The previous study demonstrated that the main

The previous study demonstrated that the main CYP isoforms present in the human HhAntag included CYP46A1, 2J2, 2U1, 1B1, 2E1 and 2D6 (Dutheil et al., 2009b, 2010). CYP1B1 and CYP2U1 were found in the mitochondrial and microsomal fractions of astrocytes in the human frontal lobe, hippocampus, substantia nigra and cerebellum. In freshly isolated human brain microvessels, CYP1B1 and CYP2U1 were the only quantifiable CYP isoforms from the 21 CYPs investigated (Shawahna et al., 2011). The in vitro study reported that CYP1B1 preferentially produced midchain HETEs, almost 55% of its total metabolites (Choudhary et al., 2004). LC–MS analysis showed that CYP2U1 was able to metabolize long chain fatty acids including the metabolism of AA to 19- and 20-HETE (Chuang et al., 2004). Glutamate is one of the most important neurotransmitters in the central nervous system. Previous study has shown the direct respond of the astrocytes to the glutamate released from synaptic terminals (Porter and McCarthy, 1996). Astrocytes have been recognized to sense glutamatergic synaptic activity over a large spatial domain. In the present study, we investigated the regulatory mechanism of brain CYP1B1 and CYP2U1 by glutamate and the changes in HETEs production in rat brain regions. This work adds to the current knowledge on the regulation of metabolic processes occurring in astrocytes by neurotransmitter-mediated signaling, and the pivotal role of astrocytes in the neurovascular unit.
Materials and methods
Results
Discussion This study demonstrated that mGlu5 receptor mediated the regulation of brain CYP1B1 and CYP2U1 by glutamate, and that glutamate changed the HETEs production within the brain. We showed that glutamate up-regulated brain CYP1B1 and CYP2U1 via CREB. Our previous work showed that glutamate affects the production of epoxyeicosanoids via brain CYP2J (Liu et al., 2017). The alteration of the CYP-mediated AA metabolism in the brain by the neurotransmitter-mediated signaling support the hypothesis that neurons and/or astrocytes may play an important role in the autoregulation of cerebral blood flow. The previous studies demonstrated that the alteration of both HETEs and EETs synthesis affected cerebral blood flow in the rat (Kehl et al., 2002b; Alkayed et al., 1996a). Brain CYPs may be the important mediators in the autoregulation of the cerebral blood flow by neurotransmitters. A sustained induction of brain CYP1B1 and CYP2U1 were observed in the cortex, hippocampus, and cerebellum from the rats that received monosodium l-glutamate before the weaning stage. The data from the fluorescent immunohistochemistry indicated the regulation of CYP1B1 and CYP2U1 in both neurons and glial cells following glutamate treatment. The expression levels of CYP1B1 mRNA and protein have been shown to be changed by triclosan in primary cultures of neocortical neurons from mouse embryos, indicating the regulation of CYP1B1 by exogenous compounds (Szychowski et al., 2016). The previous study has shown that CYP1B1 protein was present at a high level in the astrocytes, but not found in the neurons in human brain (Dutheil et al., 2009a). Our data showed the up-regulation of CYP1B1 proteins in both neurons (e.g. Purkinje cell) and astrocytes in rat brain. The in vitro data showed that glutamate can regulate CYP1B1 and CYP2U1 in both U251 cells and hCMEC/D3 cells. The co-localization of the CYPs and GFAP indicated the induction of astrocytic CYP1B1 and CYP2U1 by glutamate. As brain-specific CYP isoform, CYP2U1 proteins have been strongly detected in the astrocyte foot processes that contact microvessels (Dutheil et al., 2009b). The increased production of midchain and terminal HETEs by the neurotransmitter released from neurons may affect cerebral blood flow, especially under the pathological conditions. The mGlu5 receptor, the main mGlu receptor subtype expressed in the astrocytes, has been recognized as the mediator in the cross-talk between neurons and astrocytes (Bradley and Challiss, 2012a). In the present study, we showed that the blockade of mGlu5 receptor attenuated the induction of the phosphorylated CREB proteins in the nuclear and the binding of the CREB protein with the CYP1B1 and CYP2U1 promoters by glutamate. Both PKA and MAPK signaling pathways triggered by mGlu5 receptor were involved in the regulation of astrocytic CYP1B1 and CYP2U1, although the regulation mechanism of CYP1B1 and CYP2U1 in hCMEC/D3 cells need to be further elucidated. It has been reported that the stimulation of mGlu5 receptors leads to robust oscillatory changes in [Ca2+]i, and [Ca2+]i increases within astrocyte endfeet can lead to the induction of Ca2+-dependent phospholipase A2 activity (Bradley and Challiss, 2012b). Our data suggest that mGlu5 receptor plays a pivotal role in the regulation of AA metabolism in the neuronal cells. As HETEs were found to play an important role in vascular homeostasis(Yousif et al., 2010; Fan and Roman, 2017), the changes in the AA metabolism may lead to vascular dysfunction. Migraine is a neurovascular disorder characterized by a decrease in cerebral blood flow and a potent dilatation of cranial extracerebral blood vessels (Maassenvandenbrink and Chan, 2008; Brennan and Andrew, 2010). Glutamate and its receptors have since long been suggested in migraine pathophysiology (Chan and MaassenVanDenBrink, 2014).A recent study suggested that mGlu5 receptor was a novel therapeutic target in the treatment of migraine in human. The blockade of mGlu5 receptor attenuated neurogenic dural vasodilation in rats, while the N-methyl-d-aspartate receptor blocker MK-801 had no effect (Waung et al., 2016). Our data support the hypothesis that the blockade of mGlu5 receptor may improve the vascular dysfunction via the alteration of the vasoactive substances from AA in the neuronal cells triggered by neurotransmitter-mediated signaling.