The signaling mechanisms of long distance intercellular calcium waves (far waves)in cultured human uterine myocytes

Abstract

Cultured human myocytes exhibit intercellular calcium waves that travel farther than 100 μm (‘far waves’). This work investigates the mechanism of far wave propagation. Culture lines were initiated from myometrial biopsies of term pregnant women. Calcium green-1 was used as a fluorescence probe for intracellular free calcium. Serial imaging was performed at a frame rate of 0.83 frames/s. Intercellular calcium waves were mechanically initiated by atraumatically applying small drops of mineral oil onto the surface of the monolayer. Each intercellular calcium wave was scored using a standardized grid, and points were assigned depending upon the distance the wave traveled and the fluorescent intensity observed within each region. Experiments were performed in the presence of inhibitors of gap junctions and connexin hemichannels (octanol), ATP (apyrase and MDL 12330 A), prostaglandins (indomethacin, high concentrations of lanthanum), the prostaglandin transporter, PGT (DIDS), and transmembrane calcium flux (low concentrations of lanthanum). Octanol, apyrase and MDL 12330 A failed to modify the far waves, indicating gap junctions, connexin hemichannels and ATP do not participate in the paracrine mechanism. Indomethacin at 30, 100 and 300 μM, in a dose dependent manner, reduced the far wave score to 0, suggesting a prostaglandin was critically involved in the mechanism. DIDS reduced the far wave score, but did not fully inhibit wave propagation, suggesting the presence of PGT-dependent and -independent components to the mechanism. Lanthanum at 0.1 mM had no effect, but at 1 mM, reduced the far wave score. These results are consistent with PGF_2α and/or PGE₂ being the signal molecule for the PGT-dependent component. Taken together, these data indicate that long distance intercellular calcium waves in cultured human myocytes utilizes a paracrine signaling mechanism, but with more than one extracellular signaling compound.