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. 2015 Nov;53(5):703-11.
doi: 10.1165/rcmb.2014-0386OC.

Ryanodine receptor sensitization results in abnormal calcium signaling in airway smooth muscle cells

Huguette Croisier  1 , Xiahui Tan  2 , Jun Chen  3 , James Sneyd  4 , Michael J Sanderson  3 , Bindi S Brook  1
Affiliations

Ryanodine receptor sensitization results in abnormal calcium signaling in airway smooth muscle cells

Huguette Croisier et al. Am J Respir Cell Mol Biol. 2015 Nov.

Abstract

Intracellular Ca(2+) dynamics of airway smooth muscle cells (ASMCs) are believed to play a major role in airway hyperresponsiveness and remodeling in asthma. Prior studies have underscored a prominent role for inositol 1,4,5-triphosphate (IP3) receptors in normal agonist-induced Ca(2+) oscillations, whereas ryanodine receptors (RyRs) appear to remain closed during such Ca(2+) oscillations, which mediate ASMC contraction. Nevertheless, RyRs have been hypothesized to play a role in hyperresponsive Ca(2+) signaling. This could be explained by RyRs being "sensitized" to open more frequently by certain compounds. We investigate the implications of RyR sensitization on Ca(2+) dynamics in ASMC using a combination of mathematical modeling and experiments with mouse precision-cut lung slices. Caffeine is used to increase the sensitivity of RyRs to cytosolic Ca(2+) concentration ([Ca(2+)]i) and sarcoplasmic reticulum Ca(2+) ([Ca(2+)]SR). In ASMCs, high caffeine concentrations (>10 mM) induce a sustained elevation of [Ca(2+)]i. Our mathematical model accounts for this by the activation of store-operated Ca(2+) entry that results from a large increase in the RyR sensitivity to [Ca(2+)]SR and the associated Ca(2+) release, which leads to a reduction of [Ca(2+)]SR. Importantly, our model also predicts that: (1) moderate RyR sensitization induces slow Ca(2+) oscillations, a result experimentally confirmed with low concentrations of caffeine; and (2) high RyR sensitization suppresses fast, agonist-induced Ca(2+) oscillations by inducing substantial store-operated Ca(2+) entry and elevated [Ca(2+)]i. These results suggest that RyR sensitization could play a role in ASMC proliferation (by inducing slow Ca(2+) oscillations) and in airway hyperresponsiveness (by inducing greater mean [Ca(2+)]i for similar levels of contractile agonist).

Keywords: Ca2+ oscillations; asthma; hypersensitivity; mathematical modeling; precision-cut lung slice.

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Figures

Figure 1.
Figure 1.
Schematic diagram of the major pathways involved in the model of Ca2+ dynamics in airway smooth muscle cells (ASMCs). Solid black lines denote Ca2+ fluxes. Stimulation of cell surface G protein–coupled receptors (GPCR) leads to production of inositol 1,4,5-triphosphate (IP3) and activation of the IP3 receptor (IP3R). Subsequent release of Ca2+ from the sarcoplasmic reticulum (SR) leads to Ca2+-induced Ca2+ release (CICR) through either IP3R or ryanodine receptors (RyRs). Depletion of the SR induces the opening of store-operated Ca2+ channels (SOCCs) in the plasma membrane but also inactivates RyR. Ca2+ is removed from the cytoplasm by plasma membrane ATPase (PMCA) or by sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA). Addition of extracellular KCl leads to membrane depolarization and opening of voltage-operated Ca2+ channels (VOCCs). The consequent increase in Ca2+ influx causes (in the absence of agonist) overfilling of the SR and activation of RyR. IP3R and RyR interact via their effects on the concentration of Ca2+ ([Ca2+]) in the cytosol and the SR. Here, we focus on the predicted effects of caffeine (and other compounds), which sensitizes RyR to cytosolic and luminal Ca2+.
Figure 2.
Figure 2.
(A) Simulations of the effect of increasing levels of cytosolic RyR sensitization (rc) on cytosolic Ca2+ concentration ([Ca2+]i) dynamics, starting from (rc, rs) = (0, 1) (red dot in Figure E3A). (B) Simulations of the effect of increasing levels of luminal RyR sensitization (rs) on [Ca2+]i dynamics, starting from (rc, rs) = (0, 0) (no sensitization; black dot in Figure E3A). (C) Relative fluorescence signals indicating the [Ca2+]i responses induced by caffeine in ASMCs of mouse lung slices (see Materials and Methods) for (i) 1 mM caffeine, (ii) 3 mM caffeine, and (iii) 10 mM caffeine. (D) The predicted sarcoplasmic reticulum Ca2+ ([Ca2+]SR) associated with the [Ca2+]i dynamics modeled in B. (E) The predicted fraction of open RyRs (magenta) and open SOCCs (blue) associated with the [Ca2+]i dynamics in B.
Figure 3.
Figure 3.
(A and B) Effect of agonist application on ASMC Ca2+ dynamics with different levels of pre-existing luminal RyR sensitization (rs; increasing from top to bottom—red to blue to green), in the absence of cytosolic RyR sensitization (rc = 0). (A) Low [IP3] (p = 0.5), and (B) high [IP3] (p = 1.5), applied at t of 200 seconds (p = IP3 concentration). (C and D) Long-term mean [Ca2+]i (black) and Ca2+ oscillation frequency (red) as a function of luminal RyR sensitization for the two agonist levels in A and B.
Figure 4.
Figure 4.
Effect of simultaneous cytosolic and luminal RyR sensitization on agonist-induced Ca2+ oscillations for low [IP3] (p = 0.5). (A) Increasing levels of cytosolic sensitization (from top to bottom) with RyR luminal sensitization (rs = 3; top panel is identical to middle panel in Figure 3A). (B) Long-term mean [Ca2+]i (black curves) and oscillation frequency (red curve) as a function of luminal RyR sensitization for the two non-zero sensitization levels used in A (compare with Figure 3C).
Figure 5.
Figure 5.
Effect of different levels of RyR sensitization (increasing as indicated by the arrow) on the entire dose–response curve relating mean [Ca2+]i to [IP3] during agonist stimulation. Dashed curves correspond to Ca2+ oscillations and solid curves to steady Ca2+. Dotted vertical lines indicate the [IP3] values used in Figures 3A and 3B. The bottom (red) curve is the dose–response curve in the absence of sensitization; hence, it corresponds to the response curve in Figure E2A. Gray curves represent unstable solutions.
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