Identification of vasoactive intestinal peptide receptor subtypes in the lacrimal gland and their signal-transducing components.
OBJECTIVE
To determine the presence of vasoactive intestinal peptide (VIP) receptor (VIPR) subtypes in the lacrimal gland and to determine if the components of the VIP signaling pathway for protein secretion also are present.


METHODS
Immunofluorescence studies using conventional fluorescence microscopy or confocal microscopy were performed on fixed sections from rat lacrimal glands using antibodies raised against VIPRs types I and II, and four antibodies against five isoforms of adenylyl cyclase (AC) (II, III, IV, V/VI). Guanine nucleotide binding (G) proteins were detected by Western blotting. Changes in intracellular [Ca2+] ([Ca2+]i) were measured on fura-2-loaded acini in response to VIP. The effect of a myristoylated peptide corresponding to the pseudosubstrate sequence of protein kinase inhibitor (myr-PKI), the endogenous inhibitor of cyclic AMP (cAMP)-dependent protein kinase (PKA), was tested on VIP-stimulated peroxidase secretion.


RESULTS
The VIPRs, types I and II, were found on the basolateral membranes of acinar and ductal cells and on myoepithelial cells. Western blotting showed the presence of alpha subunits of Gs, Gi3, G0 and G beta. The AC II was found exclusively on myoepithelial cells; AC IV was located intracellularly in all cells; AC III was found on ducts and possibly nerves; no AC V/VI was detected. The VIP (10(-8) M) caused a small but significant increase in [Ca2+]i of 26 +/- 9 nM. The VIP-stimulated protein secretion was inhibited 71% by myr-PKI.


CONCLUSIONS
All components of the VIP signal transduction pathway in the lacrimal gland were present. These findings are consistent with a pathway where VIP released from parasympathetic nerves binds to VIPRs types I and II, activating G proteins, which in turn stimulate AC present on myoepithelial and acinar cells. The AC increases the intracellular cAMP concentration, which activates PKA to stimulate protein secretion. The VIP also stimulated Ca2+ influx, which could play a role in secretion.
Hodges
R R
RR
Schepens Eye Research Institute, Boston, MA 02114, USA.
Zoukhri
D
D
Sergheraert
C
C
Zieske
J D
JD
Dartt
D A
DA
D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D001485:Basement Membrane / Q000378:metabolism; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002352:Carrier Proteins / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019084:Fluorescent Antibody Technique, Indirect; D005456:Fluorescent Dyes / Q000378:metabolism; D016257:Fura-2 / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D007765:Lacrimal Apparatus / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D018613:Microscopy, Confocal; D010446:Peptide Fragments / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D018005:Receptors, Vasoactive Intestinal Peptide / Q000145:classification / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D014660:Vasoactive Intestinal Peptide / Q000494:pharmacology
D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D047908:Intracellular Signaling Peptides and Proteins; D010446:Peptide Fragments; D018005:Receptors, Vasoactive Intestinal Peptide; C015482:protein kinase modulator; D014660:Vasoactive Intestinal Peptide; D017868:Cyclic AMP-Dependent Protein Kinases; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D002118:Calcium; D016257:Fura-2