Antisense oligonucleotides against cytochrome P4502C8 attenuate EDHF-mediated Ca2+ changes and dilation in isolated resistance arteries

Using a novel vessel culture technique in combination with antisense oligon ucleotide transfection, we tested whether the endothelium-derived hyperpola rizing factor (EDHF) is a cytochrome P450 (CYP)-related compound. Isolated resistance arteries from hamster gracilis muscle (n = 19) were perfused and exposed to antisense (As), sense (S), or scrambled (Scr) oligonucleotides against the coding region of CYP2C8/9, an isoform expressed in endothelial cells. Thereafter, NO- and prostaglandin-independent, EDHF-mediated vascula r responses associated with hyperpolarization [i.e., decrease in smooth mus cle calcium (Fura 2) and vasodilation] were studied after the application o f acetylcholine (ACh). These EDHF-mediated responses were markedly attenuat ed (by 70%) by As- but not by S- or Scr-oligonucleotide treatment. However, the responses to norepinephrine (0.3 mu mol/l), the NO donor sodium nitrop russide (1 mu mol/l), and the K-Ca channel activator NS1619 (100 mu mol/l) were unaltered. As treatment, which specifically targeted the endothelial l ayer (as assessed by confocal microscopy), had no inhibitory effect on incr eases in endothelial calcium to ACh. It is concluded that a CYP2C8/9-relate d isoform functions as an EDHF synthase in hamster resistance arteries and that a product of this enzyme is an EDHF, or at least an integral part of t he signaling cascade leading to EDHF-mediated responses.