Proc. Natl. Acad. Sci. USA Vol. 87, pp. 5706-5709, August 1990 Physiology/Pharmacology Direct modulation of secretory chloride channels by arachidonic and other cis unsaturated fatty acids (tracheal epithelia/colonic epithelia/cystic fibrosis/T84 cells/asthma) TZYH-CHANG HWANG*, SANDRA E. GUGGINOt, AND WILLIAM B. GUGGINO* Departments of *Physiology and of tMedicine and Neuroscience, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205 Communicated by John W. Littlefield, May 7, 1990 ABSTRACT The effect of fatty acids on Cl- channels and bronectin (10 ,ug/ml), collagen (6 mg/ml), and bovine serum transepithelial Cl- secretion is investigated. Patch-clamp ex- albumin (100 ug/ml) and were maintained at 370C in 5% periments show that arachidonic acid blocks Cl- channels in a C02/95% air. T84 cells were grown in a 1:1 solution of dose-dependent manner. Kinetic analysis shows that the mean Dulbecco's modified Eagle's medium and Ham's F-12 me- open time is decreased 10-fold with 25 FM arachidonic acid. dium, supplemented with 10% fetal calfserum and penicillin/ There is a linear relationship between the reciprocal of mean streptomycin (100 units; 100 mg/ml). For transepithelial open time and blocker concentration within the range of 1 to 25 short circuit experiments, the cells were seeded onto collagen IAM. The reciprocal ofmean blocked time does not change with gel supports (0.2 cm2), which were prepared as described arachidonic acid concentration. Other cis unsaturated fatty (17). acids, including oleic, linoleic, and ricinoleic acids, demon- Patch-Clamp Experiments. For patch-clamp experiments, strate similar blocks. Trans unsaturated acids such as elaidic human fetal tracheal epithelial cells were transferred onto acid and saturated fatty acids, including stearic, palmitic, and glass chips coated with collagen/fibronectin/bovine serum myristic acids, do not inhibit the channel at 20 pM. Ricinoleic albumin as described above. In all experiments, single chan- acid decreases short circuit current in T84 cells, a colonic nel currents were recorded from excised apical membrane carcinoma cell line that secretes CL. Our results suggest that patches in an inside-out configuration (18). Data were re- the direct effect of arachidonic and other fatty acids on Cl- corded by an EPC-7 patch-clamp amplifier (List Electronics, secretion is to block Cl- channel current. Darmstadt, F.R.G.), filtered at 1 kHz (Frequency Devices, Haverhill, MA), digitized with a PCM-601 digital audio processor (Sony), and stored on a video cassette recorder Outwardly rectifying, secretory Cl- channels are involved in (JVC). The bath solution contained 150 mM NaCl, 2 mM fluid production in airway and intestinal epithelial cells (1). In MgCl2, 1 mM EGTA, 5 mM Hepes, and 0.5 mM CaCl2 (free epithelial cells from cystic fibrosis patients, secretory Cl- Ca2' was 110 nM measured with Fura-2). The pipette solu- channel activation by cAMP-dependent protein kinase or tion contained 150 mM NaCl, 2 mM CaCl2, and 5 mM Hepes. protein kinase C is defective (2-5). This abnormality is The pH of both solutions was adjusted to 7.3 with Tris base. thought to contribute to the production of thick mucous in Kinetic Analysis. Kinetic analysis was done with P-Clamp cystic fibrosis. Mucous plugging is also a clinical manifesta- software (version 5.05, Axon Instruments, Burlingame, CA). tion in patients with asthma (6), but the underlying mecha- The histograms were constructed by defining one-half of the nism is unknown. Arachidonic acid metabolites are respon- open channel current as the threshold. The first bin was not sible for the bronchospasm in asthma attacks (7-9), and these included in histogram fitting because of the limited band- chemical mediators are produced in many types of airway width. The long, interburst, closed events lasted from several cells (10-12). Likewise, increased arachidonic acid and ei- hundred milliseconds to minutes and varied from patch to cosanoids are a manifestation of inflammatory bowel disease patch (19). Because it was difficult to collect enough long (13-15). Because arachidonic and other fatty acids contribute closed events for kinetic analysis and because the long closed to clinical symptoms of diseases in Cl- transporting epithelia events varied between channels, we chose to analyze only and are known to be involved in signal transduction, we open and closed events within the bursts. The higher filter evaluated the role of these compounds in direct modulation frequency in our experiments (1 kHz instead of 400 Hz) of Cl- channel activity. resulted in shorter mean open and closed times than those reported by others (19). Short Circuit Current Measurement. The short circuit MATERIALS AND METHODS currents were measured on T84 cells with a DVC-1000 volt- Materials. All fatty acids except elaidic acid were pur- age-current clamp (World Precision Instruments, New Ha- chased from Sigma. Elaidic acid was from Alltech Associ- ven, CT) in modified Ussing chambers as described (20). The ates. Arachidonic acid from either Sigma or Alltech had a temperature of the bath was maintained at 37°C by a water similar effect in the present report. Diphenylamine-2-carbox- jacket. Bath solutions contained 140 mM NaCl, 2.3 mM ylate (DPC) was from Fluka. All fatty acids were dissolved in K2HPO4, 0.4 mM KH2PO4, 1.3 mM CaCl2, 1.2 mM dimethyl sulfoxide as stocks and were stored at -20°C before MgCl2-6H2O, 10 mM Hepes, and 5 mM glucose. use. The final concentration ofarachidonic acid was obtained by 1:1000 dilution ofthe stocks to keep the dimethyl sulfoxide at 0.1%. At this concentration, dimethyl sulfoxide alone has RESULTS no effect on the single channel kinetics (16). Effect of Arachidonic Acid on Channel Kinetics. Cl- chan- Cell Culture. Human fetal tracheal cells were grown in nels in excised inside-out apical membrane patches from flasks coated with a mixture containing distilled water, fi- human fetal tracheal cells were activated by depolarizing the membrane potential to values ranging from +50 to +120 mV. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: DPC, diphenylamine-2-carboxylate; I&,S short circuit in accordance with 18 U.S.C. §1734 solely to indicate this fact. current. 5706 Downloaded by guest on September 26, 2021 Physiology/Pharmacology: Hwang et al. Proc. Natl. Acad. Sci. USA 87 (1990) 5707 Control, +50 mV In the absence of fatty acid, the channel opens in bursts _Lu L. -... interspersed with long closed periods. The kinetic behavior MI~~~~Wm"' WUNo TIInt - i -- T within the bursts shows little variation from channel to 1 I' r -Trlljl- channel (19). Both open time and closed time histograms of Arachidonic acid 5 jlM events within the bursts can be fitted with one exponential (Fig. 2A). After the addition of arachidonic acid, the mean open time decreases (Fig. 2B) from 144 ± 10 ms (n = 6) in control to 11 ± 1 ms (n = 4) in the presence of 25 tkM fl arachidonic acid. In contrast, arachidonic acid has only a small effect on the mean closed time, suggesting that current interruptions induced by arachidonic acid are similar in 10 AtM duration to the normal closed events. The reciprocal of the mean open time (Fig. 3A) increases linearly over a concen- iUE tration of 1-25 LM, whereas the reciprocal ofthe closed time (Fig. 3B) is independent of the fatty acid concentration. Effect of Other Fatty Acids. Other cis unsaturated fatty 25 jAM acids, like linoleic (18:2, cis-9,12) and oleic (18:1, cis-9) acids, reduce Cl- channel activity with a potency similar to that of arachidonic acid (Fig. 3A). Ricinoleic acid, which contains an additional hydroxyl group compared to oleic acid, has a comparable potency (Fig. 3A). Trans unsaturated and long-chain saturated fatty acids do J 2 pA not affect channel kinetics. The mean open times of these 400 mAec fatty acids at 20 AM are 134 ± 32 ms (n = 3), 104 ± 13 ms (n = 4), 115 ± 10 ms (n = 3), and 140 ± 18 ms (n = 4) for elaidic FIG. 1. Dose-dependent inhibition of airway Cl- channels by (18:1, trans-9), stearic (18:0), palmitic (16:0), and myristic arachidonic acid. Top trace, channel activity in control condition. The channel is predominantly open with occasional brief closings. (14:0) acids, respectively. Because ofthe limited solubility of The other traces are recordings at different concentrations of ara- long-chain saturated fatty acids, the shorter chain, water- chidonic acid as indicated. In the presence of arachidonic acid, the soluble fatty acid butyric acid was tested. Butyrate (50 mM) number of flickery closings increases as the fatty acid concentration (4:0) does not affect channel kinetics or shift the reversal increases. Arrows indicate the closed state. The voltage is the bath potential (data not shown), suggesting that saturated fatty potential in reference to pipette ground. acids at high concentration do not affect channel gating and do not permeate the channel. Arachidonyl alcohol, with the Addition of arachidonic acid (20:4, cis-5,8,11,14) to the carboxyl group replaced by a hydroxyl group, does not block intracellular face ofthe membrane causes rapid interruptions the channel at 20 tLM. These data suggest that the structural in open channel current (Fig. 1). At 25 ,uM, the channel requirements for inhibition are a carboxyl group and a kinked opening probability decreases from 97.8% ± 0.3% (n = 6) to long carbon chain. 76.8% ± 2.2% (n = 4). However, arachidonic acid does not Short Circuit Current Experiments. The influence of fatty change the single channel conductance because current am- acids on transepithelial chloride secretion in T84 cells was plitudes at +50 mV before (2.0 ± 0.1 pA; n = 9) and after (2.1 tested.