Abstract
Purpose: :
The purpose of this study was to determine the effects of purinergic receptor agonists on the intracellular [Ca2+] ([Ca2+]i) in isolated rat lacrimal gland acinar and myoepithelial cells.
Methods: :
Rat lacrimal gland acini were isolated by collagenase digestion for 30 min. Cells were used immediately after isolation. Myoepithelial cells were prepared by repetitive cycles of collagenase digestion. Cells were placed in serum-supplemented RPMI-1640 medium until myoepithelial cells appeared, typically within 2 weeks. All cells were loaded with the calcium indicator dye fura2 for 30 min at room temperature. Calcium measurements were obtained using the InCyt Im2TM Ratio Imaging System using excitation wavelength of 340 and 380 nm. The purinergic receptor agonists 2-(methylthio)adenosine 5’-triphosphate (MeSATP), ATP, and the P2X7 receptor agonist benzoylbenzoyl adenosine 5’-triphosphate (BzATP) were used.
Results: :
In acini, MeSATP increased [Ca2+]i in a concentration-dependent manner with a maximum increase by 113 ± 78 nm at 10-4 M. ATP increased [Ca2+]i in a concentration-dependent manner with a maximum increase of 150 ± 52 nM at 10-6 M. BzATP (10-4 M) increased [Ca2+]i in the presence of Mg2+ to 80 ± 27 nM. In the absence of Mg2+, BzATP (10-4 M) increased [Ca2+]i to 204 ± 10 nM. The increase in [Ca2+]i was completely inhibited by pretreatment with two P2X7 receptor inhibitors brilliant blue G (10-5 M) and A438079 (10-5 M). In myoepithelial cells, MeSATP increased [Ca2+]i by 126 ± 67 nM while ATP (10-4 M) increased [Ca2+]i by 184 ± 102 nM. BzATP at 10-4 M also increased [Ca2+]i by 121 ± 39 nM in the absence of Mg2+ from 27 ± 12 nM in the presence of Mg2+.
Conclusions: :
We conclude that the lacrimal gland and cultured myoepithelial cells contain several purinergic receptors including P2Y1 and P2X7 receptors.
Keywords: cornea: tears/tear film/dry eye • lacrimal gland • signal transduction: pharmacology/physiology