ECOTOXICOLOCY
AND
ENVIRONMENTAL
SAFETY
22,88-93
(1991)
Effects of Lead on Kinetics of 3H-Dopamine by Rat Brain Synaptosomes’ MICHAEL J. BOYKIN, CHELLU S. CHETTY,
AND
Uptake
BETTAIYA
RAIANNA
Division of Natural Sciences, Selma University, Selma, Alabama 36701 Received September 28. 1990 The effects of lead on 3H-dopamine (‘H-DA) uptake in rat brain synaptosomes were studied. Pb inhibited ‘H-DA uptake in vitro and in vivo in a concentrationdependent manner. Altered pH versus ‘H-DA uptake demonstrated comparable inhibition in buffered acidic, neutral, and alkaline pH ranges, and higher inhibition was observed in neutral pH. Kinetic studies of ATP activation of 3H-DA uptake indicated competitive inhibition by Pb. The inhibition of ‘H-DA uptake followed the Na+ concentrations. The results indicate that the Pb inhibition of ‘H-DA uptake is pH, ATP, and Na+ dependent. o 1991 Academic PFSS, IX.
INTRODUCTION Several investigators have provided information on the cellular sites of action, reversibility of neurotoxic effects, and molecular mechanisms of behavioral effects of Pb (Sobotka and Brodie, 1975; Kober and Cooper, 1976; Carroll et al., 1977). Jason and Kellog (1977) have reported that Pb mediated alterations in the brain. Biogenic amine metabolism plays an important role in the behavior of the animal. Heavy metal ions (Cd*+, Hg*+, Mn*+, and A13+) have been shown to affect the uptake of bioamines such as dopamine, norepinephrine, choline, and serotonin in rat brain synaptosomal fractions (Ramsay et al., 1980; Lai et al., 1982). Earlier studies have also indicated that Pb disturbs some parameters associated with the neurotransmission process (Carroll et al., 1977; Bondy et al., 1979). Although the neurotoxic effects of Pb are well known, the underlying mechanism(s) are not understood (Goyer and Rhyne, 1973; Kehoe, 1976). The levels of ATP and uptake of catecholamines are interrelated. Pb has been shown to inhibit ATP synthesis and utilization and catecholamine uptake in the central nervous system (Shellenberger, 1984; Chetty et al., 1989). A parameter of synaptogenesis and neuronal differentiation of potential utility is the sodium-dependent, high affinity uptake of neurotransmitters or their precursor by nerve endings (Kuhar, 1973; Coyle and Enna, 1976). Sibergeld and Goldberg ( 1975) have correlated Pb exposure in mice with hyperactivity and a diminution of choline and dopamine uptake by the forebrain synaptosomes. In the present investigation we have studied the in vitro effects of Pb on the kinetics of )H-dopamine (3H-DA) uptake by rat brain synaptosomes as a function of pH, Na+, and ATP concentration. MATERIALS
AND
METHODS
Sprague-Dawley male rats ( 175-200 g) were obtained from Harlan Sprague Dawley, Inc., Indianapolis. Rats were housed two per cage, and glass distilled water and Purina ’ This research work was supported by NIH/NIGMS/MBRS submitted by Michael Boykin as an M.S. thesis to Alabama 0147-6513191
$3.00
Copyright 0 1991 by Academic Press, Inc. All tights of reproduction in any form resewsd.
88
Grant 08169. State University.
Part of this work
has been
EFFECTS OF Pb ON ‘H-DOPAMINE
UPTAKE
89
Rat Chow were made available ad Zibitum. 3H-DA and Aquasol were purchased from DuPont Co. (Wilmington, DE). All other chemicals used for 3H-DA uptake were purchased from Sigma Chemical Co. (St. Louis). To determine the in vivo effects of Pb, the rats were given intraperitoneal injection with Pb at concentrations of 0,25,50, and 100 mg/kg body wt daily for 2 weeks. Each treatment group consisted of five rats. Twenty-four hours after the last treatment rats were killed by decapitation, brains were removed and placed in ice-cold homogenizing medium (0.32 M sucrose and 10 mM imidazole, pH 7.5). The brains were homogenized in 9 vol of sucrose solution and the synaptosomes were prepared as per the method of Cotman and Mathews (197 1). The homogenates were centrifuged at 750 X g for 10 min. The supematant was centrifuged at 17,000 X g for 20 min. The pellet was suspended in homogenizing medium and again centrifuged at 17,000 X g for 20 min. The resulting pellet was suspended in 10 ml of 0.32 A4 sucrose and layered on a two-step discontinuous ficoll-sucrose gradient consisting of 13% (w/v) ficoll in 0.32 A4 sucrose and 7.5% (w/v) ficoll in 0.32 M sucrose. After centrifugation at 65,000 X g for 45 min, the synaptosomal fraction was obtained at the interface of the 7.5- 13% (w/v) ficoll-sucrose layer. The synaptosome band was removed, diluted with 9 vol of 0.32 M sucrose solution, and centrifuged at 17,000 X g for 30 min. The resulting pellet was suspended in 0.32 M sucrose, divided into l-ml aliquots, quick frozen in liquid nitrogen, and stored at -85°C until used. Protein was determined by the method of Lowry et al. (195 l), using bovine serum albumin as a standard. The uptake of 3H-DA was determined using procedures described by Slotkin et al. (1978) with slight modifications. A 2-ml reaction mixture contained 50 mM Tris buffer, pH 7.5; 5 mA4 MgC& ; 10 mIt4 NaCl; 5 nuI4 ATP, and 30-50 pg rat brain synaptosomal protein. The reaction mixture was incubated for 15 min at 37°C after the addition of 3H-DA. After incubation, the reaction contents were filtered through a 0.45~pm millipore filter and rinsed twice with 5 ml of Tris buffer. The filters were dissolved in 10 ml Aquasol and radioactivity was measured in a LS-6800 Beckman liquid scintillation counter. The specific uptake was determined by subtracting the nonspecific uptake from the total radioactivity and expressed as CPM/mg protein. Each point on the graphs indicates the mean of three different synaptosomal preparations, and each preparation was assayed three times. Double-reciprocal plots of
TABLE EFFWX
OF Pb ON 3H-DA UPTAKE IN RAT BRAIN SYNAP~OSOMES
IN VITRO
3H-DA uptake (CPM/mg protein) 0 10 25 50 100
I
1578 961 823 806 506
f f t + zk
178 18* 93* 88* 79*
% Inhibition 39.1 47.8 48.9 67.9
Note. Each value is mean -t SD of three different preparations, each assayedin triplicate. * Statistically significant (P < 0.05).
90
BOYKIN,
CHETTY,
AND RAJANNA
TABLE ‘H-DA
(mg,kg!&y
2
UPTAKE IN BRAIN RATS TREATED
SYNA~OSOMES WITH Pb
3H-DA (CPM/mg
wt)
Control 25 50 100
1411 1070 1058 952
uptake protein) ? 8 + 12* f 15* f 19*
OF
% Inhibition 24.2 25.0 32.5
Note. Each value is mean f SD of five samples, each assayed in triplicate. * Statistically significant (P < 0.05).
kinetic data were constructed according to the method of Lineweaver and Burk (1934). Data were subjected to regression analysis and regression lines were plotted for best straight-line fit. Data were also analyzed by Student’s t test to determine the differences between control and experimental treatments; a value of P -C 0.05 was considered significant. RESULTS The effects of in vitro and in vivo Pb on the uptake of 3H-DA were studied to delineate certain parameters of Pb toxicity to rat brain synaptosomes. The data in Table 1 suggest that Pb in vitro significantly decreased the uptake of 3H-DA. Maximum TABLE EFFECT
OF
3
pH ON Pb INHIBITION IN RAT
BRAIN
OF ‘H-DA SYNAPTOSOMES
UPTAKE
50
100
155 xk 9
96 + 6* (38.1)
82 + 16* (47.1)
6.5
170 f 13
llO& 13* (35.3)
101 + 25* (40.6)
7.0
573 + 69
206 + 42* (64.0)
165 + 16* (71.2)
7.5
1375 f 16
862 f 44* (37.3)
260 + 13* (81.1)
8.0
1084 + 46
679 + 18* (37.4)
176 + 18* (83.8)
PH
Control
6.0
Note. Each value is mean f SD of three preparations, each assayed in triplicate. Values in parentheses are percentage inhibitions over control. * Statistically significant (P < 0.05).
EFFECTS OF Pb ON ‘H-DOPAMINE
UPTAKE
91
decrease (68%) in uptake was observed with 100 PLM of Pb. A similar inhibition in uptake of 3H-DA by synaptosomes was observed in rats treated with Pb (Table 2). The inhibition of uptake was similar in the rats treated with either 25 or 50 mg/kg body wt of Pb for 14 days. Nevertheless, maximum inhibition (32.5%) was observed in rats treated with 100 mg/kg body wt of Pb. The data indicate that both in vivo and in vitro Pb exerted similar effects on uptake of 3H-DA in synaptosomes. The influence of pH on 3H-DA uptake was determined in the absence and presence of 50 and 100 PM Pb at varied pH (Table 3). In the absence of Pb, maximum 3HDA uptake was observed at 7.5 pH. On the other hand, in the presence of Pb, the uptake was significantly inhibited at all pH. Maximum inhibition of 3H-DA uptake was observed at slightly alkaline pH for both levels of Pb treatments. Activation with varying concentrations of Na+ (lo-100 mA4) showed no appreciable changes in ‘HDA uptake (Table 4). Compared with control, Pb at 50 piV decreased the 3H-DA uptake at 20 mA4 of Na+, whereas a further increase in Na+ up to 100 PM reversed the inhibitory effects. However, 100 PM Pb significantly decreased the 3H-DA uptake at 30 mM Na+. ATP-dependent (0.5-5.0 mM) 3H-DA uptake was studied in the absence and presence (50 or 100 pLM) of Pb (Fig. 1). The K, values for uptake of 3HDA were found to be 1.38, 3.09, and 4.08 mM for control, 50, and 100 PM Pb, respectively. No significant changes in V,,, were observed in the presence of Pb. DISCUSSION Pb is a recognized neurotoxicant, although its mechanism(s) of action are not well understood (Cooper et al., 1984; Shellenberger, 1984; Audesirk, 1985). The effects in TABLE EFFECTS
IN
3H-DA
VITRO OF Pb ON Na+ ACTIVATION UPTAKE (CPM/mg PROTEIN)
IN RAT BRAIN
Control
10
783 +41
30 40
50
SYNAPTOSOMES
755 +72 758 756
100
50
260 k44
753 k28
568 +48* (27.5)
473-t
432 + 38* (42.8)
526
613 f 60* (19.1)
306
654 + 49 (13.5)
329+
695
+
25
757
f
38
713k74 (5.81)
57* (35.6)
+ 117* (30.8)
f
59.6*
(59.6) 35* (56.5) 352k
(7.70)
100
OF
Pb (PM
NaCl (mM)
20
4
30* (53.2)
492k
48* (35.0)
Note. Each value is mean * SD of five different samples, each assayed in triplicate. Values in parentheses are percentage inhibition over control. * Statistically significant (P < 0.05).
92
BOYKIN,
/--c r/A-0.2 -0.6 -0.4
CHETTY,
0
I
I
0.2
AND RAJANNA
I
0.4
I
0.6
I 0.6
I 1.0
I
1.2
1
FIG. 1. Effect of Pb on the ATP-activation kinetics of 3H-DA in rat brain synaptosomes.
vitro of Pb on neuronal function have been studied to identify basic mechanisms of action (Minnema, 1986). The results of this study suggest that Pb inhibited 3H-DA uptake both in vitro and in vivo. A similar inhibition of catecholamine uptake has been reported with transition metal ions (Prakash et al., 1973; Lai et al., 1982) mercury (Rajanna and Hobson, 1985), and cadmium (Hobson et al., 1986). It has been shown that Pb and other heavy metals interfere with cellular energy metabolism by inhibiting ATP synthesis as well as ATP hydrolysis (Rajanna et al., 1983; Chetty et al., 1989). This leads to the dysfunction of energy-dependent neuronal events such as neurotransmitter uptake. The differential response of 3H-DA uptake to Pb at acidic, neutral, and alkaline pH ranges may be due to the modification of ionizable moieties or to a change in the charge pattern of the receptor protein (Na+-K+ ATPase) by Pb. This suggests that Pb inhibition of 3H-DA uptake is pH dependent. A similar modification of the ionizable moiety and charge pattern of Naf-K+ ATPase by heavy metals, organotin compounds, and chlordecone was reported earlier (Chetty et al., 1983, 1989; Prasada Rao et al., 1987). The alterations in Pb inhibition of 3H-DA uptake at different concentrations of Na+ or ATP suggest that Pb may primarily affect ATP- and Na+directed sites of the receptor protein (Na+-K+ ATPase) (Rajanna et al., 1983). The kinetic constants for ATP-dependent high affinity uptake of 3H-DA by synaptosomes in the presence of Pb suggest a competitive type of inhibition. CONCLUSIONS The results suggest that Pb both in vivo and in vitro inhibit the uptake of 3H-DA in rat brain synaptosomes. The inhibition of 3H-DA by Pb is concentration dependent. The ATP/Na+ concentration-dependent Pb inhibition of 3H-DA uptake verifies that the electrochemical potential gradient results from the ionic gradient maintained by ATP hydrolysis (Na+-K+ ATPase) and may be responsible for the uptake of dopamine in synaptosomes.
EFFECTS OF Pb ON 3H-DOPAMINE
UPTAKE
93
REFERENCES AUDESIRK, G. (1985). Effects of lead exposure on the physiology of neurons. Prog. Neurobiol. 24, 199-23 1. BONDY, S. C., HARRINGTON, M. E., ANDERSON, C. L., AND PRASAD, K. N. (1979). The effect of low concentrations of an organic lead compound on the transport and release of putative neurotransmitters. Toxicol. Lett. 3, 35-4 1. CARROLL, P. T., SILBERGELD, E. K., AND GOLDBERG, A. M. (1977). Alteration of central cholinergic function by chronic lead acetate exposure. Biochem. Pharmacol. 26, 397-402. CHETTY, C. S., ALDOUS, C. N., RASHATWAR, S. S.. AND DESAIAH, D. (1983). Effects ofchlordecone on pH and temperature dependent substrate activation kinetics of rat brain synaptosomal ATPase. Biochem. Pharmacol. 32,3205-32 11. CHETTY, C. S., RAJANNA, S., AND RAJANNA, B. (1989). Effects of lead on the kinetics of adenosine triphosphatase system and protection by thiol reagents. Toxicologist 9(l), 20. COOPER, G., SUSZKIW, J., AND MANALIS, R. (1984). Presynaptic effects of heavy metals. In Cellular and Molecular Neurotoxicology (T. Narahashi, Ed.), pp. 1-2 1. Raven Press, New York. COTMAN, C. W., AND MATHEWS, D. A. (1971). Synaptic plasma membrane from rat brain synaptosomes: Isolation and partial characterization. Biochem. Biophys. Acta 249, 380-394. COYLE, J. T., AND ENNA, S. J. (1976). Neurochemical aspects of the ontogenesis of gabanergic neurons in the rat brain. Brain Res. 111, 119-133. COYER, R. A., AND RHYNE, B. C. (1973). Pathological effectsof lead. Int. Rev. Exp. Pathol. 12, l-77. HOBSON, M., MCDONALD, M., AND RAJANNA, B. (1986). Effects of cadmium on the uptake of dopamine and norepinephrine in rat brain synaptosomes. Bull. Environ. Contam. Toxicol. 37, 421-426. JASON. K., AND KELLOG, C. K. (1977). Behavioral neurotoxicity of lead. In Lead Toxicity (R. Singhal and J. Thomas, Eds.), pp. 241-272. Urban & Schwartzenberg, Baltimore. KEHOE. R. A. (1976). Pharmacology and toxicology of heavy metals: Lead. Pharmacol. Ther. 1, 161-l 88. KOBER, T. E.. AND COOPER,G. P. (1976). Lead competitively inhibits calcium-dependent synaptic transmission in the bullfrog sympathetic ganglion. Nature (London) 262, 704-705. KUHAR, M. J. (1973). Neurotransmitter uptake: A tool in identifying neurotransmitter-specific pathways. Life Sri. 13, 1623-1634. LAI, J. C. K., LIM, L., AND DAVIDSON,A. N. (1982). Effectsof Cd*+, Mn2+ and A13+on rat brain synaptosomal uptake of noradrenaline and serotonin. J. Inorg. Biochem. 17, 2 12-225. LINEWEAVER, H., AND BURK. D. J. (1934). Determination of enzyme disassociation constant. J. Am. Chem. Sot. 56,658-666.
LOWRY, 0. H., ROSEBROUGH,N. J., FARR. A. L., AND RANDALL, R. J. (1951). Protein measurement with folin phenol reagent. J. Biol. Chem. 193, 265-275. MINNEMA, D., GREENLAND, R., AND MICHAELSON,I. A. (I 986). Effectsof in vitro inorganic lead on dopamine release from superfused rat striatal synaptosomes. Toxicol. Appl. Pharmacol. 84, 400-4 11. PRAKASH, N. J.. FONTANA, J., AND KENKIN, R. T. (1973). Effect of transitional metal ions on Na+, K+ATPase activity and the uptake of norepinephrine and choline by brain synaptosomes. Life Sci. 12, 249259.
PRASADA RAO, K. S., CHETTY, C. S., AND DESAIAH, D. (1987). Effects oftrichclohydroxytin on the kinetics of adenosine triphosphatase system and protection by thiol reagents. J. Biochem. Toxicol. 2, 125-140. RAJANNA, B., AND HOBSON, M. (1985). Influence of mercury on uptake of 13H] dopamine and [3H] uorepinephrine by rat brain synaptosomes. Toxicol. Lett. 27, 7-14. RAJANNA, B., HOBSON, M., BANSAL, S. K., AND DESAIAH, D. (1983). Effect of cadmium chloride on rat brain synaptosomal ATPases. Toxicol. Lett. 18, 331-336. RAMSAY, P. B., KRIGMAN, M. R., AND MORELL, P. (1980). Developmental studies of the uptake of choline, GABA and dopamine by crude synaptosomal preparations after in vivo or in vitro lead treatment. Brain Res. 187, 383-402.
SHELLENBERGER,M. (1984). Effects of early lead exposure on neurotransmitter systems in the brain: A review with commentary. Neurotoxicology 5, 177-2 12. SIBERGELD, E. K., AND GOLDBERG, A. M. (1975). Pharmacological and neurochemical investigations of lead-induced hyperactivity. Neuropharmacology 14,43 l-444. SLOTKIN, T. A., SALVEGGIO, M., LAU, C., AND KIRSKEY, D. F. (1978). [3H] Dopamine uptake hy synaptic storage vesicles of rat whole brain and brain regions. Life Sci’. 22, 823-830. SOBOTKA, T. J., BRODIE, R. E., AND COOK, M. P. (1975). Psychophysiologic effects of early lead exposure. Towicology 5, 17 5- 19 1.
AND
ENVIRONMENTAL
SAFETY
22,88-93
(1991)
Effects of Lead on Kinetics of 3H-Dopamine by Rat Brain Synaptosomes’ MICHAEL J. BOYKIN, CHELLU S. CHETTY,
AND
Uptake
BETTAIYA
RAIANNA
Division of Natural Sciences, Selma University, Selma, Alabama 36701 Received September 28. 1990 The effects of lead on 3H-dopamine (‘H-DA) uptake in rat brain synaptosomes were studied. Pb inhibited ‘H-DA uptake in vitro and in vivo in a concentrationdependent manner. Altered pH versus ‘H-DA uptake demonstrated comparable inhibition in buffered acidic, neutral, and alkaline pH ranges, and higher inhibition was observed in neutral pH. Kinetic studies of ATP activation of 3H-DA uptake indicated competitive inhibition by Pb. The inhibition of ‘H-DA uptake followed the Na+ concentrations. The results indicate that the Pb inhibition of ‘H-DA uptake is pH, ATP, and Na+ dependent. o 1991 Academic PFSS, IX.
INTRODUCTION Several investigators have provided information on the cellular sites of action, reversibility of neurotoxic effects, and molecular mechanisms of behavioral effects of Pb (Sobotka and Brodie, 1975; Kober and Cooper, 1976; Carroll et al., 1977). Jason and Kellog (1977) have reported that Pb mediated alterations in the brain. Biogenic amine metabolism plays an important role in the behavior of the animal. Heavy metal ions (Cd*+, Hg*+, Mn*+, and A13+) have been shown to affect the uptake of bioamines such as dopamine, norepinephrine, choline, and serotonin in rat brain synaptosomal fractions (Ramsay et al., 1980; Lai et al., 1982). Earlier studies have also indicated that Pb disturbs some parameters associated with the neurotransmission process (Carroll et al., 1977; Bondy et al., 1979). Although the neurotoxic effects of Pb are well known, the underlying mechanism(s) are not understood (Goyer and Rhyne, 1973; Kehoe, 1976). The levels of ATP and uptake of catecholamines are interrelated. Pb has been shown to inhibit ATP synthesis and utilization and catecholamine uptake in the central nervous system (Shellenberger, 1984; Chetty et al., 1989). A parameter of synaptogenesis and neuronal differentiation of potential utility is the sodium-dependent, high affinity uptake of neurotransmitters or their precursor by nerve endings (Kuhar, 1973; Coyle and Enna, 1976). Sibergeld and Goldberg ( 1975) have correlated Pb exposure in mice with hyperactivity and a diminution of choline and dopamine uptake by the forebrain synaptosomes. In the present investigation we have studied the in vitro effects of Pb on the kinetics of )H-dopamine (3H-DA) uptake by rat brain synaptosomes as a function of pH, Na+, and ATP concentration. MATERIALS
AND
METHODS
Sprague-Dawley male rats ( 175-200 g) were obtained from Harlan Sprague Dawley, Inc., Indianapolis. Rats were housed two per cage, and glass distilled water and Purina ’ This research work was supported by NIH/NIGMS/MBRS submitted by Michael Boykin as an M.S. thesis to Alabama 0147-6513191
$3.00
Copyright 0 1991 by Academic Press, Inc. All tights of reproduction in any form resewsd.
88
Grant 08169. State University.
Part of this work
has been
EFFECTS OF Pb ON ‘H-DOPAMINE
UPTAKE
89
Rat Chow were made available ad Zibitum. 3H-DA and Aquasol were purchased from DuPont Co. (Wilmington, DE). All other chemicals used for 3H-DA uptake were purchased from Sigma Chemical Co. (St. Louis). To determine the in vivo effects of Pb, the rats were given intraperitoneal injection with Pb at concentrations of 0,25,50, and 100 mg/kg body wt daily for 2 weeks. Each treatment group consisted of five rats. Twenty-four hours after the last treatment rats were killed by decapitation, brains were removed and placed in ice-cold homogenizing medium (0.32 M sucrose and 10 mM imidazole, pH 7.5). The brains were homogenized in 9 vol of sucrose solution and the synaptosomes were prepared as per the method of Cotman and Mathews (197 1). The homogenates were centrifuged at 750 X g for 10 min. The supematant was centrifuged at 17,000 X g for 20 min. The pellet was suspended in homogenizing medium and again centrifuged at 17,000 X g for 20 min. The resulting pellet was suspended in 10 ml of 0.32 A4 sucrose and layered on a two-step discontinuous ficoll-sucrose gradient consisting of 13% (w/v) ficoll in 0.32 A4 sucrose and 7.5% (w/v) ficoll in 0.32 M sucrose. After centrifugation at 65,000 X g for 45 min, the synaptosomal fraction was obtained at the interface of the 7.5- 13% (w/v) ficoll-sucrose layer. The synaptosome band was removed, diluted with 9 vol of 0.32 M sucrose solution, and centrifuged at 17,000 X g for 30 min. The resulting pellet was suspended in 0.32 M sucrose, divided into l-ml aliquots, quick frozen in liquid nitrogen, and stored at -85°C until used. Protein was determined by the method of Lowry et al. (195 l), using bovine serum albumin as a standard. The uptake of 3H-DA was determined using procedures described by Slotkin et al. (1978) with slight modifications. A 2-ml reaction mixture contained 50 mM Tris buffer, pH 7.5; 5 mA4 MgC& ; 10 mIt4 NaCl; 5 nuI4 ATP, and 30-50 pg rat brain synaptosomal protein. The reaction mixture was incubated for 15 min at 37°C after the addition of 3H-DA. After incubation, the reaction contents were filtered through a 0.45~pm millipore filter and rinsed twice with 5 ml of Tris buffer. The filters were dissolved in 10 ml Aquasol and radioactivity was measured in a LS-6800 Beckman liquid scintillation counter. The specific uptake was determined by subtracting the nonspecific uptake from the total radioactivity and expressed as CPM/mg protein. Each point on the graphs indicates the mean of three different synaptosomal preparations, and each preparation was assayed three times. Double-reciprocal plots of
TABLE EFFWX
OF Pb ON 3H-DA UPTAKE IN RAT BRAIN SYNAP~OSOMES
IN VITRO
3H-DA uptake (CPM/mg protein) 0 10 25 50 100
I
1578 961 823 806 506
f f t + zk
178 18* 93* 88* 79*
% Inhibition 39.1 47.8 48.9 67.9
Note. Each value is mean -t SD of three different preparations, each assayedin triplicate. * Statistically significant (P < 0.05).
90
BOYKIN,
CHETTY,
AND RAJANNA
TABLE ‘H-DA
(mg,kg!&y
2
UPTAKE IN BRAIN RATS TREATED
SYNA~OSOMES WITH Pb
3H-DA (CPM/mg
wt)
Control 25 50 100
1411 1070 1058 952
uptake protein) ? 8 + 12* f 15* f 19*
OF
% Inhibition 24.2 25.0 32.5
Note. Each value is mean f SD of five samples, each assayed in triplicate. * Statistically significant (P < 0.05).
kinetic data were constructed according to the method of Lineweaver and Burk (1934). Data were subjected to regression analysis and regression lines were plotted for best straight-line fit. Data were also analyzed by Student’s t test to determine the differences between control and experimental treatments; a value of P -C 0.05 was considered significant. RESULTS The effects of in vitro and in vivo Pb on the uptake of 3H-DA were studied to delineate certain parameters of Pb toxicity to rat brain synaptosomes. The data in Table 1 suggest that Pb in vitro significantly decreased the uptake of 3H-DA. Maximum TABLE EFFECT
OF
3
pH ON Pb INHIBITION IN RAT
BRAIN
OF ‘H-DA SYNAPTOSOMES
UPTAKE
50
100
155 xk 9
96 + 6* (38.1)
82 + 16* (47.1)
6.5
170 f 13
llO& 13* (35.3)
101 + 25* (40.6)
7.0
573 + 69
206 + 42* (64.0)
165 + 16* (71.2)
7.5
1375 f 16
862 f 44* (37.3)
260 + 13* (81.1)
8.0
1084 + 46
679 + 18* (37.4)
176 + 18* (83.8)
PH
Control
6.0
Note. Each value is mean f SD of three preparations, each assayed in triplicate. Values in parentheses are percentage inhibitions over control. * Statistically significant (P < 0.05).
EFFECTS OF Pb ON ‘H-DOPAMINE
UPTAKE
91
decrease (68%) in uptake was observed with 100 PLM of Pb. A similar inhibition in uptake of 3H-DA by synaptosomes was observed in rats treated with Pb (Table 2). The inhibition of uptake was similar in the rats treated with either 25 or 50 mg/kg body wt of Pb for 14 days. Nevertheless, maximum inhibition (32.5%) was observed in rats treated with 100 mg/kg body wt of Pb. The data indicate that both in vivo and in vitro Pb exerted similar effects on uptake of 3H-DA in synaptosomes. The influence of pH on 3H-DA uptake was determined in the absence and presence of 50 and 100 PM Pb at varied pH (Table 3). In the absence of Pb, maximum 3HDA uptake was observed at 7.5 pH. On the other hand, in the presence of Pb, the uptake was significantly inhibited at all pH. Maximum inhibition of 3H-DA uptake was observed at slightly alkaline pH for both levels of Pb treatments. Activation with varying concentrations of Na+ (lo-100 mA4) showed no appreciable changes in ‘HDA uptake (Table 4). Compared with control, Pb at 50 piV decreased the 3H-DA uptake at 20 mA4 of Na+, whereas a further increase in Na+ up to 100 PM reversed the inhibitory effects. However, 100 PM Pb significantly decreased the 3H-DA uptake at 30 mM Na+. ATP-dependent (0.5-5.0 mM) 3H-DA uptake was studied in the absence and presence (50 or 100 pLM) of Pb (Fig. 1). The K, values for uptake of 3HDA were found to be 1.38, 3.09, and 4.08 mM for control, 50, and 100 PM Pb, respectively. No significant changes in V,,, were observed in the presence of Pb. DISCUSSION Pb is a recognized neurotoxicant, although its mechanism(s) of action are not well understood (Cooper et al., 1984; Shellenberger, 1984; Audesirk, 1985). The effects in TABLE EFFECTS
IN
3H-DA
VITRO OF Pb ON Na+ ACTIVATION UPTAKE (CPM/mg PROTEIN)
IN RAT BRAIN
Control
10
783 +41
30 40
50
SYNAPTOSOMES
755 +72 758 756
100
50
260 k44
753 k28
568 +48* (27.5)
473-t
432 + 38* (42.8)
526
613 f 60* (19.1)
306
654 + 49 (13.5)
329+
695
+
25
757
f
38
713k74 (5.81)
57* (35.6)
+ 117* (30.8)
f
59.6*
(59.6) 35* (56.5) 352k
(7.70)
100
OF
Pb (PM
NaCl (mM)
20
4
30* (53.2)
492k
48* (35.0)
Note. Each value is mean * SD of five different samples, each assayed in triplicate. Values in parentheses are percentage inhibition over control. * Statistically significant (P < 0.05).
92
BOYKIN,
/--c r/A-0.2 -0.6 -0.4
CHETTY,
0
I
I
0.2
AND RAJANNA
I
0.4
I
0.6
I 0.6
I 1.0
I
1.2
1
FIG. 1. Effect of Pb on the ATP-activation kinetics of 3H-DA in rat brain synaptosomes.
vitro of Pb on neuronal function have been studied to identify basic mechanisms of action (Minnema, 1986). The results of this study suggest that Pb inhibited 3H-DA uptake both in vitro and in vivo. A similar inhibition of catecholamine uptake has been reported with transition metal ions (Prakash et al., 1973; Lai et al., 1982) mercury (Rajanna and Hobson, 1985), and cadmium (Hobson et al., 1986). It has been shown that Pb and other heavy metals interfere with cellular energy metabolism by inhibiting ATP synthesis as well as ATP hydrolysis (Rajanna et al., 1983; Chetty et al., 1989). This leads to the dysfunction of energy-dependent neuronal events such as neurotransmitter uptake. The differential response of 3H-DA uptake to Pb at acidic, neutral, and alkaline pH ranges may be due to the modification of ionizable moieties or to a change in the charge pattern of the receptor protein (Na+-K+ ATPase) by Pb. This suggests that Pb inhibition of 3H-DA uptake is pH dependent. A similar modification of the ionizable moiety and charge pattern of Naf-K+ ATPase by heavy metals, organotin compounds, and chlordecone was reported earlier (Chetty et al., 1983, 1989; Prasada Rao et al., 1987). The alterations in Pb inhibition of 3H-DA uptake at different concentrations of Na+ or ATP suggest that Pb may primarily affect ATP- and Na+directed sites of the receptor protein (Na+-K+ ATPase) (Rajanna et al., 1983). The kinetic constants for ATP-dependent high affinity uptake of 3H-DA by synaptosomes in the presence of Pb suggest a competitive type of inhibition. CONCLUSIONS The results suggest that Pb both in vivo and in vitro inhibit the uptake of 3H-DA in rat brain synaptosomes. The inhibition of 3H-DA by Pb is concentration dependent. The ATP/Na+ concentration-dependent Pb inhibition of 3H-DA uptake verifies that the electrochemical potential gradient results from the ionic gradient maintained by ATP hydrolysis (Na+-K+ ATPase) and may be responsible for the uptake of dopamine in synaptosomes.
EFFECTS OF Pb ON 3H-DOPAMINE
UPTAKE
93
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