Abstract
It has long been known that the conditionally essential polyunsaturated arachidonic acid (AA) regulates cerebral blood flow (CBF) through its metabolites prostaglandin E2 and epoxyeicosatrienoic acid, which act on vascular smooth muscle cells and pericytes to vasorelax cerebral microvessels. However, AA may also elicit endothelial nitric oxide (NO) release through an increase in intracellular Ca2+ concentration ([Ca2+]i). Herein, we adopted Ca2+ and NO imaging, combined with immunoblotting, to assess whether AA induces intracellular Ca2+ signals and NO release in the human brain microvascular endothelial cell line hCMEC/D3. AA caused a dose-dependent increase in [Ca2+]i that was mimicked by the not-metabolizable analogue, eicosatetraynoic acid. The Ca2+ response to AA was patterned by endoplasmic reticulum Ca2+ release through type 3 inositol-1,4,5-trisphosphate receptors, lysosomal Ca2+ mobilization through two-pore channels 1 and 2 (TPC1-2), and extracellular Ca2+ influx through transient receptor potential vanilloid 4 (TRPV4). In addition, AA-evoked Ca2+ signals resulted in robust NO release, but this signal was considerably delayed as compared to the accompanying Ca2+ wave and was essentially mediated by TPC1-2 and TRPV4. Overall, these data provide the first evidence that AA elicits Ca2+-dependent NO release from a human cerebrovascular endothelial cell line, but they seemingly rule out the possibility that this NO signal could acutely modulate neurovascular coupling.
Highlights
The 20-carbon omega-6 polyunsaturated fatty acid (PUFA) arachidonic acid (AA) is incorporated in the plasma membrane esterified to phospholipids at the second alcohol of glycerol in the sn-2 position and exerts multiple homeostatic, structural, and signalling functions [1]
AA elicited a biphasic increase in [Ca2+ ]i which was shaped by endogenous Ca2+ release through type 3 inositol-1,4,5-trisphosphate receptor isoform (InsP3 R3) and two-pore channels 1 and 2 (TPC1-2) and extracellular Ca2+ influx through transient receptor potential vanilloid 4 (TRPV4)
In order to assess whether AA induces intracellular Ca2+ signals, hCMEC/D3 cells were loaded with the Ca2+ -sensitive fluorophore, Fura-2, as described in paragraph 2.3
Summary
The 20-carbon omega-6 polyunsaturated fatty acid (PUFA) arachidonic acid (AA) is incorporated in the plasma membrane esterified to phospholipids at the second alcohol of glycerol in the sn-2 position and exerts multiple homeostatic, structural, and signalling functions [1]. Neuronal PLA2, in turn, mobilizes AA from membrane phospholipids; hereafter, AA is converted by cyclooxygenase-2 (COX-2) into prostaglandin E2 (PGE2) to relax cerebral microvessels [3]. PLA2 to stimulate the synthesis of a number of AA-derived metabolites that serve as vasodilatory mediators. These include PGE2 and epoxyeicosatrienoic acids (EETs) that are produced by COX-1 and cytochrome P450 epoxygenase [2,4], respectively. The hemodynamic response to whisker stimulation in the somatosensory cortex was dramatically impaired by inhibiting the production of PGE2 and EETs by, respectively, pyramidal neurons and astrocytes [8]. 20-HETE has been shown to induce capillary vasoconstriction in both the molecular [10] and granular [5] layer of the cerebellum, depending on the presence of nitric oxide (NO), that suppresses cytochrome P450 4A2 activity [2]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
