Enhancement of Ca2+-dependent outward current in sheep bladder myocytes by evans blue dye.

Whole-cell and inside-out patch-clamp techniques were used to assess the action of a well-known dye, Evans blue, on membrane currents in bladder isolated 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 Ca2+ current. The effect on outward current was abolished or reduced if the cells were bathed in Ca2+-free solution, iberiotoxin (5 x 10(-8) M), or charybdotoxin (5 x 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 Ca2+ concentration of 5 x 10(-7) M, Evans blue (10(-4) M) increased the open probability of large-conductance (298-pS) Ca2+-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.

Ca2+ current and Ca(2+)-activated chloride current in isolated smooth muscle cells of the sheep urethra.

1. Isolated sheep urethral cells were studied using the perforated patch clamp technique (T = 37 degrees C). Depolarizing steps ranging from -40 to -10 mV evoked an inward current that peaked within 10 ms and a slower inward current. Stepping back to the holding potential of -80 mV evoked large inward tail currents. All three currents were abolished by nifedipine (1 microM). Substitution of external Ca2+ with Ba2+ resulted in potentiation of the fast inward current and blockade of the slow current and tails. 2. Changing the chloride equilibrium potential (ECl) from 0 to +27 mV shifted the reversal potential of the tail currents from 1 +/- 1 to 27 +/- 1 mV (number of cells, n = 5). Chloride channel blockers, niflumic acid (10 microM) and anthracene-9-carboxylic acid (9AC, 1 mM), reduced the slow current and tails suggesting that these were Ca(2+)-activated Cl- currents, ICl(Ca). 4. Caffeine (10 mM) induced currents that reversed at ECl and were blocked by niflumic acid (10 microM). 5. In current clamp mode, some cells developed spontaneous transient depolarizations (STDs) and action potentials. Short exposure to nifedipine blocked the action potentials and unmasked STDs. In contrast, 9AC and niflumic acid reduced the amplitude of the STDs and blocked the action potentials. 6. In conclusion, these cells have both L-type ICa and ICl(Ca). The former appears to be responsible for the upstroke of the action potential, while the latter may act as a pacemaker current.

Tetrodotoxin-sensitive sodium current in sheep lymphatic smooth muscle.

1. Fast inward currents were elicited in freshly isolated sheep lymphatic smooth muscle cells by depolarization from a holding potential of -80 mV using the whole-cell patch-clamp technique. The currents activated at voltages positive to -40 mV and peaked at 0 mV. 2. When sodium chloride in the bathing solution was replaced isosmotically with choline chloride inward currents were abolished at all potentials. 3. These currents were very sensitive to tetrodotoxin (TTX). Peak current was almost abolished at 1 microM with half-maximal inhibition at 17 nM. 4. Examination of the voltage dependence of steady state inactivation showed that more than 90% of the current was available at the normal resting potential of these cells (-60 mV). 5. The time course of recovery from inactivation was studied using a double-pulse protocol and showed that recovery was complete within 100 ms with a time constant of recovery of 20 ms. 6. Under current clamp, action potentials were elicited by depolarizing current pulses. These had a rapid upstroke and a short duration and could be blocked with 1 microM TTX. 7. Spontaneous contractions of isolated rings of sheep mesenteric lymphatic vessels were abolished or significantly depressed by 1 microM TTX.