CELLULAR AND SUBCELLAR CALCIUM ION HOMEOSTASIS

这个项目的重点是细胞如何实现体内平衡的机制 胞浆和其他细胞内的钙离子浓度 脑室，并允许钙的变化，以响应激素和 神经递质。今年，我们利用了可获得性 膜-永久性荧光钙离子螯合剂的测定 心肌细胞悬液中线粒体内游离钙浓度 线粒体，并与心脏功能科合作，在 在单个心肌细胞中原位。在对分离的线粒体的研究中 我们测量了膜上钙离子的浓度梯度。 线粒体膜，相对于不同的线粒体外 钙以及跨膜pH梯度的变化， 这为钙从线粒体流入提供了驱动力。 在模拟的生理条件下，即在 呼吸中钠和镁离子的胞浆浓度 线粒体显示维持负的钙梯度(内部 除以外部)当线粒体外钙小于500 NM时： 在高于此值时，线粒体钙浓度 渐变变为正数。线粒体内的协同研究 在单个活细胞中原位钙是相当新的，并表明 此参数位于先前显示的调制范围内 线粒体内脱氢酶活性。此外，在二尖瓣内 游离钙因β-肾上腺素能刺激而升高 电刺激的速率，即通过 细胞，因此需要更活跃的氧化磷酸化 线粒体。

This project focuses on the mechanism whereby cells achieve homeostasis of calcium ion concentration, both within the cytosol and other cellular compartments, and allow changes in calcium in response to hormones and neurotransmitters. This year we have taken advantage of the availability of membrane-permanent fluorescent calcium ion-chelating agents to measure intramitochondrial free calcium concentrations both in suspensions of heart mitochondria and, in a collaboration with the Cardiac Function Section, in situ in a single cardiac myocyte. In the work with isolated mitochondrial membrane we have measured the concentration gradient of calcium ions across the mitochondrial membrane, with respect to varying extramitochondrial calcium and also with respect to changes in the transmembrane pH gradient, which provides the driving force for calcium afflux from the mitochondria. Under simulated physiological conditions, i.e., in the presence of cytosolic concentrations of sodium and magnesium ions, respiring mitochondria were shown to maintain a negative calcium gradient (inside divided by outside) when extramitochondrial calcium was less than 500 nM: at higher values than this, the mitochondrial calcium concentration gradient becomes positive. The collaborative studies on intramitochondrial calcium in situ in a single living cell are quite novel and have shown that this parameter lies within the range previously shown to modulate intramitochondrial dehydrogenase activity. Further, intramitochondiral free calcium rises in response to beta-adrenergic stimulation and increased rate of electrical stimulation, situations which enhance work output by the cell and thus require more active oxidative phosphorylation by the mitochondria.