kinases through its canonical pathway. However, EGF transactivation www.selleckchem.com/products/brefeldin-a.html requires long term stimulation with the agonist, and activation of p44 p42 MAPK in TIC was generated even with short incubations. thus, it was decided to analyze first the role of down stream kinases. Two of the main candidate protein kinases, PKC and PI3K, were blocked using specific pharmacologi cal tools. PI3K activation was blocked by preincubation for 30 min with the selective inhibitor 100 nM wortmanin or with 1 uM LY294002 before stimulating the cells with 10 uM UTP. under these condi tions, neither inhibitor had any effect on p44 or p42 MAPK phosphorylation. To study the possible participation of PKC, TIC cul tures were preincubated with 250 nM stauro sporine and then tested with 10 uM UTP.

Staurosporine treatment blocked completely the UTP stimulated p44 and p42 phosphorylation, strongly suggesting that phosphorylation was dependent on PKC. To support this idea, e periments were carried out in which PKC activity was downregulated by long term incubations with phorbol 12 myristate 13 acetate. Thus, TIC were pretreated for 18 h with 1 uM PMA, which did not affect the basal levels of phosphorylated p44 or p42 proteins. cells were then stimulated with 10 uM UTP. Under these conditions, PMA preincubation reduced p44 and p42 MAPK phosphorylation induced by UTP from a ma imal response without PMA of 347 58% and 299 56% for p44 and p42, respectively, to 164 16% and 210 43%. These results indicate that PKC is the main kinase responsible for the UTP induced activation of the p44 and p42 proteins.

To test the role of intracellular Ca2 during p44 and p42 MAPK phosphorylation, cell cultures were preincubated with 10 uM BAPTA AM to load the cells intrac ellularly with this Ca2 chelator. Fluorescence Brefeldin_A microscopy confirmed that this treatment prevented the calcium increase induced by UTP. In BAPTA loaded TIC, phos phorylation of MAPK elicited by UTP was strongly inhib ited, thus, in control conditions, UTP elicited a phosphorylation increase of 384 53 and 289 55% for p44 and p42, respectively, while UTP stimulated BAPTA loaded cells showed significantly lower phos phorylation increases of only 171 40 and 116 16%, respectively. This result indicates that phosphorylation was a Ca2 dependent process and provides evidence for PKC participation.

Evidence that suggests a role for purinergic signaling in TIC physiology Cell proliferation is a consequence of purinergic stimula tion in various till cell systems, here we asked whether or not P2Y stimulation of TIC induced their proliferation. For this, cell cultures were stimulated with different con centrations of UTP, ATP, or UDP. cell proliferation was estimated using thymidine incorporation and com pared with that elicited by 10% FBS. The results indicated that ATP, UTP, or UDP increased proliferation. Incubation with 10% FBS increased thymidine incorporation to 277 23% of the basal level, and a similar increase was induced by 10 uM ATP but not by