Interaction of Fluorescein Derivatives with Sulfonylurea Binding in Insulin-Secreting Cells

Abstract

Recently evidence was presented that fluorescein derivatives (e.g. phloxine B) inhibit glibenclamide binding by occupation of a nucleotide-binding site at the ATP-sensitive potassium channel (K_Atp channel). However, this conclusion was inconsistent with the results of previous studies testing the effects of nucleotides on glibenclamide binding. To elucidate the interaction mode of fluorescein derivatives with sulfonylurea binding, the effect of phloxine B on binding of [³H]glibenclamide to microsomes obtained from a pancreatic β-cell line (HIT-T15) was examined. Phloxine B inhibited specific binding of glibenclamide half-maximally at 3.2 µmol/l. The slope parameter for the displacement curve was close to one, suggesting a competitive interaction between both drugs. In accordance with this assumption 4 µmol/l phloxine B did not show an effect on the number of high-affinity binding sites but increased the apparent dissociation constant for glibenclamide by 3.1-fold and 30µmol/l phloxine B did not alter the rate of dissociation of [³H]glibenclamide. Moreover, MgATP (300 µmol/l) significantly reduced the apparent affinity for binding of phloxine B to the sulfonylurea receptor. This finding resembled the action of MgATP on binding of sulfonylureas to their receptor site. It is concluded that fluorescein derivatives inhibit glibenclamide binding due to competition for the same site at the sulfonylurea receptor

Binding of KATP channel modulators in rat cardiac membranes

“…These fits (Figure 5b) gave a high affinity component of approximately 60% of specific [ 3 H]‐P1075 binding; the IC 50 value of high affinity glibenclamide binding was 88 ± 18 nM (Table 1). Phloxine B, a fluorescein derivative, has been shown to inhibit [ 3 H]‐glibenclamide binding in HIT T1 5 insulinoma cells with IC 50 values of 2 to 3 μM (de Weille et al , 1992; Schwanstecher et al , 1995). Figure 5b shows that phloxine B inhibited [ 3 H]‐P1075 binding in a monophasic manner with an IC 50 value of 6 μM and a Hill coefficient of 1.5.…”

“…However, comparison of the high affinity component in rat cardiac membranes with the data in rat aorta shows that the potencies of glibornuride and AZ‐DF 265 are identical in the two preparations, whereas glibenclamide is four times more potent in the heart. A 160 fold potency difference between the two tissues is found for phloxine B, which available evidence in insulinoma cells suggests to bind to the glibenclamide binding site of SUR 1 (Schwanstecher et al , 1995). These differences suggest again that the [ 3 H]‐P1075 binding described here is not to the vascular smooth muscle component of the cardiac preparation.…”

Phloxine B Interacts with the Cystic Fibrosis Transmembrane Conductance Regulator at Multiple Sites to Modulate Channel Activity

“…Fluorescein derivatives inhibit glibenclamide binding to SUR (25,26). Based on these data, we were interested to learn the effect of phloxine B on glibenclamide inhibition of CFTR.…”

“…In the search for new modulators of CFTR, we tested the effect on the CFTR Cl Ϫ channel of fluorescein derivatives, a group of drugs used to investigate the function of transport ATPases and K ATP channels (23)(24)(25)(26). Like its effect on K ATP channels (25), the fluorescein derivative phloxine B both stimulated and inhibited channel activity (27, present study).…”

Potent stimulation and inhibition of the CFTR Cl− current by phloxine B

“…At μ M concentrations, phloxine B inhibited pancreatic K ATP channels; at 100 μ M , the compound also transiently reactivated channels which had reached the inactivated state in the absence of cytoplasmic ATP ( De Weille et al ., 1992 ). In addition, phloxine B has been shown to inhibit 3 H‐glibenclamide binding in membranes from insulinoma cells ( De Weille et al ., 1992 ; Schwanstecher et al ., 1995 ) and binding of the K ATP channel opener 3 H‐P1075 in membranes from skeletal ( Dickinson et al ., 1997 ) and cardiac muscle ( Löffler‐Walz & Quast, 1998 ); in all cases IC 50 values were in the low μ M range. Using the recombinant system we have recently shown that the binding site for phloxine B was located on SUR ( Hambrock et al ., 2000 ).…”