Departments of Pediatrics and.
Department of Neurological Surgery, Comprehensive Cancer Center, Wexner Medical Center, Ohio State University, Columbus, Ohio;
Am J Physiol Gastrointest Liver Physiol. 2014 Sep 1;307(5):G574-81. doi: 10.1152/ajpgi.00143.2014. Epub 2014 Jul 10.
Physiological calcium (Ca(2+)) signals within the pancreatic acinar cell regulate enzyme secretion, whereas aberrant Ca(2+) signals are associated with acinar cell injury. We have previously identified the ryanodine receptor (RyR), a Ca(2+) release channel on the endoplasmic reticulum, as a modulator of these pathological signals. In the present study, we establish that the RyR is expressed in human acinar cells and mediates acinar cell injury. We obtained pancreatic tissue from cadaveric donors and identified isoforms of RyR1 and RyR2 by qPCR. Immunofluorescence staining of the pancreas showed that the RyR is localized to the basal region of the acinar cell. Furthermore, the presence of RyR was confirmed from isolated human acinar cells by tritiated ryanodine binding. To determine whether the RyR is functionally active, mouse or human acinar cells were loaded with the high-affinity Ca(2+) dye (Fluo-4 AM) and stimulated with taurolithocholic acid 3-sulfate (TLCS) (500 μM) or carbachol (1 mM). Ryanodine (100 μM) pretreatment reduced the magnitude of the Ca(2+) signal and the area under the curve. To determine the effect of RyR blockade on injury, human acinar cells were stimulated with pathological stimuli, the bile acid TLCS (500 μM) or the muscarinic agonist carbachol (1 mM) in the presence or absence of the RyR inhibitor ryanodine. Ryanodine (100 μM) caused an 81% and 47% reduction in acinar cell injury, respectively, as measured by lactate dehydrogenase leakage (P < 0.05). Taken together, these data establish that the RyR is expressed in human acinar cells and that it modulates acinar Ca(2+) signals and cell injury.
胰腺腺泡细胞内的生理钙(Ca(2+))信号调节酶的分泌,而异常的 Ca(2+)信号与腺泡细胞损伤有关。我们之前已经确定了肌浆网中的 Ryanodine 受体(RyR)是这些病理信号的调节剂。在本研究中,我们确定 RyR 在人腺泡细胞中表达并介导腺泡细胞损伤。我们从尸体供体中获得胰腺组织,并通过 qPCR 鉴定 RyR1 和 RyR2 的同工型。胰腺的免疫荧光染色显示 RyR 定位于腺泡细胞的基底区域。此外,通过放射性标记的 Ryanodine 结合从分离的人腺泡细胞中证实了 RyR 的存在。为了确定 RyR 是否具有功能活性,用高亲和力 Ca(2+)染料(Fluo-4 AM)加载小鼠或人腺泡细胞,并以牛磺胆酸 3-硫酸盐(TLCS)(500 μM)或卡巴胆碱(1 mM)刺激。Ryanodine(100 μM)预处理降低了 Ca(2+)信号的幅度和曲线下面积。为了确定 RyR 阻断对损伤的影响,在存在或不存在 RyR 抑制剂 Ryanodine 的情况下,用病理刺激物,即胆汁酸 TLCS(500 μM)或毒蕈碱激动剂卡巴胆碱(1 mM)刺激人腺泡细胞。Ryanodine(100 μM)分别导致腺泡细胞损伤减少 81%和 47%,如乳酸脱氢酶漏出(P < 0.05)所示。总之,这些数据表明 RyR 在人腺泡细胞中表达,并调节腺泡细胞的 Ca(2+)信号和细胞损伤。