Increase of intracellular Ca2+ by purinergic receptors in cultured rat lacrimal gland myoepithelial cells.
OBJECTIVE
To isolate and characterize cultured myoepithelial cells (MECs) from rat lacrimal gland and determine which purinergic receptor subtypes are present and functional in MECs.


METHODS
Rat lacrimal glands were subjected to collagenase digestion, and MECs were grown. RT-PCR was performed for the purinergic receptors P2X(7), P2Y(1), P2Y(11), and P2Y(13) on RNA isolated from the MECs. Immunofluorescence experiments were performed with antibodies against MEC markers and P2X(7), P2Y(1), P2Y(11), and P2Y(13) purinergic receptors. Proteins from MECs were separated using Western blot analysis techniques. In addition, cells were incubated with Fura 2 tetra acetoxymethyl ester, and intracellular [Ca(2+)] ([Ca(2+)](i)) was determined in response to P2 purinergic agonists.


RESULTS
MECs expressed the MEC proteins α-smooth muscle actin, vimentin, α-actinin, and adenylyl cyclase II. RT-PCR, Western blot, and immunofluorescence techniques demonstrated the presence of the purinergic receptors P2X(7), P2Y(1), P2Y(11), and P2Y(13). The purinergic agonists ATP, benzoylbenzoyl ATP (BzATP), α,β methylene ATP, UTP, 2-methylthioATP (MeSATP), and ATPγS increased [Ca(2+)](i). As BzATP binds to the P2X(7) receptor, specific characteristics of this receptor were investigated. Neither inhibitors of P2X(7) receptors nor removal of extracellular Mg(2+) or Ca(2+) had an effect on the BzATP-stimulated increase in [Ca(2+)](i). Repeated applications of BzATP desensitized this response. Inhibitors for P2Y(1), P2Y(11), and P2Y(13) each decreased the BzATP-stimulated increase in [Ca(2+)](i) with the P2Y(1) inhibitor most effective.


CONCLUSIONS
MECs can be isolated from rat lacrimal glands, and they express P2X(7), P2Y(1), P2Y(11), and P2Y(13) purinergic receptors. Surprisingly, BzATP binds the P2Y(1) receptor, which is primarily responsible for the BzATP-stimulated increase in [Ca(2+)](i).
Schepens Eye Research Institute, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts, USA.
Kaori
K
Ohtomo
Marie A
MA
Shatos
Joanna
J
Vrouvlianis
Dayu
D
Li
Robin R
RR
Hodges
Darlene A
DA
Dartt
D000185:Actinin / Q000378:metabolism; D000199:Actins / Q000378:metabolism; D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D015415:Biomarkers / Q000032:analysis; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000502:physiology; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000378:metabolism*; D016257:Fura-2 / Q000378:metabolism; D007150:Immunohistochemistry; D007765:Lacrimal Apparatus / Q000166:cytology / Q000378:metabolism*; D008297:Male; D058910:Purinergic P2 Receptor Agonists / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D011983:Receptors, Purinergic / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014746:Vimentin / Q000378:metabolism
D000199:Actins; D015415:Biomarkers; D058910:Purinergic P2 Receptor Agonists; D011983:Receptors, Purinergic; D014746:Vimentin; D000185:Actinin; D000262:Adenylyl Cyclases; C100622:adenylyl cyclase 2; D002118:Calcium; D016257:Fura-2