Study On CaMKII And Its Downstream Signal Mechanism In Pb-exposed Hippocampus Of Rat

ObjectivePb is the common environmental neural toxicant.It's well known that exposureto Pb results in learning and memory disorder in children. In 1943,Byers and Lordfirstly reported that Pb could endanger Children cognition behavior and intelligencedevelopment in long term. Their sensibility and reactivity to neural poisonous effectof Pb isn't equal between the adults and children and the central nervous system ismore sensitive to Pb at developing stage. Pb toxicity brings about the nonreversibleimpairment of memory and attention in embryonic and infant period.At the present time, research has demonstrated that some protein kinases insynapte were activated by phosphorylation involved in learning and memory. Thereare three protein kinases at least to be activated in the form of hippocampal spatiallearning process, including Ca²⁺/Calmodulin dependent protein kinaseâ…¡(CaMKâ…¡), protein kinase A (PKA) and extracellular signal regulated kinase(ERK1/2) which maintain in long-playing activation state by self-phosphorylation.CaMKâ…¡has 8～12 subunits and is the richest kinase in the post synapticdensity of hippocampal and new cortex where are the basic structures of learningand memory. As the result of its phosphorylated Thr-286, Ca²⁺-dependent CaMKâ…¡becomes Ca²⁺-independently which explains why CaMKâ…¡can keep longtimeactivity following Ca²⁺ reduction.Mitogen activated protein kinase (MAPK) is also implicated inhippocampus-dependent long-term potentiation (LTP) and spatial learning. As anevidence, ERK inhibitor SL327 could impair spatial learning in mouse, andactivation of ERK2 was necessary in the form of LTP in rat hippocampus as well asthe formation process of association and spatial memory.Along with cAMP-induced LTP, some new proteins were synthesized which playcrucial roles in long-term memory.Ca²⁺-influx elicited by synaptic activation could activate Ca²⁺/CaM- sensitive adenylate cyclate (AC) and eventually phosphorylatedcAMP response element binding proten (CREB) leading to synthesize AMPAreceptor participating in LTP and long-term memory.In a word, these kinases including CaMKâ…¡and PICA,PKC and ERK1/2 all canaffect long-term learning and memory by regulating genes expression andsynthesizing new proteinsAlthough both CaMKâ…¡decreasing activation and Pb toxicity lead to spatiallearning disorder, their relationship is not well defined, especially, whether Pb canaffect CaMKâ…¡or not.Our team have researched the effect of Pb on nervous system signaltransduction since 1995.It was proved that Pb could increase Ca²⁺ in neuron andalter activation of PKC and ERK1/2, NOS,c-fos and c-jun, and PKC played animportant role in the form and maintenance of LTP. Based on CaMKâ…¡is alsoinvolved in learning and memory, our aim is to explore the impact of Pb on CaMKâ…¡in learning and memory and testify a certain relationship between it and ERK1/2or CREB in LTP to provide forceful evidence for the mechanism study of learningand memory, which will contribute to a good birth and care and improve citizenquality.MethodsTreatment with acute Pb exposure as follows:The healthy Wistar rats were provided by the experiment animal centre ofChinese medical university and put to death by neck dislocation. Then, thehippocampus were take out quickly and cut into 350μm slices in cold ACSFventilated with 95% O₂ and 5% CO₂. After stabilizing culture for 2 hours, thehippocampal slices were treated by different concentrations of Pb and KN 93(CaMKâ…¡special inhibitor) and cultured for desired times(0, 3, 7.5, 15, 30, 60,120min). To confirm the effects of Pb and KN93 on ERK2 and CERB, Westernblots was used to determine the active ERK2 and CERB and their total proteinexpression,and CaMKâ…¡mRNA expression was detected by RT-PCR.Treatment with chronic Pb exposure in vivo as follows:The pregnant rats were divided randomly into Pb group drinking 0.2% acetic acid lead and the control drinking Running water through to gestation and lactationperiod. Be to ensure that the child mice had identical nutrition condition, eachmother feeded 8 children at most. After ablactation at 21 d, child mice of Pb groupdrank directly 0.2% acetic acid lead as same as mothers and the child mice ofcontrol group drank Running water. To obtain chronic Pb-exposed and controlhippocampal slice, neck dislocation was uesed to put rats to death at 40 d, 60 d and80 d respectively after ablactation initiation. Western blots was to determine thetotal proein exprssion of ERK2 and the active CREB to observe the effect chronicPb exposure on ERK2 and CREB in b hippocampal slicesResults1. The effect of actue Pb exposure on active ERK2Actut exposing hippocampal slices to 20mmol/L Pb increased active ERK2 atfirst and decreased it later(minimum at 30 min) and resumed normal levelgradually.2. The effect of actue Pb exposure on total protein expression ofERK2Actut exposing hippocampal slices to 20mmol/L Pb had no effect on the totalprotein expression of ERK2.3. The effect of chronic Pb exposure on total protein expression ofERK2Chronic exposure to 0.2% Pb could reduce the total protein expression of ERK2on 20 d and 40 d, 60 and,80 d.4. The effect of actue Pb exposure on the active CREB and its totalprotein expressionActut exposure to 20mmol/L Pb could inhibit CREB activity and had no effecton the total protein expression of CREB.5. The effect of chronic Pb exposure on total CREBChronic exposure to 0.2% Pb had no effect on the total protein expression ofCREB on 20 d and 40 d, 60 and,80 d. 6. The effect of actue Pb exposure on CaMKâ…¡mRNA expressionActut exposure to Pb had no effect on CaMKâ…¡mRNA expression.7. The effect of CaMKâ…¡inhibitor KN 93 on ERK2 activityThe hippocampal slices treated with CaMKâ…¡inhibitor KN-93 could inhibit theincreasing activity of ERK2 induced by glutamic acid.8. The effect of CaMKâ…¡inhibitor KN 93 on active CREBKN-93 treatment could prevent the increasing activity of CREB induced byglutamic acid.Conclusion1. Actue Pb exposure-induced spacial learning and memory disorder wascorelative closely to its effect on the ERK2 activity.Chronic Pb exposure couldaffect the total protein expression of CREB rather than actue Pb exposure.2. Actue Pb exposure could block CREB activity.3. Actue and chronic Pb toxicosis were likely to inhibit CaMKâ…¡activity andaffect its downstream signal resulting in nervous system impairment.