Interaction of adenosine and cytochrome P450 epoxygenase pathways in neurovascular coupling during whisker stimulation

Introduction

Adenosine has been implicated in mediating functional hyperemia, but the role of specific adenosine receptors is unknown. Cerebrovascular effects of exogenous adenosine are largely mediated by adenosine A2A and A2B receptors^{1, 2}. In addition, A2A and A2B receptor activation can increase Ca in astrocytes, which may mobilize arachidonic acid. Epoxyeicosatrienoic acid (EETs) are synthesized from arachidonic acid in astrocytes by cytochrome P450 epoxygenase and open KCa channels on cerebrovascular smooth muscle, leading to dilation. Epoxygenase inhibitors MS-PPOH and miconazole reduce functional hyperemia ³ . Presently, the epoxygenase pathway was further investigated with the recently described EETs antagonists 14, 15-EEZE and its methylsulfonylimide derivative 14, 15-EEZE-mSI ⁴ . The potential interaction of the adenosine and EETs pathways were investigated by combining treatments of 14, 15-EEZE or MS-PPOH with the adenosine A2A receptor antagonist ZM241385 or the A2B antagonist alloxazine to determine if there was an additive inhibitory effect on the blood flow response to whisker stimulation.

Methods

The percent change in laser-Doppler flow (LDF) over whisker barrel cortex during 60 s whisker stimulation was measured in chloralose-anesthetized rats at baseline, at 1 h of cortical superfusion of a single antagonist, and after an additional hour of either single or combination antagonist superfusion. Responses were compared within groups by repeated measures ANOVA and paired t-test.

Results

ZM241385 (1 mg/kg iv + 1 μM CSF superfusion) had no effect on the increase in LDF during whisker stimulation (from 17±4% to 16±5% at 1 h and 17±5% at 2h; ±SD; n=6). In contrast, alloxazine (1 mg/kg iv + 1 μM superfusion; n=6) reduced the LDF response from 17±4% to 10±4% at 1 h and to 12±4% at 2 h of superfusion. Superfusion of 14, 15-EEZE (n=6) reduced the LDF response from 14±4% to 7±3% at 1 h and to 5±2% at 2 h. Similar reductions were obtained with 30 μM 14, 15-EEZE-sMI and 20 μM MS-PPOH. In another group (n=8) in which alloxazine was found to reduce the response from 15±2% to 9±3%, subsequent superfusion of both alloxazine and 14, 15-EEZE failed to significantly reduce the LDF response further (8±5%). Likewise, addition of alloxazine with MS-PPOH failed to reduce the response further (9±3%) compared to MS-PPOH alone (11±4%; n=6). 14, 15-EEZE still reduced the response (8±4%) in the presence of ZM241385 compared to ZM241385 alone (13±3%; n=6).

Conclusions

We conclude 1) that adenosine A2B receptors, rather than A2A receptors, play a significant role in coupling cortical blood flow to neuronal activity, 2) that the role of EETs previously supported by the use of epoxygenase inhibitors is strengthened by new evidence with EETs antagonists, and 3) that the epoxygenase and adenosine A2B receptor signaling pathways are not functionally additive.