&p.1:Abstract Whole-cell and inside-out patch-clamp tech- niques were used to assess the action of a well-known dye, Evans blue, on membrane currents in bladder isolat- ed smooth muscle cells from sheep. In whole cells Evans blue dose-dependently increased the outward current by up to fivefold. In contrast, Evans blue had no effect on inward Ca 2+ current. The effect on outward current was abolished or reduced if the cells were bathed in Ca 2+ -free solution, iberiotoxin (5 × 10 –8 M), or charybdotoxin (5 × 10 –8 M), but was unaffected by externally applied caffeine (5 mM) or in cells exposed to heparin (1 mg/ml) via the patch pipette. In inside-out patches bathed in a Ca 2+ concentration of 5 × 10 –7 M, Evans blue (10 –4 M) increased the open probability of large-conductance (298-pS) Ca 2+ -dependent K + channels (BK channels), shifting the half maximal-activation voltage by –70 mV. We conclude that Evans blue dye acts as an opener of BK channels. &kwd:Key words Smooth muscle · K + channel opener · Calcium-dependent potassium current&bdy: Introduction Evans blue dye, because of its strong affinity for albu- min, has been widely used both as a dilution indicator in the measurement of blood volume and in studies of vas- cular permeability. We have also previously used it as a marker to outline lymphatic vessels for cannulation in vi- vo [19]. Implicit in such studies is the assumption that Evans blue is an inert substance, having little effect on excitable membranes or the contractility of smooth mus- cle. Recently, however, there has been growing interest in this substance as a modulator of receptors and ion channels. It has, for example, been shown to modulate currents mediated via α-amino-3-hydroxy-5-methyl- isoxazole (AMPA) and kainate receptors in a variety of preparations [16, 17, 24] and to induce seizures in rats when given by intraventricular injection [9]. In the guin- ea-pig it has been shown to inhibit capsaicin-induced bronchospasm [2], while in the rat vas deferens it has been used as an antagonist of purinoceptors and ecto-nu- cleotidases [3, 4, 26]. We have recently shown that Reactive blue 2, a struc- turally unrelated dye, opens large-conductance Ca 2+ -acti- vated K channels (BK channels) in isolated smooth mus- cle cells from the sheep bladder [8]. Interestingly, this dye is also widely used as a purinoceptor antagonist. In the light of our findings with Reactive blue 2, we decid- ed to investigate the action of Evans blue on membrane currents in bladder cells to see if there were any underly- ing electrophysiological mechanisms that could explain its inhibitory actions on smooth muscle. Here we report evidence that Evans blue opens BK channels, while hav- ing little effect on inward Ca 2+ current. Materials and methods Urinary bladders of sheep of either sex were obtained from an abattoir approximately 15 min after slaughter. Pieces of detrusor (1 mm 3 ) were removed and either used immediately, or stored at 4°C for use the following day. Smooth muscle cells were isolated from the tissue pieces by incubation in a dispersal medium con- taining (per 5 ml of Hanks’ Ca 2+ -free solution): collagenase 15 mg (Sigma type 1a), protease 1 mg (Sigma type XXIV), bovine serum albumin (BSA) 10 mg (Sigma) and trypsin inhibitor 10 mg (Sig- ma) for 20 min at 35°C, after which the tissue pieces were placed in Hanks’ Ca 2+ -free solution and stirred for a further 15–30 min to release single relaxed smooth muscle cells. These were plated in Petri dishes containing Hanks’ solution (100 μM Ca 2+ ) and stored at 4°C for use within 8 h. The solutions used were of the following composition (mM): (1) Hanks’ Ca 2+ -free solution – 141 Na + , 5.8 K + , 130.3 Cl – , 15.5 HCO 3 – , 0.4 HPO 4 2– , 0.4 H 2 PO 4 – , 10 dextrose, 2.9 sucrose; 10 4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid (HEPES), pH adjusted to 7.4 with NaOH; (2) physiological salt solution (PSS) – 130 Na + , 5.8 K + , 135 Cl – , 4.16 HCO 3 – , 0.3 HPO 4 2– , 0.4 H 2 PO 4 – , 1.8 Ca 2+ , 0.9 Mg 2+ , 0.4 SO 4 2– 10 dextrose, 2.9 sucrose, 10 HEPES; (3) K + pipette solution – 110 K gluconate, 20 KCl, 0.5 MgCl 2 , 1 K 2 ATP, 0.1 Na 2 GTP, 2.5 Na 2 phosphocreatine, 5 HEPES, M.A. Hollywood · K.D. Cotton · N.G. McHale K.D. Thornbury ( ✉ ) Department of Physiology, School of Biomedical Science, The Queen’s University of Belfast, 97 Lisburn Road, Belfast BT9 7BL, Northern Ireland, UK&/fn-block: Pflügers Arch – Eur J Physiol (1998) 435:631–636 © Springer-Verlag 1998 ORIGINAL ARTICLE &roles:M.A. Hollywood · K.D. Cotton · N.G. McHale K.D. Thornbury Enhancement of Ca 2+ -dependent outward current in sheep bladder myocytes by Evans blue dye &misc:Received: 1 September 1997 / Received after revision: 18 November 1997 / Accepted: 20 November 1997