The role of phosphoinositides in vertebrate phototransduction mechanism.

受体介导的磷脂酰肌醇-4，5-二磷酸（TPI）快速水解被认为参与质膜中重要的信息转导机制。包括我们在内的几个研究小组表明，光刺激可以在感光细胞中引起这样的TPI反应，无论是无脊椎动物还是脊椎动物的眼睛。TPI的水解是由其特异性磷酸二酯酶（PDE）催化的。本文研究了蛙视杆细胞外节（ROS）中TPI特异性PDE的基本特征。通过测定从反应开始时加入的[ &lt;^（32）P&gt; ]-TPI中释放的[&lt;^（32）P&gt;]-三磷酸肌醇（I）的量，测定TPI-PDE活性<P_3>。所有操作均在完全黑暗中进行，结果如下。1)TPI-PDE具有&lt;Ca^（2+）&gt;依赖性。随着&lt;Ca^（2+）&gt;浓度从&lt;10^（-8）&gt; M增加到5 x &lt;10^（-7）&gt; M，酶活性急剧增加，并达到最大值（约10 μ mol/L）。15 nmole/min/mg蛋白质）。2)在存在&lt;10^（-7）&gt; M &lt;Ca^（2+）&gt;的条件下，光照可使TPI-PDE活性提高约130%。然而，这种光依赖性增强需要在孵育培养基中加入5%的聚乙二醇（PEG）。相反，在不存在PEG的情况下，观察到光依赖性抑制（~ 14%）。在这两种情况下，GTP <gamma>S（100 <mu>M）显示对光介导的TPI-PDE活性变化无影响。如上所述，在PEG存在下证实了光介导的TPI分解。然而，还观察到TPI-PDE的光依赖性抑制。光敏感但相互调节机制似乎控制ROS中的TPI-PDE。

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.