Tovey S C, Godfrey R E, Hughes P J, Mezna M, Minchin S D, Mikoshiba K, Michelangeli F
School of Biochemistry, University of Birmingham, UK.
Cell Calcium. 1997 Apr;21(4):311-9. doi: 10.1016/s0143-4160(97)90119-6.
PCR analysis and immunoblotting with isoform specific antibodies was used to identify the presence of type I, II and III inositol 1,4,5-trisphosphate receptors (InsP3Rs) in rat testis. PCR analysis also revealed that rat testis express both forms of the S1 splice variant (S1+ and S1-), but only the S2- from of the S2 splice variant of the type I InsP3 receptor. PCR analysis was also used to identify InsP3R isoform expression at a cellular level using myoid, Sertoli and germ cells derived from the testis of Wistar rats. The extent of [3H]-InsP3 binding was found to be 9 times lower for testicular microsomes than for cerebellar microsomes, with a Bmax of 1.4 pmoles/mg protein compared to 12.5 pmoles/mg protein for cerebellar microsomes. The Kd for InsP3 binding to its receptor in testicular microsomes was 60 +/- 10 nM which was similar to that found for cerebellar microsomes (80 +/- 20 nM). InsP3-induced Ca2+ release (IICR) in testicular microsomes was found to have an EC50 (concentration which causes a half-maximal response) of 0.5 +/- 0.03 microM, also similar to that seen for cerebellar microsomes (0.3 microM). Maximal IICR occurred at about 20 microM InsP3, with up to 4% of total intracellular Ca2+ stores being mobilized as compared to between 10-30% for cerebellar microsomes. Time resolved IICR using stopped-flow spectrofluorimetry, showed the kinetics of IICR for this testis preparation to be monophasic with a maximum rate constant of 0.15 s-1 at 30 microM InsP3. The rate constants are 7 times slower than values for cerebellar microsomes under similar conditions (approximately 1 s-1) and taken together with the binding data support the proposal that the receptor density/Ca2+ store is approximately 8 times lower than seen in cerebellar microsomal vesicles. The pharmacological properties as assessed using heparin and InsP3 analogues also confirmed similar behaviour for testicular InsP3Rs and cerebellar InsP3Rs.
采用聚合酶链反应（PCR）分析以及使用亚型特异性抗体进行免疫印迹，以鉴定大鼠睾丸中I型、II型和III型肌醇1,4,5 -三磷酸受体（InsP3Rs）的存在情况。PCR分析还显示，大鼠睾丸表达S1剪接变体的两种形式（S1 +和S1 -），但仅表达I型InsP3受体S2剪接变体的S2 -形式。PCR分析还用于在细胞水平上鉴定源自Wistar大鼠睾丸的肌样细胞、支持细胞和生殖细胞中的InsP3R亚型表达。结果发现，睾丸微粒体的[3H]-InsP3结合程度比小脑微粒体低9倍，其Bmax为1.4皮摩尔/毫克蛋白, 而小脑微粒体的Bmax为12.5皮摩尔/毫克蛋白。InsP3与睾丸微粒体中其受体结合的解离常数（Kd）为60±10 nM，这与小脑微粒体中的情况相似（80±20 nM）。发现睾丸微粒体中InsP3诱导的Ca2 +释放（IICR）的半数有效浓度（EC50，即引起最大反应一半的浓度）为0.5±0.03 microM，这也与小脑微粒体中的情况相似（0.3 microM）。最大IICR发生在约20 microM InsP3时，与小脑微粒体中10 - 30%的情况相比，此时动员的细胞内Ca2 +储存总量高达4%。使用停流荧光光谱法进行时间分辨的IICR分析表明，该睾丸制剂的IICR动力学为单相，在30 microM InsP3时最大速率常数为0.15 s-1。在相似条件下，该速率常数比小脑微粒体的值慢7倍（约1 s-1），结合结合数据支持以下观点：受体密度/Ca2 +储存比小脑微粒体囊泡中低约8倍。使用肝素和InsP3类似物评估的药理学特性也证实了睾丸InsP3Rs和小脑InsP3Rs具有相似的行为。
