Millul V, Prié D, Géniteau-Legendre M, Verpont M C, Baudouin B, Ronco P M
Institut National de la Santé et de la Recherche Médicale Unité 64, Hôpital Tenon, Paris, France.
Am J Physiol. 1996 Apr;270(4 Pt 1):C1051-60. doi: 10.1152/ajpcell.1996.270.4.C1051.
Egression of atrial natriuretic peptide (ANP)-stimulated guanosine 3', 5'-cyclic monophosphate (cGMP) was compared with that of isoproterenol-stimulated adenosine 3', 5'-cyclic monophosphate (cAMP) in a rabbit collecting duct cell line transformed with a temperature-sensitive strain of simian virus 40 (SV40). At 39.5 degrees C (inactivated large T), cells exhibit major features of principal cells, whereas at 33 degrees C (functional large T) they lose most of their specific properties. When cells were grown on plastic at 39.5 degrees C, both cyclic nucleotides were predominantly released extracellularly via probenecid-sensitive carriers. Probenecid (3mM) reduced the ratios of extracellular cGMP and cAMP by 84 and 70%, respectively. The amount of extracellular cGMP or cAMP ws linearly correlated with the time integral of the intracellular cyclic nucleotide, suggesting first-order kinetics. The apparent first-order rate constant (k) was sixfold greater for cGMP (0.139 +/- 0.037 min-1, n = 3 experiments) than for cAMP (0.022 +/- 0.003(-1), n = 3 experiments). 3-Isobutyl-1-methylxanthine markedly inhibited extrusion of cGMP (k = 0.022 +/- 0.003 min-1), whereas that of cAMP was unchanged. When cells were grown on filters at 39.5 degrees C, both nucleotides were predominantly released in the apical medium but with a greater polarity for cGMP (83 +/- 4%, n = 6 experiments) than for cAMP (60 +/- 6%, n = 3 experiments) and a prevailing apical localization of the probenecid-sensitive carrier. Activation of SV40 large T at 33 degrees C did not alter cyclic nucleotide transport characteristics but abolished the polarity of probenecid-sensitive cyclic nucleotide extrusion. These results suggest a physiological role for luminal cGMP in the rabbit collecting duct and a specific effect of large T on the probenecid-sensitive carrier polarity.
在一种用猿猴病毒40（SV40）的温度敏感株转化的兔集合管细胞系中，比较了心房利钠肽（ANP）刺激产生的鸟苷3'，5'-环磷酸（cGMP）和异丙肾上腺素刺激产生的腺苷3'，5'-环磷酸（cAMP）的流出情况。在39.5℃（失活的大T抗原）时，细胞呈现主细胞的主要特征，而在33℃（有功能的大T抗原）时，它们失去了大部分特异性特性。当细胞在39.5℃的塑料培养皿上生长时，两种环核苷酸主要通过丙磺舒敏感的载体释放到细胞外。丙磺舒（3mM）分别使细胞外cGMP和cAMP的比例降低了84%和70%。细胞外cGMP或cAMP的量与细胞内环核苷酸的时间积分呈线性相关，提示为一级动力学。cGMP的表观一级速率常数（k）（0.139±0.037 min-1，n = 3次实验）比cAMP（0.022±0.003 min-1，n = 3次实验）大6倍。3-异丁基-1-甲基黄嘌呤显著抑制cGMP的外排（k = 0.022±0.003 min-1），而cAMP的外排则不受影响。当细胞在39.5℃的滤膜上生长时，两种核苷酸主要释放到顶端培养基中，但cGMP的极性更大（83±4%，n = 6次实验），而cAMP的极性为（60±6%，n = 3次实验），且丙磺舒敏感载体主要定位于顶端。在33℃激活SV40大T抗原不会改变环核苷酸的转运特性，但消除了丙磺舒敏感的环核苷酸外排的极性。这些结果提示管腔cGMP在兔集合管中具有生理作用，且大T抗原对丙磺舒敏感载体的极性有特异性影响。
