all-trans-Retinal stimulates superoxide release and phospholipase C activity in neutrophils without significantly blocking protein kinase C.

Abstract

ABSTRACTall-trans-Retinal was previously shown to stimulate high levels of superoxide release by guinea pig neutrophils. When the cells, previously labeled with [3H]inositol, are treated with all-trans-retinal, they exhibit a decrease in the levels of [3H]inositol phospholipids and an increase in the accumulation of [3H]inositol phosphates. The maximal accumulation of inositol phosphates and the optimal rate of superoxide release occurred together at -7 min after stimulation. Retinoids are known to block the effects of phorbol esters on a wide variety of cells (6,15,16). However, we have recently reported that they stimulate high levels of O2 release by neutrophils (17). The mechanism of the stimulation by retinoids is of interest since this is a situation in which retinoids mimic, rather than inhibit, a cellular effect of phorbol esters. In this paper, we report effects of all-transretinal on phospholipase C and protein kinase C of neutrophils. (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) Ci/mmol; 1 Ci = 37 GBq) and [y-32P]ATP (2-10 Ci/mmol) were purchased from Dupont-NEN Products. all-trans-Retinal (Type XVI), phosphatidylserine, 1,2-dioleoyl-rac-glycerol, histone (type III-S), neomycin sulfate, and inositol phospholipid standards were obtained from Sigma. Anion exchange resin AG1-X8 (formate form) (200-400 mesh) was a product of Bio-Rad. Glycophase G glass beads (CPG/200) were obtained from Pierce. Sources of all other materials used have been described elsewhere (12,17). MATERIALS AND METHODS myo-[2-3H]InositolPreparation of Neutrophils. Guinea pig peritoneal neutrophils were prepared as described (18).Superoxide Release. Superoxide release by neutrophils was measured as outlined earlier (12). Solutions of all-transretinal (10 mM) were prepared in dimethyl sulfoxide immediately prior to use (17). Stock solutions of fMet-Leu-Phe and PMA were prepared in dimethyl sulfoxide and stored at -20°C until needed. These compounds were diluted with dimethyl sulfoxide so that the final concentration of solvent in the assays was 0.25% (vol/vol) in all cases. This concentration of solvent did not itself cause the effects noted.Labeling of Cells with [3H]Inositol. Neutrophil inositol phospholipids were labeled by incubating the cells (108 cells per ml) for 2 hr at 37°C in an isotonic Hepes buffer (124 mM NaCl/5 mM KCl/10 mM Hepes, pH 7.35) containing [3H]inositol (125 ,Ci/ml). The cells were washed in a modified Hanks' Hepes buffer (124 mM NaCl/5 mM KCl/0.5 mM CaCl2/0.2 mM MgCl2/10 mM Hepes, pH 7.35/5 mM glucose), resuspended in 50 ml of this buffer at a concentration of 2 x 106 cells per ml, and equilibrated to 37°C. They were stimulated with all-trans-retinal (25 ,uM), with fMetLeu-Phe (1 ,uM), or with PMA (0.05 AM) for various times, and the reactions were terminated by rapidly pouring the cell suspensions into 500-ml flasks containing 100 ml of the modified Hanks' Hepes buffer chilled to 4°C. The cells were transferred to 50-ml centrifuge tubes and collected by centrifugation for 10 min at 4°C. Cell pellets were lysed ...

Mechanism of All-trans-retinal Toxicity with Implications for Stargardt Disease and Age-related Macular Degeneration

“…NADPH oxidase has only recently been implicated as the enzymatic source of ROS generated in retinas exposed to bright light (30). Studies performed in neutrophils have demonstrated that atRAL acts as a potent stimulator of superoxide production through NADPH oxidase (33,34). In the present study, two structurally different NADPH oxidase inhibitors independently reduced ROS generation to levels similar to those in non-light exposed control mice and provided substantial protection against light-induced retinal degeneration in Abca4…”

“…NADPH oxidase is the primary catalyst involved in atRALstimulated superoxide production by neutrophils (33,34). To examine the role of NADPH oxidase in retinal cells, we added APO, a widely used NADPH oxidase inhibitor that interrupts NADPH oxidase complex assembly, to ARPE19 cells together with atRAL.…”

“…Although atRAL stimulates the production of superoxide via NADPH oxidase (33,34), there are observations that such stimulation is not the result of a direct interaction between atRAL and this enzyme (35). PLC activation reportedly occurs prior to NADPH oxidase-dependent ROS production in atRAL-treated neutrophils suggesting that products of PLC enzymatic activity, diacylglycerols and inositol 1,4,5-trisphosphate (IP 3 ), could be the intermediates involved in this pathway (33).…”

“…PLC activation reportedly occurs prior to NADPH oxidase-dependent ROS production in atRAL-treated neutrophils suggesting that products of PLC enzymatic activity, diacylglycerols and inositol 1,4,5-trisphosphate (IP 3 ), could be the intermediates involved in this pathway (33). IP 3 promotes release of Ca 2ϩ from the endoplasmic reticulum into the cytosol through binding to an intracellular IP 3 -receptor, IP 3 R (36).…”

Retinoic acid inhibits phospholipid turnover and protein kinase C activity in RA‐sensitive but not in RA‐resistant cells

“…Lochner et al [14] have demonstrated that all-trans-retinol activates PI-specific phospholipase C in neutrophils and causes only a slight decrease in PKC activity. Ponec et al [15] showed no alteration in phospholipid turnover at 3 × 10 -6 M RA in squamous cell carcinoma cells.…”