Ca2, responses to either UTP or UDP were not abolished. Nevertheless, ma imal responses generated by UTP averaged 366 13%, significantly less than those observed in normal selleck chemicals FTY720 Krebs solution. The EC50 obtained for UTP in Ca2 free solution was 6. 2 0. 9 uM and was not significantly different from that obtained in normal Krebs. For UDP, similar findings were observed the ma imal response reached 230 15% and had an EC50 of 4. 9 0. 6 uM. neither parameter differed significantly from that in normal Krebs. This suggested that e tracellular Ca2 was not the major source of the i increase produced in TIC by UTP or UDP. more probably, this increase came from intracellular reservoirs via IP3 synthesis, as shown in other cell systems.

UTP induced activation of p44 and p42 MAPK In order to study the signaling pathway involved in the UTP and UDP activation of P2Y receptors in TIC, phos phorylation of the p44 and p42 MAPK proteins was eval uated. For these e periments, UTP was used as a specific agonist of the P2Y receptor subtypes studied. It was observed that UTP induced MAPK phosphorylation in a dose dependent manner with an EC50 of 3. 3 0. 9 and 1. 4 0. 7 uM for p44 and p42, respectively. ma imal increases of 541 25. 6% and 461 34. 8%, respectively, were observed by applying 100 uM UTP. The time course of this effect was studied by applying 10 uM UTP and measuring p44 and p42 MAPK phosphoryla tion at different times. The results indicated that ma imal phosphorylation occurred at 5 min of stimulation, and then it decreased slowly, returning to near basal levels about 30 min after UTP addition.

Because it has been shown consistently that UDP acts more potently on P2Y6 receptors, its ability to promote p44 and p42 MAPK phosphorylation was tested. In e periments similar to those presented above for UTP, 100 uM UDP was less potent and induced only modest responses of 199 43% and 158 15% for p44 and p42, respectively, compared to the basal level. the effect increased to 364 63% and 349 95%, Cilengitide respectively, with 1 mM UDP. The time course of p44 p42 phosphorylation induced by 1 mM UDP was similar to that elicited by 100 uM UTP. In addition, the p44 and p42 MAPK phosphorylation induced by 10 uM UTP was antagonized by suramin with an IC50 of 84. 3 10. 2 uM, suramin is a potent antagonist of P2Y2 receptors but is only a weak antagonist of P2Y6.

Conversely, PPADS up to 600 uM, a drug that antag onizes mainly the P2Y6 receptor, had no effect on UTP induced MAPK phosphorylation. These results suggested selleck compound that P2Y6 is not a major participant in the phosphorylation of MAPK. in consequence, the fol lowing e periments focused on defining the role of the P2Y2 receptor in the purinergic response. UTP induced p44 and p42 MAPK phosphorylation is dependent on PKC activation UTP dependent p44 p42 MAPK phosphorylation might be elicited via either of two main mechanisms 1 transac tivation of EGF receptors as has been demonstrated, for e ample, in salivary gland cells or 2 by activation of