Role of protein phosphatase in exocytosis in adrenal chromaffin cells

他克莫司（FK 506）和环孢素A（CsA）是有效的免疫抑制剂，已被用于预防器官移植排斥反应。这些化合物抑制钙调磷酸酶、Ca^2+/钙调蛋白依赖性蛋白磷酸酶，从而导致抑制T淋巴细胞活化。钙调神经磷酸酶是高度保守的，广泛分布在几乎所有的细胞类型。钙调神经磷酸酶被认为调节离子通道、神经递质和激素释放的活性。在本研究中，FK 506和CsA的影响，在培养的牛肾上腺嗜铬细胞释放儿茶酚胺（CA）进行了研究。结果如下：1）在完整细胞中，FK 506（1-30μM）抑制乙酰胆碱（ACh ; 100μM）、碘化1，1-二甲基-4-苯基哌啶（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-七叶皂苷透性化的细胞中，在MgATP（2 mM）存在和不存在的情况下，FK 506抑制Ca^<2+>（1和10μM）刺激的CA释放。CsA在无Ca^<2+>条件下诱导CA释放，在MgATP存在和不存在时均增强Ca^<2 +>刺激的CA释放。因此，FK 506可能在融合阶段抑制Ca^2+依赖的胞吐作用，而CsA则诱导牛肾上腺嗜铬细胞释放CA。这也表明，它们对CA释放的影响与FK 506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.