RACK1 has the nerve to act: structure meets function in the nervous system

The receptor for activated protein kinase C 1 (RACK1) is an intracellular adaptor protein. Accumulating evidence attributes to this member of the tryptophan-aspartate (WD) repeat family the role of regulating several major nervous system pathways. Structurally, RACK1 is a seven-bladed-beta-propeller, interacting with diverse proteins having distinct structural folds. When bound to the IP3 receptor, RACK1 regulates intracellular Ca2+ levels, potentially contributing to processes such as learning, memory and synaptic plasticity. By binding to the NMDA receptor, it dictates neuronal excitation and sensitivity to ethanol. When bound to the stress-induced acetylcholinesterase variant AChE-R, RACK1 is implicated in stress responses and behavior, compatible with reports of RACK1 modulations in brain ageing and in various neurodegenerative diseases. This review sheds new light on both the virtues and the variety of neuronal RACK1 interactions and their physiological consequences.     

(-)-Epigallocatechin gallate regulates dopamine transporter internalization via protein kinase C-dependent pathway

Dopamine transporter (DAT) provides not only an integral component of dopaminergic neurotransmission but also a molecular gateway for the accumulation of some neurotoxins such as 1-methyl-4-phenylpyridinium (MPP(+)), a metabolite of 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP). Previous study reported that the neuroprotective effects of green tea polyphenols against MPP(+)-induced neurotoxicity were related to its inhibitory effect on MPP(+) uptake via DAT in dopaminergic cells. To extend the study, we investigated (-)-epigallocatechin gallate (EGCG), a monomer of green tea polyphenols, on DAT internalization in DAT-overexpressed PC12 cells. We found that EGCG (1-100 microM) can induce a dose-dependent inhibition of dopamine uptake in DAT-PC12 cells. In parallel, treatment of EGCG decreased membrane-bound DAT by 15% to 60%. Furthermore, protein kinase C (PKC) inhibitor GF109203X at 2 microM can markedly diminish the inhibitory effects of EGCG on dopamine uptake and reverse the EGCG-induced internalization of DAT. In addition, semiquantitative RT-PCR analysis indicated that EGCG did not affect DAT mRNA expression in the PC12 cells. These data suggest that EGCG exerts its inhibitory effect on DAT by modulating DAT internalization, in which PKC activation may be involved.     

Implication of protein kinase C in the orexin-induced elevation of extracellular dopamine levels and its rewarding effect

In the present study, we investigated the role of orexinergic systems in the activation of midbrain dopamine neurons. In an in vitro study, exposure to either orexin A or orexin B under superfusion conditions produced a transient increase in the intracellular Ca(2+) concentration through the phospholipase C (PLC)/protein kinase C (PKC) pathway via G(q11)alpha or Gbetagamma subunits in midbrain cultured neurons, which were shown to be tyrosine hydroxylase (TH)-positive cells, but not in purified midbrain astrocytes. Here we show that in vivo injection with a selective PKC inhibitor chelerythrine chloride or 2-{8-[(dimethylamino)methyl]-6,7,8,9-tetrahydropyrido[1,2-a]indol-3-yl}-3-1-methyl-1H-indol-3-ylmaleimide HCl (Ro-32-0432) into the ventral tegmental area (VTA) significantly suppressed the place preference and increased levels of dopamine in the nucleus accumbens (NAcc) induced by intra-VTA injection of orexins. These results strongly support the idea that activation of the orexin-containing neuron in the VTA leads to the direct activation of mesolimbic dopamine neurons through the activation of the PLC/PKC pathway via G(q11)alpha or Gbetagamma-subunit activation, which could be associated with the development of its rewarding effect.     

Involvement of Ca2+- and cyclic adenosine monophosphate-mediated signaling pathways in photodynamic injury of isolated crayfish neuron and satellite glial cells

To investigate the mechanisms of oxidative injury of neurons and glia, we studied the photodynamic effect on isolated stretch receptor that consists of only two sensory neurons enwrapped by satellite glial cells. Photodynamic therapy (PDT), a potent inducer of oxidative stress, is a prospective method for destruction of brain tumors. PDT induced functional inactivation and necrosis of neurons, necrosis, apoptosis, and proliferation of glial cells. The roles of calmodulin, calmodulin-dependent kinase II, phospholipase C, protein kinases A and C, and phosphodiesterase in these processes were studied by using their inhibitors: fluphenazine, KN-93, D-609, H89, staurosporine, and papaverine, respectively. PDT-induced firing abolishment was enhanced by H89 or papaverine, whereas staurosporine acted oppositely. Fluphenazine or KN-93 reduced necrosis of neurons and glial cells. H89 enhanced necrosis of neurons, whereas staurosporine enhanced necrosis of glial cells. Inhibition of protein kinases A and C enhanced PDT-induced glial apoptosis. Photodynamic gliosis was prevented by KN-93 or staurosporine. These data indicate possible involvement of calmodulin and calmodulin-dependent kinase II in photoinduced necrosis of neurons and glia. Protein kinase C could protect glial cells from necrosis and apoptosis and participate in photoinduced gliosis and loss of neuronal activity. Protein kinase A maintained neuronal firing and protected neurons from photoinduced necrosis and glial cells from apoptosis. Phosphodiesterase reduced necrosis of photosensitized neurons and glia. Thus, Ca(2+)- and cAMP-mediated signaling pathways were involved in photooxidative injury of neurons and glia. Their pharmacological modulation may differently change the efficacy of photodynamic injury of neurons and glial cells.     

Changes in the composition of detergent-resistant membrane domains of cultured neurons following protein kinase C activation

Changes in the composition of cell fractions, and in particular of detergent-resistant membranes (DRM) isolated from cultured rat cerebellar granule cells, were taken as possible changes in lipid raft composition during a signal transduction event. After activation of protein kinase C (PKC) with phorbol esters (PMA) or glutamate, the content of PKC and of proteins highly enriched (GAP43, Fyn, and PrP(c)) or not (MARCKS) in DRM was followed. PKC activation strongly increased its association with membranes (from 2% to 75%), causing its enrichment within DRM; the substrate GAP43, enriched in DRM, remained membrane associated, but its proportion in DRM dramatically decreased (from about 40% to 2.5%), suggesting its shift from raft to nonraft membranes, possibly as a consequence of phosphorylation by PKC. The distribution of Fyn and PrP(c) (DRM-enriched) and of MARCKS (present mainly outside DRM) did not change. PKC activation was followed by an increase of GAP43 and MARCKS phosphorylation (about 7- and 8-fold, respectively). Noteworthy was that, after cell treatment with the lipid raft-disrupting drug methyl-beta-cyclodextrin, PKC activation occurred normally, followed by MARCKS phosphorylation, but GAP43 phosphorylation did not occur. Taken altogether, these data suggest that the integrity of lipid rafts is necessary for PKC to affect GAP43 and catalyze its phosphorylation.     

New aspects of the role of hydroxyeicosatetraenoic acids in cell growth and cancer development

Lipoxygenase (LOX) pathway leads to the formation of leukotrienes and also catalyses the conversion of arachidonic acid (AA) to hydroperoxyeicosatetraenoic acids that are then reduced to hydroxyeicosatetraenoic acids (HETE) by glutathione peroxidase. There are four mammalian LOXs that produce 5-, 8-, 12- and 15-HETE, respectively. Cytochrome P-450 isozymes are also capable of metabolising AA to HETEs either by bis-allylic oxidation (lipoxygenase-like reaction) to generate 5-, 8-, 9-, 11-, 12- and 15-HETE; or by ?/?-1 hydroxylation to yield 16-, 17-, 18-, 19- and 20-HETEs.It is now widely recognised that HETEs have important physiological and pathological functions that modulate ion transport, renal and pulmonary functions, vascular tone and reactivity, and inflammatory and growth responses. They can be released during the action of growth factors and cytokines, reaching physiological concentrations higher than that of prostanoids and modulating the functions of these factors. Their effects can occur through receptor or non-receptor mechanisms. Recent reviews have summarised the effects of HETEs in vascular homeostasis or lung and renal physiology. The present review focuses on the emerging effects of HETEs on cell signalling and physiological cell growth. It also discusses current observations regarding the role of HETEs in apoptosis, angiogenesis, the proliferation of cancer cells and metastasis, which constitute a potential area for successful therapeutic intervention.     

Phosphodiesterase isozymes involved in regulation of HCO3- secretion in isolated mouse duodenum in vitro

We examined the effects of various isozyme-selective PDE inhibitors on HCO(3)(-) secretion in the mouse duodenum in vitro and investigated which type(s) of phosphodiesterase (PDE) isozymes are involved in the response to PGE(2) and NO. The duodenal mucosa of male DDY mice was stripped of the muscle layer and mounted on an Ussing chamber, and HCO(3)(-) secretion was measured at pH 7.0 by a pH-stat method using 2mM HCl. Both PGE(2) and NOR-3 (NO donor) increased HCO(3)(-) secretion in the mouse duodenum in vitro, and the response to PGE(2) was inhibited by both EP3 and EP4 antagonists but not EP1 antagonist, while that to NOR-3 was inhibited by methylene blue. IBMX, a nonselective PDE inhibitor, significantly increased basal HCO(3)(-) secretion and potentiated the responses to both PGE(2) and NOR-3. Likewise, vinpocetine (PDE1 inhibitor) and cilostamide (PDE3 inhibitor) also increased the basal secretion at high doses and potentiated the HCO(3)(-) response to PGE(2) at doses that had no effect by themselves on the basal secretion. By contrast, the HCO(3)(-) stimulatory action of NOR-3 was significantly potentiated by vinpocetine but not cilostamide. Inhibitors of other PDE subtypes had no effect on the HCO(3)(-) secretion under basal or stimulated conditions. Both PDE1 and PDE3 mRNAs were expressed in the duodenal mucosa. These results suggested that PDE1 and PDE3 are involved in the regulation of duodenal HCO(3)(-) secretion and that the response to PGE(2) is associated with both PDE1 and PDE3, while the response to NO is mainly modulated by PDE1.     

Mechanisms for prostaglandin E2 formation caused by proteinase-activated receptor-1 activation in rat gastric mucosal epithelial cells

Proteinase-activated receptor-1 (PAR1), a thrombin receptor, plays a protective role in gastric mucosa via prostanoid formation. Thus, we studied effects of PAR1 stimulation on prostaglandin E(2) (PGE(2)) formation in rat normal gastric mucosal epithelial RGM1 cells and analyzed the underlying signal transduction mechanisms. The PAR1-activating peptide (PAR1-AP) and thrombin increased PGE(2) release from RGM1 cells for 18h, an effect being suppressed by inhibitors of COX-1, COX-2, MEK, p38 MAP kinase (p38 MAPK), protein kinase C (PKC), Src and EGF receptor-tyrosine kinase (EGFR-TK), but not JNK and matrix metalloproteinase (MMP)/a disintegrin and metalloproteinases (ADAMs). PAR1-AP caused persistent (6h or more) and transient (5min) phosphorylation of ERK and p38 MAPK, respectively, followed by delayed reinforcement at 18h. PAR1-AP up-regulated COX-2 in a manner dependent on MEK and EGFR-TK, but not p38 MAPK. The PAR1-mediated persistent ERK phosphorylation was reduced by inhibitors of Src and EGFR-TK. PAR1-AP actually phosphorylated EGF receptors and up-regulated mRNA for heparin-binding-EGF (HB-EGF), the latter effect being blocked by inhibitors of Src, EGFR-TK and MEK. Heparin, an inhibitor for HB-EGF, suppressed PAR1-mediated PGE(2) formation and persistent ERK phosphorylation. These results suggest that PAR1 up-regulates COX-2 via persistent activation of MEK/ERK that is dependent on EGFR-TK activation following induction of HB-EGF, leading to PGE(2) formation. In addition, our data also indicate involvement of COX-1, PKC and p38 MAPK in PAR1-triggered PGE(2) formation. PAR1, thus stimulates complex multiple signaling pathways responsible for PGE(2) formation in RGM1 cells.