NO-cGMP mediated galanin expression in NGF-deprived or axotomized sensory neurons.

Leukaemia inhibitory factor (LIF) and nerve growth factor (NGF) are well characterized regulators of galanin expression. However, LIF knockout mice containing the rat galanin 5' proximal promoter fragment (- 2546 to + 15 bp) driving luciferase responded to axotomy in the same way as control mice. Also, LIF had no effect on reporter gene expression in vitro, neither in the presence or absence of NGF, suggesting that other factors mediate an axotomy response from the galanin promoter. We then addressed the role of nitric oxide (NO) using NGF-deprived rat dorsal root ganglion (DRG) neuron cultures infected with viral vectors containing the above-mentioned construct, and also studied endogenous galanin expression in axotomized DRG in vivo. Blocking endogenous NO in NGF-deprived DRG cultures suppressed galanin promoter activity. Consistent with this, axotomized/NGF-deprived DRG neurons expressed high levels of neuronal NO synthase (nNOS) and galanin. Further, using pharmacological NOS blockers, or adenoviral vectors expressing dominant-negative either for nNOS or soluble guanylate cyclase in vivo and in vitro, we show that the NO-cGMP pathway induces endogenous galanin in DRG neurons. We propose that both LIF and NO, acting at different promoter regions, are important for the up-regulation of galanin, and for DRG neuron survival and regeneration after axotomy.

Distinct roles for protein kinase C isoforms in regulating platelet purinergic receptor function.

ADP is a critical regulator of platelet activation, mediating its actions through two G protein-coupled receptors (GPCRs), P2Y1 and P2Y12. We have shown previously that the receptors are functionally desensitized, in a homologous manner, by distinct kinase-dependent mechanisms in which P2Y1 is regulated by protein kinase C (PKC) and P2Y12 by G protein-coupled receptor kinases. In this study, we addressed whether different PKC isoforms play different roles in regulating the trafficking and activity of these two GPCRs. Expression of PKCalpha and PKCdelta dominant-negative mutants in 1321N1 cells revealed that both isoforms regulated P2Y1 receptor signaling and trafficking, although only PKCdelta was capable of regulating P2Y12, in experiments in which PKC was directly activated by the phorbol ester phorbol 12-myristate 13-acetate (PMA). These results were paralleled in human platelets, in which PMA reduced subsequent ADP-induced P2Y1 and P2Y12 receptor signaling. PKC isoform-selective inhibitors revealed that novel, but not conventional, isoforms of PKC regulate P2Y12 function, whereas both novel and classic isoforms regulate P2Y1 activity. It is also noteworthy that we studied receptor internalization in platelets by a radioligand binding approach showing that both receptors internalize rapidly in these cells. ADP-induced P2Y1 receptor internalization is attenuated by PKC inhibitors, whereas that of the P2Y12 receptor is unaffected. Both P2Y1 and P2Y12 receptors can also undergo PMA-stimulated internalization, and here again, novel but not classic PKCs regulate P2Y12, whereas both novel and classic isoforms regulate P2Y1 internalization. This study therefore is the first to reveal distinct roles for PKC isoforms in the regulation of platelet P2Y receptor function and trafficking.