Griffith D A, Jarvis S M
Research School of Biosciences, University of Kent, Canterbury, UK.
Biochim Biophys Acta. 1996 Oct 23;1284(2):213-20. doi: 10.1016/s0005-2736(96)00132-0.
The involvement of protein kinase C (PKC) in the regulation of Na(+)-dependent and -independent hypoxanthine transport was investigated by exposing confluent monolayers of LLC-PK1 renal epithelia cells to the PKC activator, phorbol 12-myristate 13-acetate (PMA). Chronic exposure (> 2 h) of LLC-PK1 monolayers to 16 nM PMA resulted in approximately 75% inhibition of Na(+)-dependent hypoxanthine influx occurring maximally at 8 h and persisting for 72 h. In contrast, PMA had little effect on Na(+)-independent hypoxanthine influx at 8 h, but longer exposure resulted in stimulation of influx (approximately 3-fold) that peaked at 24 h and thereafter declined to control levels at 72 h. The effects of PMA were dose-dependent and were associated with changes in Vmax of transport (2-4-fold) with no significant change in apparent K(m). 4 alpha-Phorbol, a phorbol ester that does not activate PKC, had no effect on hypoxanthine transport by LLC-PK1 cells. The diacylglycerol kinase inhibitor, R59022 (10 microM), partially inhibited (28%) Na(+)-dependent hypoxanthine influx. In addition, the PMA-induced effects on hypoxanthine transport were reversed by Ro-31-8220 (1 and 5 microM) and calphostin C (50 nM), potent and selective inhibitors of PKC. The increase in Na(+)-independent hypoxanthine influx following exposure to PMA was blocked by the protein synthesis inhibitor, cycloheximide (20 microM), and correlated with an increase in LLC-PK1 cell proliferation. The PMA-induced decrease in Na(+)-dependent hypoxanthine transport was independent of PMA effects on cell proliferation and not dependent on protein synthesis. These results are consistent with the proposal that the PMA-induced effects on hypoxanthine transport are due to PKC activation.
通过将LLC - PK1肾上皮细胞的汇合单层暴露于蛋白激酶C（PKC）激活剂佛波醇12 - 肉豆蔻酸酯13 - 乙酸酯（PMA），研究了PKC在调节Na⁺依赖性和非依赖性次黄嘌呤转运中的作用。将LLC - PK1单层长期（> 2小时）暴露于16 nM PMA导致Na⁺依赖性次黄嘌呤内流在8小时时最大程度地受到约75%的抑制，并持续72小时。相比之下，PMA在8小时时对Na⁺非依赖性次黄嘌呤内流几乎没有影响，但更长时间的暴露导致内流刺激（约3倍），在24小时达到峰值，此后在72小时降至对照水平。PMA的作用是剂量依赖性的，并且与转运的Vmax变化（2 - 4倍）相关，而表观K(m)没有显著变化。4α - 佛波醇，一种不激活PKC的佛波醇酯，对LLC - PK1细胞的次黄嘌呤转运没有影响。二酰基甘油激酶抑制剂R59022（10 μM）部分抑制（28%）Na⁺依赖性次黄嘌呤内流。此外，PMA对次黄嘌呤转运的诱导作用被Ro - 31 - 8220（1和5 μM）和钙磷蛋白C（50 nM）逆转，它们是PKC的强效和选择性抑制剂。暴露于PMA后Na⁺非依赖性次黄嘌呤内流的增加被蛋白质合成抑制剂环己酰亚胺（20 μM）阻断，并且与LLC - PK1细胞增殖增加相关。PMA诱导的Na⁺依赖性次黄嘌呤转运减少与PMA对细胞增殖的影响无关，也不依赖于蛋白质合成。这些结果与PMA对次黄嘌呤转运的诱导作用是由于PKC激活的提议一致。
