While additional studies are required to address these possibilities, the data in this study identify a novel function for sEH in regulating axonal growth
While additional studies are required to address these possibilities, the data in this study identify a novel function for sEH in regulating axonal growth. That EETs are specifically targeting axonal growth and not increasing axonal growth via nonspecific effects on general cell viability or enhanced survival of neuronal subpopulations is suggested by the finding that addition of exogenous EETs and/or pharmacologic inhibition of sEH had no effect on mitochondrial activity as determined using the MTT assay. 11,12-EET. 14,15-EET also promotes axon outgrowth in cultured cortical neurons. Co-exposure to EETs and either of two structurally diverse pharmacological inhibitors of sEH potentiate the axon-enhancing activity of EETs in sensory and cortical neurons. Mass spectrometry indicates that sEHi significantly increase EETs and significantly decrease dihydroxyeicosatrienoic acid metabolites in neuronal cell cultures. These data show that EETs enhance axon outgrowth and suggest that axonal sEH activity regulates EETs-induced axon outgrowth. These findings suggest a novel therapeutic use of sEH inhibitors in promoting nerve regeneration. 2004, Iliff 2010, Inceoglu 2007). While production of EETs in the brain was demonstrated soon after the initial discovery of these novel arachidonic acid metabolites (Snyder 1983), much of the research around the biological activity of these novel eicosanoids has focused on regulatory functions of EETs in cardiovascular and renal physiology and pathology. Recently, however, EETs have emerged as important players in regulating central and peripheral nervous system function. EETs signaling has been linked to regulation of cerebral blood flow, modulation of neuronal pain processing in the brain stem, control of neurohormone release, and influence on synaptic transmission (Iliff et al. 2010, Inceoglu et al. 2007, Inceoglu 2008). The biological activity of EETs is usually rapidly terminated via their metabolism through multiple pathways (Iliff et al. 2010, Inceoglu et al. 2007), with the predominant metabolic pathway being hydration by soluble epoxide hydrolase (sEH, EC 3.3.2.10) to form the less active dihydroxyeicosatrienoic acids (DHETs) (Chacos 1983, Spector et al. 2004). Our recent work has exhibited that sEH is usually highly abundant and active in the central and peripheral nervous system. Immunohistochemical analyses of mouse brain revealed sEH immunoreactivity in the cerebral cortex that was predominantly localized to neurons (Zhang 2007). Within cortical neurons, sEH immunoreactivity was observed in neuronal cell body and processes but the most striking obtaining was the localization of sEH immunoreactivity in axons in the neuropil and nerve fiber bundles within gray and white matter. Additional studies show that sensory neurons of the trigeminal ganglia and parasympathetic neurons of the sphenopalatine ganglia are also immunoreactive for sEH as are nerve fibers emanating from these ganglia (Iliff 2009). The functional relevance of sEH expression in axons is not known. We hypothesize that sEH regulates axonal growth via modulation of the local levels of epoxy fatty acids and particularly EETs. To test this novel hypothesis, we examined the subcellular localization of sEH in main cultures of central (cortical) and peripheral (sympathetic and sensory) neurons and decided whether experimentally increasing levels of EETs by addition of exogenous EETs and/or pharmacologic inhibition of sEH influenced axonal growth 1999). 1-(1-Methylsulfonyl-piperidin-4-yl)-3-(4-trifluoromethoxy-phenyl)-urea (TUPS) was synthesized and purified according to recently published methods (Tsai 2010). Unless otherwise noted, all other chemical reagents were purchased N-Bis(2-hydroxypropyl)nitrosamine from Sigma Chemical Organization (St. Louis, MO). A mixture of EET-methyl esters as well as individual EET regioisomers (free acid form) were synthesized and purified as explained earlier (Campbell 1991). To determine the ratio of individual N-Bis(2-hydroxypropyl)nitrosamine EET-methyl ester regioisomers in the combination, the methyl esters were base hydrolyzed as explained (Newman 2002) and the producing free fatty acid EETs combination was analyzed by LC-MS/MS (as explained below under quantification of EETs). The regioisomeric EET-methyl ester combination contained 14,15-, 11,12-, 8,9- and 5,6-EET at a ratio of 10:10:10:1. The relatively low level of 5,6-EET in this combination likely displays its susceptibility to conversion to the lactone derivative under the conditions utilized for LC-MS/MS. N-Bis(2-hydroxypropyl)nitrosamine Aliquots of stock solutions of the individual 8,9-, 11,12- and 14, 15-EET preparations used in axon outgrowth experiments were analyzed by LC-MS/MS to confirm purity and concentration. Animals All procedures involving animals were performed according to protocols approved by the Institutional Animal Care and Use Committees of Oregon Health & Science University or college and UC Davis. Timed-pregnant Sprague Dawley rats were purchased from Charles River Laboratories (Hollister, CA) and housed individually in standard plastic.The MRM transitions used were 14(15)-EET (319/219) and 14,15-DHET (337/207). in neuronal cell cultures. These data show that EETs enhance axon outgrowth and suggest that axonal sEH activity regulates EETs-induced axon outgrowth. These findings suggest a novel therapeutic use of sEH inhibitors in promoting nerve regeneration. 2004, Iliff 2010, Inceoglu 2007). While production of EETs in the brain was demonstrated soon after the initial discovery of these novel arachidonic acid metabolites (Snyder 1983), much of the research around the biological activity of these novel eicosanoids has focused on regulatory functions of EETs in cardiovascular and renal physiology and pathology. Recently, however, EETs have emerged as important players in regulating central and peripheral nervous system function. EETs signaling has been linked to regulation of cerebral blood flow, modulation of neuronal pain processing in the brain stem, control of neurohormone release, and influence on synaptic transmission (Iliff et al. 2010, Inceoglu et al. 2007, Inceoglu 2008). The biological activity of EETs is usually rapidly terminated via their metabolism through multiple pathways (Iliff et al. 2010, Inceoglu et al. 2007), with the predominant metabolic pathway being hydration by soluble epoxide hydrolase (sEH, EC 3.3.2.10) to form the less active dihydroxyeicosatrienoic acids (DHETs) (Chacos 1983, Spector et al. 2004). Our recent work has exhibited that sEH is usually highly abundant and active in the central and peripheral nervous system. Immunohistochemical analyses of mouse brain revealed sEH immunoreactivity in the cerebral cortex that was predominantly localized to neurons (Zhang 2007). Within cortical neurons, sEH immunoreactivity was observed in neuronal cell body and processes but the most striking obtaining was the localization of sEH immunoreactivity in axons in the neuropil and nerve fiber bundles within gray and white matter. Additional studies show that sensory neurons of the trigeminal ganglia and parasympathetic neurons of the sphenopalatine ganglia are also immunoreactive for sEH as are nerve fibers emanating from these ganglia (Iliff 2009). The functional relevance of sEH expression in axons is not known. We hypothesize that sEH regulates axonal growth via modulation of the local levels of epoxy fatty acids and particularly EETs. To test this novel hypothesis, we examined the subcellular localization of sEH in main cultures of central (cortical) and peripheral (sympathetic and sensory) neurons and decided whether experimentally increasing levels of EETs by addition of exogenous EETs and/or pharmacologic inhibition of sEH influenced axonal growth 1999). 1-(1-Methylsulfonyl-piperidin-4-yl)-3-(4-trifluoromethoxy-phenyl)-urea (TUPS) was synthesized and purified according to recently published methods (Tsai 2010). Unless normally noted, all other chemical reagents were purchased from Sigma Chemical Organization (St. Louis, MO). A mixture of EET-methyl esters as well as individual EET regioisomers (free acid form) N-Bis(2-hydroxypropyl)nitrosamine were synthesized and purified as explained earlier (Campbell 1991). To determine the ratio of individual EET-methyl ester regioisomers in the combination, the methyl esters were base hydrolyzed as explained (Newman 2002) and the producing free fatty acid EETs combination was analyzed by LC-MS/MS (as explained below under quantification of EETs). The regioisomeric EET-methyl ester combination contained 14,15-, 11,12-, 8,9- and 5,6-EET at a ratio of N-Bis(2-hydroxypropyl)nitrosamine 10:10:10:1. The relatively low level of 5,6-EET in this blend likely demonstrates its susceptibility to transformation towards the lactone derivative beneath the conditions useful for LC-MS/MS. Aliquots of share solutions of the average person 8,9-, 11,12- and 14,15-EET Rabbit Polyclonal to ADORA1 arrangements found in axon outgrowth tests were examined by LC-MS/MS to verify purity and focus. Animals All methods involving animals had been performed relating to protocols authorized by the Institutional Pet Care and Make use of Committees of Oregon Wellness & Science College or university and UC Davis. Timed-pregnant Sprague Dawley rats had been bought from Charles River Laboratories (Hollister, CA) and housed separately in standard plastic material cages with Alpha-Dri bed linen (Shepherd Specialty Documents, Watertown, TN) inside a temperatures (222C) controlled space on the 12h invert light-dark cycle. Water and food were offered 2003). Under these tradition circumstances, immunocytochemical analyses indicated that ~95% from the cell inhabitants was immunoreactive for the neuronal marker, neurofilament, and ~5% from the cell inhabitants was immunoreactive for the astrocytic marker, glial fibrillary acidic proteins. Sympathetic neurons had been dissociated through the excellent cervical ganglia (SCG) of embryonic day time 21 (E21) rat pups and taken care of in the lack of glial cells in serum-free moderate supplemented with nerve development element (-NGF, 100ng/ml, Harlan.