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

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.