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ABSTRACT. Light-mediated hydrolysis of phosphatidylinositol-4,5-bisphosphate (TPI) to 1,2-diacylglycerol and D-myo-inositol 1,4,5-triphosphate (IP3) has been reported in the visual photoreceptor cells. We have investigated the localization of the TPI antigenic sites in dark-and light-adapted rat retinas using rabbit anti TPI antibodies (Ab). Sprague-Dawley rats were dark-, or light-adapted, or were exposed to a light flash. The eyes were fixed immediately and the tissue sections stained with the rabbit anti TPI Ab. The peroxidase-antiperoxidase method was used to find the localization of the TPI antigenic site. Image analysis of the sections was performed to obtain optical density profiles of the stain. Dark-adapted retinas showed intense staining of the rod outer segment (OS) layer but much less staining of the rod inner segment layer. Compared with the OS of dark-adapted retinas, those of the flash-bleached retinas were stained much less. The OS of fully bleached retinas showed little or no staining. The anti TPI Ab-protein A-gold conjugate intensely stained disks from dark-adapted retina but those from bleached retina much less. Our results suggest that rapid hydrolysis of TPI in rat rod outer segments occurs in vivo in response to light.Increased phosphatidylinositol turnover in response to extracellular stimuli seems to be a general mechanism of signal transmission in various receptor systems (3,16). The hydrolysis of phosphatidylinositol-4,5-bisphosphate (TPI) gives rise to two intracellular effectors; 1,2-diacylglycerol which activates protein kinase C (17) and inositol 1,4,5-triphosphate (IP3) which causes calcium flux across the cell membrane (3).Light stimulates the rapid hydrolysis of TPI in vertebrate (8, 11) and invertebrate (5, 23) photoreceptors. TPI turnover also seems to function in Drosophila photoreceptors (26). Recent evidence indicates that IP3 mimics the effect of light in modulating the membrane potential of the photoreceptors. Intracellular injections of IP3 in the dark induce the depolarization of Limulus ventral photoreceptors reversibly

ABSTRACT. Immunocytochemical localization of phosphatidylinosito1-4,5-bisphosphate (PIP2) in the rat rod photoreceptor outer segments (OS) was investigated with rabbit antiPIP2 antibodies. The OS of the light-adapted rat eye showed little or no staining, whereas the OS of the dark-adapted eye were intensely stained for PIP2. The immunoreactivity of photoreceptor PIP2 in the eye exposed to a brief flash of light was markedly reduced. However, subsequent dark-adaptation of the flash-bleached eye resulted in a rapid recovery of PIP2 immunoreactivity; dark-adaptation for 5 min was sufficient for recovery to the fully dark-adapted level. In dark-adapted eyes exposed to graded light intensities, the PIP2 immunostaining varied with light levels and was correlated with unbleached rhodopsin concentrations. These results suggest that PIP2 in the rat photoreceptor cells is rapidly hydrolyzed upon light exposure and rapidly synthesized in the dark and that the decrease of PIP2 level is triggered by photic bleaching of rhodopsin.Light-stimulated turnover of polyphosphoinositides in vertebrate (5, 6, 10) and invertebrate (2, 13, 14) photoreceptor cells has been studied by several investigators. A key step in the agonist-elicited phosphoinositide turnover is the hydrolysis of phosphatidylinosito1-4,5-bisphosphate (PIP2) to two intracellular effectors : 1,2-diacylglycerol which activates protein kinase C (7) and inositol 1,4,5-trisphosphate (IP3) which mobilizes calcium (1). IP3 has been proposed to be an intracellular messenger for invertebrate photoreceptors (13).In our previous study, we presented immunocytochemical evidence suggesting that PIP2 in retinal rod outer segment membranes of dark-adapted rat eyes is quickly decreased upon exposure of the animal to light (3). A similar observation was made in cone-cell dominant chicken retina (8). The cellular level of PIP2 is determined by

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