Volume and agonist-induced regulation of myosin light-chain phosphorylation in glomerular mesangial cells.
Volume and agonist-induced regulation of myosin light-chain phosphorylation in glomerular mesangial cells.
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DOI：
10.1152/ajprenal.1993.264.3.f421
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发表时间：
1993-03
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影响因子：
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通讯作者：
Michio Takeda;T. Homma;M. Breyer;N. Horiba;Richard L. Hoover;S. Kawamoto;I. Ichikawa;Valentina Kon-Vale
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作者：
Michio Takeda;T. Homma;M. Breyer;N. Horiba;Richard L. Hoover;S. Kawamoto;I. Ichikawa;Valentina Kon-Vale
我们研究了细胞体积减少本身是否可以激活导致系膜细胞收缩的细胞内机制。为此，我们应用高渗应激培养肾小球系膜细胞，并检查对肌球蛋白轻链（MLCP）磷酸化的影响。与对照细胞相比，通过NaCl或棉子糖获得的高渗胁迫（390 mosmol/kg）分别使MLCP显著增加至140.7 +/- 7.0%（n = 5）和134.8 +/- 7.7%（n = 4），同时细胞体积减少。这种增加与通过以下激动剂实现的增加相当：精氨酸加压素（AVP，100 nM; n = 5）和内皮素-1（ET，10 nM; n = 5）。通过使用二维胰蛋白酶磷酸肽图谱，肌球蛋白轻链激酶（MLCK）和蛋白激酶C（PKC）观察到的磷酸化的贡献进行了检查，通过鉴定丝氨酸-19（MLCK）和丝氨酸-1或丝氨酸-2（PKC）的磷酸化。在静息条件下，MLCK和PKC位点之间磷酸化的相对分布为60.1 +/-8.4%和39.9 +/-8.4%。在高渗应激或激动剂刺激期间，这些酶的相对贡献保持相似。由于胞浆Ca 2+浓度（[Ca 2 +]i）是MLCP的重要决定因素，因此我们还在这些环境中检查了[Ca 2 +]i。而AVP和ET-诱导的[Ca ~（2+）]i的特征性的瞬时峰，高渗胁迫引起[Ca ~（2+）]i的逐渐和适度的增加。这些研究表明，在肾小球系膜细胞中，细胞体积的减少通过与激动剂诱导事件不同的机制诱导MLCP。
We investigated whether cell volume decrease per se can activate intracellular mechanisms leading to mesangial cell contraction. For this purpose, we applied hyperosmotic stress to cultured glomerular mesangial cells and examined the effects on phosphorylation of myosin light chain (MLCP). Compared with control cells, hyperosmotic stress (390 mosmol/kg) attained by either NaCl or raffinose significantly increased MLCP to 140.7 +/- 7.0% (n = 5) and 134.8 +/- 7.7% (n = 4), respectively, in parallel with a decrease in the cell volume. This increase was comparable to that achieved by the following agonists: arginine vasopressin (AVP, 100 nM; n = 5) and endothelin-1 (ET, 10 nM; n = 5). By using two-dimensional tryptic phosphopeptide mapping, contribution of myosin light-chain kinase (MLCK) and protein kinase C (PKC) to the observed phosphorylation was examined by identifying phosphorylation at serine-19 (by MLCK) and at serine-1 or serine-2 (by PKC). Under resting conditions, relative distribution of phosphorylation between MLCK and PKC sites was 60.1 +/- 8.4 and 39.9 +/- 8.4%. The relative contribution by these enzymes remained similar during hyperosmotic stress or agonist stimulation. Since cytosolic Ca2+ concentration ([Ca2+]i) is an important determinant of MLCP, we also examined [Ca2+]i in these settings. While AVP and ET-induced a characteristic transient spike in [Ca2+]i, hyperosmotic stress caused a gradual and modest increase in [Ca2+]i. These studies show that, in mesangial cells, reduction in cell volume induces MLCP through mechanisms distinct from those involved in agonist-induced events.