Eldridge R, Casida J E
Department of Environmental Science, Policy, and Management, University of California, Berkeley 94720.
Toxicol Appl Pharmacol. 1995 Jan;130(1):95-100. doi: 10.1006/taap.1995.1013.
Our earlier studies indicated that the action of cantharidin (CA) in mice is associated with binding to protein phosphatase 2A in liver cytosol and inhibition of its phosphorylase a phosphatase activity. In this investigation, we find that CA totally inhibits the phosphorylase a phosphatase activity in mouse liver, muscle, and skin cytosol at 5000 nM, with IC50s of 110-250 nM. About 50% of the phosphorylase a phosphatase activity of brain cytosol is sensitive to CA with an IC50 of approximately 80 nM and the remaining half is not inhibited even at 5000 nM. Intraperitoneal treatment of mice with CA leads to a dose-dependent decrease in phosphorylase a phosphatase activity with the aforementioned tissues displaying differential CA sensitivity. At 60 min after a 10 mg/kg CA dose, there is 90-95% inhibition of phosphorylase a phosphatase activity in liver and skin cytosol, 50% in muscle cytosol, and almost no inhibition in brain cytosol. The phosphorylation state of several phosphoproteins examined with tissue cytosol and [gamma-32P]ATP is increased by CA, in a concentration-dependent manner, as follows: endogenous glycogen phosphorylase a in muscle both in vitro and in vivo, and unidentified phosphoproteins in brain (approximately 34 and approximately 75 kDa) and skin (approximately 34 kDa) in vitro. These findings confirm the importance of protein phosphatases as primary targets of CA action in a variety of mouse tissues and, more generally, the possible use of CA and its analogs to investigate and potentially control some processes modulated by the reversible phosphorylation of proteins.
我们早期的研究表明，斑蝥素（CA）在小鼠体内的作用与它与肝细胞质中蛋白磷酸酶2A的结合以及对其磷酸化酶a磷酸酶活性的抑制有关。在本研究中，我们发现CA在5000 nM时能完全抑制小鼠肝脏、肌肉和皮肤细胞质中的磷酸化酶a磷酸酶活性，IC50为110 - 250 nM。脑细胞质中约50%的磷酸化酶a磷酸酶活性对CA敏感，IC50约为80 nM，即使在5000 nM时，其余一半也不受抑制。给小鼠腹腔注射CA会导致磷酸化酶a磷酸酶活性呈剂量依赖性降低，上述组织对CA的敏感性存在差异。在给予10 mg/kg CA剂量60分钟后，肝脏和皮肤细胞质中的磷酸化酶a磷酸酶活性受到90 - 95%的抑制，肌肉细胞质中受到50%的抑制，而脑细胞质中几乎没有抑制。用组织细胞质和[γ - 32P]ATP检测的几种磷蛋白的磷酸化状态会因CA而以浓度依赖性方式增加，具体如下：体外和体内肌肉中的内源性糖原磷酸化酶a，以及体外脑（约34 kDa和约75 kDa）和皮肤（约34 kDa）中未鉴定的磷蛋白。这些发现证实了蛋白磷酸酶作为CA在多种小鼠组织中作用的主要靶点的重要性，更普遍地说，证实了CA及其类似物在研究以及潜在控制一些由蛋白质可逆磷酸化调节的过程中的可能用途。
