Boadu E, Vaskinn S, Sundkvist E, Jaeger R, Sager G
Department of Pharmacology, Institute of Medical Biology, Medical Faculty, University of Tromsø, N-9037, Tromsø, Norway
Biochem Pharmacol. 2001 Aug 15;62(4):425-9. doi: 10.1016/s0006-2952(01)00682-7.
The cellular extrusion of guanosine 3',5'-cyclic monophosphate (3',5'-cGMP) is a unidirectional ATP-dependent process that is inhibited by probenecid, a non-selective transport inhibitor of organic anions. In the present study, various cGMP analogues were tested for their ability to inhibit 3',5'-cGMP efflux and stimulate the cGMP-selective ATPase in human erythrocytes. The difference in uptake of 1 microM [(3)H]3',5'-cGMP to inside-out vesicles in the presence and absence of 1 mM ATP at 37 degrees was defined as active transport. Two ATP-dependent components were detected for unlabelled 3',5'-cGMP (0.01--100 microM) with respective K(i) of 1.3 +/- 0.2 and 280 +/- 50 microM (mean +/- SEM, N = 3). The high-affinity transport was inhibited by the analogues with a typical pattern: Rp-monophosphorothioate guanosine 3',5'-cyclic monophosphate (Rp-cGMPS) > 3',5'-cGMP > 2'-O-monobutyryl guanosine 3',5'-cyclic monophosphate (O-mb-cGMP) approximately N(2)-monobutyryl guanosine 3',5'-cyclic monophosphate (N-mb-cGMP) > or = N(2),2'-O-dibutyryl guanosine 3',5'-cyclic monophosphate (Db-cGMP) approximately 8'-bromo guanosine 3',5'-cyclic monophosphate (Br-cGMP) approximately Guanosine 2',3'-cyclic monophosphate (2'3'-cGMP) > Sp-monophosphorothioate guanosine 3',5'-cyclic monophosphate (Sp-cGMPS). A concentration-dependent inhibition was found for the low-affinity transport, but no distinct order of potency was identified. Analysis according to Lineweaver--Burk of active [(3)H]3',5'-cGMP transport (0.2--2 microM) gave a K(m) value of 1.5 +/- 0.1 microM (mean +/- SEM, N = 3). The presence of 10 microM cGMP analogues did not change the ordinate intercept, but made the slopes steeper with a typical order: Rp-cGMPS > 3',5'-cGMP > N-mb-cGMP approximately O-mb-cGMP approximately db-cGMP approximately 8-Br-cGMP > 2',3'-cGMP > Sp-cGMPS. Only 3',5'-cGMP and 2',3'-cGMP were able to activate the cGMP-specific ATPase, 640 +/- 200% and 430 +/- 160% (mean +/- SEM, N = 5) above basal levels, respectively. The present data show that the binding is less selective than ATPase activation of the cellular cGMP transport system.
鸟苷 3',5'-环磷酸（3',5'-cGMP）的细胞外排是一个单向的、依赖 ATP 的过程，该过程会被丙磺舒抑制，丙磺舒是一种非选择性有机阴离子转运抑制剂。在本研究中，测试了各种 cGMP 类似物抑制 3',5'-cGMP 外排以及刺激人红细胞中 cGMP 选择性 ATP 酶的能力。在 37℃下，存在和不存在 1 mM ATP 时，1 μM [(3)H]3',5'-cGMP 向内翻外囊泡的摄取差异被定义为主动转运。对于未标记的 3',5'-cGMP（0.01 - 100 μM）检测到两个依赖 ATP 的成分，其各自的 K(i) 为 1.3 ± 0.2 和 280 ± 50 μM（平均值 ± 标准误，N = 3）。高亲和力转运被类似物以典型模式抑制：Rp - 单磷酸硫代鸟苷 3',5'-环磷酸（Rp - cGMPS）> 3',5'-cGMP > 2'-O - 单丁酰鸟苷 3',5'-环磷酸（O - mb - cGMP）≈ N(2)-单丁酰鸟苷 3',5'-环磷酸（N - mb - cGMP）≥ N(2),2'-O - 二丁酰鸟苷 3',5'-环磷酸（Db - cGMP）≈ 8'-溴鸟苷 3',5'-环磷酸（Br - cGMP）≈ 鸟苷 2',3'-环磷酸（2'3'-cGMP）> Sp - 单磷酸硫代鸟苷 3',5'-环磷酸（Sp - cGMPS）。对于低亲和力转运发现了浓度依赖性抑制，但未确定明显的效价顺序。根据 Lineweaver - Burk 分析主动 [(3)H]3',5'-cGMP 转运（0.2 - 2 μM）得到的 K(m) 值为 1.5 ± 0.1 μM（平均值 ± 标准误，N = 3）。10 μM cGMP 类似物的存在没有改变纵坐标截距，但使斜率更陡，具有典型顺序：Rp - cGMPS > 3',5'-cGMP > N - mb - cGMP ≈ O - mb - cGMP ≈ db - cGMP ≈ 8 - Br - cGMP > 2',3'-cGMP > Sp - cGMPS。只有 3',5'-cGMP 和 2',3'-cGMP 能够激活 cGMP 特异性 ATP 酶，分别比基础水平高 640 ± 200%和 430 ± 160%（平均值 ± 标准误，N = 5）。目前的数据表明，与细胞 cGMP 转运系统的 ATP 酶激活相比，结合的选择性较低。
