Interplay between P2X7 and P2Y1 receptors controls intracellular [Ca2+] signals in fibroblasts of the human subcutaneous connective tissue

D Paramos1, AR Pinheiro1, F Ferreirinha1, MA Costa1,2, P Correia-de-Sá1. 1Instituto de Ciências Biomédicas Abel Salazar - Universidade do Porto (ICBAS-UP), Laboratório de Farmacologia e Neurobiologia, Portugal, 2Instituto de Ciências Biomédicas Abel Salazar - Universidade do Porto (ICBAS-UP), Laboratório de Química, Portugal

Several P2 purinoceptor-based mechanisms likely contribute to the ability of ATP to alter nociceptive sensitivity following tissue injury. Activation of P2X3, P2X2/3, P2X4, P2X7, and P2Y receptors is able to modulate pain via direct (neuronal excitability) and/or indirect (neural-glial and neural-inflammatory cell interactions) mechanisms. Recently, P2X7 receptors have been subject of many studies as they might play an important role in chronic inflammation and pain. Moreover, it appears that these receptors may form functional heteromeric receptors with other purinoceptors (e.g. P2X4/7), which might complicate data interpretation regarding their involvement in chronic inflammation and pain (reviewed by Carroll et al., 2009, Purinergic Signal. 5:63-73). Considering that subcutaneous connective tissue is richly innervated by afferent neurons, we aimed at investigating the expression and functional role of P2X7 and P2Y1 receptors on human fibroblasts as we hypothesized that these cells may process and amplify signals between inflammatory cells and sensory nerves. Fibroblasts were isolated from subcutaneous connective tissue of human organ donors. Cells were obtained by the explant technique and experiments were performed using the first subculture. Intracellular calcium oscillations were monitored in the time-lapse-mode using a laser scanning confocal microscope (Olympus FV, Japan) after loading the cells with Fluo-4NW (2.5 μM, 45 min at 37ºC). Immunolocalization studies confirmed the expression of P2X7 and P2Y1 receptors on human fibroblasts. The ATP analogue exhibiting some degree of selectivity for P2X7 receptors, BzATP, increased intracellular calcium ([Ca2+]i); the effect of BzATP (500 µM, n=163) peaked 120 s after drug application and decreased thereafter until removal of the drug from the bathing solution (6 min incubation). BzATP-induced [Ca2+]i transients were significantly (P<0.05) attenuated by the selective P2X7 receptor antagonist, A438079 (3 µM, n=86). Previous studies provided information demonstrating that BzATP may act as a partial agonist of metabotropic ADP-sensitive P2Y1 receptors. The selective P2Y1 receptor antagonist, MRS 2179 (0.3 µM, n=23), attenuated the peak [Ca2+]i response of BzATP (500 µM, n=163), without significantly affecting the kinetics of the rising phase. The opposite occurred when BzATP (500 µM, n=163) was applied after pre-incubation with ADP; in these conditions, ADP (1 mM, n=23) delayed BzATP-induced [Ca2+]i rise. In contrast with BzATP (500 µM), ADP (1 mM, n=23)-induced [Ca2+]i response was faster and brief, attaining a maximum 60 s after drug application. Surprisingly, when cells where incubated with ADP (1 mM, n=75) after pre-incubation with BzATP (500 µM), the kinetics of fibroblasts [Ca2+]i responses became similar to that observed with BzATP alone. This result rules out the possibility that, under the present circumstances, BzATP acts as a partial agonist of P2Y1 receptors. Data suggest that activation of P2X7 and P2Y1 may cooperate to increase [Ca2+]i in fibroblasts of the human subcutaneous connective tissue. Whether these receptors form a novel heteromeric complex giving rise to distinct pharmacological profiles or whether BzATP activates P2X7 receptors to release ATP which then acts on P2Y1 receptors deserves further investigations.

Supported by FCT (FEDER funding, PTDC/SAU-FCF/108263/2008 and UMIB-215/94). AR Pinheiro is in receipt of a FCT PhD Studentship (SFRH/BD/47373/2008).