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Developmental Lead Exposure and Two-Way Active Avoidance Training Alter the Distribution of Protein Kinase C Activity In the Rat Hippocampus

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Long-term exposure to a low level of lead is associated with learning deficits. Several types of learning have been correlated to hippocampal protein kinase C (PKC) activation. This study was designed to determine if there is a correlation between
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  Neurochemical Research, Vol. 22, No. 9, 1997, pp. 1119-1125 Developmental Lead Exposure and Two-way ActiveAvoidance Training Alter the Distribution of Protein Kinase C Activity in the Rat Hippocampus Hwei-Hsien Chen, 1 Tangeng Ma, 1 Ian A. Paul, 1 James L. Spencer, 1 and Ing K. Ho 1 ' 2 (Accepted March 10, 1997) Long-term exposure to a low level of lead is associated with learning deficits. Several types of learning have been correlatedto hippocampal protein kinaseC (PKC) activation. This studywasdesigned to determine if there is a correlation between the effects of lead on hippocampal PKCactivation and those on learning performance. Rats were exposed to 0.2% (w/v) lead acetate at different developmental stages including a maternally exposed group, a postweaning exposedgroup, and a continuously exposed group. The continuously lead exposed rats tended to avoid less frequently and not respond more frequently in two-way active avoidance training than did controls.This training process was associated with translocation of hippocampal PKC activity from cytosolto membrane. Two-way analysis of variance of data indicates that there is a significant trainingand lead treatment interaction in the ratio of membrane to cytosolic PKC activity (F 332 = 3.013; p = 0.044). The interaction is attributable to the absence of the training-induced PKC translocation in the continuously lead exposed rats. In addition, no significant changes were observed in learningperformanceand training-induced hippocampal PKC activation after maternal and postweaninglead exposure. Continuous and longer duration of lead exposure appears to affect the learning performance and hippocampal PKC activation. These data suggest that a change in the activationof hippocampal PKC may be involved in the lead-induced deficit in learning. KEY WORDS: Lead; protein kinase C; avoidance learning. INTRODUCTION Long-term exposure to a low level of lead is as- sociated with learning and behavioral deficits in humanand experimental animals (1,2). However, the mecha-nism of the effects of lead on learning and memory still remains unknown. Even though there are various param-eters of neuronal function affected by lead, few of them 1 Department of Pharmacology and Toxicology University of Missis-sippi Medical Center 2500 North State Street Jackson, Mississippi 39216. 2 Address reprint requeststo: Dr. Ing K. Ho,DepartmentofPharma- cology and Toxicology, University of Mississippi Medical Center, 2500 North State Street, Jackson, Mississippi 39216. Tel.: 1-601-984- 1602 Fax: 1-601-984-1637. occur in exposures to low concentrations of lead whichhave been postulated to cause disturbance of brain de-velopment and function in young children. The brainlead levels were determined in the micromolar range inthe chronic lead exposed animals (3-5). Most of the lead inthe brain binds with high affinity to glycosoaminog-lycans (6); therefore,the free lead concentrationinbrainshould be far less than micromolar. Brain PKC has beenreported to be activated by lead at subnanomolar con-centrations (10~ 14 to10~'°M) in thepresenceofphos-phatidylserine (PS) and diacylglycerol (DAG) (7). Directmeasurement of free lead and calcium in the PKC re-action mixture indicates that free lead activates PKC in the range of 1Q-" to 1Q- 8 M (8). So far, PKC is the only cellular target affected by lead where the range of 1119 0364-3190/97/0900-1119$12.50/0 O 1997 Plenum Publishing Corporation  1120Chen, Ma, Paul, Spencer, and Ho free lead concentration is about 2-3 X lO' 11 M (9,10).However, Murakami et al. (11) reported that rather thanactivating PKC, lead exerts an inhibitory effect (IC 50 of ~2-10 |0,M) on the individual PKC subtypes (I, II, and III which correspond to 7, fil + (311, and a) purified from the ratbrain.Thestudiesoflead effects on PKCconducted in vitro are not consistent. Furthermore, theintracellular free lead concentration in the brain of leadexposed individual cannot be determined. Measurement of the PKC activation in vivo is a more relevant way than in vitro approaches to clarify the effects of lead on PKC in physiological conditions. Although direct dem- onstration of PKC activation in vivo is not possible,'translocation' of the enzyme provides an indirect indi-cator of enzyme activation. The intracellular transloca- tion of PKC from cytosol to membrane has beenproposed to reflect the state of enzyme activation(12,13). The long-term activation of PKC, which results in long-term alteration in the state of phosphorylation ofphosphoproteins,hasbeen proposedas abiochemicalmechanism involved in the regulation of signal trans-duction in theprocessoflearningandmemory (14,15). The hippocampus is an important site of learning and memory, and the learning and memory deficits producedby hippocampal lesions are similar to those observed inlead-exposed rats (16). Inaddition, hippocampalPKCactivation has been correlated with performance in sev- eral learning tasks (14,17-23). A lead-induced relativechange in the activation of hippocampal PKC in vivocould explain, in part, the lead-induced deficits of learn- ing and memory.This study was designed to determine if a change in the hippocampal PKC activation is involved in theputative lead-induced deficit in learning. The following experiments were planned: (1) the effects of lead on learning performance in the two-way active avoidance task; (2) the effects of lead on hippocampal PKC acti-vation by monitoring the PKC activity in the membrane and cytosol; and (3) the effects of lead and two-wayactive avoidance training on hippocampal PKC activa-tion. EXPERIMENTAL PROCEDURE Materials: Sprague-Dawley rats were obtained from Harlan (In- dianapolis, IN). The protein kinase C enzyme assay system (RPN77) and [7- 32 P]ATP were purchased from Amersham International (Ar- lington Heights, IL). Tris-HCl, ethylenediaminetetra-acetate (EDTA),ethyleneglycol-bis-(p-aminoethyl ether) N,N,N',N'-tetra-acetate (EGTA),p-mercaptoethanol, leupetin, and phenylmethylsulphonyl fluoride (PMSF) were obtained from Sigma Chemical Co. (St. Louis, Mo). Bicinchonic acid (BCA) protein assay reagent was obtained fromPierce (Rockford, IL). Animal Treatment Protocols. All animal use procedures were in strict accordance with the NIH Guide for the Care and Use of Labo-ratory Animals and were approved by the Institutional Animal Careand Use Committee. Timed pregnant female Sprague-Dawley rats (1-2 daygestation) were purchased, housedinindividual nesting cageandmaintained on a controlled 12-hr light/dark cycle, with free access to food and water. Lead treated groups received 0.2% (w/v) lead acetate[(C 2 H 3 O 2 ) 2 Pb • 3H 2 O] (si000 ppm) in their drinking water. Controlanimals received 0.145% (w/v) sodium acetate (the same number of acetate molecules as 0.2% lead acetate) under the same condition.Animals were checked each morning and afternoon for litters, and theday on which a litter was obtained was taken as PNO. On PN1 thepups were sexed, and cross-fostered with other litters within 24 hoursof birth. Each dam received 8-10 male pups.Animals were divided into 4experimental groups:acontrolgroup; a maternally exposed group which was exposed to lead in utero, via maternal milk ended at PN21; a postweaning exposed group whichwas exposed to lead from drinking water started at PN21 and contin-ued until PN56; a continuously lead exposed group which comprised of maternal and postweaning lead exposure. Two-Way Active Avoidance TrainingEquipment. Two-way active avoidance training was conducted intwo identical metal shuttleboxes equipped with wire grid floors foradministering shock (Columbus Instruments, Columbus, OH). Eachshuttlebox had two chambers, which were separated by a metal barrier with an open doorway. A small incandescent light bulb was mountedon the top panel in each chamber. A speaker and sound generator weremounted above thebarrier. Duringtheexperiment,the light and a 3kHz tone signaled shocks. Shock intensitywas set at 0.5 mA. Infrared beams on both sides of the barrier registered crossing between cham-bers. Stimulus programming, shock delivery, and data recording werecomputer-controlled. Training Procedure. Animals were movedto thetesting roomforat least2hours before each experimental session.At thestartof atraining session, the room lights were turned off, and the programstarted. Rats were acclimatizedto thetesting chamberfor 2minutes.During acclimatization, rats were allowed to freely explore both cham- bers of the shuttlebox. At the start of each trial, the light in the shut-tleboxwasswitchedon. Thetonein theshuttleboxwasswitchedon3 seconds later. If the rat crossed to the other chamber within 6 sec- onds,thelightandtone were switch off,and a 30 seconds intertrialinterval began. If the rat did not cross within 6 seconds, shock was delivered until either the animal crossed or for the maximum 10 sec- onds. Trial was followed by a 30 seconds intertrial interval. Each ratwasgiven60trialsatPN53andPN54and 30trialsatPN55. Latencies to cross from one side of the chamber to the other were recorded and rated as "avoids", "escapes", and "no responses". An avoid was recordedif theanimal crossed less than6 seconds after thelightwaspresented. A latency greater than 6 seconds but less than 16 secondswas scored as an escape, and a trial in which the animal did not cross was scored as a no response. Tissue Preparation. All rats were sacrificed by decapitation atPN56. The hippocampus was rapidly dissected, placed in a plasticmicrocentrifuge tube and stored at -70°C (24). This freezing proce-dure was necessary to reduce the variability normally associated withthe assay of PKC activity when the samples could not be collected atthe same time. On the day of assay for PKC activity, the hippocampuswas allowed to thaw at room temperature for 1 min and then placed  Lead Exposure and Protein Kinase C Activity 1121 into 1.5 ml ice-cold homogenization buffer (50 mM Tris-HCl, pH 7,5, 5 mM ethylenediaminetetra-acetate (EDTA), 10 mM ethyleneglycol-bis-(p-aminoethyl ether) N,N,N',N'-tetra-acetate (EGTA), 0.3% (w/v)fl-mercaptoethanol, 10 tig/ml leupetin and 1 mM phenylmethylsul- phonyl fluoride (PMSF)). Tissues were homogenized and then centri- fuged at 100,000 g for 30 min to yield supernatant and pellet fractions. The supernatant contained the cytosolic fraction. The pellets were re-suspended in 1.5 ml homogenization buffer containing 0.1% (v/v) Tri-ton X-100, rehomogenizedbyPolytron(4 x5s), shakenfor 30 minat 4°C, and centrifuged at 100,000 g for 30 min. The supernatantcontained solubilized membrane fraction. The membrane fractions and the previously saved cytosolic fractions were diluted to 1-3 jig pro-tein/25 nl with 50 mM Tris-HCl, pH 7.5, and assayed for PKC activity. Measurement of Protein Kinase C Activity. PKC activity wasmeasured by the transfer of phosphate from [7- J2 P]ATP (5,000 Ci/mmol) into substrate peptide (Amersham PKC assay systemRPN77) at 37°C for 15 minutes. The basic reaction mixture contained 1-3 |lg sample protein, 102 (iM substrate peptide, 2.7 ng/ml of <*- phosphatidyl-L-serine (PS), 2.7 ng/ml phorbol 12-myristate 13-acetate(PMA), 1.36mMcalcium acetate,6.5 mMmagnesium acetate, 0.11 mM ATP, and 0.2 nCi [«/- 32 P]ATP (6 nM) in a final volume of 55 ul in 50 mM Tris-acetate, pH 7.5. Nonstimulated PKC activity was de- termined without Ca 2 *, PS and PMA. The reaction was terminated byadding 10 ul of 300 mM orthophosphoric acid containing carmosinered. A 35 fll aliquot was spotted onto Whatman p81 phosphocellulose paper. The paper was washed with 5% (v/v) acetic acid (at least 10ml per paper), transferred to vials, and counted in a Packard liquid scintillation analyzer (2200 CA, Grove, IL). Results were expressed as pmol 32 P incorporated into peptide/min/fig protein. PKC activity wascalculatedas the difference between PMA and PS-stimulated and non-stimulated activity. Protein concentrationwasdeterminedby the BCA method (25). Determination of Blood Lead. Blood samples were collected after decapitation of the animals. Concentration of lead in the blood for each animal was performed by anodic stripping voltammetry (LeadAnalyzer Model 3010B, ESA Inc. Bedford, MA). Statistical Analysis. Data were analyzed by analysis of variance(ANOVA) followed by Student-Newman-Keuls method. The effects of training and lead exposure at different stages were compared using two factors, exposure stage and training. Differences were deemed significant when p < 0.05. RESULTS Body Weight and the Levels of Blood Lead, Leadexposure during gestation did not produce any signifi- cant effect in litter size and birth weight. The weightgain in control rats and in lead-exposed rats was similar,consistent with previous report (26). The blood concen-trations of maternally exposed rats were lower than the postweaning and continuously exposed rats (Table I). Effects of Lead onTwo-Way Active AvoidanceLearning. The performances of rats in two-way active avoidance task are shown in Fig. 1 by scattering plot.Data analyzed by one-way ANOVA indicated that there was a significant difference in the number of no response (F 318 = 5.285, P = 0.009), but no significant difference Table I. Blood Lead Concentrations ofRats Exposed to Lead at Different Developmental Stages Control <2 Maternal 3.8 ±0.2Postweaning25.3 ±1.9Continuous29.9+1.3Values are expressed as mean ± SEM in unit of |ig/dl. Maternal: maternally exposed group in which a lead acetate exposure concentration of 0.2% was administered in utero, via maternal milk till PN21 and sacrificed at PN56.Postweaning: postweaning exposed group in which the lead exposure was started at PN21 and continued until PN56.Continuous: Continuously exposed group in which lead exposure wereadministered in utero, via maternal milk till PN21, then through drink- ing water till PN56. in the number of avoidance (F 3|18 = 2.698, P = 0.076) and escape (F 318 = 0.213, P = 0.886). Post hoc com-parison revealed that the difference in the number of no response was due to the fact that the continuously ex- posed rats were more unresponsive than other threegroups. Effects of Lead on Hippocampal Protein Kinase C Activation. Since the PKC activity assay was performed in the optimal concentrations of cofactors such as Ca 2+ ,PS, andPMA,PKCactivity reportedin theliteratureis correlated to the amount of PKC protein (18,27). How-ever, the protein concentrations in the extracts of mem-brane and cytosolic fractions are not the same.Considering the difference in protein distribution, the data were normalized as percentages of total PKC activ- ityto be representative of the distribution of PKC in the membrane and cytosolic fractions. Half of the rats exposedtoleadat different devel-opmental stages were trained by two-way active avoid-ance learning task. Since training process affected the PKC distribution, the results from untrained rats and allrats (untrained and trained) were not consistent. It ismore reasonable to use the data from untrained rats to present the effects of lead on PKC activation. One-way ANOVA indicated that there was no significant differ- ence between groups in cytosolic PKC activity (F 3i , 6 =0.846, P = 0.489), but a significant difference existed in membrane PKC activity (F 3 , I6 = 4.948, P = 0.013).Lower membrane PKC activity was shown in maternallyandpostweaning exposed groups (Table II). Whenwecompared the distribution of PKC activity, postweaningexposed rats showed lower percentage of total activity associated with membrane and higher percentage in cy- tosol compared to control rats (Table III), Effects of Lead and Two-Way Active AvoidanceLearning on Hippocampal PKC Activity. Rats that had been trained showed higher PKC activity in the mem-brane compared to untrained rats (F 332 = 84.879, P <  1122Chen, Ma, Paul, Spencer, and Ho Fig. 1. Effects of lead exposure during different developmental stages on the performances in two-way active avoidance learning task. Scatter plots of (A) number of avoidance, (B) number of escape, (C) number of no response. Total of 150 trials/rat were performed. *P < 0.05 in Newman-Keuls test after ANOVA. Table II. The PKC Activity in the Rats With and Without Two- Way Active Avoidance Training Untrained Cytosol Membrane Trained CytosolMembraneControl 14,46±0.79 18.26 ±0.78 13.82±0.4621.79 + 0.61*Maternal14.48 + 0.8815.59 + 0.67°15.09 ±0.70 21.12±0.20*Postweaning14.39 + 0.78 14.07 + 0.94° 15.14+1.18°19.93 + 0.50"Continuous15.09 + 0.7016.93 ±0.83 19.40 ±0.57°*19.40 ±0.52"'*Values are expressed as mean±SEM, pmol 32 P incorporated per min- ute per microgram protein. "Significantlydifferent from control group at p<0.05 in Newman- Keuls test after ANOVA. 'Significantly different from untrained group at p<0.05 in Newman- Keuls test after ANOVA. 0.001), but not in the cytosol (F 3>32 = 3.593, P = 0.067). When we compared the distribution of PKC activity us- ing the ratio of membrane to cytosolic PKC activity, two-way ANOVA analysis indicated that there was a significant interaction between training and lead treat-ment (F 332 = 3.013; P = 0.044). Training caused the shift of PKC activity from cytosol to membrane in con- trol, maternally, and postweaning exposed groups, but no significant difference between untrained and trainedrats in continuously exposed group (Fig. 2). The inter-action is due to the lack of the training-induced PKC translocation in the continuously lead exposed rats. DISCUSSION Blood-lead concentrations in postweaning and con- tinuously lead exposed rats were approximately 30 Hg/dl. The Center for Disease Control has establishedthe blood lead levels ranging from 30 to 80 ug/dl to beassociated with "subclinical lead poisoning" in lead- exposed children. The exposure level used in this inves-tigation is in the range of lead present in the environmentthat may cause low-level toxicity in children. The blood lead levels of preweaning exposed rats were significantly lower than the postweaning and continuously exposed rats. The results from the performance of two-way activeavoidance learning task showed a tendency of a lowernumberofavoidanceand significantly higher numberof no response in continuously exposed rats, but not in ma- ternally and postweaning exposed rats. The blood levels of maternally exposed rats were low (3.8 ± 0.2 |Ag/dl) at PN56. Although we did not measure the lead levels in the brain, the brain lead levels of chronic lead-ex-posed animal are correlated with the blood lead levels  Lead Exposure and Protein Kinase C Activity1123 Table III. The Distribution of PKC Activity in the Rats With and Without Two- Way Active Avoidance Training Untrained CytosolMembrane Trained CytosolMembraneControl 40.75 ± 1.05(%) 59.25 ± 1.05(%) 35.56 ± 1.1 3(%) 64.44 ± 1.1 3(%) Maternal 44.61 ± 1.86(%) 55.39 ± 1.86(%) 38.24± 1.03(%) 61.76 ± 1.03(%) Postweaning47.16 ± 1.45(%) 52.84 ± 1.45(%) 39.58 ± 2.2 1(%) 60.42 ± 2.2 1(%)Continuous 44.97 ± 1.81(%) 55.03 ± 1.81(%) 46.52 ± 1.29(%) 53.48 ± 1.29(%)Values are expressed as mean ± SEM, % of total PKC activity. (3-5). In addition, the half life of lead in thehippocampus is about 21 days (3), and the levels of lead in the brains of maternally exposed rats when lead ex- posure was stopped at PN21 should be low at PN56. Itis possible that some lead-induced functional changes had recovered in the maternally exposed rats. It has beendemonstratedthatthe PKCactivityandexpression pat-tern of rats at PN21 is similar to those in adult rats (28,29). The already developed brain is more resistant to lead exposure than the developing brain, therefore, no impairment was observed in the performance of two-way active avoidance learning task in the postweaningexposed group which lead exposure started at PN21. Thedata revealed that continuous and longer durationof leadexposure isrequiredtoinduce impairmentinlearningperformance.Several studies have correlated hippocampal PKC with performance in learning tasks. For example, clas- sical conditioning of the nictitating membrane response in rabbits has been correlated with increased phorbol es- ter binding, which indicates that membrane-associatedPKC is increased in the hippocampus (18,19). Spatiallearning performances of rats (20,21) and mice (22,23)in the hidden-platform Morris water maze task have alsobeen correlated with hippocampal PKC redistribution.Our studies showed that two-way active avoidance train- ing also induced an PKC activation in hippocampus. Only in thecontinuously lead-exposed rats,themem-brane associated PKC after training did not increase. The absence of PKC activation and translocation in the hip-pocampusisconsistent withtheimpairmentofbehav-ioral performance in continuously lead-exposedrats. The assay of PKC activity was conducted underoptimal conditions and, thus, its activity is considered to reflect the amount of enzyme protein. When a change isobserved in the PKC activity, it is assumed that the amount of PKC protein has also changed. In addition to redistributionof PKC from cytosoltomembrane,thelead or training-induced changes in PKC activity maybe the results of the alteration in its expression. How- Fig. 2. Effects of lead exposure during different developmental stagesand two-way active avoidance training on the distribution of hippo-campal PKC activity. The PKC activity and protein concentration ofmembrane and cytosol fractions were measured as described in ex-perimental procedure. Data were normalized to the ratio of membrane to cytosol PKC activity. *P < 0.05, in Newman-Keuls test after two-way ANOVA. ever, we can not rule out the possibility of direct lead-induced changes in enzyme activity.Markovacand Goldstein (7) were the first to sug-gest that lead may interact with PKC. They demon-strated that lead activates brain PKC activity atpicomolar concentrations. It is interesting that, in a sim-ilar study carried out in microvessel preparations, Mar-kovac and Goldstein (30) observed that micromolarconcentrations of lead were required to activate PKC.The results of the studies suggest that microvessel prep-arations might contain a PKC subtype with lower sen-sitivity to lead. However, contradictory results indicatingthat lead inhibits (IC 50 ~2-10 (iM) rather than activatesthree subtypes (y, (31 + {ill, and a) of PKC activity werereported by Murakami et al. (11). Tomsig and Suszkiw(31) found that lead is a partial agonist of PKC in ad- renal chromaffin cells, thus, lead activates the enzyme in the picomolar range and inhibits the enzyme in the nanomolar to micromolar range. These studies were allconducted in vitro. Based on the present study, contin-uous lead exposure resulted in an impairment of the two-
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