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Ahn, Jae Suk,Camden, Jean M.,Turner, John T. Korean Academy of Oral Biology and the UCLA Dental 2001 International Journal of Oral Biology Vol.26 No.1
We have reported previously that when dispersed cell aggregates from rat submandibular glands (SMG) are placed in culture, the expression and activity of the P2Y_2 nucleotide receptor increase as a function of time. These results, combined with additional findings in the SMG and in other organ systems, suggest that changes in gland cell organization and structure induce up-regulation of P2Y_2 receptors. As part of a study to define intracellular signaling path ways contributing to receptor up-regulation, we investigated the possible involvement of the extracellular signal-related kinase (ERK) signaling cascade. The effects of PD 98059, a specific inhibitor of ERK phosphorylation and activation, on P2Y_2 receptor expression and activity during short-term culture were evaluated. The intracellular free calcium concentration ([Ca^2+]_i) in SMG cells in response to the P2Y_2 receptor agonist, UTP (100 μM), was increased following 24 h of culture. This increase was blocked, in a concentration-dependent manner, up to 60% by PD 98059. The up-regulation and increased activity of P2Y_2 receptors following 24 h in culture in the absence of PD 98059 was not reversed by subsequent incubation with the inhibitor for an additional 24 h, although further up-regulation was prevented. Changes in ([Ca^2+]_i) in response to the muscarinic receptor agonist, carbachol (100 μM), were unaffected by time in culture or inclusion of PD 98059. RT-RCR indicated that P2Y_2 receptor mRNA was increased about 14-fold after 24 h of culture. This increase also was attenuated by treatment with 100 μM PD 98059. Furthermore, UTP increased the phosphorylation of ERK in SMG cells cultured for 24 h - this increase was inhibited by PD 98059. Together, these results implicate the ERK pathway in up-regulation of P2Y_2 receptors in rat SMG cells during short-term culture and in signaling by the P2Y_2 receptor following its up-regulation.