Action potentials and net membrane currents of isolated smooth muscle cells (urinary bladder of the guinea-pig)

Abstract

Cells were isolated by incubating chunks of tissue from the urinary bladder of the guinea-pig in a high potassium, low chloride medium containing 0.2 mM calcium plus the enzymes collagenase and pronase. After isolation, the cells were superfused with a physiological salt solution (PSS) containing 150 mM NaCl, 3.6 mM CaCl2 and 5.4 mM KCl (35 degrees C). Patch electrodes filled with an isotonic KCl-solution were used for whole cell recordings. With a single electrode voltage clamp we measured a capacitance of 50 +/- 5 pF per cell, an input resistance of 200 +/- 25 kOhm X cm2 and a series resistance of 44 +/- 4 Ohm X cm2. The cells had resting potentials of -52 +/- 2 mV. They did not beat spontaneously but responded to stimuli with single action potentials (APs) which rose from the threshold (-38 mV) with a maximal rate of 6.5 +/- 1.8 V/s to an overshoot of 22 +/- 3 mV. The AP lasted for 36 +/- 4 ms (measured between threshold and -40 mV). Continuous cathodal current produced repetitive activity, a pacemaker depolarization followed the AP and preceded the next upstroke. Net membrane currents evoked by clamp steps to positive potentials were composed of an inward and an outward component. The inward component generating the upstroke of the AP was carried by Ca ions (iCa, Klöckner and Isenberg 1985). The repolarization resulted from a potassium outward current iK. Ca-channel blockers (5 mM NiCl2) reduced iK suggesting that (part of) iK was Ca-activated. iK rose within about 100 ms to a peak of 40-200 muA/cm2 from which it inactivated slowly and incompletely. The inactivating iK followed a bell-shaped voltage-dependence, the noninactivating iK an outwardly rectifying one. Both parts had similar steady state inactivation curves with a half maximal inactivation potential at -36 mV and a slope of 9 mV. Repolarization to -50 mV induced outward tail currents which reversed polarity at -85 mV (the calculated potassium equilibrium potential). The amplitude and the time course of the envelope of the tail currents varied in proportion to iK during the prestep. Thus, the tail current is suggested to reflect the turning off of a potassium conductance which had been activated during the prepulse. iK was largely reduced but not blocked by 20 or 150 mM tetraethylammonium (TEA). TEA did not significantly change the resting potential, but it prolonged the AP and facilitated upstroke and overshoot.(ABSTRACT TRUNCATED AT 400 WORDS)

Pharmacological characterization of the inwardly‐rectifying current in the smooth muscle cells of the rat bladder

“…The tissue was then triturated in Kraftbruhe (Klockner & Isenberg, 1985) with a wide bore, smooth-tipped pipette. The cells were used for experiments within 10 h of separation, during which time they were stored at 6'C in Kraftbruhe.…”

Ionic channels in smooth muscle studied with patch-clamp methods.

“…On depolarization of the membrane, an outward current generally increases to a peak which was reached after 100-300 ms and then slowly decayed (the guinea-pig urinary bladder [31]). The transient part of this current is sensitive to Ca and considered to flow through the "Ca-activated K channels."…”

“…Using a pipette filled with Cs ion instead of K, the outward current is largely eliminated. 4-Aminopyridine (4-AP 1-5 mM) had no effect on the membrane current in the muscle cells from guinea-pig urinary bladder [31] and ileum [49]. However, it was found in the rabbit pulmonary artery that outward currents were inhibited by external application of 4-AP, by shifting the V -I curve towards the depolarizing direction [50].…”

Identification of the major membrane currents in freshly dispersed single smooth muscle cells of guinea‐pig ureter.

“…Both iCa and a component of iK are abolished upon blockade of Ca2+ entry, using organic ions such as nickel, manganese or cadmium (Walsh & Singer, 1981, 1987Klockner & Isenberg, 1985a;Bolton et al 1985;Droogmans & Callewaert, 1986). This Ca2+-activated component of iK is also sensitive to the blocking actions of tetraethylammonium (TEA) (Ohya et al 1986;Walsh & Singer, 1987).…”

“…This Ca2+-activated component of iK is also sensitive to the blocking actions of tetraethylammonium (TEA) (Ohya et al 1986;Walsh & Singer, 1987). The K+ current present after Ca2+ removal (voltage activated) is generally less sensitive to TEA (Klockner & Isenberg, 1985a;Ohya et al 1986), but can be sensitive to a direct blockade by Ca2+-entry blockers such as verapamil, diltiazem and nicardipine (Terada, Kitamura & Kuriyama, 1987). This pattern of current flow upon depolarization is not evident in all smooth muscles.…”