Physiological evidence for a P2Y receptor responsive to diadenosine polyphosphates in human lung via Ca 2+ release studies in bronchial epithelial cells

Abstract

P2Y 2 receptors that are activated by the extracellular nucleotides ATP or UTP mediate Cl − secretion via an increase in [Ca 2+] i (intracellular calcium concentration). Therefore, in the lung of patients suffering from cystic fibrosis, inhalation of aerosolized UTP offers a way to circumvent the defect in Cl − secretion by the cystic fibrosis transmembrane conductance regulator. A possible alternative for the relatively unstable UTP in inhalation therapy is the more resistant diadenosine tetraphosphate (Ap 4A). In human and rat lung membranes, Ap 4A binds to P2 receptor sites coupled to G proteins. Here, we showed that Ap 4A caused an increase in [Ca 2+] i with an ec 50 of 17 μM in human bronchial epithelial cells (HBE1). The [Ca 2+] i rise evoked by ATP and UTP was completely, but that induced by Ap 4A only partially, caused by release of Ca 2+ from internal stores. Moreover, the potency of Ap 4A to mobilize Ca 2+ was lower than that of ATP and UTP ( ec 50 1.5 and 1.8 μM, respectively), and the maximal increase in [Ca 2+] i was considerably smaller than that after ATP or UTP. In accordance with our previous results providing evidence for a common binding site for various diadenosine polyphosphates in lung membranes, all Ap nA analogues tested (n = 3 to 6) caused a comparable [Ca 2+] i increase. Homologous or heterologous prestimulation largely diminished the increase in [Ca 2+] i found after a second pulse of either UTP or Ap 4A. Although specific binding characteristics and functional responses of Ap 4A on lung cells are in favor of a distinct receptor for Ap 4A, the cross-talk between UTP and Ap 4A in HBE1 cells and the only slight differences in Ca 2+ mobilization by ATP or UTP and Ap 4A render it impossible at this point to state unequivocally whether there exists a distinct P2Y receptor specific for diadenosine polyphosphates in lung epithelia or whether Ap 4A activates one of the nucleotide receptors already described.