Feher J J, Lee K N, Wu Q Y
Department of Physiology, Medical College of Virginia, Virginia Commonwealth University, Richmond 23298-0551, USA.
Mol Cell Biochem. 1998 Dec;189(1-2):9-17. doi: 10.1023/a:1006800328118.
Thapsigargin is a natural product that specifically inhibits all known SERCA calcium pumps with high affinity. We investigated the effects of thapsigargin on cardiac sarcoplasmic reticulum (SR) by measuring the oxalate-supported calcium uptake rate in the unfractionated homogenate and in the isolated SR fraction. The uptake rate in both the isolated SR and unfractionated homogenate are stimulated about two-fold by preincubation with high concentrations of ryanodine, which closes the SR efflux channel. Thapsigargin stoichiometrically and completely inhibited the calcium uptake rate in the isolated SR, both in the presence and absence of SR channel blockade. In contrast, thapsigargin nearly completely inhibited the homogenate calcium uptake only in the absence of SR channel blockade; in the presence of blockade, about 20% of the uptake activity was insensitive to thapsigargin. This result unmasks a thapsigargin-insensitive, ryanodine-sensitive component of calcium uptake in the heart. This activity is in an oxalate-permeable pool and is inhibited by cyclopiazonic acid, another inhibitor of the SERCA calcium pumps. There was no TG-insensitive activity in the rat EDL muscle homogenate. The absence of thapsigargin-insensitive uptake activity in the isolated SR can be attributed to its inactivation during the isolation of the SR. The oxalate permeability and ryanodine sensitivity suggest that the TG-insensitive calcium uptake activity is closely related to the classical SR. The different thapsigargin sensitivities suggests the existence of two kinds of intracellular calcium pumps in the heart.
毒胡萝卜素是一种天然产物，它能以高亲和力特异性抑制所有已知的肌浆网钙泵（SERCA）。我们通过测量未分级匀浆和分离的肌浆网部分中草酸盐支持的钙摄取率，研究了毒胡萝卜素对心肌肌浆网（SR）的影响。在高浓度的兰尼碱预孵育后，分离的肌浆网和未分级匀浆中的摄取率均受到约两倍的刺激，兰尼碱会关闭肌浆网外流通道。无论是否存在肌浆网通道阻断，毒胡萝卜素都能按化学计量比完全抑制分离的肌浆网中的钙摄取率。相比之下，毒胡萝卜素仅在不存在肌浆网通道阻断时几乎完全抑制匀浆中的钙摄取；在存在阻断的情况下，约20%的摄取活性对毒胡萝卜素不敏感。这一结果揭示了心脏中存在对毒胡萝卜素不敏感但对兰尼碱敏感的钙摄取成分。这种活性存在于草酸盐可渗透的池中，并受到另一种SERCA钙泵抑制剂环匹阿尼酸的抑制。大鼠趾长伸肌匀浆中不存在对毒胡萝卜素不敏感的活性。分离的肌浆网中不存在对毒胡萝卜素不敏感的摄取活性，这可能归因于其在肌浆网分离过程中的失活。草酸盐通透性和兰尼碱敏感性表明，对毒胡萝卜素不敏感的钙摄取活性与经典肌浆网密切相关。毒胡萝卜素敏感性的差异表明心脏中存在两种细胞内钙泵。
