Ca 2+ ‐activated K + channels of small and intermediate conductance participate in the control of eNOS phosphorylation at threonine 495

Ca 2+ ‐activated K + channels of small (SK Ca ) and intermediate (IK Ca ) conductance are involved in the NO‐dependent control of vasomotor tone. To evaluate the participation of SK Ca and IK Ca channels in the NO signaling activated in mesenteric vessels by 1‐min stimulation with 100nM ACh, we analyzed the changes in NO (chemiluminiscence), superoxide (dihydroethidium) and eNOS phosphorylation at serine 1177 (p‐eNOS S1177 ) and threonine 495 (p‐eNOS thr495 ). NO production activated by ACh was abolished by simultaneous blockade of SK Ca and IK Ca channels with 500nM apamin and 10μM TRAM‐34, respectively. Apamin, TRAM‐34 or ACh did not affect p‐eNOS S1177 . However, ACh stimulation in presence of apamin resulted in an increase in p‐eNOS thr495 . TRAM‐34 alone or in combination with apamin also elicited an increment in p‐eNOS thr495 , which was not modified by ACh. In presence of apamin and TRAM‐34, ACh evoked an increased in superoxide production that was sensitive to 100μM apocynin. In addition, blockade of NADPH oxidase with apocynin precluded the increase in p‐eNOS thr495 and the inhibition of ACh‐induced NO production observed during the treatment with apamin and TRAM‐34. These results indicate that blockade of SK Ca and IK Ca channels leads to a NADPH oxidase‐dependent superoxide production, which in turn inhibits NO production by increasing the eNOS phosphorylation at p‐eNOS thr495 . FONDECYT 1100850 and 1090757, Anillos ACT‐71