By simultaneously measuring intraluminal SR ([Ca²⁺]_L) and cytosolic ([Ca²⁺]_i) calcium concentration in single uterine smooth muscle cells, we have found (Shmigol et al. 2001) that during application of agonists [Ca²⁺]_L decreased and [Ca²⁺]_i rose. These data are consistent with an InsP₃-mediated SR calcium release mechanism. There was, however, in the maintained presence of agonist, a partial re-accumulation of [Ca²⁺]_L. The aim of the present study was to investigate this re-accumulation, and its contribution to subsequent agonist-induced Ca²⁺ changes, in both the SR and cytosol.

Nineteen- to 21-day pregnant rats were killed by cervical dislocation after CO₂ anaesthesia. Single uterine cells were prepared by enzymatic digestion and loaded with mag-fluo-4 and fura-2 for SR and cytosolic Ca²⁺ measurements, respectively (see Shmigol et al. 2001).

We found that repetitive applications of agonist (carbachol and ATP) to cells bathed in Ca²⁺-free solution, evoked progressively decreasing [Ca²⁺]_i transients (n = 27). This occurred despite the fact that [Ca²⁺]_L was not depleted and in fact was partially restored during each application. The decrease was not due to desensitisation of the plasmalemmal receptors, as intercalating applications of different agonists led to the same effect. Moreover, increasing the SR Ca content did not potentiate the agonist induced Ca²⁺ release (n = 6), as illustrated in Fig. 1.In these experiments, carbachol was applied initially in the presence of extracellular Ca²⁺. This activated both SR Ca²⁺ release and Ca²⁺ entry from outside the cell. As a result, [Ca²⁺]_L was increased at the end of application. The rest of the experiment was performed in Ca²⁺-free solution. As can be seen the second application of carbachol was not potentiated. A third application of carbachol and then ATP application produced little effect, despite the fact that SR contained plenty of Ca²⁺. This was confirmed by the application of 1 µM ionomycin. Preliminary, rapid confocal imaging of the [Ca²⁺]_L during agonist application, suggests that release and re-accumulation sites in uterine smooth muscle cells may be spatially and functionally separated from each other. Such a conclusion would also be consistent with the above data.This work was supported by the Medical Research Council.

Figure 1. Changes in cytosolic and luminal Ca2+ levels evoked by 100 µM carbachol (dashed bar) and 100 µM ATP (continuous bar) in normal Krebs and in Ca2+-free solution (indicated by the open bar). Top trace: mag-fluo-4 fluorescence expressed as F/Fo, where Fo is the fluorescence intensity before stimulation. Bottom trace: the cytosolic Ca2+ calculated from fura-2 ratio.

Shmigol, A.V., Eisner, D.A. & Wray, S. (2001). J. Physiol. 531, 707-713. abstract