inositol-1-4-5-trisphosphate and Retinal-Degeneration

To investigate the effect of carbachol on the phagocytosis of photoreceptor outer segments (OS) in cultures of normal Long-Evans and dystrophic Royal College of Surgeons (RCS) rat retinal pigment epithelial (RPE) cells.. Retinal pigment epithelial cells from normal and RCS rats were grown in tissue culture. On reaching confluence, they were presented with OS suspended in Krebs-Henseleit buffer in the presence or absence of carbachol and LiCl. The number of bound and ingested OS was quantitated using double immunofluorescence staining.. LiCl inhibited the ingestion of OS by more than 90% but had no effect on the binding of OS by Long-Evans RPE cells. The addition of carbachol further reduced OS ingestion. Carbachol alone decreased OS ingestion by normal RPE cells by 30% but had no effect on OS binding. The effect of LiCl and carbachol on RCS RPE cells was similar to their effect on normal RPE cells.. Carbachol does not increase OS phagocytosis in normal or RCS rat RPE cells. The phagocytic defect in RCS rat RPE cannot be reversed or overcome by stimulation of the IP3 pathway by carbachol. LiCl strongly inhibits the ingestion of OS by normal and by RCS RPE cells, and this effect is enhanced by carbachol.

Topics: Animals; Carbachol; Cells, Cultured; Inositol 1,4,5-Trisphosphate; Lithium Chloride; Phagocytosis; Pigment Epithelium of Eye; Rats; Rats, Mutant Strains; Retinal Degeneration; Rod Cell Outer Segment

Using the whole-cell configuration of the patch-clamp technique, we studied the conditions necessary for the activation of Cl--currents in retinal pigment epithelial (RPE) cells from rats with retinal dystrophy (RCS) and nondystrophic control rats. In RPE cells from both rat strains, intracellular application of 10 microM inositol-1, 4,5-triphosphate (IP3) via the patch pipette led to a sustained activation of voltage-dependent Cl- currents, blockable by 1 mm 4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS). IP3 activated Cl- currents in the presence of a high concentration of the calcium chelator BAPTA (10 mM) in the pipette solution, but failed to do so when extracellular calcium was removed. Intracellular application of 10(-5)M Ca2+ via the patch pipette also led to a transient activation of Cl- currents. When the cells were preincubated in a bath solution containing thapsigargin (1 microM) for 5 min before breaking into the whole-cell configuration, IP3 failed to activate voltage-dependent currents. Thus, IP3 led to release of Ca2+ from cytosolic calcium stores. This in turn activated an influx of extracellular calcium into the submembranal space by a mechanism as yet unknown, leading to an activation of calcium-dependent chloride currents. In RPE cells from RCS rats, which show an increased membrane conductance for calcium compared to normal rats, we observed an accelerated speed of Cl--current activation induced by IP3 which could be reduced by nifedipine (1 microM). Thus, the increased membrane conductance to calcium in RPE cells from RCS rats changes the response of the cell to the second messenger IP3.

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; Animals; Calcium; Chelating Agents; Chloride Channels; Chlorides; Egtazic Acid; Electric Stimulation; Inositol 1,4,5-Trisphosphate; Intracellular Fluid; Microinjections; Patch-Clamp Techniques; Pigment Epithelium of Eye; Rats; Rats, Mutant Strains; Retinal Degeneration; Second Messenger Systems

To measure outer segment phagocytosis in cultures of Royal College of Surgeons (RCS) rat retinal pigment epithelium (RPE) that have been treated with carbachol. Carbachol treatment of RCS RPE results in an increase in the second messenger inositol triphosphate, which mimics that observed in normal RPE after interaction with rod outer segments (ROS).. Cultures of RCS RPE were phagocytically challenged with isolated rat ROS for 2 hours. Carbachol (1 mM) was added to some cultures to stimulate inositol triphosphate synthesis, and incubation continued for 30 minutes at 37 degrees C. Inositol triphosphate concentration was measured by radioreceptor assay. Bound and ingested outer segments were quantified by double immunofluorescent staining. Ingestion of outer segment membranes was confirmed by electron microscopy and immunogold staining.. Carbachol treatment was associated with a rapid and temporary increase in inositol triphosphate levels. Royal College of Surgeons rat RPE phagocytically challenged with outer segments and treated with carbachol showed significantly higher ingestion (34%) compared to untreated RCS RPE (9%) (P < 0.05).. Exposure of cultured RCS RPE to carbachol increases the intracellular concentration of inositol triphosphate and enhances phagocytosis of bound ROS. These results support the hypothesis that the phagocytic defect in RCS RPE is related to an abnormality in the generation of inositol triphosphate as a second messenger after outer segment recognition and binding.

Topics: Animals; Carbachol; Cells, Cultured; Fluorescent Antibody Technique; Immunohistochemistry; Inositol 1,4,5-Trisphosphate; Phagocytosis; Pigment Epithelium of Eye; Radioligand Assay; Rats; Rats, Mutant Strains; Retinal Degeneration; Rhodopsin; Rod Cell Outer Segment

To evaluate the generation of the second messenger molecule inositol triphosphate (IP3) in response to phagocytic challenge in cultures of retinal pigment epithelium (RPE) prepared from normal Long Evans or dystrophic Royal College of Surgeons (RCS) rats.. RPE cultures were phagocytically challenged with isolated rat rod outer segments (ROS) or polystyrene latex spheres (PSL). Carbachol was added to some cultures as a positive control to stimulate the IP3 pathway. Inositol triphosphate levels in RPE cells were analyzed by high-pressure liquid chromatography and radioreceptor assay. Incorporation of 3H-myoinositol into phosphatidylinositol lipids was analyzed by thin layer chromatography and autoradiography.. Long Evans RPE phagocytically challenged with ROS or exposed to carbachol showed a significant increase in IP3 levels compared to unchallenged control cultures or cultures phagocytically challenged with PSL. RCS RPE cells did not show an increase in IP3 levels upon phagocytic challenge with ROS or PSL. Carbachol treatment of RCS RPE produced an increase in IP3 levels, demonstrating that the components of the IP3 pathway are present and the pathway can be activated. Phagocytic challenge of Long Evans RPE with ROS was associated with a decrease in the IP3 precursor, phosphatidylinositol bisphosphate (PIP2). No decrease in PIP2 was observed in RCS RPE incubated with ROS.. These results show that phagocytic challenge of Long Evans RPE with ROS is associated with PIP2 hydrolysis and subsequent generation of IP3 as a second messenger. In RCS RPE, ROS stimulation of the IP3 pathway is absent.

Topics: Animals; Carbachol; Cells, Cultured; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Inositol 1,4,5-Trisphosphate; Microspheres; Phagocytosis; Phosphotransferases (Alcohol Group Acceptor); Pigment Epithelium of Eye; Radioligand Assay; Rats; Rats, Mutant Strains; Retinal Degeneration; Rod Cell Outer Segment