Parkinson S J, Waldman S A
Department of Medicine, Division of Clinical Pharmacology, Thomas Jefferson University, Phildelphia, Pennsylvania 19107, USA.
Biochemistry. 1996 Mar 12;35(10):3213-21. doi: 10.1021/bi9524326.
Guanylyl cyclase C (GCC), the receptor for the Escherichia coli heat-stable enterotoxin (ST), is inhibited by 2-substituted adenine nucleotides in an allosteric fashion. In confluent cultures of Caco-2 intestinal epithelial cells, extracellular 2-methylthioadenosine triphosphate (2MeSATP) had no effect on basal or ST-stimulated cyclic GMP (cGMP) accumulation. However, this nucleotide inhibited cGMP accumulation in digitonin-permeabilized Caco-2 human colon carcinoma cells, demonstrating that allosteric inhibition of GCC by adenine nucleotides is mediated by an intracellular adenine nucleotide binding site rather than purinergic receptors. The role of guanine nucleotides in the regulation of GCC by adenine nucleotides was examined. Increasing GTP concentrations from 5 to 100 microM increased the potency of 2MeSATP inhibition of GCC 20-fold, with a shift in the Ki from 447 to 22 microM, respectively. Also, the hydrolysis-resistant analogue, guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS), supported 2MeSATP inhibition of GCC with a potency which was 10-fold greater than GTP. In addition, GTP alone, in the absence of adenine nucleotides and at concentrations greater than 1 mM, inhibited GCC through a mechanism convergent with 2MeSATP. Guanine nucleotides supported adenine nucleotide inhibition of GCC at low concentrations and directly inhibited this enzyme at high concentrations when these studies were conducted with receptors expressed in Caco-2 cells, native rat intestine, or cloned rat GCC heterologously expressed in 293 monkey kidney cells. These observations demonstrate that adenine nucleotide inhibition of GCC is mediated through an intracellular mechanism which is guanine nucleotide-dependent.
鸟苷酸环化酶C（GCC）是大肠杆菌热稳定肠毒素（ST）的受体，可被2-取代的腺嘌呤核苷酸以变构方式抑制。在Caco-2肠上皮细胞的汇合培养物中，细胞外2-甲硫基腺苷三磷酸（2MeSATP）对基础或ST刺激的环鸟苷酸（cGMP）积累没有影响。然而，这种核苷酸抑制了洋地黄皂苷通透的Caco-2人结肠癌细胞中的cGMP积累，表明腺嘌呤核苷酸对GCC的变构抑制是由细胞内腺嘌呤核苷酸结合位点而非嘌呤能受体介导的。研究了鸟嘌呤核苷酸在腺嘌呤核苷酸对GCC调节中的作用。将GTP浓度从5微摩尔增加到100微摩尔可使2MeSATP对GCC的抑制效力增加20倍，其抑制常数（Ki）分别从447微摩尔变为22微摩尔。此外，抗水解类似物鸟苷5'-O-（3-硫代三磷酸）（GTPγS）支持2MeSATP对GCC的抑制，其效力比GTP高10倍。此外，在不存在腺嘌呤核苷酸且浓度大于1毫摩尔时，单独的GTP通过与2MeSATP趋同的机制抑制GCC。当使用在Caco-2细胞、天然大鼠肠道或在293猴肾细胞中异源表达的克隆大鼠GCC中表达的受体进行这些研究时，鸟嘌呤核苷酸在低浓度时支持腺嘌呤核苷酸对GCC的抑制，而在高浓度时直接抑制该酶。这些观察结果表明，腺嘌呤核苷酸对GCC的抑制是通过一种依赖鸟嘌呤核苷酸的细胞内机制介导的。
