Blockade of swelling‐induced chloride channels by phenol derivatives

Abstract

1 In NIH3T3 fibroblasts, the chloride channel involved in regulatory volume decrease (RVD) was identified as ACln, a protein isolated from a cDNA library derived from Madin Darby canine kidney (MDCK) cells. ICI5 expressed in Xenopus laevis oocytes gives rise to an outwardly rectifying chloride current, sensitive to the extracellular addition of nucleotides and the known chloride channel blockers, DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid) and NPPB (5-nitro-2-(3-phenylpropylamino)-benzoic acid). We set out to study whether substances structurally similar to NPPB are able to interfere with RVD. 2 RVD in NIH3T3 fibroblasts and MDCK cells is temperature-dependent. 3 RVD, the swelling-dependent chloride current and the depolarization seen after reducing extracellular osmolarity can be blocked by gossypol and NDGA (nordihydroguaiaretic acid), both structurally related to NPPB. 4 The cyclic AMP-dependent chloride current elicited in CaCo cells is less sensitive to the two substances tested while the calcium-activated chloride current in fibroblasts is insensitive. 5The binding site for the two phenol derivatives onto ICan seems to be distinct but closely related to the nucleotide binding site identified as G x G x G, a glycine repeat located at the predicted outer mouth of the Icn channel protein.

The Promoter for Constitutive Expression of the Human ICln Gene CLNS1A

“…When whole-cell patch-clamp experiments are made using symmetrical CsCl concentrations, a chloride current can be measured after the reduction of the extracellular osmolality (5,20,21). Similar findings have been reported for a variety of different cells (2).…”

Molecular and functional aspects of anionic channels activated during regulatory volume decrease in mammalian cells*

“…As shown in Fig. 3, even within a given cell line, current rectification and voltage-dependent inactivation show a considerable biological bandwidth [102,105,107,274,343,352], which makes it difficult to accept that these two highly variable biophysical properties should be taken as fingerprints for RVDC [214]. Current inactivation, if present, seems to occur at the single-channel level [184], since positive potentials lead to a stepwise closure of single channels over time -an effect that can be reversed by subsequent hyperpolarizing voltage steps [134,136].…”

Hyperosmolar Solution Effects in Guinea Pig Airways. III. Studies on the Identity of Epithelium-Derived Relaxing Factor in Isolated Perfused Trachea Using Pharmacological Agents

“…The mechanism by which this effect occurred is not clear, because gossypol inhibits endothelium-dependent relaxation factor-mediated relaxation (Radermacher et al, 1990), lipoxygenase (Kulkarni and Sajan, 1997), phospholipase A 2 (Soubeyrand et al, 1997), protein kinase C (Pelosin et al, 1990), gap junctions (Ye et al, 1990), and Ca 2ϩ chan- nels (Sgaragli et al, 1993;Bai and Shi, 2002). Of relevant importance, gossypol inhibits channels and transporters that are involved in cell volume regulation, i.e., taurine channels (Ballatori et al, 1995), myo-inositol uptake (Strange et al, 1993) and volume-activated Cl Ϫ channels that are also sensitive to 4,4Ј-diisothiocyano-2,2Ј-stilbene disulfonate (Gschwentner et al, 1996;Szucs et al, 1996), an agent that blocks EpDRF-induced relaxation . It is tempting to speculate that our results are explained by inhibition of epithelial Cl Ϫ channels.…”