Am J Physiol Cell Physiol AJP: Gastrointestinal and Liver Physiology
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Am J Physiol Cell Physiol 291: C946-C956, 2006. First published June 7, 2006; doi:10.1152/ajpcell.00014.2006
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RECEPTORS AND SIGNAL TRANSDUCTION

Effects of {alpha}1D-adrenergic receptors on shedding of biologically active EGF in freshly isolated lacrimal gland epithelial cells

LiLi Chen,1 Robin R. Hodges,1 Chika Funaki,1 Driss Zoukhri,2 Robert J. Gaivin,3 Dianne M. Perez,3 and Darlene A. Dartt1

1Schepens Eye Research Institute and Department of Ophthalmology, Harvard Medical School, Boston; 2Tufts University School of Dental Medicine, Boston, Massachusetts; and 3Lerner Research Institute, The Cleveland Clinic, Cleveland, Ohio

Submitted 13 January 2006 ; accepted in final form 25 May 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
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 {alpha}1-adrenergic receptors (ARs), namely, {alpha}1B- and {alpha}1D-ARs. {alpha}1D-ARs mediate phenylephrine (an {alpha}1-adrenergic agonist)-induced protein secretion and activation of p42/p44 MAPK, because the {alpha}1D-AR inhibitor BMY-7378, but not the {alpha}1A-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, {alpha}1-adrenergic agonists activate the {alpha}1D-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.

epidermal growth factor ectodomain shedding; protein secretion; signal transduction

THREE TYPES OF {alpha}1-ADRENERGIC RECEPTORS (ARs), {alpha}1A-, {alpha}1B-, and {alpha}1D-subtypes, have been identified using biochemical and molecular biology techniques (23). These receptors are members of the G protein-coupled receptor (GPCR) superfamily. The signaling pathways used by these subtypes are cell specific, but the agonists generally activate either phospholipases C or A2 (23). There are, however, differences in coupling efficiencies of the receptors to the signaling pathways (23). {alpha}1-ARs are expressed in many tissue types, most notably in vascular tissues, where they typically regulate smooth muscle contraction (7). In particular, the only function directly attributed to {alpha}1D-adrenergic receptors is as a mediator of contraction of large arteries such as the aorta, femoral, iliac, and superior mesenteric arteries (23).

Similar to other members of its family, EGF is synthesized as a glycosylated membrane-anchored precursor protein (precursor EGF) that is proteolytically cleaved to release the soluble form (proEGF). ProEGF is further cleaved to release the mature 6-kDa form of EGF. All forms of the proteins are thought to be biologically active (16). The process by which EGF family members are released is known as ectodomain shedding and is mediated mainly by matrix metalloproteinases (MMP). In particular, ADAM10 (a disintegrin and MMP) has been shown to be the MMP involved in shedding of EGF and betacellulin (26), whereas ADAM17 (also known as TACE) has been shown to be responsible for the shedding of transforming growth factor-{alpha} (TGF-{alpha}), amphiregulin, and heparin-binding EGF (HB-EGF) (3, 11, 22, 26). GPCRs have been shown to activate the MMPs that then cleave the precursor form of the growth factors. The precursor growth factor can then bind to and activate the EGF receptor (24). This mode of EGF receptor activation is known as the "triple membrane passing signal" model (24).

Numerous studies have documented the effects of activation of GPCRs and the subsequent transactivation of the EGF receptor through the triple membrane passing signal model (17, 21, 27, 37). Ectodomain shedding of two members of the EGF family, TGF-{alpha} and HB-EGF, occurs in multiple tissues with stimulation by a variety of GPCR agonists (25). In contrast, there are only two reports to date documenting the ectodomain shedding of EGF (13, 26). In addition, few studies have quantified the amount of the released growth factors as a result of activation of GPCRs. Those that have quantified the amount of growth factors have relied on transfected cells expressing labeled growth factors (6, 13, 26, 32) or cells grown in culture (17). There have been no studies quantifying the shedding of growth factors from nontransformed, noncultured, freshly isolated cells.

The exocrine lacrimal gland is the major contributor to the aqueous component of the tear film. As a result, protein, electrolyte, and water secretion from this gland is tightly regulated by parasympathetic and sympathetic nerves that innervate the gland (9). Our group (9) has previously shown that {alpha}1-adrenergic agonists released from sympathetic nerves are potent stimuli of protein secretion from the lacrimal gland. {alpha}1-Adrenergic agonists increase cGMP, which stimulates lacrimal gland protein secretion (10). It is also known that phenylephrine (an {alpha}1-adrenergic agonist in the lacrimal gland) activates protein kinase C (PKC)-{epsilon}, which stimulates protein secretion, and PKC-{alpha} and -{delta}, which inhibit secretion. In addition, we have shown that {alpha}1-adrenergic agonists transactivate the EGF receptor, recruiting Shc and Grb2 to activate p42/p44 MAPK. When p42/p44 MAPK activation is inhibited by U0126 (an inhibitor of MEK), {alpha}1-adrenergic agonist-stimulated protein secretion is increased. Thus activation of p42/p44 MAPK reduces protein secretion and acts to negatively modulate protein secretion from the lacrimal gland (21). Therefore, {alpha}1-adrenergic agonists activate two antagonistic pathways, resulting in a net increase in protein secretion.

Because lacrimal gland acini are polarized with tight junctions present between cells, the location of EGF is of particular interest. Any EGF molecule located in the apical membrane of acinar cells would be shed into the ducts and then into the tears upon stimulation. Thus this EGF would be interacting with any EGF receptors on the cells of the ocular surface. In contrast, EGF located in the basal and/or lateral membrane of acinar cells would be shed upon stimulation into the extracellular space and would bind to the EGF receptor on acinar cells, thus having effects on the acinar cells themselves. Our group (34) has shown previously that addition of exogenous EGF stimulates protein secretion from the lacrimal gland. In this study, we identify the {alpha}1-adrenergic receptor subtypes present in the lacrimal gland and their effects on the shedding of EGF from freshly isolated epithelial cells of the lacrimal gland.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. [125I]HEAT {(±)-beta-([125I]iodo-4-hydroxyphenyl)ethyl-aminomethyl-tetralone} was purchased from PerkinElmer (Boston, MA). Rat EGF was obtained from Biomedical Technologies (Stoughton, MA). Collagenase type III was obtained from Worthington Biochemical (Lakewood, NJ). RNA extraction reagent (TRIzol) was from GIBCO BRL (Grand Island, NY), and the Reverse Transcription System and PCR Core System II were purchased from Promega (Madison, WI). Amplex red was purchased from Molecular Probes (Eugene, OR). Enhanced chemiluminescent substrate for detection of horseradish peroxidase (HRP) was obtained from Pierce (Rockford, IL). GM6001 {N-[(2R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl]-L-tryptophan methylamide}, its negative control N-t-butoxycarbonyl-L-leucyl-L-tryptophan methylamide, and TAPI-1 {N-(R)-[2-(hydroxyaminocarbonyl)methyl]-4-methylpentanoyl-L-naphthylalanyl-L-alanine, 2-aminoethylamide} were purchased from EMD Biosciences (San Diego, CA). EGF was purchased from Peprotech (Rocky Hill, NJ). All other reagents were obtained from Sigma Chemical (St. Louis, MO).

Antibodies. Anti-rat EGF polyclonal antibody immunohistochemical grade was purchased from Biomedical Technologies (Stoughton, MA). A second anti-rat EGF polyclonal antibody raised against a peptide corresponding to the carboxy terminus of the mature form of EGF of rat origin was obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and used in Western blot analyses. Anti-human EGF receptor polyclonal antibody used for immunohistochemistry was obtained from Cell Signaling Technology (Beverly, MA). Anti-human EGF receptor polyclonal antibody used for Western blot analyses was obtained from Santa Cruz Biotechnology. Anti-EGF receptor used for immunoprecipitation was obtained from Upstate (Charlottesville, VA). Anti-EGF receptor (pY1086) phosphospecific polyclonal antibody was obtained from BioSource International (Camarillo, CA). Anti-actin monoclonal antibody was acquired from Chemicon International (Temecula, CA). Monoclonal antibodies generated against p42 MAPK and phosphorylated p42/p44 MAPK, which specifically reacts with Tyr-204 of phosphorylated ERK1 and ERK2, were obtained from Santa Cruz Biotechnology. The secondary antibody for immunohistochemistry, fluorescein isothiocyanate (FITC)-conjugated donkey anti-rabbit IgG, was purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). HRP-conjugated secondary antibodies for Western blot analyses were obtained from Santa Cruz Biotechnology.

Animals. Male Sprague-Dawley rats weighing between 125 and 150 g were obtained from Taconic Farms (Germantown, NY). They were maintained in constant temperature rooms with fixed light-dark intervals of 12 h and were fed ad libitum. The rats were anesthetized for 1 min in CO2 and decapitated, and both exorbital lacrimal glands were removed. All experiments were approved by the Schepens Eye Research Institute Animal Care and Use Committee.

Immunohistochemistry. Lacrimal glands were fixed by immersion in 4% formaldehyde diluted in phosphate-buffered saline (PBS: 145 mM NaCl, 7.3 mM Na2HPO4, and 2.7 mM NaH2PO4, pH 7.2) at 4°C overnight. After cryopreservation in 10–30% sucrose dissolved in PBS, the tissue was frozen in OCT embedding medium (Tissue Tek; Sakura, Torrance, CA). Cryostat sections (6 µm) were placed on slides and air-dried. The sections were rinsed in PBS and permeabilized in PBS containing 1% Triton X-100, and nonspecific binding of IgG was blocked by preincubation with PBS containing 10% bovine serum albumin (BSA). The sections were then incubated with the primary antibody for 2 h at room temperature. The secondary antibody was applied for 1 h at room temperature. Negative controls consisting of omission of the primary antibody were performed with each experiment. The sections were viewed with a Nikon Eclipse E800 microscope, and pictures were taken with a SPOT Diagnostic Instruments camera.

Preparation of lacrimal gland acini. Lacrimal glands were trimmed, fragmented, and incubated with collagenase in Krebs-Ringer bicarbonate (KRB) buffer (119 mM NaCl, 4.8 mM KCl, 1 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 25 mM NaHCO3, 10 mM HEPES, and 5.5 mM glucose) and 0.5% BSA. Lobules were subjected to gentle pipetting through tips of decreasing diameter, filtered through nylon mesh (150-µm pore size), and centrifuged briefly. The pellet was washed twice by centrifugation (50 g, 2 min) through a 4% BSA solution made in KRB buffer. The dispersed acini were allowed to recover for 60 min in fresh KRB with 0.5% BSA.

Membrane binding studies. Freshly isolated acini were homogenized in homogenization buffer [30 mM Tris·HCl, pH 7.5, 10 mM EDTA, 5 mM EGTA, 1 mM dithiothreitol, and 250 mM sucrose supplemented with 10 µl/ml protease inhibitor cocktail (Sigma)] and centrifuged at 100,000 g for 60 min. The membrane fraction was resuspended in cold buffer consisting of 20 mM HEPES, pH 7.4, 1.4 mM EDTA, and 12.5 mM MgCl2 containing protease inhibitors, rehomogenized with a motorized Potter-Elvehjem PTFE Tissue Grinder (Fisher Scientific, Pittsburgh, PA), and diluted to a protein concentration of 60 µg per assay tube with cold HEM buffer (20 mM HEPES, pH 7.4, 1.5 mM EGTA, and 12.5 mM MgCl2).

Saturation studies were performed by incubating the diluted membranes with increasing concentrations (35–600 pM) of [125I]HEAT in the presence and absence of 0.1 mM phentolamine for 1 h at 22°C. Competition studies were performed using either the {alpha}1A-AR inhibitor 5-methylurapidil (5-MU) or the {alpha}1D-AR inhibitor BMY-7378 from 10 pM to 1 mM in the presence of 90 pM [125I]HEAT for 1 h at 22°C. Samples were then filtered on a Brandel Cell Harvester through Whatman GF/C filter paper that had been pretreated with 0.3% (vol/vol) polyethylenimine/distilled H2O and 0.1% (wt/vol) BSA and washed with cold HEM buffer. Filters were counted on a gamma counter for 1 min, and data were analyzed using Prism 3.0 (GraphPad Software, San Diego, CA) by nonlinear regression. Determination of one vs. two site curves was determined using an F-test.

Measurement of protein secretion. Lacrimal gland acini were preincubated in KRB buffer containing 0.5% BSA in the presence of 5-MU, BMY-7378, GM6001, its negative control compound, or TAPI-1 at varying concentrations for 20 min at 37°C, followed by the addition of the {alpha}1-adrenergic agonist phenylephrine (10–4 M) for 20 min. Peroxidase (our marker for protein secretion) was measured using Amplex red. The amount of fluorescence was quantified using a fluorescence microplate reader (model FL600; Bio-Tek, Winooski, VT) with 530-nm excitation wavelength and 590-nm emission wavelength. The amount of peroxidase in the supernatant (secreted) was expressed as a percentage of the total peroxidase (amount of peroxidase present in the supernatant plus the amount remaining in the cells) (21).

Western blot analyses. Lacrimal gland acini or tissue pieces were incubated at 37°C in the absence or presence of phenylephrine at 10–4 M in KRB buffer with 0.5% BSA for 10–120 min. In some experiments, the ADAM17 inhibitor TAPI-1 (10–4–10–6 M) was added 20 min before addition of phenylephrine. The acini or tissue pieces were then homogenized in RIPA buffer (10 mM Tris·HCl, pH 7.4, 150 mM NaCl, 1% sodium deoxycholate, 1% Triton X-100, 0.1% SDS, and 1 mM EDTA) in the presence of a protease inhibitor cocktail (Sigma) and centrifuged at 20,000 g for 30 min. Proteins in the supernatant were separated using SDS-PAGE on a 10% gel or 4–20% linear gradient gel. Separated proteins were transferred onto nitrocellulose membranes, blocked in 5% nonfat dried milk in buffer containing 10 mM Tris·HCl, pH 8.0, 150 mM NaCl, and 0.05% Tween-20 (TBST) for 1 h at room temperature, and incubated with the primary antibody made up in 5% dried milk-TBST overnight at 4°C. After being washed, the membranes were incubated with the appropriate HRP-conjugated secondary antibody for 1 h. Immunoreactive bands were detected using the enhanced chemiluminescence method. The films were digitally scanned and analyzed using NIH Image software (version 1.63; Bethesda, MD). The amount of phosphorylated MAPK in each sample was standardized to the amount of total MAPK.

RNA extraction and RT-PCR. Total RNA was isolated from lacrimal gland acini or brain by using the extraction reagent Trizol according to the manufacturer's protocol. One microgram of purified total RNA was used for complementary DNA (cDNA) synthesis with the use of the Reverse Transcription System as described in the manufacturer's instructions. The absence of genomic DNA in RNA preparations was verified by omitting the reverse transcriptase in the reaction. The cDNA was amplified by PCR in a thermal cycler (PCR Sprint; Thermo Hybaid, Ashton, UK). The sense and antisense oligonucleotide primers were derived from previously published sequences and are given respectively as follows: {alpha}1A-AR, 5'-GTA GCC AAG AGA GAA AGC CG-3' and 5'-CAA CCC ACC ACG ATG CCC AG-3', 212-base pair product; {alpha}1B-AR, 5'-GCT CCT TCT ACA TCC CGC TCG-3', 5'-AGG GGA GCC AAC ATA AGA TGA-3', 300-base pair product; and {alpha}1D-AR, 5'-CGT GTG CTC CTT CTA CCT ACC-3' and 5'-GCA CAG GAC GAA GAG ACC CAC-3', 304-base pair product. The glyceraldehyde-3-phosphate dehydrogenase (G3PDH) gene was used as the internal control. Primers for G3PDH were purchased from Clonetech Laboratories (Palo Alto, CA). Each PCR reaction consisted of 0.5 µM each of sense and antisense primer, 200 µM each of dNTP, 1.5 mM MgCl2, 1.25 units of Taq polymerase, and 1 µl of cDNA. For {alpha}1-adrenergic receptors, PCR was performed after a 5-min hot start at 94°C, followed by 35 cycles of denaturation for 1 min, annealing for 1.5 min at 55°C, and extension for 1.5 min at 72°C. The final elongation time step was performed at 72°C for 7 min. For G3PDH, 30 cycles of denaturation for 1 min at 94°C, annealing for 1 min at 55°C, and extension for 1.5 min at 72°C were performed. The final elongation time step was performed at 72°C for 7 min. Sample with no cDNA was also amplified as negative control in every reaction. After amplification, 10 µl of products were separated using electrophoresis on a 1.2% agarose gel and visualized using ethidium bromide staining.

Measurement of EGF release by enzyme-linked immunosorbent assay. Lacrimal gland tissue pieces were incubated at 37°C in the absence or presence of phenylephrine at 10–4 M in KRB buffer containing 0.5% BSA for 10–120 min. The supernatant was removed, and the tissue was homogenized in homogenization buffer. The amount of EGF protein in both the supernatant and the tissue homogenate was measured in duplicate with enzyme-linked immunosorbent assay (ELISA). Microtiter plates (Nunc Maxisorp) were coated with the medium or homogenate diluted in 50 mM carbonate buffer at pH 9.6 and incubated overnight at 4°C. After the target protein solution was removed, nonspecific protein binding sites were blocked by incubating with PBS containing 1% BSA for 2 h at room temperature, followed by incubation with the anti-rat EGF polyclonal antibody for 1 h at room temperature. After being washed with PBS containing 0.05% Tween 20, HRP-conjugated donkey anti-goat IgG antibody diluted in blocking buffer was added for 1 h at room temperature. The plates were washed three times in 0.05% Tween 20-PBS and once in 50 mM Tris·HCl (pH 8.0), and peroxidase activity was measured in duplicate using Amplex red. The amount of fluorescence was quantified as described for secretion assays. EGF release was expressed as the percentage of EGF protein secreted into the medium compared with total EGF protein in the tissue homogenate plus the medium.

Data presentation and statistical analysis. Data are expressed as the relative increase above basal value, which was standardized to 1.0. Results are expressed as means ± SE. Data were analyzed using one-way ANOVA followed by Bonferroni/Dunn test to compare basal and poststimulation values or by Student's t-test. P < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Identification of {alpha}1-AR subtype in lacrimal gland acini. To determine which {alpha}1-adrenergic receptors are present in rat lacrimal gland, we isolated membranes from acinar cells by centrifugation. Saturation and competition curves were generated using [125I]HEAT, the {alpha}1A-AR inhibitor 5-MU, and the {alpha}1D-AR inhibitor BMY-7378 (38). 5-MU is 50–100 times more selective for {alpha}1A-ARs than {alpha}1B - or {alpha}1D-ARs, whereas BMY-7378 is 100 times more selective for {alpha}1D-ARs than {alpha}1A- or {alpha}1B-ARs. As shown in Fig. 1A, saturation studies were performed by incubating the membranes with increasing concentrations (35–600 pM) of [125I]HEAT in the presence and absence of 0.1 mM phentolamine. These experiments (Fig. 1) indicated that lacrimal gland acini contain 0.206 ± 0.033 pmol of adrenergic receptors per milligram of protein. Data obtained from competition studies with 5-MU (Fig. 1B) show a one-site binding model of low affinity, indicating that {alpha}1A-ARs are not present in lacrimal gland acini. Competition studies performed with BMY-7378 (Fig. 1C) show a two-site binding model, indicating that a majority (~90%) of {alpha}1-adrenergic receptors present are low-affinity {alpha}1B-ARs, whereas the remainder of the {alpha}1-ARs are high-affinity {alpha}1D-ARs.


Figure 1
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  		Fig. 1. Binding, competition, and RT-PCR studies. A: saturation studies were performed by incubating lacrimal gland membranes with increasing concentrations of [125I]HEAT in the presence or absence of 0.1 mM phentolamine. Specific binding is shown for a representative curve. Maximum binding = 0.206 ± 0.033 pmol/mg membrane protein and Kd = 107.8 ± 10.7 pM, from 3 independent experiments performed in triplicate. B and C: competition studies were performed using the {alpha}1A-adrenergic receptor (AR) selective inhibitor 5-methylurapidil (5-MU; B) or the {alpha}1D-AR selective inhibitor BMY-7378 (C) from concentrations of 10 pM to 1 mM in the presence of 90 pM [125I]HEAT. Various symbols represent data from individual experiments (n = 4) performed in duplicate. D: RT-PCR was performed on lacrimal gland acini and brain mRNA with primers specific to {alpha}1A-, {alpha}1B-, {alpha}1D-ARs or glyceraldehyde-3-phosphate dehydrogenase (G3PDH). Lanes 1, 2, and 3 correspond to individual animals, and lane 4 corresponds to RT-PCR performed with each primer using brain mRNA. NC, negative control.

 
The presence of {alpha}1B- and {alpha}1D-AR subtypes in the lacrimal gland was confirmed using RT-PCR. RNA was isolated from lacrimal gland acini from three individual animals and from the brain of one of the animals, and cDNA was synthesized. PCR was then performed using primers specific to rat {alpha}1A-, {alpha}1B-, and {alpha}1D-ARs and the housekeeping gene G3PDH. As shown in Fig. 1D, PCR products of the expected sizes were obtained using the primers for {alpha}1B- and {alpha}1D-ARs, whereas no product was detected using {alpha}1A-AR primers. As a positive control, PCR products for {alpha}1A-, {alpha}1B-, and {alpha}1D-adrenergic receptors were detected in brain. As a control for the quality of the cDNA, PCR was also performed at the same time with the same cDNA used for the {alpha}1-ARs by using primers for G3PDH. A single band of the appropriate molecular weight was detected using this primer. In addition, PCR was performed in the absence of cDNA (negative control).

{alpha}1D-ARs mediate lacrimal gland functions. Because phenylephrine is a potent stimulator of lacrimal gland secretion, we determined which of the {alpha}1-AR subtypes mediates phenylephrine-induced protein secretion. Acini were preincubated for 20 min with BMY-7378 from 10–11–10–5 M at 37°C before the addition of phenylephrine (10–4 M) for 20 min. The amount of peroxidase secreted into the media was then measured as described. As shown in Fig. 2A, preincubation with BMY-7378 significantly inhibited phenylephrine-induced peroxidase secretion a maximum of 82.7 ± 6.5% at 10–7 M. Interestingly, this response is biphasic and, indeed, secretion following preincubation with 10–5 M BMY-7378 was significantly different from that with 10–7 M. BMY-7378 alone had no significant effect on peroxidase secretion at any concentration (data not shown). With the specific {alpha}1A-adrenergic inhibitor 5-MU, phenylephrine induced-secretion was reduced, though not significantly, a maximum of 30.0 ± 15% at 10–8 M (data not shown).


Figure 2
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  		Fig. 2. Effect of inhibition of {alpha}1D-ARs on lacrimal gland functions. Lacrimal gland acini were preincubated for 20 min with the specific {alpha}1D-AR inhibitor BMY-7378 (10–11–10–5 M) before addition of the {alpha}1-adrenergic agonist phenylephrine (10–4 M). A: peroxidase secretion was measured after 20 min. Data are means ± SE of percentage of response from 3–6 independent experiments. B: p42/p44 MAPK was measured after 5 min. Data are means ± SE of percentage of response from 4–8 independent experiments. Blot shown in B is a representative experiment. *Statistically significant difference from no BMY-7378. #Statistically significant difference from 10–7 M BMY-7378.

 
Our group (21) has previously shown that phenylephrine stimulates the activation of p42/p44 MAPK in lacrimal gland acinar cells through transactivation of the EGF receptor. To determine whether p42/p44 MAPK activation is also mediated by {alpha}1D-ARs, we preincubated acini with BMY-7378 from 10–11 to 10–5 M for 20 min at 37°C and then stimulated with phenylephrine (10–4 M) for 5 min. The amount of phosphorylated p42/p44 MAPK was determined using Western blot techniques. BMY-7378 significantly inhibited phenylephrine-induced p42/p44 MAPK activation by 66.0 ± 15.0, 81.0 ± 10.0, 92.0 ± 7.0, 86.3 ± 11.8, and 100 ± 0% at 10–9, 10–8, 10–7, 10–6, and 10–5 M, respectively (Fig. 2B). BMY-7378 alone had no significant effect of MAPK activation at any concentration (data not shown). These results imply that two phenylephrine-stimulated functions, namely, peroxidase secretion and p42/p44 MAPK activation, are mediated by the high-affinity {alpha}1D-ARs.

Transactivation of EGF receptor by {alpha}1D-ARs occurs via MMPs. Previous work by our group (21) showed that phenylephrine-induced activation of MAPK occurs via transactivation of the EGF receptor, although the precise mechanism was not determined. Because it has been shown in other cell types that EGF, HB-EGF, and TGF-{alpha} are cleaved by MMPs into their soluble forms, which transactivate the EGF receptor (13, 17, 24), we tested whether, in the lacrimal gland, transactivation of EGF receptor by phenylephrine occurs via MMP cleavage of a member of the EGF family. The effects of a broad spectrum MMP inhibitor GM6001, its negative control, and an inhibitor specific for ADAM17, TAPI-1 (20, 29), on peroxidase secretion were determined. Because phenylephrine activation of MAPK decreases secretion, inhibition of MAPK stimulation will increase secretion. Thus, if GM6001 or TAPI-1 were to inhibit EGF release, phenylephrine-stimulated secretion would increase. Lacrimal gland acini were preincubated with GM6001 (10–6 M), its negative control (10–6 M), or TAPI-1 (10–4–10–6 M) for 20 min before stimulation with phenylephrine (10–4 M) for 20 min. GM6001 significantly increased phenylephrine-stimulated peroxidase secretion by 143.6 ± 14.8%. The negative control compound for GM6001 did not significantly increase phenylephrine-induced peroxidase secretion (Fig. 3A). Because GM6001 is a broad spectrum inhibitor for MMPs, we also tested the effects of an inhibitor to ADAM17, TAPI-1. TAPI-1 significantly increased phenylephrine-stimulated peroxidase secretion at 10–5 M by 120.2 ± 6.3% (Fig. 3B). TAPI-1 alone had no effect on peroxidase secretion (data not shown).


Figure 3
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  		Fig. 3. Effect of inhibition of matrix metalloproteinases on lacrimal gland functions. A and B: lacrimal gland acini were preincubated for 20 min with either GM6001 (GM; A), its negative control (neg; 10–6 M; A), or the ADAM17 inhibitor TAPI-1 (TAPI; 10–4–10–6 M; B) before addition of the {alpha}1-adrenergic agonist phenylephrine (Ph; 10–4 M), and peroxidase secretion was measured after 20 min. Data are means ± SE from 6 independent experiments. C and D: representative blots. E and F: acini were preincubated for 20 min with either GM6001, its negative control (10–6 M; E) or the ADAM17 inhibitor TAPI-1 (10–4–10–6 M; F) before addition of the {alpha}1-adrenergic agonist phenylephrine (10–4 M), and p42/p44 MAPK was measured after 5 min. Data are means ± SE from 6–7 independent experiments. *Statistically significant difference from agonist alone.

 
Lacrimal gland acini were preincubated with GM6001 (10–6 M), its negative control (10–6 M), and TAPI-1 (10–4–10–6 M) for 20 min before stimulation with phenylephrine (10–4 M) for 5 min. p42/p44 MAPK activation was then measured using Western blot techniques. Phenylephrine significantly increased p42/p44 MAPK activation (Fig. 3, C and D). Incubation with GM6001 reduced the amount of activated MAPK, whereas the negative control compound did not (Fig. 3C). TAPI-1 (10–5 and 10–4 M) also reduced the amount of activated MAPK (Fig. 3D). Analysis of results of seven independent experiments shows that GM6001 significantly reduced the amount of activated MAPK by 57.1 ± 21.8%. TAPI-1 at 10–5 and 10–4 M significantly reduced phenylephrine-induced p42/p44 MAPK by 32.7 ± 15.8 and 81.1 ± 13.9%, respectively, in six independent experiments (Fig. 3F). TAPI-1 alone had no effect on activation of p42/p44 MAPK (data not shown).

It is not clear why TAPI-1 at 10–4 M did not further increase protein secretion, because p42/p44 MAPK activation was inhibited by 80%. Although we do not understand this finding, we believe that because MAPK activation is upstream from protein secretion, it is possible that additional factors come into play between the upstream effector (MAPK) and the end point (secretion), accounting for the loss of effect. This is particularly likely given that phenylephrine activates specific pathways that stimulate secretion (e.g., PKC-{epsilon}) and others that inhibit secretion (e.g., PKC-{alpha}, -{delta}) (39). These data indicate that phenylephrine activates an MMP, perhaps ADAM17, to release a member of the EGF family, which activates the EGF receptor and MAPK, ultimately leading to an inhibition of protein secretion.

Location of EGF in lacrimal gland. EGF is synthesized in a precursor form (~170 kDa) that is cleaved to the proEGF form (~150 kDa) and the mature form (~6 kDa). To determine which form of EGF is present in the lacrimal gland, we homogenized the lacrimal gland in RIPA buffer or isolated acini before homogenization in RIPA buffer. As shown in Fig. 4A, the lacrimal gland homogenate and acini contained a single band at ~170 kDa, indicating the presence of precursor EGF and not pro- or mature EGF. As a control, commercially available rat EGF and submandibular gland (which contains only the mature form of EGF) (15, 28) were also analyzed using Western blot analysis. The submandibular gland contained only the mature form (6 kDa) of EGF (Fig. 4A).


Figure 4
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  		Fig. 4. Presence of EGF in the lacrimal gland. A: proteins from either lacrimal gland homogenate or acini were separated on 4–20% polyacrylamide gel, and EGF was detected in whole lacrimal gland, isolated lacrimal gland acini, commercially available rat EGF, and whole submandibular gland using an anti-EGF antibody. Arrow indicates the 6-kDa EGF form of EGF. B: localization of EGF was determined by immunofluorescence techniques in lacrimal gland. Asterisks indicate the presence of EGF in lacrimal gland ducts; arrows indicate the presence of EGF in the apical and lateral membranes of lacrimal gland acinar cells. Inset shows the presence of EGF in apical and lateral membranes of lacrimal gland acinar membranes. A schematic diagram of the structure of a lacrimal gland acinus is shown at bottom with the apical membrane highlighted in red and a red arrowhead; green indicates basal membranes, whereas black indicates lateral membranes. C: localization of EGF was determined using immunofluorescence techniques in submandibular gland. Inset shows EGF in secretory granules in submandibular glands. A schematic diagram of the structure of a submandibular gland acinar cell is shown at bottom with the basal membrane shown in green; black indicates secretory granules. D: negative control. Magnification, x600. Inset, magnification in B and C, x1,000.

 
The location of EGF in the lacrimal gland is of particular importance because of the highly polarized nature of the acinar cells. EGF located in the apical membrane would be shed into the ducts and onto the ocular surface. EGF located on the basolateral membranes would enter the intracellular space and exert any effects on the acinar cells themselves. To determine the location of EGF in lacrimal gland acini, we examined sections (6 µm) of lacrimal gland by using immunofluorescence techniques with the antibody directed against EGF used in Western blot analysis in Fig. 4A. EGF was predominately located in the ducts and in apical and basolateral membranes of acini of the lacrimal gland (Fig. 4B). We did not detect EGF in secretory granules, which is the location seen in the submandibular gland (Fig. 4C). The negative control was devoid of staining (Fig. 4D). These data indicate that EGF is located in the apical and lateral membranes of acinar cells. Thus shedding of EGF could influence the acinar cells themselves.

{alpha}1-Adrenergic agonists stimulate ectodomain shedding of EGF. To determine whether {alpha}1-adrenergic agonists increase the shedding of EGF, we incubated lacrimal gland pieces with phenylephrine (10–4 M) for 0–120 min. At the end of the incubation period, acini were centrifuged, the supernatant was removed, and tissue was homogenized. ELISA analysis for EGF was performed on the supernatant, whereas Western blot analysis was performed on the homogenate by using an antibody against EGF. Phenylephrine decreased the amount of precursor EGF in lacrimal gland homogenate (Fig. 5A) and caused a statistically significant increase in EGF in the supernatant at 30 min (1.4 ± 0.2 times greater than control, Fig. 5B).


Figure 5
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  		Fig. 5. Effect of {alpha}1-adrenergic agonists on ectodomain shedding of EGF. A: lacrimal gland pieces were incubated with the {alpha}1-adrenergic agonist phenylephrine (10–4 M) for 0–120 min. Tissue was homogenized, proteins were separated on 4–20% polyacrylamide gel, and Western blot analysis was performed using an antibody against either EGF or F-actin. B: Western blots from A were scanned and quantified, and the amount of precursor EGF was standardized to the amount of F-actin. After incubation with phenylephrine, the incubation medium was collected and the amount of EGF was determined using ELISA with the same antibody used in A (secreted EGF). Data are means ± SE of 4 independent experiments. *Statistically significant difference from time 0. C: acini were preincubated with TAPI-1 for 20 min before stimulation with phenylephrine for 30 min. The amount of EGF present in the medium was determined using ELISA with the same antibody used in A. Data are means ± SE of 4 independent experiments. *Statistically significant difference from phenylephrine alone.

 
This shedding was statistically significantly inhibited by 55.0 ± 20.9% when acini were pretreated with TAPI-1 (10–6 M) for 20 min before stimulation with phenylephrine (10–4 M) for 30 min (Fig. 5C). These results indicate that {alpha}1D-adrenergic agonists cause the shedding of EGF.

EGF receptor is activated by conditioned medium. To determine whether the molecules, including EGF, shed by phenylephrine are biologically active, we stimulated lacrimal gland pieces in KRB with 0.5% BSA with or without phenylephrine (10–4 M) for 30 min at 37°C. The conditioned KRB (c-KRB) was removed and incubated with freshly isolated acini that had been pretreated for 20 min with BMY-7378 (10–7 M). BMY-7378 was added to block any effect that phenylephrine (from the first incubation) might have. The amount of phosphorylated EGF receptor was measured after a 1-min incubation with c-KRB. In acini pretreated with BMY-7378 and stimulated with c-KRB from lacrimal gland pieces incubated with phenylephrine, the amount of phosphorylated EGF receptor was increased above that seen in acini preincubated with BMY-7378 and incubated with c-KRB from untreated pieces (Fig. 6A). Analysis of results of four independent experiments shows that the amount of phosphorylated EGF receptor was significantly increased 1.23 ± 0.04 times that of cells incubated with c-KRB from untreated lacrimal gland pieces (Fig. 6B). When acini were preincubated with the EGF receptor inhibitor AG1478 (10–7 M) in addition to BMY-7378 for 20 min, the amount of phosphorylated EGF receptor was decreased (Fig. 6A). Analysis of results of three independent experiments shows that the decrease (85.0 ± 23.0%) was statistically significant (Fig. 6C). AG1478 alone did not have a significant effect on basal MAPK activity (0.91 ± 0.24 times that of basal). These results imply that the EGF receptor is activated by molecules shed in response to phenylephrine.


Figure 6
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  		Fig. 6. EGF shed by {alpha}1-adrenergic agonists is biologically active and interacts with the EGF receptor. Lacrimal gland pieces were incubated with either no additions or with the {alpha}1-adrenergic agonist phenylephrine (10–4 M) for 30 min. The conditioned Krebs-Ringer bicarbonate medium (c-KRB) was removed and added to acini pretreated with the {alpha}1D-AR inhibitor BMY-7378 (10–7 M) for 1 min. The amount of EGF receptor phosphorylation was measured using Western blot analysis. A representative blot is shown in A. Experiments were quantified, and the amount of EGF receptor was standardized to actin and is shown in B. Data are means ± SE of 4 independent experiments. *Statistically significant difference from no phenylephrine. Acini were preincubated with AG1478 (AG; 10–7 M) for 20 min. c-KRB was added for 1 min. Experiments were quantified, and the amount of EGF receptor was standardized to actin and is shown in C. Data are means ± SE of 3 independent experiments. *Statistically significant difference from no AG1478.

 
Active EGF is shed by {alpha}1-adrenergic agonists and interacts with EGF receptor to activate p42/p44 MAPK. To determine whether the shed EGF and other molecules cause the phos-phorylation of p42/p44 MAPK through the activation of the EGF receptor, we stimulated lacrimal gland pieces in KRB with 0.5% BSA containing phenylephrine (10–4 M) for 30 min at 37°C. Again, the c-KRB was removed and incubated with freshly isolated acini that had been pretreated for 20 min with BMY-7378 (10–7 M). These acini were stimulated for 1 min with the c-KRB. The amount of phosphorylated p42/p44 MAPK increased with the addition of c-KRB from lacrimal gland pieces stimulated with phenylephrine over that seen in acini incubated with medium from pieces incubated in the absence of phenylephrine (Fig. 7A). Analysis of results from five experiments shows that the increase in phosphorylated p42/p44 MAPK was significantly increased 2.38 ± 0.7 times that of basal (Fig. 7B). When acini were preincubated with AG1478 (10–7 M) in addition to BMY-7378 for 20 min, the amount of phosphorylated p42/p44 MAPK was decreased by 91 ± 7.4% compared with acini that were not treated with AG1478 (Fig. 7A). This decrease was statistically significant (Fig. 7C).


Figure 7
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  		Fig. 7. EGF shed by {alpha}1-adrenergic agonists activates p42/p44 MAPK. Lacrimal gland pieces were incubated with either no additions or with the {alpha}1-adrenergic agonist phenylephrine (10–4 M) for 30 min. c-KRB was removed and added to freshly isolated acini pretreated for 20 min with BMY-7378 (10–7 M). The amount of p42/p44 MAPK activation was measured after 1 min by using Western blot analysis. A representative blot is shown in A. Experiments were quantified, and the amount of phosphorylated p42/p44 MAPK was standardized to total p42/p44 MAPK and is shown in B. Data are means ± SE of 5 independent experiments. *Statistically significant difference from no phenylephrine. Acini were preincubated with AG1478 (10–7 M) for 20 min before stimulation with c-KRB. Experiments were quantified, and the amount of p42/p44 MAPK activation was measured using Western blot analysis and is shown in C. Data are means ± SE of 3 independent experiments. *Statistically significant difference from no AG1478.

 
To confirm the identity of the EGF shed in response to {alpha}1-adrenergic agonist stimulation, we preincubated the c-KRB from lacrimal gland pieces incubated with phenylephrine for 15 min with 20 µg/ml of an EGF-neutralizing antibody. Acini were then incubated with the neutralizing antibody mixture for 1 min, and activation of p42/p44 MAPK was measured in acini preincubated with BMY-7378 and incubated with c-KRB. As shown in Fig. 8A, activation of p42/p44 MAPK by incubation with c-KRB was decreased by the EGF neutralizing antibody. Analysis of results of five independent experiments shows that the EGF neutralizing antibody significantly inhibited phosphorylation by 64.6 ± 18.8% (Fig. 8B).


Figure 8
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  		Fig. 8. Biologically active EGF shed by {alpha}1-adrenergic agonists. Lacrimal gland pieces were incubated with either no additions or with the {alpha}1-adrenergic agonist phenylephrine (10–4 M) for 30 min. c-KRB was removed and incubated for 15 min with an EGF neutralizing antibody (Ab; 20 µg/ml) before addition to freshly isolated acini pretreated for 20 min with BMY-7378 (10–7 M). A representative blot is shown in A. Experiments were quantified, and the amount of p42/p44 MAPK activation was measured using Western blot analysis and is shown in B. Data are means ± SE of 5 independent experiments. *Statistically significant difference from no antibody. As a control for the antibody specificity, exogenous EGF (10–7 M) was preincubated for 15 min with the EGF neutralizing antibody (20 µg/ml) before addition to freshly isolated acini. A representative blot is shown in C. Experiments were quantified, and the amount of p42/p44 MAPK activation was measured using Western blot analysis and is shown in D. Data are means ± SE of 5 independent experiments. *Statistically significant difference from no antibody.

 
To confirm the specificity of the EGF neutralizing antibody, we preincubated commercially available EGF for 15 min with the EGF neutralizing antibody used in the previous experiments. The EGF-EGF neutralizing antibody mixture was then added to freshly isolated acinar cells for 1 min. EGF alone increased the phosphorylation of p42/p44 MAPK, which was inhibited by incubation with the antibody mixture (Fig. 8C). Analysis of results of five independent experiments shows that the EGF neutralizing antibody significantly inhibited phosphorylation by 66.6 ± 15.8% (Fig. 8D). These results indicate that EGF is shed from the lacrimal gland as a result of stimulation with {alpha}1-adrenergic agonists and interacts with the EGF receptor to activate p42/p44 MAPK.


   DISCUSSION
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In freshly isolated lacrimal gland acini, {alpha}1D-ARs have a dual role in protein secretion to both stimulate and reduce it. These receptors produce nitric oxide and increase intracellular cGMP concentrations, leading to an increase in protein secretion (10). They also activate Gq{alpha}, presumably leading to activation of an as yet unidentified phospholipase (19), leading to activation of three isoforms of PKC, namely, PKC-{alpha}, -{delta}, and -{epsilon}. Interestingly, PKC-{alpha} and -{delta} reduce phenylephrine-induced protein secretion from these cells, whereas activation of PKC-{epsilon} stimulates protein secretion. In addition to the classic GPCR pathway, {alpha}1D-ARs also transactivate the EGF receptor by activating an MMP, probably ADAM17, to cause the shedding of proEGF. The proEGF binds to the EGF receptor to recruit Shc, Grb2, and SOS, leading to the activation of the kinase cascade, culminating in the activation of p42/p44 MAPK (21). Activation of p42/p44 MAPK plays a role in modulating phenylephrine-induced protein secretion by reducing it. This probably acts to control protein secretion.

It is worth noting that the inhibition of {alpha}1D-adrenergic receptor-induced protein secretion with BMY-7378 is a biphasic response in that inhibition of secretion reaches a maximum level at 10–7 M and is reduced with higher concentrations of BMY-7378. This is in contrast to inhibition of {alpha}1D-adrenergic receptor-induced MAPK activation with BMY-7378. In our laboratory (39), we have observed similar results with other inhibitors of {alpha}1D-adrenergic receptor-induced protein secretion, namely, with inhibitors of PKC-{alpha} and -{delta}. It is unclear why this response is biphasic.

The lacrimal gland contains both {alpha}1B- and {alpha}1D-adrenergic receptors. No {alpha}1A-adrenergic receptors were detected by either binding or RT-PCR studies. Because there are no specific {alpha}1B-adrenergic receptor inhibitors, we were unable to determine the relative role that these receptors play in phenylephrine-induced lacrimal gland functions. However, the {alpha}1D-AR inhibitor BMY-7378 completely inhibited both phenylephrine-stimulated protein secretion and p42/p44 MAPK activation at concentrations previously shown to inhibit contraction of rat tail artery (12). It is therefore unlikely that {alpha}1B-ARs are involved in these processes. Of interest, {alpha}1D-ARs have been shown to be localized intracellularly (4). Hague et al. (8) subsequently showed that in HEK-293 cells, coexpression of {alpha}1B-ARs with {alpha}1D-ARs results in heterodimers, leading to the expression of {alpha}1D-ARs on the cell surface. In addition, the formation of these heterodimers results in an increase in the coupling of {alpha}1D-ARs to intracellular Ca2+ release (8). It is possible that the {alpha}1B-ARs present in the lacrimal gland play a role in the localization of the {alpha}1D-ARs. Lack of specific antibodies for the AR subtypes makes it difficult to examine this possibility in freshly isolated cells.

{alpha}1D-ARs are present in a variety of tissues (1, 14, 18). The lacrimal gland contains 0.206 pmol of {alpha}1D-adrenergic receptors per milligram of protein. This is in comparison to rat tail artery, which has 0.138 pmol of {alpha}1D-adrenergic receptors per milligram of protein, and rat aorta, which has 0.527 pmol of {alpha}1D-adrenergic receptors per milligram of protein (31). Despite widespread distribution, there is limited knowledge regarding the role of {alpha}1D-ARs in cellular functions. Few studies have linked activation of {alpha}1D-ARs to functions outside of the vascular tissue. {alpha}1D-ARs have been implicated in smooth muscle contraction in large arteries, including the aorta, iliac, and femoral arteries (14). In addition, {alpha}1D-ARs have been shown to activate p42/p44 MAPK, leading to increased protein synthesis in both arterial smooth muscle (36) and fibroblasts (35). However, both studies utilized cultured cells overexpressing the {alpha}1D-ARs. In this study, using freshly isolated cells, we have shown that the {alpha}1D-ARs mediate phenylephrine-stimulated protein secretion and p42/p44 MAPK activation.

It is important to note that these experiments were performed on freshly isolated lacrimal gland acini. These cells have not been modified to overexpress any proteins. It is known that proteins that are overexpressed can result in protein misfolding, changes in rates of degradation, and interactions between proteins that would not normally occur (5, 33). Thus it is vital that interactions between proteins be confirmed in wild-type, nontransformed cells.

It is well established that EGF is synthesized as a large membrane-bound precursor molecule with a molecular mass of ~170 kDa. Proteolytic cleavage releases the proEGF in a process known as ectodomain shedding, which is mediated by a MMP of the ADAM family (13). This process is similar to those described for other members of the EGF family, namely, HB-EGF, TGF-{alpha}, and amphiregulin (2, 11, 22, 26, 30). The proEGF released is not only soluble but also, because it contains EGF domains, is biologically active. Additional proteases present in the extracellular space can further cleave proEGF to the mature 6-kDa form of EGF. In the lacrimal gland, we confirmed previous findings from Marechal et al. (15) that EGF is present as only precursor EGF. We also demonstrated that it is not present in the secretory granules of the lacrimal gland. This implies that EGF is not secreted but rather is shed by an MMP from the cells from the apical membrane and released into the tears and from the basolateral membranes and released into the extracellular space surrounding acinar cells. Immunolocalization demonstrated that EGF was present on both the apical membranes, indicating shedding into the tears, thereby possibly exerting its effects on the cornea and conjunctiva, and on the basolateral membranes, indicating that EGF can also exert its effects on acinar cells themselves. Although we cannot determine from these experiments whether EGF is shed from the apical or basolateral membranes, this study does demonstrate that EGF is shed from lacrimal gland acini, that the shed EGF is biologically active, and that acinar cells are able to respond to this EGF.

In this study we also showed that upon stimulation with {alpha}1-adrenergic agonists, precursor EGF is cleaved. ProEGF is then shed from the membrane through the actions of a MMP and is released into the medium. TAPI-1, a specific inhibitor of ADAM17, reduces phenylephrine-induced activation of p42/p44 MAPK, implicating this MMP in the shedding of EGF in these cells. However, TAPI-1 at 10–4 M did not further increase protein secretion, because p42/p44 MAPK activation was substantially inhibited. It is possible that because MAPK activation is upstream of protein secretion, downstream proteins play a role in phenylephrine-stimulated protein secretion. However, activation of ADAM17 is in contrast to the study performed by Sahan et al. (26), who showed that ADAM10 was the main MMP involved in shedding of EGF from fibroblasts from mice in which different combinations of ADAMs were knocked out. This could be due to a difference in cell types. In addition, we showed that the proEGF released by {alpha}1-adrenergic agonists is biologically active, because the conditioned buffer can be added to freshly isolated cells and EGF receptor and MAPK can be activated in these cells. Activation of MAPK is partially blocked by an EGF receptor inhibitor and, more importantly, by an EGF neutralizing antibody, confirming the fact that proEGF released binds to the EGF receptor to activate MAPK. It is likely that additional growth factors other than EGF also are shed in response to {alpha}1-adrenergic agonists, and they could be responsible for the remainder of the MAPK activation not blocked by the EGF receptor inhibitor or the neutralizing antibody.

In summary, in freshly isolated lacrimal gland epithelial cells, {alpha}1D-ARs stimulate protein secretion in addition to the shedding of a biologically active proEGF, leading to the transactivation of the EGF receptor and activation of p42/p44 MAPK, which in turn negatively modulates protein secretion.


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This work was supported by National Eye Institute Grant EY-06177.


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. R. Hodges, Schepens Eye Research Institute, 20 Staniford St., Boston, MA 02114 (e-mail: hodges{at}vision.eri.harvard.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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