Developmental studies of the uptake of choline, gaba and dopamine by crude synaptosomal preparations after in vivo or in vitro lead treatment

Brain Research, 187 (1980) 383-402 ~('~Elsevier/North-Holland Biomedical Press

383

D E V E L O P M E N T A L STUDIES OF THE U P T A K E OF CHOLINE, GABA A N D DOPAM1NE BY C R U D E S Y N A P T O S O M A L P R E P A R A T I O N S A F T E R IN VIVO OR IN VITRO LEAD T R E A T M E N T

PEGGY B RAMSAY*, MARTIN R. KRIGMAN and PIERRE MORELL** Btologtcal Sctence~ Research Center and Department oJ Blochetms try and Nutrttlon, School of Medtcme, UntverstO' o! North Catohna, Chapel Htll, N C 27514

(Accepted July 5th, 1979)

Key woJds, transmitter uptake - - transmitter release -- synaptosomes - - choline - - GABA --

dopamme

SUMMARY The kinetics of sodmm dependent, high affimty uptake of choline and dopamme by striatal synaptosomal preparations and of GABA (gamma-aminobutyric acid) by cortical synaptosomal preparations have been examined during the development of Long-Evans control and lead-treated rats. Chohne uptake was very low until 12 days postnatally, then the Vmax increased and approached adult values of 29 pmol/mg prot/rain w~thm a week. GABA uptake was somewhat elevated at birth and only after three weeks did ~t decrease to the adult value of 0.7 nmol/mg prot./mm. Dopamme uptake was low at birth, developed linearly with age and by 30 days postnatally approached the adult value of 68 pmoles/mg prot./min. The h~gh affinity uptake constants (chohne, 0.66 # M ; GABA, 4.4/~M, and dopamine, 0.31/~M) did not change markedly during development. S~mflar studies were conducted with rats treated at the h~ghest lead dosage which d~d not result m weight loss (100 #g lead as lead acetate/g body werght/day via intubatlon). Blood and brain lead determinations confirmed a substantial lead exposure. Such chromc exposure did not markedly affect the amount or developmental pattern of uptake of the putative neurotransmitters. The effect of 2.5 "~, 10-5 M lead acetate in vitro on the kinetics of h~gh affinity uptake of these compounds into preparations from 20-26-day-old rats was investigated. When uptake was assayed m the absence of calcium, lead caused a 20"o Present address Pathology Research 151B, Veterans Administration Hospital, 3801 Miranda Avenue, Palo Alto, Cahf 94304, U S A. ** Fo whom reprint requests and correspondence should be sent at' Biological Sciences Research Center, 220H, D~vlslon of Health Affairs, University of North Carolina, Chapel HJII, N C 27514

384 increase m the Vmax for dopamme. Th~s st~mulatton was reduced if samples were assayed m the presence of 1 mM CaCI~,. The K,, lbr high affimty uptake of these neurotransmltter-related compounds was not affected by lead. In other studies, crude synaptosomal preparations were preloaded w~th neurotransmitter by premcubation with radmact~ve chohne, GABA, or dopamine. Release of radmact~ve neurotransm~tter, e~ther spontaneous or m response to potassmm depolanzatmn, was quantitated and correlated with the presence or absence of 2.5 10-'5 M lead and/or 10-3 M calcmm ions. Lead shghtly inhibited calcium dependent spontaneous release of dopamme. Lead also appeared to partially substitute for calcium m the potassium depolarized release of dopamme and GABA, although subtraction of the spontaneous (potassium independent) component reduced the magnitude of the lead effect.

INTRODUCTION

The neurotoxmologlcal effects of lead exposure are well known although the underlying mechanisms are not understood 15,-'z. The greater frequency and the severity of symptoms among children suggest a greater susceptibility to lead l'~ and has prompted an interest in possible subclinical indicators of lead exposure m children 51. In an effort to elucidate the neurobiological basis of these symptoms, animal models have been established to study the effects of chronic and acute lead exposure 1,~,'-'' 38.42,4b,49.

A parameter of synaptogenesls and neuronal differentiation of potentml utlhty is the sodium dependent, high affinity uptake of neurotransmitters or their precursor by nerve endings 7,z7. Of particular relevance to th~s study is a report by Sdbergeld and Goldberg 4a, associating lead exposure m mice with hyperactivity and with a diminution of choline and dopamme uptake by the forebram synaptosomes isolated from treated ammals. Several studies also indicate that lead added in wtro perturbs some parameters associated with the neurotransmlss~on process 3,4,23,3~,37-4~ In the present study, we have pursued these observatmns from other laboratories by making a detailed study of the kinetic constants of choline, dopamine and GABA uptake by crude synaptosomal preparations from tissue from normal and chronically lead-treated Long-Evans rats during the first 30 days of postnatal development. The sodium dependent, high affinity uptake of choline and dopamme was measured m synaptosomal preparatmns from the corpus stnatum ; uptake of GABA wa~ measured in synaptosomal preparations from the cortex. These regions were selected because they are enriched in the high affinity uptake sites which were to be studiedS,3'z.4~,:"L We also studied the effect o f m vitro application of lead on several parameters of transmitter metabolism. This allows for the use of lead concentrations much higher than can be achieved in vlvo without the complicating secondary effects of undernutnhon. Uptake of choline, GABA and dopamme were studied as a function of the presence or absence of lead and/or calcium, which were set up to be analogous to the in vlvo investigations. The effect of calcium was studied because of the possibility that

385 toxic effects of lead are brought about by competition with calcmm at critical sites. Another parameter studied in vitro was the depolarization coupled release of transmitter from synaptosomes. In these studies elevated potassium was used to bring about depolarization and the influence of calcium and lead on this release was investigated. MATERIALS AND METHODS Materials

The [methyl-3H]chohne was obtained from Amersham Searle (Arlington Heights, Ill.) at a specific activity of 1.0 Cl/mmol Portions of the choline stock were evaporated to dryness and redlssolved m distilled water ~mmedmtely before use. Dopamme, [ethyl-l-3H]dlhydroxyphenylethanolamme and [2,3-3H]y-aminobutyric acid were obtained from New England Nuclear (Boston, Mass) at specific activities of 21.421 Ci/mmol and 36.73 C~/mmol, respectively. These radioactive compounds were periodically chromatographed to assure their purity. Choline was apphed to cellulose plates (GI44D Carl Schhecher and SchueIl, Dassel, G.F.R.) which were developed with n-butanol:ethanol:acetic acld:H20 (8:2:1:3). GABA, also applied to cellulose plates, was chromatographed in n-butanol :acetic acid :H20 (25:4.10). Dopamine was chromatographed on Quantum silica gel plates (Kontes, Vineland, N.J.) with a solution of n-butanol:methylene chloride:acetone:methanol:formic acid:H20 (6.20:4:8.12). The compounds were visuahzed by standard techmques 39,~8. All non-radioactive neurotransmitters, metabolites and inhlbitors used in the study were the best available grades and obtained from Sigma Chemical Co. (St. Louis, Mo.) except that hem~cholinium-3 and pargyline were obtained from Aldrich Chemical Co. (Milwaukee, Wlsc.) and Abbott Laboratories (Chicago, Ill.), respectlvely. The sucrose used throughout the study was a special lead-free preparation (Beckman, Palo Alto, Cahf.). The uptake assay medium was Hanks Balanced Salt Solution (Glbco, Long Island, N.Y.) which contained 136.9 mM NaC1, 5.4 mM KCI, 0.3 mM Na2HPOa and 0 4 mM KH2PO4; it was supplemented with I 0 mM CaCl2, 1.8 mM MgSOa.7H20 and 25 mM glucose. The pH was adjusted to 7.4 with NaHCO3. All other routinely used reagents were locally available reagent grade chemicals. Antmal care and treatment

Long-Evans hooded rats (Charles River Laboratories, W~lmington, Mass.) were maintained on Purina Laboratory Rat Chow and tap water ad lib~tum. Litters were adjusted to 8 rats within 1 day of birth. The lead treatment began on day 2 via gastric intubatlon of a solution of lead acetate (18.3 mg/ml) equivalent to 100/zg lead per gram of body weight. Paired control litters received an equivalent amount ofdelonized water by the same method. The rats were dosed six days per week for up to 30 days. For experiments requiring the addition of lead m vitro, naive Long-Evans rats, 20-26 days old were used to prepare the tissues. Subcellular fraetwnatwn

Rats were killed by decapitation and the cortex and corpus striatum were rapidly

386 removed and dissected on a chilled glass plate. Tissue from several rats was pooled, quickly blotted and weighed, then homogenized 15 strokes at 400 rpm m 20 volumes of 0.32 M sucrose. Potter-Elveh}em tissue grinders, with a clearance of 0.025 cm, were utilized (special order items from Kontes (Vineland, N.J.)). The crude homogenate was transferred to Corex tubes and was centrifuged at 1000 ~, g at 4 ~'C for l0 mm in the SS34 rotor of a Sorvall RC2B centrifuge. The supernatant fraction was decanted and recentrifuged at 17,000 ~: g for 15 rain at 4 "C. The resultant P2 pellet (crude synaptosomal fraction) was covered with the original ~olume of ice-cold 0.32 M sucrose and was held on ice. Immediately prior to initiation of the uptake experiments, the pellet was resuspended in the sucrose by gentle agitation and homogemzation for five strokes to obtain a uniform suspension.

Analysis of neurotransmitter uptake Uptake experiments were imtlated by adding 0. l ml of a synaptosome suspension containing 0.8-1.5 mg protein to 0.9 ml of supplemented Hanks balanced salt solution, and transfer o f the incubation tube to an Eberbach shaking water bath at 37 °C. Pargyline (0.017 g/liter) and ascorbate (0.2 g/liter) were added to the medium for dopamine uptake. Blanks were treated identically except that the NaCI in the m e d m m was replaced with sucrose. Experiments where lead was added m vitro were identical. except that 0.010 ml of a freshly prepared solution of lead acetate at the indicated concentration was added to the incubation medium of the samples immediately prior to the addition of the synaptosome preparation. Each sample was preincubated for 5 minutes to allow the temperature to equilibrate at which time 0.01 ml of a solution containing 1-2 Ci/ml of [aH]neurotransmitter was added to begin the uptake process. After an incubation of 2-4 rain, each sample was removed and uptake was terminated by dilution with 5 ml of ice-cold medium and placed on ~ce until 24 samples were collected for centrifugatlon. Samples were centrifuged in the SM 24 rotor of a Sorvall RC2B at 17,000 / g for 15 min at 4 °C (incubations and centrifugation were carried out in the 10 ml polyethelene tubes for this rotor). The supernatant was discarded and the surface of the pellet was gently washed with 5 ml ice-cold saline. After the addition of 0.5 ml of Protosol (New England Nuclear), the samples were incubated at 37 °C for at least 15 mm to dissolve the pellets. Samples were then transferred from the tubes to scintillation vials with two 5-ml rinses of scintillation cocktail (3 g of 2,5-&phenyloxazole per liter of xylene) and radioactivity determined in a Beckman LSC-100 scintillation spectrophotometer. Routinely, an experiment conSlSted of duplicate samples and duplicate blanks at each of six neurotransmitter concentratlons. The data obtained from each experiment were used to obtain Km and Vmax values; a linear regression analysis was used to obtain the best straight line fit to the data on a Lineweaver-Burk plot.

Assay for neurotransmttter release In principle, the system used was that of Haycock et al. 18 with minor modifications. The crude synaptosomal fraction was prepared in the same manner as for an

387 uptake assay. Then 2 ml of the resuspended Pz pellet were added to 8 ml of the uptake medmm m 30 ml Corex tubes and 1-4 ~tC~ of trmated neurotransmitter was added. The tubes were then covered and reverted several t~mes to thoroughly m~x the contents. After 10 mm of incubation, the contents of the tubes were poured into Erlenmeyer flasks contaimng 90 ml of ice-cold calcmm-free medium. The flasks were shaken briefly to mix the synaptosomes prior to the withdrawal of each sample. A Milhpore glass filtration apparatus (Mdhpore, Bedford, Mass.) containing Whatman GFA glass fiber filters (Fisher Scientific Co., Raleigh, N. C ) was utlhzed to collect and hold the synaptosomes for the release studies. Two milhhters of regular medium minus calcium at room temperature were passed through the glass fiber filter. Then 2 ml of the cooled and diluted uptake m~xture containing the preloaded synaptosomes were placed on the filter and most of the liqmd was removed by the apphcatmn of gentle vacuum. Two mllhhters of wash solution (uptake medmm lacking CaCI.,) were added and after 0.3 rain, gentle vacuum was apphed for 0 2 ram. A thin film of hqmd was left on the filter to insure that the synaptosomes d~d not dry out. Th~s procedure was repeated 5 more times so that the synaptosomes were washed 6 t~mes w~thin 3 ram. The seventh 2 ml p o m o n of medmm was the test solution and contained some combination of CaCle, Pb(C~_H30.~),, and elevated KCI. This rinse was collected m a scmtlllatmn vial and the filter was carefully placed m a separate vial. When studying the effect of lead on release, the last 2 of the 6 precollected rinses as well as the seventh portion contained 2.5 z 10-~ M Pb(C,,H302),,. Scmtlverse (Fisher, Raleigh, N. C.) was added to the wals; it was necessary to add some water to the vmls containing filters to avoid phase separation. Samples were kept overmght to allow chemiluminescence to subside before quantltatlon of radioactlwty m a Beckman LSC-100 scintillation counter. The amount of neurotransm~tter released was expressed as per cent release and was determined by: o~ Release =

CPM filtrate CPM filter + CPM filtrate

," 100

The radJoactlwty measured m release experiments involwng preloadlng w~th radmactive GABA or dopamme was assumed to be GABA or dopamme, respectively based on detailed studies of this system by others6, zg. In the case of preloading with radioactive choline, we assume that the label was converted to acetylcholine and released as such, since this has been demonstrated in other laboratonesl7, ~4. However, since the acetylchohne released is rapidly cleaved by acetylchohnesterase 3.5, the released material is referred to as ace@choline (chohne) since we did not quantitate the distribution of label between these compounds.

Lead determinations Individual samples of brain regmn homogenates did not contain enough lead for satisfactory measurement. Therefore, the tissue from 4 rats was combined for lead determmatmn m regional homogenates and corresponding synaptosomal fractmns. Blood samples were drawn before sacrifice. Lead determinations on both blood and

388 b r a i n samples were carried o u t by a t o m i c a b s o r p t i o n spectroscopy using a modified Delves cup m e t h o d lz. Blanks were p o r t i o n s of 0.32 M sucrose used to homogenize a n d centrifuge the tissue. Protein determinations

The a m o u n t of protein in the crude s y n a p t o s o m a l p r e p a r a t i o n s was estimated by the m e t h o d o f Lowry et al. al. RESULTS Control experiments

Several c o n t r o l experiments were performed to establish the characteristics of the s o d i u m dependent, high affinity uptake a n d to c o m p a r e those characteristics with the descriptions of the uptake systems f o u n d in the literature. Electron micrographs o f the crude s y n a p t o s o m a l fraction (P2 fraction) resembled those p u b h s h e d by others 11,t3,16. The linearity o f u p t a k e with respect to time a n d p r o t e i n c o n c e n t r a t i o n was established for each c o m p o u n d for y o u n g a n d a d u l t animals. O n the basis o f these TABLE I Inhibition of high affinity uptake by spectfic inhibitors and ouabain Neurotransmitter

Age

Choline

22 days

.

.

.

Htghest concentration*

15 days Adult

Dopamine

Uptake (prnol/mg/mtn) .

Adult

GABA

Inhibitor

15 days Adult

None 0.05/~M HC-3*** 0.1 mM ouabam None 0.05/~M HC-3 0.1 mM ouabam

1.21 0 0 29.7 0 0

.

.

.

.

.

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Lowest eoncentratton**

3.5 0.7 0 8.0 0 0.19

None 0.1 mM DAB 0.1 mM ouabam None 0.1 mM DAB 0.1 mM ouabaln

1541 0 397.6 I 188 0 0

96.2 0 37 2 78 0 0

None 0.1/,M DAS 0 1 mM ouabam None 0.I/~M DAS 0.1 mM ouabain

67.9 44 8 17.4 164.3 47 9 35.1

4I 19 0.3 53 I9 1.9

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Htghest concerttration*

Lowest concentration**

100 100

80 100

100 100

100 98

100 74

100 61

100 100

100 t00

34 74

53 92

71 79

65 65

* Highest concentrations: chohne, 2.5 #M ; GABA, 10/~M; dopamine, 1.0/tM, * * Lowest concentrations: choline, 0.1 #M, GABA, 0.4 #M, dopamine, 0.02 ItM *** Abbreviations: HC-3 = hemicholinium-3 ; DAB = l-diaminobutyric acid ; DAS - D-amphetamine sulfate.

389 experiments, the uptake of chohne and GABA were measured at 4 min and dopamine uptake was measured at 2 rain, well wzthm the linear range for both parameters. Protein concentrations ranged from 0.5 to 2.0 mg/tube for different experiments. Other control experiments established the sensitivity of the high affinity uptake to ouabam inhibition (Table I). The uptake of each compound was also appropriately inhiblted by compounds (Table I) reported by other investigators to be specific with regard to site of action z°,28,45,53. In some experiments the preloaded crude synaptosomal fraction was further subfractionated as described by E~chberg et al. 13 The percentage of label migrating with the sedimentation properties expected of synaptosomes was 80, 83 and 64 for choline, GABA and dopamine, respectively, in young animals, and over 80 ~o in each case for adult animals. Developmental studies with normal rats During the developmental studies, body weights and brain weights were recorded (Fig. 1). In addition, the lead content of the blood and brain fractnons was measured (Table II). Control rats showed low, but consistent blood lead levels indicating some environmental exposure. The lead content of the fractions was below the detection limits of 2-5 Fg Pb/mg protein. Nerve ending fractions obtained from the various litters were used to define the kinetic constants for high affinity uptake of choline, GABA and dopamine. An example of the data obtained in a single experiment at a single age point is shown for i

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390 TABLE I1 Concentration o f lead measured tn the blood and brain oJ ratJ at various age,s Age, day I0

20

30

7 . 2 (3)* 234 + 63 (8)

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Strmta Homogenate Crude synaptosomal preparatmn Cortex Homogenate Crude synaptosomal preparauon

2 I 27

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c h o h n e (Fig. 2), G A B A (Fig. 3), and d o p a m m e (F~g. 4). The kinetic constants o b t a i n e d f r o m m a n y such experiments are plotted as a function o f age for each o f the rad i o act i v e c o m p o u n d s (Figs. 5-7). T h e a c c u m u l a t i o n o f high affimty uptake sites, as m e a s u r e d by Vm~x, differs for each n e u r o t r a n s m i t t e r during d e v e l o p m e n t C h o h n e u p t a k e in the stmatum developed rapidly between 12 and 14 days (Fig. 5). A f t e r 16 days, there was only a gradual increase with the Vm~x at 30 days, shghtly less than the average adult value o f 29.3 + 2.8 p m o l / m g / m m . Vm,~x between 10 an d 30 days was 18.9 -t- 1.1 p m o l / m g / m i n . T h e Vmax for high affimty G A B A

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different preparations from cortex was vamable but clearly displayed two peaks when plotted against age (Fig. 6). One peak occurred shortly after birth (within the first week) and the other occurred during the third week. Average Vmax values during these times were well above the adult average of 0.67:1:0.04 nmol/mg/mm. Dopamme uptake in the strmtum (Fig. 7) increased linearly from 4 days and approximated the adult value (68.3 ~ 1.5 pmol/mg/min) by 30 days. There was httle varmtion m the Km values for any of the neurotransmmers during development. For each neurotransmltter, the Km data fit a straight line generated by a linear regression analysis. There was, however, a slight, but significant (0.025 < P < 0.05), decrease in the Km for choline with age (Fig. 5). The average Km

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between 16 and 31 days was 0.66 ~ 0.04 #M as compared with the average adult Km of 0.54 ± 0.04 yM. The Km of GABA tended to increase with age during the first month (Fig. 6). Statistical analys~s indicated that the slope was significantly different from zero (P < 0.001). However, the average adult Km was 3.48 +_ 0.02 # M which was less than the average Km of 4.40 ±: 0.04 #M observed during the first 30 days. The Kr, of dopamine also appeared to decrease slightly during the first month; however, the slope of the line through the points was not significantly different from zero. During the development period the average Km was 0.31 ~ 0.04/~M in contrast to a Km of 0.21 ~ 0.02 y M in adulthood.

Developmental studies with lead-treated rats Data for body and brain weights for the lead-treated rats are shown In Fig. [. The lead dosage was chosen on the basis of previous studies z6 as the highest which does not cause weight loss and, as expected, the weights of control and treated rats d~d not differ significantly. Blood and brain lead concentrations were, however, markedly higher than those found in the control population (Table II). The blood lead concentraUons, initially very high as compared with those of controls, dropped considerably by 30 days.

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,

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,

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Fig. 7. Developmental uptake of dopamme into stnatal synaptosomes. Each Km and V ~ point was the result of a single kmeUc experiment and was determined by linear regresston analysis of duplicate samples and blanks at six concentrations of dopamlne ranging from 0.02-1 0 ttM. The lines through the data were generated by hnear regression analysis Similar plots of the Km values were analyzed (Figs. 5-7). The mean Km for choline, 0.77 ~ 0.14 # M , was somewhat higher than that observed m the controls, but neither the difference between the means nor the difference in slopes was significant. The Km values obtained for G A B A uptake were the same (4.4 _i_- 0.08 /~M). The apparently positive slope of the regression line through the data from lead-treated rats was not significantly different from the slope for the control data. Lead treatment appeared to increase the Km for dopamine. However, as with the Vmax data, the difference was not clearly significant (0.05 <-P <. 0.06). The most noticeable effect of in vivo lead treatment on the uptake of these neurotransmitter related compounds was to increase the variability of both the Km and Vmax determinations. When the choline and dopamine uptake data were fitted to straight lines, s~gnificantly more varmtion was apparent for lead-treated animal data as compared with control data (F test). The G A B A Km data, but not the Vmax data, from lead-treated rats were significantly more variable (F test). Uptake studies with lead added tn vitro The kinetics of uptake of choline and dopamine by stnatal ~ynaptosome preparations (Pz fraction) and of G A B A uptake by cortical synaptosomes was studied with lead added in vitro. Each determmation of Km and Vma~ was on the basis of measurement of uptake at five concentrations of substrate carried out Ln duplicate,

395 TABLE Ill Synaptosomal neurotransm~tter uptake effect of lead and calcium m vitro Neurotransmitter

Age ( dav,s )

10 "~ 1VI C a '~+

N o Ca '-+ N o P b 2+

25 p b 2+

10 5 M

No PN"

25 "10 pb2~

6M

l/max, plllol/'tllg,l ltllll *

Chohne GABA Dopamme

23-26 23 26 20-22

21.9 ± 3 4 829 ± 141 40 3 ± 5 2

15 4 ± 0 7 703 ~ 109 53 I 5- 4 6**

17 9 ~ 1 4 1054 ± 184 36 5 ± 6 5

17.65 16 1022 ± 178 44.6± 51

Chohne(4) GABA (5) Dopamme(5)

23-26 23-26 20 22

0 69 ± 0 13 3.12±077 0.12- 001

0 44 ± 007 267±038 015±002

0 57 5- 0 09 322±060 019±005

0.49 ± 0.04 3 13 -~- 0.66 0 20 ± 0.02

The values reported are means ± S E M ' ~ Significantly different with respect to lead treatment by paired t-test (P ~- 0 05)

with a p p r o p r m t e blanks at each concentration. All assays were carried out m regions o f hnearlty o f uptake w~th respect to protein c o n c e n t r a t i o n a n d t~me. The concentration of lead acetate used, 2.5 ~ 10 5 M, is the h~ghest t h a t does n o t f o r m a visible precipitate m these medm. Since m e d i a containing this c o n c e n t r a t i o n o f lead became cloudy on standing overmght, the lead was a d d e d to the m e d i a 10-15 m m before the u p t a k e experiments were conducted. This concentration o f lead is an o r d e r o f magnitude higher t h a n that achieved by m vwo lead t r e a t m e n t C a l c m m had no stat~stlcally s~gmficant effect on the u p t a k e p a r a m e t e r s o f any o f the neurotransm~tters studied (Table IIl). The only s~gmficant effect of lead was an increase in the Vm~x of d o p a m i n e u p t a k e m the absence of c a l c m m (values of 53.1 c o m p a r e d to 40.3 in Table Ill). W h e n calcium was added, the trend for elevated Vrn~x m the presence o f lead a p p e a r e d to still be present, but was n o t statistically s~gmficant by t-test analysis A l t h o u g h n o t o f statistical significance, the t r e n d to r e d u c t i o n o f the average Vma~ o f choline u p t a k e m the presence o f lead when calcium was omitted from the m e d i u m is relevant to the observations m the hterature (see D~scussion). The a d d i t i o n o f calcium to the m e d m m a p p e a r e d to overcome a n y possible lead effect.

Release studtes with lead added in vitro A f t e r p r e l o a d m g o f nerve endings with radJoactlve n e u r o t r a n s m l t t e r , there ~s a low level o f s p o n t a n e o u s (m the absence o f d e p o l a r i z a t i o n ) release o f r a d l o a c t w i t y (Table IV) The a d d i t i o n o f I m M CaC12 to the m e d i u m stimulated the s p o n t a n e o u s release o f the n e u r o t r a n s m i t t e r s : m o r e than 70°,,', in the case of d o p a m m e a n d acetylchohne (chohne). D e p o l a r i z a t i o n (reduced by raising the K C I c o n c e n t r a t i o n to 65 m M a n d m a i n t a i n i n g o s m o l a r i t y b y reducing the NaC1 c o n c e n t r a t i o n ) caused only slight changes m the t r a n s m i t t e r release pattern relative to the expected great p o t a s s i u m d e p o l a r i z a t i o n increase d e p e n d e n t on the presence o f 1 m M Ca "~ (Table IV).

396 TABLE IV The e f f e c t o f lead m vttro on the release o t neurotransmitter,s Neurotransmitter

2,5 , 10 -'~ M P e r c e n t release* Lead acetate . . . . . . . . . . . . S p o n t a n e o u s release K ~ d e p o l a r i z e d release 83 m M N a 142 m M N a 4 ; 6 m M _ _ K 0 Ca e~

0 Ca ~ 1 mM Ca"

,65 m M K

1 m M Ca -'~ 1~ ~ s n m -

¢ ) , z . de-

tdated * * release

pendent* * * K ' s'timulated release

Acetylcholine - (choline) t

1.8±0 3 2.1 -L0 2

3.5±0.5 3,4±0.2

2.3~0 I 24~-0 2

0.5±0.3 0.3±0 2

6 3~+0.2 4.0~i-0 3 6 0 ~ 0 . t 3.640 5

GABA

--

1.4-L0 1 I 5401

2.0±02 1 8-L-0.1

24±0.1 3 0~-0.3§

I 0±0.1 15:£0.2

47-i0,I 2.~-t 0.2 5,2~_-0.2~227_04

Dopamme

--

3.15-0.3 5,3±0.2 43~0.1 32 k-0.1 4.3:L0,2~ 5 3 £ 0 2 ~

1.2_-£04 10.9±0 7 6,63 0.8 2,1~02~§ 123-f0.5~ 70 =04

* The values reported are means i S E.M for eight determinations ** The spontaneous release (142 mM Na +, 6 mM K +, no Ca 2+) was used as a blank *** The K+-stlmulated release (65 mM K ÷, 83 mM Na +, no Ca 2÷) was used as a blank § P < 0.05 with respect to lead treatment by students t-test. §§ 0,05 < P < 0.10 with respect to lead treatment by students t-test.

The influence o f 2 5 < 10 _5 m M Pb 2~ on the s p o n t a n e o u s a n d p o t a s s i u m d e p o l a r i z e d release o f n e u r o t r a n s m i t t e r was investigated (Table IV). Lead did n o t significantly affect the calcium i n d e p e n d e n t s p o n t a n e o u s release o f a n y o f the neurotransmitters. T h e presence o f P b z~ significantly decreased the C a ~"~ d e p e n d e n t spont a n e o u s release o f d o p a m m e ( P ~ 0.025 by S t u d e n t ' s t-test), b u t d i d n o t influence release o f the o t h e r n e u r o t r a n s m i t t e r s . U n d e r c o n d i t i o n s o f K + i n d u c e d d e p o l a r i z a t i o n m the absence o f C a 2+, lead caused a n increase in the release o f G A B A a n d d o p a m i n e ( P < 0.05, S t u d e n t ' s t-test). I f the s p o n t a n e o u s c o m p o n e n t was s u b t r a c t e d f r o m the p o t a s s i u m s t i m u l a t e d release (Table IV), the significance o f the difference was m a r g i n a l (0.05 ~.. P < 0.10). This analysis does n o t negate the significance of the o b s e r v a t i o n t h a t lead causes a n increase in the release o f G A B A a n d d o p a m i n e ; tt is r e p o r t e d since it m a y influence i n t e r p r e t a t i o n o f future w o r k c o n c e r n i n g the m e c h a nism involved. The p o t a s s i u m s t i m u l a t e d release o f acetylcholine (choline) was not significantly altered b y the presence o f lead. U n d e r c o n d i t i o n s o f o p t i m a l n e u r o t r a n s m i t t e r release, p o t a s s i u m d e p o l a r i z a t i o n in the presence o f calcium, it also a p p e a r e d t h a t release o f G A B A a n d d o p a m m e was further s t i m u l a t e d by lead ( P < 0.05). H o w e v e r , when only the c a l c i u m - d e p e n d e n t p o r t i o n o f the p o t a s s i u m - s t i m u l a t e d release was considered, by s u b t r a c t i n g the p o t a s s i u m - d e p e n d e n t release w i t h o u t calcium, there were no differences between lead a n d c o n t r o l values (Table IV). A c e t y l c h o l i n e (choline) release a p p e a r e d to be unaffected by lead.

397 DISCUSSION

Development of uptake mechanisms Choline uptake by striatal synaptosomes is first s~gmficant by 12 days of age as measured by Vmax determination. In a few days, adult values for Vmax are approached. The rapid development at th~s age is slmdar to that observed for preparations from rat strlatal tissue by Sormachi and Kataoka 47 and Coyle and Yamamura TM. These other laboratories report that some uptake (less than 10~o of adult values) is found using animals less than 12 days of age. This minor d~fference in interpretation may be due to the stricter kinetic criteria we used (there is some uptake before 12 days of age but the kinetic analyses we used suggested this m~ght be due to the preexisting low affimty uptake system) The kinetic constants determined from t~ssue obtained from adult rats agree well with those reported by Simon and Kuhar 4z, Sorimach~ and Kataoka 47, and Yamamura and Snyder 54. The trend for shght decline m the Km for chohne during development is also observable in the results reported by Sorimach~ and Kataoka aT. For GABA uptake an initmlly high Vmax was observed during the first week, followed by a dechne during the second week and then another peak which then declined to adult values. These results are compatible with the report of Coyle and Enna 8, m which uptake was reported to be greater at early ages than in adult ammals. Not enough age points were taken m th~s earher study to define a two peak pattern of the type we observed. The adult Vma× values obtained in our study are s~milar to those reported by Martin 32 and Coyle and Enna 8. It should be noted that originally ~t was anticipated that the Vmax of GABA would indicate the development of GABA neurons, m the same manner as choline and dopamine uptake reflect the development of cholinergic and aminergic neurons 2v. However, development of high affinity uptake does not correlate well with the development of functioning GABA medmted synapses since the amount of active uptake does not correlate well with other measurements of functioning synapses 40. We have not found any literature concermng developmental studies of dopamme uptake by rat strlatal synaptosomes. The pattern of V~a~x obtained m this study is s~mllar to the development pattern of norepinephrine uptake into the hypothalamus 7. It should be noted that in vitro norepinephrine and dopamine are accumulated by the same neurons, as indicated by competition studies, although the kinetic constants vary according to the predominant neurotransmitter~9.4L The adult values for the kinetic parameters for dopamine agree well with those presented in other reports 9.'~, 45,50

A study of the uptake of a number of compounds, including choline, GABA, and dopamine by nerve endings of developing chick brain has been conductedL Although the results from this different system cannot be compared directly to our work on the rat, the developmental plots e~h~bit similarities.

Effects of in viro lead treatment The lead dosage (100/~g Pb/g body wt/day) did not cause undernutrition. Lead

398 dosages of 200 #g Pb/g body wt/day by this method of treatment result m about a 10 °,,, body weight loss, whde double that amount often results in death ~6 In evaluating neurobiologlcal studies of toxic compounds, weight matched controls are essential since undernutritton brings about a decreased cell number and impaired neuronal development 5z, which could clearly influence neurotransmitter metabohsm. Although the weights were unaltered by lead treatment, the blood and brain lead concentrations revealed that the treated rats did have a much higher body burden of lead than the control rats. The very low blood lead level at 30 days probably reflected the developmental decrease m absorption which occurs at 20-22 days and the concomitant development of excretory processes 1~. Brain lead concentratmns showed no such dechne, underscoring the increased retention of lead by this organ However. the possibility that lead might be locahzed in the nerve endings was not supported by these data. Developmental patterns of neurotransmitter uptake were only slightly different after lead exposure. Using the rat system we d~d not observe any decrease in chohne uptake by nerve endings after treatment: such a decrease has been reported for mice b) Silbergeld and Goldberg 43. In fact, the observed trend for our data was m the direction of stimulation of uptake in preparations from lead-treated animals, although this was not statistically significant. Our data are more in accord with a second report from that laboratory 4, indicating that high affinity uptake of choline by cortical minces from chronically lead-treated mice was not different from uptake by untreated tlssue~ Studies of dopamine uptake by preparatmns from lead-treated animals gave results analogous to those obtained with choline - - no statistically significant d~fferences in Vmax values relative to controls although the trend was in the dlrectmn of increased uptake in lead-treated rats. These results are d~fferent from the decreased uptake of dopamme reported for lead-treated m~ce relative to controls 43. The affinity constants (K,~) were not significantly different after lead treatment as compared with controls. Finally, GABA uptake was not significantly altered by m VlVO lead treatment. The lack of perturbation of choline and dopamme uptake by nerve endings m response to m vivo lead treatment in rats, in contrast to the decrease m m~ce reported by Silbergeld and Goldberg 43, might be due to the more stringent kinetic cmerm for transmitter uptake used in the present studies, or might reflect species d~fferences. A third possibility ~s that in the mouse studies, the observed perturbations in neurotransmitter metabolism were a consequence of undernumtion 3° rather than primarily related to the lead treatment. In any case, although we had hoped to obtain ewdence suggesting a possibly specific effect of lead treatment on neurotransmltter metabolism, our data do not support such a hypothesis One general effect observed was the greater variatmn in data obtained from leadtreated rats, in accord with a recent report from another laboratory 5. The variation in data obtained after lead treatment was significantly greater (F test) for both the K,,~ and Vma~ constants determined for choline and dopamme uptake. Perhaps the normal animal-to-animal variability, which occurs during development, and ~s reduced a~ ammals reach the more stable mature state, is amplified by various forms of stress such as lead treatment. These effects may be on some bmlogical parameters which are not directly connected with the presynapt~c actions of neurotransmltters

399

Studws of lead treatment in vitro There are several reasons for study of lead addition in in vitro experiments as an adjunct to m wvo treatment studies. (1) such experiments control for the possibility that lead, present in the region of nerve endings after in vJvo treatment, ~s washed out during the preparation of the crude synaptosoma! fraction; (2) much higher concentrations of lead can be reached than are possible w~th m wvo treatment, which m~ght magmfy any possible effects of lead that are seen at lower lead concentrations; (3) the action of lead d~rectly on the uptake or release system can be studied independently of other factors which may be reduced by m vivo treatment (e.g. undernutntlon or developmental lag affecting the central nervous system primarily at some other s~te with a secondary alteration of synaptic function). Because both lead and calcium are d~valent cations, any effect of lead may be due to an alteration in the calcmm function. Others have investigated this poss~bllity m experiments m both the peripheral ~'3 and central T M nervous systems with results which support the hypothesis. Therefore, when mvesttgatmg the effect of lead addition in vitro we also studied the effect of calcium Although the concentration of lead m vitro was much higher than that attained m vivo ag, there was no marked effect on high affi.nlty uptake of neurotransm~tter related compounds. Although not of stahstlcal s~gmficance, the chohne Vmax data correspond m direction with the trend reported by Sllbergeld al. However, our results on the effect of lead m vitro to increase the Vm~ of dopamme is m a d~rectJon opposite to that observed in that same study 41. No stahstlcally significant effect of lead or calcium on G A B A uptake parameters was observed. The relatively small effects observed in response to challenge by h~gher concentrat~ons of lead indicate that lead has httle direct effect on the high affimty uptake of these compounds. However, it is possible that the stimulation of dopamme uptake by lead, m the absence of calcmm, ~s of biological sigmficance m cases where lead exposure ~s combined w~th a calcium deficiency. Spontaneous release of neurotransmltters (that taking place m the absence of electrically or potassium reduced depolarization) was also investigated. The spontaneous release is slightly stimulated by the presence of calcium. Lead added m wtro had no effect on the spontaneous release of acetylchohne (chohne) or GABA with or without calcium in the medium. However, the calcium-dependent component of the spontaneous release of dopamine was inhibited significantly by lead. This result is opposite in direction to that reported by Silbergeld al who, In a similar study on rat preparations, reported that lead enhanced the calcmm stimulated release o f d o p a m m e . However, m that study effects were observed at higher calcium and lead concentrahons than were used in our work (m fact, the lead concentration used in that study was such that it precipitated from our medium). The depolarization stimulated release of all three neurotransm~tter related compounds exhibited the expected calcium dependenceaS.3a,a~. Depolarized release of GABA and dopamme was stimulated m the presence of lead This is ~n agreement with recent work of Bondy et al. a, who demonstrated a shght increase m dopamine release by lead from adult mouse brain nerve ending preparations (the mare point of that publication was the much more profound effects

400 on neurotransmttter metabolism brought about by organic lead compounds relatwe to lead acetate). Lead had no effect on the calcmm dependent portion of the release. suggesting that the added lead may have substituted for calcium at h~gh potassium concentrations. It may be that many divalent cations can substitute for calcium to trigger release since Murrin et al. 35 have shown that barium or strontium can effectively replace calcium. This explanation does not account for the lack of lead effect on the depolarized release of acetylcholine (choline). There is, however, potential for error m the acetylchohne studies since there is a report that under certain conditions the potassmm induced release of choline may vary independently of acetylchohne release 4. The combined evidence of the developmental study with lead treatment m vlvo and the experiments concerning addition of lead in vitro do not support the idea that inorganic lead significantly alters the presynaptlc actions of choline or GABA m the central nervous system. There is a suggestion of some stimulation of dopammergic presynaptic functions and further investigations designed on the bas~s of the developmental data which we have presented might prove fruitful. ACKNOWLEDGEMENTS We thank B. J. Loudin for her excellent technical assistance, V. Knopp for the care and treatment of the rats, Ms. Barbara Glover and Dr. Paul Mushak for carrying out the lead determinations, and Dr. Robert Horton for discussions. The research described m this manuscript represents partial fulfillment of the requirements for the Ph. D. degree from the Department of Biochemistry at N o r t h Carolina State Umversity. Th~s research was supported by USPHS Grants ES-01 104; NS-11615 and HD-03ll0.

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