scholarly journals Effects of α1D-adrenergic receptors on shedding of biologically active EGF in freshly isolated lacrimal gland epithelial cells

2006 ◽  
Vol 291 (5) ◽  
pp. C946-C956 ◽  
Author(s):  
LiLi Chen ◽  
Robin R. Hodges ◽  
Chika Funaki ◽  
Driss Zoukhri ◽  
Robert J. Gaivin ◽  
...  
Keyword(s):  
Growth Factors ◽  
Protein Secretion ◽  
Lacrimal Gland ◽  
Adrenergic Receptors ◽  
Egf Receptor ◽  
Cultured Cells ◽  
Biologically Active ◽  
Ectodomain Shedding ◽  
Egf Receptors ◽  
Isolated Cells

Transactivation of EGF receptors by G protein-coupled receptors is a well-known phenomenon. This process involves the ectodomain shedding of growth factors in the EGF family by matrix metalloproteinases. However, many of these studies employ transformed and/or cultured cells that overexpress labeled growth factors. In addition, few studies have shown that EGF itself is the growth factor that is shed and is responsible for transactivation of the EGF receptor. In this study, we show that freshly isolated, nontransformed lacrimal gland acini express two of the three known α1-adrenergic receptors (ARs), namely, α1B- and α1D-ARs. α1D-ARs mediate phenylephrine (an α1-adrenergic agonist)-induced protein secretion and activation of p42/p44 MAPK, because the α1D-AR inhibitor BMY-7378, but not the α1A-AR inhibitor 5-methylurapidil, inhibits these processes. Activation of p42/p44 MAPK occurs through transactivation of the EGF receptor, which is inhibited by the matrix metalloproteinase ADAM17 inhibitor TAPI-1. In addition, phenylephrine caused the shedding of EGF from freshly isolated acini into the buffer. Incubation of freshly isolated cells with conditioned buffer from cells treated with phenylephrine resulted in activation of the EGF receptor and p42/p44 MAPK. The EGF receptor inhibitor AG1478 and an EGF-neutralizing antibody blocked this activation of p42/p44 MAPK. We conclude that in freshly isolated lacrimal gland acini, α1-adrenergic agonists activate the α1D-AR to stimulate protein secretion and the ectodomain shedding of EGF to transactivate the EGF receptor, potentially via ADAM17, which activates p42/p44 MAPK to negatively modulate protein secretion.

Protein secretion induced by isoproterenol or pentoxifylline in lacrimal gland: Ca2+ effects

1984 ◽  
Vol 246 (1) ◽  
pp. C37-C44 ◽  
Author(s):  
P. Mauduit ◽  
G. Herman ◽  
B. Rossignol
Keyword(s):  
Protein Secretion ◽  
Lacrimal Gland ◽  
Adrenergic Receptors ◽  
Time Course ◽  
Phosphodiesterase Inhibitor ◽  
Extracellular Calcium ◽  
Maximal Response ◽  
Beta Adrenergic Receptors ◽  
Beta Adrenergic ◽  
Camp Analogue

In exorbital lacrimal glands, pentoxifylline (a methylxanthine) induces labeled protein secretion in a dose-related manner: the half-maximal and maximal stimulations are at 4 and 10 mM, respectively. In the presence of papaverine (10(-5) M), a phosphodiesterase inhibitor, labeled protein discharge is strongly stimulated by isoproterenol, via beta-adrenergic receptors: the maximal response is at 10(-6) M. l-Propranolol specifically inhibits the secretory stimulation to isoproterenol in a dose-related manner: for 5 X 10(-6) M isoproterenol in the presence of 10(-5) M papaverine, the half-maximal and maximal inhibitions are at 3 X 10(-7) and 10(-5) M, respectively. The beta-adrenergic response is mimicked by the adenosine 3',5'-cyclic monophosphate (cAMP) analogue dibutyryl cAMP (DBcAMP) at a 10(-3) M concentration. The time course of labeled protein secretion induced by pentoxifylline, DBcAMP, and isoproterenol shows a latency. In the presence or absence of extracellular calcium, pentoxifylline and isoproterenol immediately increase the cAMP intracellular level. Extracellular calcium omission increases the observed latency and also affects the maximal rate of protein secretion. As opposed to the cholinergic agonist, pentoxifylline has only a slight but sustained effect on 45Ca efflux, whereas isoproterenol has none. These data suggest that labeled protein secretion, such as that of peroxidase, can also be stimulated in rat exorbital lacrimal gland, through beta-adrenergic receptors; in the stimulation evoked by a beta-adrenergic agonist, DBcAMP, or methylxanthine, calcium could play a key role.

scholarly journals Characterization of a Drosophila protein that binds both epidermal growth factor and insulin-related growth factors.

1987 ◽  
Vol 105 (1) ◽  
pp. 449-456 ◽  
Author(s):  
J V Garcia ◽  
M P Stoppelli ◽  
K L Thompson ◽  
S J Decker ◽  
M R Rosner
Keyword(s):  
Growth Factors ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Transforming Growth Factor Beta ◽  
Egf Receptor ◽  
Transforming Growth Factor ◽  
Dissociation Constants ◽  
Egf Receptors ◽  
Drosophila Protein ◽  
Epidermal Growth

The identification of a novel protein from Drosophila melanogaster that binds both mammalian epidermal growth factor (EGF) and insulin has been reported (Thompson, K. L., S. J. Decker, and M. R. Rosner, 1985, Proc. Natl. Acad. Sci. USA., 82:8443-8447). This 100-kD protein (designated dp100) is also recognized by an antiserum against the human EGF receptor. To further characterize the properties of this protein, we have determined the binding spectrum, glycosylation state, and cellular distribution of dp100. Our results indicate that dp100 binds to other insulin-like and EGF-like growth factors with dissociation constants ranging from 10(-6) to 10(-9) M, and these ligands compete with each other for binding to dp100. All other ligands tested, including platelet-derived growth factor, transforming growth factor-beta, nerve growth factor, and glucagon, either did not bind or bound with a Kd greater than 10(-6) M. Unlike the Drosophila insulin receptor, dp100 does not bind to wheat germ agglutinin and is present in a cytoplasmic as well as a membrane-bound form that cannot be differentiated by two-dimensional PAGE. Further, dp100 is the sole transforming growth factor-alpha-binding protein detected by affinity labeling in Drosophila Kc cells. These results indicate that dp100 shares properties in common with, but distinct from, the Drosophila homologues of the insulin and EGF receptors.

scholarly journals Purification and characterization of heparin-binding growth factors from porcine uterus

1990 ◽  
Vol 266 (1) ◽  
pp. 273-282 ◽  
Author(s):  
D R Brigstock ◽  
R B Heap ◽  
P J Barker ◽  
K D Brown
Keyword(s):  
Growth Factors ◽  
Amino Acid ◽  
Growth Factor ◽  
Reproductive Tract ◽  
Egf Receptor ◽  
Reversed Phase ◽  
Exchange Chromatography ◽  
Biologically Active ◽  
Heparin Binding ◽  
Heparin Binding Growth Factors

Heparin-binding growth factors present in pig uterine tissue were purified by approx. 50,000-fold using a combination of ammonium sulphate precipitation, ion-exchange chromatography and heparin-affinity chromatography. Purification of the uterus-derived growth factors (UDGFs) was monitored by the stimulation of [3H]thymidine incorporation into Swiss 3T3 cells and by a radioreceptor assay using 125I-labelled epidermal growth factor (EGF) as the ligand. The latter was shown to be a novel, rapid and reliable assay for heparin-binding growth factors which utilizes their trans-modulation of EGF receptor affinity. UDGFs exhibit strong affinity for immobilized heparin and two forms, named alpha UDGF and beta UDGF, were distinguished by salt gradient elution from heparin-agarose affinity columns. beta UDGF activity was eluted from heparin-agarose between 1.5 M- and 1.8 M-NaCl, and was correlated with the elution of a protein doublet of 17.2 kDa and 17.7 kDa. Immunoblotting of heparin-purified beta UDGF indicated that the beta UDGF doublet is immunologically related to the 146-amino-acid form of bovine basic fibroblast growth factor (bFGF), and that the 17.2 kDa component is an N-terminally truncated form of the 17.7 kDa component. After purification by C4 reversed-phase h.p.l.c., this doublet was biologically active and greater than 95% pure as assessed by silver-stained SDS/PAGE. Amino acid composition and sequence analysis confirmed that these beta UDGF polypeptides were microheterogeneous forms of bFGF. Fractions containing alpha UDGF activity were eluted from heparin-agarose in 1.3 M-NaCl. These fractions contained a 16.5 kDa protein which co-migrated on SDS/polyacrylamide gels with recombinant human acidic FGF (aFGF) and which which cross-reacted with an antiserum raised against aFGF. The identification of heparin-binding growth factors in porcine uterus at the time of implantation raises the possibility that they function in the reproductive tract during early pregnancy.

Stimulation of rabbit lacrimal gland secretion with biologically active peptides

1988 ◽  
Vol 254 (3) ◽  
pp. G300-G306
Author(s):  
D. A. Dartt ◽  
M. Shulman ◽  
K. L. Gray ◽  
S. R. Rossi ◽  
C. Matkin ◽  
...  
Keyword(s):  
Protein Secretion ◽  
Lacrimal Gland ◽  
Excretory Duct ◽  
Biologically Active ◽  
Gland Secretion ◽  
Dependent Manner ◽  
Active Peptides ◽  
Biologically Active Peptides ◽  
Dose Dependent ◽  
Stimulation Of

To determine whether biologically active peptides can stimulate lacrimal gland secretion, we measured fluid and protein secretion from the cannulated lacrimal gland excretory duct of anesthetized rabbits after arterial injection of various peptides. Vasoactive intestinal peptide (VIP, 0.003-3 nmol) and porcine histidine isoleucine-containing peptide (PHI, 0.01-3 nmol) stimulated fluid and protein secretion in a dose-dependent manner. Cholecystokinin octapeptide (CCK-8, 0.01-3 nmol) stimulated fluid but not protein secretion. Neither bombesin nor eledoisin in doses as high as 3 nmol stimulated fluid or protein secretion. When combinations of high or low doses of VIP and the cholinergic agonist acetylcholine (ACh) were injected simultaneously, fluid and protein secretion was additive. We concluded that VIP and PHI stimulated secretion of lacrimal gland fluid and protein, CCK-8 stimulated secretion of fluid, and bombesin and eledoisin did not stimulate either fluid or protein secretion. VIP and ACh, both found in lacrimal gland nerve endings, stimulate lacrimal gland fluid and protein secretion by separate pathways.

α1-Adrenergic and cholinergic agonists activate MAPK by separate mechanisms to inhibit secretion in lacrimal gland

2003 ◽  
Vol 284 (1) ◽  
pp. C168-C178 ◽  
Author(s):  
Isao Ota ◽  
Driss Zoukhri ◽  
Robin R. Hodges ◽  
José D. Rios ◽  
Vanja Tepavcevic ◽  
...  
Keyword(s):  
Protein Secretion ◽  
Lacrimal Gland ◽  
Egf Receptor ◽  
Mitogen Activated Protein Kinase ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Signaling Mechanisms ◽  
Mitogen Activated Protein ◽  
Secretion Inhibition

The purpose of this study was to determine the role of p42/p44 mitogen-activated protein kinase (MAPK) in α1-adrenergically and cholinergically stimulated protein secretion in rat lacrimal gland acinar cells and the pathways used by these agonists to activate MAPK. Acini were isolated by collagenase digestion and incubated with the α1-adrenergic agonist phenylephrine or the cholinergic agonist carbachol, and activation of MAPK and protein secretion were then measured. Phenylephrine and carbachol activated MAPK in a time- and concentration-dependent manner. Inhibition of MAPK significantly increased phenylephrine- and carbachol-induced protein secretion. Inhibition of EGF receptor (EGFR) with AG1478, an inhibitor of the EGFR tyrosine kinase activity, significantly increased phenylephrine- but not carbachol-induced protein secretion. Whereas phenylephrine-induced activation of MAPK was completely inhibited by AG1478, activation of MAPK by carbachol was not. Phenylephrine stimulated tyrosine phosphorylation of the EGFR, whereas carbachol stimulated p60Src, and possibly Pyk2, to activate MAPK. We conclude that, in the lacrimal gland, activation of MAPK plays an inhibitory role in α1-adrenergically and cholinergically stimulated protein secretion and that these agonists use different signaling mechanisms to activate MAPK.

Electron Probe Microanalysis of Frozen Hydrated Kidneys, Isolated Cells, and Cultured Cells

1982 ◽  
Vol 40 ◽  
pp. 402-403 ◽  
Author(s):  
Claude Lechene
Keyword(s):  
Liquid Nitrogen ◽  
Electron Probe Microanalysis ◽  
Electron Probe ◽  
Cultured Cells ◽  
Liquid Nitrogen Temperature ◽  
Elemental Content ◽  
Isolated Cells ◽  
Renal Tubular Cells ◽  
Diamond Saw ◽  
Saw Blade

Electron probe microanalysis of frozen hydrated kidneysThe goal of the method is to measure on the same preparation the chemical elemental content of the renal luminal tubular fluid and of the surrounding renal tubular cells. The following method has been developed. Rat kidneys are quenched in solid nitrogen. They are trimmed under liquid nitrogen and mounted in a copper holder using a conductive medium. Under liquid nitrogen, a flat surface is exposed by sawing with a diamond saw blade at constant speed and constant pressure using a custom-built cryosaw. Transfer into the electron probe column (Cameca, MBX) is made using a simple transfer device maintaining the sample under liquid nitrogen in an interlock chamber mounted on the electron probe column. After the liquid nitrogen is evaporated by creating a vacuum, the sample is pushed into the special stage of the instrument. The sample is maintained at close to liquid nitrogen temperature by circulation of liquid nitrogen in the special stage.

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