本文論述了細胞外鈣調素對花粉細胞內鈣離子的影響及其信號傳遞途徑的研究。作者： 中國農業大學的尚忠林著。
基本介紹
- 中文名：細胞外鈣調素對花粉細胞內鈣離子的影響及其信號傳遞途徑的研究
- 論文作者：尚忠林著
- 學位級別：d 2001n
- 導師 ：孫大業,王學臣教授指導
- 學位授予單位：中國農業大學
中文摘要,外文摘要,
中文摘要
以川百合花粉為材料，建立了改進的低溫裝載方法，在完整的花粉粒中裝載了鈣離子螢光指示劑fluo-3，利用雷射共聚焦顯微技術研究了細胞外鈣調素對花粉細胞內游離鈣離子濃度的影響，並對其信號轉導途徑進行了探索。結果如下：
在完整花粉粒細胞內裝載酯化形式的螢光指示劑時，克服細胞壁中的非特異性脂酶是保證成功裝載的關鍵。在常溫條件下裝載鈣離子螢光指示劑成功率比較低、效果不穩定，而利用低溫裝載的方法則可以通過抑制細胞壁中的脂酶收到良好的裝載效果。
外加純化鈣調素可以促進花粉細胞內游離鈣離子水平提高，以10〓mol/L的鈣調素作用效果最強，低於或超過這一濃度，鈣調素的作用效果均有所降低；說明外源純化鈣調素可以作為一種信號，刺激鈣離子進入花粉細胞質，作為第二信使發揮作用。不能透過細胞膜的鈣調素抗血清和大分子鈣調素拮抗劑W〓-agarose處理後細胞內鈣離子水平降低，免疫前血清對花粉細胞內鈣離子濃度沒有明顯影響；進一步支持了上述結果，並且表明內源細胞外鈣調素在維持和提高細胞內鈣離子濃度方面發揮作用。
花粉細胞質膜上異三聚體G蛋白的活性影響花粉細胞內鈣離子濃度。G蛋白的激活劑霍亂毒素（CTX）處理後，花粉細胞內鈣離子濃度升高；而其抑制劑百日咳毒素（PTX）處理則使細胞內鈣離子水平降低。在G蛋白活性受到PTX的抑制後，鈣調素處理不能對花粉細胞內鈣離子濃度產生明顯影響；而鈣調素的大分子拮抗劑處理後，再加入CTX處理，細胞內鈣離子濃度迅速升高，表明細胞外鈣調素的拮抗劑對CTX的作用效果沒有影響。上述結果表明G蛋白在細胞外鈣調素促進細胞內鈣離子水平升高的過程中發揮重要作用，而且在信號轉導過程中位於鈣調素的下游。
細胞質膜上鈣離子通道的活性影響細胞內鈣離子水平，經電壓門控鈣離子通道抑制劑異搏定（verapamil）處理後，細胞內鈣離子水平降低，而另一種鈣離子通道抑制劑尼群地平（nifedipine）對花粉細胞內鈣離子水平沒有影響。表明花粉細胞質膜或細胞內膜上對異搏定敏感、對尼群地平不敏感的鈣離子通道參與正常情況下花粉細胞鈣離子內流。異搏定處理後再加入CTX，細胞內鈣離子水平升高的幅度相當於單獨用CTX處理的細胞；而異搏定處理後再加入外源鈣調素，細胞內鈣離子水平只有小幅度回升。表明細胞質膜上鈣離子通道可能作為鈣調素激活的G蛋白的下游效應器，在鈣調素促進細胞內鈣離子水平過程中發揮重要作用。但異搏定並沒有完全抑制鈣調素促進的鈣離子升高，表明除了鈣通道之外，另外的鈣離子內流途徑也可能參與了鈣離子內流。
利用肝素處理後沒有發現正常萌發花粉細胞內鈣離子水平明顯變化，有可能在沒有外來刺激的條件下，細胞內的肌醇磷脂信號途徑的活性很低，達不到可檢測水平。肝素處理後再進行CTX處理，細胞內鈣離子亦沒有明顯變化，說明CTX主要通過肌醇磷脂信號途徑起作用。肝素處理後再加入外源鈣調素，細胞內鈣離子濃度升高，但是升高幅度明顯低於單獨鈣調素處理。說明細胞外鈣調素刺激產生的[Ca〓]i升高與CTX不同，除了肌醇磷脂信號系統外，還有其他途徑（如異搏定依賴的鈣離子通道）參與了細胞外鈣調素對細胞內鈣離子影響的信號傳遞過程。
在完整花粉粒細胞內裝載酯化形式的螢光指示劑時，克服細胞壁中的非特異性脂酶是保證成功裝載的關鍵。在常溫條件下裝載鈣離子螢光指示劑成功率比較低、效果不穩定，而利用低溫裝載的方法則可以通過抑制細胞壁中的脂酶收到良好的裝載效果。
外加純化鈣調素可以促進花粉細胞內游離鈣離子水平提高，以10〓mol/L的鈣調素作用效果最強，低於或超過這一濃度，鈣調素的作用效果均有所降低；說明外源純化鈣調素可以作為一種信號，刺激鈣離子進入花粉細胞質，作為第二信使發揮作用。不能透過細胞膜的鈣調素抗血清和大分子鈣調素拮抗劑W〓-agarose處理後細胞內鈣離子水平降低，免疫前血清對花粉細胞內鈣離子濃度沒有明顯影響；進一步支持了上述結果，並且表明內源細胞外鈣調素在維持和提高細胞內鈣離子濃度方面發揮作用。
花粉細胞質膜上異三聚體G蛋白的活性影響花粉細胞內鈣離子濃度。G蛋白的激活劑霍亂毒素（CTX）處理後，花粉細胞內鈣離子濃度升高；而其抑制劑百日咳毒素（PTX）處理則使細胞內鈣離子水平降低。在G蛋白活性受到PTX的抑制後，鈣調素處理不能對花粉細胞內鈣離子濃度產生明顯影響；而鈣調素的大分子拮抗劑處理後，再加入CTX處理，細胞內鈣離子濃度迅速升高，表明細胞外鈣調素的拮抗劑對CTX的作用效果沒有影響。上述結果表明G蛋白在細胞外鈣調素促進細胞內鈣離子水平升高的過程中發揮重要作用，而且在信號轉導過程中位於鈣調素的下游。
細胞質膜上鈣離子通道的活性影響細胞內鈣離子水平，經電壓門控鈣離子通道抑制劑異搏定（verapamil）處理後，細胞內鈣離子水平降低，而另一種鈣離子通道抑制劑尼群地平（nifedipine）對花粉細胞內鈣離子水平沒有影響。表明花粉細胞質膜或細胞內膜上對異搏定敏感、對尼群地平不敏感的鈣離子通道參與正常情況下花粉細胞鈣離子內流。異搏定處理後再加入CTX，細胞內鈣離子水平升高的幅度相當於單獨用CTX處理的細胞；而異搏定處理後再加入外源鈣調素，細胞內鈣離子水平只有小幅度回升。表明細胞質膜上鈣離子通道可能作為鈣調素激活的G蛋白的下游效應器，在鈣調素促進細胞內鈣離子水平過程中發揮重要作用。但異搏定並沒有完全抑制鈣調素促進的鈣離子升高，表明除了鈣通道之外，另外的鈣離子內流途徑也可能參與了鈣離子內流。
利用肝素處理後沒有發現正常萌發花粉細胞內鈣離子水平明顯變化，有可能在沒有外來刺激的條件下，細胞內的肌醇磷脂信號途徑的活性很低，達不到可檢測水平。肝素處理後再進行CTX處理，細胞內鈣離子亦沒有明顯變化，說明CTX主要通過肌醇磷脂信號途徑起作用。肝素處理後再加入外源鈣調素，細胞內鈣離子濃度升高，但是升高幅度明顯低於單獨鈣調素處理。說明細胞外鈣調素刺激產生的[Ca〓]i升高與CTX不同，除了肌醇磷脂信號系統外，還有其他途徑（如異搏定依賴的鈣離子通道）參與了細胞外鈣調素對細胞內鈣離子影響的信號傳遞過程。
外文摘要
ABSTRACT
In this paper, lilium daviddi pollen as material, the calcium sensitive fluorescence indicator fluo-3 AM was loaded into intact pollen grain cells, confocal laser scanning microscopy was used as main method, the effect of extracellular calmodulin on cytosolic calcium concentration and its signal transduction pathway were investigated. The result is as below:
When cytosolic calcium is being measured with acetyl methyl ester of calcium sensi...>> 詳細
In this paper, lilium daviddi pollen as material, the calcium sensitive fluorescence indicator fluo-3 AM was loaded into intact pollen grain cells, confocal laser scanning microscopy was used as main method, the effect of extracellular calmodulin on cytosolic calcium concentration and its signal transduction pathway were investigated. The result is as below:
When cytosolic calcium is being measured with acetyl methyl ester of calcium sensi...>> 詳細
ABSTRACT
In this paper, lilium daviddi pollen as material, the calcium sensitive fluorescence indicator fluo-3 AM was loaded into intact pollen grain cells, confocal laser scanning microscopy was used as main method, the effect of extracellular calmodulin on cytosolic calcium concentration and its signal transduction pathway were investigated. The result is as below:
When cytosolic calcium is being measured with acetyl methyl ester of calcium sensitive fluorescence indicator, eliminating the effect of extracellular lipases is the critical step for successful dye loading. Loading indicator at room temperature was unsuccessful, but low temperature may inhibit the extracellular lipases, indicators can be loaded successfully.
Exogenous purified calmodulin can elevate intracellular calcium ion concentration, the optimum concentration of CaM is 10〓mol/L. The effect of higher or lower concentration CaM all reduced. The result show that as a signal, purified exogenous CaM can stimulate cytosolic Ca〓, then Ca〓 may modulate metabolism in cytoplasm as a secondary messager. Cell membrane nonpermeable inhibitor of calmodulin -W〓-agarose- and the anti-serum of calmodulin can decrease the cytosolic calcium level, the results show that the endogeneous extracellular calmodulin may play an important role in maintaining and accelerating the cytosolic calcium level in pollen grain cell.
The activity of heterotrimeric G protein in cell membrane affect the cytosolic calcium. After treated by CTX, activator of heterotrimeric G protein, the cytosolic calcium elevated markedly. In pollen cells treated with PTX, the inhibitor of heterotrimeric G protein, cytosolic calcium decreased. When the pollen cells which pre-treated with PTX were treated by exogenous calmodulin, calmodulin can not effect cytosolic calcium level. In pollen cells which had been treated by anti-serum of CaM, CTX can elevate the cytosolic Ca〓 markedly. The result indicate that heterotrimeric G protein play an important role in the effect of exogenous calmodulin on cytosolic calcium, in this signal transduction chain, heterotrimeric G protein locate at the downstream of extracellular calmodulin.
The activity of calcium channels in plasma membrane affect the cytosolic calcium, verapamil-an inhibitor of voltage-dependent calcium channels in plasma membrane-treatment can decrease the cytosolic calcium, but nifedipine had no effect. The result indicate that the calcium channel which sensitive to verapamil but insensitive to niedipine participate in maintaining calcium influx into cytoplasm of pollen cells under normal condition. The pollen cells pre-treated with verapamil were treated with CTX or CaM consequently, the cytosolic calcium elevated. The elevation amplitude in pollen cells treated by CTX is just the same as singly CTX treatment, but in pollen cells which treated by CaM, the elevation amplitude is lower than cells which treated with CaM singly. It is deduced that the calcium channel may be as the downstream effector of heterotrimeric G protein which modulated by extracellular calmodulin, play an important role in the effect of extracellular calmodulin on cytosolic calcium in pollen cells. At same time, the effect of CaM or CTX were not totally eliminated by verapamil, there may be other calcium influx pathway participate in the effect of extracellular calmodulin and CTX on cytosolic calcium in pollen cells.
When pollen cells were treated with heparin, no obvious change of cytosolic calcium was found, it is deduced that in pollen cells without treatment, the activity of lipositol signal transduction pathway is low and cannot be detected. CTX had no effect on cytosolic calcium concentration in pollen cells pretreaterd with heparin, the result show that lipositol signal transduction pathway participate in the effect of CTX on cytosolic calcium in pollen cells, maybe a main pathway. Pollen cells pre-treated by heparin were treated by CaM, cytosolic calcium elevated, but the elevation amplitude was lower than single CaM treatment. The lipositol signal transduction system maybe participate in the effect of extracellular calmodulin on cytosolic calcium in pollen cells. But the effect of CaM was not totally eliminated by heparin, there may be other calcium influx pathway participate in the effect of extracellular calmodulin on cytosolic calcium in pollen cells.
In this paper, lilium daviddi pollen as material, the calcium sensitive fluorescence indicator fluo-3 AM was loaded into intact pollen grain cells, confocal laser scanning microscopy was used as main method, the effect of extracellular calmodulin on cytosolic calcium concentration and its signal transduction pathway were investigated. The result is as below:
When cytosolic calcium is being measured with acetyl methyl ester of calcium sensitive fluorescence indicator, eliminating the effect of extracellular lipases is the critical step for successful dye loading. Loading indicator at room temperature was unsuccessful, but low temperature may inhibit the extracellular lipases, indicators can be loaded successfully.
Exogenous purified calmodulin can elevate intracellular calcium ion concentration, the optimum concentration of CaM is 10〓mol/L. The effect of higher or lower concentration CaM all reduced. The result show that as a signal, purified exogenous CaM can stimulate cytosolic Ca〓, then Ca〓 may modulate metabolism in cytoplasm as a secondary messager. Cell membrane nonpermeable inhibitor of calmodulin -W〓-agarose- and the anti-serum of calmodulin can decrease the cytosolic calcium level, the results show that the endogeneous extracellular calmodulin may play an important role in maintaining and accelerating the cytosolic calcium level in pollen grain cell.
The activity of heterotrimeric G protein in cell membrane affect the cytosolic calcium. After treated by CTX, activator of heterotrimeric G protein, the cytosolic calcium elevated markedly. In pollen cells treated with PTX, the inhibitor of heterotrimeric G protein, cytosolic calcium decreased. When the pollen cells which pre-treated with PTX were treated by exogenous calmodulin, calmodulin can not effect cytosolic calcium level. In pollen cells which had been treated by anti-serum of CaM, CTX can elevate the cytosolic Ca〓 markedly. The result indicate that heterotrimeric G protein play an important role in the effect of exogenous calmodulin on cytosolic calcium, in this signal transduction chain, heterotrimeric G protein locate at the downstream of extracellular calmodulin.
The activity of calcium channels in plasma membrane affect the cytosolic calcium, verapamil-an inhibitor of voltage-dependent calcium channels in plasma membrane-treatment can decrease the cytosolic calcium, but nifedipine had no effect. The result indicate that the calcium channel which sensitive to verapamil but insensitive to niedipine participate in maintaining calcium influx into cytoplasm of pollen cells under normal condition. The pollen cells pre-treated with verapamil were treated with CTX or CaM consequently, the cytosolic calcium elevated. The elevation amplitude in pollen cells treated by CTX is just the same as singly CTX treatment, but in pollen cells which treated by CaM, the elevation amplitude is lower than cells which treated with CaM singly. It is deduced that the calcium channel may be as the downstream effector of heterotrimeric G protein which modulated by extracellular calmodulin, play an important role in the effect of extracellular calmodulin on cytosolic calcium in pollen cells. At same time, the effect of CaM or CTX were not totally eliminated by verapamil, there may be other calcium influx pathway participate in the effect of extracellular calmodulin and CTX on cytosolic calcium in pollen cells.
When pollen cells were treated with heparin, no obvious change of cytosolic calcium was found, it is deduced that in pollen cells without treatment, the activity of lipositol signal transduction pathway is low and cannot be detected. CTX had no effect on cytosolic calcium concentration in pollen cells pretreaterd with heparin, the result show that lipositol signal transduction pathway participate in the effect of CTX on cytosolic calcium in pollen cells, maybe a main pathway. Pollen cells pre-treated by heparin were treated by CaM, cytosolic calcium elevated, but the elevation amplitude was lower than single CaM treatment. The lipositol signal transduction system maybe participate in the effect of extracellular calmodulin on cytosolic calcium in pollen cells. But the effect of CaM was not totally eliminated by heparin, there may be other calcium influx pathway participate in the effect of extracellular calmodulin on cytosolic calcium in pollen cells.
