Massheimer V, Fernandez L M, Boland R, de Boland A R
Departamento de Biologia, Universidad Nacional del Sur, Bahia Blanca, Argentina.
Mol Cell Endocrinol. 1992 Mar;84(1-2):15-22. doi: 10.1016/0303-7207(92)90066-f.
Experiments were carried out to obtain information about the mechanism underlying the fast action of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in skeletal muscle. N-2'-o-dibutyryladenosine-3',5'-cyclic monophosphate (dbcAMP), similarly as 1,25(OH)2D3 (5 x 10(-10) M), rapidly increased 45Ca uptake by soleus muscle from vitamin D-deficient chicks (+25% and +98% at 3 min and 10 min, respectively) in a dose-dependent manner. The effects of the cAMP analog (10 microM) and 1,25(OH)2D3 could be abolished by the Ca(2+)-channel blocker nifedipine and the calmodulin antagonist flufenazine. Calmodulin binding by two muscle microsomal proteins of 28 kDa and 30 kDa was stimulated within 1 min of exposure of the tissue to 1,25(OH)2D3. Direct effects of the sterol on membrane calmodulin binding were shown with isolated microsomes. The 1,25(OH)2D3-mediated rise of [125I]calmodulin binding to microsomal membranes was dependent on the presence of medium ATP. Forskolin (10 microM) and cAMP (10 microM) also increased [125I]calmodulin binding (+75% and +64%, respectively, with respect to controls). Pretreatment of microsomal membranes with cAMP-dependent protein kinase inhibitor (1 microgram/ml) or addition of alkaline phosphates (1 U/ml) after hormonal treatment caused complete inhibition of 1,25(OH)2D3-induced [125I]calmodulin binding to microsomal membrane proteins. These results imply modifications of membrane protein phosphorylation through the cAMP signal pathway and in turn of calmodulin binding in the mechanism by which 1,25(OH)2D3 rapidly stimulates skeletal muscle Ca2+ uptake.
开展了多项实验，以获取有关1,25 - 二羟基维生素D3（1,25(OH)2D3）在骨骼肌中快速作用机制的信息。N - 2'-O - 二丁酰腺苷 - 3',5'-环磷酸单酯（dbcAMP）与1,25(OH)2D3（5×10⁻¹⁰ M）类似，能以剂量依赖方式使维生素D缺乏雏鸡的比目鱼肌对⁴⁵Ca的摄取迅速增加（在3分钟和10分钟时分别增加25%和98%）。环磷酸腺苷（cAMP）类似物（10 μM）和1,25(OH)2D3的作用可被钙通道阻滞剂硝苯地平和钙调蛋白拮抗剂氟奋乃静消除。在组织暴露于1,25(OH)2D3的1分钟内，28 kDa和30 kDa的两种肌肉微粒体蛋白与钙调蛋白的结合受到刺激。用分离的微粒体显示了该甾醇对膜钙调蛋白结合的直接作用。1,25(OH)2D3介导的[¹²⁵I]钙调蛋白与微粒体膜结合的增加依赖于培养基中ATP的存在。福斯可林（10 μM）和cAMP（10 μM）也增加了[¹²⁵I]钙调蛋白的结合（相对于对照分别增加75%和64%）。用cAMP依赖性蛋白激酶抑制剂（1 μg/ml）预处理微粒体膜或在激素处理后加入碱性磷酸酶（1 U/ml）可完全抑制1,25(OH)2D3诱导的[¹²⁵I]钙调蛋白与微粒体膜蛋白的结合。这些结果表明，在1,25(OH)2D3快速刺激骨骼肌Ca²⁺摄取的机制中，通过cAMP信号通路对膜蛋白磷酸化进行了修饰，进而对钙调蛋白结合进行了修饰。
