A glutathione conjugate of hepoxilin A3: formation and action in the rat central nervous system.

Abstract

Incubation of (8R)-and (8S)-[1-'4C]hepoxilin A3 [where hepoxilin A3 is 8-hydroxy-11,12-epoxyeicosa-(5Z,9E,14Z)-trienoic acid] and glutathione with homogenates of rat brain hippocampus resulted in a product that was identified as the (8R) and (8S) diastereomers of 11-glutathionyl hepoxilin A3 by reversed-phase high performance liquid chromatographic comparison with the authentic standard made by total synthesis. Identity was further confirmed by cleavage of the isolated product with y-glutamyltranspeptidase to yield the corresponding cysteinylglycinyl conjugate that was identical by reversed-phase high performance liquid chromatographic analysis with the enzymic cleavage product derived from the synthetic glutathionyl conjugate. The glutathionyl and cysteinylglycinyl conjugate are referred to as hepoxilin A3-C and hepoxilin A3-D, respectively, by analogy with the established leukotriene nomenclature. Formation of hepoxilin A3-C was greatly enhanced with a concomitant decrease in formation of the epoxide hydrolase product, trioxilin A3, when the epoxide hydrolase inhibitor trichloropropene oxide was added to the incubation mixture demonstrating the presence of a dual metabolic pathway in this tissue involving hepoxilin epoxide hydrolase and glutathione S-transferase processes. Hepoxilin A3-C was tested using intracellular electrophysiological techniques on hippocampal CA1 neurons and found to be active at concentrations as low as 16 nM in causing membrane hyperpolarization, enhanced amplitude and duration of the postspike train afterhyperpolarization, a marked increase in the inhibitory postsynaptic potential, and a decrease in the spike threshold. These findings suggest that these products in the hepoxilin pathway of arachidonic acid metabolism formed by the rat brain may function as neuromodulators.

Mammalian soluble epoxide hydrolase is identical to liver hepoxilin hydrolase

“…Quit unexpected were the large amounts of HxB 3 , particularly, in the livers of sEH Ϫ / Ϫ mice whereas HxA 3 did not accumulate to that extent. HxA 3 has been shown to be a substrate for glutathione-S-transferases and the glutathione-conjugated metabolite maintains biologic activity ( 32,37 ). Due to the high expression level of GSTs in the liver, one would expect a lack of hepoxilin accumulation, which is only seen for the HxA 3 regioisomer ( Fig.…”

“…In the brain, hepoxilins modulate synaptic neurotransmission and neuronal excitability, mostly through stimulation of intracellular calcium release or increased calcium infl ux into the cell (34)(35)(36)(37). Hepoxilins are considered pro-infl ammatory because increased hepoxilin and trioxilin levels have been found in psoriatic lesions ( 38 ) and HxA 3 was shown to regulate neutrophil migration in response to infl ammation ( 39,40 ).…”

Mammalian epoxide hydrolases in xenobiotic metabolism and signalling

“…Hepoxilins formed in rat pancreatic islets enhance the glucose dependant secretion of insulin (Pace-Asciak and Martin 1984). Hepoxilins facilitate calcium transport (Derewlany et al 1984), and they are further formed in the brain with synaptic neuromodulatory actions on hippocampal neurons by hyperpolarisation and enhancement of the inhibitory postsynaptic potential (Pace-Asciak et al 1990. Hepoxilin signalling is most likely receptor mediated, an assumption based on the identiWcation of a hepoxilin binding protein in human neutrophils (Reynaud et al 1999).…”

Hepoxilin A3 is the endogenous lipid mediator opposing hypotonic swelling of intact human platelets.

“…Further characterization ofthe material in the fraction at [16][17][18] Hepoxilins possess an epoxide moiety which is subject to hydrolysis by epoxide hydrolases in platelets (7). TCPO inhibits hepoxiin epoxide hydrolase (8). When hepoxilin A3 was tested in the presence of 1 mM TCPO, its activity was found to be 4-to 5-fold higher (Fig.…”

“…Our data correlate well with these studies and show that, like neurons, platelets apparently possess potassium channels that are activated by hepoxilins. Hepoxilins have been shown to exert a number of biological actions-e.g., release of insulin (11), potentiation of norepinephrine-induced aortic vasoconstriction (12), potentiation of vascular permeability in the skin (13), activation of neutrophils (14), and modulation of neurotransmission in the mammalian central nervous system (8,15) and the Aplysia brain (16 …”