Panarelli M, Holloway C D, Mulatero P, Fraser R, Kenyon C J
Medical Research Council Blood Pressure Unit, Western Infirmary, Glasgow, Scotland.
J Clin Endocrinol Metab. 1994 Apr;78(4):872-7. doi: 10.1210/jcem.78.4.8157714.
Glucocorticoids inhibit translation of the lysozyme gene. This effect may be the basis of an improved method of measuring glucocorticoid responsiveness in human tissues. We have compared lysozyme synthesis in various types of white blood cells and examined the specificity of inhibitory responses to various steroid hormones. The dose-related effects of the glucococorticoid receptor antagonist RU486 on dexamethasone responses were also assessed. Glucocorticoid receptor binding in mononuclear leukocytes (HML) was characterized by homologous displacement of [3H]dexamethasone and compared with the dose-related inhibitory effect of dexamethasone on lysozyme synthesis. Lysozyme activity was measured photometrically as the ability to cause lysis of Micrococcus lysodeikticus in the medium. The greatest effect of dexamethasone was observed after 72 h of culture. Qualitatively similar effects of dexamethasone were observed on cell lysozyme content and lysozyme activity in the medium, but for convenience, activity in medium, rather than cell content, was measured in subsequent assays. Lysozyme activities in various cell types prepared from the blood of healthy volunteers were ranked as follows: polymorphonuclear cells > monocytes > mononuclear cells > lymphocytes. However, dexamethasone inhibited lysozyme synthesis to a similar degree for all types. As mononuclear cells are more conveniently prepared in greater yield compared with other cells, this HML fraction formed the basis of a method of assessing glucocorticoid responsiveness and sensitivity. Lysozyme activity from HML was not significantly affected by incubation with 1 mumol/L estradiol, progesterone, dehydroepiandrosterone, or aldosterone. Dexamethasone and cortisol at 1 mumol/L both inhibited release by 45-50%. Although RU486 when added alone partially inhibited lysozyme activity, the same concentration (1 mumol/L) antagonized glucocorticoid responses and shifted the IC50 and threshold values for the effect of dexamethasone from 1.2 nmol/L to more than 1 mumol/L and from less than 1.0 to 19 nmol/L, respectively. The equilibrium dissociation constants (Kd) for dexamethasone binding to the glucocorticoid receptor ranged from 2.8-12.5 nmol/L and were positively correlated with dexamethasone IC50 values for lysozyme synthesis (r = 0.57; P = 0.002). In conclusion, the inhibition of lysozyme synthesis by dexamethasone in human mononuclear cells is a convenient and specific method of measuring responsiveness to glucocorticoids.
糖皮质激素抑制溶菌酶基因的翻译。这一效应可能是一种改进的测量人体组织中糖皮质激素反应性方法的基础。我们比较了不同类型白细胞中溶菌酶的合成，并研究了对各种类固醇激素抑制反应的特异性。还评估了糖皮质激素受体拮抗剂RU486对地塞米松反应的剂量相关效应。通过[3H]地塞米松的同源置换来表征单核白细胞（HML）中的糖皮质激素受体结合，并与地塞米松对溶菌酶合成的剂量相关抑制效应进行比较。溶菌酶活性通过光度法测量，即其在培养基中导致溶壁微球菌裂解的能力。培养72小时后观察到地塞米松的最大效应。在地塞米松对细胞溶菌酶含量和培养基中溶菌酶活性的影响在质量上相似，但为方便起见，在后续实验中测量的是培养基中的活性，而非细胞含量。从健康志愿者血液中制备的各种细胞类型中的溶菌酶活性排序如下：多形核细胞＞单核细胞＞单个核细胞＞淋巴细胞。然而，地塞米松对所有类型的溶菌酶合成抑制程度相似。由于与其他细胞相比，单个核细胞更容易大量制备，因此这个HML部分构成了一种评估糖皮质激素反应性和敏感性方法的基础。用1μmol/L雌二醇、孕酮、脱氢表雄酮或醛固酮孵育HML的溶菌酶活性没有受到显著影响。1μmol/L的地塞米松和皮质醇均抑制释放45% - 50%。虽然单独添加RU486时部分抑制溶菌酶活性，但相同浓度（1μmol/L）可拮抗糖皮质激素反应，并将地塞米松作用的IC50值和阈值分别从1.2 nmol/L转变为超过1μmol/L以及从小于1.0转变为19 nmol/L。地塞米松与糖皮质激素受体结合的平衡解离常数（Kd）范围为2.8 - 12.5 nmol/L，并且与地塞米松抑制溶菌酶合成的IC50值呈正相关（r = 0.57；P = 0.002）。总之，地塞米松对人单核细胞中溶菌酶合成的抑制是一种测量对糖皮质激素反应性的简便且特异的方法。
