Abstract
Purpose :
Lacrimal gland myoepithelial cells (MEC) play a key role in lacrimal fluid secretion. We recently reported that when MEC are stimulated with oxytocin (OXT), the phospholipase C/calcium pathway is activated, causing these cells to contract. In other tissues, OXT is also known to activate adenylate cyclase to generate the secondary messenger cyclic adenosine monophosphate (cAMP). In this study we aimed to investigate the involvement of cAMP and its downstream effectors in OXT-mediated lacrimal gland MEC contraction.
Methods :
Lacrimal gland MEC were isolated and propagated from α-smooth muscle actin (SMA)-GFP mice, in which MEC express GFP making them easily identifiable. RNA and protein samples were prepared to analyze the mRNA and protein expression of adenylate cyclase coupling G proteins (Gαs, Gαo, and Gαi) by RT-PCR and Western blotting, respectively. To increase intracellular cAMP concentration, the following agents were used: forskolin (FKN, a direct activator of adenylate cyclase), 3-isobutyl-1-methylxanthine (IBMX, an inhibitor of the phosphodiesterase that hydrolyzes cAMP), or dibutyryl-cAMP (db-cAMP). In addition, selective inhibitors were used to investigate the role of protein kinase A (PKA) and exchange protein activated by cAMP (EPAC) in OXT-induced MEC contraction. MEC contraction was monitored in real time and changes in cell size were quantified using Fiji software.
Results :
The adenylate cyclase coupling G proteins, Gαs, Gαo, and Gαi, are expressed in lacrimal gland MEC at both the mRNA and protein levels. FKN, IBMX and db-cAMP significantly stimulated MEC contraction, and preincubation of cells with either myr-PKI (PKA inhibitor) or ESI09 (EPAC inhibitor) resulted in almost complete inhibition of OXT- and FKN-stimulated MEC contraction.
Conclusions :
We conclude that cAMP agonists modulate lacrimal gland MEC contraction via PKA and EPAC and also play a role in oxytocin-induced MEC contraction.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.