Modeling of Ca2+ transients initiated by GPCR agonists in mesenchymal stromal cells

Abstract

The integrative study that included experimentation and mathematical modeling was carried out to analyze dynamic aspects of transient Ca²⁺ signaling induced by brief pulses of GPCR agonists in mesenchymal stromal cells from the human adipose tissue (AD-MSCs). The experimental findings argued for IP₃/Ca²⁺-regulated Ca²⁺ release via IP₃ receptors (IP₃Rs) as a key mechanism mediating agonist-dependent Ca²⁺ transients. The consistent signaling circuit was proposed to formalize coupling of agonist binding to Ca²⁺ mobilization for mathematical modeling. The model properly simulated the basic phenomenology of agonist transduction in AD-MSCs, which mostly produced single Ca²⁺ spikes upon brief stimulation. The spike-like responses were almost invariantly shaped at different agonist doses above a threshold, while response lag markedly decreased with stimulus strength. In AD-MSCs, agonists and IP₃ uncaging elicited similar Ca²⁺ transients but IP₃ pulses released Ca²⁺ without pronounced delay. This suggested that IP₃ production was rate-limiting in agonist transduction. In a subpopulation of AD-MSCs, brief agonist pulses elicited Ca²⁺ bursts crowned by damped oscillations. With properly adjusted parameters of IP₃R inhibition by cytosolic Ca²⁺, the model reproduced such oscillatory Ca²⁺ responses as well. GEM-GECO1 and R-CEPIA1er, the genetically encoded sensors of cytosolic and reticular Ca²⁺, respectively, were co-expressed in HEK-293 cells that also responded to agonists in an "all-or-nothing" manner. The experimentally observed Ca²⁺ signals triggered by ACh in both compartments were properly simulated with the suggested signaling circuit. Thus, the performed modeling of the transduction process provides sufficient theoretical basis for deeper interpretation of experimental findings on agonist-induced Ca²⁺ signaling in AD-MSCs.

Keywords: Agonist-induced Ca2+ signaling; Ca2+-induced Ca2+ release; IP3 receptors; Mathematical modeling; Mesenchymal stromal cells.

Graphical abstract

Fig. 1

Dose dependencies of agonist responses. (A–С) Representative monitoring of intracellular Ca²⁺ in three different AD-MSCs, which were serially stimulated by noradrenaline (A), ATP (B), or adenosine (С) at indicated concentrations. (D) Summary of noradrenaline responses of 10 cells exhibiting the same threshold concentration of 0.15 μM. The dose-response curve was generated by averaging normalized Ca²⁺ responses at a given noradrenaline concentration within 0.03–1 μM. In each case, Ca²⁺ transients were normalized to a cell response to 1 μM noradrenaline. (E) Dose-response curves for ATP (triangles) and adenosine (squares) responses. The data were overaged over 9 cells responsive to ATP with the threshold of 1 μM and 7 adenosine-responsive cells exhibiting the 0.3-μM threshold. In each case, responses to 10 μM ATP and 5 μM adenosine were normalizing. (F) Representative Ca²⁺ transients elicited by adenosine at 100 nM (near-threshold concentration) and 2 μM in the same cell. These adenosine responses were delayed relative to the moment of agonist application by 97 s and 32 s, respectively. The characteristic time of the response delay (τ_d) was calculated as a time interval necessary for a Ca²⁺ transient to reach the half-magnitude. (G) Response lag versus agonist concentration (mean ± S.D.). The data were collected solely from robust noradrenaline-, ATP-, and adenosine-responsive cells (n=10, 16, and 21, respectively), which exhibit negligible responsivity rundown and tolerated the serial stimulation by the particular agonist at all indicated doses. In (A-D), ΔF = F-F₀, F is the instant intensity of cell fluorescence, F₀ is the averaged intensity of cell fluorescence in the very beginning of a recording.

Fig. 2

Involvement of the phosphoinositide cascade and CICR in agonist transduction. (A) Removal of bath Ca²⁺ weakly affected Ca²⁺ responses to 1 μM noradrenaline (18 cells). The PLC inhibitor U73122 (1 μM) suppressed AD-MSC responsivity to noradrenaline, while its much less effective analogue U73343 (1 μM) was ineffective (18 cells). (B, C) Evidence for Ca²⁺-induced Ca²⁺ release mediated by IP₃R. ATP (3 μM) and IP₃ uncaging by a 2-s UV flash elicited similar responses in a cell loaded with caged-Ins(145)P3/PM (36 cells) (B). In MSCs loaded with NP-EGTA and responsive to ATP (3 μM), 2-s Ca²⁺ uncaging elicited a small Ca2+ jump relaxing exponentially, while a 4-s UV flash elicited a large biphasic Ca²⁺ transient that was similar to an ATP response (13 cells) (C). Because a UV laser employed for uncaging was in fact a biharmonic light source emitting at 351 and 527 nm, a light stimulus caused an optical artifact that was seen as a marked overshoot in the fluorescence traces acquired at 535 ± 25 nm. (D) Ryanodine (50 μM) affected neither AD-MSC responsiveness to 0.5 μM noradrenaline nor Ca²⁺ transients elicited by Ca²⁺ uncaging by a 4-s UV flash (11 cells). In contrast, 50 μM 2-APB completely abolished biphasic agonist-like responses to Ca²⁺ uncaging by 4-s UV flashes. In the experiments with Ca²⁺ uncaging (C, D), emission of a UV laser was weakened by the factor ten, so that Ca²⁺ uncaging should have lasted for 4 s to liberate as many Ca²⁺ ions as necessary for stimulating CICR. This more gradual release of caged Ca²⁺ slowed the rising phase of a biphasic Ca²⁺ transient produced by CICR, thereby making a lag between a UV flash and a light response clearly visible. In (A-D), intracellular Ca²⁺ was monitored in different AD-MSCs.

Fig. 3

Kinetic model of agonist transduction in AD-MSCs. Substances: A, agonist; R, receptor; R*, activated receptor formed by reversible binding of A to R; G, G-protein; G*, activated G-protein; PIP₂, phosphatidylinositol (4,5)-bisphosphate; IP₃, inositol (1,4,5)-trisphosphate; Ca²⁺_c,cytosolic Ca²⁺; Ca²⁺_s, stored Ca²⁺ releasable by IP₃; Ca²⁺_e, extracellular Ca²⁺ postulated to be constant. The arrows with symbols denote +g: formation of G* catalyzed by R*. -g: inactivation of G* to G due to GTPase activity. +s: synthesis of PIP₂. -s: removal of PIP₂ by dephosphorylation and phosphorylation. plc: reaction catalyzed by phospholipase C. met: IP₃ metabolism 1. Ca²⁺ release through the IP₃ receptor 2. Refilling of Ca²⁺ store by reticular Ca²⁺-ATPase (SERCA). 3. Ca²⁺entry mediated by Ca²⁺ channels operating in the plasma membrane. 4. Ca²⁺ extrusion by plasma membrane Ca²⁺-ATPase (PMCA). The dashed red and blue arrows denote activation and inhibition, respectively.

Fig. 4

Simulations of Ca²⁺ transients in stimulated AD-MSCs. (A) The deviation of cytosolic Ca²⁺ on serial 60-s stimulation by an agonist at three different normalized ([A]/K_D) doses of 0.1, 0.2, and 1. (B) Model response magnitude (ο) versus normalized agonist dose ([A]/K_D). The dose-response curve for noradrenaline from Fig. 1D (•) is presented for comparison. The noradrenaline concentrations were normalized to 1 μM. (C) Model response lag (ο) versus normalized agonist concentration. The dose-response curve for noradrenaline from Fig. 1G (•) is presented for comparison. Agonist concentrations were normalized as in (B). (D) Simulation of Ca²⁺ uncaging by a step-like increase of intracellular Ca²⁺ from 100 nM to 180 and 300 nM. While the 80 nM jump elicited the small exponentially relaxing Ca²⁺ transient, the 200 nM jump produced the agonist-like Ca²⁺ response.

Fig. 5

Dynamics of the key mediators and cytosolic/reticular Ca²⁺ during agonist transduction. The kinetics of G*, PIP₂ and IP₃ (variables g, s, and p), cytosolic Ca²⁺ (C), and reticular Ca²⁺ (C_s) were simulated using Eqs. (2)–(6) at the basic parameter set (Table 1S). The sequential stimuli of increased concentrations (a=0.1, 0.2, and 1) were applied as indicated. G*, PIP₂, and IP₃ are presented in arbitrary units.

Fig. 6

Effect of exogeneous Ca²⁺ buffer on agonist response. (A) Loading of AD-MSCs with NP-EGTA affected AD-MSC responsiveness to 3 μM ATP, while the photolysis of this exogeneous Ca²⁺ buffer reversed effects of NP-EGTA. The recordings from cells 1–3 exemplify the diversity of NP-EGTA effects. (B) Simulations of effects of exogeneous saturable NP-EGTA-like Ca²⁺ buffers on agonist-induced Ca²⁺ transients using the Eqs. (2)–(5), 6b), ((8), and (9) with increasing B_t. Parameter values were as in Table 1S, and K_bD=0.08 μM, k_-b=0.5 s^-1.

Fig. 7

Oscillatory Ca²⁺ signals elicited by agonist. (A) Representative (43 cells) monitoring of GEM-GECO1 and R-CEPIA1er fluorescence in a transfected HEK-293 cell. The upper curve reflects the behavior of cytosolic Ca²⁺ upon cell stimulation with ACh at doses varied as indicated. The evolution of reticular Ca²⁺ is depicted by the bottom trace. To evaluate a relative change in reticular Ca²⁺ elicited by ACh, in the end of the recording, Ca²⁺ store was emptied by ionomycin (5 μM ) applied with 260 nM Ca²⁺ in the bath. For GEM-GECO1 or R-CEPIA1er, the fluorescence traces were presented as ΔF/F₀, where ΔF = F₀-F, or ΔF = F-F₀, respectively, F is the instant intensity of cell fluorescence, F₀ is the averaged intensity of cell fluorescence in the very beginning of a recording. The R-CEPIA1er signal was corrected for photobleaching (Supplementary Fig. 1S). (B) Simulations of oscillatory Ca²⁺ signals elicited by an agonist at two doses in the cytosol (upper curves) and in Ca²⁺ store (bottom curves). The computations were performed using the Eqs. (2)-(6) with the basic parameter set (Table 1S) except for m=3, K_i=0.4 μM, and a_c=70.