ATP Induces Interleukin-8, Intracellular Calcium Release, and ERK1/2 Phosphorylation in Bovine Endometrial Cells, Partially through P2Y Receptors

TL;DR: In this article , the effect of adenosine triphosphate (ATP) on the inflammatory response in bovine endometrial cells has been investigated using RT-qPCR.

Abstract: Simple Summary Bovine uterine infections are common in the postpartum period and are associated with economic losses in dairy herding. Adequate immune function is key to preventing disease. Adenosine triphosphate (ATP) is a known activator of the inflammatory response, and can be produced in the endometrial microenvironment. In this study, we observed that ATP increased the proinflammatory responses in bovine endometrial cells, such as release of chemokine interleukin-8 (IL-8), intracellular calcium mobilization, and ERK1/2 phosphorylation. Additionally, we demonstrated the presence of a subtype of purinergic receptors (P2Y), specifically, high levels of P2Y1 and P2Y2 receptors. The inhibition of P2Y receptors reduced the proinflammatory responses induced by ATP. The results suggest that P2Y receptors play a role in endometrial inflammatory activation, which could be useful as a therapeutic strategy to regulate uterine inflammation through the modulation of P2Y receptors. Abstract The bovine endometrium has an important defensive role in the postpartum period that acts when an inflammatory process associated with tissue damage or infection by bacteria is produced. Endometrial cells release cytokines and chemokines that recruit inflammatory cells, which release danger-associated molecular patterns (DAMPs), such as adenosine triphosphate (ATP), and initiate and regulate the inflammatory response. However, the role of ATP in bovine endometrial cells is unclear. The aim of this study was to determine the effect of ATP on interleukin-8 (IL-8) release, intracellular calcium mobilization, ERK1/2 phosphorylation, and the role of P2Y receptors, in bovine endometrial cells. Bovine endometrial (BEND) cells were incubated with ATP and the IL-8 release was determined by the ELISA assay. ATP of 50 and 100 μM significantly increased IL-8 released in BEND cells (50 μM: 23.16 ± 3.82 pg/mL, p = 0.0018; 100 μM: 30.14 ± 7.43 pg/mL, p = 0.0004). ATP (50 μM) also induced rapid intracellular calcium mobilization in Fura-2AM-loaded BEND cells, as well as ERK1/2 phosphorylation (ratio 1.1 ± 0.04, p = 0.0049). Suramin (50 μM), a pan-antagonist of P2Y receptors, partially reduced the intracellular calcium mobilization, ERK1/2 phosphorylation (ratio 0.83 ± 0.08, p = 0.045), and IL-8 release (9.67 ± 0.02 pg/mL, p = 0.014) induced by ATP. Finally, BEND cells expressed higher mRNA levels of P2Y1 and P2Y2 purinergic subtype receptors, and lower levels of P2Y11 and P2Y12 receptors, as determined by RT-qPCR. In conclusion, these results showed that ATP activates pro-inflammatory responses in BEND cells, which are partially mediated via P2Y receptors, and BEND cells express the mRNA of subtypes of P2Y receptors, which could have a key role in bovine endometrial inflammation.