Effects of mercury and lead on rubidium uptake and efflux in cultured rat astrocytes

Brain

Research

Bulletin.

Vol. 26, pp. 639442.

(1361.9230191 $3.00 + .OO

0 Pergamon Press plc, 1991. Printed in the U.S A

RAPID COMMUNICATION

Effects of Mercury and Lead on Rubidium Uptake and Efflux in Cultured Rat Astrocytes MICHAEL

ASCHNER,”

and fDivision

RICHARD

CHEN* AND HAROLD

K. KIMELBERG*l-

*Department of Pharmacology and Toxicology of Neurosurgery, Albany Medical College, Albany, NY 12208 Received 27 September 1990

M., R. CHEN AND H. K. KIMELBERG. Effects of mercu~ and lead on rubidium uptake and eflux in cultured rat BRAIN RES BULL 26(4) 639-642, 1991 .-Astrocytes readily sequester lead and mercury (8, 10, 19, 22). Accordingly, studies were undertaken to assess the effects of lead and mercury on homeostatic functions in neonatal rat brain primary astrocyte cultures. Both inorganic and organic mercury, but not lead, significantly inhibited the initial rate (5 min) of uptake of “6RbCl, used as a tracer for K+ , at concentrations of lo-100 JLM. Mercury and to a lesser extent lead also stimulated the efflux of intracellular *6Rb+ at 10-500 PM. These observations suggest that the astrocyte plasma membrane may be an important target for lead and mercury, and that relatively low concentrations of these heavy metals should inhibit the ability of astrocytes to maintain a transmembmne K‘ gradient. ASCHNER,

astroqtes.

Lead

Mercuric

mercury

Methylmercury

Rubidium

Nat -K + -ATPase

Astrocytes

Rat

cells (13,20). The plasma membrane is thought to be the target organelle for these effects, although both mercury and lead are also potent inhibitors of intracellular enzymes and metabolic processes (16,25). The present study was undertaken to examine the effects of mercury and lead on astrocytic function under the assumption that interference with the active “pumping” process of K+ may represent a neurotoxic mechanism for mercury- and lead-induced injury.

A predisposition of astrocytes for lead- and mercury-inflicted damage offers a possible explanation for the neurotoxicity of these metals, since both accumulate within glial cells (8, 10, 19, 22). It has been suggested that sequestration of heavy metals within astrocytes may constitute a reservoir for their continuous release into the extracellular fluid and contribute to the cytotoxicity of adjacent neurons (5,lO). However, a direct toxic effect on astrocytes themselves would lead to failure of astrocyte homeostatic functions resulting in neuronal impairment, injury and death. The existence of ion gradients between the intra- and extracellular fluids is important to the optimal function of many cells. Like almost all cells astrocytes have a high internal concentration of K’ but their plasma membranes have an unusually high selectivity for KC, leading to membrane potentials that are more negative than most neurons (14). According to the concept of “K+ spatial buffering” (14), localized K+ release from neurons selectively depolarizes neighboring astrocytic membranes resulting in an electric potential between this and more distant region of the astrocytic syncytium (14). This difference in electric potential results in a membrane current flow, carrying K+ from its extracellular site of release into the astrocyte and out again at more distant regions due to the high selectivity of the membrane to Kt ions. In maintaining the correct balance of K+ ,the astrocytes are serving to maintain a proper milieu for neuronal function (11). It has long been noted that mercury and lead compounds can influence ion, water, and nonelectrolyte transport in a variety of

METHOD

86RbC1 was purchased from Amersham Corporation (Arlington Heights, IL). Methylmercuric chloride was purchased from K & K Labs, ICN Pharmaceuticals (Plainview, NY). Cold vapor atomic absorption analysis by the method of Magos and Clarkson (15) indicated that >99% of the total Hg was in the organic form. Other compounds were of the highest analytical grade and were purchased from the Sigma Chemical Company (St. Louis, MO). Tissue Culture Cultures were prepared according to the method by Frangakis and Kimelberg (7). Briefly, the cerebral cortices were removed, the meninges were carefully dissected off, and the brain dissociated using a neutral protease (Dispase; Boehringer Mannheim, Indianapolis, IN). The cells were seeded at 8 X lo4 viable cells/ml

639

ASCHNER.CHEN ANDKIMELBER(r

640

120

0

A

. 100 it

witout I mM ouabain with I mM ouabain I

l

with

1 mM ouabain

80 -

60 -

40 -

20 * 0

;:

,

. , . , ,.*.,

. . . ,.,., I

*

,

, ,.,C

[Ml MeHg

IMI H&I2

FIG. 1. Effects of 30 tnin incubation of cultured astrocytes with O-5 X lob4 M MeHgCl (A) and HgCI, (B), on the 5 min uptake of *‘Rb+ Uptake was measured in normal HEPES buffer (see tbe Method section) in tbe presence or absence of 1 mM ouabain. Ouabain and the mercurials were added at the same time as the s6Rb. Statistical analysis was carried out by one-way analysis of variance (ANOVA; p
in 12well trays (1 ml of cell suspension and 4 cm’ growing area per well) and grown at 37°C in a 5% CO,/951 0, atmosphere in Eagle’s minimum essential medium with 10% horse serum (GIBCO, Grand Island, NY) plus 100 units penicillin and 100 bg streptomycin per ml. About 2-3% of the seeded cells attach to the culture dishes and attain a saturation density of 2-3 X lo4 viable cells/cm2 by 3-4 weeks. These cultures show >95% staining of the cells for the astrocyte marker, glial fibrillary acidic protein (GFAP). Experimental Protocol for Rb + Uptake The growth medium was removed and the cells were rinsed rapidly with 4 x 1 ml of a HEPES-buffered Ringer’s solution containing 122 mM NaCl, 3.3 mM KCl, 1.2 mM KH,PO,, 1.3 mM CaCl,, 0.4 mM MgSO,, 10 mM D-glucose, and 25 mM HEPES acid adjusted to pH 7.4 with NaOH (total [Na+] 140 mM). The total osmolality was 300 mOsm. Cells were incubated in 1 ml medium containing MeHg (O-5 x loo4 M), at 37°C for 30 minutes in a CO, incubator prior to the actual transport measurements. Uptake was initiated by aspirating the medium and adding 0.5 ml of prewarmed buffer containing radiolabeled 86RbC1(specific activity 1 @i&l; l-8 mCi/mg rubidium). Uptake was terminated by rapid aspiration of the medium, followed by 4 X 1 ml washes with 0.29 M mannitol solution (4°C) buffered with 10 mM Tris/rris nitrate, and also containing 0.5 mM calcium nitrate to maintain cell adhesion. The solution was titrated to pH 7.4 with HNO,. Cells were dissolved in 1 ml 1 N NaOH and aliquots (800 ~1) were removed for counting and protein determination (100 ~1) according to the rapid method of Goldschmidt and Kimelberg (9). Experimental Protocol for Rb+ Eflux

For these measurements it is desirable to achieve isotopic equilibrium with the intracellular concentration such that the total specific activity inside the cell equals the specific activity in the outside medium, i.e., all intracellular compartments are equil-

ibrated. This was done by allowing cells to take up s6Rb for 90 min, as described in the preceding section, and then wash the cells to remove external radioactivity. The efflux was measured by removing 20 ~1 aliquots of medium at various time points up to 120 min. The experiments were terminated by aspirating the medium followed by the same mannitol wash as described above. The cells were then dissolved and cell protein content and radioactivity were determined as described in the preceding section. The initial intracellular content of g6Rb+ at zero time was determined from the sum of the counts leaving the cells during the time of the experiment plus those remaining in the astrocytes at the end of the experiment. The data were calculated in cpm/mg protein, and expressed as the percent 86Rb retained in the cells at each time point. RESULTS The effects of MeHg, and HgCl, on the initial rate (5 min) of 86Rb+ uptake is shown in Fig. 1. *6Rb+ uptake was measured in cells incubated in either HEPES-buffer or in HEPES-buffer containing 1 mM ouabain added at the same time as 86Rb’. The difference between 86Rb+ uptake in the presence and absence of ouabain, reflects transport via the Na+-dependent component of 86Rbf (K+) transport. MeHf (Fig. 1A) and HgCl, (Fig. 1B) added at the same time as s RbCl, inhibited the initial rate of 86Rbf uptake in a dose-dependent fashion. Statistical analysis of the data was carried out by one-way ANOVA (p
MERCURY

641

AND LEAD TOXICITY IN ASTROCYTES

TABLE 1 n

control IO PM 100 pM

0

500 MM

El l

SUMMARY OF THE lC,, OF MERCURIALS AND LEAD ON “Rb + UPTAKE AND EFFLUX IN CULTURED ASTROCYTES

MeHg + Hg+ + Pb+ +

80 60

120 ]C

0

-I

control

l

IO pM

.

100 PM 500 PM

0 80 60 40 20 0 0

I

I

50

100

150

time (mio)

FIG. 2. Time course of s6Rb+ efflux in primary astrocytes incubated with O-5 X 10mJ M MeHgCl (A), HgCl, (B), and PbCl, (C). Astrocytes were loaded for 90 min with *6Rb+ by incubating with a tracer concentration of 86RbCl (1 @i/ml). They were then washed, and at time 0 the metal was added in HEPES buffer. Values shown are means of quadruplicate cell preparations (S.E.M.
DISCUSSION

In theory, the cell membrane is the fist barrier to encounter mercury or lead. Since it is both very accessible and functionally important, it is not surprising that many cellular effects of these toxins can be ascribed to interactions at cellular membranes. Many of these effects appear to be associated with ion transport processes across membranes. While the inhibitory actions of lead and mercury on membrane transport processes have been studied in several cell types (22,23), little is known about their effects on astrocyte ion transport. Since astrocytes seem crucial in maintain-

Uptake 04)

Efflux (Ml

-2x10-5 <10v5 no effect

10-5-10 a -5x10 4 5x10 T

ing the ion homeostasis of the brain (11) any such effects could be related to the neurotoxicity of mercury and lead. A strong case may be made for the membrane toxicity theory for both lead and mercury. In virtually every tissue that has been studied, both organic and inorganic mercury compounds appear to inhibit Naf-K+-ATPase (2, 4. 21). MeHg inhibits both the active, ouabain-sensitive cation flux, and the Na+-Kf-ATPase activity measured in cell homogenates or membrane fractions (4.17). The lack of time lag for the onset of inhibition of Na+K+-ATPase activity suggests that the critical inhibitory site is in the plasma membrane itself (13). Pb+ + has also been reported to inhibit Nat-K+-ATPase activity in rat brain homogenates (3). and brain synaptosomes (6). Increased membrane permeability to cations would be expected to rapidly depolarize the membrane potential and reduce transmembrane ion gradients resulting in the inhibition of transport processes which are dependent upon such gradients. Thus, one would predict that Hg+ ‘~, MeHg’ , and Pb + + would affect a wide range of membrane transport systems secondary to Na ’ K+-ATPase inhibition or the dissipation of transmembrane ion gradients. This prediction is supported by previous observations of the inhibitory effect of both organic (1) and inorganic mercury (5) on a Naf -dependent transport system prevalent in astrocytesthe uptake of L-glutamate. It is further corroborated by a dose-dependent increase in the cell volume of astrocytes exposed to MeHg, presumably due to influx of NaCl ( 1). Since astrocytes are thought to take up K ’ by spatial buffering of KC1 uptake or Nat-K+ pump driven uptake (12). the loss of Naf -K+-ATPase activity and transmembrane Kt gradients can lead to loss of the rigorously controlled K’ concentration in the extracellular fluid compartment resulting in enhanced neuronal firing and transmission. In addition. functional impairment of Na+-dependent processes, such as the uptake of L-glutamate from the extracellular compartment may lead to an excitotoxic on N-methyl-D-aspartate receptor-containing neuresponse, rons (18). The present study demonstrates that at concentrations attained in the CNS, both Hg+ + and MeHg + affect K ’ uptake and efflux in astrocytes. Among the many homeostatic roles proposed for astrocytes are acid-base control, uptake and inactivation of extracellular glutamate and other transmitters, and control of blood-brain barrier permeability (11). Interference with one or more of these functions may lead to neuronal impairment. Accordingly, the potential involvement of astrocytes in the neurotoxicity of mercury warrants further consideration.

ACKNOWLEDGEMENTS

This project was supported in part by BRSG S07RR05394-26 and the National Institute of Environmental Health Sciences ES 05223 awarded to M.A., and National Institute of Neurological Diseases and Stroke NS 23750 awarded to H.K.K.

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ASCHNER,

CHEN

.AND KIMELBERO

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