Role of protein phosphatase in exocytosis in adrenal chromaffin cells

他克莫司(FK506)和环孢菌素A(CsA)是预防器官移植排斥反应的有效免疫抑制药物。这些化合物抑制钙调神经磷酸酶、钙调素依赖的蛋白磷酸酶，从而抑制T淋巴细胞的激活。钙调神经磷酸酶高度保守，在几乎所有类型的细胞中广泛分布。钙调神经磷酸酶被认为可以调节离子通道、神经递质和激素的释放。本研究观察了FK506和CsA对牛肾上腺嗜铬细胞儿茶酚胺(CA)释放的影响。结果如下：1)在完整细胞中，FK506(1-30μM)可抑制乙酰胆碱(ACh；100μM)、1，1-二甲基-4-苯基哌啶碘(DMPP；10μM)或高K~+(40 MM)刺激的CA释放。CsA(1-30μM)对ACh或DMPP刺激的CA释放有轻微的抑制作用，但对高K~+刺激的CA释放有促进作用。2)在β-七叶皂苷通透性细胞中，FK506在有或没有MgATP2 mM的情况下抑制Ca~(2+)和Gt；(1和10μM)刺激的CA释放。在肾上腺嗜铬细胞中，环孢素A在无钙条件下诱导CA释放，在有或无镁离子的情况下增强钙刺激的CA释放。众所周知，肾上腺嗜铬细胞的钙依赖性胞吐至少包括两个不同的步骤，即需要ATP的启动阶段和不依赖于ATP的融合阶段。因此，推测FK506可能在融合阶段抑制依赖Ca~(2+)的胞吐作用，而CsA则诱导牛肾上腺嗜铬细胞释放CA。提示它们对CA释放的影响与FK506或CsA对钙调神经磷酸酶的抑制作用无关。

Tacrolimus (FK506) and cyclosporin A (CsA) are potent immunosuppressant drugs that have been used in the prophylaxis of allograft rejection in organ transplantation. These compounds inhibit calcineurin, Ca^<2+>/calmodulin-dependent protein phosphatase, thereby resulting in the inhibition of T-lymphocyte activation. Calcineurin is highly conserved and widely distributed in virtually all cell types. Calcineurin has been suggested to regulate the activity of ion channels, neurotransmitter and hormone release. In the present study, the effects of FK506 and CsA on catecholamine (CA) release were examined in cultured bovine adrenal chromaffin cells. The results obtained are as follows.1)In intact cells, FK506 (1-30μM) inhibited CA release stimulated by acetylcholine (ACh ; 100μM), 1,1-dimethyl-4-phenyl-piperadinium iodide (DMPP ; 10μM) or high K^+ (40mM). CsA (1-30μM) had a little inhibitory effect on the ACh- or DMPP-stimulated CA release, whereas it enhanced the high K^+ -stimulated CA release.2)In β-escin-permeabilized cells, FK506 inhibited CA release stimulated by Ca^<2+> (1 and 10μM) in the presence and absence of MgATP (2mM). CsA induced CA release under Ca^<2+> free condition and enhanced the Ca^<2+>-stimulated CA release in the presence and absence of MgATP.It is known that the Ca^<2+>-dependent exocytosis involves at least two distinct steps, ATP-requiring priming stage and ATP-independent fusion step in adrenal chromaffin cells. Therefore, it is suggested that FK506 inhibits the Ca^<2+>-dependent exocytosis probably at the fusion step whereas CsA induces CA release from bovine adrenal chromaffin cells. It is also suggested that their effects on CA release is not related to the inhibitory action of FK506 or CsA on calcineurin.