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Properties of the Ryanodine‐sensitive Release Channels that Underlie Caffeine‐induced Ca 2+ Mobilization from Intracellular Stores in Mammalian Sympathetic Neurons
Author(s) -
HernándezCruz A.,
DíazMuñoz M.,
GómezChavarín M.,
CanñedoMerino R.,
Protti D. A.,
Escobar A. L.,
Sierralta J.,
SuárezIsla B. A.
Publication year - 1995
Publication title -
european journal of neuroscience
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.346
H-Index - 206
eISSN - 1460-9568
pISSN - 0953-816X
DOI - 10.1111/j.1460-9568.1995.tb00690.x
Subject(s) - ryanodine receptor , caffeine , calcium , chemistry , intracellular , biophysics , calcium in biology , fura 2 , voltage dependent calcium channel , medicine , endocrinology , biochemistry , biology , cytosol , enzyme , organic chemistry
Abstract The most compelling evidence for a functional role of caffeine‐sensitive intracellular Ca 2+ 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 Ca 2+ release channels to examine calcium release from these intracellular stores in rat sympathetic neurons from the superior cervical ganglion. Intracellular Ca 2+ measurements showed that these cells possess caffeine‐sensitive intracellular Ca 2+ stores capable of releasing the equivalent of 40% of the calcium that enters through voltage‐gated calcium channels. The efficiency of caffeine in releasing Ca 2+ showed a complex dependence on [Ca 2+ ] i . Transient elevations of [Ca 2+ ] i by 50–500 nM were facilitatory, but they became less facilitatory or depressing when [Ca 2+ ] i reached higher levels. The caffeine‐induced Ca 2+ release and its dependence on [Ca 2+ ] 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 [ 3 H]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 [Ca 2+ ] 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 [Ca 2+ ] rose, with an optimum at [Ca 2+ ] between 100 and 500 nM and a 50% decrease between 1 and 10 μM. At 100 μM [Ca 2+ ], caffeine and ATP enhanced [ 3 H]ryanodine binding by 35 and 170% respectively, while binding was reduced by >90% with ruthenium red and MgCl 2 . High‐conductance (240 pS) Ca 2+ release channels present in ganglionic microsomal membranes were incorporated into planar phospholipid bilayers. These channels were activated by caffeine and by micromolar concentrations of Ca 2+ from the cytosolic side, and were blocked by Mg 2+ 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/Ca 2+ release channels present in mammalian peripheral neurons can account for the properties of caffeine‐induced Ca 2+ release. Our data also suggest that the release of Ca 2+ by caffeine has a bell‐shaped dependence on Ca 2+ in the physiological range of cytoplasmic [Ca 2+ ].