Klauke N, Plattner H
Faculty of Biology, University of Konstanz, P.O. Box 5560, D-78434 Konstanz, Germany.
J Membr Biol. 1998 Jan 1;161(1):65-81. doi: 10.1007/s002329900315.
Caffeine causes a [Ca2+]i increase in the cortex of Paramecium cells, followed by spillover with considerable attenuation, into central cell regions. From [Ca2+]resti approximately 50 to 80 nm, [Ca2+]acti rises within </=3 sec to 500 (trichocyst-free strain tl) or 220 nm (nondischarge strain nd9-28 degrees C) in the cortex. Rapid confocal analysis of wildtype cells (7S) showed only a 2-fold cortical increase within 2 sec, accompanied by trichocyst exocytosis and a central Ca2+ spread during the subsequent >/=2 sec. Chelation of Ca2+o considerably attenuated [Ca2+]i increase. Therefore, caffeine may primarily mobilize cortical Ca2+ pools, superimposed by Ca2+ influx and spillover (particularly in tl cells with empty trichocyst docking sites). In nd cells, caffeine caused trichocyst contents to decondense internally (Ca2+-dependent stretching, normally occurring only after membrane fusion). With 7S cells this usually occurred only to a small extent, but with increasing frequency as [Ca2+]i signals were reduced by [Ca2+]o chelation. In this case, quenched-flow and ultrathin section or freeze-fracture analysis revealed dispersal of membrane components (without fusion) subsequent to internal contents decondensation, opposite to normal membrane fusion when a full [Ca2+]i signal was generated by caffeine stimulation (with Ca2+i and Ca2+o available). We conclude the following. (i) Caffeine can mobilize Ca2+ from cortical stores independent of the presence of Ca2+o. (ii) To yield adequate signals for normal exocytosis, Ca2+ release and Ca2+ influx both have to occur during caffeine stimulation. (iii) Insufficient [Ca2+]i increase entails caffeine-mediated access of Ca2+ to the secretory contents, thus causing their decondensation before membrane fusion can occur. (iv) Trichocyst decondensation in turn gives a signal for an unusual dissociation of docking/fusion components at the cell membrane. These observations imply different threshold [Ca2+]i-values for membrane fusion and contents discharge.
咖啡因会使草履虫细胞皮层中的[Ca2+]i升高，随后会有相当程度的衰减，并扩散到细胞中央区域。从约50至80纳米的[Ca2+]resti开始，[Ca2+]acti在≤3秒内升至500纳米（无刺丝泡菌株tl）或220纳米（非放电菌株nd9 - 28℃）的皮层水平。对野生型细胞（7S）的快速共聚焦分析显示，在2秒内皮层仅增加2倍，同时伴有刺丝泡胞吐作用，且在随后≥2秒内Ca2+向中央扩散。细胞外Ca2+的螯合显著减弱了[Ca2+]i的升高。因此，咖啡因可能主要动员皮层Ca2+库，同时叠加有Ca2+内流和扩散（特别是在刺丝泡对接位点为空的tl细胞中）。在nd细胞中，咖啡因导致刺丝泡内容物在内部解聚（Ca2+依赖性伸展，通常仅在膜融合后发生）。对于7S细胞，这种情况通常只在小范围内发生，但随着细胞外Ca2+螯合使[Ca2+]i信号减弱，发生频率增加。在这种情况下，淬灭流动以及超薄切片或冷冻断裂分析显示，在内部内容物解聚后膜成分发生分散（无融合），这与咖啡因刺激产生完整的[Ca2+]i信号（细胞内Ca2+和细胞外Ca2+均存在）时正常的膜融合情况相反。我们得出以下结论。（i）咖啡因可独立于细胞外Ca2+的存在从皮层储存中动员Ca2+。（ii）为产生正常胞吐作用的足够信号，在咖啡因刺激期间Ca2+释放和Ca2+内流都必须发生。（iii）[Ca2+]i升高不足会导致咖啡因介导的Ca2+进入分泌内容物，从而在膜融合发生之前导致其解聚。（iv）刺丝泡解聚进而为细胞膜上对接/融合成分的异常解离发出信号。这些观察结果意味着膜融合和内容物释放具有不同的[Ca2+]i阈值。
