Effect of serotonin on ciliary beating and intracellular calcium concentration in identified populations of embryonic ciliary cells
Effect of serotonin on ciliary beating and intracellular calcium concentration in identified populations of embryonic ciliary cells
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DOI：
10.1242/jeb.00924
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发表时间：
2004-03-01
影响因子：
2.8
通讯作者：
Goldberg, JI
中科院分区：
文献类型：
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作者：
Doran, SA;Koss, R;Goldberg, JI
池塘蜗牛Helisoma trivolvis的胚胎在发育早期的胚胎表面表达三种已知的纤毛细胞亚型：脚细胞、背外侧细胞和散在的单个纤毛细胞(SSCCs)。足部和背侧纤毛细胞由一对5-羟色胺能感觉运动神经元支配，负责产生最早的全动物行为，即在卵囊内旋转。以往对未知睫状细胞的细胞培养研究表明，5-羟色胺(5-羟色胺；5-羟色胺)可显著增加大部分睫状体细胞的纤毛搏动频率(CBF)。钙离子内流和一种独特的蛋白激酶C(PKC)亚型都参与了纤毛兴奋性反应的信号转导通路。本研究的目的是描述三种已知的浅层睫状细胞群之间的解剖学和生理学差异。足细胞和背外侧纤毛细胞具有共同的结构特征，包括扁平的形态、致密的纤毛和侧方副纤毛根。与之相比，SSCCs形态呈立方体，纤毛数量减少，纤毛长度增加，无侧方附根。在含有未知纤毛细胞的培养中，钙/钙调蛋白依赖的酶抑制剂卡米达唑铵(2mumol 1(-1))可阻断5-羟色胺(100mumol 1(-1))对CBF的刺激作用。此外，50%的未鉴定培养细胞对5-羟色胺(100mumol 1(-1))有反应，[Ca~(2+)](I)升高。为了便于个体群体的功能分析，我们开发了一种方法来培养已鉴定的纤毛亚型，并表征它们对5-羟色胺的纤毛和钙反应。在含有足或背外侧睫状体细胞的培养中，5-羟色胺(100mumol 1(-1))可使所有细胞的CBF迅速增加，[Ca~(2+)]i增加较慢。100mumol 1(-1)5-羟色胺对SSCCs的CBF和[Ca~(2+)](I)无明显影响。最近从Helisoma中克隆的两个可能的5-HT1受体的免疫组织化学显示，脚细胞和背外侧睫毛细胞持续表达5-HT1Hel蛋白。5-HT7Hel免疫反应仅见于足底和睫状背外侧细胞的一小部分。邻近SSCCs的细胞表达5-HT1Hel和5-HT7Hel免疫反应，而纤毛细胞本身不表达。这些结果表明，足细胞和背外侧纤毛细胞是一种同质的生理亚型，这将有助于阐明5-羟色胺诱导纤毛兴奋的信号转导机制。
Embryos of the pond snail Helisoma trivolvis express three known subtypes of ciliary cells on the surface of the embryo early in development: pedal, dorsolateral and scattered single ciliary cells (SSCCs). The pedal and dorsolateral ciliary cells are innervated by a pair of serotonergic sensory-motor neurons and are responsible for generating the earliest whole-animal behaviour, rotation within the egg capsule. Previous cell culture studies on unidentified ciliary cells revealed that serotonin (5-hydroxytryptamine; 5-HT) produces a significant increase in the ciliary beat frequency (CBF) in a large proportion of ciliary cells. Both Ca2+ influx and a unique isoform of protein kinase C (PKC) were implicated in the signal transduction pathway underlying the cilio-excitatory response to 5-HT. The goal of the present study was to characterize the anatomical and physiological differences between the three known populations of superficial ciliary cells. The pedal and dorsolateral ciliary cells shared common structural characteristics, including flat morphology, dense cilia and lateral accessory ciliary rootlets. By contrast, the SSCCs had a cuboidal morphology, reduced number of cilia, increased ciliary length and absence of lateral accessory rootlets. In cultures containing unidentified ciliary cells, the calcium/ calmodulin-dependent enzyme inhibitor calmidazolium (2 mumol 1(-1)) blocked the stimulatory effect of 5-HT (100 mumol 1(-1)) on CBF. In addition, 50 % of unidentified cultured cells responded to 5-HT (100 mumol 1(-1)) with an increase in [Ca2+](i). To facilitate the functional analyses of the individual populations, we developed a method to culture identified ciliary subtypes and characterized their ciliary and calcium responses to 5-HT. In cultures containing either pedal or dorsolateral ciliary cells, 5-HT (100 mumol 1(-1)) produced a rapid increase in CBF and a slower increase in [Ca2+]i in all cells examined. By contrast, the CBF and [Ca2+](i) of SSCCs were not affected by 100 mumol 1(-1) 5-HT. Immunohistochemistry for two putative 5-HT receptors recently cloned from Helisoma revealed that pedal and dorsolateral ciliary cells consistently express the 5-HT1Hel protein. Intense 5-HT7Hel immunoreactivity was observed in only a subset of pedal and dorsolateral ciliary cells. Cells neighboring the SSCCs, but not the ciliary cells themselves, expressed 5-HT1Hel and 5-HT7Hel immunoreactivity. These data suggest that the pedal and dorsolateral ciliary cells, but not the SSCCs are a homogeneous physiological subtype that will be useful for elucidating the signal transduction mechanisms underlying 5-HT induced cilio-excitation.