Thrombin-induced growth cone collapse: involvement of phospholipase A(2) and eicosanoid generation

Abstract

The studies presented here explore intracellular signals resulting from the action of repellents on growth cones. Growth cone challenge with thrombin or thrombin receptor-activating peptide (TRAP) triggers collapse via a receptor-mediated process. The results indicate that this involves activation of cytosolic phospholipase A(2) (PLA(2)) and eicosanoid synthesis. The collapse response to repellents targets at least two functional units of the growth cone, the actin cytoskeleton and substratum adhesion sites. We show in a cell-free assay that thrombin and TRAP cause the detachment of isolated growth cones from laminin. Biochemical analyses of isolated growth cones reveal that thrombin and TRAP stimulate cytosolic PLA(2) but not phospholipase C. In addition, thrombin stimulates synthesis of 12- and 15-hydroxyeicosatetraenoic acid (HETE) from the released arachidonic acid via a lipoxygenase (LO) pathway. A selective LO inhibitor blocks 12/15-HETE synthesis in growth cones and inhibits thrombin-induced growth cone collapse. Exogenously applied 12(S)-HETE mimics the thrombin effect and induces growth cone collapse in culture. These observations indicate that thrombin-induced growth cone collapse occurs by a mechanism that involves the activation of cytosolic PLA(2) and the generation of 12/15-HETE.

Fig. 1.

Effects of thrombin, thrombin plus LO inhibitor, and 12(S)-HETE on growth cone configuration. Phase-contrast micrographs are shown of cultures of embryonic day 18 rat cortical neurons that were either pretreated with vehicle alone or inhibitor for 45 min before challenge with thrombin or 12(S)-HETE. Numbers in thetop right corners indicate time in minutes of exposure to collapsing reagent. Top to bottom, 100 nm thrombin, 10 μm CDC pretreatment (30 min) followed by 100 nm thrombin, and 10⁻⁷m 12(S)-HETE. The large growth cone in CDC+Thrombin exhibits at 0 min (which is a CDC-alone control) and at 0.5 min vertically extended veils. By 5 min they have disappeared.

Fig. 2.

Effects of thrombin, TRAP, thrombin or TRAP plus LO inhibitor, and 12(S)-HETE on filamentous actin in growth cones. Cultured El8 rat cortical neurons were fixed and stained with Texas Red-conjugated phalloidin after the following experimental treatments: control (vehicle alone), thrombin (100 nm) for 7 min, TRAP (100 μm) for 7 min, 45 min pretreatment with CDC (10 μm) followed by thrombin (100 nm) or TRAP (100 μm) for 7 min, and 12(S)-HETE (10⁻⁷m) for 12 min. Thrombin and TRAP cause collapse, but the TRAP effect, although clear-cut, is not as dramatic as that of thrombin; CDC inhibits or significantly attenuates collapse, and 12(S)-HETE mimics the effects of thrombin and TRAP.

Fig. 3.

The LO inhibitor CDC inhibits thrombin-induced shape change of the growth cone but may allow redistribution of the actin cytoskeleton. A, Growth cones fixed and stained with Texas Red-conjugated phalloidin after control (vehicle alone) incubation. B, 30 min CDC (10 μm) pretreatment followed by thrombin challenge (100 nm) for 7 min. Small arrows indicate intact filopodia with diminished actin staining. The large arrow denotes “clumped” actin filaments in the proximal growth cone.

Fig. 4.

Quantitative analysis of growth cone collapse induced by thrombin (with or without CDC pretreatment), TRAP, or 12(S)-HETE. Collapse status was assessed on fixed neural cultures having undergone the following treatments:C, control, vehicle alone; THR, thrombin (100 nm) for 7 min; CDC/THR, CDC (10 μm) pretreatment for 30 min, followed by thrombin (100 nm) for 7 min; TRAP (100 μm) for 7 min; and 12(S)-HETE (10⁻⁷m) for 10 min. For each condition at least 200 growth cones from at least four independent experiments were scored as described in Materials and Methods. Data are presented as percent of total growth cones observed. Error bars indicate SEM; n, number of independent experiments.

Fig. 5.

Thrombin- and TRAP-induced detachment of GCPs from a laminin substratum. GCPs plated on laminin were exposed for 20 min to thrombin (THR, 100 or 200 nm), to vehicle with or without cytochalasin D (CYTO D, 1 μm) or BDM (20 mm) pretreatment, or to TRAP (100 μm). Detachment was measured as pelletable protein collected in the supernatant. Data are net increases ± SEM above untreated controls, expressed as percent of total GCP protein bound initially. In control conditions detachment was 15.2 ± 6.9% (average ± SD) of total GCPs plated. Numbers inparentheses indicate numbers of independent experiments.

Fig. 6.

Dose–response curves of PLA₂activation by thrombin (A) and TRAP (B) in GCPs. GCPs were incubated for 10 min on ice with varying concentrations of thrombin (A) or TRAP (B) and then for 10 min at 37°C in the reaction mixture in the presence of 10 μmCaCl₂. The substrate was [¹⁴C]AA-PI. The data presented are net values in triplicate. Error bars represent SD; where not present error bars were too small to be indicated.

Fig. 9.

Thrombin inhibits growth cone PLC in a calcium-dependent manner. GCPs were incubated alone (control) or in the presence of 100 nm thrombin for 10 min on ice and then combined with substrate ([¹⁴C]AA-PI) for 10 min at 37°C in the presence of EGTA or varying micromolar concentrations of CaCl₂ (0 [Ca], no addition of Ca²⁺ or EGTA). DG release was measured as described in Materials and Methods. Data presented are triplicates ± SD.

Fig. 10.

IGF-1 stimulation of PLC and PLA₂ in growth cones. GCPs were assayed for both PLA₂ and PLC activity as described, in the presence of 10 μmCaCl₂ and varying concentrations of IGF-1, using [¹⁴C]AA-PI as substrate and recovering [¹⁴C]AA and [¹⁴C]AA-DG as products. Data are from a representative experiment done in triplicate. Error bars indicate SD; where no error bars appear they were too small to register.

Fig. 11.

Thrombin-stimulated release of AA and HETE. GCPs were assayed for both PLA₂ and LO activity as described. Inhibitor (0.156 μm CDC or 0.2 μmindomethacin) was introduced to some GCP samples for 15 min on ice before the addition of 100 nm thrombin. After an additional 10 min incubation on ice, the GCP mixture was then incubated with either [¹⁴C]AA-PC (A, B) or [³H]AA (C) in the presence of 10 μm CaCl₂ for 15 min at 37°C.A, B, Thrombin-stimulated release of AA and HETE in assays using the PC substrate, shown as actual amounts (A) and fold increase (B).C, Thrombin stimulation of HETE directly from the AA substrate. In A and B, values for control and thrombin-stimulated conditions were averaged from six experiments, all done in triplicate, whereas the values for CDC and indomethacin-treated GCPs were averaged from two experiments, done in triplicate. Data in C are from one representative experiment, done in triplicate. Error bars indicate SD; where not seen, they were too small to be shown.

Fig. 12.

Growth cones synthesize 12- and 15-HETE, as demonstrated by LC–MS–MS. Lipid extracts from growth cones incubated with equal molar quantities of unlabeled and deuterium-labeled (d₈) arachidonic acid were resolved by reverse-phase HPLC, and the eluted fractions were analyzed by tandem MS. A, LC–MS–MS corresponding to the collisional activation of m/z 319, yielding the ion at m/z 219, which corresponds to 15-HETE.B, Collisional activation ofm/z 327, yielding product ions atm/z 226, which correspond to D₈-15-HETE. C, Collisional activation ofm/z 319, yielding product ions atm/z 179, which correspond to 12-HETE.D, Collisional activation ofm/z 327, yielding product ions atm/z 184, which correspond to D₈-12-HETE.

Fig. 13.

Western blot of GCP polypeptides probed with an antibody to leukocyte 12/15-LO. Equal amounts of protein (30 μg/lane) of LSS and pelleted GCPs were analyzed. The arrowheadindicates a 75 kDa band corresponding to the molecular weight of 12/15-LO. Note that the 75 kDa band is enriched in the GCP lane.