The Journal of biological chemistry
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The capsaicin receptor, VR1, is a sensory neuron-specific ion channel that serves as a polymodal detector of pain-producing chemical and physical stimuli. It has been reported that ATP, one of the inflammatory mediators, potentiates the VR1 currents evoked by capsaicin or protons and reduces the temperature threshold for activation of VR1 through metabotropic P2Y(1) receptors in a protein Kinase C (PKC)-dependent pathway, suggesting the phosphorylation of VR1 by PKC. In this study, direct phosphorylation of VR1 upon application of phorbol 12-myristate 13-acetate (PMA) was proven biochemically in cells expressing VR1. ⋯ Patch clamp analysis of the point mutants where Ser or Thr residues were replaced with Ala in the total 16 putative phosphorylation sites showed that two Ser residues, Ser(502) and Ser(800) were involved in the potentiation of the capsaicin-evoked currents by either PMA or ATP. In the cells expressing S502A/S800A double mutant, the temperature threshold for activation was not reduced upon PMA treatment. The two sites would be promising targets for the development of substance modulating VR1 function, thereby reducing pain.
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Glial cell line-derived neurotrophic factor (GDNF) is known for its potent effect on neuronal survival, but its role in the development and function of synapses is not well studied. Using Xenopus nerve-muscle co-cultures, we show that GDNF and its family member neurturin (NRTN) facilitate the development of the neuromuscular junction (NMJ). Long-term application of GDNF significantly increased the total length of neurites in the motoneurons. ⋯ Imaging analysis showed that the size of acetylcholine receptor clusters at synapses increased in muscle cells overexpressing GDNF. Neurturin had very similar effects as GDNF. These results suggest that GDNF and NRTN are new neuromodulators that regulate the development of the neuromuscular synapse through both pre- and postsynaptic mechanisms.
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Binding of nerve growth factor (NGF) to the p75 neurotrophin receptor (p75) in cultured hippocampal neurons has been reported to cause seemingly contrasting effects, namely ceramide-dependent axonal outgrowth of freshly plated neurons, versus Jun kinase (Jnk)-dependent cell death in older neurons. We now show that the apoptotic effects of NGF in hippocampal neurons are observed only from the 2nd day of culture onward. This switch in the effect of NGF is correlated with an increase in p75 expression levels and increasing levels of ceramide generation as the cultures mature. ⋯ The acid ceramidase inhibitor, (1S,2R)-d-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol, enhanced cell death, supporting a role for ceramide itself and not a downstream lipid metabolite. Finally, scyphostatin inhibited NGF-induced Jnk phosphorylation in hippocampal neurons. These data indicate an initiating role of ceramide generated by neutral sphingomyelinase in the diverse neuronal responses induced by binding of neurotrophins to p75.
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Peroxisome proliferator-activated receptor alpha (PPARalpha) is a nuclear receptor transcription factor that has an important role in controlling cardiac metabolic gene expression. We determined whether mice lacking PPARalpha (PPARalpha (-/-) mice) have alterations in cardiac energy metabolism. Rates of palmitate oxidation were significantly decreased in isolated working hearts from PPARalpha (-/-) hearts compared with hearts from age-matched wild type mice (PPARalpha (+/+) mice), (62 +/- 12 versus 154 +/- 65 nmol/g dry weight/min, respectively, p < 0.05). ⋯ In contrast, the expression and activity of acetyl-CoA carboxylase, which synthesizes malonyl-CoA and 5'-AMP-activated protein kinase, which regulates acetyl-CoA carboxylase, were not altered. Glucose transporter expression (GLUT1 and GLUT4) was not different between PPARalpha (-/-) and PPARalpha (+/+) hearts, suggesting that the increase in glycolysis and glucose oxidation in the PPARalpha null mice was not due to direct effects on glucose uptake but rather was occurring secondary to the decrease in fatty acid oxidation. This study demonstrates that PPARalpha is an important regulator of fatty acid oxidation in the heart and that this regulation of fatty acid oxidation may in part occur due to the transcriptional control of malonyl-CoA decarboxylase.
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Messenger RNA (mRNA) regulatory elements often form helices specifically distorted by loops or bulges, which control protein synthesis rates in vitro. Do such three-dimensional RNA structures form in vivo? We now observe formation of the internal loop/bulge (IL/B structure) in the IRE (iron-responsive element) of ferritin mRNA expressed in HeLa cells, using radical cleavage with Cu-phen (Cu-1,10-phenantholine), and protection of the loop/bulge by the regulatory protein (IRP), expressed by cotransfection. Cu-phen, a metal coordination complex (MC) selected because of binding and cleavage at the IL/B in solution, recognized the same site in mRNA in HeLa cells. ⋯ Selective RNA IL/B recognition by Cu-phen in vivo is emphasized by resistance to cleavage of a mutated, IL/B IRE in ferritin mRNA. Development of small MCs even more selective than Cu-phen can exploit three-dimensional mRNA or viral RNA structures in vivo to manipulate RNA function. Formation in vivo of the IL/B in the ferritin IRE, which is associated in vitro with greater repression than single IRE structures in other mRNAs, likely contributes to larger derepression of ferritin synthesis in vivo triggered by signals for the IRE/IRP system.