ions during a 30-min interval, Each point is the average of three different experiments fS.D.

TABLE 111 Variation of Leu and MeAIB uptake (V-J in brain microvessels in

the presence or absence of NH: ions after predigestion with collagenase

Data shown are the mean f S.D. of three different experiments.

Control (undigested mi- After digestion with crovessels) collagenase (0.6 mJml)

pmol. min" . mg pro- tein"

pmol. min" . mg pro- tein"

Leucine MeAIB

293 & 12 387 * 11 583 2 9 877 118 4182 16 409+ 15 151 f 10 1 5 4 f 7 ~ _ _ ~ _ _ _ _ _ _ ____

I I I

0 5 15 30 TIME t m h l

FIG. 6. Effect of 2,4-dinitrophenol on the ["CILeu uptake. After 20-min incubation at 37 "C, in the presence (0) or absence of NH,' ions (0) or upon addition of 0.25 mM NH,' ions plus 2,4- dinitrophenol (A), the labeled amino acid was added and the subse- quent uptake followed for 30 min. Data shown are the mean of three different determinations f S.D.

TABLE V Gln synthetase activity in isolated brain microvessels

The enzyme activity was determined before and after collagenase treatment of the isolated microvessels (means f S.D. of three exper- iments).

nmol. mg protein". min"

Gray matter 22.0 f 0.1 Isolated microvessels

Control 2.6 f 0.9 Collagenase-treated 2.8 f 0.4

-~ __ _ _ ~ -

TABLE VI Effect of 20-min incubation in the presence or absence of NH' 4 mm .

on the intracellular Gln levels in isolated brain microvessels The intracellular Gln content was determined in microvessels

isolated in Na+-containing or Na+-free buffer or after 20-min prein- cubation with MSO. Data shown are the average of several (numbers in parentheses) determinations & S.D.

122 mM 5 mM 0.25 mM Na+ ions MSO NHdHCO, Gln

nmol/mg protein - - 3.33 f 0.10 (6) +

MSO NH4HCOs (mean ? S.D.) ____ 10 s 5 min 15min 30min

-~ __ mM mM % of control

0.25 82 f 3 178k3" 154 f 17" 142 f 14" 0.5 113k 16 113 C 3 123 & 17 110t17 0.5 0.25 1 0 6 k 2 9 107 f 6 8 2 t 6 9 0 t 16

(I Differs significantly from control, p < 0.01. __ ~__-

Correlation with Gln Levels-As shown in Table V, detect- able Gln synthetase activity was present in our microvessel preparations and remained at the same levels even after extensive collagenase digestion.* The intracellular levels of

2 p. Cardelli-Cangiano, C. Cangiano, F. Barberini, and R. Strom, _ ~ _ _ ~ __ ~

unpublished data.

GIn, measured after 20-min incubation at 37 "C, either in the presence or in the absence of Na' and/or NH: ions, were significantly higher when both Na+ and NH: ions were pres- ent in the incubation mixture (Table VI). The absence of Na' ions or pretreatment with MSO caused instead a depletion of the intracellular Gln levels even when microvessels were incubated in NH:-containing buffer (Table VI).

Intracellular ATP Levels-The intracellular ATP levels, measured after 20-min incubation at 37 "C in Na+-containing buffer, were about 500 pmol'mg of protein" and were only slightly modified by the presence of NH: ions (Fig. 7A) . When microvessels were suspended either in Na+-free buffer, with or without NH: ions, or in the presence of 2,4-dinitrophenol, the ATP concentration dropped to about 75 pmol.mg of protein" (Fig. 7B).

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Effect of Ammonia on Amino Acid Uptake by Brain Microvessels 5299 18

i n

J,A< I , , I ,

ATP pmoles- mg pmt.~i’

FIG. 7. Determination of intracellular ATP levels under different experimental conditions. A, the intracellular ATP con- centration was determined after 20-min incubation at 37 “C in Na+- containing buffer in the presence (W) or absence (0) of 0.25 mM NH: ions or upon addition of 2,4-dinitrophenol (A); B shows the ATP levels found in the Na+-free buffer after 20-min incubation at 37 “C in the presence (B) or absence (0) of NH: ions. Data shown are the means of three determinations f S.D. On the ordinate axis are reported the potential differences (normalizedper mg of protein) recorded on the luminometer when the trichloroacetic acid extract from microvessels was tested with the luciferin-luciferase system, either as such or after addition of exogenous ATP standards (the values of these standards being reported on the abscissa axis). The ATP levels existing in the microvessels result from the intersection, on the negative side of the x axis, of the resulting straight lines.

DISCUSSION

Moderately high concentrations (0.25 mM) of NH1; ions, similar to those found in severe hepatic failure and/or after portacaval anastomosis, caused i n vitro a stimulation of the L-system-mediated uptake of large neutral amino acids and BCH by brain microvessels without exerting any effect on other amino acid transport systems. The significant rise of the uptake by microvessels of the nonmetabolizable amino acid cycloleucine, observed in the presence of NH:, suggests that this ammonia-related effect cannot be due to a stimula- tion of the intracellular amino acid metabolism.

Kinetic analysis of the initial rate of uptake indicates that the presence of 0.25 mM NH: ions primarily increased the maximal influx ( VmSJ of the saturable component of a neutral amino acid L-system-mediated transport, whereas neither the K,,, nor the nonsaturable component was apparently changed. When the concentration of NH: ions was increased to 1 mM, the overall uptake of Leu did not differ from controls; this finding apparently results anyhow from a decrease of the carrier affinity which counteracts a further rise of the V,,,,., whereas the nonsaturable component of neutral amino acid uptake was only slightly modified.

As for the mechanism involved in the stimulatory effect of NH: ions, the synthesis of Gln appears to play a crucial role.

First of all, when microvessels were exposed to 0.25 mM NH: ions, the significant stimulation of the L-system-me- diated neutral amino acid transport was always consistent with an evident increase of the intracellular Gln levels. Pre- vious data from these laboratories (10) have indicated that high intracellular concentrations of Gln, obtained by sus- pending the brain microvessels in Gln-containing buffer, stimulate the uptake of large neutral amino acids. This effect appeared to be proportional to the internal concentration of Gln and was due to a selective stimulation of the rate of transport ( VmJ by the L-system. In the same series of exper- iments it had also been demonstrated that Gln was predomi- nantly taken up from the incubation mixture by the concen- trative A-system and subsequently released by the exchanging activity of the L-system resulting in a stimulation of the uptake of other neutral amino acids (10).

The essential role of Gln synthesis results also from the experiments in which the stimulatory effect of NH’ Ions ’ on neutral amino acids uptake was abolished by addition of MSO or of 2,4-dinitrophenol, as well as by the use of Na+-free buffer. Although MSO inhibits y-glutamylcysteine synthetase (25 ) , only the inhibition of Gln synthetase is known to persist after MSO removal. Moreover, in our experiments, MSO does not modify the neutral amino acids uptake in the absence of NH: ions, but its inhibitory action is limited to the NH:- induced stimulation of neutral amino acids uptake. The cru- cial role of Na’ ions for the stimulatory effect of NH:, which could seem to contradict the assignment to the Na’-inde- pendent L-system of transport, can be explained by the drop of intracellular ATP levels, which are essential for the activity of Gln synthetase, in Na+-free buffer; this is confirmed by the inhibitory effect of 2,4-dinitrophenol.

Data reported from different laboratories using immuno- histochemical procedures have indeed shown that most of the Gln synthetase activity in the brain has an astrocytic local- ization (26). Nonetheless, a recent investigation (27 ) on the cerebral uptake and metabolism of 13N-ammonia using a freezing-blowing technique in normal rats has shown that, 5 s after intracarotid injection of a I3N-ammonia bolus, approx- imately 60% of the radioactivity recovered in the brain is already incorporated into Gln, so that, in uiuo, the half-time for conversion of ammonia into Gln can be estimated not to exceed 3 s. Our finding shows that a detectable Gln synthetase activity is still present in isolated brain microvessels even after extensive collagenase digestion and support the idea that this enzyme is also present inside brain microvessel endothe- lial cells and may contribute to the “rapid conversion of ammonia into Gln.

In conclusion, in contradiction of a recent i n uiuo report (28), our results indicate that NH: ions increase neutral amino acid uptake by brain microvessels not through a direct “toxic” nonspecific mechanism on the endothelial cell mem- brane permeability, but through an elevation of intracellular concentration of Gln. Our in vitro data, as well as data obtained in vivo (27 ) , suggest indeed that brain microvessel endothelial cells participate, together with glial cells, in the small glutamate pool of the brain. Extrapolating our results to the i n vivo situation, it can be thus hypothesized that high ammonia levels result in a rise of the brain microvessel intracellular levels of Gln which may be either synthesized in situ or taken up from brain extracellular fluid uiu the concen- trative A-system (10). This will essentially lead to an in- creased efflux of Gln from the brain (29) and in turn to an overall enhanced exchange of brain Gln for blood neutral amino acids via the L-system (10).

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5300 Effect of Ammonia on Amino Acid Uptake by Brain Microvessels Acknowledgments-We thank L. Edwards and V. Peresempio for

skilled technical assistance.

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P Cardelli-Cangiano, C Cangiano, J H James, F Ceci, J E Fischer and R StromEffect of ammonia on amino acid uptake by brain microvessels.

1984, 259:5295-5300.J. Biol. Chem. 

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