Brostrom M A, Brostrom C O, Wolff D J
J Biol Chem. 1979 Aug 25;254(16):7548-57.
The Ca2+ content of glial tumor (C6) cells was reduced approximately 5-fold by repeated treatment with media containing ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA) without loss of cellular viability. The ability of the cells to accumulate cAMP in response to beta-adrenergic agonists was reduced 60 to 70% following Ca2+ depletion. Ca2+ did not affect the apparent KACT for norepinephrine, nor did it change the concentration of propranolol required to produce 50% inhibition of the maximal norepinephrine response. Phentolamine did not alter the Ca2+ dependence of the response. The binding of dihydroalprenolol by intact C6 cells was not influenced by Ca2+. Furthermore, pretreatment with norepinephrine did not affect the Ca2+ dependence of cAMP accumulation. The effects of Ca2+, therefore, appeared to be exerted on components of the adenylate cyclase system other than the catecholamine receptor. Micromolar free Ca2+ concentration in the extracellular medium were sufficient to restore a maximal norepinephrine response to Ca2+-depeleted cells. The effect of Ca2+ on cAMP accumulation in response to hormone was immediate and was rapidly reversible upon the addition of EGTA in excess of the cation. Cells in media containing Ca2+ exhibited a characteristic biphasic time course of cAMP accumulation; with Ca2+-depleted cells cAMP was accumulated more slowly and the subsequent decline in cAMP content was also reduced. Verapamil, an inhibitor of plasmalemmal Ca2+ influx, decreased the Ca2+-dependent component of the cAMP accumulation when added prior to the cation. The effect of Ca2+ on cAMP accumulation was reduced more extensively by pretreatment of cells at 45 degrees C under Ca2+-depleted (80% loss) than under Ca2+-restored (30% loss) conditions. Trifluoperazine at micromolar concentrations decreased the Ca2+-dependent increment in accumulation of cAMP in Ca2+-restored cells. This inhibition was not overcome by increasing concentrations of norepinephrine or of extracellular Ca2+.
用含有乙二醇双（β-氨基乙基醚）N，N'-四乙酸（EGTA）的培养基反复处理，神经胶质瘤（C6）细胞的Ca2+含量降低了约5倍，而细胞活力并未丧失。Ca2+耗竭后，细胞对β-肾上腺素能激动剂积累cAMP的能力降低了60%至70%。Ca2+不影响去甲肾上腺素的表观KACT，也不改变产生最大去甲肾上腺素反应50%抑制所需的普萘洛尔浓度。酚妥拉明不改变反应对Ca2+的依赖性。完整C6细胞对二氢阿普洛尔的结合不受Ca2+影响。此外，用去甲肾上腺素预处理不影响cAMP积累对Ca2+的依赖性。因此，Ca2+的作用似乎施加于腺苷酸环化酶系统中除儿茶酚胺受体以外的成分上。细胞外培养基中微摩尔浓度的游离Ca2+足以使对Ca2+耗竭细胞的最大去甲肾上腺素反应恢复。Ca2+对激素刺激的cAMP积累的影响是即时的，并且在加入过量阳离子EGTA后迅速可逆。含有Ca2+的培养基中的细胞表现出cAMP积累的特征性双相时间进程；Ca2+耗竭的细胞中cAMP积累较慢，随后cAMP含量的下降也减少。维拉帕米是一种质膜Ca2+内流抑制剂，在阳离子之前加入时会降低cAMP积累的Ca2+依赖性成分。在Ca2+耗竭（损失80%）条件下于45℃预处理细胞，比在Ca2+恢复（损失30%）条件下，Ca2+对cAMP积累的影响降低得更广泛。微摩尔浓度的三氟拉嗪降低了Ca2+恢复细胞中cAMP积累的Ca2+依赖性增加。增加去甲肾上腺素或细胞外Ca2+的浓度并不能克服这种抑制作用。
