The identification of a caffeine-induced Ca2+ influx pathway in rat primary sensory neurons

Abstract

Caffeine-induced Ca2+ transients (CICTs) in rabbit nodose ganglion neurons (NGNs) are produced by two distinct mechanisms: release from intracellular stores via ryanodine receptors and Ca2+ influx across the plasma membrane, due to activation of an unknown receptor. In isolated rat NGNs, we used single-cell microfluorimetry to measure changes in intracellular Ca2+ and to test whether TRPV1 receptors underlie the Ca2+ influx pathway. Caffeine (10 mM) evoked CICTs in all NGNs tested (n = 47) averaging 365 +/- 32 nM. CICTs were partially dependent upon a Ca2+ influx pathway that ranged between 33% and 98% of the total Ca2+ transient. Application of two selective TRPV1 antagonists significantly attenuated CICTs. The peak average amplitudes of CICTs in Ca2+-free Locke solution and Ca2+-free Locke solution with IRTX or with BCTC were not significantly different from one another (n = 5 and 7, respectively). These observations suggest that caffeine can induce Ca2+ influx by activating TRPV1 channels.

Fig. 1

Caffeine-induced Ca²⁺ transients (CICTs) are partially dependent upon extracellular Ca²⁺. a Reproducibility of CICTs. Three representative CICTs evoked by three 15-s pulses of 10 mM caffeine in normal Locke solution. The CICTs had an average peak amplitude of 218 ± 20, 221 ± 24 and 212 ± 34 nM (n = 4) for the first, second and third application of caffeine, respectively. b CICTs elicited by caffeine (10 mM) in the presence or in the absence of extracellular Ca²⁺. The first and third CICTs were evoked by caffeine in normal Locke solution. The middle CICT was evoked by caffeine in a Locke solution containing nominally zero Ca²⁺. The difference in amplitude between the averaged control CICTs and the second CICTs represents the magnitude of Ca²⁺ influx across the plasma membrane. The amplitude of the CICTs recorded in nominally Ca²⁺-free Locke solution averaged 45 ± 9% of the amplitude of control CICTs (n = 6) recorded in normal Lock solution. Solid squares depict time of caffeine application; hatched bar depicts time when the neurons were superfused with nominally Ca²⁺-free Locke solution

Fig. 2

Effect of IRTX, a TRPV1 selective antagonist on the Ca²⁺ influx component of caffeine-induced Ca²⁺ transients (CICTs). a CICTs elicited by 10 mM caffeine in the presence or absence of IRTX (100 nM). IRTX significantly attenuated CICTs. The average CICT amplitude recorded in IRTX was 45 ± 8% of the control CICTs recorded in normal Ca²⁺ Locke solution (P = 0.001, n = 9). b CICTs elicited by 10 mM caffeine in nominally Ca²⁺-free Locke containing IRTX. Addition of IRTX did not significantly reduce the amplitude of CICT beyond that produced by nominally Ca⁺-free Locke solution alone (n = 5)