Sulfonylureas Rapidly Cross Phospholipid Bilayer Membranes by a Free-Diffusion Mechanism

Abstract

Because sulfonylureas directly activate the exocytotic machinery, we were interested in the extent to which these compounds penetrate the ␤-cell plasma membrane and the underlying molecular mechanism(s). We now provide evidence that sulfonylureas cross phospholipid bilayer membranes rapidly and effectively by a free-diffusion mechanism. Two sulfonylurea compounds investigated by 1 H nuclear magnetic resonance spectroscopy, glibenclamide and tolbutamide, were found to incorporate into phospholipid bilayers, with the ionizable sulfonamide exposed to the aqueous interface and its apparent dissociation constant (pK a ) increased to ϳ7.0. Diffusion of weak amphiphilic acids across membranes is associated with a measurable change in pH. Thus, by using a fluorescencebased pH assay, we could investigate the diffusion of sulfonylurea compounds across phospholipid bilayer membranes. A fluorescent pH indicator (pyranin or [2,7-bis (2-carboxyethyl)-5(6)-carboxyfluorescein] [BCECF]) was trapped in egg phosphatidylcholine vesicles. Addition of glibenclamide decreased internal pH (pH in ), and addition of albumin reversed this drop by 50%. With the same amount of tolbutamide, the decrease in pH in was much smaller, primarily because of the lower partitioning of tolbutamide into phospholipid bilayers. Using similar protocols, we also demonstrated diffusion by the same mechanism across the ␤-cell plasma membrane. Thus, we now provide a molecular mechanism by which sulfonylureas can penetrate the plasma membrane and reach intracellular sites regulating exocytosis. Diabetes 52:2526 -2531, 2003 S ulfonylureas are drugs that stimulate secretion of insulin from the pancreatic ␤-cells (1,2) and are therefore used extensively in the treatment of type 2 diabetes. It is well established that sulfonylureas stimulate insulin release by interacting with the high-affinity 140-kDa SUR1 protein of the ATP-regulated K ϩ channel at the cytoplasmic leaflet of the plasma membrane. This interaction closes the channel, causing membrane depolarization, the opening of voltage-gated L-type Ca 2ϩ channels, an increase in cytoplasmic-free Ca 2ϩ concentration, and the activation of the secretory machinery (3,4). We have also shown that sulfonylureas stimulate insulin exocytosis by directly interacting with the secretory machinery and not through closure of the plasma membrane ATP-regulated K ϩ channel (5-7). This effect may constitute part of the therapeutic benefits of sulfonylureas and contribute to their hypoglycemic action in diabetes. Although this direct effect of sulfonylureas on insulin exocytosis is now well established and suggestions for underlying molecular mechanisms have been put forward (8), the physiological relevance of these findings are still in question. This skepticism originates primarily from the difficulty in envisaging how the sulfonylureas bypass the ␤-cell plasma membrane rapidly and effectively, thereby interacting with intracellular binding sites involved in the regulation of exocytosis. Indeed, earlier studies suggested that ...

Glibenclamide Treatment Recruits β-Cell Subpopulation Into Elevated and Sustained Basal Insulin Synthetic Activity

“…At variance with first-generation sulfonylureas, the compounds also generate a secretory activity after their removal from the extracellular medium (22). This sustained effect coincides with cellular accumulation of the drug, primarily in membranes of secretory vesicles and mitochondria (22)(23)(24)(25). It has been attributed to a protein kinase C activation following glibenclamide stimulation of diacylglycerol synthesis (21,26).…”

Actions of glucagon-like peptide-1 on KATP channel-dependent and -independent effects of glucose, sulphonylureas and nateglinide

“…The binding of sulphonylureas to the sulphonylurea receptor (SUR)1 subunit of these channels results in closure of the Kir6.2 pore, which in turn evokes membrane depolarisation, influx of extracellular calcium and, ultimately, exocytosis of insulin (Ashcroft & Gribble 1999, Bryan et al 2005. In addition to this mechanism, several studies have reported direct intracellular effects of sulphonylureas upon exocytosis independent of K ATP -channel function (Flatt et al 1994, Eliasson et al 1996, Tian et al 1998, Ball et al 2000a, Kamp et al 2003.…”

Diazoxide Improves Hormonal Counterregulatory Responses to Acute Hypoglycemia in Long-standing Type 1 Diabetes

“…Moreover, they were able to detect diazoxide in the cerebrospinal fluid of rodents after oral ingestion reaching levels of 0.26 6 0.06 mg/mL 1 h after gavage and 0.78 6 0.03 mg/mL by 4 h, providing convincing evidence that diazoxide penetrates the blood-brain barrier (BBB) (35). Species differences may affect BBB permeability to diazoxide; however, diazoxide contains an ionizable sulfonyl group that makes it extremely lipid soluble and therefore able to partition into the lipid bilayer for penetration through the BBB (36). These studies support the hypothesis that diazoxide acts to amplify the CRR to acute hypoglycemia through a direct action in the brain.…”