GABA-induced calcium signaling in cultured enteric neurons is reinforced by activation of cholinergic pathways

Abstract

GABA is an important inhibitory transmitter in the CNS. In the enteric nervous system, however, both excitatory and inhibitory actions have been reported. Here, we investigated the effects of GABA on the intracellular Ca 2+ concentration of guinea-pig myenteric neurons (at 35°C) using Fura-2-AM. Neurons were identified by 75mM K + depolarization (5 s), which evoked a transient intracellular Ca 2+ concentration increase. GABA (10 s) induced a dose dependent (5nM–1μM) transient intracellular Ca 2+ concentration rise in the majority of neurons (500nM GABA: 251±17nM, n=232/289). Interestingly, the response to 5μM GABA ( n=18) lasted several minutes and did not fully recover. GABA response amplitudes were significantly ( P<0.001) reduced by GABA A and GABA B receptor antagonists (10μM) bicuculline and phaclofen. The GABA A agonist isoguvacine (10μM) and GABA B agonist baclofen (10μM) induced similar responses as 50nM GABA, while the GABA C agonist cis-4-aminocrotonic acid (CACA) (10μM) only elicited small responses in a minority of neurons. Removal of extracellular Ca 2+ abolished all responses while depletion of intracellular Ca 2+ stores by thapsigargin (5μM) did not alter the responses to 500nM GABA ( n=13), but reduction of Ca 2+ influx through voltage-dependent Ca 2+ channels did. The nicotinic antagonist hexamethonium (100μM) also reduced GABA responses by almost 70% suggesting that GABA stimulates cholinergic pathways, while the purinergic receptor blocker pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS) and the 5-HT 3 receptor blocker ondansetron only had minor effects. Conclusion: GABA elicits transient intracellular Ca 2+ concentration responses in the majority of myenteric neurons through activation of GABA A and GABA B receptors and much of the response can be attributed to facilitation of ACh release. Thus GABA may act mainly as a modulator that sets the state of excitability of the enteric nerve network. A concentration of 5μM GABA, although frequently used in pharmacological experiments, seems to cause a detrimental response reminiscent of the neurotoxic effects glutamate has in the CNS.