A synthetic prostone activates apical chloride channels in A6 epithelial cells

Abstract

The bicyclic fatty acid lubiprostone (formerly known as SPI-0211) activates two types of anion channels in A6 cells. Both channel types are rarely, if ever, observed in untreated cells. The first channel type was activated at low concentrations of lubiprostone (<100 nM) in >80% of cell-attached patches and had a unit conductance of approximately 3-4 pS. The second channel type required higher concentrations (>100 nM) of lubiprostone to activate, was observed in approximately 30% of patches, and had a unit conductance of 8-9 pS. The properties of the first type of channel were consistent with ClC-2 and the second with CFTR. ClC-2's unit current strongly inwardly rectified that could be best fit by models of the channel with multiple energy barrier and multiple anion binding sites in the conductance pore. The open probability and mean open time of ClC-2 was voltage dependent, decreasing dramatically as the patches were depolarized. The order of anion selectivity for ClC-2 was Cl > Br > NO(3) > I > SCN, where SCN is thiocyanate. ClC-2 was a "double-barreled" channel favoring even numbers of levels over odd numbers as if the channel protein had two conductance pathways that opened independently of one another. The channel could be, at least, partially blocked by glibenclamide. The properties of the channel in A6 cells were indistinguishable from ClC-2 channels stably transfected in HEK293 cells. CFTR in the patches had a selectivity of Cl > Br >> NO(3) congruent with SCN congruent with I. It outwardly rectified as expected for a single-site anion channel. Because of its properties, ClC-2 is uniquely suitable to promote anion secretion with little anion reabsorption. CFTR, on the other hand, could promote either reabsorption or secretion depending on the anion driving forces.

Stimulation of mucosal secretion by lubiprostone (SPI-0211) in guinea pig small intestine and colon

“…Our results with lubiprostone at the whole organ level are consistent with the interpretation of results acquired by others at the cellular level with ClC-2-transfected HEK-293 cells and confluent T84 and A6 cell monolayers (2,16). Lubiprostone opens epithelial Cl Ϫ channels, and this was reflected as stimulation of I sc in our Ussing chamber studies with both small and large intestinal preparations.…”

“…The concentration dependence and EC 50 values in the low nanomolar range for stimulation of I sc by lubiprostone in our study are reminiscent of the results of Bao et al and Cuppoletti et al (2,16), who reported that application of low nanomolar concentrations of lubiprostone to confluent epithelial cell monolayers stimulated electrogenic Cl Ϫ secretion and I sc across the monolayers. Our finding that application of lubiprostone to either the mucosal or submucosal side of the preparations stimulated I sc was unexpected because the ClC-2 channels, on which lubiprostone is believed to act, are reported to be expressed in the apical membrane and should be opened by mucosal but not serosal application.…”

“…A second pair of electrodes was connected to an automated voltage-clamp apparatus, which compensated for the solution resistance between the PD-sensing bridges. The flat-sheet preparations were mounted between halves of Ussing chambers, which had a total cross-sectional area of 0.64 cm 2 . The tissues were bathed on both sides with 10 ml of Krebs solution and maintained at 37°C by circulation from a temperature-controlled water bath.…”

“…Stimulation of I sc across confluent T84 cell monolayers occurs with an EC 50 of 18 nM, and activation of the ClC-2 channels in transfected HEK-293 cells occurs also with a low EC 50 of 17 nM. Low concentrations of lubiprostone Ͻ100 nM activate the ClC-2 channel in cultured cellular monolayers from Xenopus kidney (A6 cells), whereas concentrations greater than 100 nM activate the CFTR Cl Ϫ channel in the same monolayers (2). Human recombinant ClC-2 channel currents recorded in HEK-293 expression systems are stimulated by agents, such as forskolin, that elevate intracellular cAMP and stimulate protein kinase A (PKA).…”

Engineered Membranes and Transporters for Useful Devices

“…The ClC-2 channel is known to be activated by a drug, lubiprostone, that is currently used in treatment of constipation (14,15) and by a large variety of fatty acids, including arachidonic acid (13). In ongoing work, we have been searching for the binding region for lubiprostone and other fatty acids.…”