Expression Alternation Of Secretory Pathway Ca²⁺-ATPase（SPCA1） Induced By Global Cerebral Ischemia Reperfusion In Sprague-Dawley Rats And Its Relationship With Calcium Overload

ObjectiveAnalyze expression pattern of SPCA1responding to global cerebral ischemia reperfusion and explore its possible involvement in cytosolic Ca2+regulation.MethodsThe four-vessel occlusion rat were used as model, which is established by occluding20min and then reperfusion for different times, such as3h,6h,24h,3d and7d. Methods of Nissl staining and hematoxylin-eosin (H&E) staining were utilized to study morphological and amount alternations of neurons in different brain tissues. Western Blot and immuno-fluorescence hiological analysis were applied to test the expression of SPCA1protein in cortex of cerebrum. And fluorospectrophotometry were used to determine concentration of Ca2+in cytoplasm and Golgi vesicles of nerve cells by using Fura2-AM uploading. All datas derived from experiments were analyzed in SPSS15.0. ResultsResults of H&E staining and Nissl staining showed that brain tissues were injured in large area by ischemia and then reperfusion, the morphological alternation possess characteristics of delayed neuron death for cytoplasm shrinkage, nuclear condensation and so on. And the amount of neurons decreased dramatically during the process of ischemia and reperfusion.Fluore-immunological location and determination of SPCA1protein in ischemia and reperfusion rat brain tissues indicated that SPCA1expressed widely in different tissues such as cerebrum cortex and hippocampus, and its expression got to the top point at reperfusion for6hours. And alternation of SPCA1manifested a trends in low-high-low, just similar to changes of calcium in Golgi vesicles. In the same, results of western blot analysis also gave the identical trends.Results of analysis on concentration of Ca2+in cytoplasm occurred a keeping calcium overload state, especially, it got to the peak at reperfusion for3hours, and the value was eight-time higher than control and sham-operation tissues. Thus, concentration in Golgi vesicles manifested a contrary alternation, it means when cytosolic concentration got to the peak, the Golgi vesicles decreased to the lowest value, and when cytosolic concentration recoveried, concentration in Golgi vesicles increased to higher level. ConclusionsAt first, SPCA1expression is tightly responded to ischemia and reperfusion in cerebrum nerve cells, and is manifested typical stress reaction.In the second, calcium overload sustained in the procedure of ischemia and reperfusion, yet the calcium concentration declined slowly in the late reperfusion. Golgi apparatus, as a calcium store, take part in the establish and decreasing of calcium overload.At last, according to the relationship between SPCA1expression and calcium concentration in cytoplasm and Golgi vesicles occurred in our experiments, it can be concluded that SPCA1, as a calcium pump located only on the Golgi membrane, played a important role on calcium signal regulation, and can be a marker of Golgi stress respond to ischemia and reperfusion.