Study on role of protein phosphorylation in sialagogue-stimulated parotid gland.

采用无血清199培养基上漂浮的硅化晶状体纸培养的大鼠腮腺外植体，研究了唾液刺激下腮腺中鸟氨酸脱羧酶（ODC）和原癌基因蛋白磷酸化和mRNA水平的变化。结果总结如下：1)异丙烯醇（IPR）增强了表观分子量分别为17K、20K、31K和32K的4种蛋白丝氨酸/苏氨酸残基的磷酸化，羧基醇（CC）刺激了31K和32K蛋白的磷酸化。这些蛋白磷酸化先于ODC活性的增加。莫能菌素选择性地抑制知识产权依赖性ODC的诱导和磷酸化，而莫能菌素不抑制cc依赖性ODC的诱导和磷酸化，而莫能菌素不抑制cc依赖性ODC的诱导和磷酸化。莫能菌素和多粘菌素B均不能抑制IPR或cc刺激的淀粉酶分泌。2) Northern/dot和Western blot分析显示，IPR、CC、More methoxamine等唾液诱变剂分别在刺激后2 h和6 h使ODC mRNA和ODC蛋白的稳态水平达到最大值。这种增加大致与ODC活性的增加成正比，表明唾液诱导依赖酶的诱导在转录水平上受到调节。在刺激后30分钟（c-fos）或60分钟（c-jun， c-myc和c-src），检测的9个原癌基因中的4个mrna显示唾液刺激物依赖性增加至最大值。这些升高都是短暂的，在60分钟内恢复到未刺激的水平。3)酪氨酸激酶抑制剂，如herbimycin A，染料木素和甲基2,5-二羟基肉桂酸抑制唾液诱导的ODC诱导，但增强唾液诱导的淀粉酶分泌。酪氨酸磷酸酶抑制剂Na_3 VO_4增加ODC活性，而这种增加被herbimycin A抑制。这些结果表明，丝氨酸/苏氨酸和酪氨酸磷酸化的蛋白参与了唾液诱导ODC的诱导，蛋白酪氨酸磷酸化对淀粉酶的分泌有抑制作用。少

Changes in protein phosphorylation and mRNA levels of ornithine decarboxylase (ODC) and proto-oncogenes in sialagogue-stimulated parotid gland were investigated in rat parotid explants cultured on siliconized lens paper floating on serum-free 199 medium.The results were summarized as follows;1) Isoproterenol (IPR) enhanced the phosphorylation of serine/threonine residues of four proteins with apparent molecular weights of 17K, 20K, 31K and 32K and carbachol (CC) stimulated the phosphorylation of31K and 32K proteins. These protein phosphorylations preceded the increase of ODC activity. IPR-dependent ODC induction and phosphorylation of the proteins were selectively suppressed by monensin but not by polymyxin B, whereas CC-dependent ODC induction and protein phosphorylation were inhibited by polymyxin B but not by monensin. Neither monensin nor polymyxin B suppressed IPR- or CC-stimulated amylase secretion.2) Northern/dot and Western blot analyses revealed that the sialagogues (IPR, CC, … More methoxamine) elevated the steady-state levels of ODC mRNA and ODC protein to maxima at 2 h and 6 h, respectively, after stimulation. The increases were roughly proportional to those in ODC activity, suggesting that sialagogue-dependent enzyme induction is regulated at the transcriptional level. The mRNAs of four of nine proto-oncogenes examined showed sialagogue-dependent increases to maxima at 30 min (c-fos) or 60 min (c-jun, c-myc and c-src) after stimulation. These increases were all transient, with the levels returning to the unstimulated levels within 60 min.3) Tyrosine kinase inhibitors such as herbimycin A, genistein and methyl 2,5-dihydroxycinnamate inhibited a sialagogue-stimulated ODC induction but enhanced a sialagogue-induced amylase secretion. A tyrosine phosphatase inhibitor, Na_3 VO_4, increased ODC activity and this increase was suppressed by herbimycin A.These results suggest that serine/threonine and tyrosine phosphorylations of proteins participate in a sialagogue-stimulated ODC induction, and that protein tyrosine phosphorylation shows an inhibitory effect on amylase secretion. Less