Study Of Pb²⁺ On [³H]ryanodine Binding With RyRs Of The Brain’s Endoplasmic Reticulum

Objective:The toxic metallic, lead,is widely distributed in the environment and a two valent metal as the same as calcium. lead is believed to mimic or interfere the calcium dependent biochemical process and cause multi system damage to the nervous system of the body. The previous basis showed that the lead can cause an increase in resting calcium concentration in hippocampal neurons through the Ry Rs. Then whether or not lead directly to the Ry Rs channel opening effecta nd how to effect can make nothing of it. Therefore, this study will be through the receptor ligand binding assay of lead with endoplasmic reticulum [3H]ryanodine on the brain, and study the impact of lead on open Ry Rs channel function. Methods:(1) Adult rats were anesthetized to take the brain directly,then homogenize with the lysates after the brains were removed.(2) We adopt the method of density gradient ultracentrifugation to get the endoplasmic reticulum of rat brain.And identify endoplasmic reticulum components by transmission electron microscopy and Western blotting.(3) Experimental study on radioactive ligand receptor binding: A. Effect of lead on cerebral endoplasmic reticulum [3H]ryanodine binding, including dose response and time response relationship; B. Effect of interaction between lead and calcium on [3H]ryanodine binding; C. The interaction effect of lead and other regulators of Ry Rs combined with [3H]ryanodine. Results:(1) The extraction of endoplasmic reticulum contains rough endoplasmic reticulum and smooth endoplasmic reticulum and Ry R2, Ry R3 and endoplasmic reticulum marker protein GRP78 all expressed.(2) [Pb2+]f in the 0~100μM range, the lead ion inhibits [3H]ryanodine binding with "V" type.(3) The overall level of 0.39 μM lead ion calcium dependent [3H]ryanodine was inhibited, but inhibition of calcium dependent curves of lead was not in a concentration dependent manner; the presence of calcium concentration make lead have more obvious inhibition.(4) The presence of lead influence or change the role of regulators such as PKA, DTT and Mg2+ on Ry Rs. Conclusion:The brain endoplasmic reticulum radioactive ligand receptor binding experiment showed that lead may act on multiple sites of action of Ry Rs and inhibit [3H]ryanodine binding with "V" type.