Regulation by P2 agonists of the intracellular calcium concentration in epithelial cells freshly isolated from rat trachea

Epithelial cells were isolated from rat trachea by incubation of the organ in a calcium-free medium. The intracellular concentration of calcium ([Ca2+]i) was measured with the calcium-sensitive fluorescent dye fura2. In resting conditions, the cells maintained a low [Ca2+]i in spite of the presence of millimolar concentration of calcium in the incubation medium. These cells had retained intracellular stores of calcium which were emptied after exposure of the cells to thapsigargin, an inhibitor of intracellular calcium ATPases. Substance P (125 nM) transiently increased 2.5-fold the [Ca2+]i. ATP (1 mM) doubled the [Ca2+]i after a few seconds and further induced a sustained increase of the [Ca2+]i. Coomassie blue fully blocked the response to ATP and extracellular magnesium only inhibited the delayed response to ATP. Among purinergic analogs, only benzoyl-ATP (Bz-ATP), an agonist on P2X ionotropicpurinergic receptors, reproduced the response to ATP. UTP and 2-methylthioATP (two agonists on P2Y metabotropic purinergic receptors) transiently increased the [Ca2+]i. Thapsigargin, ATP and Bz-ATP increased the uptake of extracellular calcium. RT–PCR analysis revealed that two metabotropic receptors (P2Y1 and P2Y2) and two ionotropic receptors (P2X4 and P2X7) were expressed by the cells present in the suspension. It is concluded that purinergic agonists can modulate the response of rat tracheal epithelial cells by several mechanisms. The activation of metabotropic receptors should mobilize intracellular IP3-sensitive calcium pools. The activation of the ionotropic receptors should not only open a non-specific cation channel leading to the entry of calcium but should also induce the formation of pores in cells expressing the P2X7 receptors, which could be deleterious to these cells.