Purinergic Ca²⁺ signaling in myenteric neurons via P₂ purinoceptors

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 'micropuff' application of ATP or ATPγS caused an increase in [Ca²⁺](i) in 99% of 200 multipolar neurons. The potency profile of agonists for the rise in [Ca²⁺](i) was ATPγS = ATP >> ADP >> AMP, adenosine, 5'-(N-ethylcarboxamido)adenosine, and 2-chloro- N⁶-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 peak ATP 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.