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-sensiti ve intracellular Ca2+ reservoirs in nerve cells derives from experimen ts on peripheral neurons. However, the properties of their ryanodine r eceptor calcium release channels have not been studied. This work comb ines single-cell fura-2 microfluorometry, [H-3]ryanodine binding and r ecording of Ca2+ release channels to examine calcium release from thes e intracellular stores in rat sympathetic neurons from the superior ce rvical ganglion. Intracellular Ca2+ measurements showed that these cel ls possess caffeine-sensitive intracellular Ca2+ stores capable of rel easing the equivalent of 40% of the calcium that enters through voltag e-gated calcium channels. The efficiency of caffeine in releasing Ca2 showed a complex dependence on [Ca2+](i). Transient elevations of [Ca 2+](i) by 50-500 nM were facilitatory, but they became less facilitato ry or depressing when [Ca2+](i) reached higher levels. The caffeine-in duced Ca2+ release and its dependence on [Ca2+](i) was further examine d by [H-3]ryanodine binding to ganglionic microsomal membranes. These membranes showed a high-affinity binding site for ryanodine with a dis sociation constant (K-D = 10 nM) similar to that previously reported f or brain microsomes. However, the density of [H-3]ryanodine binding si tes (B-max = 2.06 pmol/mg protein) was at least three-fold larger than the highest reported for brain tissue. [H-3]Ryanodine binding showed a sigmoidal dependence on [Ca2+] in the range 0.1-10 mu M that was fur ther increased by caffeine. Caffeine-dependent enhancement of [H-3]rya nodine binding increased and then decreased as [Ca2+] rose, with an op timum at [Ca2+] between 100 and 500 nM and a 50% decrease between 1 an d 10 mu M. At 100 mu M [Ca2+], caffeine and ATP enhanced [H-3]ryanodin e binding by 35 and 170% respectively, while binding was reduced by > 90% with ruthenium red and MgCl2. High-conductance (240 pS) Ca2+ relea se channels present in ganglionic microsomal membranes were incorporat ed 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 mu M) s lowed channel gating and elicited a long-lasting subconductance state while 10 mM ryanodine closed the channel with infrequent opening to th e subconductance level. These results show that the properties of the ryanodine receptor/Ca2+ release channels present in mammalian peripher al neurons can account for the properties of caffeine-induced Ca2+ rel ease. Our data also suggest that the release of Ca2+ by caffeine has a bell-shaped dependence on Ca2+ in the physiological range of cytoplas mic [Ca2+].