Abstract

An increase in intracellular free Ca2+ concentration ([Ca 2+]i) has been shown to be involved in the increase in ciliary beat frequency (CBF) in response to ATP; however, the signaling pathways associated with inositol 1,4,5-trisphosphate (IP3) receptor-dependent Ca 2+ mobilization remain unresolved. Using radioimmunoassay techniques, we have demonstrated the appearance of two IP3 peaks occurring 10 and 60 s after ATP addition, which was strongly correlated with a release of intracellular Ca2+ from internal stores and an influx of extracellular Ca2+, respectively. In addition, ATP-dependent Ca 2+ mobilization required protein kinase C (PKC) and Ca2+/ calmodulin-dependent protein kinase II activation. We found an increase in PKC activity in response to ATP, with a peak at 60 s after ATP addition. Xestospongin C, an IP3 receptor blocker, significantly diminished both the ATP-induced increase in CBF and the initial transient [Ca 2+]i component. ATP addition in the presence of xestospongin C or thapsigargin revealed that the Ca2+ influx is also dependent on IP3 receptor activation. Immunofluorescence and confocal microscopic studies showed the presence of IP3 receptor types 1 and 3 in cultured ciliated cells. Immunogold electron microscopy localized IP 3 receptor type 3 to the nucleus, the endoplasmic reticulum, and, interestingly, the plasma membrane. In contrast, IP3 receptor type 1 was found exclusively in the nucleus and the endoplasmic reticulum. Our study demonstrates for the first time the presence of IP3 receptor type 3 in the plasma membrane in ciliated cells and leads us to postulate that the IP3 receptor can directly trigger Ca2+ influx in response to ATP.