K. changed as cultures progressed from monolayer to stratified. Cross-linking Tiaprofenic acid gels demonstrated the presence of homo- and heterotrimers. We examined epithelium from age matched diabetic and non-diabetic corneas patients and detected a 4-fold increase in P2X7mRNA from diabetic corneal epithelium compared to nondiabetic controls and an increased trend in expression of P2X7variant mRNA. Taken together, these data indicate that corneal epithelial cells express full-length and truncated forms of P2X7, which ultimately allows P2X7to function as a multifaceted receptor that can mediate cell proliferation and migration or cell Tiaprofenic acid death. == Introduction == Wound closure and response to disease involve a series of complex biologic events. Rapid and long-term signals are generated in response to the release of nucleotides from cells upon cell stress, mechanical stimulation, ligand binding or injury. The stimulation can lead to an increase in cytosolic calcium generated by the activation of purinergic receptors (P2Y or P2X) through distinct mechanisms. The P2Y receptors are G-protein coupled, while the ionotropic P2X receptors form cation channels and allow for the influx of extracellular Tiaprofenic acid Ca2+[1],[2]. Both are present in corneal epithelial cells where the distinct mechanisms of increasing intracellular Ca2+are displayed[3]. For example stimulation of the P2Y receptors attenuates the injury induced calcium wave, while after activation of the P2X7receptor with BzATP it is not diminished[3]. The canonical P2X7receptor (defined as full-length receptor) contains an extended C-terminus compared to the other P2X receptors[4]. While the protein was initially hypothesized to be a cell death inducing receptor, it is now implicated in both proliferative and death processes, which reflect the expression of the truncated or full-length receptor respectively. One of the features of the full-length receptor is usually its ability to induce the formation of large nonselective pores in the cell membrane in response to prolonged or repeated stimulation. The non-specific pore allows for the passage of organic cations up to 900 Da into the cell and can be monitored by the uptake of fluorescent dyes[4][8]. In addition many of the functions of the canonical P2X7receptor such as membrane blebbing, formation of large pores and a cytotoxic response depend on protein interactions with specific domains in the C-terminus[9][11]. In contrast the lack of pore formation, dye uptake and cytotoxicity is usually defined as non-canonical activity and attributed to P2X7splice variants[10][12]. The cell death induced by activation of the canonical receptor occurs via import of lethal intracellular Ca2+levels, activation of caspases, and ultimately apoptosis or necrosis[13][18]. In this regard the P2X7receptor is usually implicated in inflammation Tiaprofenic acid and is expressed in immune cells and cells of the central nervous system[13],[19]where it is a mediator of a number of interleukins that may serve to integrate the response[5],[17],[20],[21]. This is supported by data demonstrating that P2X7/mice are resistant to inflammation[22]. However, its activation has been shown to yield a number of other activities including the growth of human neuroblastoma cells[23], the increase in mitogenic activity in peripheral blood lymphocytes[20]and enhanced expression in cervical cancer[11],[12]. In addition, activation of the receptor has been shown to result in the phosphorylation of MAP kinase[25][27]. Still other work has shown that epithelial adhesion to basal lamina and corneal wound repair in P2X7/mice is usually attenuated[28]. These data Rabbit Polyclonal to LDLRAD2 indicate that this receptor can play several different roles that may be attributed to receptor variants. In this study, we show that corneal epithelial cells display both canonical and non-canonical responses. BzATP induces a canonical slow onset Ca2+wave, which is absent in Ca2+free media and is attenuated in the presence of Zn2+or Mg2+. In contrast, cell migration is usually enhanced with BzATP, and reduced significantly in cells transfected with siRNA to the P2X7receptor. Furthermore cytotoxic responses and dye uptake were not detected in confluent cultures upon P2X7activation, which correlates with the expression of the P2X7Jvariant mRNA and the expression of both 75 and 42 kDa forms. However, in stratified cell cultures there is a decrease in the expression of the truncated form, which correlates with Tiaprofenic acid uptake of ToPro-3 in the most apical cells. In addition we demonstrate that the P2X7mRNA is increased in diabetic corneal epithelium compared to control. Together these data indicate that the expression of full length and truncated forms.