Sulfonylurea binding to a low-affinity site inhibits the Na/K-ATPase and the KATP channel in insulin-secreting cells.

Abstract

A B ST RA C T We have used hamster insulinoma tumor (HIT) cells, an insulin-secreting tumor cell line, to investigate modulation of the Na/K-ATPase and of the ATP-sensitive K channel (KATe) by the sulfonylurea glyburide. Membrane proteins from cells cultured in RPMI with 11 mM glucose have at least two glyburide receptor populations, as evidenced by high and low binding affinity constants, (Kd = 0.96 and 91 nM, respectively). In these cells KATP channel activity was blocked by low glyburide concentrations, IC50 = 5.4 nM. At 12.5 nM glyburide the inhibition developed slowly, "r = 380 s, and caused reduction of channel activity by 75%. At higher concentrations, however, inhibition occurred at a fast rate, -r = 42 s at 100 nM, and was almost complete. Na/K-ATPase activity measured enzymatically and electrophysiologically was also suppressed by glyburide, but higher concentrations were needed, ICs0 = 20-40 nM. Inhibition occurred rapidly, T = 30 s at 50 nM, when maximum, activity was reduced by 40%. By contrast, cells cultured in RPMI supplemented with 25 mM glucose exhibit a single receptor population binding glyburide with low affinity, Kd = 68 nM. In these cells inhibition of the Na/K-ATPase by the sulfonylurea was similar to that observed in cells cultured in 11 mM glucose, but KAT P channel inhibition was markedly altered. Inhibition occurred only at high concentrations of glyburide and at a fast rate; maximum inhibition was observed at ~100 nM. Based on these data, we propose that glyburide binding to the high affinity site affects primarily KAT P channel activity, while interaction with the low affinity site inhibits both Na/K-ATPase and KAT P channel activities. The latter observation suggests possible functional interactions between the Na/K-ATPase and the KATP channel.

Interactions of K+ ATP channel blockers with Na+/K+-ATPase

“…Evidently, such balance also exists in the amphiphilic structure of glyburide, allowing its interaction with the postulated amphiphilic binding site on the enzyme [9]. Mechanistic issues aside, our present data indicate that depending on the prevailing ATP concentrations, Na + /K + -ATPase may indeed be either activated or inhibited by glyburide concentrations, up to 500 lM, that are often used for the presumed specific inhibition of the plasma membrane K ATP channels in various cell types [10,29]. This, and the possibility that plasma membrane K ATP channels of different cells may have different sulfonylurea sensitivities [23], suggest the necessity of caution in the use of glyburide in studies where the proposed interaction between Na + /K + -ATPase and any K ATP channel is examined.…”

“…Since this drug has been used in a number of studies to explore the mechanism of suggested functional interaction between Na + /K + -ATPase and the plasma membrane K ATP channels of renal epithelia and other cell types [29], the possibility of its direct interaction with Na + /K + -ATPase has been considered before [10][11][12]. However, when glyburide effects on Na + /K + -ATPase activities of crude isolated membranes or homogenates have been attempted, either inhibition [10] or lack of effect [11] have been reported. The present data on the purified kidney enzyme clearly show both activating and inhibitory effects of Membranes were prepared and assays were done at 5 mM ATP as indicated in Materials and methods.…”

“…Due to conflicting reports on the direct inhibitory effect of glyburide on Na + /K + -ATPase [10][11][12], we examined the effects of varying concentrations of this drug on the purified enzyme activity, using optimal and suboptimal substrate concentrations: 5 mM ATP and 50 lM ATP. This hydrophobic sulfonylurea, like most fatty acids and related amphiphiles, exhibited both activating and inhibitory effects depending on substrate concentration.…”

“…The primary aim of this work, therefore, was to examine the effects of 5-HD on purified Na + /K + -ATPase. Because of the reported disagreements [10][11][12] on the interaction of Na + /K + -ATPase with another widely used K ATP channel blocker, glyburide (glybenclamide), we have also examined the effects of this compound on the purified Na + /K + -ATPase.…”

Glibenclamide Effects on Renal Function and Histology after Acute Hemorrhage in Rats under Sevoflurane Anesthesia

“…Glibenclamide, as an intervention agent, also works in other sites, antagonizing thromboxane A2 receptors and inhibiting Na+/K+ ATPase. [29] The understanding of the interactive nature of tissue protection mechanisms (cellular, inflammatory, endothelial, tubular, etc.) is still imprecise and of difficult distinction.…”