Kaushik D. Meisheri

Vascular pharmacology of ATP-sensitive K+ channels : interactions between glyburide and K+ channel openers

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.

Nicorandil-induced vasorelaxation: functional evidence for K+ channel-dependent and cyclic GMP-dependent components in a single vascular preparation.

Using a series of functional criteria, we wished to evaluate the K‘ conductance mechanism and the cyclic GMP mechanism implicated in the actions of nicorandil (NIC) as a vasodilator. In rabbit isolated superior mesenteric artery, NIC exhibited two relaxation dose-response curves (DRCs): one with a lower IC50 of 4.8 x 10-6 M for norepinephrine (NE 5

A sensitive in vitro functional assay to detect K+-channel-dependent vasodilators

mUM) contraction, and another with a higher IC50 of 1.4 x 10-4 M for 80 mM K+ contraction K+ channel blockers (TEA 1–10 mM), Ba2+ (0.1–0.5 mM), glyburide (1

Coronary Vasorelaxation by Nitroglycerin: Involvement of Plasmalemmal Calcium-Activated K+ Channels and Intracellular Ca++ Stores

mUM), and increased [K+]ex (20 mM), all caused significant attenuations in the ability of NIC to relax NE contraction, but did not influence the ability of NIC to relax high-K+ contraction. Pretreatment with 5

Enzymatic and non-enzymatic sulfation mechanisms in the biological actions of minoxidil

mUM methylene blue, a guanylate cyclase inhibitor, produced a pronounced inhibition of nitroglycerine (NTG) relaxation, but only a marginal inhibitory effect on the NIC relaxation DRC for NE contraction. Functional studies demonstrated that the inhibitory effect of NIC on NE-sensitive intracellular Ca2+ release occurred in the same concentration range as that required for relaxation of 80 mM K+ contractions (105-10 3M). Furthermore, NIC also caused increases in cellular cyclic GMP levels at this higher concentration range. Finally, NIC relaxation of NE contraction was not prone either to self-tolerance (30 mM NIC preexposure) or cross-tolerance (0.55 mM NTG preexposure) development. In contrast, a modest but significant degree of self-tolerance to NIC could be demonstrated under high-K+ contraction condition. These studies thus show the existence of both cellular mechanisms for NIC in the same vascular preparation and further show that these two mechanistic components are separate and independent. The K+ channel-dependent component occurs at lower concentrations, is blocked by K+ channel blockers, is not inhibited by methylene blue, is not associated with increases in cyclic GMP, and is not prone to tolerance development. In this, NIC resembles other K+ channel openers. The cyclic GMP-dependent component is evident at relatively higher concentrations, is associated with inhibition of [Ca2+]i release, is associated with increases in cyclic GMP levels, and is prone to tolerance development. In this, NIC resembles other nitrovasodilators. A combination of these characteristics of the actions of NIC may contribute to the differences in the acute versus chronic hemodynamic profile of NIC.

4-morpholinecarboximidine-N-1-adamantyl-N'-cyclohexylhydrochloride (U-37883A): pharmacological characterization of a novel antagonist of vascular ATP-sensitive K+ channel openers.

This study describes a sensitive in vitro relaxation assay using isolated rabbit mesenteric artery to detect the activity of a vasodilator as a K(+)-channel activator. Thus, comparison of several known K(+)-channel activators was made with other vasodilators known to work via various cellular mechanisms. The vasodilators used were minoxidil sulfate (MNXS; 5 microM), BRL-34915 (cromakalim, 0.1 microM), nicorandil (10 microM), pinacidil (1 microM), diazoxide (100 microM), sodium nitroprusside (10 microM), forskolin (1 microM), D600 (0.5 and 10 microM), hydralazine (10 microM), and viprostal (PGE1 analog, 5 microM). The concentrations chosen were equipotent to produce greater than 80% relaxation of the maximal norepinephrine (NE) (5 microM) contraction. At these concentrations, MNXS, cromakalim, pinacidil, nicorandil, and diazoxide were found to be ineffective in producing relaxation of 80 mM K(+)-contractions. Subsequently, pretreatment of tissues with 20 mM K+ before NE contraction was found to attenuate relaxation significantly by these agents, but had not effect on the relaxations by forskolin or D600. These initial criteria helped to establish cromakalim, pinacidil, nicorandil, and diazoxide as compounds acting similarly to MNXS as K(+)-channel-dependent. In another set of experiments, the effects of tetraethylammonium (TEA) (10 mM), Ba2+ (0.5 mM), and glyburide (1 microM) as K(+)-channel blockers were examined. Again it was found that these blockers had the most inhibitory effect on the class of compounds identified as K(+)-channel activators. Additionally, it was found that these K(+)-channel activators were without any significant effect on the NE-sensitive intracellular Ca2+ release as studied by contraction in a Ca2(+)-free solution. Thus, this series of functional criteria clearly show that the profile of these K(+)-channel activators is distinctly different from the vasodilators working via other mechanisms such as cyclic AMP (cAMP) (forskolin), cyclic GMP (cGMP) (nitroprusside), and Ca2+ antagonists (D600). It is suggested that appropriately defined, systematic functional studies, such as the one described here, can provide a sensitive and reproducible vascular model to discover and delineate the role of pharmacologically relevant mechanisms for vasodilation.