Caffeine-induced calcium release from internal stores in cultured rat sensory neurons

Abstract

Free intracellular calcium concentration [Ca 2+] in was recorded at 22°C by means of Indo-1 or Fura-2 single-cell microfluorometry in cultured dorsal root ganglion neurons obtained from neonatal rats. The resting [Ca 2+] in in dorsal root ganglion neurons was 73 ± 21nM (mean ± S.D., n = 94). Fast application of 20 mM caffeine evoked [Ca 2+] in transient which reached a peak of 269 ± 64nM within 5.9 ± 1.1 s. After reaching the peak the [Ca 2+] in level started to decline in the presence of caffeine and for 87.2 ± 10.6s cytoplasmic calcium returned to an initial resting value. In 40% of neurons tested [Ca 2+] in decreased to subresting levels following the washout of caffeine (the so-called post-caffeine undershoot). On average, the undershoot level was 19 ± 2.5nM below the resting [Ca 2+] in value. Prolonged exposure of caffeine depleted the caffeine-sensitive stores of releasable Ca 2+; the degree of this depletion depended on caffeine concentration. The depletion of the caffeine-sensitive internal stores to some extent was linked to calcium extrusion via La 3+-sensitive plasmalemmal Ca 2+-ATPases. The stores could be partially refilled by the uptake of cytoplasmic Ca 2+, but the complete recovery of releasable Ca 2+ content of the caffeine-sensitive pools required the additional calcium entry via voltage-operated calcium channels. Caffeine-evoked [Ca 2+] in transients were effectively blocked by 10 μM ryanodine, 5mM procaine, 10 μM dantrolene or 0.5 mM Ba 2+, thus sharing the basic properties of the Ca 2+-induced-Ca 2+ release from endoplasmic reticulum. Pharmacological manipulation with caffeine-sensitive stores interfered with the depolarization-induced [Ca 2+] in transients. In the presence of low caffeine concentration (0.5–1 mM) in the extracellular solution the rate of rise of the depolarization-triggered [Ca 2+] in transients significantly increased (by a factor 2.15 ± 0.29) suggesting the occurrence of Ca 2+-induced Ca 2+ release. When the caffeine-sensitive stores were emptied by prolonged application of caffeine, the amplitude and the rate of rise of the depolarization-induced [Ca 2+] in transients were decreased. These facts suggest the involvement of internal caffeine-sensitive calcium stores in the generation of calcium signal in sensory neurons.