Figure 5.

Translocation of PKCε to neuronal surface membrane caused by thrombin. A. Translocation of PKCε to neuronal surface membrane in control conditions (left) and following exposure to thrombin (100 nM, 30 and 60 sec). PKCε translocated rapidly to the surface membrane following application of thrombin (arrow in middle panel) and at longer times became progressively internalised (arrowhead in middle panel and right panel). Adult mouse neurons cultured in 10% FBS in absence of NGF and neurturin. Scale bars 5 μm.

B. Percentage of neurons showing translocation to the plasma membrane as a function of time of exposure to thrombin (number of neurons > 2000 for each point).

C. Peak percentage of neurons in which PKCε was translocated, as a function of thrombin concentration (number of neurons > 2000 for each point). Continuous curve shows a Hill equation with n = 0.7 and K1/2 = 2 nM.

D. Size distribution of thrombin-responsive neurons. Grey bars show size distribution of overall neuronal population, and black bars show neurons in which PKCε translocation was observed following exposure to thrombin (100 nM, 30 s).

E. Activation of PKCε translocation by proteases and specific PAR activator peptides. Thrombin, trypsin and cathepsin G (all 100 nM, 30 s) caused translocation of PKCε in a similar percentage of adult mouse neurons but tryptase and collagenase IV were ineffective. *, p < 0.05, ***, p < 0.001, t test compared to thrombin. PAR1-AP (TFLLR, 100 mM) caused translocation similar that of thrombin. PAR4-AP (AYPGKF, 200 mM, bar 2) caused translocation in a significantly smaller proportion of neurons when compared to PAR1-AP. Increased concentrations of activating peptides did not cause increased translocation (not shown). The effects of PAR1-AP and PAR4-AP (both at 100 mM) were partially but not completely additive. PAR2-AP (SLIGRL-NH2) had no effect. *, p < 0.05, ***, p < 0.001, t test compared to PAR1 alone.

Vellani et al. Molecular Pain 2010 6:61   doi:10.1186/1744-8069-6-61
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