Interaction of Metals with Muscarinic Cholinoceptor and Adrenoceptor Binding, and Agonist-Stimulated Inositol Phospholipid Hydrolysis in Rat Brain

Comp. Biochem. Physiol. Vol. 116C, No. 2, pp. 111–116, 1997 Copyright  1997 Elsevier Science Inc.

ISSN 0742-8413/97/$17.00 PII S0742-8413(96)00165-X

Interaction of Metals with Muscarinic Cholinoceptor and Adrenoceptor Binding, and Agonist-Stimulated Inositol Phospholipid Hydrolysis in Rat Brain B. Rajanna,1 C. S. Chetty,2 S. Rajanna,3 E. Hall, S. Fail, and P. R. Yallapragada4 1

Division of Biological Sciences, Alcorn State University, Lorman, MS 39096, 2 Department of Biology, Savannah State University, Savannah, GA 31404, U.S.A. 3 Division of Computer Science and Mathematics, Alcorn State University, Lorman, MS 39096, U.S.A. and 4 Department of Zoology, Andhra University, Waltair 530 003, India ABSTRACT. In vitro mercury (Hg) or lead (Pb) effectively inhibited the binding of 3 H-quinuclidinyl-benzilate (QNB) (a muscarinic cholinoceptor antagonist) and 3 H-prazosin (an α1-adrenoceptor antagonist) to their receptors in cerebellar and cerebral cortex membranes in a concentration-dependent manner. Hg was more potent than Pb. When the rats were treated with Hg (5 mg/kg body wt) or Pb (25 mg/kg body wt) for 24 hr, a decrease in 3 H-prazosin and an increase in 3 H-QNB receptor binding were observed in cerebral cortex. There was no alteration in 3 H-prazosin binding in cerebellum with the above treatment of metals, but 3 H-QNB binding in cerebellum was significantly inhibited by Hg. However, both 3 H-prazosin and 3 H-QNB receptor bindings were significantly decreased in cerebellum of rats treated for 7 days with Hg (1 mg/kg body wt/day) or Pb (25 mg/ kg body wt/day). But in cerebral cortex of rats treated with these metals for 7 days, a decrease in 3 H-prazosin and an increase in 3 H-QNB receptor binding activities were noticed. There was a significant decrease in phospholipid content in cerebral cortex but not in cerebellum of rats treated with these metals for 7 days. At 100 µM concentration carbachol or acetylcholine or norepinephrine stimulated 3 H-inositol incorporation and 3 H-inositol phosphate (IP) formation in rat cerebral cortical slices. Hg or Pb in vitro though increased the agonist-stimulated 3 H-inositol incorporation, 3 H-IP formation was not significantly altered. The present investigation demonstrates the differential responses by α1-adrenoceptor and muscarinic cholinoceptor in cerebellum and cerebral cortex of rat to in vitro and in vivo effects of Hg or Pb. comp biochem physiol 116C;2:111–116, 1997.  1997 Elsevier Science Inc. KEY WORDS. Mercury, lead, inositol phospholipid hydrolysis, muscarinic cholinoceptor, α 1-adrenoceptor

INTRODUCTION The neurotoxicity of heavy metals such as mercury (Hg) and lead (Pb) is well established (10,11,19,26,30,31,33). Heavy metals interfere with many cellular functions of brain including conduction and neurotransmitter (NT) metabolism (15,32). However, it is still obscure as to which biochemical or physiological pathways are primarily affected by the metals. The underlying mechanism(s) of neurological signs caused by heavy metals have been correlated to alterations in cholinergic, adrenergic and gabaergic transmission leading to impairment of neural transmission (28). It has been shown that the heavy metals alter calcium homeostasis (1,14,33,34) and this may adversely affect the NT release, phosphorylation of proteins and activity of proteAddress reprint requests to: B. Rajanna, Department of Biological Sciences, Alcorn State University, 1000 ASU Drive, PO Box #870, Lorman, MS 39096, U.S.A. Tel. 601-877-6681; Fax 601-877-6256. E-mail: [email protected]

ases (7,24). Evidences suggest that alteration in calcium fluxes may be an important step in the process of neuronal damage and death (23,24). It has been established that the primary messengers such as hormones and NTs interact with their selective receptors on the cell surface and stimulate the hydrolysis of phosphoinositides (PI) resulting in the formation of second messengers such as inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (2,29). IP3 plays an important role in mobilizing intracellular calcium whereas diacylglycerol activates protein kinase C (2,3). G-protein serves as an intermediary to connect the surface receptor with phospholipase C (5,12). Thus the whole chain of signal transduction is regulated by the agonists such as hormones and NTs. The cerebellum and cerebral cortex have high density of IP3 and IP4 receptors. Any modulation in the receptors on the cell surface by metals will have a significant impact on the signal transduction mechanism altering the formation of second messengers. In view of this we propose to study the in vitro and

B. Rajanna et al.

112

in vivo effects of heavy metals such as Hg and Pb on the binding of agonists to their specific receptors and the in vitro interaction of these metals (Hg and Pb) with agonist-stimulated PI hydrolysis in cerebral cortical slices of rat brain. MATERIALS AND METHODS Materials The radioactive chemicals 3H-prazosin (Specific activity: 76.2 Ci/mmol; 98% purity), 3 H-Quinuclidinyl-benzilate (QNB) (Specific activity: 45.4 Ci/mmol; 98% purity), myo(2-3H) inositol (Specific activity: 20 Ci/mmol; 95% purity) and Aquasol were purchased from New England Nuclear Corporation, Boston, MA. All biochemicals including mercuric chloride (Hg) and lead acetate (Pb) were obtained from Sigma Chemical Co., St Louis, MO. Male SpragueDawley rats (175–200 g) were purchased from Harlan Sprague-Dawley Inc., Indianapolis, IN. They were housed for 1 week in our controlled animal facility at a temperature of 21°C, 50–80% relative humidity and 12 hr photoperiod conditions. A commercial diet (Purina Rat Chow) and water were provided to the animal ad libitum. In Vitro Studies The stock solutions of Hg and Pb were prepared in distilled water and 10 µl were added to the reaction mixture in order to get the desired concentration of the respective metals. The reaction mixture was incubated with the metal for 10 min prior to the initiation of the reaction. The controls received 10 µl of either sodium chloride or sodium acetate. In Vivo Studies One batch of rats were treated (0.5 ml, i.p.) for 24 hr with a dose of 5 mg/kg body wt of mercuric chloride or 25 mg/ kg body wt of lead acetate. Another batch of rats were given 1.0 mg/kg body wt/day of mercuric chloride or 25 mg/kg body wt/day of lead acetate for a period of 7 days. Controls received 0.5 ml of sodium chloride or sodium acetate. The rats were sacrificed by decapitation and the brains were quickly removed. The cerebral cortex and cerebellum were isolated on ice. 3

H-Prazosin

The binding of α1-adrenoceptor in cerebellum and cerebral cortex was studied using 3 H-prazosin as a selective ligand following the method described by Nicoletti et al. (22). Briefly, the cerebellum or cerebral cortex was homogenized in 30 volumes of ice-cold buffer A (pH 7.4) containing 0.25 M sucrose, 1 mM MgCl2, 5 mM Tris-HCl, and 0.05% ascorbic acid. The homogenate was centrifuged at 750 g for 8 min at 4°C. The pellet obtained was suspended in icecold buffer B (50 mM Tris-HCl, 10 mM MgCl2, 0.05%

ascorbic acid, pH 7.4). The reaction mixture (250 µl) consisting of buffer B and 300–400 µg of membrane protein was incubated at 25°C with 0.5 µCi of 3H-prazosin for 25 min. The reaction was terminated by adding 3 ml of icecold buffer B and filtered rapidly through Whatman GF/B glass fiber filters. Then the filters were washed three times with 5 ml of ice-cold buffer B and suspended in scintillation vials containing Aquasol. The radioactivity was determined using Beckman Scintillation Counter (LS 6800). Non-specific binding in the presence of 2 µM phentolamine was subtracted from the total to get specific 3 H-prazosin binding which was represented as fmols/mg protein. The membrane protein was estimated by Lowry et al. (16) using bovine serum albumin as standard. 3

H-QNB

The characteristic muscarinic receptor binding was studied in cerebellum and cerebral cortex using its antagonist 3HQNB following the method of Nordberg and Winblad (25). Briefly, the cerebellum or cerebral cortex was homogenized in 50 volumes of buffer (50 mM Tris-HCl, pH 7.4, 1 mM EDTA), pelleted by centrifugation (35,000 g for 10 min), washed twice in buffer and resuspended in 50 volumes of buffer. The 50 µl reaction mixture consisted of buffer (50 mM Tris-HCl, pH 7.4) and 200–400 µg of membrane protein. It was incubated with 0.25 µCi of 3 H-QNB at 4°C for 40 min and the reaction was terminated by centrifugation in microfuge. The pellet was extracted and the radioactivity was determined using a Liquid Scintillation Counter (LS 6800). Non-specific binding was measured by preincubating the reaction mixture with 1.5 µM atropine at 4°C for 60 min and subtracted from total to get specific binding. The results were presented as fmols/mg protein. PI Hydrolysis The method of Batty et al. (37) was used to determine the PI hydrolysis. Cerebral cortex slices (350 3 350 µm) were prepared using the McIlwain tissue chopper. The slices were transferred to screw-topped bottles containing a slightly modified Krebs-Hensleit buffer (118 mM NaCl, 4.7 mM KCl; 1.3 mM CaCl2, 1.2 mM KH2PO4, 1.2 MgSO4, 25 mM NaHCO3, 11.7 mM glucose) equilibrated with 95% O2/CO2 to a final pH of 7.4. The slices were agitated at 37°C in a shaking water bath for 1 hr with two intermediate changes of buffer. Samples (100 µl) of gravity packed tissue were then labelled with 5 µCi of myo-2-( 3 H) inositol for 30 min in a final volume of 300 µl of buffer containing 5 mM LiCl. To this 10 µl of metal was added and incubated for 10 min. Subsequently the samples were challenged with 100 µM (10 µl) agonist for 10 min and the incubation was stopped by addition of 880 µl chloroform/methanol (1: 2). To this 310 µl chloroform and 310 µl water were added to separate the phases. The phases were centrifuged at 1000 g for 5 min

Metals and Inositol Phospholipid Hydrolysis

113

and 750 µl of the upper aqueous phase was taken for assay of 3H-inositol phosphates. Two hundred µl of the lower phase was removed, dried overnight at room temperature, and dissolved in scintillation fluid for estimation of 3 H-inositol incorporation. The total 3 H-inositol phosphates (IP) were determined by the method of Berridge et al. (4). In the ‘‘Batch’’ for assay of total labelled inositol phosphates, 750 µl aliquots of the aqueous phase (obtained above) were diluted to 3 ml with water, and a 50% (W/V) slurry of Dowex-1 (0.5 ml: 100–200 mesh: 3 8) in the formate form was added to bind the phosphates. After four washes with 3 ml of 5 mM myo-inositol, the phosphates were eluted with 500 µl of 1 M ammonium formate/0.1 formic acid. Two hundred µl of this elute was then added to 5.0 ml of scintillation fluid for estimation of total 3 H-IP formation. FIG. 1. Effect of varying concentrations of Hg and Pb on 3H-

Phospholipids The phospholipids were estimated in cerebral cortex and cerebellum of control and treated rats colorimetrically by following the method of Stewart (35). The tissue was homogenized in 0.25 M sucrose and a protein concentration of approximately 10 mg/ml was extracted with chloroform/ methanol. One ml of the extract was removed with a syringe and concentrated to complete dryness in a stream of air at 50°C. The dried extract was then dissolved in 2 ml of chloroform in a test tube. To this 2 ml of 0.1 N ammonium ferrothiocyanate was added and mixed on a rotamixer for one minute. The lower chloroform phase was removed with a Pasteur pipet and the optical density was measured at 488 nm in a spectrophotometer. Dipalmitoyl lecithin was used as standard for calibration.

Statistical Analysis

prazosin binding in rat cerebellum (C) and cerebral cortex (CC) in vitro. (C: 259 6 15 fmols/mg protein; CC: 293 6 12 fmols/mg protein). Each value represents mean of at least four independent observations done in triplicate. *Significantly different from control. P , 0.05.

observed in cerebral cortex and cerebellum (except 24 hr treatment) of rats treated with Hg or Pb (Fig. 2). At micromolar concentrations Hg or Pb in vitro also inhibited 3 H-QNB receptor binding in both cerebellum and cerebral cortex in a concentration-dependent manner (Fig. 3). The data on IC50 showed that the muscarinic cholinoceptors in cerebellum (Hg: 20 µM; Pb: 82 µM) as compared to cerebral cortex (Hg: 37 µM; Pb: 96 µM) are more sensitive to Hg or Pb. From the results it is also evident that Hg or Pb are more potent (P , 0.05) on 3H-QNB than 3Hprazosin receptor binding both in cerebellum and cerebral cortex. No appreciable changes were observed in the receptor binding of 3 H-QNB in cerebellum of rats treated with

Data were expressed as the mean 6 SEM. All studies requiring statistical evaluation represent a minimum of four preparations each assayed in triplicate. Statistical significance was calculated by using ANOVA and two-sample t-test for independent samples. A value of P , 0.05 was accepted as statistically significant. IC50 values were determined using Slide Write Plus 3 (SWP3) program.

RESULTS The results from the in vitro effect of metals revealed that Hg and Pb at micromolar concentrations effectively inhibited 3 H-prazosin binding in a concentration-dependent manner. However, the IC50 values indicate that Hg was more potent (P , 0.05) in inhibiting the 3 H-prazosin receptor binding in cerebral cortex (42 µM) as compared to cerebellum (93 µM). Whereas, Pb elicited higher inhibition in cerebellum (143 µM) than in cerebral cortex (225 µM) (Fig. 1). Similar decrease in 3 H-prazosin binding was also

FIG. 2. In vivo effect of mercury (Hg) and lead (Pb) on 3H-

prazosin binding in cerebellum and cerebral cortex of rats treated with the metal for 24 hr and 7 days. *Significantly different from control. P , 0.05.

B. Rajanna et al.

114

FIG. 3. Effect of varying concentrations of Hg and Pb on 3 H-

FIG. 5. In vivo effect of mercury (Hg) and lead (Pb) on phos-

QNB binding in rat cerebellum (C) and cerebral cortex (CC) in vitro. (C: 322 6 12 fmols/mg protein; CC: 548 6 14 fmols/ mg protein). Each value represents mean of at least four independent observations done in triplicate. *Significantly different from control. P , 0.05.

pholipid content in cerebellum and cerebral cortex of rats treated with the metal for 7 days. *Significantly different from control. P , 0.05.

Hg or Pb for 24 hr or 7 days. However, a significant increase in 3 H-QNB binding was observed in the cerebral cortex of rats treated for 24 hr but not 7 days (Fig. 4). At 100 µM concentration, the agonists such as carbachol (157%), acetylcholine (56%) and norepinephrine (104%) significantly stimulated incorporation of 3 H-inositol in cerebral cortical slices (data not presented). Subsequent challenge with 300 µM Pb or Hg also further enhanced agonist-stimulated incorporation of 3 H-inositol. Pb elicited 272, 192 and 88% increases in the 3H-inositol incorporation when the cerebral cortical slices were preincubated with carbachol, norepinephrine, and acetylcholine, respectively. Similar increases (204, 153, and 76% for carbachol, norepinephrine and acetylcholine preincubated slices respectively) in 3 H-

FIG. 4. In vivo effect of mercury (Hg) and lead (Pb) on 3 H-

QNB binding in cerebellum and cerebral cortex of rats treated with the metal for 24 hr and 7 days. *Significantly different from control. P , 0.05.

inositol incorporation were also observed in presence of 300 µM Hg was added. At concentrations below 300 µM Hg or Pb did not elicit any appreciable effect on 3H-inositol incorporation in cerebral cortical slices (data not presented). A greater (72–78%) portion of the 3 H-inositol incorporated in presence of agonists was used in the formation of 3 H-inositol phosphates (IP). Similar rates of 3H-IP formation was also observed in the slices challenged with Pb (76– 79%) or Hg (80–86%). A significant decrease in phospholipid levels were observed in cerebral cortex of rats treated with Hg (46%) or Pb (26%) for 7 days (Fig. 5). However, the cerebellum of either Hg or Pb treated rats did not show significant change in the levels of phospholipids. DISCUSSION The data on 3H-prazosin binding indicate that heavy metals such as Hg and Pb in vitro effectively inhibit α1-adrenoceptor and muscarinic cholinoceptor binding in rat brain. The responses of α1-adrenergic receptors to in vitro and in vivo exposure of metals were found to be similar indicating an inhibition in their receptor binding activity. A decrease in 3 H-QNB receptor binding activity in cerebellum and a stimulation in cerebral cortex by Hg or Pb in vitro further confirm the earlier reports of regional differences in muscarinic receptor binding in rat brain of treatment with metals. Moingeon et al. (18) and Widmer et al. (36) reported an increase in muscarinic receptor binding in cortex and striatum on treatment with Pb. In other regions such as olfactory bulb and visual cortex, there was a significant reduction in the activity of these receptors on exposure to Pb (36). In contrast, Bondy and Agrawal (6) reported inhibition of muscarinic receptors by Hg or Pb. Recently Schulte et al. (39) demonstrated that the muscarinic receptors are insen-

Metals and Inositol Phospholipid Hydrolysis

sitive to Pb in vitro as well as in vivo in mouse brain. However, in the present study the muscarinic cholinoceptors in cerebral cortex exhibited differential response to in vitro and in vivo effects of Hg or Pb. These differential responses of the receptor binding could be attributed to changes in neurotransmitter levels during metal-induced toxicity (8, 13,27,28). In vitro inhibition of receptor binding may be due to interaction of Hg or Pb with the receptor protein resulting in conformational change. Additional investigations are required to understand the changes in receptor density and affinity in the presence of these metals. In general, a close relationship exists between the degree of occupancy of receptors by agonists and the extent of stimulation of PI hydrolysis (9). Receptor cloning studies revealed the existence of five different, but highly homologous subtypes of muscarinic receptors M1 to M5 (20). The M1 and M3 receptors are coupled to PI turnover whereas the M2 receptor is coupled to cAMP second messenger system. There is also evidence that one group of α1-subtypes are coupled to PI turnover and the other group is linked to activation of adenylate cyclase (17). Stimulation of 3 H-inositol incorporation in cerebral cortical slices by agonists such as carbachol, acetylcholine and norepinephrine observed in the present investigation confirms the earlier reports (21,29). Though Hg or Pb increased agonist-stimulated 3H-inositol incorporation, the rate of 3 H-IP formation was not significantly affected by these metals. This suggests that Hg or Pb did not alter PIP2 hydrolysis appreciably. Phosphoinositides constitute 10% of total phospholipids and their metabolic rates are higher when compared with other phospholipids (9). However, the decrease in phospholipid content observed in cerebral cortex of Pb or Hg treated rats might be partly due to the lipoperoxidative damage (38,40).

115

7. 8. 9. 10. 11.

12. 13.

14. 15. 16. 17.

18. 19.

This research work was supported by a NIH/NIGMS/MBRS Grant # GM 08169 awarded to Dr. B. Rajanna as a faculty in the Division of Natural and Applied Sciences at Selma University, Selma, Al. 36701, USA. Miss Elizabeth Hall and Miss Sandra Fail were Undergraduate Research Participants at Selma University.

20.

21.

References 1. Atchison, W.D.; Hare, M.F. Mechanisms of methylmercuryinduced neurotoxicity. FASEB J. 8(9):622–629;1994. 2. Berridge, M.J. Inositol trisphosphate and diacylglycerol: Two interacting second messengers. Ann. Rev. Biochem. 56:159– 193;1987. 3. Berridge, M.J. Inositol trisphosphate and calcium signaling. Nature 361:315–325;1993. 4. Berridge, M.J.; Downes, C.P.; Hanley, M.R. Lithium amplified agonist-dependent phosphatidylinositol responses in brain and salivary glands. Biochem. J. 206:587–595;1982. 5. Birnbaumer, L.; Abramowitz, J.; Yatani, A.; Okabe, K.; Mattera, R.; Graf, R.; Sanford, J.; Codina, J.; Brown, A.M. Roles of G proteins in coupling of receptors to ionic channels and other effector systems. CRC. Crit. Rev. Biochem. Mol. Biol. 25:225–244;1990. 6. Bondy, S.C.; Agrawal, A.K. The inhibition of cerebral high

22. 23.

24. 25. 26.

affinity receptor sites by lead and mercury compounds. Arch. Toxicol. 46:249–256;1980. Browning, M.D.; Huganir, R.; Greengard, P. Protein phosphorylation and neural function. J. Neurochem. 45:11–23; 1985. Bressler, J.P.; Goldstein, G.W. Mechanisms of lead neurotoxicity. Biochem. Pharmacol. 41:479–484;1991. Chuang, D.M. Neurotransmitter receptors and phosphoinositide turnover. Ann. Rev. Pharmacol. Toxicol. 29:71–110; 1989. Cooper, G.; Suszkiw, W.J.; Manalis, R. Presynaptic effects of heavy metals. In: Nahroski, T. (ed). Cellular and Molecular Neurotoxicology. New York: Raven Press; 1984:1–21. Cory-Slechta, D.A. Relationships between lead-induced learning impairments and changes in dopaminergic, cholinergic, and glutamatergic neurotransmitter system functions. Ann. Rev. Pharmacol. Toxicol. 35:391–415;1995. Hall, A. Signal transduction through small GTPases—A tale of two GAPs. Cell 69:389–391;1992. Komulainen, H. Neurotoxicity of methyl mercury: Cellular and subcellular aspects. In: Bondy, S.C.; Prasad, K.N. (eds). Metal Neurotoxicity. Boca Raton, FL: CRC Press; 1988:167 – 182. Komulainen, H.; Bondy, S.C. Increased free intracellular Ca21 by toxic agents: An index of potential neurotoxicity? Trends Pharmacol. Sci. 9:154–156;1988. Lai, J.C.K.; Lim, L.; Davidson, A.N. Effects of Cd21, Mn21 , and Al31 on rat brain synaptosomal uptake of noradrenaline and serotonin. J. Inorg. Biochem. 17:212–225;1982. Lowry, O.H.; Rosebrough, N.J.; Farr, A.L.; Randall, R.J. Protein measurement with the Folin-phenol reagent. J. Biol. Chem. 193:265–275;1951. Minneman, K.P.; Johnson, R.D. 3 α-adrenergic receptors linked to inositol phosphate and cyclic AMP accumulation in rat brain (Abstract). Abstracts of the Sixth European Society of Neurochemistry Meeting, pp. 103, European Society of Neurochemistry, Prague. Moingeon, P.H.; Bidart, J.M.; Alberici, G.F.; Boudene, C.; Bohuon, C. In vivo modulation of muscarinic receptors in rat brain by acute lead intoxication. Toxicol. 31:135–142;1984. Moore, M.R.; McIntosh, M.J.; Bushnell, W.R. The neurotoxicity of lead. Neurotoxicol. 7:541–556;1986. Mutschler, E.; Moser, U.; Wess, J.; Lambrecht, G. Muscarinic receptor subtypes—Pharmacological, molecular, biological and therapeutical aspects. Pharm. Acta. Helv. 69:115–119; 1995. Myles, M.E.; Fain, J.N. Carbachol but not norepinephrine, NMDA, ionocycin, ouabain or phorbol myristate acetate, increases inositol 1,3,4,5-tetrakisphosphate accumulation in rat brain cortical slices. J. Neurochem. 62:2333–2339;1994. Nicoletti, F.; Barbaccia, M.L.; Iadarola, M.J.; Pozzi, O.; Laird, H.E. Abnormality of α1-adrenergic receptors in the frontal cortex of epileptic rats. J. Neurochem. 46:270–273;1986. Nicotera, P.; McCokey, D.; Svensson, S.A.; Bellomo, G.; Orrenius, S. Correlation between cytosolic Ca21 concentration and cytotoxicity in hepatocytes exposed to oxidative stress. Toxicol. 52:55–63;1988. Nicotera, P.; Bellomo, G.; Orrenius, S. Calcium-mediated mechanisms in chemically induced cell death. Ann. Rev. Pharmacol. Toxicol. 32:449–470;1992. Nordberg, A.; and Winblad, B. Reduced number of 3 H-nicotine and 3 H-acetylcholine binding sites in the frontal cortex of Alzheimer brains. Neurosci. Lett. 72:115–119;1986. Pounds, J.G. Effect of lead intoxication on calcium homeostasis and calcium mediated cell function: A review. Neurotoxicol. 5:295–332;1984.

B. Rajanna et al.

116

27. Rajanna, B.; Hobson, M. Influence of mercury on uptake of 3 H-dopamine and 3 H-norepinephrine by rat brain synaptosomes. Toxicol. Lett. 27:7–14;1985. 28. Ramsay, P.B.; Krigman, M.R.; Morell, P. Developmental studies of the uptake of choline, GABA and dopamine by crude synaptosomal preparation after in vivo or in vitro lead treatment. Brain Res. 187:383–402;1980. 29. Rana, R.S.; Hokin, L.E. Role of phosphoinositides on transmembrane signalling. Physiol. Rev. 70:115–164;1990. 30. Rehul, K.R.; Chang, L.W. Effects of methylmercury on the development of the nervous system: A review. Neurotoxicol. 1:21–55;1979. 31. Rocha, J.B.T.; Pereira, M.E.; Emanuelli, T.; Christofari, R.S.; Souza, D.O. Effect of treatment with mercury chloride and lead acetate during the second stage of rapid postnatal brain growth on aminolevulinic acid dehydratase (ALA-D) activity in brain, liver, kidney and blood of suckling rats. Toxicol. 100: 27–37;1995. 32. Shellenberger, M. Effects of early lead exposure on neurotransmitter systems in the brain: A review with commentary. Neurotoxicol. 5:117–122;1984. 33. Simons, T.J.B. Lead-calcium interactions in cellular lead toxicity. Neurotoxicol. 14:77–86;1993. 34. Singh, A.K. Neurotoxicity in rats chronically exposed to lead

35. 36.

37.

38.

39. 40.

ingestion: Measurement of intracellular concentrations of calcium and lead ions in resting or depolarized brain slices. Neurotoxicol. 16:133–138;1995. Stewart, J.C.M. Colorimetric determination of phospholipids with ammonium ferrothiocyanate. Anal. Biochem. 104:10– 14;1980. Widmer, H.R.; Vedder, H.; Schlumpf, M.; Lichtensteiger, W. Concurrent changes in regional cholinergic parameters and nest odor preference in the early postnatal rat after lead exposure. Neurotoxicol. 13:615–624;1992. Batty, J.R.; Nahorski, S.R.; Irvine, R.F. Rapid formation of inositol-1,2,4,5-tatrakisphosphate following muscarinic receptor stimulation of rat cerebral slices. Biochem. J. 232:211– 215;1985. Sandhir, R.; Julka, D.; Gill, K.D. Lipoperoxidative damage on lead exposure in rat brain and its applications on membrane bound enzymes. Pharmacol. Toxicol. 74:66–71; 1994. Schulte, S.; Miller, M.S.; Friedberg, K.D. In vitro exposure to lead does not influence mascarinic receptors in the frontal cortex of the mouse brain. Toxicol. 93:99–112;1994. Skoczynska, A.; Smolik, R.; Jelen, M. Lipid abnormalities in rats given small doses of lead. Arch. Toxicol. 67:200–204; 1993.