The role of caffeine-sensitive calcium stores in the regulation of the intracellular free calcium concentration in rat sympathetic neurons in vitro

S. A. Thayer, L. D. Hirning, R. J. Miller

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Intracellular Ca2+ stores were studied in sympathetic neurons grown in primary culture from the superior cervical ganglion of the rat. The [Ca2+](i) was measured in single cells using the fluorescent Ca2+ indicator fura-2 and a sensitive microfluorimeter. Superfusion of the cells with 10 mM caffeine elicited a rapid and transient increase in [Ca2+](i) in the absence of extracellular Ca2+, indicating the presence of a caffeine-sensitive intracellular Ca2+ storage site. After depletion of the store by mobilization of Ca2+ with caffeine, it could be refilled by elevating [Ca2+](i), allowing multiple caffeine-induced [Ca2+](i) transient to be elicited from a single neuron. Ryanodine (1 μM), an alkaloid that promotes Ca2+ release from the sarcoplasmic reticulum, was an effective inhibitor of the caffeine-induced [Ca2+](i) transients in sympathetic neurons. Exposure to ryanodine in the presence of caffeine was required to produce a subsequent inhibition of the caffeine-induced response, suggesting a 'use-dependent' inhibition that may result from depletion of the Ca2+ stores. In contrast, dantrolene Na (10 μM), an agent known to interfere with Ca2+ release from the sarcoplasmic reticulum, also blocked the caffeine-induced [Ca2+](i) transients, but in a time-dependent rather than a use-dependent manner. Electrophysiological measurements using the whole cell version of the patch-clamp technique were made simultaneously with [Ca2+](i) microfluorimetric recordings. The magnitude of the [Ca2+](i) transients elicited by step depolarizations closely paralleled the magnitude of Ca2+ influx via voltage-sensitive Ca2+ channels, regardless of whether the magnitude of the Ca2+ current was modified by varying the test pulse duration or potential. The relationship between the magnitude of Ca2+ influx and the resulting increase in [Ca2+](i) saturated at large Ca2+ influxes resulting from long depolarizations, consistent with the activation of a large capacity, low affinity [Ca2+](i) buffering mechanism. Caffeine (10 mM) and ryanodine (10 μM), applied singly or together, produced a small and variable decrease in the [Ca2+](i) transient resulting from cell depolarization using the whole-cell patch-clamp technique. We conclude that mammalian sympathetic neurons possess intracellular Ca2+ stores with pharmacological characteristics that closely resemble those found in muscle but that these are relatively small and produce little amplification of [Ca2+](i) transients resulting from Ca2+ influx through voltage-sensitive Ca2+ channels.

Original languageEnglish (US)
Pages (from-to)664-673
Number of pages10
JournalMolecular pharmacology
Issue number5
StatePublished - 1988

ASJC Scopus subject areas

  • Molecular Medicine
  • Pharmacology


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