Rhythmic expression of cytochrome P450 epoxygenases CYP4x1 and CYP2c11 in the rat brain and vasculature.

Mammals have circadian variation in blood pressure, heart rate, vascular tone, thrombotic tendency, and cerebral blood flow (CBF). These changes may be in part orchestrated by circadian variation in clock gene expression within cells comprising the vasculature that modulate blood flow (e.g., fibroblasts, cerebral vascular smooth muscle cells, astrocytes, and endothelial cells). However, the downstream mechanisms that underlie circadian changes in blood flow are unknown. Cytochrome P450 epoxygenases (Cyp4x1 and Cyp2c11) are expressed in the brain and vasculature and metabolize arachidonic acid (AA) to form epoxyeicosatrienoic acids (EETs). EETs are released from astrocytes, neurons, and vascular endothelial cells and act as potent vasodilators, increasing blood flow. EETs released in response to increases in neural activity evoke a corresponding increase in blood flow known as the functional hyperemic response. We examine the hypothesis that Cyp2c11 and Cyp4x1 expression and EETs production vary in a circadian manner in the rat brain and cerebral vasculature. RT-PCR revealed circadian/diurnal expression of clock and clock-controlled genes as well as Cyp4x1 and Cyp2c11, within the rat hippocampus, middle cerebral artery, inferior vena cava, hippocampal astrocytes and rat brain microvascular endothelial cells. Astrocyte and endothelial cell culture experiments revealed rhythmic variation in Cyp4x1 and Cyp2c11 gene and protein expression with a 12-h period and parallel rhythmic production of EETs. Our data suggest there is circadian regulation of Cyp4x1 and Cyp2c11 gene expression. Such rhythmic EETs production may contribute to circadian changes in blood flow and alter risk of adverse cardiovascular events throughout the day.

Adenosine can mediate its actions through generation of reactive oxygen species.

Adenosine is an important cerebral vasodilator, but mediating mechanisms are not understood. We investigated the expression of adenosine receptor subtypes in isolated cerebral arterial muscle cells (CAMCs), and their role in adenosine-induced superoxide (O(2)(-)) generation and reduction in cerebral arterial tone. Reverse transcriptase-PCR, western blotting, and immunofluorescence studies have shown that CAMCs express transcript and protein for A1, A(2A), A(2B), and A(3) adenosine receptors. Stimulation of CAMCs with adenosine or the A(2A) agonist CGS-21680 increased the generation of O(2)(-) that was attenuated by the inhibition of A(2A) and A(2B) adenosine receptor subtypes, or by the peptide inhibitor of nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase gp91ds-tat, or by the mitochondria uncoupler 2,4-dinitrophenol. Application of adenosine or CGS-21680 dilated pressure-constricted cerebral arterial segments that were prevented by the antioxidants superoxide dismutase (SOD) conjugated to polyethylene glycol (PEG) and PEG-catalase or by the A(2B) adenosine receptor antagonist MRS-1754, or by the mixed A(2A) and A(2B) antagonist ZM-241385. Antagonism of the A(2A) and A(2B) adenosine receptors had no effect on cerebral vasodilatation induced by nifedipine. These findings indicate that adenosine reduces pressure-induced cerebral arterial tone through stimulation of A(2A) and A(2B) adenosine receptors and generation of O(2)(-) from NADPH oxidase and mitochondrial sources. This signaling pathway could be one of the mediators of the cerebral vasodilatory actions of adenosine.

Loss of Nocturnin, a circadian deadenylase, confers resistance to hepatic steatosis and diet-induced obesity.

The mammalian circadian system consists of a central oscillator in the suprachiasmatic nucleus of the hypothalamus, which coordinates peripheral clocks in organs throughout the body. Although circadian clocks control the rhythmic expression of a large number of genes involved in metabolism and other aspects of circadian physiology, the consequences of genetic disruption of circadian-controlled pathways remain poorly defined. Here we report that the targeted disruption of Nocturnin (Ccrn4l) in mice, a gene that encodes a circadian deadenylase, confers resistance to diet-induced obesity. Mice lacking Nocturnin remain lean on high-fat diets, with lower body weight and reduced visceral fat. However, unlike lean lipodystrophic mouse models, these mice do not have fatty livers and do not exhibit increased activity or reduced food intake. Gene expression data suggest that Nocturnin knockout mice have deficits in lipid metabolism or uptake, in addition to changes in glucose and insulin sensitivity. Our data support a pivotal role for Nocturnin downstream of the circadian clockwork in the posttranscriptional regulation of genes necessary for nutrient uptake, metabolism, and storage.