Reshkin S J, Guerra L, Bagorda A, Debellis L, Cardone R, Li A H, Jacobson K A, Casavola V
Department of General and Environmental Physiology, University of Bari, 70126 Bari, Italy.
J Membr Biol. 2000 Nov 15;178(2):103-13. doi: 10.1007/s002320010018.
We have previously demonstrated that in A(6) renal epithelial cells, a commonly used model of the mammalian distal section of the nephron, adenosine A(1) and A(2A) receptor activation modulates sodium and chloride transport and intracellular pH (Casavola et al., 1997). Here we show that apical addition of the A(3) receptor-selective agonist, 2-chloro-N(6)-(3-iodobenzyl)-adenosine-5'-methyluronamide (Cl-IB-MECA) stimulated a chloride secretion that was mediated by calcium- and cAMP-regulated channels. Moreover, in single cell measurements using the fluorescent dye Fura 2-AM, Cl-IB-MECA caused an increase in Ca(2+) influx. The agonist-induced rise in Ca(2+) was significantly inhibited by the selective adenosine A(3) receptor antagonists, 2,3-diethyl-4, 5-dipropyl-6-phenylpyridine-3-thiocarboxylate-5-carboxylate (MRS 1523) and 3-ethyl 5-benzyl 2-methyl-6-phenyl-4-phenylethynyl-1, 4-(+/-)-dihydropyridine-3,5-dicarboxylate (MRS 1191) but not by antagonists of either A(1) or A(2) receptors supporting the hypothesis that Cl-IB-MECA increases Ca(2+) by interacting exclusively with A(3) receptors. Cl-IB-MECA-elicited Ca(2+) entry was not significantly inhibited by pertussis toxin pretreatment while being stimulated by cholera toxin preincubation or by raising cellular cAMP levels with forskolin or rolipram. Preincubation with the protein kinase A inhibitor, H89, blunted the Cl-IB-MECA-elicited Ca(2+) response. Moreover, Cl-IB-MECA elicited an increase in cAMP production that was inhibited only by an A(3) receptor antagonist. Altogether, these data suggest that in A(6) cells a G(s)/protein kinase A pathway is involved in the A(3) receptor-dependent increase in calcium entry.
我们之前已经证明，在A(6)肾上皮细胞（一种常用的哺乳动物肾单位远曲小管模型）中，腺苷A(1)和A(2A)受体的激活可调节钠和氯的转运以及细胞内pH值（卡萨沃拉等人，1997年）。在此我们表明，顶端添加A(3)受体选择性激动剂2-氯-N(6)-(3-碘苄基)-腺苷-5'-甲基脲酰胺（Cl-IB-MECA）可刺激由钙和cAMP调节通道介导的氯分泌。此外，在使用荧光染料Fura 2-AM的单细胞测量中，Cl-IB-MECA导致Ca(2+)内流增加。选择性腺苷A(3)受体拮抗剂2,3-二乙基-4,5-二丙基-6-苯基吡啶-3-硫代羧酸盐-5-羧酸盐（MRS 1523）和3-乙基-5-苄基-2-甲基-6-苯基-4-苯乙炔基-1,4-(+/-)-二氢吡啶-3,5-二羧酸盐（MRS 1191）可显著抑制激动剂诱导的[Ca(2+)]i升高，但A(1)或A(2)受体拮抗剂则无此作用，这支持了Cl-IB-MECA仅通过与A(3)受体相互作用来增加[Ca(2+)]i的假说。百日咳毒素预处理对Cl-IB-MECA诱导的Ca(2+)内流无显著抑制作用，而霍乱毒素预孵育或用福斯可林或咯利普兰提高细胞cAMP水平则可刺激Ca(2+)内流。用蛋白激酶A抑制剂H89预孵育可减弱Cl-IB-MECA诱导的[Ca(2+)]i反应。此外，Cl-IB-MECA可引起cAMP生成增加，且仅被A(3)受体拮抗剂抑制。总之，这些数据表明，在A(6)细胞中，G(s)/蛋白激酶A途径参与了A(3)受体依赖性钙内流增加。
