Cytosolic Ca²⁺ clearance plays a key role in Ca²⁺ signalling. The aim of this study was to use both theoretical and experimental approach to determine the influence of the sarco-endoplasmic Ca²⁺-ATPase (SERCA) and mitochondrial Ca²⁺ uptake on cytosolic Ca²⁺ clearance in airway myocytes.

Experimental studies were performed on myocytes freshly isolated from rat trachea. Rats were humanely killed according to national guidelines. [Ca²⁺]_i responses were measured by microspectrofluorimetry using the Ca²⁺-sensitive fluorescent dye Indo-1. Data are mean ± S.E.M. Statistical comparisons were done by Student t tests or ANOVA for repeated measurements, and considered as significant when P < 0.05.

Stimulation by caffeine (CAF) for 30 s induced a concentration-graded response characterised by a transient peak followed by a progressive decay to a plateau phase. The peak and plateau values in response to 30 s stimulation by 5 mM CAF were 697 ± 59 nM and 43 ± 4.0 nM, respectively (n = 19). For 1 s CAF stimulation, the amplitude of the peak was not modified (695 ± 82 nM, n = 35), but the decay phase was accelerated, indicating ryanodine receptor (RyR) closure. A second CAF stimulation 15 s after the first one induced a second peak smaller than the first one (206 ± 26 nM, n = 35). In Na⁺-Ca²⁺-free medium containing 0.5 mM La³⁺, the [Ca²⁺]_i response pattern to CAF 30 s and 1 s caffeine stimulation was not significantly modified, indicating no involvement of transplasmalemmal Ca²⁺ fluxes. On these experimental basis, we built a mathematical model describing the mechanism of Ca²⁺ handling upon RyR stimulation that included sarcoplasmic reticulum (SR), mitochondria and cytosolic proteins as Ca²⁺ buffers. Our model predicts that after Ca²⁺ release from the SR, Ca²⁺ is first buffered by cytosolic proteins and mitochondria, and pumped back into the SR after a time delay.To test the model predictions, we studied the effect of cyclopiazonic acid (CPA), a reversible inhibitor of SERCA, and FCCP, a mitochondrial inhibitor, on 1 s CAF stimulation. 30 s exposure to 10 microM CPA or 5 microM FCCP did not significantly modified the resting [Ca²⁺]_i. When cells were stimulated 30 s after the beginning of CPA exposure, the first peak was not modified (96.4 ± 6.9 % control, n = 21), but the Ca²⁺ response to the second stimulation was greatly decreased (49.2 ± 68.1 % control), indicating SERCA inhibition by CPA. However, the [Ca²⁺]_i decay following Ca²⁺ increase was not modified compared to control. When cells were stimulated in the presence of FCCP, the amplitude of the peak was not modified (102.4 ± 6.2 % control, n = 25). The subsequent [Ca²⁺]_i decay down to baseline persisted but was significantly slowed down.

This study indicate that, after Ca²⁺ release from the SR, the shape of the [Ca²⁺]_i decay does not primarily depend on SERCA activity but on mitochondrial Ca²⁺ uptake, in parallel with other buffering processes that may be, according to the model predictions, cytosolic Ca²⁺ binding proteins.