Role of Ca2+ in apoptosis evoked by human amylin in pancreatic islet β-cells

被引:47
作者
Bai, JZ
Saafi, EL
Zhang, SP
Cooper, GJS
机构
[1] Univ Auckland, Sch Biol Sci, Auckland 1, New Zealand
[2] Univ Auckland, Sch Med, Dept Med, Auckland, New Zealand
关键词
fibril; islet amyloid; type 2 diabetes mellitus;
D O I
10.1042/0264-6021:3430053
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The objective of these studies was to clarify the role of Ca2+ in the mechanism of death evoked by human amylin (hA) in islet beta-cells, hA forms fibrils in vitro and islet amyloid in vivo. Here we show that pure synthetic hA aggregated in solution, formed fibrils and evoked death in cultured RINm5F islet p-cells in a time-dependent (0-24 h) and concentration-dependent (0-20 mu M) manner. Dying cells underwent shrinkage of the nucleus, with clumping and segregation of chromatin into masses that lay against the nuclear envelope, and internucleosomal DNA fragmentation. These cells therefore show many features of apoptosis, although aspects of the morphology might be characteristic of this particular cell type rather than of a general apoptotic nature. Aurintricarboxylic acid, an inhibitor of both Ca2+-dependent and Ca2+-independent nucleases, suppressed this DNA fragmentation and inhibited apoptosis at concentrations between 25 and 200 mu M. Direct measurements of the cytoplasmic free Ca2+ concentration ([Ca2+](i)) in fura-2 acetoxymethyl ester (AM)-loaded beta-cells showed that neither hA nor its non-cytotoxic homologue, rat amylin were effective in raising [Ca2+](i). Modulators of Ca2+ regulation were tested for their effects on hA-induced beta-cell apoptosis. Ca2+ ionophore (A23187) and thapsigargin tan inhibitor of endoplasmic reticular Ca2+-ATPase activity) by themselves evoked apoptosis accompanied by increased [Ca2+](i). Neither the Ca2+ channel blocker verapamil, the extracellular Ca2+ chelator EGTA nor the cytosolic Ca2+ buffer bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid ('BAPTA')/AM protected beta-cells from hA-evoked apoptosis. Prolonged incubation of beta-cells with a lethal dose of hA altered neither the basal [Ca2+](i) nor the thapsigargin-induced release of Ca2+ from intracellular stores. Furthermore, (CaCl2)-Ca-45 uptake by RINm5F cells did not differ in the presence or absence of hA. These results suggest that, whereas alterations in cytosolic Ca2+ homoeostasis do have a significant role in certain forms of beta-cell death, they do not contribute to the pathway of apoptosis evoked by hA in islet beta-cells.
引用
收藏
页码:53 / 61
页数:9
相关论文
共 54 条
[1]  
ALNEMRI ES, 1990, J BIOL CHEM, V265, P17323
[2]   THE ROLE OF DNA FRAGMENTATION IN APOPTOSIS [J].
BORTNER, CD ;
OLDENBURG, NBE ;
CIDLOWSKI, JA .
TRENDS IN CELL BIOLOGY, 1995, 5 (01) :21-26
[3]   AMYLIN AND THE AMYLIN GENE - STRUCTURE, FUNCTION AND RELATIONSHIP TO ISLET AMYLOID AND TO DIABETES-MELLITUS [J].
COOPER, GJS ;
DAY, AJ ;
WILLIS, AC ;
ROBERTS, AN ;
REID, KBM ;
LEIGHTON, B .
BIOCHIMICA ET BIOPHYSICA ACTA, 1989, 1014 (03) :247-258
[4]   AMYLIN HORMONE [J].
COOPER, GJS ;
WILLIS, AC ;
LEIGHTON, B .
NATURE, 1989, 340 (6231) :272-272
[6]   PURIFICATION AND CHARACTERIZATION OF A PEPTIDE FROM AMYLOID-RICH PANCREASES OF TYPE-2 DIABETIC-PATIENTS [J].
COOPER, GJS ;
WILLIS, AC ;
CLARK, A ;
TURNER, RC ;
SIM, RB ;
REID, KBM .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1987, 84 (23) :8628-8632
[7]   AMYLIN STIMULATES OSTEOBLAST PROLIFERATION AND INCREASES MINERALIZED BONE VOLUME IN ADULT MICE [J].
CORNISH, J ;
CALLON, KE ;
COOPER, GJS ;
REID, IR .
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 1995, 207 (01) :133-139
[8]   A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD [J].
Enari, M ;
Sakahira, H ;
Yokoyama, H ;
Okawa, K ;
Iwamatsu, A ;
Nagata, S .
NATURE, 1998, 391 (6662) :43-50
[9]  
FURUYA Y, 1994, CANCER RES, V54, P6167
[10]   IDENTIFICATION OF PROGRAMMED CELL-DEATH INSITU VIA SPECIFIC LABELING OF NUCLEAR-DNA FRAGMENTATION [J].
GAVRIELI, Y ;
SHERMAN, Y ;
BENSASSON, SA .
JOURNAL OF CELL BIOLOGY, 1992, 119 (03) :493-501