Facchinetti M M, Boland R, de Boland A R
Departamento de Biologia, Bioquimica y Farmacia, Universidad Nacional del Sur, San Juan, Bahia Blanca, Argentina.
Mol Cell Endocrinol. 1998 Jan 15;136(2):131-8. doi: 10.1016/s0303-7207(97)00221-9.
We have examined the effects in vitro of calcitriol [1,25(OH)2D3], the hormonal form of vitamin D3, on the breakdown of membrane phosphoinositides in skeletal muscle from young (3 months) and aged (24 months) rats. Calcitriol (10(-9) M) induced a rapid and transient release of IP3/inositol phosphates and diacylglycerol (DAG) from muscle slices/membranes prelabeled with [3H]myo-inositol and [3H]arachidonate, respectively. Inositol phosphate release was maximal at 15 s and then declined. The effects of hormone specificity exhibited as the closely related derivatives of vitamin D3, 25OHD3, 1alphaOHD3 and 24,25(OH)2D3 did not alter muscle inositol phosphate levels. The stimulation of DAG was biphasic, the early phase (15 s) being abolished by neomycin (0.5 mM), an inhibitor of phosphoinositide hydrolysis, similar to IP3 formation and consistent with a role of phospholipase C (PLC) in intracellular signal generation. Neomycin had no effect on the second DAG peak (2 min) induced by calcitriol, suggesting that the late phase of DAG formation is independent from the hydrolysis of phosphoinositides. Higher basal inositol phosphate and DAG levels were detected in muscle from aged rats thereby reducing the effects of the hormone on second messenger generation ( -80 and -60% for IP3 and DAG, respectively). Calcitriol stimulation of PLC was mimicked, in both young and old rats, by GTPgammaS, a non-hydrolyzable analogue of GTP, while GDPbetaS, a G protein inhibitor, suppressed the effect of the hormone. The early effects of calcitriol and GTPgammaS were not additive. Bordetella pertussis toxin abolished by 85% the effects of calcitriol on inositol phosphate release in young rats but was without effect in aged animals. These results demonstrate that calcitriol activates phosphoinositide-PLC in rat skeletal muscle by a mechanism which involves a pertussis-sensitive G protein and that the effects of the hormone are altered with ageing.
我们研究了维生素D3的激素形式骨化三醇[1,25(OH)2D3]对年轻（3个月）和老年（24个月）大鼠骨骼肌中膜磷酸肌醇分解的体外作用。骨化三醇（10(-9) M）分别诱导预先用[3H]肌醇和[3H]花生四烯酸标记的肌肉切片/膜快速、短暂地释放肌醇三磷酸/肌醇磷酸和二酰甘油（DAG）。肌醇磷酸的释放在15秒时达到最大值，然后下降。作为维生素D3密切相关衍生物的骨化三醇、25羟维生素D3、1α羟维生素D3和24,25(OH)2D3的激素特异性作用并未改变肌肉中肌醇磷酸水平。DAG的刺激是双相的，早期阶段（15秒）被新霉素（0.5 mM）消除，新霉素是磷酸肌醇水解的抑制剂，类似于肌醇三磷酸的形成，并且与磷脂酶C（PLC）在细胞内信号产生中的作用一致。新霉素对骨化三醇诱导的第二个DAG峰值（2分钟）没有影响，表明DAG形成的后期阶段与磷酸肌醇的水解无关。在老年大鼠的肌肉中检测到较高的基础肌醇磷酸和DAG水平，从而降低了激素对第二信使产生的作用（肌醇三磷酸和DAG分别降低80%和60%）。在年轻和老年大鼠中，GTPγS（一种不可水解的GTP类似物）模拟了骨化三醇对PLC的刺激，而G蛋白抑制剂GDPβS则抑制了激素的作用。骨化三醇和GTPγS的早期作用不是相加的。百日咳博德特氏菌毒素消除了年轻大鼠中骨化三醇对肌醇磷酸释放作用的85%，但对老年动物没有影响。这些结果表明，骨化三醇通过一种涉及百日咳敏感G蛋白的机制激活大鼠骨骼肌中的磷酸肌醇-PLC，并且激素的作用随年龄增长而改变。
