How does beta-adrenergic stimulation increase the heart rate? The role of intracellular Ca2+ release in amphibian pacemaker cells.

1. The mechanism by which sympathetic transmitters increase the firing rate of pacemaker cells was explored in isolated cells from the sinus venosus of the cane toad Bufo marinus. Intracellular calcium concentration ([Ca2+]i) was measured with indo-1 and membrane potential and currents were recorded with the nystatin perforated-patch technique. 2. Adrenaline or isoprenaline (2 microM) increased the transient rise in [Ca2+]i and increased the firing rate; these effects were blocked by propranolol (2 microM). 3. To determine whether the changes in [Ca2+]i might influence the firing rate we studied agents which affect either the loading or the release of Ca2+ from the sarcoplasmic reticulum (SR). Rapid application of caffeine (10 mM) to spontaneously firing cells caused a large Ca2+ release from the SR and the cells were then quiescent for 24 s. In the presence of beta-adrenergic stimulation the caffeine-induced [Ca2+]i was 14 % larger but the period of quiescence after application was reduced to 12 s. 4. Ryanodine, at either low (1 microM) or high (> 10 microM) concentration, stopped firing. However, when the SR store content of Ca2+ was tested with caffeine, at low ryanodine concentration the SR Ca2+ store was empty whereas at the high concentration the SR store was still loaded with Ca2+. beta-Adrenergic stimulation was not able to restore firing at the low concentration of ryanodine but did restore firing at the high ryanodine concentration. 5. An SR Ca2+ pump blocker, 2, 5-di(tert-butyl)-1,4-hydroquinone (TBQ) which depletes the SR store of Ca2+, also rapidly and reversibly stopped spontaneous firing. 6. The relation between the amplitude of the [Ca2+]i transient and firing rate established in the presence of ryanodine was similar when firing was restored by beta-stimulation. 7. In both spontaneously firing and voltage-clamped cells, depleting the SR store with either ryanodine or TBQ suggested that about half of the Ca2+ which contributes to the calcium transient is released from the SR. 8. These results show that the amplitude of the [Ca2+]i transient is an important factor in the firing rate of toad pacemaker cells and consequently agents which modify SR Ca2+ release influence firing rate. The effects of beta-stimulation on firing rate seem to be largely mediated by changes in amplitude of the [Ca2+]i transient.