Calcium Influx, But Not Intracellular Calcium Release, Supports PACAP-Mediated ERK Activation in HEK PAC1 Receptor Cells

In HEK cells expressing GFP-tagged PAC1Hop1 receptors, PACAP augments ERK phosphorylation through two parallel pathways: one through PACAP/PAC1 receptor internalization/endosome MEK/ERK signaling and the other through PLC/DAG/PKC activation. We examined whether elevation of intracellular calcium ([Ca 2+ ] i ) was required for either of the PACAP/PAC1 receptor-mediated ERK activation mechanisms. The PACAP (25 nM)-induced elevation of [Ca 2+ ] i was greater with cells maintained in Ca 2+ -containing than in Ca 2+ -deficient solution, suggesting that both calcium release from internal stores and calcium influx contributed to the rise in [Ca 2+ ] i . A thapsigargin-induced increase in [Ca 2+ ] i also was greater with calcium in the external solution. OAG, the cell permeable analogue of DAG, increased [Ca 2+ ] i , but only in Ca 2+ -containing solution. Decreasing external calcium or depleting internal calcium stores did not block PACAP-induced PAC1 receptor internalization. Omission of calcium from the external solution, but not thapsigargin pretreatment, significantly blunted PACAP-stimulated ERK phosphorylation. The PKC inhibitor BimI decreased PACAP-mediated ERK activation in both Ca 2+ -containing or Ca 2+ -deficient solutions. In contrast, following Pitstop 2 pretreatment to block endocytic mechanisms, PACAP activated ERK only when calcium was present in the external solution. We conclude that the endosome signaling pathway is largely calcium-independent whereas calcium influx appears necessary for the PLC/DAG/PKC component of PACAP-induced ERK activation.