Xi D, Van Dolah F M, Ramsdell J S
Marine Biomedical and Environmental Sciences, Medical University of South Carolina, Charleston 29412.
J Biol Chem. 1992 Dec 15;267(35):25025-31.
Maitotoxin (MTX) is a water-soluble polyether, isolated from the marine dinoflagellate Gambierdiscus toxicus, that stimulates hormone release and Ca2+ influx. We have investigated the action by which MTX induces Ca2+ influx and stimulates prolactin (PRL) release from GH4C1 rat pituitary cells. PRL release elicited by MTX is abolished in a concentration-dependent manner by nimodipine, a dihydropyridine (DHP) antagonist of type L voltage-dependent calcium channels (L-VDCC), indicating that MTX-enhanced PRL release occurs via activation of type L-VDCC. As an initial approach to determine whether MTX interacts directly with VDCC, we examined whether MTX affects the binding of [3H]PN 200-110, a DHP class antagonist, in intact GH4C1 cells. MTX increased the Bmax of [3H]PN 200-110 binding to intact GH4C1 cells from 4.6 +/- 0.03 to 12.5 +/- 2.2 fmol/10(6) cells, without changing the Kd. This indicates that MTX does not bind to the DHP site, but rather suggests that MTX may have an allosteric interaction with the DHP binding site. The effect of MTX on DHP binding was largely (65%) calcium-dependent. We next examined whether MTX alters the membrane potential of GH4C1 cells using the potential sensitive fluorescent dye bisoxonol. Addition of 100 ng/ml MTX to GH4C1 cells caused a membrane depolarization within 2.5 min which reached a plateau at 5 min. The MTX-induced depolarization was not prevented by substitution of impermeant choline ions for Na+. It was similarly unaffected by K+ channel blockers or by depleting the K+ chemical concentration gradient with gramicidin, a monovalent cation pore-forming agent. By contrast, low extracellular Ca2+ totally abolished the depolarization response, and nimodipine at 100 nM substantially reduced the MTX-induced membrane depolarization. These results indicate that the predominant effect of MTX on depolarization is Ca2+ influx through L-VDCC. Taken together, our results indicate that MTX-enhanced PRL release occurs exclusively via activation of type L-VDCC in GH4C1 cells. We suggest that MTX induces an initial slow calcium conductance, possibly via an allosteric interaction with a component of the VDCC complex, which, in turn, initiates a positive feedback mechanism involving calcium-dependent membrane depolarization and voltage-dependent activation of calcium channels.
maitotoxin（MTX）是一种水溶性聚醚，从海洋双鞭毛藻甘比毒藻中分离得到，可刺激激素释放和Ca2+内流。我们研究了MTX诱导Ca2+内流并刺激GH4C1大鼠垂体细胞释放催乳素（PRL）的作用机制。MTX引发的PRL释放可被尼莫地平以浓度依赖的方式阻断，尼莫地平是L型电压依赖性钙通道（L-VDCC）的二氢吡啶（DHP）拮抗剂，这表明MTX增强PRL释放是通过激活L型VDCC实现的。作为确定MTX是否直接与VDCC相互作用的初步方法，我们检测了MTX是否影响完整GH4C1细胞中[3H]PN 200-110（一种DHP类拮抗剂）的结合。MTX使[3H]PN 200-110与完整GH4C1细胞的结合Bmax从4.6±0.03增加到12.5±2.2 fmol/10(6)细胞，而Kd不变。这表明MTX不与DHP位点结合，而是提示MTX可能与DHP结合位点存在变构相互作用。MTX对DHP结合的影响在很大程度上（65%）依赖于钙。接下来，我们使用电位敏感荧光染料双羟萘酚检测MTX是否改变GH4C1细胞的膜电位。向GH4C1细胞中添加100 ng/ml MTX在2.5分钟内导致膜去极化，并在5分钟时达到平台期。MTX诱导的去极化不受用不透性胆碱离子替代Na+的影响。它同样不受K+通道阻滞剂的影响，也不受用短杆菌肽（一种单价阳离子成孔剂）消耗K+化学浓度梯度的影响。相比之下，低细胞外Ca2+完全消除了去极化反应，100 nM的尼莫地平显著降低了MTX诱导的膜去极化。这些结果表明，MTX对去极化的主要作用是通过L-VDCC的Ca2+内流。综上所述，我们的结果表明，MTX增强PRL释放仅通过激活GH4C1细胞中的L型VDCC实现。我们认为，MTX可能通过与VDCC复合物的一个成分发生变构相互作用诱导初始的缓慢钙电导，进而启动一个涉及钙依赖性膜去极化和钙通道电压依赖性激活的正反馈机制。
