Rhythmic expression of vasoactive compounds in the rat brain and cerebral vasculature

Abstract

In mammals, circadian variation is evident in hormone levels, arterial pressure, heart rate and cerebral blood flow (CBF). In humans there is an increased stroke incidence in the morning. CYP2C11 and CYP4X1 are cytochrome P450 epoxygenases expressed in rat brain which convert arachidonic acid (AA) to vasodilatory epoxyeicosatrienoic acids (EETs). We have shown that these genes are expressed in a circadian or biphasic circadian manner in rat brain and vasculature. Here we investigate rhythms in cultured astrocytes and cerebral microvascular endothelial cells over 48 hours using RT‐PCR. Using Western blotting, we measured CYP2C11 and CYP4X1 protein levels over 24 hours and CYP450 enzyme activity by measuring EETs and 20‐HETE formation following addition of the substrate AA. We show Cyp2c11, Cyp4x1 and Cyp4a2 mRNA expression is rhythmic with a 12 hour period although the phase differs between genes. This pattern was also apparent at the protein level. Preliminary P450 activity data suggest rhythmic formation of EETs and 20‐HETE with 12 and 24 hour periods. Our data indicate CYP450 enzymes have rhythmic mRNA and protein expression and activity levels, suggesting their involvement in regulation of rhythmic physiological phenomena, such as CBF. Our findings will lead to a better understanding of the mechanisms underlying increased morning stroke incidence in humans.