The role of phosphoinositides in vertebrate phototransduction mechanism.

Research Project

Project/Area Number	60580214
Research Category	Grant-in-Aid for General Scientific Research (C)
Allocation Type	Single-year Grants
Research Field	分子遺伝学・分子生理学
Research Institution	Kobe University
Principal Investigator	AMAKAWA Taisaku Associate Professor Dept. Biology, College of General Education, Kobe Univ., 教養部, 助教授 (70031359)
Co-Investigator(Kenkyū-buntansha)	HAYASHI Fumio Assistant Dept. Biology, College of General Education, Kobe Univ., 教養部, 助手 (80093524)
Project Period (FY)	1985 – 1986
Project Status	Completed (Fiscal Year 1986)
Budget Amount *help	¥2,200,000 (Direct Cost: ¥2,200,000) Fiscal Year 1986: ¥600,000 (Direct Cost: ¥600,000) Fiscal Year 1985: ¥1,600,000 (Direct Cost: ¥1,600,000)
Keywords	Phototransduction / Phosphoinositides / Phospholipase C / Calcium / GTP-binding protein / Inositoltrisphosphate / Frog / イノシトール3リン酸
Research Abstract	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.