Ca²⁺ waves in smooth muscle, from sarcolemma agonist activity, play an important role in normal physiological function. Yet the mechanisms underlying wave propagation are unclear. One proposal is that a small catalytic Ca²⁺ release from the InsP₃-gated channel, on the sarcoplasmic reticulum, activates neighbouring ryanodine receptors (RyR) to generate a more robust Ca²⁺ release. Ca²⁺ diffuses from this latter site to activate nearby RyR (Ca²⁺-induced Ca²⁺ release) and so the cycle of release and diffusion continues through the cell by repeated activation of RyR. However, experimental studies on the dual roles of InsP₃ and RyR in the generation of Ca²⁺ waves, in intact cells, have been hampered by the inability to evoke rapid and reproducible step changes in InsP₃ in subcellular regions. As a result, our understanding of the generation of waves in smooth muscle derives, predominantly, from studies using pharmacological approaches, which may lack specificity in intact cells. In the present study the initiation and propagation of Ca²⁺ waves, in single voltage-clamped smooth muscle cells, was examined using methods enabling elevations in InsP₃ to occur in subcellular regions in the intact cell. The cytoplasmic Ca²⁺ concentration ([Ca²⁺]_c) was imaged (with fluo-3) simultaneously at high temporal resolution (100 frames per second; 562 nm pixels at the cell, 160 X 160 pixel array).

From male guinea-pigs stunned by a blow to the head and killed by exsanguination, following the guidelines of the Animal (Scientific Procedures) Act, 1986, a segment of colon was removed and single smooth muscle cells prepared (McCarron & Muir, 1999). Depolarisation (-70 mV to 0 mV) increased [Ca²⁺]_c uniformly throughout the cell; the magnitudes and time courses were similar in all regions. The rate of decline of [Ca²⁺]_c, after the depolarisation, though slower than the rise, was also similar throughout. In contrast, the [Ca²⁺]_c increase by sarcolemma agonists, which generate InsP₃, was not uniform but often appeared as a localised rise which then moved through the cell with approximately constant amplitude though with a variable velocity (a Ca²⁺ wave). The contribution of RyR to wave propagation was examined. Local increases in InsP₃ (in a 5 mm diameter region) were evoked by spot flash photolysis in single cells (V_M -70 mV; mean bulk average [Ca²⁺]_c 99 ± 21 nM, mean ± S.E.M., n = 7) and produced an increase in [Ca²⁺]_c of a magnitude similar to that evoked by the agonist, but did not generate a propagating Ca²⁺ wave; [Ca²⁺]_c declined in amplitude from the site of release. In the same cell, after depolarising to -20 mV, which elevated bulk average [Ca²⁺]_c (276 ± 32 nM, mean ± S.E.M., n = 7) and activated RyR, as evidenced by the occurrence of STOCs, local InsP₃ increases still failed to evoke a propagating Ca²⁺ wave. The failure of InsP₃ itself to evoke a Ca²⁺ wave could not be accounted for by a requirement for Ca²⁺ release at a specific initiating site, since focal release of InsP₃ at different 5 mm diameter locations throughout the cell evoked approximately equal increases in [Ca²⁺]_c, which declined in amplitude from the release site. However, in the presence of a low concentration of a sarcolemma agonist that generated InsP₃ (which itself did not evoke an increase in [Ca²⁺]_c) focal release of InsP₃ generated a propagating Ca²⁺ wave. PKC activation did not enable wave propagation since indolactam did not significantly alter the release evoked by InsP₃. On the other hand dialysing the cell with low concentrations of free InsP₃ permitted local increases in [Ca²⁺]_c, from the release of caged InsP₃, to evoke a propagating Ca²⁺ wave. Thus low concentrations of InsP₃ may increase the Ca²⁺ sensitivity of the InsP₃ receptor. These results suggest that a global subthreshold increase in InsP₃ throughout the cell, by agonists, may allow a propagating Ca²⁺ wave to occur by Ca²⁺-induced Ca²⁺ release acting on the InsP₃ receptor rather than RyR.

This work was supported by The Wellcome Trust and British Heart Foundation.

All procedures accord with current UK legislation.