IP3-induced Ca2+ Signals at the Cytoplasm and Nucleus in HL-1 Atrial Cells: Possible Roles of IP3 Receptor Subtypes

Abstract

HL-1 cells are the only adult cardiac cell line available that continuously divides while maintaining an atrial phenotype. We examined the expression and localization of inositol 1,4,5-trisphosphate receptor (IP3R) subtypes and their functional roles in the local Ca2+ signaling of HL-1 cells. RT-PCR and western blot analyses of IP3R revealed significant expression of type 1 (IP3R1) and type 2 IP3R (IP3R2) in HL-1 and isolated atrial cells. IP3R1 was more abundant in HL-1 cells than atrial cells, while IP3R2 protein band was darker in intact atrial cells than HL-1 cells. Immunostaining of the IP3R subtypes in HL-1 and intact atrial cells demonstrated that IP3R1 localized to nuclear envelope and that IP3R2 was distributed at the cytoplasm as a punctate form. Extracellular application of 1 mM ATP, known to generate IP3, in intact HL-1 cells elicited Ca2+ rise with oscillation, while 10 mM caffeine produced a Ca2+ transient with no oscillation. Exposure of saponin-permeabilized cells to IP3 in the presence of tetracaine (1 mM) elicited transient Ca2+ increases. The percentage of cells with the IP3 response, the magnitude of IP3-induced Ca2+ rise, and propensity of Ca2+ oscillations were dependent on the concentrations of IP3. The IP3-induced Ca2+ oscillations were more pronounced in the cytoplasm than the nucleus, such that they developed faster and lasted longer at the cytoplasm, and that their magnitudes were larger. In contrast, IP3-induced nuclear Ca2+ signal showed more prolonged and larger increase in the basal level with less oscillation. The IP3-induced Ca2+ changes were prevented by the blockers of IP3Rs, heparin and 2-APB. These results suggest that specific subcellular localization of IP3R subtypes may be responsible for distinct temporal properties of cytoplasmic and nuclear Ca2+ signaling.