It was important to investigate whether Ca
2+ mobilization originated from intracellular stores or whether it was caused by influx from the extracellular medium. P2X receptors are ligand-gated ion channels, some of which are permeable to Ca
2+. Therefore, extracellular Ca
2+ contributes to the initial Ca
2+ peak when the receptor is activated. P2Y receptors are G-protein-coupled receptors, and the initial increase in [Ca
2+]
i results from release of Ca
2+ from intracellular stores. Removing extracellular Ca
2+ can, therefore, determine whether agonists act through P2X or P2Y receptor subtypes by indication of whether this initial increase in [Ca
2+]
i is from the extracellular medium or is from an intracellular source. All agonists were capable of eliciting increases in [Ca
2+]
i in the absence of extracellular Ca
2+ to the same magnitude as responses observed in the presence of extracellular Ca
2+ (Figs. 4A 4B 4C 4D 4E) . This indicated that the receptors mediating the [Ca
2+]
i increase were P2Y receptors and that the initial peak originated from intracellular Ca
2+ stores. In addition, these experiments showed differences in the response profiles by different agonists. The rate of decrease of [Ca
2+]
i occurred more rapidly in response to ATP and UTP in the absence of extracellular Ca
2+ than in the presence of extracellular Ca
2+ (Figs. 4A 4B) ; however, no significant difference was noted when comparing ADP, 2MeSATP, and UDP responses in Ca
2+-containing medium compared with Ca
2+-free medium
(Figs. 4C 4D 4E) . Hence, ATP and UTP have a secondary Ca
2+ influx component to their response—leading to the observed biphasic Ca
2+ response—whereas the other agonists do not. This suggests that ATP and UTP activate different receptor subtypes to ADP, 2MeSATP, and UDP. To enable a more detailed comparison of the agonist responses, dose-response characteristics were investigated.