Ransjö M, Lerner U H
Department of Oral Pathology, University of Umeå, Sweden.
Biochim Biophys Acta. 1987 Oct 1;930(3):378-91. doi: 10.1016/0167-4889(87)90011-5.
We have utilized the adenylate cyclase stimulator, cholera toxin, as a tool to test the role of cyclic AMP as a mediator of the effects on bone resorption by the calcium-regulating hormones, parathyroid hormone (PTH) and calcitonin. The effects on bone resorption were studied in an organ culture system using calvarial bones from newborn mice. Cyclic AMP response was assayed in calvarial bone explants and isolated osteoblasts from neonatal mouse calvaria. Cholera toxin caused a dose-dependent cAMP response in calvarial bones, seen at and above approx. 1-3 ng/ml and calculated half-maximal stimulation (EC50) at 18 ng/ml. The stimulatory effect of cholera toxin could be potentiated by the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX, 0.2 mmol/l). Cyclic AMP accumulation in the bones was maximal after 4-6 h, and thereafter declined. However, activation of the adenylate cyclase was irreversible and the total amount (bone + medium) of cAMP produced, in the presence of IBMX (0.2 mmol/l), increased with time, for at least 48 h. In osteoblast-like cells cholera toxin (1 microgram/ml) stimulated the cellular levels of cAMP with a peak after 60-120 min, which could be potentiated with IBMX. The total cAMP accumulation indicated an irreversible response. In short-term bone organ cultures (at most, 24 h) cholera toxin, at and above 3 ng/ml, inhibited the stimulatory effect of PTH (10 nmol/l) on 45Ca release from prelabelled calvarial bones. The inhibitory effect of cholera toxin (0.1 microgram/ml) on 45Ca release was significant after 6 h and the calculated IC50 value at 24 h was 11.2 ng/ml. Cholera toxin (0.1 microgram/ml) also inhibited PTH-stimulated (10 nmol/l) release of Ca2+, inorganic phosphate (Pi), beta-glucuronidase, beta-N-acetylglucosaminidase and degradation of organic matrix (release of 3H from [3H]proline-labelled bones) in 24 h cultures. 45Ca release from bones stimulated by prostaglandin E2 (1 mumol/l) and 1 alpha-hydroxyvitamin D3 (0.1 mumol/l) was also inhibited by cholera toxin (0.3 microgram/ml) in 24-h cultures. The inhibitory effect of cholera toxin on bone resorption was transient, and in long-term cultures (120 h) cholera toxin caused a dose-dependent, delayed stimulation of mineral mobilization (Ca2+, 45Ca, Pi), degradation of matrix and release of the lysosomal enzymes beta-glucuronidase and beta-N-acetylglucosaminidase.(ABSTRACT TRUNCATED AT 400 WORDS)
我们利用腺苷酸环化酶刺激剂霍乱毒素作为工具，来测试环磷酸腺苷（cAMP）作为钙调节激素甲状旁腺激素（PTH）和降钙素对骨吸收作用的介质的作用。在一个使用新生小鼠颅骨的器官培养系统中研究了对骨吸收的影响。在颅骨外植体和从新生小鼠颅骨分离的成骨细胞中测定了cAMP反应。霍乱毒素在颅骨中引起了剂量依赖性的cAMP反应，在约1 - 3 ng/ml及以上浓度时可见，计算得出的半最大刺激浓度（EC50）为18 ng/ml。磷酸二酯酶抑制剂异丁基甲基黄嘌呤（IBMX，0.2 mmol/l）可增强霍乱毒素的刺激作用。骨中cAMP积累在4 - 6小时后达到最大值，此后下降。然而，腺苷酸环化酶的激活是不可逆的，并且在存在IBMX（0.2 mmol/l）的情况下，产生的cAMP总量（骨 + 培养基）随时间增加，至少持续48小时。在成骨样细胞中，霍乱毒素（1微克/毫升）刺激细胞内cAMP水平在60 - 120分钟后达到峰值，这可被IBMX增强。总的cAMP积累表明是不可逆反应。在短期骨器官培养（最长24小时）中，霍乱毒素在3 ng/ml及以上浓度时，抑制了PTH（10 nmol/l）对预先标记的颅骨中45Ca释放的刺激作用。霍乱毒素（0.1微克/毫升）对45Ca释放的抑制作用在6小时后显著，24小时时计算得出的IC50值为11.2 ng/ml。霍乱毒素（0.1微克/毫升）在24小时培养中还抑制了PTH刺激（10 nmol/l）的Ca2 +、无机磷酸盐（Pi）、β - 葡萄糖醛酸酶、β - N - 乙酰氨基葡萄糖苷酶的释放以及有机基质的降解（从[3H]脯氨酸标记的骨中释放3H）。在24小时培养中，前列腺素E2（1 μmol/l）和1α - 羟基维生素D3（0.1 μmol/l）刺激的骨中45Ca释放也被霍乱毒素（0.3微克/毫升）抑制。霍乱毒素对骨吸收的抑制作用是短暂的，在长期培养（120小时）中，霍乱毒素引起了剂量依赖性的、延迟的矿物质动员（Ca2 +、45Ca、Pi）、基质降解以及溶酶体酶β - 葡萄糖醛酸酶和β - N - 乙酰氨基葡萄糖苷酶的释放。（摘要截短至400字）
