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

Publisher Website
Google Scholar
Abstract
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, [3H]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+]iby 50–500 nM were facilitatory, but they became less facilitatory or depressing when [Ca2+]ireached higher levels. The caffeine‐induced Ca2+release and its dependence on [Ca2+]iwas further examined by [3H]ryanodine binding to ganglionic microsomal membranes. These membranes showed a high‐affinity binding site for ryanodine with a dissociation constant (KD= 10 nM) similar to that previously reported for brain microsomes. However, the density of [3H]ryanodine binding sites (Bmax= 2.06 pmol/mg protein) was at least three‐fold larger than the highest reported for brain tissue. [3H]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 [3H]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 [3H]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+].