Role of ADP-ribose in 11,12-EET-induced activation of KCa channels in coronary arterial smooth muscle cells

We recently reported that cADP-ribose (cADPR) and ADP-ribose (ADPR) play an important role in the regulation of the Ca2+-activated K+ (K-Ca) channel activity in coronary arterial smooth muscle cells (CASMCs). The present study determined whether these novel signaling nucleotides participate in 11,12-epoxyeicosatrienoic acid (11,12-EET)-induced activation of the K-Ca channels in CASMCs. HPLC analysis has shown that 11,12-EET increased the production of ADPR but not the formation of cADPR. The increase in ADPR production was due to activation of NAD glycohydrolase as measured by a conversion rate of NAD into ADPR. The maximal conversion rate of NAD into ADPR in coronary homogenate was increased from 2.5 +/- 0.2 to 3.4 +/- 0.3 nmol.min(-1).mg protein(-1) by 11,12-EET. The regioisomers of 8,9-EET, 11,12-EET, and 14,15-EET also significantly increased ADPR production from NAD. Western blot analysis and immunoprecipitation demonstrated the presence of NAD glycohydrolase, which mediated 11,12- EET-activated production of ADPR. In cell-attached patches, 11,12-EET (100 nM) increases K-Ca channel activity by 5.6-fold. The NAD glycohydrolase inhibitor cibacron blue 3GA (3GA, 100 muM) significantly attenuated 11,12-EET-induced increase in the KCa channel activity in CASMCs. However, 3GA had no effect on the K-Ca channels activity in inside-out patches. 11,12-EET produced a concentration-dependent relaxation of precontracted coronary arteries. This 11,12-EET-induced vasodilation was substantially attenuated by 3GA (30 muM) with maximal inhibition of 57%. These results indicate that 11,12-EET stimulates the production of ADPR and that intracellular ADPR is an important signaling molecule mediating 11,12- EET-induced activation of the K-Ca channels in CASMCs and consequently results in vasodilation of coronary artery.