Trueta Citlali, Méndez Bruno, De-Miguel Francisco F
Departamento de Biofísica, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, D.F., México.
J Physiol. 2003 Mar 1;547(Pt 2):405-16. doi: 10.1113/jphysiol.2002.030684. Epub 2003 Jan 17.
We studied somatic exocytosis of serotonin and its mediation by L-type calcium (Ca2+) channels in cultured Retzius neurones of the leech. Exocytosis was induced by trains of impulses at different frequencies or by depolarisation with 40 mM potassium (K+), and was quantified by use of the fluorescent dye FM 1-43. Stimulation increased the membrane fluorescence and produced a pattern of FM 1-43 fluorescent spots of 1.28 +/- 0.01 microm in diameter, provided that Ca2+ was present in the bathing fluid. Individual spots lost their stain during depolarisation with 40 mM K+. Electron micrographs showed clusters of dense core vesicles, some of which were in contact with the cell membrane. Presynaptic structures with clear vesicles were absent from the soma. The number of fluorescent spots per soma, but not their diameter or their fluorescence intensity, depended on the frequency of stimulation. Trains at 1 Hz produced 19.5 +/- 5 spots per soma, 77.9 +/- 13.9 spots per soma were produced at 10 Hz and 91.5 +/- 16.9 spots per soma at 20 Hz. Staining patterns were similar for neurones in culture and in situ. In the presence of the L-type Ca2+ channel blocker nimodipine (10 microM), a 20 Hz train produced only 22.9 +/- 6.4 spots per soma, representing a 75 % reduction compared to control cells (P < 0.05). Subsequent incubation with 10 mM caffeine to induce Ca2+ release from intracellular stores increased the number of spots to 73.22 +/- 12.5. Blockers of N-, P-, Q- or invertebrate Ca2+ channels did not affect somatic exocytosis. Our results suggest that somatic exocytosis by neurones shares common mechanisms with excitable endocrine cells.
我们研究了水蛭培养的Retzius神经元中5-羟色胺的体细胞胞吐作用及其由L型钙（Ca2+）通道介导的过程。通过不同频率的脉冲串或用40 mM钾（K+）进行去极化来诱导胞吐作用，并使用荧光染料FM 1-43进行定量。只要浴液中存在Ca2+，刺激就会增加膜荧光并产生直径为1.28±0.01微米的FM 1-43荧光斑点模式。在用40 mM K+去极化期间，单个斑点会失去其染色。电子显微镜照片显示有密集核心囊泡簇，其中一些与细胞膜接触。胞体中不存在具有清亮囊泡的突触前结构。每个胞体的荧光斑点数量，而非其直径或荧光强度，取决于刺激频率。1 Hz的脉冲串每个胞体产生19.5±5个斑点，10 Hz时每个胞体产生77.9±13.9个斑点，20 Hz时每个胞体产生91.5±16.9个斑点。培养的神经元和原位神经元的染色模式相似。在存在L型Ca2+通道阻滞剂尼莫地平（10 microM）的情况下，20 Hz的脉冲串每个胞体仅产生22.9±6.4个斑点，与对照细胞相比减少了75%（P<0.05）。随后用10 mM咖啡因孵育以诱导细胞内钙库释放钙，使斑点数量增加到73.22±12.5。N-、P-、Q-或无脊椎动物Ca2+通道的阻滞剂不影响体细胞胞吐作用。我们的结果表明，神经元的体细胞胞吐作用与可兴奋内分泌细胞具有共同机制。
