Electroporation loading and photoactivation of caged InsP3: tools to investigate the relation between cellular ATP release in response to intracellular InsP3 elevation.

Photolytic liberation of InsP(3) in single cells triggers cell-to-cell propagating calcium changes that are communicated by a gap junctional and a paracrine purinergic pathway involving InsP(3)-triggered ATP release. We investigated the relation between the InsP(3) stimulus and the resulting ATP release in ECV304 cells using UV photolysis of caged compounds and bioluminescent ATP measurements. Careful consideration of all steps, starting from caged InsP(3) loading into the cells by electroporation, up to photoliberation upon UV exposure, allowed to derive a dose-response relation that revealed a first part with a flattening ATP release response in the below 10microM InsP(3) concentration range and a second phase of steeply increasing ATP release in response to above 10microM InsP(3) stimulation. ATP release triggered by below 10microM InsP(3) concentrations attained a level in the order of 30% above baseline ATP release, while the steeply increasing response to high InsP(3) concentrations attained values in the order of 150% above baseline. Our data indicate the involvement of low affinity InsP(3) receptor sites in the pathway leading to triggered ATP release, with activation of these receptors causing the release of 1-2% of the total cellular ATP pool.