Proceedings of The Physiological Society

University College Dublin (2009) Proc Physiol Soc 15, PC177

Poster Communications

Multiple purinergic receptors regulate anion secretion in the bovine oviduct epithelium

N. Keating1, L. Quinlan1

1. Physiology, NUI Galway, Galway, Ireland.

Introduction: Fluid within the oviduct provides the appropriate environment essential for gamete transport, fertilization and early embryo development. However, little is known about the potential mechanisms underlying the formation of oviduct fluid. Our previous studies [1] have demonstrated a role for basolateral purinoceptor activation in promoting chloride secretion across the bovine oviduct epithelium. The present study explored the regulation of anion secretion by luminal purinergic receptors. Methods: Ion secretion was measured as changes in short-circuit current (ISC(μA/cm2) across voltage-clamped polarized oviduct epithelial cell monolayers. Changes in intracellular calcium were measured by fluorescence microscopy using fura-2. mRNA expression was confirmed by reverse transcriptase PCR (RT-PCR). Results are expressed as mean ± SEM and statistical analyses were made by one way ANOVA and the Bonnferroni/Dunn post hoc test Results and Discussion: ATP (100μM) application to the luminal surface of polarized bovine oviduct epithelial cells induced a rapid and transient increase in current of 28.82 ±1.7 μA/cm2 followed by a sustained current of 1.91±0.1μA/cm2 (n=18). Removal of extracellular chloride or bicarbonate ions or both reduced the ATP-induced ISC response by 30%, 40% and 70% respectively (n=5). Furthermore, the ATP response was preserved in the presence of amiloride but was reduced by 60% in the presence of the CFTR inhibitor, glybenclamide (p<0.001,n=4) and almost completely abolished in the presence of thapsigargin (p<0.001, n=4).Ca2+ measurements revealed that ATP also induced a rapid transient increase in intracellular Ca2+ which was sensitive to thapsigargin, U73122 and BAPTA-AM (p<0.001, n=4). Other P2Y agonists also rapidly activated anion secretion with a potency profile of ATP = UTP > ADP suggesting the involvement of the P2Y2 subtype of purinoceptor. In addition a range of P2X agonists rapidly activated anion secretion with a potency profile of ATP = 2MeSATP >α,β meATP > BzATP, suggesting the presence of multiple functional P2X receptors on the luminal membrane. P2X1, P2X4 and P2X7 receptor expression was confirmed by RT-PCR. Finally the P1 receptor agonist, adenosine, also induced a rapid transient increase in ISC by 7.46 ± 0.86 μA/cm2 which was associated with a subsequent sustained current of 4.31 ± 0.38 μA/cm2 (n=4). In conclusion, we have demonstrated that luminal ATP rapidly activates Cl- and HCO3- secretion across the bovine oviduct, in a Ca2+ dependent fashion most likely through P2Y2 receptor activation. Furthermore activation of P2X and P1 receptor subtypes on the luminal membrane are also capable of promoting anion secretion. The purinergic regulation of anion secretion in the oviduct provides an important method of regulating oviduct fluid formation and optimizing conditions for fertilization and early embryo development.

Where applicable, experiments conform with Society ethical requirements