Surprisingly, inhibition of membrane bound NTPDases with POM 1 also decreased the plat eau phase of bradykinin induced i response in human subcutaneous fibroblasts. The result obtained with POM 1 was confirmed when we tested the effect of bradykinin on i oscillations in the ab sence of Mg2, an ion that must be present in millimolar together concentration in the extracellular fluid for maximum ac tivity of ectonucleotidases. These find ings provide the first evidence that the plateau phase of bradykinin induced i accumulation by human subcutaneous fibroblasts requires the release of ATP and its subsequent conversion into other biologically active metabolites, most probably ADP, by ectonucleotidases. The kinetics of the extracellular catabolism of adenine nucleotides and formation of me tabolites in fibroblasts cultured from the human subcuta neous tissue is shown in Figure 5B.

Average half lives of ATP, ADP and AMP were respectively 12. 6 4. 2 min, Inhibitors,Modulators,Libraries 27. 0 1. 6 min and 1. Inhibitors,Modulators,Libraries 5 0. 2 min, when the substrates were used in a 3 uM concentration. ATP was sequentially Inhibitors,Modulators,Libraries metabolized into ADP, adenosine, inosine and hypoxan thine. AMP was rapidly and sequentially converted into ADO and INO respectively by ecto 5 nucleotidase and adenosine deaminase, which might explain why AMP accumulation Inhibitors,Modulators,Libraries was almost negligible when ATP and ADP were used as substrates. The analysis of the corresponding half life time values clearly indicates that the extracellular catabolism of ADP into AMP is the rate limiting step to generate ADO from exogenously added adenine nucleotides in cultured human subcutaneous fibroblasts.

Therefore, transient accumulation of ADP in the cultures Inhibitors,Modulators,Libraries is in favor of a preferential activation of ADP sensitive P2Y purinoceptors. To investigate the contribution of ADP sensitive P2Y purinoceptors activation to bradykinin induced i response in human subcutaneous fibroblasts, we tested its effect in the pres ence of selective P2Y1, P2Y12 and P2Y13 receptors antago nists. Selective blockade of the P2Y12 receptor with AR C 66096 significantly attenuated the plateau phase of i rise caused by bradykinin without much affecting the magnitude of the initial i rise. No significant differences were observed in the presence of MRS 2179 and MRS 2211 which selectively antagonize P2Y1 and P2Y13 receptors, respectively. None of these antagonists modified per se i in human subcutaneous fibroblasts. The expression of the P2Y12 receptor in cultured human subcutaneous fibroblasts was confirmed by immunocytochemistry. mostly Adenine nucleotides, but not adenosine, increase the influx of Ca2 in human subcutaneous fibroblasts Changes in i in human subcutaneous fibroblasts treated with adenine nucleotides in the presence and in the absence of extracellular Ca2 are shown in Figure 6A.