Thévenod F, Gasser K W, Hopfer U
Department of Physiology and Biophysics, School of Medicine, Case Western Reserve University, Cleveland, OH 44106.
Biochem J. 1990 Nov 15;272(1):119-26. doi: 10.1042/bj2720119.
The regulation of Cl- conductance by cytoplasmic nucleotides was investigated in pancreatic and parotid zymogen granules. Cl- conductance was assayed by measuring the rate of cation-ionophore-induced osmotic lysis of granules suspended in iso-osmotic salt solutions. Both inhibition and stimulation were observed, depending on the type and concentration of nucleotide. Under optimal conditions, the average inhibition measured in different preparations was 1.6-fold, whereas the average stimulation was 4.4-fold. ATP was inhibitory at 1-10 microM but stimulated Cl- conductance above 50 microM. Stimulation by ATP was more pronounced in granules with low endogenous Cl- conductance. The potency of nucleotides in terms of inhibition was ATP greater than adenosine 5'-[gamma-thio]triphosphate (ATP[S]) greater than UTP much greater than or equal to CTP much greater than or equal to GTP much greater than or equal to guanosine 5'-[gamma-thio]triphosphate (GTP[S]) much greater than or equal to ITP. The potency with respect to stimulation had the following order: adenosine 5'-[beta gamma-methylene]triphosphate (App[CH2]p) greater than ATP greater than guanosine 5'-[beta-thio]diphosphate (GDP[S]). Adenosine 5'-[beta gamma-imido]triphosphate (App[NH]p) was also stimulatory, and was more potent than ATP in the parotid granules, but less potent in the pancreatic granules. Aluminium fluoride stimulated Cl- conductance maximally at 15-30 microM-Al3+ and 10-15 mM-F. F was less effective at higher concentrations. Protein phosphorylation by kinases was apparently not involved, since the nucleotide effects (1) could be mimicked by non-hydrolysable analogues of ATP and GTP, (2) showed reversibility, and (3) were not abolished by the protein kinase inhibitors 1-(5-isoquinolinesulphonyl)-2-methylpiperazine (H-7) or staurosporine. The data suggest the presence of at least two binding sites for nucleotides, whereby occupancy of one induces inhibition and occupancy of the other induces stimulation.
在胰腺和腮腺的酶原颗粒中研究了细胞质核苷酸对氯离子电导的调节作用。通过测量阳离子载体诱导的悬浮在等渗盐溶液中的颗粒的渗透裂解速率来测定氯离子电导。根据核苷酸的类型和浓度，观察到了抑制和刺激两种作用。在最佳条件下，不同制剂中测得的平均抑制倍数为1.6倍，而平均刺激倍数为4.4倍。ATP在1 - 10微摩尔时具有抑制作用，但在50微摩尔以上时刺激氯离子电导。ATP对氯离子电导的刺激作用在具有低内源性氯离子电导的颗粒中更为明显。就抑制作用而言，核苷酸的效力顺序为：ATP大于腺苷5'-[γ-硫代]三磷酸（ATP[S]）大于UTP远大于或等于CTP远大于或等于GTP远大于或等于鸟苷5'-[γ-硫代]三磷酸（GTP[S]）远大于或等于ITP。就刺激作用而言，效力顺序如下：腺苷5'-[βγ-亚甲基]三磷酸（App[CH2]p）大于ATP大于鸟苷5'-[β-硫代]二磷酸（GDP[S]）。腺苷5'-[βγ-亚氨基]三磷酸（App[NH]p）也具有刺激作用，在腮腺颗粒中比ATP更有效，但在胰腺颗粒中效力较低。氟化铝在15 - 30微摩尔 - Al3 +和10 - 15毫摩尔 - F时最大程度地刺激氯离子电导。在较高浓度下F的效果较差。激酶介导的蛋白质磷酸化显然不涉及其中，因为核苷酸的作用（1）可以被ATP和GTP的不可水解类似物模拟，（2）表现出可逆性，并且（3）不被蛋白激酶抑制剂1 -（5 - 异喹啉磺酰基）- 2 - 甲基哌嗪（H - 7）或星形孢菌素消除。数据表明存在至少两个核苷酸结合位点，占据其中一个会诱导抑制，而占据另一个会诱导刺激。
