Different types of blockers of the intermediate-conductance outwardly rectifying chloride channel in epithelia

Abstract

Epithelial chloride channels can be blocked by various inhibitors, which show considerable differences in their molecular structure. In the present patch-clamp study, we compared different blockers of one type of epithelial Cl- channel with respect to their inhibitory potency. We applied the blockers to excised inside-out-or outside-out-oriented membrane patches of cultured HT29 colon carcinoma and respiratory epithelial cells (REC) containing the outwardly rectifying intermediate-conductance (ICOR) chloride channel. Four types of inhibitory compounds were tested: stilbene disulphonate derivatives, indanyloxyacetic acid, amidine, and arylaminobenzoates. The concentrations for half-maximal inhibition (IC50) for the different channel blockers were (mumol/l): 4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulphonic acid 100; 4,4'-diisothiocyanato-stilbene-2,2'-disulphonic acid 80; indanyloxyacetic acid 9; 4,4'-dinitrostilbene-2,2'-disulphonic acid 8; amidine 8 and 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) 0.9. All compounds, when applied to the cytosolic side of the channel, induced a flicker-type block of the ICOR Cl- channel at lower concentrations and a complete channel inhibition at higher concentrations. The inhibitory potency of NPPB was much higher when it was added to the external surface of the channel in outside-out-oriented membrane patches. At 1 mumol/l the inhibition was complete. All blocker effects were fully reversible. The probe with the highest affinity (NPPB) and a closely related compound 5-nitro-2-(3-phenylethylamino)-benzoate (NPEB) were used to construct macromolecular probes by linking these blockers to aminopolyethyleneglycol (PEG) or amino-ethyl-O-dextran (5 kDa).2+ These macromolecular NPPB and NPEB derivatives inhibited the ICOR Cl- channels only from the outside but had no effect on the cytosolic side.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of an outwardly rectifying Cl‐ conductance in single proximal tubule cells isolated from frog kidney.

“…However, in contrast to all but one of these channels, GC1 does not show inactivation at positive potentials. Additionally, the values for half-maximal inhibition of GCc by DIDS, 41 to 9/M, are well within the range for inhibition of DIDS on a number of Cl-channels (Tilmann et al 1991;Lewis et al 1993;Diaz et al 1993). …”

Chloride Channel Inhibition Blocks the Protection of Ischemic Preconditioning and Hypo-Osmotic Stress in Rabbit Ventricular Myocardium

“…Control, preconditioned, and oxygenated baseline (240-minute incubation in an O 2 atmosphere at 37°C) myocytes were simultaneously treated with either of 2 selective Cl -channel blockers that have distinct molecular structures and inhibitory mechanisms, 10 mol/L IAA-94 or 1 mol/L 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) (Research Biochemicals International Inc), 30,31 or they were treated with the vehicle of either drug (0.03% and 0.09% ethanol for IAA-94 and NPPB, respectively). The blocker was initially added to the myocyte suspension either 10 minutes before IP or just before the long SI (IAA-94 only).…”

“…IAA-94 was added either 10 minutes before IP or just before the long SI, whereas NPPB was added before IP only. Myocyte viability was assessed at the following time points ( * ): end of stabilization, before long SI, during long SI alone (30,60,90,120, and 180 minutes), and at the end of SI (45 minutes) and during SR (15,60, and 120 minutes) in the ischemia/reperfusion protocol. channels play a role in the protection of IP during the long ischemia, hearts were subjected to the same control and IP protocols with a 10-minute exposure to IAA-94 or NPPB, at the same concentrations used in the isolated myocyte model, before the long ischemia.…”

P‐glycoprotein regulates a volume‐activated chloride current in bovine non‐pigmented ciliary epithelial cells.

“…Pharmacology of the CO-current NPPB has been reported to be a most potent blocker for intermediate-conductance outwardly rectifying Cl-channel in HT29 epithelial cells (Tilmann et al 1991) and also was reported to block the Cl-pathways involved in the regulatory volume decrease in intestinal 407 cells (Kubo & Okada, 1992) and virus-transformed ciliary epithelial cells . In our study the volume-sensitive Cl currents showed a high sensitivity to NPPB with an IC50 of 13-4 uM, which compares with 25 uM for a volumeregulatory Cl-channel in cultured human intestinal 407 epithelial cells (Kubo & Okada, 1992).…”