Manganese (Mn) transport across the rat blood-brain barrier: Saturable and transferrin-dependent transport mechanisms

Brarn Research Bulletin, Vol. 33, pp. 345-349, Printed in the USA. All rights reserved.

1994 Copyright

0361-9230194 $6.00 + MI 0 1993 Pergamon Press Ltd.

Manganese (Mn) Transport Across the Rat Blood-Brain Barrier: Saturable and Transferrindependent Transport Mechanisms MICHAEL

Department

of Pharmacology

ASCHNER’

AND

and Toxicology,

Received

7 April

MAUREEN

GANNON

A-136, Albany Medical

1993; Accepted

12 August

College, Albany,

NY 12208

1993

ASCHNER, M. AND M. GANNON. Manganese (Mn) transport across the rat blood-brain barrier: Saturable and transferrindependent transport mechanisms. BRAIN RES BULL 33(3) 345-349, 1994.-Using a single capillary pass technique, the transport of manganese (Mn) across the rat blood-brain barrier (BBB) was characterized. Initial rate measurements (15 s) of Mn’+ [O1000 PM] accumulation in rat brains clearly indicated saturation kinetics by both l/v vs. l/s plots, and plots of v vs. [s]. Common carotid injection of freshly mixed Mn*+ with transferrin at a 1:lO molar ratio did not result in a significant change in the initial rate of Mn brain levels compared with injection of Mn*+ alone. However, when Mn*’ was incubated at 25°C in the presence of transferrin at a 1:lO ratio for up to 5 days prior to common carotid injection, the initial rate of Mn uptake by brain was incubationtime-dependent, increasing linearly with prolonged incubations. These findings suggest that the saturable component of divalent Mn transport into brain represents but one of the transport mechanisms for Mn across the BBB. A second transport system for Mn may occur by a transferrin-conjugated Mn transport system. Manganese

Blood-Brain

barrier

Transport

Rat

Transferrin

MANGANESE (Mn) is an essential metal which is especially critical during development (19). Both in the adult and the developing fetus, the brain normally contains a small amount of Mn (12). Manganese deficiency during development is associated with convulsive disorders (38,39,45). Exposure to elevated levels of Mn in adulthood results in an irreversible brain disease characterized by prominent psychologic and neurologic disturbances (6212). Manganese functions as an integral component of several enzymes important to the central nervous system (CNS), such as the mitochondrial protein, superoxide-dismutase (SOD). Another manganoprotein is glutamine synthetase (48). Glutamate is an excitotoxic amino acid (37) and one major route of its inactivation is by a high affinity uptake system into astrocytes, where glutamine synthetase, an enzyme found exclusively or predominantly in astrocytes (34) catalyzes its conversion to glutamine. Glutamine synthetase contains 8 Mn ions per octamer (49) and accounts for approximately 80% of total Mn in the CNS (48). An important determinant in the neurotoxicologic outcome of metal exposure is the rate and means of transport from plasma into the brain across the blood-brain barrier (BBB). To cross this barrier, metal ions or their complexes must be either highly lipid soluble, or possess affinity for specific carrier-mediated transport systems within the endothelial plasma membrane (7).

’ To whom requests

for reprints

Manganese enters the CNS after both oral and systemic administration (10,15,22). Employing magnetic resonance spectroscopy techniques, several research groups have described area-specific accumulation of Mn within the CNS (27,31). The dependence of Mn uptake into the CNS on iron homeostasis has also been reported (3,28). Information about the nature of Mn-binding ligands in plasma and serum and its transport mechanism across the BBB is sparse. Studies to date have focused on distribution, excretion, and accumulation of intravenous and intraperitoneal solutions of soluble divalent salts of Mn. The finding that Mr-?+ is very rapidly cleared from the blood and efficiently excreted in bile is misleading, as it appears that Mn”, and not Mn”, is the toxic species that is transported across membranes (2). Indeed, compared to divalent Mn, Mn3’ has a slower elimination rate (16) and, therefore, may have a greater tendency to accumulate in tissues. Furthermore, in view of the dependence of Mn accumulation within the CNS on iron homeostasis (3,28), the oxidation state of Mn may represent a key determinant in the differential distribution, accumulation and secretion profiles of Mn, a fact that has received little attention in experimental toxicology. Accordingly, in this study of the distribution and membrane transport of Mn, two critical issues are addressed: (a) the importance of the oxidation state of Mn as it governs the affinity of Mn to endogenous ligands, and (b) the importance of the reaction of Mn’+ with

should be addressed.

345

346

ASCHNER AND GANNON

transferrin, the iron carrying protein in the plasma. In this study we report, for the first time, on transport kinetics of Mn across the BBB and the putative role of transferrin in the transport of Mn across the restrictive BBB. METHODS The modified method of Oldendorf (36) that was utilized here permits a convenient measurement of 54Mn uptake in brain following a single capillary pass in the CNS. Radiolabeled carrierfree MnClz (specific radioactivity 148.5 acing) was purchased from New England Nuclear (Wilmington, DE). All other chemicals were of the highest analytical grade and were obtained from the Sigma Chemical Company (St. Louis, MO). Carrier Free 54MnC12(2 &i/rat) and transferrin were dissolved in a saline buffer adjusted to pH 7.4. Male rats (175-200 grams, Long-Evans, Blue Spruce Farms, ~t~ont, NY) were used in the study. Under ~ntob~bital anesthesia (45 mg/kg) rats were placed in a supine position and the anterior neck skin was incised. The common carotid artery was punctured with a 30 gauge needle, and a mixture of Mn alone (O-1000 PM) or Mn complexed with transferrin (1:lO molar ratios, respectively) was injected over a 2-3 s interval. Each injection solution also contained 2 &i of [U]-‘4C-sucrose (specific activity 10 mCi/m mole) and 2 &i of 3HOH (specific activity 5 Cilm mole), both purchased from Amersham Corporation (Arlington Heights, IL). [U]-‘4C-sucrose when injected as a bolus will exhibit a negligible penetration of the BBB and binding to erythrocytes during a single capillary passage through the brain (35). Thus, it establishes a background level for tracer trapped in the cerebrovasculature to which the uptake of Mn can be compared. By using ‘HOH, which enters the brain and distributes in the course of one capillary passage to a rapidly exchangeable space that is much larger than the capillary space, it was possible to assess whether the treatment procedure altered cerebral blood flow. Animals were sacrificed by decapitation I5 s after injection. This sacrifice time was selected on the basis of sequentially timed in&in studies (35). It is the earliest time possible for minimize tissue washout of 3HOH that is used to define the amount of the injected bolus which passes through the brain tissue, and late enough to assume that all of the nonextracted substance has been washed out of the brain. The brains were removed from the calvaria and dissolved in 8 ml Protosol (Du Pont, NEN Research Products, Boston, MA) and heated to 60°C for 24 h. 54Mn radioactivity was determined by ~-titillation spectrometry (1272 Cl~-G~ma, Pharmacia LKB Nuclear, Inc., Gaithersburg, MD), and 14C and 3H were determined by means of &spectroscopy (Beckman LS 3801, Beckman Instruments Division, Irvine, CA). To compute the brain uptake index (BUI) of Mn the 54Mn/3H ratio in the tissue was divided by the same ratio in the injected vehicle (Fig. 1). When multiplied by 100, it provides the total brain uptake index of the test substance as a percentage of 3HOH uptake. Similarly, the i4C/3H ratio in the injection solution provides the *4Cuptake index relative to the percentage of 3HOH. The difference between the two indices provides the net BUI for S4Mn over the 15-s injection period.

RESULTS

Following intra carotid injections of [U]-‘4C-sucrose, the residual sucrose in the cerebrovascular space corresponded to 0.24% (S.E. = t 0.01) of the total injected dose. Thus, mean values exceeding 0.24% represent distribution of J4Mn beyond the vascular space and into the brain parenchyma. Manganese had no effect on the diffusion of [U]-‘4C-sucrose refuting the possibility that Mn may alter the physical integrity of the BBB.

Arterial inflow contains: 54 NKIZ, 30, and 14cs”crose.

BRAIN UPTAKE INDEX =

TISSUE

54Mn,

TISSUE 3H x I.00

MIX !j4 ml, MIX '8

TISSUE MIX

1“sucrose, I4 sucrose,

TISSUE

nrx

3R

3,

x ioo

FIG. 1. Schematic representation of the Oldendorf single capillary pass. Radiolabeled Mn, sucrose and water were injected into the common carotid artery. Animals were sacriticed at 1.5 s postinjection, the brains removed and subjected to routine digestion and preparation for b- and g-scintillation counting. For the methodology employed to calculate the brain uptake index of Mn please refer to the text.

The 15-s initial rate of Mn uptake across the rat BBB is shown in Fig. 2A. A plot of uptake rate vs. [Mn] demonstrates saturation kinetics. The initial rate of Mn uptake in brain increases sharply at Mn concentrations between O-100 PM, leveling off at concentrations between 500-1000 PM. Saturation kinetics is also clearly indicated by the reciprocal plot of these data (2A) with a positive intercept on the l/v axis (Fig. 2B, inset), and a calculated K,,, of 0.85 pmol. Addition of transfetrin at a 1O:l molar ratio to Mn (lOO:lO, 50050, and 1ooO:100 PM, respectively), and immediate injection into the common carotid artery had no significant effect on the 15-s initial rate of Mn uptake across the BBB (Fig. 3). The data for “Mn uptake were compared by an overall test of significance using an F ratio derived from one- way analysis of variance (ANOVA). However, when Mn*+ was incubated at 25°C in the presence of transferrin at a 1:lO ratio (10 and 100 PM, respectively) for up to 5 days prior to common carotid injection, the initial rate (15 s) of Mn uptake by brain was incubation-timedependent (Fig. 4). %Mn brain levels increased linearly with prolonged incubations at 25”C, attaining, following a 5-day incubation time, levels that are approximately 3 fold greater than those observed upon the immediate injection of freshly mixed Mn and transferrin. DISCUSStON Although only a small portion of systemic Mn is transported into the CNS, it has a defined role both in CNS borneostasis and pathology (13,40). Accordingly, understanding of Mn uptake across the BBB is essential for providing the framework for understanding its metabolism and disposition in mammalian CNS. The mechanism of Mn transport into rat brain has been established by measuring the initial rate of Mn uptake, demonstrating

Mn UPTAKE

ACROSS

THE BLOOD-BRAIN

347

BARRIER

(4

0

A$, , , , , , 0

200

l/s T

400

,

,

600

1

/

400

[Mnl ,

,

,

,

1000

FIG. 2. (A)Concentration dependence of 54MnZ+ uptake at 15 s after common carotid injection. Each point represents (B) Reciprocal plot of “‘Mn2+ uptake data from A. Solid line represents least squares fit to data.

saturation kinetics. The 15-s uptake data conform to MichaelisMenten kinetics with a K,,, for transport closely approximating the K,,, of Mn transport in astrocytes (5). The relatively high K, (0.85 pmol) is consistent with an efficient uptake mechanism for Mn” on capillary endothelial membrane. In contrast to its binding affinity for albumin, Mn binds to transferrin more avidly than Zr?+ and Cd’+ (3,43). Manganese binding to transferrin is time-dependent, with a greater fraction of radioactive 54Mn associated with the chromatographic peak of transferrin upon prolonged incubations of the two compounds (43). When complexed with transferrin, Mn is exclusively present in the trivalent oxidation state, with 2 metal ions tightly bound to each transferrin molecule (1). A bicarbonate-binding site is activated for each bound Mn ion, a step requiring carbonic anhydrase-the enzyme converting CO2 and Hz0 to bicarbonate. In plasma, oxidizing agents such as ceruloplasmin may play a role in the oxidation of Mn*+ to Mn3’ (16). Other ligands for Mn*+ must exist, however, because plasma that is saturated with Fe*‘. Zn*‘, and Cd*+ will bind Mn*’ (43). Inasmuch, as the concentration of Mn*+ m normal peripheral blood is less than 0.1 PM and the level of free Mn*+ in blood is infinitesimally small (less than 0.02 ,uM; 44) the uptake of Mn*’ by the CNS will increase as a linear function of concentration for a considerable range of Mn values. Furthermore, the specificity of the carrier ensures no interference from the major divalent cations of the blood. It is likely that minute amounts of Mn*’ in plasma exist, according to the Law of Mass Action, as the chloride complex. While the amount of this complex in plasma at any one time must be small, the Law of Mass Action states that a definite infinitesimal amount is always present. It also holds that if any Mn*’ should leave the system by dissolving in a lipid membrane, the protein-Mn*+ complex should dissociate to maintain equilibrium. The plausibility that this mechanism is involved in the transport of Mn across the membrane remains open for in situ experimentation.

1

1

12li”

the mean i- SEM of 4-8 animals.

In view of the slow oxidation of divalent Mn to the trivalent species (43) the contribution of the latter species to Mn accumulation in the brain must be addressed. Our findings clearly suggest that the saturable component of divalent Mn transport into brain represents but one of the transport mechanisms for Mn across the BBB (29) and that a second transport system for this metal may occur by a transferrin-conjugated Mn transport system. The role of transferrin in Mn transport across the BBB is favored by several lines of evidence. In the rat brain, the return of Mn to control levels is significantly slower when Mn is administered beyond days 18-20 of life (41) a developmental period which coincides with the appearance of immunocytochemical localization of transferrin (1 l), and carbonic anhydrase (42) in the brain. Ontogenic studies in rats suggest an age-related retention and distribution of ingested Mn. Newborn rat pups younger than 18 days show 4 fold greater brain levels of Mn (s4MnC12) compared with adults (28). Manganese uptake in the brain of 6-day-old suckling rats is 34 times higher compared with adult females (26). Brain concentrations of Mn from a single ingested dose of Mn304 are easily detected in infants but not in mature animals (9). The retention of Mn is also a function of age at exposure. High levels of Mn in brain tissue are found in preweanling rats dosed daily with Mn304 for 12-27 days postnatally. These observations may be linked to the immaturity of the blood-brain barrier (8). There is additional theoretical and experimental evidence that transferrin, the principal Fe-carrying protein of the plasma, functions prominently in Mn transport across the blood-brain barrier. In the absence of Fe, the binding sites of transferrin can accommodate a number of other metals including gallium, copper, chromium, cobalt, vanadium, aluminum, terbium and plutonium, raising the possibility that transferrin functions in vivo as a transport agent for many of these metals. Fe” is taken up by cells after transferrin binds to a specific cell surface receptor (20,46), and the transferrin receptor complex is internalized (23,25). Trans-

348

ASCHNEK AND GANNON

NoTransferrin ransferrin at 10 x [Mn] 4-1

FIG. 3. Relationships between 10 fold molar excess of transferrin in the presence of 3 different concentrations of Mn on the initial rate (15 s) uptake of %&In’+in rat CNS. Each point represents the mean 2 SEM of 4-8 animals.

ferrin receptors

have been demonstrated

on numerous

cell types

(33), including CNS capillaries (21). Transferrin has also been shown to enter brain endothelial cells via receptor-mediated endocytosis and to subsequently enter the brain (14). At normal plasma Fe concentrations (0.9-2.8 &ml), normal iron binding capacity (2.5-4 /.&nl), and at normal transferrin concentration in plasma, 3 mg/ml, with 2 metal-ion-binding sites per molecule (M, 77000) of which only 30% are occupied by Fe3+stransferrin has available 50 p mole of unoccupied Mn3+ binding sites per liter (24). Accordingly, analogous to the situation with aluminum, Mn may not need to displace Fe to bind to transferrin. It is

3

2

: 1

0 0

1

2

3

4

5

6

Day

FIG. 4. The incubation-time-dependence (up to 5 days) of the initial rate of brain Mn uptake (10 PM) in the presence of 10 fold molar excess of transfeti (100 PM). Each point represents the mean -+ SEM of 4-8 animals.

also notable, that Mn3’ has a similar ionic radius and chelating behavior to Fe3+ (31). Examination of the distribution of transferrin receptors in relationship to Mn accumulation in the brain is intriguing. Manganese concentrations are highest in the pallidum, thalamic nuclei, and substantia nigra (8). Interestingly, the highest Fe concentrations are found in these structures as well (17). The areas with the highest transferrin receptor distribution (18) are not identical to those which concentrate Mn. However, Mn-accumulating areas are efferent to areas of high transferrin receptor density, suggesting that perhaps these sites may accumulate Mn through neuronal transport. For example, the Mn rich areas of the ventral-pallidum, globus pallidus, and substantia nigra receive input from the nucleus accumbens and the caudate-putamen (30,47)-two areas abundantly rich in transferrin receptors. The presence of transferrin-receptors in the neuropil may provide the anatomical substrate for the “internalization” of Mn3’-transferrin conjugates into neurons. Furthermore, just as a dissociation exists between Mn and transferrin with regard to localization, a dissociation could also exist with regard to transport rates, analogous to Fe and transferrin transport into the CNS (4). In summary, these studies provide an estimate for the initial rates of Mn uptake into the rat CNS. The system for divalent Mn operates in the linear range of l/s vs. l/v, and a second putative transport system for trivalent Mn as a transferrin conjugate is postulated. The relative contributions of each transport mechanism to CNS-Mn accumulation should be critically evaluated. ACKNOWLEDGEMENTS

This project was supported in part by the National Institute of Environmental Health Sciences 05223, and the Biomedical Research Support Grant Program !#7RRO5394-26.

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Mn

UPTAKE

8. Bradbury, 9.

IO.

Il.

12.

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