title: Multiple target of hAmylin on rat primary hippocampal neurons.
abstract: Alzheimer's disease (AD) and type II diabetes mellitus (DM2) are the most common aging-related diseases and are characterized by β-amyloid and amylin accumulation, respectively. Multiple studies have indicated a strong correlation between these two diseases. Amylin oligomerization in the brain appears to be a novel risk factor for developing AD. Although amylin aggregation has been demonstrated to induce cytotoxicity in neurons through altering Ca2+homeostasis, the underlying mechanisms have not been fully explored. In this study, we investigated the effects of amylin on rat hippocampal neurons using calcium imaging and whole-cell patch clamp recordings. We demonstrated that the amylin receptor antagonist AC187 abolished the Ca2+response induced by low concentrations of human amylin (hAmylin). However, the Ca2+response induced by higher concentrations of hAmylin was independent of the amylin receptor. This effect was dependent on extracellular Ca2+. Additionally, blockade of L-type Ca2+channels partially reduced hAmylin-induced Ca2+response. In whole-cell recordings, hAmylin depolarized the membrane potential. Moreover, application of the transient receptor potential (TRP) channel antagonist ruthenium red (RR) attenuated the hAmylin-induced increase in Ca2+. Single-cell RT-PCR demonstrated that transient receptor potential vanilloid 4 (TRPV4) mRNA was expressed in most of the hAmylin-responsive neurons. In addition, selective knockdown of TRPV4 channels inhibited the hAmylin-evoked Ca2+response. These results indicated that different concentrations of hAmylin act through different pathways. The amylin receptor mediates the excitatory effects of low concentrations of hAmylin. In contrast, for high concentrations of hAmylin, hAmylin aggregates precipitated on the neuronal membrane, activated TRPV4 channels and subsequently triggered membrane voltage-gated calcium channel opening followed by membrane depolarization. Therefore, our data suggest that TRPV4 is a key molecular mediator for the cytotoxic effects of hAmylin on hippocampal neurons.
authors.affiliation: Department of Pharmacology, Institute of Chinese Integrative Medicine, Hebei Medical University, 361 East Zhongshan Road, Shijiazhuang, Hebei Province, 050017 China; Department of Pharmacy, Hebei North University, Zhangjiakou, Hebei Province, 075000, China.
authors.forename: Nan
authors.initials: N
authors.lastname: Zhang
authors.affiliation: Department of Pharmacology, Institute of Chinese Integrative Medicine, Hebei Medical University, 361 East Zhongshan Road, Shijiazhuang, Hebei Province, 050017 China; Present Address: Department of Physiology, Hebei University of Chinese Medicine, 3 Xingyuan Road, Shijiazhuang 050020, Hebei, China.
authors.forename: Shengchang
authors.initials: S
authors.lastname: Yang
authors.affiliation: Department of Anatomy, Hebei Medical University, 361 East Zhongshan Road, Shijiazhuang, Hebei Province, 050017 China.
authors.forename: Chang
authors.initials: C
authors.lastname: Wang
authors.affiliation: Department of Pharmacology, Institute of Chinese Integrative Medicine, Hebei Medical University, 361 East Zhongshan Road, Shijiazhuang, Hebei Province, 050017 China.
authors.forename: Jianghua
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Department of Pharmacology, Institute of Chinese Integrative Medicine, Hebei Medical University, 361 East Zhongshan Road, Shijiazhuang, Hebei Province, 050017 China.
authors.forename: Lifang
authors.initials: L
authors.lastname: Huo
authors.affiliation: Department of Pharmacology, Institute of Chinese Integrative Medicine, Hebei Medical University, 361 East Zhongshan Road, Shijiazhuang, Hebei Province, 050017 China.
authors.forename: Yiru
authors.initials: Y
authors.lastname: Cheng
authors.affiliation: Department of Pharmacology, Hebei Medical University, 361 East Zhongshan Road, Shijiazhuang, Hebei Province, 050017 China.
authors.forename: Chuan
authors.initials: C
authors.lastname: Wang
authors.affiliation: Department of Pharmacology, Hebei Medical University, 361 East Zhongshan Road, Shijiazhuang, Hebei Province, 050017 China.
authors.forename: Zhanfeng
authors.initials: Z
authors.lastname: Jia
authors.affiliation: Department of Pharmacology, Institute of Chinese Integrative Medicine, Hebei Medical University, 361 East Zhongshan Road, Shijiazhuang, Hebei Province, 050017 China. Electronic address: ren-leiming@263.net.
authors.forename: Leiming
authors.initials: L
authors.lastname: Ren
authors.affiliation: Department of Anatomy, Hebei Medical University, 361 East Zhongshan Road, Shijiazhuang, Hebei Province, 050017 China. Electronic address: jiepouky@163.com.
authors.forename: Lin
authors.initials: L
authors.lastname: Kang
authors.affiliation: Department of Pharmacology, Institute of Chinese Integrative Medicine, Hebei Medical University, 361 East Zhongshan Road, Shijiazhuang, Hebei Province, 050017 China. Electronic address: weizhang@hebmu.edu.cn.
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D005260:Female; D057809:HEK293 Cells; D006801:Humans; D058228:Islet Amyloid Polypeptide / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology; D010446:Peptide Fragments / Q000494:pharmacology; D011247:Pregnancy; D051381:Rats; D017207:Rats, Sprague-Dawley; D050916:TRPV Cation Channels / Q000172:deficiency / Q000378:metabolism*
chemical_list: D058228:Islet Amyloid Polypeptide; D010446:Peptide Fragments; D050916:TRPV Cation Channels; C486577:Trpv4 protein, rat; C087166:AC 187; D002118:Calcium
keywords: Calcium; Hippocampal neurons; TRPV4; hAmylin