The role of phosphoinositides in vertebrate phototransduction mechanism.

受体介导的磷脂酰肌醇4,5-二磷酸（TPI）的快速水解被认为参与了质膜中一个重要的信息转导机制。包括我们在内的几个研究小组表明，无论无脊椎动物还是脊椎动物的眼睛，光刺激都可以在光感受器细胞中引起这种tpi反应。然而，tpi反应在光感受器细胞中的生理作用尚未明确。TPI的水解是由其特异性磷酸二酯酶（PDE）催化的。在本文中，我们报道了蛙杆外段（ROS）中tpi特异性PDE的基本特征。用EGTA-Ringers溶液(<10^（-9)> M <Ca^(2+)>）搅拌视网膜制备粗ROS悬液。通过测定在反应开始时加入的[<^(32)P>]- tpi释放的[<^(32)P>]-肌醇三磷酸（I < p3 >）的量来测定TPI-PDE活性。所有的操作都是在完全黑暗中进行的。得到以下结果：1) TPI-PDE <Ca^(2+)> -依赖。随着<Ca^(2+)>浓度的增加，活性急剧增加，从<10^(-8)>m增加到5 × <10^(-7) >m，并达到最大值（约为10^(-7)>m）。15 nmol /min/mg蛋白)。2)在<10^(-7)>、<Ca^(2+)>的存在下，TPI-PDE活性在光作用下提高到130%左右。然而，这种光依赖性增强需要5%的聚乙二醇（PEG）在孵育介质中。相反，在没有PEG的情况下，观察到光依赖性抑制（- 14%）。在这两种情况下，GTP < γ > S （100 <mu> M）对光介导的TPI-PDE活性变化没有影响。如上所述，在PEG存在下证实了光介导的TPI击穿。然而，TPI-PDE的光依赖性抑制也被观察到。在ROS中，TPI-PDE似乎受到光敏但相互调节机制的控制。

Receptor-mediated rapid hydrolysis of phosphatidylinositol-4,5-bisphosphate (TPI) is believed to be involved in an important information transduction mechanism in the plasma membrane. Several research groups including us showed that light stimulus can evoke such a TPI-response in photoreceptor cells irrespective of invertebrate or vertebrate eyes. However, the physiological role of the TPI-response in photoreceptor cells has not been clarified yet.The hydrolysis of TPI is catalyzed by its specific phospho- diesterase (PDE). In the present paper, we report studies on the fundamental characteristics of the TPI-specific PDE in frog rod outer segments (ROS).Crude ROS suspension was prepared by agitating the retinas in EGTA-Ringers solution ( <10^(-9)> M <Ca^(2+)> ). TPI-PDE activity was assayed by measuring the amount of [ <^(32)P> ]- inositoltrisphosphate (I <P_3> ) released from [ <^(32)P> ]-TPI which was added at the reaction start. All manipulations were done in complete darkness.Following results were obtained. 1) The TPI-PDE was <Ca^(2+)> - dependent. The activity steeply increased with the increase in <Ca^(2+)> concentration from <10^(-8)> M to 5 x <10^(-7)> M and reached to maximum level (approx. 15 nmole/min/mg protein). 2) The TPI-PDE activity was enhanced to about 130 % by light in the presence of <10^(-7)> M <Ca^(2+)> . However, this light-dependent enhancement required 5 % polyethylenglycol (PEG) in the incubation medium. In the absence of PEG, on the contrary, a light-dependent inhibition (-14 %) was observed. In both cases, GTP <gamma> S (100 <mu> M) revealed no effect on the light-mediated TPI-PDE activity changes.As mentioned above, the light-mediated TPI breakdown was confirmed in the presence of PEG. However, the light-dependent inhibition of the TPI-PDE was also observed. Light-sensitive but reciprocal regulation mechanisms seem to control the TPI-PDE in the ROS.