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The effects of beta-adrenergic stimulation on the Ca2(+)-insensitive transient outward current (Ito) in rabbit ventricular myocytes were examined. Exposure to isoproterenol (ISO; 1 microM) activated a time-dependent current at positive membrane potentials. To determine whether this ISO-induced current was associated with Ito, sensitivity to the K+ channel antagonist, 3,4-diaminopyridine (DAP; 200 microM) was compared before and after exposure to ISO. The DAP-sensitive current was not enhanced by ISO, suggesting that the ISO-induced current was not a component of Ito. Ito and the ISO-induced current could also be dissociated by changing the membrane holding potential. Positive holding potentials, which produced significant inactivation of Ito, had little effect on the ISO-induced membrane current. Furthermore, the ISO-induced current could be observed when K+ was replaced by Cs+. The reversal potential of the ISO-induced current agreed with the predicted Cl- equilibrium potential, and exposure to Cl(-)-free extracellular solutions eliminated the response to ISO. Therefore, we conclude that ISO does not directly activate Ito in rabbit ventricular myocytes, but instead, activates a time-independent chloride current (ICl) similar to that recently described in guinea pig ventricular myocytes and shown to be regulated by adenylate cyclase (R. D. Harvey and J. R. Hume. Science Wash. DC 244: 983-985, 1989).

It has been reported that D 600 blocks the high-threshold Ca2+ channel (L-type) from the outside in isolated vascular and ileal smooth muscle cells of the rabbit (Y. Ohya, K. Terada, K. Kitamura, and H. Kuriyama. Pfluegers Arch. 408: 80-82, 1987). We have reinvestigated this hypothesis by comparing the effects of external and internal applications of D 600 and the permanently charged quaternary derivative D 890 on the whole cell Ca2+ current (Ica) recorded in vascular smooth muscle cells isolated from the rabbit portal vein. At low frequencies of stimulation (0.05 Hz), externally applied D 600 inhibited Ica in a dose-dependent fashion, with a complete block occurring at 10(-4) M. D 600 was approximately 1,000 times more potent than D 890 for causing inhibition of Ica using this protocol. During a train of stimulations at 0.5 Hz, D 600 (10(-6) M) produced a minor additional frequency-dependent block of Ica, as shown in other preparations. During superfusion with D 890 (10(-4) M), a similar protocol produced little if any decline in the amplitude of Ica. No evidence of block could be detected during intracellular dialysis of D 600 (10(-4) M). At the same concentration, intracellular application of D 890 produced a slow block of Ica. To test whether D 600 could be effectively dialysed using a patch micropipette, similar experiments were performed in cardiac ventricular myocytes. In this preparation, intracellular dialysis of D 600 induced a rapid inhibition of the Ica.(ABSTRACT TRUNCATED AT 250 WORDS)

1. Currents through Ca2+ channels of single guinea‐pig ventricular myocytes were studied using patch electrodes for whole‐cell recording. Currents through Na+ and K+ channels were suppressed by the application of drugs or the substitution of impermeant ions. 2. Inactivation of the Ca2+ current (ICa) was investigated using a two‐pulse protocol. The amount of inactivation left behind by a pre‐pulse appeared to be related to current magnitude, as others have reported. The dependence of inactivation on the pre‐pulse potential was partially U‐shaped, as the amount of inactivation peaked at 0 mV and then declined with more positive pre‐pulses. 3. Non‐specific current carried by monovalent ions through Ca2+ channels (Ins) was induced by lowering the extracellular Ca2+ concentration with EGTA. Ins peaked in an inward direction at ‐20 mV, reversed direction at +22 mV, and became a large outward current at more positive potentials. 4. Ins inactivated with a slow time course. The inactivation was not due to accumulation or depletion phenomena. Studies using two‐pulse protocols showed that the amount of inactivation left by a pre‐pulse was directly related to the pre‐pulse potential. 5. The addition of micromolar amounts of free Ca2+ to the external solution induced outward rectification of Ins. Inward currents were small or absent, while larger outward currents could still be seen at very positive potentials. Ca2+‐channel inactivation still occurred under these conditions, even in the absence of any significant ionic movement. 6. The time courses of Ins inactivation and recovery were studied. The half‐time of Ins inactivation decreased with larger depolarizations. Recovery of Ins was very slow, but could be accounted for by changes in the surface charge of the membrane. 7. It is concluded that Ins inactivation is due solely to a voltage‐dependent inactivation process which is intrinsic to myocardial Ca2+ channels. Voltage‐dependent inactivation appears to account for a significant proportion of total Ca2+‐channel inactivation at negative potentials, and appears to account for almost all of the inactivation at very positive potentials, even in the presence of millimolar concentration of external Ca2+.

1. Single smooth muscle cells isolated from rabbit portal vein were voltage clamped at room temperature using the whole‐cell configuration of the patch‐clamp technique. These cells exhibited a mean resting potential of ‐47.9 mV and a mean input resistance of 376 M omega. 2. Using small tip diameter micropipettes (to avoid dialysis of the cells), depolarizing voltage‐clamp pulses from a holding potential of ‐50 mV elicited two distinct outward currents: a quasi‐instantaneous background current and a time‐dependent current that did not appear to inactivate (delayed rectifier). Upon return to the holding potential, an outward tail current decaying back to the holding current was observed. 3. The time course of development of the tail current as estimated from envelopes of tail current protocols followed the kinetics of activation of the delayed rectifier elicited during the preceding test pulse. The tail current reversed close to the equilibrium potential for K+ ions indicating that it is mainly carried by potassium ions. 4. Using large tip diameter micropipettes to internally dialyse the cells (EGTA = 0.1 mM; ATP = 5 mM), two additional outward currents having transient kinetics were revealed: a smooth transient outward current (Ito) and spontaneous transient outward currents (STOCs). Ito was found to be mainly selective for K+ ions and exhibited voltage‐dependent inactivation with half‐maximal availability near ‐40 mV. 5. Removal of calcium from the bathing solution significantly reduced the background current and abolished both Ito and STOCs. The delayed rectifier current appeared to be insensitive to this procedure. The two types of transient outward currents were never recorded when EGTA was elevated to 5 mM inside the micropipette whereas the background and delayed rectifier currents were not affected. These results suggested that Ito and the spontaneous transient outward currents are activated by internal calcium. 6. External application of TEA (0.5‐20 mM) blocked all four outward currents. Calcium replacement by barium significantly reduced the background current and Ito, and had small effects on the delayed rectifier current. When potassium was replaced with caesium (130 mM) and TEA (20 mM) inside the pipette, none of the outward currents described was ever observed. In about 60% of the cells dialysed with this solution a small inward Ca2+ current was revealed. 7. External application of caffeine (5 mM) abolished STOCs in cells in which this activity was present under control conditions. In cells lacking this type of activity under control conditions caffeine induced and later abolished this type of current.(ABSTRACT TRUNCATED AT 400 WORDS)

Single myocardial cells were enzymatically dispersed from guinea‐pig atria and ventricles. At 25 degrees C, atrial cell action potentials differed significantly from ventricular cell action potentials in duration (atrial = 141 ms, ventricular = 497 ms) and over‐shoot (atrial = +36 mV, ventricular = +42 mV). Action potentials of atrial and ventricular cells responded differently to changes in external K+ concentration ([K+]o). Elevation of [K+]o from 6 to 11 mM depolarized atrial cells but produced no significant change in action potential duration; similar changes in [K+]o depolarized ventricular cells and produced a significant shortening of the action potential duration. Voltage‐clamp experiments were performed to investigate the ionic basis underlying the different action potential configurations of single atrial and ventricular myocytes. A single‐micropipette voltage‐clamp technique was used, employing either extremely small‐tip diameter pipettes, without internal cell dialysis (Hume & Giles, 1983), or larger tip diameter pipettes, with internal dialysis (Hamill, Marty, Neher, Sakmann & Sigworth, 1981). Two significant differences in background K+ conductance in single atrial and ventricular myocytes were observed: (i) the isochronal (5 s) current‐voltage relationship of single ventricular myocytes exhibited a region of prominent negative slope conductance and elevation of [K+]o produced cross‐over; a negative slope conductance region was absent in atrial cells and elevation of [K+]o produced very little cross‐over of isochronal current‐voltage relationships, and (ii) hyperpolarizing voltage pulses applied from holding potentials of ‐50 mV elicited inward current in ventricular cells which decayed with time; similar voltage‐clamp pulses in atrial cells elicited inward currents which fail to decay. Single K+ channel current measurements confirmed the existence of different resting K+ channel properties in single atrial and ventricular myocytes. Resting K+ channels in both cell types had similar single channel conductances (30‐32 pS with [K+]o = 145 mM) but ventricular K+ channels had significantly slower gating kinetics compared to atrial K+ channels (ventricular K+ channel mean open time = 223 ms; atrial K+ channel mean open time = 1 ms at Vr (resting membrane potential) ‐20 mV). The plateau and duration of the guinea‐pig ventricular action potential was insensitive to high concentrations of tetrodotoxin (3 X 10(‐5) M) but extremely sensitive to external Ca2+ concentration ([Ca2+]o). The second inward Ca2+ current (iCa) density was estimated in small atrial and ventricular myocytes of similar diameter and length.(ABSTRACT TRUNCATED AT 400 WORDS)

The effects of cromakalim (BRL 34915) and its (-) optical isomer, lemakalim (BRL 38227) on the activity of 265-pS Ca2+-activated K+ channels (BK channels) were examined in cell-attached and inside-out patches from canine colonic myocytes. In cell-attached patches lemakalim increased the open probability (Po) of BK channels. Mean NPo, where N is the number of channels per patch, at + 50 mV increased from 0.08 to 0.26 (20 µM lemakalim). In inside-out patches, cromakalim and lemakalim increased channel NPo rapidly and reversibly. This increase in NPo was due to a shift in half-maximal activation. Glyburide (20 µM) prevented the increase in NPo caused by lemakalim in cell-attached patches and reversed the increase in NPo in inside-out patches. Under conditions where Ca2+-activated K+ channels were maximally activated, lemakalim failed to increase current or induce a second type of K+ channel activity. When tetraethylammonium (200 µM) was added to the pipette solution to block the BK channel half maximally, lemakalim also failed to induce a second type of channel. Adenosine triphosphate (1 or 2 mM) applied to the inner surface of inside-out patches had no effect on Po of BK channels. Finally, the effects of lemakalim on ensemble average currents, constructed from multiple openings of BK channels in cell-attached patches was found to successfully mimic the effects of the drug on whole-cell membrane currents. We conclude that cromakalim and lemakalim activate BK channels in canine colonic cells. Whether this action participates in the membrane hyperpolarization and the decrease in frequency and duration of slow waves produced by these compounds in intact colonic muscles remains to be investigated.