This study in isolated rabbit superior artery (RMA) investigated the interactions between glyburide, a known blocker of vascular ATP-sensitive K+ channels (KATP), and several chemically diverse potassium channel openers (PCOs): minoxidil sulfate (MNXS; 5 microM), pinacidil (1 microM), cromakalim (0.5 microM) and RP-49356 (1 microM; a PCO from Rhône Poulenc). Relaxation time courses for these PCOs were obtained in norepinephrine (NE; 5 microM)-precontracted RMA, and the concentrations of PCOs found to be equipotent to each other in terms of the degree of maximum relaxation (about 80%) and the time course of relaxation (within 15 min) were chosen for further study. This was taken as a functional indicator of a similar degree as well as similar kinetics of K+ channel opening by these PCOs. Pretreatment with glyburide (10-500 nM) produced a dose-dependent inhibition of the PCO relaxation time course. The glyburide IC50s against pinacidil, MNXS and RP-49356 were statistically similar and ranged from 72-79 nM. The glyburide IC50 against cromakalim was a modest 2-fold higher, at 148 nM. In contrast, pretreatment with charybdotoxin (200 nM) produced no significant inhibition of the maximum relaxation produced by these PCOs. Furthermore, glipizide, a sulfonylurea that is 10- to 25-fold less potent than glyburide for insulin secretion, was found to be 20- to 30-fold less potent than glyburide as a vascular KATP antagonist. These data suggest a mechanistic model in which these structurally diverse PCOs share a common critical step in the sequence of events leading to the KATP opening, and that glyburide interferes with this common critical step to produce a similar type of blockade against all four PCOs. Interaction studies with glyburide and pinacidil demonstrated 15 min to be the optimal pretreatment time for glyburide to produce maximal inhibition. Glyburide also reversed existing pinacidil relaxation regardless of the degree of pre-existing relaxation. These data suggest that glyburide is able to produce its blockade regardless of the state of K+ channel activation. Studies on the effect of pH (6.4 vs. 7.3) showed that at acidic pH, pinacidil became less effective and the effectiveness of glyburide was significantly enhanced, whereas the actions of D600 remained unchanged. These data suggest the effects of both openers and blockers of the KATP are strongly pH dependent.