Characterization of P2X7 purinergic receptors and their function in rat lacrimal gland.
OBJECTIVE
To characterize the effects of P2X(7) purinergic receptors on lacrimal gland function.


METHODS
P2X(7) purinergic receptors were identified by RT-PCR, Western blot analysis, and immunofluorescence techniques. Rat lacrimal gland acini were isolated by collagenase digestion. Acini were incubated with the fluorescent indicator molecule fura 2, and [Ca(2+)](i) was measured by a fluorescence imaging system. Protein secretion was measured with a fluorescence assay system. Activation of ERK 1/2 was determined by Western blot analysis with an antibody against phosphorylated (active) ERK 1/2.


RESULTS
P2X(7) receptors were present in the lacrimal gland by RT-PCR and Western blot analysis. These receptors were located in the membranes of acinar and ductal cells and the cytoplasm of acinar cells. Activation of P2X(7) receptors with (benzoylbenzoyl)adenosine 5'-triphosphate increased [Ca(2+)](i), peroxidase secretion, and ERK 1/2 activation, each of which was inhibited by the P2X(7) receptor inhibitors Brilliant Blue G or A 438079.


CONCLUSIONS
P2X(7) purinergic receptors are present in rat lacrimal gland and when stimulated increase [Ca(2+)](i), protein secretion, and ERK 1/2 activation.
Hodges
Robin R
RR
Schepens Eye Research Institute, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts 02114, USA. robin.hodges@schepens.harvard.edu
Vrouvlianis
Joanna
J
Shatos
Marie A
MA
Dartt
Darlene A
DA
D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D004789:Enzyme Activation; D019084:Fluorescent Antibody Technique, Indirect; D016257:Fura-2 / Q000378:metabolism; D007765:Lacrimal Apparatus / Q000378:metabolism*; D008297:Male; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D010766:Phosphorylation; D051381:Rats; D017207:Rats, Sprague-Dawley; D018048:Receptors, Purinergic P2 / Q000378:metabolism*; D058486:Receptors, Purinergic P2X7; D020133:Reverse Transcriptase Polymerase Chain Reaction
C546470:P2rx7 protein, rat; D018048:Receptors, Purinergic P2; D058486:Receptors, Purinergic P2X7; C535149:Mapk1 protein, rat; D019950:Mitogen-Activated Protein Kinase 1; D002118:Calcium; D016257:Fura-2