Alzheimer disease(AD) and typeⅡdiabetes mellitus(DM2) are the most common disease in aging people,with β-amyloid and amylin accumulation respectively.Studies have shown more and more correlations between these two diseases,and amylin oligomerization in the brain provided a novel risk target for developing AD.Although cumulative studies reported that amylin aggregation induced cytotoxicity in pancreatic beta cells by altering Ca2+homeostasis,fewer studies investigated the effect of amylin on hippocampal neuron.To address this question,it was investigated the effect of amylin on primary cultured rat hippocampal neurons by calcium imaging and whole-cell patch clamp recording in this study,while the results revealed that human amylin(hAmylin) but not rat amylin or pramlintide(hAmylin analgue) produced a rapid increase in intracellular calcium in a dose dependent manner.This effect relied on extracellular calcium and not abolished by amylin receptor antagonist AC187.Additionally,the calcium increase induced by hAmylin was dependent onvoltage-gated Ca2+channels,especially L-type Ca2+channel activation.In whole-cell recording hAmylin could depolarize membrane potential and increase the cell exitability.Moreover,application of transient receptor potential vanilloid(TRPV) antagonist ruthenium red could abolish part of the intracellular calcium increase.Single cell RT-PCR revealed that TRPV4 mRNA expressed in most of the reactive neuron and selective TRPV4 antagonist HC067047 inhibited the intacellular calcium increasing.These results indicated that hAmylin aggregation precipitating on the neuron membrane activated TRPV4 channels and then triggered membrane voltage gated calcium channel opening followed by membrane depolarization,expressing that TRPV4 is a key molecular target for the cytotoxic effect of hAmylin on cultured neurons.
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