Sahai A, Fadda G Z, Sandler R S, Tannen R L
Department of Medicine, University of Southern California School of Medicine, Los Angeles.
Kidney Int. 1994 Oct;46(4):1069-73. doi: 10.1038/ki.1994.368.
A novel variant of the LLC-PK1 cell line was used to examine directly the mechanism whereby PGF2 alpha and TPA inhibit renal ammoniagenesis. The variant cells, which exhibit a growth pattern and morphology similar to the parent cell line, were isolated by a self selection process utilizing long-term cultures of parent cells maintained under conditions of continuous gentle rocking of the media fluid. Incubation of both parent and variant LLC-PK1 cells for one hour in a glutamine supplemented Krebs-Hensleit media of low pH (pH 6.8) increased ammonia and alanine production in comparison to the basal rates at pH 7.4. The phorbol ester TPA and also PGF2 alpha inhibited the low pH-induced increases in ammonia and alanine formation in parent cells; however, neither TPA nor PGF2 alpha inhibited ammonia or alanine metabolism in variant cells. TPA and PGF2 alpha activated PKC similarly in the parent and variant cells as demonstrated by a significant increase in membrane bound enzyme activity. BCECF labeling of cells indicated that the parent and variant cells possess an amiloride sensitive Na+/H+ antiporter of comparable activity. Exposure of parent cells to PGF2 alpha or TPA resulted in the activation of Na+/H+ antiporter activity. By contrast, neither compound stimulated antiporter activity in variant cells. These studies strongly suggest that PKC mediated activation of the Na+/H+ antiporter accounts for the inhibition of ammonia production produced by both PGF2 alpha and TPA. In addition, this novel variant of LLC-PK1 cells should provide a valuable tool to investigate various normal and pathophysiological functions involving mediation by PKC and/or Na+/H+ antiporter activity.
一种新型的LLC-PK1细胞系变体被用于直接研究PGF2α和TPA抑制肾氨生成的机制。这些变体细胞呈现出与亲代细胞系相似的生长模式和形态，是通过一种自我选择过程分离得到的，该过程利用在培养基持续温和摇晃条件下维持的亲代细胞长期培养物。与pH 7.4时的基础速率相比，将亲代和变体LLC-PK1细胞在低pH（pH 6.8）的谷氨酰胺补充的Krebs-Hensleit培养基中孵育一小时，会增加氨和丙氨酸的产生。佛波酯TPA以及PGF2α抑制了亲代细胞中低pH诱导的氨和丙氨酸形成的增加；然而，TPA和PGF2α均未抑制变体细胞中的氨或丙氨酸代谢。如膜结合酶活性显著增加所表明的，TPA和PGF2α在亲代和变体细胞中同样激活了PKC。细胞的BCECF标记表明，亲代和变体细胞具有活性相当的氨氯吡脒敏感的Na+/H+逆向转运体。将亲代细胞暴露于PGF2α或TPA会导致Na+/H+逆向转运体活性的激活。相比之下，这两种化合物均未刺激变体细胞中的逆向转运体活性。这些研究强烈表明，PKC介导的Na+/H+逆向转运体激活解释了PGF2α和TPA对氨产生的抑制作用。此外，这种新型的LLC-PK1细胞变体应该为研究涉及PKC和/或Na+/H+逆向转运体活性介导的各种正常和病理生理功能提供一个有价值的工具。
