Electroporation loading and photoactivation of caged InsP 3: tools to investigate the relation between cellular ATP release in response to intracellular InsP 3 elevation

Abstract

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 10 μM InsP 3 concentration range and a second phase of steeply increasing ATP release in response to above 10 μM InsP 3 stimulation. ATP release triggered by below 10 μM 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.