Berl T, Siriwardana G, Ao L, Butterfield L M, Heasley L E
Department of Medicine, University of Colorado School of Medicine, Denver 80262, USA.
Am J Physiol. 1997 Mar;272(3 Pt 2):F305-11. doi: 10.1152/ajprenal.1997.272.3.F305.
Inner medullary collecting duct (IMCD) cells adapt to a hypertonic environment by synthesizing transporters that allow for accumulation of organic osmolytes. To examine for activation of additional mitogen-activated protein (MAP) kinases, extracts of IMCD-3 cells subjected to a hypertonic medium (600 mosmol/kgH2O) for 15 min were fractionated by Mono Q fast-performance liquid chromatography and assayed with the epidermal growth factor receptor [EGFR-(662-681)] peptide as substrate. Three peaks of activity were identified. Western blotting revealed that these peaks coincided with Jun NH2-terminal kinase (JNK), extracellular signal-regulated protein kinases, ERK1 and ERK2, and p38 MAP kinase. To assess the functional significance of ERK2 activation in IMCD-3 cells, the effect of PD-098059, an inhibitor of the upstream regulatory protein kinase MAP/ERK kinase (MEK) was assessed. PD-098059 inhibited ERK activation by hypertonicity. Yet, the stimulation of inositol uptake, a marker of adaptation, after 16 h was unaltered. Direct measurements of JNK activity [phosphorylation of GST-cJun-(1-79)] revealed a marked (20- to 40-fold) increase in activity as medium osmolality was increased from 300 to 900 mosmol/kgH2O with either NaCl or mannitol. Urea induced a more modest increase in activity. The response is prompt and detected as early as 2 min after exposure, reaching a maximum activation at 10-15 min. Downregulation of cellular protein kinase C (PKC) by chronic exposure to phorbol esters only minimally attenuated the JNK response to hyperosmolality, indicating a lack of involvement of PKC. We conclude that, in IMCD-3 cells, inhibition of ERK activation by hyperosmolality does not prevent osmoregulatory increase in inositol transport. This is not consistent with a role for ERKs in the response. The roles for JNK and p38 have not been ruled out, and these pathways may represent the initiating event in the subsequent transcription of organic osmolyte transporter genes and adaptation to extracellular hypertonicity.
髓质内集合管（IMCD）细胞通过合成转运蛋白来适应高渗环境，这些转运蛋白能够促进有机渗透溶质的积累。为了检测其他丝裂原活化蛋白（MAP）激酶的激活情况，将在高渗培养基（600 mosmol/kgH₂O）中处理15分钟的IMCD - 3细胞提取物，通过Mono Q快速高效液相色谱进行分级分离，并用表皮生长因子受体[EGFR-(662 - 681)]肽作为底物进行检测。鉴定出三个活性峰。蛋白质免疫印迹法显示，这些峰与Jun氨基末端激酶（JNK）、细胞外信号调节蛋白激酶ERK1和ERK2以及p38 MAP激酶一致。为了评估ERK2激活在IMCD - 3细胞中的功能意义，评估了上游调节蛋白激酶MAP/ERK激酶（MEK）的抑制剂PD - 098059的作用。PD - 098059抑制了高渗引起的ERK激活。然而，16小时后作为适应性标志物的肌醇摄取刺激未改变。对JNK活性的直接测量[GST - cJun-(1 - 79)的磷酸化]显示，随着培养基渗透压从300 mosmol/kgH₂O增加到900 mosmol/kgH₂O（使用NaCl或甘露醇），活性显著增加（20至40倍）。尿素引起的活性增加较为适度。该反应迅速，最早在暴露后2分钟即可检测到，在10 - 15分钟达到最大激活。通过长期暴露于佛波酯使细胞蛋白激酶C（PKC）下调，仅轻微减弱了JNK对高渗的反应，表明PKC未参与其中。我们得出结论，在IMCD - 3细胞中，高渗对ERK激活的抑制并不妨碍肌醇转运的渗透调节增加。这与ERK在该反应中的作用不一致。JNK和p38的作用尚未排除，并且这些途径可能代表随后有机渗透溶质转运蛋白基因转录和适应细胞外高渗的起始事件。
