August 2005
Volume 46, Issue 8
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Cornea  |   August 2005
Nitric Oxide and cGMP Mediate α1D-Adrenergic Receptor–Stimulated Protein Secretion and p42/p44 MAPK Activation in Rat Lacrimal Gland
Author Affiliations
  • Robin R. Hodges
    From the Schepens Eye Research Institute, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts.
  • Marie A. Shatos
    From the Schepens Eye Research Institute, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts.
  • Rachel S. Tarko
    From the Schepens Eye Research Institute, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts.
  • Joanna Vrouvlianis
    From the Schepens Eye Research Institute, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts.
  • Jian Gu
    From the Schepens Eye Research Institute, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts.
  • Darlene A. Dartt
    From the Schepens Eye Research Institute, Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts.
Investigative Ophthalmology & Visual Science August 2005, Vol.46, 2781-2789. doi:10.1167/iovs.05-0022
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      Robin R. Hodges, Marie A. Shatos, Rachel S. Tarko, Joanna Vrouvlianis, Jian Gu, Darlene A. Dartt; Nitric Oxide and cGMP Mediate α1D-Adrenergic Receptor–Stimulated Protein Secretion and p42/p44 MAPK Activation in Rat Lacrimal Gland. Invest. Ophthalmol. Vis. Sci. 2005;46(8):2781-2789. doi: 10.1167/iovs.05-0022.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. To determine whether α1-adrenergic receptors use the nitric oxide (NO)/cGMP pathway to stimulate protein secretion by rat lacrimal gland.

methods. Identification and cellular location of endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS) were determined by Western blot and immunofluorescence techniques, respectively. Rat lacrimal gland acini were isolated by collagenase digestion, and protein secretion stimulated by phenylephrine, an α1-adrenergic agonist, was measured with a fluorescence assay system. Acini were preincubated with inhibitors for 20 minutes before addition of phenylephrine (10−4 M). NO and cGMP were measured in response to phenylephrine stimulation. Activation of p42/p44 MAPK was determined by Western blot analysis with an antibody against phosphorylated (active) p42/p44 MAPK.

results. eNOS and nNOS were both present in lacrimal gland. eNOS appeared to be localized with caveolae, whereas nNOS was present in the nerves surrounding the acini. Inhibition of eNOS with N G-nitro-l-arginine methyl ester (l-NAME; 10−6 M) completely inhibited phenylephrine-stimulated protein secretion, whereas the inactive isomer d-NAME and inhibition of nNOS with S-methyl-l-thiocitrulline did not. Phenylephrine increased NO production in a time- and concentration-dependent manner, but the increase was abolished by the α1D-adrenergic receptor inhibitor BMY-7378. Inhibition of guanylate cyclase with oxadiazoloquinoxalin (ODQ) also inhibited phenylephrine-induced protein secretion, whereas phenylephrine caused a 2.2-fold increase in cGMP. In addition, preincubation with l-NAME and ODQ inhibited phenylephrine-stimulated p42/p44 MAPK activation.

conclusions. α1D-Adrenergic agonists stimulate eNOS to produce NO, leading to production of cGMP by guanylate cyclase, to transduce the extracellular signal through the cell and stimulate protein secretion in rat lacrimal gland.

Nitric oxide (NO) is a small, diffusible gaseous molecule that has been identified as a mediator in a variety of cellular functions including secretion, inflammation, and blood flow. 1 2 3 NO is synthesized from arginine and molecular oxygen by nitric oxide synthase (NOS), resulting in NO and l-citrulline. Nitric oxide synthase is a family of enzymes consisting of three known isoforms, endothelial nitric oxide synthase (eNOS or NOS-3), neuronal nitric oxide synthase (nNOS or NOS-1), and inducible nitric oxide synthase (iNOS or NOS-2). Despite their names, both eNOS and nNOS have been shown to be present in many different tissues. 4 eNOS and nNOS are constitutively expressed; hence, they are referred to as constitutive enzymes. These two isoforms are activated by intracellular calcium and calmodulin. iNOS is not present in resting cells and is induced in a variety of cells by cytokines, infection, or lipopolysaccharides. This isoform is calcium independent and constitutively active. 5 Induction of iNOS results in a large, rapid increase in NO that can have detrimental effects on surrounding tissue. In contrast, activation of eNOS and nNOS results in a smaller, slower increase in NO, which interacts with a variety of signaling pathways. 
The effects of NO can be classified as either cGMP dependent or cGMP independent. NO interacts with many different types of proteins; however, interactions with heme-containing proteins such as ryanodine receptors or guanylate cyclase (GC) are well documented. 4 6 Ryanodine receptors are sarcoplasmic reticulum calcium-release channels, whereas GCs convert GTP to cGMP. 7 Signal transduction by cGMP is dependent on its synthesis by GCs, its targeting, and its degradation by cGMP-dependent phosphodiesterases (PDEs). Once produced, cGMP interacts with protein kinase G (PKG) to phosphorylate downstream proteins (many of which have yet to be identified) that activate a variety of cellular functions. 
The lacrimal gland is an exocrine gland responsible for producing the majority of the aqueous portion of the tear film. 8 Acinar cells are the major cell type present in the lacrimal gland. In addition, myoepithelial and ductal epithelial cells are present. If lacrimal gland secretion is altered in either amount or composition, a spectrum of diseases called dry eye syndrome results. Therefore, secretion from the lacrimal gland is tightly regulated. Accordingly, parasympathetic and sympathetic nerves extensively innervate the lacrimal gland. Stimulation of the afferent sensory nerves in the cornea triggers tear secretion through the efferent parasympathetic and sympathetic nerves that innervate the lacrimal gland. 8 Receptors for these neurotransmitters are located on the basolateral side of the acinar cells. 8 Activation of these receptors initiates signal transduction pathways that culminate in secretion of proteins, electrolytes, and water across the apical membranes into the lumen and onto the cornea and conjunctiva. 
We have shown that cholinergic agonists released from parasympathetic nerves and α1-adrenergic agonists released from sympathetic nerves are potent stimuli of protein secretion from the lacrimal gland. The signal transduction pathways used by cholinergic agonists have been well characterized. 8 In contrast, the pathways used by α1-adrenergic agonists have remained elusive. We have also shown that α1-adrenergic receptors mediate both protein secretion and activation of p42/p44 MAPK. 9 Despite this information, the G protein(s) involved in transduction of this signal are unknown, though Meneray and Fields 10 have shown that inhibition of Gαq is responsible for approximately 32% of the α1-adrenergic-agonist–induced protein secretion. The phospholipase involved also has not been identified, though it is known that neither phospholipase C nor D is involved. 11 12 It is known that phenylephrine (an α1-adrenergic agonist in the lacrimal gland) activates PKC-ε, which stimulates protein secretion and PKCα and -δ to inhibit secretion. 13 We have also shown that α1-adrenergic agonists transactivate the epidermal growth factor receptor (EGFR), recruiting Shc and Grb2, which leads to activation of p42/p44 MAPK. Activation of p42/p44 MAPK inhibits protein secretion and may attenuate overall protein secretion from the lacrimal gland or terminate stimulated secretion. 
It is known that the lacrimal gland contains many of the proteins necessary for the production of NO and cGMP. nNOS has been shown to be present in the lacrimal gland surrounding ducts, blood vessels, and acinar cells. 14 15 Exogenous addition of NO, through the use of NO donors, has been shown to increase total protein secretion, which is inhibited by guanylate cyclase inhibitors in cultured lacrimal gland acinar cells. 3 16 In addition, NO donors that increase cGMP induce an increase in intracellular [Ca2+] ([Ca2+]i) through the release of Ca2+ from intracellular stores. 17 Jorgensen et al. 18 demonstrated that an α1-adrenergic-agonist–induced increase in [Ca2+]i is inhibited by inhibitors of GC and cGMP-dependent protein kinase Ia. 18 It is also known that when total IgGs and purified autoantibodies against M3 muscarinic receptors from patients who have the autoimmune disease Sjögren’s syndrome were added to freshly prepared rat lacrimal gland slices, NOS activity was increased. 19 However, there has been no evidence to support a role for α1-adrenergic, β-adrenergic, or muscarinic agonists in stimulating regulated protein secretion by freshly isolated lacrimal gland acini through the NO/cGMP pathway. 
In the present study, we hypothesized that the α1-adrenergic agonist phenylephrine, activates NOS to increase NO production from freshly isolated rat lacrimal gland acinar cells—in turn, activating GC to increase cGMP levels that lead to an increase in protein secretion and p42/p44 MAPK induction. 
Materials and Methods
Materials
Rabbit polyclonal antibodies directed against eNOS, nNOS, and caveolin-1 were purchased from Transduction Laboratories (San Jose, CA). A monoclonal antibody against α-smooth muscle actin was purchased from Diagnostic Biosystems (Pleasanton, CA); the MAP-2 antibody from Roche Molecular Biochemicals (Indianapolis, IN); antibodies to phosphorylated p42/p44 and total p42 MAPK from Santa Cruz Biotechnology (Santa Cruz, CA); l- and d-NAME, S-methyl-l-thiocitrulline, l-thiocitrulline, and oxadiazoloquinoxalin (ODQ) from Calbiochem (San Diego, CA); and NO and cGMP assay kits from Biomol Research Laboratories (Plymouth Meeting, PA). A fluorescent molecule (Amplex Red) was acquired from Molecular Probes (Eugene, OR). All other reagents were obtained from Sigma-Aldrich (St. Louis, MO). 
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 hours and were fed ad libitum. The rats were anesthetized for 1 minute in CO2 and decapitated, and both exorbital lacrimal glands were removed. All experiments were in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Schepens Eye Research Institute Animal Care and Use Committee. 
Preparation of Rat Lacrimal Gland Acini
Acini were prepared using a modification of a method developed by Herzog et al. 20 In brief, lacrimal glands were trimmed, fragmented, and incubated with collagenase CLSIII (100 U/mL) in Krebs-Ringer bicarbonate (KRB) buffer (119 mM NaCl, 4.8 mM KCl, 1 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, and 25 mM NaHCO3) supplemented with 10 mM HEPES, 5.5 mM glucose (KRB-HEPES), and 0.5% BSA (pH 7.4). 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 (50g, 2 minutes) through a 4% BSA solution made in KRB-HEPES buffer. The dispersed acini were allowed to recover for 60 minutes in fresh KRB-HEPES buffer containing 0.5% BSA. 
Western Blot Analysis
Lacrimal glands were homogenized in RIPA buffer (9.1 mM dibasic sodium phosphate, 1.7 mM monobasic sodium phosphate, 150 mM NaCl [pH 7.4] 1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS). Proteins in the supernatant were separated by SDS-PAGE on an 8% gel. Separated proteins were transferred onto nitrocellulose membranes, which were blocked overnight at 4°C in 5% nonfat dried milk in buffer containing 10 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 0.05% Tween-20 (TBST) and then incubated with the primary antibody (1:200 for eNOS and 1:100 for nNOS) for 2 hours at room temperature. After the membranes were washed three times in TBST, they were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody. Immunoreactive bands were detected by the enhanced chemiluminescence method. 
Immunohistochemistry
The 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]) for 4 hours at 4°C. After cryopreservation overnight at 4°C in 30% sucrose dissolved in PBS, the tissue was frozen in optimal cutting temperature embedding compound. Cryostat sections (6 μm) were placed on gelatin-coated slides, air dried for 2 hours, and rinsed for 5 minutes in TBS (10 mM Tris-HCl [pH 8.0] and 150 mM NaCl). Nonspecific sites were blocked in PBS containing 10% normal goat serum, 1% bovine serum albumin (BSA), and 0.2% Triton X-100 for 45 minutes. After they were rinsed for 5 minutes in TBS, the sections were then incubated with the primary antibody for 2 hours at room temperature in a humidified chamber. Antibodies to eNOS, nNOS, α-smooth muscle actin, or caveolin-1 were used at a concentration of 1:50 diluted in TBS, and the antibody to microtubule-associated protein (MAP)-2 was used at 1:500 diluted in TBS. The secondary antibodies, conjugated to either FITC (1:150) or Cy3 (1:300), diluted in TBS with 1% BSA, were applied for 1 hour at room temperature. Coverslips were mounted with a medium consisting of glycerol and paraphenylenediamine. In negative control experiments, the primary antibody was omitted. The sections were viewed by microscope (Eclipse E80i; Nikon, Tokyo, Japan) and micrographs were taken with a digital camera (Spot; Diagnostic Instruments, Inc., Sterling Heights, MI). 
Measurement of Peroxidase Secretion
Lacrimal gland acini were incubated in 0.5% BSA-KRB-HEPES buffer in duplicate for 20 minutes at 37°C in the absence or presence the α1-adrenergic agonist phenylephrine (10−4 M). Inhibitors were added 20 minutes before addition of phenylephrine. After a brief centrifugation, supernatant was collected, and peroxidase activity was measured in duplicate in both the supernatant and the pellet fraction, by using a fluorescent molecule (Amplex Red; Molecular Probes, Inc.). Oxidation of this reagent by peroxidase in the presence of hydrogen peroxide produces a highly fluorescent molecule, resorufin. The amount of fluorescence was quantified by a fluorescence microplate reader (model FL600; Bio-Tek, Winooski, VT) with an excitation wavelength of 530 nm and an emission wavelength of 590 nm. Peroxidase secretion, an index of lacrimal gland protein secretion, was expressed as the percentage of peroxidase secreted into the medium (supernatant) compared with total peroxidase in the cells (pellet) and the medium. 
Measurement of NO
Lacrimal gland acini were incubated with phenylephrine (10−4 M) for 0 to 30 seconds or with increasing concentrations of phenylephrine (10−7–10−3 M) for 20 seconds. The supernatant was collected and filtered through 10,000 molecular-weight-cutoff filters to remove proteins. The amount of NO was determined with a NO assay kit, by reducing nitrate to nitrite with nitrate reductase. Nitrite was determined by the Griess reaction, and the color reaction was read at 540 nm, according to the manufacturer’s protocol. 
Measurement of cGMP
Lacrimal gland acini were preincubated for 10 minutes with the phosphodiesterase inhibitor 3-isobutylmethylxanthine (10−3 M) before stimulation with phenylephrine (10−4 M) for 0 to 30 minutes. The reaction was stopped by brief centrifugation, the supernatant removed, and the acini lysed in 0.1 N HCl. The cells were sonicated briefly and centrifuged for 5 minutes at 5000g at room temperature. The amount of cGMP in the supernatant was measured with a cGMP assay kit, according to the manufacturer’s instructions. 
Measurement of MAPK Activity
The activation of p42/44 MAPK was examined by Western blot techniques. Acini were preincubated with the inhibitors l-NAME, its inactive isomer d-NAME, and ODQ for 20 minutes before addition of phenylephrine (10−4 M) for 5 minutes. To terminate incubation, ice-cold KRB buffer was added and centrifuged briefly. The pellet was homogenized in RIPA buffer (10 mM Tris-HCl [pH 7.4] 150 mM NaCl, 1% sodium deoxycholate, 1% Triton X-100, 0.1% SDS, 1 mM EDTA, 100 μg/mL phenylmethylsulfonyl fluoride, 30 μL/mL aprotinin, and 1 mM Na3VO3), sonicated, and centrifuged at 20,000g for 30 minutes. As described for Western blot analysis, the proteins in the supernatant were separated by SDS-PAGE, transferred onto nitrocellulose membrane, and probed with either total p42 MAPK (1:1000) or phosphorylated p42/44 MAPK (1:100) followed by HRP-conjugated secondary antibody. Immunoreactive bands were digitally scanned and analyzed with ImageJ (available by ftp at zippy.nimh.nih.gov/ or at http://rsb.info.nih.gov/nih-image/; developed by Wayne Rasband, National Institutes of Health, Bethesda, MD). The amount of phosphorylated p42/p44 MAPK in each sample was standardized to the amount of total p42 MAPK. 
Data Presentation and Statistical Analysis
Data are expressed as the x-fold increase over the basal value, which was standardized to 1.0. Results are expressed as the mean ± SEM. Data were analyzed by Student’s t-test. P < 0.05 was considered statistically significant. 
Results
Presence of eNOS and nNOS in the Lacrimal Gland
To confirm the presence of nNOS and determine whether eNOS is present in rat lacrimal gland, either the whole lacrimal gland or acini was homogenized in RIPA buffer and the proteins separated by SDS-PAGE as described in the Methods section. Western blot analysis was then performed with antibodies to the eNOS and nNOS isoforms. Homogenates from liver, aorta, and brain were used in positive control experiments. In lacrimal gland, eNOS was present at the same molecular weight as that in the brain and liver. The major band present in lacrimal gland homogenate was of a slightly higher molecular weight than the major band present in the lacrimal gland acini. eNOS expressed in the aorta was of a lower molecular weight than either the liver, brain, or lacrimal gland (Fig. 1)
nNOS was also present in both the lacrimal gland homogenate and acini. The major bands in both lacrimal gland homogenate and acini were of the same molecular weight as the nNOS in liver and brain (Fig. 1) . An additional, lower-molecular-weight band was also observed in the brain, lacrimal gland acini, and homogenate and may represent a different phosphorylation state. 21  
These data indicate that the lacrimal gland expresses the eNOS and nNOS isoforms of NOS. 
Localization of eNOS and nNOS in the Lacrimal Gland
To determine the localization of eNOS, lacrimal gland sections were incubated with an antibody directed against eNOS, and immunofluorescence microscopy was performed. eNOS was present in a cobblestone pattern, with immunoreactivity present in the basal membranes of acinar cells (Fig. 2A) . As eNOS has been shown to be present in caveolae, 22 23 lacrimal gland sections were also labeled with an antibody against caveolin-1 to determine whether the eNOS expressed in the lacrimal gland was also present in the caveolae. Caveolin-1 was located on the basal membranes of acinar cells in a pattern similar to that of eNOS (Fig. 2B) . When sections were double-labeled with both eNOS and caveolin-1, there was significant colocalization of both proteins in the basal membranes (Fig. 2C)
As NO is a diffusible molecule and myoepithelial cells are present in the lacrimal gland and are believed to play a role in secretion, we determined whether eNOS was also present in these cells. Myoepithelial cells contain an abundance of α-smooth muscle actin, which can be used to identify these cells. 24 Myoepithelial cells were abundant throughout the gland and present surrounding the acinar cells (Fig. 2E) . Double labeling with eNOS antibody showed no apparent colocalization, indicating that myoepithelial cells do not contain eNOS (Fig. 2F) . Negative controls were devoid of staining (Figs. 2G 2H)
The localization of nNOS was also determined with an antibody against nNOS in immunofluorescence experiments. nNOS surrounded a population of acini near the basal membrane (Fig. 3A) . It has been localized to nerve filaments in many different tissues 25 26 27 including the lacrimal gland 6 14 15 28 ; therefore lacrimal gland sections were also double-labeled with MAP-2, which is highly expressed in mammalian nerves. 29 Localization of MAP-2 was observed in nerve filaments (Fig. 3B) . When the images of nNOS and MAP-2 staining were overlaid, significant colocalization of nNOS and MAP-2 was observed (Fig. 3C)
These data indicate that eNOS is present in the acinar cells, specifically localized to caveolae, whereas nNOS is present in the nerve fibers of the rat lacrimal gland. 
Effect of Inhibition of eNOS and nNOS on α1-Adrenergic-Agonist–Induced Protein Secretion
As the α1-adrenergic agonist phenylephrine is a potent stimulator of protein secretion, we determined the effects of inhibitors of eNOS and nNOS on phenylephrine-stimulated lacrimal gland secretion. Acini were preincubated with either the eNOS inhibitor l-NAME, its inactive isomer d-NAME, or the nNOS-specific inhibitors S-methyl-l-thiocitrulline or l-thiocitrulline for 20 minutes. Phenylephrine (10−4 M) was then added for an additional 20 minutes. Phenylephrine increased peroxidase secretion by 2.9 ± 0.7-fold above control (Fig. 4) . l-NAME, which has an IC50 of 0.5 × 10−6 M for eNOS 30 and is reportedly 2000 times more selective of eNOS than nNOS in an allergic rat model, 31 completely inhibited phenylephrine-induced protein secretion at 10−6 M. The inactive isomer, d-NAME had no effect on phenylephrine-induced protein secretion (Fig. 4A) . In addition, neither l-thiocitrulline nor S-methyl-l-thiocitrulline, which is more selective for nNOS than eNOS, 1 32 had any effect on phenylephrine-stimulated protein secretion (Fig. 4B)
As a control, acini were also incubated with the cholinergic agonist carbachol (Cch), which has been shown to stimulate peroxidase secretion through the Ca2+/PKC pathway. 8 Cch (10−4 M) increased secretion 1.2 ± 0.03-fold above the basal level (data not shown). Cch-stimulated secretion was unchanged after preincubation with l-NAME or d-NAME (10−6 M, data not shown). 
These data imply that phenylephrine stimulates eNOS, but not nNOS, to activate peroxidase secretion. 
Effect of α1-Adrenergic Agonists on NO Production
Since an inhibitor of NOS inhibited phenylephrine-induced protein secretion, we wanted to determined whether phenylephrine increases NO production. Acini were incubated with phenylephrine (10−4 M) for 0 to 30 seconds, and the amount of NO was measured in the supernatant, as described earlier. Phenylephrine significantly increased the production of NO above basal level in a time-dependent manner (2.1 ± 0.6- and 2.8 ± 0.6-fold, after 20 seconds and 30 seconds, respectively; Fig. 5A ). 
Phenylephrine also increased NO in a concentration-dependent manner. Acini were incubated for 20 seconds with phenylephrine concentrations ranging from 10−7 to 10−3 M. Phenylephrine at 10−4 and 10−3 M significantly increased NO (1.9 ± 0.3- and 2.7 ± 0.6-fold, respectively) above basal level (Fig. 5B)
To determine whether the phenylephrine-induced increase in NO is mediated through the α1D-adrenergic receptor, acini were preincubated with the α1D-adrenergic receptor antagonist BMY-7378 (10−8 M) for 20 minutes before addition of phenylephrine (10−4 M) for 20 seconds. BMY-7378 completely inhibited the phenylephrine-induced increase in NO (Fig. 5C)
These data indicate that phenylephrine, acting through the α1D-adrenergic receptor, increases NO production in a time- and concentration-dependent manner. 
Effect of NO Donors and cGMP Analogues on Lacrimal Gland Protein Secretion
As a positive control, lacrimal gland acini were incubated with the NO donor S-nitroso-N-acetyl penicillamine (SNAP). SNAP (1.7 × 10−4 M) was added for 20 minutes, and peroxidase secretion was measured. SNAP significantly increased (1.4 ± 0.2-fold) above basal level (Fig. 6) . This result is compared to phenylephrine-stimulated protein secretion, which increased peroxidase secretion by 1.6 ± 0.2-fold above basal level. 
One possible consequence of an increase in the production of NO production is to stimulate the activity of guanylate cyclase to produce cGMP. To determine whether an increase in intracellular concentrations of cGMP had an effect on peroxidase secretion, acini were incubated with the cell-permeable analogue of cGMP, 8-bromo-cGMP (8-Br-CGMP), and peroxidase secretion was measured. 8-Br-cGMP (10−3 M) significantly increased peroxidase to 1.2 ± 0.1-fold above basal level (Fig. 6)
These data indicate that exogenous addition of NO or cGMP is sufficient to increase peroxidase secretion in lacrimal gland acini. 
Effect of Guanylate Cyclase Inhibitors on α1-Adrenergic-Agonist–Stimulated Protein Secretion
Since phenylephrine stimulation increases NO production through activation of NOS, to stimulate protein secretion, and addition of exogenous cGMP also increases protein secretion, we next investigated the effects of inhibition of guanylate cyclase, the enzyme that synthesizes cGMP from GTP, on phenylephrine-stimulated peroxidase secretion. Lacrimal gland acini were preincubated for 20 minutes with ODQ (10−10–10−7 M) before incubation with phenylephrine (10−4 M) for 20 minutes. Phenylephrine significantly increased peroxidase secretion (1.7 ± 0.1-fold) above basal level (Fig. 7) . ODQ significantly inhibited phenylephrine-induced peroxidase secretion (by 64.3% ± 23.5%, 43.7% ± 24.1%, and 90.7% ± 7.9% to 1.1% ± 0.2%, 1.3% ± 0.1%, and 1.0% ± 0.1%) at 10−9, 10−8, and 10−7 M, respectively (Fig. 7) . As a control, acini were also stimulated with Cch (10−4 M) for 20 minutes. Cch stimulated secretion 1.2 ± 0.1-fold above basal level (data not shown). Similar to l-NAME and d-NAME, ODQ (10−9 M) did not have any effect on Cch-stimulated protein secretion (data not shown). 
These data indicate that guanylate cyclase is activated by α1-adrenergic agonists to stimulate protein secretion. 
Effect of Phenylephrine on cGMP Concentration
Since phenylephrine generates NO and inhibition of guanylate cyclase leads to decreased peroxidase secretion, we next determined whether phenylephrine increases the cGMP concentration within the acinar cells. Acini were incubated with phenylephrine (10−4 M) for 0 to 30 minutes, and the amount of cGMP was determined by enzyme immunoassay, as described in the Methods section. Phenylephrine significantly increased the amount of cGMP (2.2-fold) above control (from 0.05 ± 0.02 to 0.11 ± 0.01 picomoles/mL cGMP) after 5 minutes (Fig. 8) . The amount of cGMP declined to 0.10 ± 0.04 picomoles/mL after 10 minutes and returned to basal level 15 minutes after addition of phenylephrine (Fig. 8)
These data indicate that phenylephrine increases cGMP concentration in lacrimal gland acini. 
Effect of Inhibition of NOS and Guanylate Cyclase on α1-Adrenergic-Agonist–Stimulated MAPK Activation
We have shown that phenylephrine transactivates the EGFR to activate p42/p44 MAPK. 10 Activation of p42/p44 MAPK negatively modulates peroxidase secretion. 10 To determine whether generation of NO and cGMP play a role in the transactivation of the EGFR, acini were incubated with either the NOS inhibitor l-NAME (10−6 M), its inactive isomer d-NAME (10−6 M), or the guanylate cyclase inhibitor ODQ (10−9 M) for 20 minutes before stimulation with phenylephrine (10−4 M) for 5 minutes. Western blot analysis was performed with antibodies against phosphorylated (active) and total p42/p44 MAPK. Phenylephrine significantly increased phosphorylation of p42/p44 MAPK above basal level (1.2 ± 0.1-fold; data not shown). The amount of phosphorylated p42/p44 MAPK stimulated with phenylephrine, in the presence of l-NAME was significantly reduced, to 19.5% ± 16.9% of the response obtained with phenylephrine alone (Fig. 9) . The inactive isomer d-NAME decreased phosphorylated p42/p44 MAPK, though the response was not statistically significant. Neither l-NAME nor d-NAME alone had an effect on basal p42/p44 MAPK activity (data not shown). 
Similar to l-NAME, the activation of p42/p44 MAPK in the presence of phenylephrine and ODQ was significantly reduced, to 14% ± 14% of the response to phenylephrine alone (Fig. 9) . ODQ alone did not have any effect on basal p42/p44 MAPK activity (data not shown). 
These data indicate that generation of NO and cGMP is necessary for phenylephrine-induced activation of p42/p44 MAPK. 
Discussion
In this study we showed that α1-adrenergic agonists, acting through the α1D-adrenergic receptors, stimulate NOS, probably eNOS, to generate NO, leading to production of cGMP. Activation of this pathway by α1-adrenergic agonists leads to increased protein secretion and activation of p42/p44 MAPK from freshly isolated rat lacrimal gland acini. 
We have identified the presence and localization of eNOS and confirmed the presence and location of nNOS in the lacrimal gland. Many studies have shown that eNOS is localized to caveolae, 22 23 33 and in vitro studies have shown that interactions between caveolin-1 and eNOS lead to an inhibition of eNOS activity. 34 Caveolae are microdomains of the plasma membrane in which numerous signaling molecules are targeted. 34 In endothelial cells, eNOS translocates from the caveolae to the cytoplasm on stimulation and is presumably activated. 34 Although activation of eNOS on stimulation has not been shown in epithelial cells, it is known that many agonists stimulate eNOS activation. In the lacrimal gland, similar to other tissues, eNOS colocalizes with caveolin-1 in unstimulated cells. Thus, it is possible that addition of α1-adrenergic agonists translocates eNOS from the caveolae to the cytoplasm. 
Studies have demonstrated the presence and localization of nNOS in the mouse lacrimal gland with nNOS immunoreactivity appearing in nerve fibers and surrounding a few acini. 14 In the present study, nNOS immunoreactivity was abundantly present surrounding numerous acini of the rat lacrimal gland. In addition, nNOS was present in nerves, as this immunoreactivity colocalized with MAP-2, a protein abundant in mammalian nerves. It is possible that either a species difference exists, such that the rat lacrimal gland contains more nNOS surrounding acini than the mouse. It is also possible that different antibodies used in each study account for the differences between the previous and present studies. 
We did not investigate the role of iNOS in protein secretion. iNOS is known as an inducible enzyme, and its expression is stimulated by inflammation and bacterial products. 5 It is unlikely that iNOS would be involved in α1-adrenergic-agonist–stimulated protein secretion, as this stimulation is a very fast process occurring in seconds, whereas induction of iNOS would be a longer-term process. Beauregard et al. 16 showed that maximum expression of iNOS occurred 4 hours after addition of interleukin-1β to cultured rabbit lacrimal gland acinar cells. 
In the lacrimal gland, l-NAME was the only NOS inhibitor to inhibit α1-adrenergic–induced protein secretion. The specificity of NOS inhibitors, including l-NAME, is controversial, as some studies have indicated that l-NAME is more selective for eNOS than nNOS or iNOS. Others have determined that l-NAME is more selective for the constitutive NOS isoforms (eNOS and nNOS) than the inducible form (iNOS). 35 In rat aorta, l-NAME reverses the vasodilative effects of acetylcholine, with an IC50 of 0.5 μM. 36 In the freshly prepared lacrimal gland acini, inhibition of NOS with l-NAME completely inhibited α1-adrenergic-agonist–stimulated protein secretion, whereas the inhibitors l-thiocitrulline and S-methyl-l-thiocitrulline, which have greater selectivity for nNOS than eNOS, 1 had no effect on this secretion. These data imply that eNOS is primarily responsible for the increased generation of NO by α1-adrenergic agonists that leads to protein secretion. eNOS has been shown to be activated by α2-adrenergic receptors to inhibit NaCl absorption in the thick ascending limb of the loop of Henle 37 and to increase mucus secretion in estradiol-stimulated cervical epithelial cells. 38  
The effects of the NOS inhibitor l-NAME and the guanylate cyclase inhibitor ODQ are specific to α1-adrenergic receptors, as neither inhibitor had any effect on cholinergic-agonist–stimulated secretion. Bacman et al. 39 showed that Cch increases activity of NOS and stimulates the production of cGMP in rat lacrimal gland. They did not link the increase in cGMP to a specific cellular function, however. In the present study, we measured the effects of NOS and guanylate cyclase inhibitors on a cellular function—namely, protein secretion. Thus, it is possible that cholinergic agonists increase NOS activity and stimulate the production of cGMP, but these molecules do not play a role in cholinergic-agonist–stimulated protein secretion. 
Generation of NO leads to increased protein secretion in the lacrimal gland, as demonstrated by Beauregard et al. 3 In that study, the investigators measured the effects of NO generators on total protein release in cultured rabbit lacrimal gland acinar cells. Measurement of total protein secretion can be problematic, as this method measures not only proteins secreted in a regulated manner such as peroxidase, but also constitutively released proteins and proteins that can be released by ectodomain shedding, as occurs with growth factors such as epidermal growth factor. Almost 4% of cell surface proteins can be released by ectodomain shedding. In the present study, we showed that NO production could occur through agonist-induced receptor activation, leading to regulated protein secretion in freshly isolated acinar cells. Receptor-linked generation of NO also occurs in other exocrine glands, such as the pancreas, where cholecystokinin and carbachol stimulate NO production and amylase release, and the submandibular glands, where Cch-stimulated amylase secretion is inhibited by ODQ. 2 40  
NO and cGMP production has also been linked to release of calcium from rat lacrimal gland. 17 18 Jorgensen et al. 18 reported that inhibitors of guanylate cyclase and cGMP-dependent protein kinase Ia inhibits phenylephrine induced-Ca2+ release. In addition, NO donors also stimulated release of Ca2+ and NO meditated the release of Ca2+ stimulated by β-adrenergic, but not α1-adrenergic, agonists. 17 41 Addition of cGMP analogues did not increase [Ca2+]i, nor did they have any effect on cyclic ADP-ribose–induced Ca2+ release, nor did α1-adrenergic agonists increase NO production. 17 In these studies, NO was measured with the fluorescent molecule DAF-2. However, it is not possible to use the ratio method with DAF-2 as it is used with fura2. 41 Thus, its fluorescence is dependent on the amount of dye entering the cells, the extent of ester hydrolysis within the cell altering the nonfluorescent DAF-2 DA to the fluorescent DAF-2, and the number of cells in each preparation. These factors change with each cell preparation, making this method unreliable. In the present study, α1-adrenergic agonists caused a significant increase in NO, as determined by reducing nitrate to nitrite and measuring the amount of nitrite using the Griess reaction, a well-established method. As studies have shown that α1-adrenergic-agonist–induced protein secretion is dependent on an increase in [Ca2+]i, 12 it is possible that the synthesis of cGMP by NOS leads to an activation of guanylate cyclase followed by an increase in [Ca2+]i which ultimately leads to protein secretion. We did not examine the role of NO in the release of Ca2+ in this study. 
In addition to protein secretion, NO/cGMP production was also linked to another lacrimal gland function—activation of p42/p44 MAPK. The NOS inhibitor l-NAME and the guanylate cyclase inhibitor ODQ both inhibited α1-adrenergic-agonist–induced activation of p42/p44 MAPK. 
In another study, we showed that PKCε, but not PKCα or -δ, plays a significant role in α1-adrenergic-agonist–stimulated protein secretion. 13 Inhibition of PKCε blocks phenylephrine-induced protein secretion by approximately 80%. As inhibition of eNOS also substantially inhibits phenylephrine-induced protein secretion, it is likely that there is an interaction between PKCε and eNOS. PKCε has been shown to bind to and phosphorylate both Akt and eNOS in murine heart tissue, leading to an increase in activation of both molecules. 42 Although we did not examine the interactions between PKCε and eNOS, it is possible that a similar interaction also occurs in the lacrimal gland. 
In summary, we have shown that α1-adrenergic agonists, acting through α1D-adrenergic receptors, stimulate NOS, and probably eNOS, to generate NO and lead to production of cGMP. Activation of this pathway by α1-adrenergic agonists leads to increased protein secretion and induction of p42/p44 MAPK from freshly isolated rat lacrimal gland acini. 
 
Figure 1.
 
Western blot analysis of eNOS and nNOS in rat lacrimal gland. Rat liver, cultured rat aorta cells, rat lacrimal gland acini, whole rat lacrimal gland, and rat brain were homogenized and the proteins separated by SDS-PAGE and transferred to nitrocellulose. Blots were probed with antibodies against eNOS and nNOS. Blots are representative of results in three independent experiments.
Figure 1.
 
Western blot analysis of eNOS and nNOS in rat lacrimal gland. Rat liver, cultured rat aorta cells, rat lacrimal gland acini, whole rat lacrimal gland, and rat brain were homogenized and the proteins separated by SDS-PAGE and transferred to nitrocellulose. Blots were probed with antibodies against eNOS and nNOS. Blots are representative of results in three independent experiments.
Figure 2.
 
Localization of eNOS in rat lacrimal gland. Rat lacrimal gland sections were double labeled with antibodies against (A) eNOS and (B) caveolin-1. (C, yellow) Colocalization of eNOS and caveolin-1. Sections were also double labeled with (D) eNOS and (E) α-smooth muscle actin to label myoepithelial cells. Overlay of (D) and (E) is shown in (F). (G) Negative control for Cy3; (H) negative control for FITC. Micrographs are representative of results in three independent experiments. Magnification, ×400.
Figure 2.
 
Localization of eNOS in rat lacrimal gland. Rat lacrimal gland sections were double labeled with antibodies against (A) eNOS and (B) caveolin-1. (C, yellow) Colocalization of eNOS and caveolin-1. Sections were also double labeled with (D) eNOS and (E) α-smooth muscle actin to label myoepithelial cells. Overlay of (D) and (E) is shown in (F). (G) Negative control for Cy3; (H) negative control for FITC. Micrographs are representative of results in three independent experiments. Magnification, ×400.
Figure 3.
 
Localization of nNOS in rat lacrimal gland. Rat lacrimal gland sections were double labeled with antibodies against (A) nNOS and (B) MAP-2. (C, yellow) Colocalization of nNOS and MAP-2. Micrographs are representative of results in three independent experiments. Magnification, ×400.
Figure 3.
 
Localization of nNOS in rat lacrimal gland. Rat lacrimal gland sections were double labeled with antibodies against (A) nNOS and (B) MAP-2. (C, yellow) Colocalization of nNOS and MAP-2. Micrographs are representative of results in three independent experiments. Magnification, ×400.
Figure 4.
 
Effect of inhibition of eNOS and nNOS on α1-adrenergic-agonist–stimulated protein secretion. (A) Lacrimal gland acini were preincubated with the eNOS inhibitor l-NAME or its inactive isomer d-NAME at the indicated concentration for 20 minutes before stimulation with the α1-adrenergic agonist phenylephrine (10−4 M) for 20 minutes. Peroxidase secretion was then measured. Data are the mean ± SEM of results in four to six independent experiments. (B) Acini were preincubated with the nNOS inhibitors l-thiocitrulline or methyl-l-thiocitrulline at the indicated concentration for 20 minutes before stimulation with the α1-adrenergic agonist phenylephrine (10−4 M) for 20 minutes. Peroxidase secretion was then measured. Data are the mean results from two independent experiments. *Statistically significant difference from basal level; #statistically significant difference from phenylephrine alone.
Figure 4.
 
Effect of inhibition of eNOS and nNOS on α1-adrenergic-agonist–stimulated protein secretion. (A) Lacrimal gland acini were preincubated with the eNOS inhibitor l-NAME or its inactive isomer d-NAME at the indicated concentration for 20 minutes before stimulation with the α1-adrenergic agonist phenylephrine (10−4 M) for 20 minutes. Peroxidase secretion was then measured. Data are the mean ± SEM of results in four to six independent experiments. (B) Acini were preincubated with the nNOS inhibitors l-thiocitrulline or methyl-l-thiocitrulline at the indicated concentration for 20 minutes before stimulation with the α1-adrenergic agonist phenylephrine (10−4 M) for 20 minutes. Peroxidase secretion was then measured. Data are the mean results from two independent experiments. *Statistically significant difference from basal level; #statistically significant difference from phenylephrine alone.
Figure 5.
 
Effect of α1-adrenergic-agonist–stimulated on NO production. Rat lacrimal gland acini were stimulated with phenylephrine (10−4 M) for various times (A) or at various concentrations of phenylephrine for 20 seconds (B), and the amount of NO was measured. (C) Acini were also preincubated with BMY-7378 (10−8 M) for 20 minutes before addition of phenylephrine (10−4 M) for 20 seconds. Data are the mean ± SEM of results in three to nine independent experiments. *Statistically significant difference from basal level; #statistically significant difference from phenylephrine alone.
Figure 5.
 
Effect of α1-adrenergic-agonist–stimulated on NO production. Rat lacrimal gland acini were stimulated with phenylephrine (10−4 M) for various times (A) or at various concentrations of phenylephrine for 20 seconds (B), and the amount of NO was measured. (C) Acini were also preincubated with BMY-7378 (10−8 M) for 20 minutes before addition of phenylephrine (10−4 M) for 20 seconds. Data are the mean ± SEM of results in three to nine independent experiments. *Statistically significant difference from basal level; #statistically significant difference from phenylephrine alone.
Figure 6.
 
Effect of NO donors and cGMP on lacrimal gland protein secretion. Rat lacrimal gland acini were incubated with either the NO donor SNAP (1.7 × 10−4 M), the α1-adrenergic agonist phenylephrine (10−4 M), or 8-Br-cGMP (10−3 M) for 20 minutes, and protein secretion was measured. Data are the mean ± SEM of results in six to nine independent experiments. *Statistically significant difference from basal level.
Figure 6.
 
Effect of NO donors and cGMP on lacrimal gland protein secretion. Rat lacrimal gland acini were incubated with either the NO donor SNAP (1.7 × 10−4 M), the α1-adrenergic agonist phenylephrine (10−4 M), or 8-Br-cGMP (10−3 M) for 20 minutes, and protein secretion was measured. Data are the mean ± SEM of results in six to nine independent experiments. *Statistically significant difference from basal level.
Figure 7.
 
Effect of inhibition of guanylate cyclase on α1-adrenergic-agonist–stimulated protein secretion. Lacrimal gland acini were preincubated with the guanylate cyclase inhibitor ODQ from 10−10 to 10−7 M for 20 minutes either alone or before addition of the α1-adrenergic agonist phenylephrine (10−4 M) for 20 minutes. Data are the mean ± SEM of results in three independent experiments. *Statistically significant difference from basal level; #statistically significant difference from phenylephrine alone.
Figure 7.
 
Effect of inhibition of guanylate cyclase on α1-adrenergic-agonist–stimulated protein secretion. Lacrimal gland acini were preincubated with the guanylate cyclase inhibitor ODQ from 10−10 to 10−7 M for 20 minutes either alone or before addition of the α1-adrenergic agonist phenylephrine (10−4 M) for 20 minutes. Data are the mean ± SEM of results in three independent experiments. *Statistically significant difference from basal level; #statistically significant difference from phenylephrine alone.
Figure 8.
 
Effect of α1-adrenergic agonists on cGMP concentration. Lacrimal gland acini were incubated with the α1-adrenergic agonist phenylephrine (10−4 M) for 0 to 30 minutes. cGMP concentration was then measured. Data are the mean ± SEM of results in three independent experiments. *Statistically significant difference from basal level.
Figure 8.
 
Effect of α1-adrenergic agonists on cGMP concentration. Lacrimal gland acini were incubated with the α1-adrenergic agonist phenylephrine (10−4 M) for 0 to 30 minutes. cGMP concentration was then measured. Data are the mean ± SEM of results in three independent experiments. *Statistically significant difference from basal level.
Figure 9.
 
Effect of inhibition of eNOS and guanylate cyclase on α1-adrenergic-agonist–stimulated p42/p44 MAPK. Lacrimal gland acini were incubated for 20 minutes with the NOS inhibitor l-NAME (10−6 M), the inactive isomer d-NAME (10−6 M), or the guanylate cyclase inhibitor ODQ (10−9 M) before stimulate with the α1-adrenergic agonist phenylephrine (10−4 M) for 5 minutes. Activated p42/p44 MAPK was determined by Western blot analysis, with an antibody directed against phosphorylated (activated) p42/p44 MAPK. Data are the mean ± SEM of results in three independent experiments. *Statistically significant difference from phenylephrine.
Figure 9.
 
Effect of inhibition of eNOS and guanylate cyclase on α1-adrenergic-agonist–stimulated p42/p44 MAPK. Lacrimal gland acini were incubated for 20 minutes with the NOS inhibitor l-NAME (10−6 M), the inactive isomer d-NAME (10−6 M), or the guanylate cyclase inhibitor ODQ (10−9 M) before stimulate with the α1-adrenergic agonist phenylephrine (10−4 M) for 5 minutes. Activated p42/p44 MAPK was determined by Western blot analysis, with an antibody directed against phosphorylated (activated) p42/p44 MAPK. Data are the mean ± SEM of results in three independent experiments. *Statistically significant difference from phenylephrine.
The authors thank Diane Darland for providing the cultured aorta cell homogenate and Driss Zoukhri for many helpful discussions and a reading of the manuscript. 
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Figure 1.
 
Western blot analysis of eNOS and nNOS in rat lacrimal gland. Rat liver, cultured rat aorta cells, rat lacrimal gland acini, whole rat lacrimal gland, and rat brain were homogenized and the proteins separated by SDS-PAGE and transferred to nitrocellulose. Blots were probed with antibodies against eNOS and nNOS. Blots are representative of results in three independent experiments.
Figure 1.
 
Western blot analysis of eNOS and nNOS in rat lacrimal gland. Rat liver, cultured rat aorta cells, rat lacrimal gland acini, whole rat lacrimal gland, and rat brain were homogenized and the proteins separated by SDS-PAGE and transferred to nitrocellulose. Blots were probed with antibodies against eNOS and nNOS. Blots are representative of results in three independent experiments.
Figure 2.
 
Localization of eNOS in rat lacrimal gland. Rat lacrimal gland sections were double labeled with antibodies against (A) eNOS and (B) caveolin-1. (C, yellow) Colocalization of eNOS and caveolin-1. Sections were also double labeled with (D) eNOS and (E) α-smooth muscle actin to label myoepithelial cells. Overlay of (D) and (E) is shown in (F). (G) Negative control for Cy3; (H) negative control for FITC. Micrographs are representative of results in three independent experiments. Magnification, ×400.
Figure 2.
 
Localization of eNOS in rat lacrimal gland. Rat lacrimal gland sections were double labeled with antibodies against (A) eNOS and (B) caveolin-1. (C, yellow) Colocalization of eNOS and caveolin-1. Sections were also double labeled with (D) eNOS and (E) α-smooth muscle actin to label myoepithelial cells. Overlay of (D) and (E) is shown in (F). (G) Negative control for Cy3; (H) negative control for FITC. Micrographs are representative of results in three independent experiments. Magnification, ×400.
Figure 3.
 
Localization of nNOS in rat lacrimal gland. Rat lacrimal gland sections were double labeled with antibodies against (A) nNOS and (B) MAP-2. (C, yellow) Colocalization of nNOS and MAP-2. Micrographs are representative of results in three independent experiments. Magnification, ×400.
Figure 3.
 
Localization of nNOS in rat lacrimal gland. Rat lacrimal gland sections were double labeled with antibodies against (A) nNOS and (B) MAP-2. (C, yellow) Colocalization of nNOS and MAP-2. Micrographs are representative of results in three independent experiments. Magnification, ×400.
Figure 4.
 
Effect of inhibition of eNOS and nNOS on α1-adrenergic-agonist–stimulated protein secretion. (A) Lacrimal gland acini were preincubated with the eNOS inhibitor l-NAME or its inactive isomer d-NAME at the indicated concentration for 20 minutes before stimulation with the α1-adrenergic agonist phenylephrine (10−4 M) for 20 minutes. Peroxidase secretion was then measured. Data are the mean ± SEM of results in four to six independent experiments. (B) Acini were preincubated with the nNOS inhibitors l-thiocitrulline or methyl-l-thiocitrulline at the indicated concentration for 20 minutes before stimulation with the α1-adrenergic agonist phenylephrine (10−4 M) for 20 minutes. Peroxidase secretion was then measured. Data are the mean results from two independent experiments. *Statistically significant difference from basal level; #statistically significant difference from phenylephrine alone.
Figure 4.
 
Effect of inhibition of eNOS and nNOS on α1-adrenergic-agonist–stimulated protein secretion. (A) Lacrimal gland acini were preincubated with the eNOS inhibitor l-NAME or its inactive isomer d-NAME at the indicated concentration for 20 minutes before stimulation with the α1-adrenergic agonist phenylephrine (10−4 M) for 20 minutes. Peroxidase secretion was then measured. Data are the mean ± SEM of results in four to six independent experiments. (B) Acini were preincubated with the nNOS inhibitors l-thiocitrulline or methyl-l-thiocitrulline at the indicated concentration for 20 minutes before stimulation with the α1-adrenergic agonist phenylephrine (10−4 M) for 20 minutes. Peroxidase secretion was then measured. Data are the mean results from two independent experiments. *Statistically significant difference from basal level; #statistically significant difference from phenylephrine alone.
Figure 5.
 
Effect of α1-adrenergic-agonist–stimulated on NO production. Rat lacrimal gland acini were stimulated with phenylephrine (10−4 M) for various times (A) or at various concentrations of phenylephrine for 20 seconds (B), and the amount of NO was measured. (C) Acini were also preincubated with BMY-7378 (10−8 M) for 20 minutes before addition of phenylephrine (10−4 M) for 20 seconds. Data are the mean ± SEM of results in three to nine independent experiments. *Statistically significant difference from basal level; #statistically significant difference from phenylephrine alone.
Figure 5.
 
Effect of α1-adrenergic-agonist–stimulated on NO production. Rat lacrimal gland acini were stimulated with phenylephrine (10−4 M) for various times (A) or at various concentrations of phenylephrine for 20 seconds (B), and the amount of NO was measured. (C) Acini were also preincubated with BMY-7378 (10−8 M) for 20 minutes before addition of phenylephrine (10−4 M) for 20 seconds. Data are the mean ± SEM of results in three to nine independent experiments. *Statistically significant difference from basal level; #statistically significant difference from phenylephrine alone.
Figure 6.
 
Effect of NO donors and cGMP on lacrimal gland protein secretion. Rat lacrimal gland acini were incubated with either the NO donor SNAP (1.7 × 10−4 M), the α1-adrenergic agonist phenylephrine (10−4 M), or 8-Br-cGMP (10−3 M) for 20 minutes, and protein secretion was measured. Data are the mean ± SEM of results in six to nine independent experiments. *Statistically significant difference from basal level.
Figure 6.
 
Effect of NO donors and cGMP on lacrimal gland protein secretion. Rat lacrimal gland acini were incubated with either the NO donor SNAP (1.7 × 10−4 M), the α1-adrenergic agonist phenylephrine (10−4 M), or 8-Br-cGMP (10−3 M) for 20 minutes, and protein secretion was measured. Data are the mean ± SEM of results in six to nine independent experiments. *Statistically significant difference from basal level.
Figure 7.
 
Effect of inhibition of guanylate cyclase on α1-adrenergic-agonist–stimulated protein secretion. Lacrimal gland acini were preincubated with the guanylate cyclase inhibitor ODQ from 10−10 to 10−7 M for 20 minutes either alone or before addition of the α1-adrenergic agonist phenylephrine (10−4 M) for 20 minutes. Data are the mean ± SEM of results in three independent experiments. *Statistically significant difference from basal level; #statistically significant difference from phenylephrine alone.
Figure 7.
 
Effect of inhibition of guanylate cyclase on α1-adrenergic-agonist–stimulated protein secretion. Lacrimal gland acini were preincubated with the guanylate cyclase inhibitor ODQ from 10−10 to 10−7 M for 20 minutes either alone or before addition of the α1-adrenergic agonist phenylephrine (10−4 M) for 20 minutes. Data are the mean ± SEM of results in three independent experiments. *Statistically significant difference from basal level; #statistically significant difference from phenylephrine alone.
Figure 8.
 
Effect of α1-adrenergic agonists on cGMP concentration. Lacrimal gland acini were incubated with the α1-adrenergic agonist phenylephrine (10−4 M) for 0 to 30 minutes. cGMP concentration was then measured. Data are the mean ± SEM of results in three independent experiments. *Statistically significant difference from basal level.
Figure 8.
 
Effect of α1-adrenergic agonists on cGMP concentration. Lacrimal gland acini were incubated with the α1-adrenergic agonist phenylephrine (10−4 M) for 0 to 30 minutes. cGMP concentration was then measured. Data are the mean ± SEM of results in three independent experiments. *Statistically significant difference from basal level.
Figure 9.
 
Effect of inhibition of eNOS and guanylate cyclase on α1-adrenergic-agonist–stimulated p42/p44 MAPK. Lacrimal gland acini were incubated for 20 minutes with the NOS inhibitor l-NAME (10−6 M), the inactive isomer d-NAME (10−6 M), or the guanylate cyclase inhibitor ODQ (10−9 M) before stimulate with the α1-adrenergic agonist phenylephrine (10−4 M) for 5 minutes. Activated p42/p44 MAPK was determined by Western blot analysis, with an antibody directed against phosphorylated (activated) p42/p44 MAPK. Data are the mean ± SEM of results in three independent experiments. *Statistically significant difference from phenylephrine.
Figure 9.
 
Effect of inhibition of eNOS and guanylate cyclase on α1-adrenergic-agonist–stimulated p42/p44 MAPK. Lacrimal gland acini were incubated for 20 minutes with the NOS inhibitor l-NAME (10−6 M), the inactive isomer d-NAME (10−6 M), or the guanylate cyclase inhibitor ODQ (10−9 M) before stimulate with the α1-adrenergic agonist phenylephrine (10−4 M) for 5 minutes. Activated p42/p44 MAPK was determined by Western blot analysis, with an antibody directed against phosphorylated (activated) p42/p44 MAPK. Data are the mean ± SEM of results in three independent experiments. *Statistically significant difference from phenylephrine.
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