Date

April 2010

Document Type

Dissertation

Degree Name

Ph.D.

Department

Dept. of Physiology and Pharmacology

Institution

Oregon Health & Science University

Abstract

Chapter 1: Introduction. Cerebral blood flow is controlled through three levels of regulation: intrinsic vasomotor regulation, neurovascular coupling, and regulation by perivascular vasomotor nerves. Epoxyeicosatrienoic acids (EETs) are arachidonic acid metabolites of cytochrome P450 (CYP). In the cerebral vasculature, EETs are vasodilators that contribute to intrinsic vasomotor regulation and to neurovascular coupling. In the present series of studies, I tested the overall hypothesis that EETs contribute to the regulation of the cerebral vasculature by perivascular vasodilator nerves. Chapter 2. The hypothesis that parasympathetic and trigeminal vasodilator nerves surrounding the cerebral vasculature express EETs-synthetic CYP epoxygenase and EETs-regulatory sEH enzymes was tested. RT-PCR and Western blot revealed the expression of CYP-2J3 and CYP-2J4 epoxygenases and sEH in the rat sphenopalatine and trigeminal ganglia. Immunofluorescence double-labeling indicated that CYP-2J and sEH localize to neurons within both ganglia. These findings demonstrate that parasympathetic and trigeminal perivascular neurons possess the biochemical machinery for the synthesis and regulation of vasodilator EETs. Chapter 3. The hypothesis that CYP-2J epoxygenases are constitutively active in primary rat trigeminal ganglion neurons was tested. LC-MS/MS detected the presence of 5,6-DHET, 8,9-, 11,12-, and 14,15-EETs regio-isomers within whole-cell extract from primary trigeminal ganglion neurons. These findings demonstrate the CYP-2J3 is functional and EETs are endogenously present in primary trigeminal ganglion neurons. Chapter 4. The hypothesis that EETs are released from rat primary trigeminal ganglion neurons was tested. EETs release from trigeminal neurons following stimulation with depolarizing K+ or capsaicin was assayed by LC-MS/MS. No evidence for EETs release was observed. The regulation of neuropeptide release from trigeminal ganglion neurons by endogenous EETs was then evaluated. Capsaicin stimulation resulted in a ~5-fold increase in CGRP release measured by ELISA. Pre-treatment with the EETs antagonist 14,15-EEZE (10 μmol/L) and the CYP epoxygenase inhibitor MS-PPOH (10 μmol/L) significantly attenuated neuropeptide release. Similar findings were observed for K+-stimulated CGRP release. These results demonstrate that endogenous EETs regulate neuropeptide release from primary trigeminal ganglion neurons. Chapter 5. The hypothesis that EETs contribute to the regulation of cerebral blood flow by perivascular vasodilator nerves was tested in vivo. Electrical ethmoidal nerve stimulation increased cerebral blood flow measured by laser Doppler flowmetry via a closed cranial window. This effect was inhibited by the EETs antagonist 14,15-EEZE (10 μmol/L) from a baseline of 23±4% with 20 Hz stimulation to 7±1% following 14,15-EEZE treatment. Chemical stimulation of oral trigeminal fibers with capsaicin increased cerebral blood flow, and this effect was also inhibited by 14,15-EEZE. These findings demonstrate that EETs contribute to the regulation of cerebral blood flow by perivascular vasodilator nerves. Chapter 6: Summary and Conclusions. In the present study, the expression and constitutive activity of EETs-synthetic CYP-2J epoxygenase is demonstrated in parasympathetic sphenopalatine and trigeminal ganglia neurons. The release of vasodilator EETs from primary trigeminal ganglion neurons could not be detected, however, endogenous EETs were demonstrated to regulate the release of vasoactive neuropeptides from these neurons. This provides a mechanistic basis for the functional role of EETs in the regulation of cerebral blood flow by perivascular vasodilator nerves in vivo.

Identifier

doi:10.6083/M4CN71WH

School

School of Medicine

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