Abstract
1. Ionic mechanisms related to the caffeine-induced current (Icaffeine) were examined in the single isolated sympathetic neurones of the bull-frog. We used the 'concentration-jump' technique in combination with intracellular perfusion and a rapid external solution change, under single-electrode voltage-clamp conditions. 2. Icaffeine was pharmacologically separated into a tetraethylammonium (TEA)-sensitive transient outward current (ITO), a picrotoxin (PTX)-sensitive transient inward current (ITI) and a TEA- and PTX-insensitive sustained inward current (ISI). At low concentrations of caffeine, a sustained outward current (ISO) was observed instead of ISI. 3. All components of Icaffeine were abolished by intracellular perfusion of 30 mM-EGTA. Pre-treatment with A23187 or ryanodine or the simultaneous application of procaine either reduced or abolished all the components of Icaffeine in a dose-dependent manner. The concentration causing 50% inhibition (IC50) was 10(-8) M for A23187 and 2 mM for procaine. 4. The peak response of ITO increased abruptly at caffeine concentrations between 3 and 6 mM followed by saturation above 30 mM. A notch was observed on the rising phase of ITO. 5. The reversal potential (Ecaffeine) of ITO shifted 58 mV for a tenfold change of the extracellular K+ concentration. External application of TEA blocked ITO with an IC50 of 1 mM. ITO was relatively insensitive to apamin, 4-aminopyridine and muscarine. 6. In external solution containing 2 mM-Ca2+, ITO induced by 10 mM-caffeine recovered completely within 3 min from a previous exposure to caffeine. In the absence of extracellular Ca2+, there was little such recovery. A 5 min treatment in a Ca2+-free solution reduced ITO induced by the first application of caffeine by 5%. With a continuous application of 3 mM-caffeine, the amplitude of ITO induced by 10 mM-caffeine reduced in 1 min, and showed a partial recovery in 3 min. The amplitude of ITO increased by increasing the concentration of intracellular Cl-. 7. ITI was activated around the peak of ITO and was rapidly inactivated. ITI was evoked at caffeine concentrations of about 6-10 mM. When the intracellular Cl- concentration was changed, the amplitude of ITI behaved like a Cl- electrode. The Ecaffeine of ITI was close to the Cl- equilibrium potential (ECl). 8. ISI was a 'plateau' response and persisted for over 3 min. ISI was due to a decrease in K+ conductance. In the presence of muscarine (3 x 10(-5) M), ISI was occluded.(ABSTRACT TRUNCATED AT 400 WORDS)