title: Properties of the ryanodine-sensitive release channels that underlie caffeine-induced Ca2+ mobilization from intracellular stores in mammalian sympathetic neurons.
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+]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 [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 microM 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 microM. At 100 microM [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 microM) 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+].
authors.affiliation: Departamento de Neurociencias, D.F. Mexico.
authors.forename: A
authors.initials: A
authors.lastname: Hernández-Cruz
authors.forename: M
authors.initials: M
authors.lastname: Díaz-Muñoz
authors.forename: M
authors.initials: M
authors.lastname: Gómez-Chavarín
authors.forename: R
authors.initials: R
authors.lastname: Cañedo-Merino
authors.forename: D A
authors.initials: DA
authors.lastname: Protti
authors.forename: A L
authors.initials: AL
authors.lastname: Escobar
authors.forename: J
authors.initials: J
authors.lastname: Sierralta
authors.forename: B A
authors.initials: BA
authors.lastname: Suárez-Isla
mesh_terms: D000320:Adrenergic Fibers / Q000502:physiology*; D000818:Animals; D002110:Caffeine / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000187:drug effects*; D005260:Female; D016257:Fura-2; D008297:Male; D011188:Potassium / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D012433:Ryanodine / Q000494:pharmacology*; D013997:Time Factors
chemical_list: D015220:Calcium Channels; D012433:Ryanodine; D002110:Caffeine; D011188:Potassium; D002118:Calcium; D016257:Fura-2