High-affinity P2Y2 and low-affinity P2X7 receptor interaction modulates ATP-mediated calcium signaling in murine osteoblasts

The P2 purinergic receptor family implicated in many physiological processes, including neurotransmission, mechanical adaptation and inflammation, consists of ATP-gated non-specific cation channels P2XRs and G-protein coupled receptors P2YRs. Different cells, including bone forming osteoblasts, express multiple P2 receptors; however, how P2X and P2Y receptors interact in generating cellular responses to various doses of [ATP] remains poorly understood. Using primary bone marrow and compact bone derived osteoblasts and BMP2-expressing C2C12 osteoblastic cells, we demonstrated conserved features in the P2-mediated Ca2+ responses to ATP, including a transition of Ca2+ response signatures from transient at low [ATP] to oscillatory at moderate [ATP], and back to transient at high [ATP], and a non-monotonic changes in the response magnitudes which exhibited two troughs at 10−4 and 10−2 M [ATP]. We identified P2Y2 and P2X7 receptors as predominantly contributing to these responses and constructed a mathematical model of P2Y2R-induced inositol trisphosphate (IP3) mediated Ca2+ release coupled to a Markov model of P2X7R dynamics to study this system. Model predictions were validated using parental and CRISPR/Cas9-generated P2Y2 and P2Y7 knockouts in osteoblastic C2C12-BMP cells. Activation of P2Y2 by progressively increasing [ATP] induced a transition from transient to oscillatory to transient Ca2+ responses due to the biphasic nature of IP3Rs and the interaction of SERCA pumps with IP3Rs. At high [ATP], activation of P2X7R modulated the response magnitudes through an interplay between the biphasic nature of IP3Rs and the desensitization kinetics of P2X7Rs. Moreover, we found that P2Y2 activity may alter the kinetics of P2X7 towards favouring naïve state activation. Finally, we demonstrated the functional consequences of lacking P2Y2 or P2X7 in osteoblast mechanotransduction. This study thus provides important insights into the biophysical mechanisms underlying ATP-dependent Ca2+ response signatures, which are important in mediating bone mechanoadaptation.

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High-affinity P2Y2 and low-affinity P2X7 receptor interaction modulates ATP-mediated calcium signaling in murine osteoblasts

10.1101/2021.03.16.435624 ◽

2021 ◽

Author(s):

Nicholas Mikolajewicz ◽

Delaney Smith ◽

Svetlana V. Komarova ◽

Anmar Khadra

Keyword(s):

Mathematical Model ◽

Calcium Signaling ◽

P2 Receptors ◽

Receptor Interaction ◽

Receptor Subtype ◽

Dependent Manner ◽

P2 Receptor ◽

P2x7 Receptors ◽

High Affinity ◽

Kinetics Of

1AbstractP2 purinergic receptor family implicated in many physiological processes, including neurotransmission, mechanical adaptation and inflammation, consist of ATP-gated non-specific cation channels P2XRs and G-protein coupled receptors P2YRs. Different cells, including bone forming osteoblasts, express multiple P2 receptors; however, how P2X and P2Y receptors interact in generating cellular responses to various doses of [ATP] remains poorly understood. Using primary bone marrow and compact bone derived osteoblasts and BMP2-expressing C2C12 osteoblastic cells, we demonstrated conserved features in the P2-mediated Ca2+ responses to ATP, including a transition of Ca2+ response signatures from transient at low [ATP] to oscillatory at moderate [ATP], and back to transient at high [ATP], and a non-monotonic changes in the response magnitudes which exhibited two troughs at 10−4 and 10−2 M [ATP]. We identified P2Y2 and P2X7 receptors as predominantly contributing to these responses, and constructed a mathematical model of P2Y2R-induced inositol trisphosphate (IP3) mediated Ca2+ release coupled to a Markov model of P2X7R dynamics to study this system. Model predictions were validated using parental and CRISPR/Cas9-generated P2Y2 and P2Y7 knockouts in osteoblastic C2C12-BMP cells. Activation of P2Y2 by progressively increasing [ATP] induced a transition from transient to oscillatory to transient Ca2+ responses due to the biphasic nature of IP3Rs and the interaction of SERCA pumps with IP3Rs. At high [ATP], activation of P2X7R modulated the response magnitudes through an interplay between the biphasic nature of IP3Rs and the desensitization kinetics of P2X7Rs. Moreover, we found that P2Y2 activity may alter the kinetics of P2X7 towards favouring naïve state activation. Finally, we demonstrated the functional consequences of lacking P2Y2 or P2X7 in osteoblast mechanitransduction. This study thus provides important insights into the biophysical mechanisms underlying ATP-dependent Ca2+ response signatures, which are important in mediating bone mechanoadaptation.2Author SummaryATP-sensitive purinergic receptors comprise a network of cell-surface receptors that activate upon ATP binding, allowing them to transmit information in a tissue- and context-dependent manner. In bone, mechanically-stimulated osteoblasts release ATP that stimulates low- and high-affinity P2 receptors in neighboring cellular populations, inducing appropriate physiological responses. P2 receptor signaling is characterized by elevations in intracellular calcium levels. When simultaneously stimulated by their common ligand, ATP, the contribution of each P2 receptor subtype gives rise to a complex calcium response, exhibiting oscillatory characteristics and biphasic dose-dependent behaviours. Here we used experimental and computational modeling approaches to determine the underlying dynamics of ATP-mediated calcium signaling in osteoblasts. The latter was done by developing a mathematical model that was comprised of a subset of low-(P2X7) and high-(P2Y2) affinity P2 receptors, reflecting the conserved P2 expression observed across different osteoblast models. We demonstrated that this model recapitulates experimental recordings of ATP-induced calcium signaling in osteoblasts and describes the dynamic interplay between P2Y2 and P2X7 receptors in the P2 receptor network.

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Pregnancy-enhanced Ca2+ responses to ATP in uterine artery endothelial cells is due to greater capacitative Ca2+ entry rather than altered receptor coupling

Journal of Endocrinology ◽

10.1677/joe.1.06635 ◽

2006 ◽

Vol 190 (2) ◽

pp. 373-384 ◽

Cited By ~ 18

Author(s):

Shannon M Gifford ◽

Fu-Xian Yi ◽

Ian M Bird

Keyword(s):

Endothelial Cells ◽

Uterine Artery ◽

Purinergic Receptor ◽

Cell Types ◽

P2y Receptors ◽

Receptor Expression ◽

Receptor Subtype ◽

Initial Transient ◽

Receptor Coupling ◽

Specific Variation

Uterine artery endothelial cells (UAEC) derived from pregnant (P-UAEC) and nonpregnant (NP-UAEC) ewes retain pregnancy-specific differences in cell signaling as well as vasodilator production through passage 4. In particular, when P- and NP-UAEC are stimulated with ATP over a 2.5 min recording period, they exhibit similar initial transient peaks in the intracellular free Ca2+ concentration ([Ca2+]i), but the P-UAEC show a heightened sustained phase. In order to establish whether thiswas due to an altered subclass of purinergic receptor (P2), both the dose dependencyof [Ca2+]i responses to ADP and UTP and the profile of purinergic receptor expression are determined in NP- and P-UAEC. Our findings indicate that while several isoforms of P2X and P2Y receptors are present, it is P2Y2 that is responsible for the ATP-induced initial transient peak in both cell types. We also characterized several key components of the ATP-induced Ca2+ signaling cascade, including the inositol 1,4,5-trisphosphate receptor and G-proteins, but could not confirm any pregnancy-specific variation in the protein expression that correlated with pregnancy-specific differences in prolonged Ca2+ signaling. We thus investigated whether such a difference may be inherent to the cell itself rather than specific to the purinergic receptor-signaling pathway. Using thapsigargin (Tg), we were able to demonstrate that the initial Tg-sensitive intracellular pool of Ca2+is nearly identical with the capacity in both cell types, but the P-UAEC is nonetheless capable of greater capacitative Ca2+ entry (CCE) than NP-UAEC. Furthermore, CCE induced by Tg could be dramatically inhibited by 2-aminoethoxydiphenyl borate, suggesting a role for store-operated channels in the ATP-induced [Ca2+]i response. We conclude that changes at the level of capacitative entry mechanisms rather than switching of receptor subtype or coupling to phospholipase C underlies pregnancy adaptation of UAEC at the level of Ca2+signaling.

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