Phospholipase Cγ₁ in Bovine Rod Outer Segments: Immunolocalization and Light‐Dependent Binding to Membranes

Abstract

We have investigated the isozymes of a phosphoinositide‐specific phospholipase C (PLC) in bovine retina using several monoclonal antisera to PLCβ1, γ1, and δ1. Immunoblot analysis showed that all three isozymes were present in the retina. Immunocytochemical localization in frozen bovine retina sections showed that PLCγ1 was present in the photoreceptor cell layer, outer plexiform cell layer, inner plexiform cell layer, and ganglion cell layer. Immunoreaction within the photoreceptor cell layer was dependent on dark/light adaptation state of retinas. Immunoblot analysis of rod outer segments (ROS) with monoclonal or polyclonal antibodies to PLCγ1 showed the presence of an immunoreactive band of 140 kDa. ROS prepared from retinas light‐adapted in vitro had more PLCγ1 on immunoblots than ROS from dark‐adapted retinas. PLC enzyme activity in ROS from light‐adapted retinas was 69 and 46% higher than ROS from dark‐adapted retinas, when assayed in the presence and absence of ATP, respectively. This increase in enzyme activity was observed at [Ca2+]free between 0.32 and 100 µM. These results demonstrate the presence of PLCγ1 in bovine ROS and show that ROS prepared from light‐adapted retinas are enriched in this isozyme, suggesting that light may promote the binding of this isozyme to bleached ROS membranes.

Lipid second messengers and related enzymes in vertebrate rod outer segments

“…A neuroprotective role associated with PI3K pathway activation was suggested ( 212 ). As was previously mentioned (see above), LROS were found to have higher PLC ␥ 1 activity than DROS and a higher PLC ␥ 1 was associated with LROS ( 69 ). These results are in accordance with results from Ilincheta de Boschero and Giusto ( 74 ) who found that membranes obtained from DROS washed in dark with low ionic strength buffer (5Dm), show higher polyphosphoinositide [PI(4)P and PI(4,5)P 2 ] phosphorylation with respect to that found in membranes obtained from DROS exposed to light and washed in light with low ionic strength buffer (5Lm).…”

“…The activities of enzymes involved in ROS phospholipid turnover such as PLC ( 3,46,68,69 ), phospholipase A2 (PLA2) ( 70 ), phosphatidylethanolamine N-methyltransferase (PEMT) ( 71 ), diacylglycerol kinase (DAGK) (72)(73)(74)(75), lipid phosphate phosphatase (LPP) ( 76 ), diacylglycerol lipase (DAGL) ( 77,78 ), PI synthase ( 79 ), phosphoinositide-3-kinase (PI3K) ( 80,81 ), and PLD ( 82 ) have been reported to be modulated by light. Detailed studies on PI3K signaling are described by Rajala in the thematic review in this series on PI3K in the vertebrate retina.…”

“…Experimental conditions favoring PKC phosphorylation inhibit LPP in purifi ed ROS ( 78 ). Light stimulates DAG generation by PI(4,5)P 2 -PLC activity ( 69 ) and may thus activate ROS PKC. This could be at least one of the light-induced mechanisms involved in LPP modulation.…”

“…Vertebrate retinal photoreceptor cells have an active phosphoinositide metabolism and several steps in the PI cycle are stimulated by light ( 10,46,48,68,69,72,74,79,83,84 ). The two pathways that deplete the PI(4,5)P 2 normally present in membranes (PLC-and PI3K-mediated pathways) are lightactivated in vertebrate ROS, although the physiological consequences of their activities are not yet fully clear.…”

Light-dependent Association of Src with Photoreceptor Rod Outer Segment Membrane Proteinsin Vivo

“…Our data suggest that Src is a light-activated tyrosine kinase in photoreceptor cells and may, in part, be responsible for the increase in tyrosine phosphorylation in ROS membranes. Several putative Tyr(P) substrates have been identified in ROS, including phosphatidylinositol 3-kinase (28), SHP-2 (29), transducin ␣ (19), and PLC␥ 1 , which binds to bleached ROS membranes in vitro and translocates from the inner and outer segments of photoreceptors in response to light adaptation in vitro (30). The possible regulation of the former enzymes by light in vivo is currently under investigation.…”