Modeling Interactions among Individual P2 Receptors to Explain Complex Response Patterns over a Wide Range of ATP Concentrations

Abstract

Extracellular ATP acts on the P2X family of ligand-gated ion channels and several members of the P2Y family of G protein-coupled receptors to mediate intercellular communication among many cell types including bone-forming osteoblasts. It is known that multiple P2 receptors are expressed on osteoblasts (P2X2,5,6,7 and P2Y1,2,4,6). In the current study, we investigated complex interactions within the P2 receptor network using mathematical modeling. To characterize individual P2 receptors, we extracted data from published studies of overexpressed human and rodent (rat and mouse) receptors and fit their dependencies on ATP concentration using the Hill equation. Next, we examined responses induced by an ensemble of endogenously expressed P2 receptors. Murine osteoblastic cells (MC3T3-E1 cells) were loaded with fluo-4 and stimulated with varying concentrations of extracellular ATP. Elevations in the concentration of cytosolic free calcium ([Ca2+]i) were monitored by confocal microscopy. Dependence of the calcium response on ATP concentration exhibited a complex pattern that was not explained by the simple addition of individual receptor responses. Fitting the experimental data with a combination of Hill equations from individual receptors revealed that P2Y1 and P2X7 mediated the rise in [Ca2+]i at very low and high ATP concentrations, respectively. Interestingly, to describe responses at intermediate ATP concentrations, we had to assume that a receptor with a K1∕2 in that range (e.g. P2Y4 or P2X5) exerts an inhibitory effect. This study provides new insights into the interactions among individual P2 receptors in producing an ensemble response to extracellular ATP.

Transmission of mechanical information by purinergic signaling

“…We showed that the total amount of ATP released depended on both the severity of mechanically-induced injury 0 and rate of membrane repair 1/2 , while the peak extracellular ATP concentration was determined by the severity of the injury alone. Since the P2 receptor network consists of 15 receptor subtypes with ATP affinities covering over a million-fold range of ATP concentrations (16,17), the peak ATP release will determine the degree of activation of low-affinity P2 receptors [e.g. P2X7; ec50 = 1.9 mM (human), ec50 = 130 µM (rat) (16)].…”

“…Since the P2 receptor network consists of 15 receptor subtypes with ATP affinities covering over a million-fold range of ATP concentrations (16,17), the peak ATP release will determine the degree of activation of low-affinity P2 receptors [e.g. P2X7; ec50 = 1.9 mM (human), ec50 = 130 µM (rat) (16)]. In contrast, the total amount of ATP released will determine the area over which high-affinity P2 receptors are activated [e.g.…”

“…In contrast, the total amount of ATP released will determine the area over which high-affinity P2 receptors are activated [e.g. P2Y2; ec50 = 200 nM (human), ec50 = 2.9 µM (rat) (16)]. Thus, the P2 receptors network can decipher the information about the extent of the injury and the rate of repair encoded in the dynamics of ATP release.…”

“…Several P2 receptors have been implicated in the mechano-adaptive response in bone (13)(14)(15), however it is uncertain why so many receptors are required and how they integrate mechanical information. While individual P2 receptors are sensitive to ATP concentrations over 2-3 orders of magnitude, the entire P2 receptor network covers over a million-fold range of ATP concentrations (16,17). In addition, many P2Y receptors are differentially sensitive to ADP and ATP.…”

Systematic Characterization of Dynamic Parameters of Intracellular Calcium Signals

“…P2 receptors have been demonstrated to play an important role on bone physiology (Lenertz et al, 2015). Since individual bone cells commonly express multiple active P2 receptors (Gallagher and Buckley, 2002), responses to purinergic stimulation result in complex, concentration-dependent [Ca 2+ ] i transients (Xing et al, 2016). While it remains difficult to experimentally isolate the contribution of individual receptors, a number of studies have demonstrated that various P2 receptor subtypes have distinct calcium response kinetics and signatures.…”