Purinergic Ca2+ signaling in myenteric neurons via P2 purinoceptors

Abstract

Fura 2 microfluorimetry was used to test the hypothesis that ATP acts at P1 and P2 purinoceptors to elevate cytosolic free Ca2+ concentrations [Ca2+]i) in calbindin-immunoreactive cultured myenteric neurons from adult guinea pig small intestine. Local "micro-puff" application of ATP or ATP(gamma)S caused an increase in [Ca2+]i in 99% of 200 multipolar neurons. The potency profile of agonists for the rise in [Ca2+]i was ATP(gamma)S = ATP >> ADP >> AMP, adenosine, 5'-(N-ethylcarboxamido)adenosine, and 2-chloro-N(6)-cyclopentyladenosine. Tetrodotoxin-sensitive synaptic transmission could contribute as much as 25% to the ATP response. The P1 antagonist 8-cyclopentyl-1,3-dipropylxanthine blocked 50% of the peakATP Ca2+ response. P2 antagonists blocked the ATP response: pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid > reactive blue 2 > suramin. Suramin enhanced the ATP response in 27.5% of neurons. Some neurons (<15%) displayed distinct multiphasic Ca2+ signatures. About 54% of ATP-responsive neurons expressed calbindin. The data support the following hypotheses: 1) two distinct P2 purinoceptors are linked to the rise in [Ca2+]i in myenteric neurons; 2) purinergic Ca2+ signaling is not restricted to one neuronal phenotype; and 3) intraneuronal Ca2+ is not involved in adenosinergic hyperpolarization in AH/type 2 neurons.