Properties of the Ryanodine-sensitive Release Channels that Underlie Caffeine-induced Ca2+ Mobilization from Intracellular Stores in Mammalian Sympathetic Neurons

The most compelling evidence for a functional role of caffeine-sensitive intracellular Ca²⁺ reservoirs in nerve cells derives from experiments on peripheral neurons. However, the properties of their ryanodine receptor calcium release channels have not been studied. This work combines single-cell fura-2 microfluorometry, ³ H]ryanodine binding and recording of Ca²⁺ release channels to examine calcium release from these intracellular stores in rat sympathetic neurons from the superior cervical ganglion. Intracellular Ca²⁺ measurements showed that these cells possess caffeine-sensitive intracellular Ca²⁺ stores capable of releasing the equivalent of 40% of the calcium that enters through voltage-gated calcium channels. The efficiency of caffeine in releasing Ca²⁺ showed a complex dependence on Ca²⁺]_i. Transient elevations of Ca²⁺]_i by 50–500 nM were facilitatory, but they became less facilitatory or depressing when Ca²⁺]_i reached higher levels. The caffeine-induced Ca²⁺ release and its dependence on Ca²⁺]_i was further examined by ³ H]ryanodine binding to ganglionic microsomal membranes. These membranes showed a high-affinity binding site for ryanodine with a dissociation constant (K_D= 10 nM) similar to that previously reported for brain microsomes. However, the density of ³H]ryanodine binding sites (B_max= 2.06 pmol/mg protein) was at least three-fold larger than the highest reported for brain tissue. ³ H]Ryanodine binding showed a sigmoidal dependence on Ca²⁺] in the range 0.1–10 μM that was further increased by caffeine. Caffeine-dependent enhancement of ³ H]ryanodine binding increased and then decreased as Ca²⁺] rose, with an optimum at Ca²⁺] between 100 and 500 nM and a 50% decrease between 1 and 10 μM. At 100 μM Ca²⁺], caffeine and ATP enhanced ³ H]ryanodine binding by 35 and 170% respectively, while binding was reduced by >90% with ruthenium red and MgCl₂. High-conductance (240 pS) Ca²⁺ release channels present in ganglionic microsomal membranes were incorporated into planar phospholipid bilayers. These channels were activated by caffeine and by micromolar concentrations of Ca²⁺ from the cytosolic side, and were blocked by Mg²⁺ and ruthenium red. Ryanodine (2 μM) slowed channel gating and elicited a long-lasting subconductance state while 10 mM ryanodine closed the channel with infrequent opening to the subconductance level. These results show that the properties of the ryanodine receptor/Ca²⁺ release channels present in mammalian peripheral neurons can account for the properties of caffeine-induced Ca²⁺ release. Our data also suggest that the release of Ca²⁺ by caffeine has a bell-shaped dependence on Ca²⁺ in the physiological range of cytoplasmic Ca²⁺].