Agonist-Induced Ca Signaling in HEK-293-Derived Cells Expressing a Single IP Receptor Isoform.

In mammals, three genes encode IP receptors (IPRs), which are involved in agonist-induced Ca signaling in cells of apparently all types. Using the CRISPR/Cas9 approach for disruption of two out of three IPR genes in HEK-293 cells, we generated three monoclonal cell lines, IP3R1-HEK, IP3R2-HEK, and IP3R3-HEK, with the single functional isoform, IPR1, IPR2, and IPR3, respectively. All engineered cells responded to ACh with Ca transients in an "all-or-nothing" manner, suggesting that each IPR isotype was capable of mediating CICR.

Modeling of Ca transients initiated by GPCR agonists in mesenchymal stromal cells.

The integrative study that included experimentation and mathematical modeling was carried out to analyze dynamic aspects of transient Ca signaling induced by brief pulses of GPCR agonists in mesenchymal stromal cells from the human adipose tissue (AD-MSCs). The experimental findings argued for IP/Ca-regulated Ca release via IP receptors (IPRs) as a key mechanism mediating agonist-dependent Ca transients. The consistent signaling circuit was proposed to formalize coupling of agonist binding to Ca mobilization for mathematical modeling.

Calcium signaling mediated by aminergic GPCRs is impaired by the PI3K inhibitor LY294002 and its analog LY303511 in a PI3K-independent manner.

The phosphoinositide 3-kinase (PI3K) inhibitor LY294002 (LY294) and its much less active analog LY303511 (LY303) constitute the paired probe that is commonly used to demonstrate the involvement of PI3K in intracellular signaling. We studied effects of LY294 and LY303 on Ca signaling initiated by certain GPCR agonists in cells of several lines, including CHO cells expressing the recombinant serotonin receptor 5-HT2C and mesenchymal stromal cells derived from the human adipose tissue (AD-MSCs) and umbilical cord (UD-MSCs). The LY294/LY303 pair exerted apparently specific effects on responsiveness of AD-MSCs to ATP, suggesting the involvement of PI3K in ATP transduction.