The key target of neuroprotection after the onset of ischemic stroke: secretory pathway Ca(2+)-ATPase 1

Abstract

The regulatory mechanisms of cytoplasmic Ca(2+) after myocardial infarction-induced Ca(2+) overload involve secretory pathway Ca(2+)-ATPase 1 and the Golgi apparatus and are well understood. However, the effect of Golgi apparatus on Ca(2+) overload after cerebral ischemia and reperfusion remains unclear. Four-vessel occlusion rats were used as animal models of cerebral ischemia. The expression of secretory pathway Ca(2+)-ATPase 1 in the cortex and hippocampus was detected by immunoblotting, and Ca(2+) concentrations in the cytoplasm and Golgi vesicles were determined. Results showed an overload of cytoplasmic Ca(2+) during ischemia and reperfusion that reached a peak after reperfusion. Levels of Golgi Ca(2+) showed an opposite effect. The expression of Golgi-specific secretory pathway Ca(2+)-ATPase 1 in the cortex and hippocampus decreased before ischemia and reperfusion, and increased after reperfusion for 6 hours. This variation was similar to the alteration of calcium in separated Golgi vesicles. These results indicate that the Golgi apparatus participates in the formation and alleviation of calcium overload, and that secretory pathway Ca(2+)-ATPase 1 tightly responds to ischemia and reperfusion in nerve cells. Thus, we concluded that secretory pathway Ca(2+)-ATPase 1 plays an essential role in cytosolic calcium regulation and its expression can be used as a marker of Golgi stress, responding to cerebral ischemia and reperfusion. The secretory pathway Ca(2+)-ATPase 1 can be an important neuroprotective target of ischemic stroke.

Keywords: Ca2+ pump; Golgi Ca2+; Golgi apparatus; Golgi stress; NSFC grant; brain injury; cytoplasmic Ca2+; global cerebral ischemia; homeostasis; nerve regeneration; neural protection; neural regeneration; secretory pathway Ca2+-ATPase 1.

Figure 1

Changes in Ca²⁺ concentration in cytoplasm and separated Golgi vesicles in nerve cells during cerebral ischemia and reperfusion. (A) Changes in cytoplasmic Ca²⁺, which increased rapidly during ischemia and early reperfusion and then slightly but appreciably declined. (B) Changes in Golgi Ca²⁺, which showed an opposite trend to cytoplasmic Ca²⁺. C: Control group; S: sham operation group; I: ischemia group; R: reperfusion group; h: hours; d: days. The data are expressed as the mean ± SEM (calculated from data of five pieces of brain section) and oneway analysis of variance followed by the least significance difference test was used.

Figure 2

Data of fluorescent density of SPCA1 primary antibody during ischemia and reperfusion as detected by fluorescence immunohistochemistry. *P < 0.05, **P < 0.01, vs. C and S. The data are expressed as the mean ± SEM (calculated from data of five pieces of brain section) and oneway analysis of variance and the least significance difference test were used. C: Control group; S: sham operation group; I: ischemia group; R: reperfusion group; SPCA1: secretory pathway Ca²⁺ ATPase 1; h: hours; d: days.

Figure 3

Broad expression of SPCA1 in the brain (immunofluorescence, fluorescence microscope). (A) Image of specimen without SPCA1 antibody, used as a reference. No fluorescent signals are detected. (B, C) SPCA1 expression in the brain, particularly in the cortex and hippocampus neurons, at 6 hours after reperfusion. SPCA1-immunoreactive cells show blue fluorescence. SPCA1: Secretory pathway Ca²⁺ ATPase 1.

Figure 4

Fluorescent immunoreactivity of SPCA1 during ischemia and reperfusion (immunofluorescence, fluorescence microscope). SPCA1 expression was analyzed by fluorescence in situ immunohistochemistry. Neuronal cells expressing SPCA1 are marked by blue fluorescence. (A, B) Images of control and sham operation groups; (C) ischemia group; (D–H) reperfusion group, R3 h, R6 h, R24 h, R3 d and R7 d. SPCA1-immunoreactive cells show blue fluorescence. Scale bars: 20 μm. R: Reperfusion; SPCA1: secretory pathway Ca²⁺ ATPase 1; h: hours; d: days.

Figure 5

Western blot assay of SPCA1 during ischemia and reperfusion. In C and S, almost no difference was detected in SPCA1 expression. In I and R3 h, an obvious decline occurred. In R6 h, a dramatic increase appeared. After R24 h, the expression decreased very slowly. C: Control group; S: sham operation group; I: ischemia group; R: reperfusion group; SPCA1: secretory pathway Ca²⁺ ATPase 1; h: hours; d: days.