Whalen M M, Crews J D
Department of Chemistry, Tennessee State University, Nashville, TN 37209, USA.
Biochem Pharmacol. 2000 Aug 15;60(4):499-506. doi: 10.1016/s0006-2952(00)00369-5.
Natural killer (NK) cells are lymphocytes that are capable of destroying tumor cells and virally infected cells (cytolysis) without prior sensitization. When cyclic AMP (cAMP) is elevated artificially in NK cells, it is a potent inhibitor of their cytolytic function. Recently, we have shown that when NK cells are exposed to a range of lysis-sensitive (LS) tumor target cells, there is an increase in intracellular cAMP levels in the NK cells over a 60-min period. There is no increase in NK-cell cAMP in response to lysis-resistant (LR) tumor target cells. We determined that this cAMP elevation is due, in part, to an LS target-induced activation of adenylyl cyclase (AC), and that the AC-activation component appears to require a protein tyrosine kinase (PTK) activity. In the present study, we demonstrated that an LS target-induced inhibition of phosphodiesterase (PDE) is also contributing to the overall elevation of cAMP. Direct measurement of PDE activity showed an inhibition in lymphocytes that were exposed to LS targets but not in those exposed to LR targets. The inhibition of PDE activity was maximal by 30 min. Lymphocytes were exposed to targets and then lysed, so that PDE activity could be measured. Addition of class-selective inhibitors of PDE (at levels sufficient to completely block that class of PDE) to the lysate focused the measurement of PDE activity on those classes of PDE that were unaffected by the selective inhibitor. Using the PDE IV selective inhibitor rolipram and the PDE III selective inhibitors trequinsin and milrinone, we showed that a PDE III is being inhibited in lymphocytes by exposure to LS targets. As PDE III is known to be inhibited by elevated cyclic GMP (cGMP) levels, increased cGMP in NK cells following exposure to LS targets was a possible mechanism by which a PDE III in NK cells might be inhibited. However, when we measured cGMP levels in control and LS target-stimulated lymphocytes, we saw no change.
自然杀伤（NK）细胞是一种淋巴细胞，能够在未经预先致敏的情况下破坏肿瘤细胞和病毒感染细胞（细胞溶解）。当环磷酸腺苷（cAMP）在NK细胞中人工升高时，它是其细胞溶解功能的有效抑制剂。最近，我们发现当NK细胞暴露于一系列对裂解敏感（LS）的肿瘤靶细胞时，在60分钟内NK细胞内的cAMP水平会升高。对裂解抗性（LR）肿瘤靶细胞无反应时，NK细胞的cAMP不会增加。我们确定这种cAMP升高部分是由于LS靶标诱导的腺苷酸环化酶（AC）激活，并且AC激活成分似乎需要蛋白酪氨酸激酶（PTK）活性。在本研究中，我们证明LS靶标诱导的磷酸二酯酶（PDE）抑制也有助于cAMP的整体升高。PDE活性的直接测量显示，暴露于LS靶标的淋巴细胞中有抑制作用，而暴露于LR靶标的淋巴细胞中则没有。PDE活性的抑制在30分钟时最大。淋巴细胞暴露于靶标后裂解，以便测量PDE活性。向裂解物中添加PDE的类别选择性抑制剂（浓度足以完全阻断该类PDE），可将PDE活性的测量集中在不受选择性抑制剂影响的那些PDE类别上。使用PDE IV选择性抑制剂咯利普兰和PDE III选择性抑制剂曲喹辛和米力农，我们发现暴露于LS靶标会抑制淋巴细胞中的PDE III。由于已知PDE III会被环磷酸鸟苷（cGMP）水平升高所抑制，因此暴露于LS靶标后NK细胞中cGMP增加可能是NK细胞中PDE III被抑制的一种机制。然而，当我们测量对照和LS靶标刺激的淋巴细胞中的cGMP水平时，未发现变化。
