Abstract
Purpose: :
To determine the types of purinergic receptors present in rat lacrimal gland myoepithelial cells in culture and their function.
Methods: :
Rat lacrimal glands were dissociated by repetitive cycles of digestion in collagenase. Cells were placed in serum-supplemented RPMI-1640 medium until myoepithelial cells (MECs) appeared. Cells were evaluated for expression of the myoepithelial cell markers. Immunofluorescence microscopy (IM) was performed using antibodies to P2Y1, P2Y2, P2Y4, P2Y11 and P2Y13 receptors. For intracellular calcium ([Ca2+]i) measurement, cells were loaded with the calcium indicator dye fura2 in a HEPES-buffered solution for 1hr at 37°C. The purinergic receptor agonists ATP (P2Y2, P2Y11 agonist), UTP (P2Y2, P2Y4 agonist), 2’(3’)-O-(4-benzoylbenzoyl) adenosine 5’ -triphosphate (BzATP, P2Y11 and P2Y13 agonist), adenosine 5’-O-(3-thiotriphosphate) (ATPγS, P2Y11 agonist) were used. U73122 was used as a phospholipase C inhibitor, NF157 as a P2Y11 receptor inhibitor and MRS2211 as P2Y13 receptor inhibitor. Type I collagen was used for a gel contraction assay. Reconstituted collagen solution was poured into culture dishes and incubated for 30min at 37ºC. MECs were then plated on the top of the gel in RPMI-1640 medium. After 48 hours of attachment, gels were released from the culture dishes and agonists were added. 20min after stimulation, gel size was measured with NIH image J.
Results: :
α-SMA, α-actinin and adenylyl cyclase II were expressed in the cultured lacrimal gland myoepithelial cells. IM confirmed the presence of P2Y2, P2Y4, P2Y11 and P2Y13 receptors on these cells. ATP increased [Ca2+]i in a concentration-dependent manner with maximum increase of 360±32nM at 10-3M. UTP increased [Ca2+]i in a concentration-dependent manner with maximum increase of 351±7nM at 10-4M. BzATP increased [Ca2+]i in a concentration-dependent manner with maximum increase of 220±19nM at 10-3M. ATPγS increased [Ca2+]i in a concentration-dependent manner with maximum increase of 260±38nM at 10-4M. Pretreatment with U73122(10-5M) significantly inhibited the UTP and BzATP-induced increase in [Ca2+]i by 72% and 81% respectively (p<0.05). The BzATP-induced increase in [Ca2+]i was inhibited 56% and 25% by pretreatment with NF157 (10-4M) and MRS2211 (10-4M) respectively. UTP triggered collagen gel contraction indicating myoepithelial cell contraction.
Conclusions: :
P2Y2, P2Y4, P2Y11 and P2Y13 receptors are present and increase [Ca2+]i in lacrimal gland myoepithelial cells. Activation of P2Y receptors by UTP causes myoepithelial cell contraction that could alter functioning of lacrimal acinar cells.
Keywords: lacrimal gland • calcium • receptors: pharmacology/physiology