April 2007
Volume 48, Issue 4
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Cornea  |   April 2007
The α1- and β1-Adrenergic Modulation of Lacrimal Gland Function in the Mouse
Author Affiliations
  • Chuanqing Ding
    From the Department of Cell and Neurobiology, University of Southern California, Los Angeles, California; the
  • Benjamin Walcott
    Department of Neurobiology and Behavior, State University of New York at Stony Brook, Stony Brook, New York; the
    Vision Science Centre RSBS, Australian National University, Canberra, ACT, Australia; and the
  • Kent T. Keyser
    Vision Science Research Center, University of Alabama at Birmingham, Birmingham, Alabama.
Investigative Ophthalmology & Visual Science April 2007, Vol.48, 1504-1510. doi:https://doi.org/10.1167/iovs.05-1634
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      Chuanqing Ding, Benjamin Walcott, Kent T. Keyser; The α1- and β1-Adrenergic Modulation of Lacrimal Gland Function in the Mouse. Invest. Ophthalmol. Vis. Sci. 2007;48(4):1504-1510. https://doi.org/10.1167/iovs.05-1634.

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

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Abstract

purpose. To determine the expression patterns of α1- and β1-adrenergic receptors in the mouse exorbital lacrimal gland (LG). An α- and β-receptor agonist and antagonist were used to elucidate the receptors’ relevance to protein secretion.

methods. Mouse LGs were processed for single- and double-labeled indirect immunofluorescence studies and examined with confocal scanning microscopy. Protein secretion was measured from gland fragments in response to adrenergic agonists.

results. Extensive α1-immunoreactivity (IR) was found on the surface and cytoplasm of acinar cells and much more α1-IR in the interstitial areas. In contrast, more β1-IR was found in the LG, and most β1-IR appeared to concentrate in the cytoplasm of acinar cells, with almost no β1-IR in the interstitial areas. The protein secretion in response to phenylephrine and isoproterenol showed that direct stimulation of either the α1- or β1-receptor could induce significant protein secretion from LGs. The specificity of this stimulation was further indicated by the effects of adrenergic antagonists. No synergism was observed between α1- and β-receptor-mediated protein secretions.

conclusions. The results support the notion that there is extensive adrenergic control in the mouse LG. The adrenergic receptors may be a better choice of markers, compared with tyrosine hydroxylase and dopamine β-hydroxylase, to reflect the extent of adrenergic control because circulating norepinephrine in the bloodstream should be taken into consideration. Both confocal microscopy observations and protein secretion data suggest the presence of α1- and β1-mediated pathways in the mouse LG.

The adrenergic system in the mammalian lacrimal gland (LG) has been assumed to play an indirect and minor role in lacrimal function, mostly by regulating the blood flow and distribution to the gland. 1 2 However, increasing evidence indicates that the adrenergic system may also play a direct and significant role in regulating tear secretion. 3 4  
Electrophysiological recordings from mouse lacrimal acinar cells have shown that isoproterenol (a β-adrenergic agonist) markedly enhances ATP-induced currents, which are blocked by propranolol (a β-adrenergic antagonist). 5 Iontophoretic application of epinephrine to mouse lacrimal acinar cells increased potassium permeability which was similar to the change induced by acetylcholine. 6 Similar findings have been obtained from the rat. 7 In the rabbit, adrenergic stimulation enhances lacrimal flux. 1 2 8 These data suggest that the adrenergic stimulation may have a direct and significant influence on lacrimal acinar cells. 
Most investigations have used markers such as tyrosine hydroxylase (TH) or dopamine β-hydroxylase to visualize the adrenergic innervation in LGs. 9 10 11 However, adrenergic fibers in the LG proper are not the only source of catecholamines, which can be released by other tissues or organs into the bloodstream and serve as systemic neurotransmitters. 
For those acini that are in close association with adrenergic nerves, catecholamines released from nerve terminals may influence secretory cells directly. However, as catecholamines are inactivated quickly on release and only a few acini are directly innervated by adrenergic nerves, 12 13 it seems unlikely that the catecholamines would diffuse across long distances to the acinar cells and still reach the threshold concentration to stimulate secretion. 
It is our working hypothesis that adrenergic receptors within the lacrimal gland may be better indicators of the extent of adrenergic control and of the important role that the adrenergic system plays in lacrimal protein secretion. Although there were many pharmacological studies that support the existence of an adrenergic system and its role in lacrimal secretion, 3 4 7 14 we were unable to find any literature with direct immunocytochemical observations of adrenergic receptors in the LG of any species. Also, most of the previous studies regarding adrenergic regulation of lacrimal secretion were performed in the rat. 4 7 14 Because there are significant differences between species, and due to the increasing popularity of using mouse models for Sjögren’s syndrome and dry eye studies, it is critical to understand the adrenergic regulation in the LG of normal mice. Therefore, in the present study, we used antibodies against α1- and β1-adrenergic receptors to determine the distribution of these receptors in the mouse LG and to determine the level of protein secretion induced by various adrenergic agonists. 
Materials and Methods
Chemicals
Carbamylcholine chloride (carbachol), isoproterenol bitartrate, norepinephrine bitartrate, l-phenylephrine hydrochloride, phentolamine hydrochloride, and dl-propranolol hydrochloride, were from Sigma-Aldrich (St. Louis, MO). All reagents were of the highest purity available. 
Animals
C57 female mice (∼18 g body weight), aged from 2 to 12 months, were purchased from commercial vendors (Taconic Farms, Germantown, NY, or Charles River, Wilmington, MA). All animals were kept in a 12-hour light-dark cycle and maintained in an accredited animal facility with freely available food and water. They were managed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
Immunohistochemistry
Animals were killed with an overdose of halothane around midday, and the LGs were removed and fixed in freshly prepared 4% buffered paraformaldehyde. After 3 to 4 hours of fixation at 4°C, the tissue was placed in 0.1 M phosphate buffer containing 30% sucrose at pH 7.4 for at least 12 hours at 4°C. The glands were then placed in “optimal cutting temperature” (OCT) embedding medium (Sakura Finetek USA, Torrance, CA), serially sectioned at 10 μm on a cryostat (Leica, Deerfield, IL), and collected on slides and stored at −20°C until used. 
The antibodies used were rabbit anti-α1-receptor polyclonal antibody (Oncogene, San Diego, CA), at a dilution of 1:100; rabbit anti-β1-receptor polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA), dilution 1:1000; and sheep anti-TH polyclonal antibody (Chemicon, Temecula, CA), dilution 1:800. 
Sections were incubated in primary antibody diluted with 0.1 M sodium phosphate buffer (PBS) for 48 hours at 4°C. In control sections, primary antibodies were omitted. The secondary antibodies used were fluorescein isothiocyanate (FITC)-conjugated donkey anti-rabbit IgG, at a dilution of 1:100, and rhodamine red-X-conjugated donkey anti-sheep IgG (Jackson ImmunoResearch, West Grove, PA), dilution 1:200. The secondary antibodies were applied for an hour at room temperature. The slides were then washed with three changes of PBS, followed by one change of 4 mM sodium carbonate (pH 10.0), and coverslipped. The slides were examined with a laser-scanning microscope (TCS SP; Leica) and the images analyzed on a computer (PhotoShop; Adobe Systems, Mountain View, CA). At least 20 sections were examined per gland to examine the whole gland. 
Protein Secretion
LGs were weighed before being cut into fragments of 1 to 2 mm with a scalpel blade. The fragments were washed in 5 mL of saline solution (116 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 0.81 mM MgCl2, 1.01 mM NaH2PO4, 26.2 mM NaHCO3, and 5.6 mM dextrose [pH 7.4]) that was maintained at 37°C and was vigorously bubbled with 95% O2 and 5% CO2 in a beaker for 10 minutes. The solution was changed three times and discarded. The gland fragments were then incubated in 1 mL saline for 10 minutes, the saline was then removed and replaced with fresh medium. This process was repeated three times, and the saline was collected after each of the 10-minute incubations. The proteins in these samples represent the basal secretion from the glands. In the last exchange, the medium that was added contained one of the drugs at a specific concentration. After another 10-minute incubation, the medium was removed and saved, and the proteins in these solutions, representing the stimulated secretion in response to various drugs, were recorded. One gland was used in each experiment. 
Although isoproterenol is subject to inactivation by oxidation in oxygenated medium, our relatively short incubation period (10 minutes) did not cause a significant change in concentration. 3 Also, though isoproterenol becomes brownish pink on oxidation, the color did not become visually perceptible until the concentration exceeded 10−4 M. However, to ensure that the discoloration did not decrease accuracy, control media without tissue fragments with corresponding isoproterenol concentrations were included in each experiment, and corrections were made at various concentrations. 
Theoretical additivity was the sum of the experimental responses obtained in the presence of each agonist alone with the basal value subtracted. A response was defined as additive if the theoretical additivity did not differ significantly from the experimental additivity. This was interpreted to mean that the effects of the two agonists are modulated by separate pathways, but that the pathways interacted. A response was not considered additive if the experimental additivity was significantly lower than the theoretical additivity, which was interpreted as the two agonists activating the same pathway. 15  
Protein Assay
The samples were analyzed for their total protein by using a Coomassie protein assay kit (Pierce, Rockford, IL). Bovine serum albumin (BSA) was used as a standard protein, and standards were run with each assay. Protein concentrations were determined from the standard curves measured with each assay. The assays were performed on a microplate reader (EL 808; Bio-Tek Instruments, Winooski, VT), read at 595 nm. Both samples and standards were read in duplicate in 96-well flat-bottomed microplates (Costar; Corning Inc., Corning, NY). Total protein concentration was determined using the software provided by the manufacturer (KC4; Bio-Tek). Proteins secreted in response to various agonists (stimulated secretion) were the difference between total and basal secretions. The readings were then converted to micrograms per milliliter per gram tissue per minute (μg/[mL · g] per minute). 
Statistical Analysis
Data were expressed as the mean ± SEM. Student’s t-tests were performed with commercial software (SigmaPlot; SPSS Inc., Chicago, IL). P < 0.05 was considered to be significant. 
Results
Immunohistochemistry Observations
The mouse LG had extensive α1-immunoreactivity (α1-IR) and β1-IR. Some α1-IR was on the surface of acinar cells, whereas much of the α1-IR appeared to be inside the acinar cells (Figs. 1B 1C) . In addition to the α1-receptors that were associated with acinar cells, much more α1-IR was found in the interstitial areas, in association with blood vessels and ducts (Fig. 1B) . α1-IR on the acinar cells’ surface appeared as small punctate labeling, whereas that in the interstitial areas appeared as larger punctate labeling (Fig. 1B) . Negative control sections showed no such punctate staining (Figs. 1A 1D) . Double-labeling results showed that although some of the α1-IR appeared colocalized or in close association with TH-immunoreactive nerve fibers, most was not (Figs. 2A 2B)
Compared with the extent of α1-IR, there was more β1-IR in the LG. Some β1-IR was evident on the basolateral surfaces, whereas most appeared to concentrate in the central areas of the acinar cells (Figs. 1E 1F) . Unlike the distribution of α1-IR, almost no β1-IR could be observed in the interstitial areas (Figs. 1E 1F) . Double-labeling results indicated that none of the β1-IR colocalized with TH-immunoreactive nerves (Figs. 2C 2D)
Dose-Response Curves for Autonomic Agonists
The dose dependency of protein secretion in response to carbachol, norepinephrine, and phenylephrine stimulation was similar (Fig. 3) . Protein secretion rates rose with increasing agonist concentration and reached their maxima at 10−5 M. Moreover, the secretory responses to carbachol in the concentration range of 10−8 to 10−6 M and norepinephrine in the range of 10−7 to 10−5 M were almost linear. As the concentration increased further, the secretion rates reached a plateau or decreased slightly. Although carbachol, norepinephrine, and phenylephrine showed similar patterns and exhibited the same maximal responses, the threshold concentration of carbachol that elicited protein secretion was approximately 10 times lower than that of norepinephrine or phenylephrine. The half-maximum concentrations (EC50), the concentration at which half of the maximum response was elicited, were: 3 × 10−7 M for carbachol, 5 × 10−7 M for phenylephrine, and 10−6 M for norepinephrine. 
The protein secretion rate increased almost linearly with increasing isoproterenol concentration (Fig. 3) . At the highest dose used (10−4 M), it appeared that isoproterenol still did not achieve its maximum effect, which may be higher than that for carbachol, norepinephrine, and phenylephrine. However, by using the secretion rate in response to isoproterenol at 10−4 M as the maximum response, the EC50 of isoproterenol was estimated to be 3 × 10−5 M. 
The stimulated protein secretion rate induced by each agonist was significantly greater (P < 0.05) than its corresponding basal rate at all concentrations. However, at 10−5 M (the concentration of carbachol, norepinephrine, and phenylephrine that induced maximum secretion), no significant differences (P > 0.05) existed among the regulated secretion rates. 
Protein Secretion in Response to Agonists with the Presence of Antagonists
To explore further the specificity of the adrenergic agonists, we exposed the gland fragments to agonists at their EC50 in the presence of specific antagonists. 
Phentolamine, a general α-receptor antagonist, inhibited phenylephrine-induced secretion in a concentration-dependent manner. At the lowest concentration used, 10−8 M, phentolamine caused 50% inhibition. As the concentration of phentolamine increased, secretory inhibition also increased. A maximum inhibition of approximately 80% occurred at 10−5 M. As the phentolamine concentration further increased to 10−4 M, the inhibitory effect was slightly lower than that at 10−5 M (Fig. 4)
Propranolol, a general β-receptor antagonist, also inhibited protein secretion induced by isoproterenol in a concentration-dependent manner. Generally, the inhibitory effect was proportional to the concentration of propranolol, and maximum inhibition of 52% occurred at 10−5 M. As with phentolamine, the inhibitory effect of propranolol at 10−4 M was slightly lower than that at 10−5 M (Fig. 5)
Since norepinephrine can stimulate both α- and β-receptors and is naturally present in the body, we also used antagonists to explore the relative contribution of the α- and β-receptor-mediated pathways. Norepinephrine was used at 10−6 M, the concentration that induced maximum protein secretion. Antagonists were used at 10−5 M, the concentration that induced the maximum inhibitory response to the corresponding agonists. Norepinephrine and antagonists were added simultaneously. The addition of phentolamine decreased norepinephrine-induced protein secretion by approximately 67%, whereas propranolol caused a 47% decrease (Fig. 6) . Neither phentolamine nor propranolol alone caused a change in the protein secretion rate. 
Protein Secretion to α- and β-Receptors Agonists Added Together
Phenylephrine and isoproterenol were used at their EC50. Protein secretions in response to these two agonists applied simultaneously was slightly larger than that induced by either agonist alone. However, the response was much lower than the theoretical addition of the secretion elicited by each agonist alone, and the difference was significant (Fig. 7)
Discussion
Our confocal microscopy observations showed that there was extensive α1-IR and β1-IR in the mouse LG, suggesting the presence of both α1- and β1-mediated pathways. Although both α1-IR and β1-IR were in close association with acinar cells, they exhibited different distribution patterns in the interstitial areas. Extensive α1-IR was in the interstitial areas, in association with blood vessels and ducts, and less was in contact with acinar cells. This suggests that the α1 pathway, in addition to regulating protein secretion from acinar and ductal cells, also plays a role in modulating blood flow within the gland. Vasoconstriction in the LG is mediated by α-receptors, 16 and in cat and rabbit, vascular tone in the LG correlates with tear flow. 2 17 Other studies demonstrated that blood flow in the submandibular gland influences secretory function. 18 19 In contrast, our study showed that most of the β1-IR is in association with acinar cells rather than with blood vessels and ducts, suggesting that the β1 pathway may be involved only in the secretory process at the acinar cell level. 
Even in the acinar cells, α1-IR and β1-IR appeared to have different distribution patterns. α1-IR was relatively evenly distributed on the surfaces and inside the cytoplasm, whereas much of the β1-IR appeared to concentrate in the central areas of cytoplasm, with only some labeling on the membrane. 
Lacrimal acinar cells perform constitutive vesicle-mediated transport between their basolateral plasma membranes and an endomembrane system that includes recycling, transcytotic, and degradative compartments. 20 There is ongoing recycling of β-receptors between a large intracellular pool (recycling endosome) and a small surface-expressed pool, 21 and their residence time at the basal-lateral membranes is quite brief, 22 consistent with the distribution pattern of β1-receptors that we observed. The unique expression pattern of β1-receptors may explain the gland’s low relative responsiveness to the agonist at low concentrations and robust absolute responsiveness at higher concentrations (i.e., more β1-receptors are transported to the membrane when the cells were stimulated with agonist at higher concentration, whereas only a small number of receptors were on the membrane when the agonist concentration was low). The LG’s robust response to phenylephrine at low concentration may be explained by the abundance of α1-receptors on the membranes, although acinar cells also have a large intracellular pool of α1-receptors. 
The internalized β-receptors in the early endosome may be sorted to the lysosome for degradation or to the recycling endosome for return to the plasma membrane, 23 24 through the highly coordinated intracellular trafficking that is regulated by many factors at distinct organelles. 25 26 27 This possibility suggests that enhanced lacrimal protein secretion, as a therapeutic approach for lacrimal dysfunction, may be achieved by facilitating the recycling of internalized β-receptors through modifying components of the vesicular transport machinery. 
Protein secretion studies using α1- and β1-adrenergic agonists suggested that activation of either of these pathways could stimulate protein secretion. Our dose-response curves of carbachol, norepinephrine, and phenylephrine were very similar to those in rat LG, which measured either newly synthesized protein 28 or peroxidase secretion. 29 30 31 Between 10−6 and 10−4 M, norepinephrine and phenylephrine induced similar protein secretion to that of carbachol, suggesting that adrenergic stimulation was as effective as the cholinergic activation. Although carbachol, norepinephrine and phenylephrine all induced similar amounts of protein secretion between 10−6 and 10−4 M, carbachol was more effective at lower concentrations (<10−6 M). In fact, the threshold of carbachol-induced protein secretion was approximately 10 times lower than that for norepinephrine and phenylephrine, and at least 100 times lower than that for isoproterenol. By comparing each EC50, the order of potency of these agonists can be estimated: carbachol (EC50 = 3 × 10−7 M) > phenylephrine (EC50 = 5 × 10−7 M) > norepinephrine (EC50 = 10−6 M) > isoproterenol (EC50 = 3 × 10−5 M). 
The dose-response curve for isoproterenol indicates that although isoproterenol failed to induce protein secretion at concentrations <10−5 M, it significantly increased protein secretion at concentrations >10−5 M. The isoproterenol-induced protein secretion increased almost linearly with the increase of agonist concentration. It appeared that isoproterenol had not yet achieved its maximum effect at 10−4 M, which may be higher than that of norepinephrine, phenylephrine, or carbachol. However, the EC50 of isoproterenol was approximately 100 times that of carbachol and 60 times that of phenylephrine. These results are in contrast to those from rabbit LG 3 and rat salivary gland. 32 In rabbit LG, the threshold for isoproterenol-induced protein secretion (3 × 10−8 M) was 100 times lower than that for carbachol (3 × 10−6 M). 3 In rat isolated submandibular cells, isoproterenol induced IgA secretion at a lower threshold and showed a larger increase of secretion at higher concentrations compared with that elicited with carbachol. 32  
Phentolamine inhibited phenylephrine-induced protein secretion in a dose-dependent manner. Although the inhibitory effect of phentolamine appeared incomplete (maximum inhibition was 80% at 10−5 M phentolamine), protein secretion was decreased by 50% at 10−8 M, the lowest concentration used. In rat lacrimal acini, maximum inhibition by phentolamine on phenylephrine (10−4 M)-induced peroxidase secretion was 90%, which was considered to be complete inhibition, whereas the half inhibition was achieved at 10−6 M. 33 34 Our data suggest an α1-mediated protein secretion pathway in the mouse LG, and the immunofluorescence observations suggest that this pathway may be of the α1 type. 
Propranolol also inhibited isoproterenol-induced protein secretion in a dose-dependent manner, although the inhibition was only partial. Maximum inhibition of 52% was achieved at 10−5 M and with 20% inhibition at 10−8 M. In the rat, propranolol achieves complete inhibition of isoproterenol-induced protein secretion. 35 Our data demonstrated the presence of the β-mediated protein secretory pathway in the mouse LG, and the observation of β1-IR in the gland suggests that the β pathway may be of the β1 type. 
Both immunofluorescence and protein secretion findings suggest that there is an α1-adrenergic pathway in the mouse LG. Confocal microscopy demonstrated that extensive α1-IR was present on the surface and inside the acinar cells and that α1-IR was found in the interstitial areas. Phenylephrine, the specific α1 agonist, induced a significant amount of protein secretion from the gland fragments. The inhibitory effects of phentolamine on phenylephrine-induced protein secretion provide further evidence of the presence of an α-mediated protein secretory pathway. The findings are in agreement with a previous report that α1-receptors are present in the mouse LG. 36  
In rat LG, α-receptors were shown to be located on the acinar cells. Activation of these cells by phenylephrine increases potassium permeability and release, and this effect was almost completely blocked by phentolamine. 4 Phenylephrine has been reported to stimulate peroxidase secretion from rat lacrimocytes and this effect was suppressed by phentolamine, even though radioligand-binding assays have indicated that the gland did not possess a substantial number of α1- and α2-receptors. 33  
Isoproterenol-induced protein secretion suggests the presence of a β-mediated secretory pathway in the mouse LG, a conclusion that is further supported by the inhibitory effect of propranolol on the isoproterenol-induced response. It is known that isoproterenol markedly enhances ATP-induced inward and outward currents, which are blocked by propranolol. 5 These findings are in agreement with our results. However, other studies in mouse LG have indicated that the mouse LG contains only the α1-receptor, 36 and intracellular recordings from acinar cells failed to detect any isoproterenol-induced membrane potential and resistance changes. 6 Data supporting the β-receptor-mediated modulation of LG function has been reported in the rat: isoproterenol stimulated peroxidase secretion 35 37 38 and increased potassium permeability. 4 However, other reports claimed that isoproterenol had no effect on inducing radiolabeled protein 39 and peroxidase 14 secretions. In rabbit, in vitro studies showed the existence of β-receptors and their association with protein secretion. 3  
Taken together, the results of the present study showed three lines of evidence in support of the involvement of the β-adrenergic system in the regulation of protein secretion in the mouse LG: (1) Isoproterenol stimulated protein secretion from the gland fragments; (2) the isoproterenol-induced secretion was specifically inhibited by propranolol, the nonselective β antagonist; and (3) β1-IR was observed in close association with acinar cells and interstitial areas. The microscopy findings further suggested that the β-pathway in the mouse LG was of the β1 type. 
From the dose-response curves, phenylephrine, at concentrations of 10−6 to 10−4 M, appeared to act similarly to norepinephrine in eliciting protein secretion. Phenylephrine specifically activates an α1 pathway, to stimulate lacrimal protein secretion. 3 At 10−5 M, our data showed that protein secretion induced by phenylephrine was approximately 90% of that induced by norepinephrine and 97% of that at 10−4 M. At 10−6 M, phenylephrine-induced secretion was 1.6 times of that of norepinephrine, which was a significant difference. The adrenergic neurotransmitter norepinephrine can activate both α- and β-receptors. Our data indicate that phentolamine decreased norepinephrine-induced protein secretion by 67%, and propranolol achieved 47% inhibition. These data suggest that most of the adrenergic activation in the mouse LG is mediated by α-receptors and that β activation plays a less important role. Our data are in agreement with those obtained in the rat, 37 but are contrary to the data from rabbit LG, in which phentolamine reduced norepinephrine-evoked protein secretion by 47%, whereas propranolol achieved 71% inhibition. 3  
When the LG fragments were exposed to both phenylephrine and isoproterenol, the protein secretion rate was higher than that evoked by either agonist. However, the rate was significantly lower (P < 0.05) than the theoretical addition of the rates resulting from exposure to each agonist separately. Based on the definition of synergism, 15 37 it appears that there is no synergistic effect between the α1 and β activation in the mouse LG. Agonist binding to the α1-receptors partially activates the Ca2+/diacylglycerol (DAG)/cGMP-dependent pathway, whereas binding to β-receptors activates the cAMP-dependent pathway. 12 31 40 A report of experiments in the rat described synergism between α1 and β stimulation 37 ; and, in the rabbit, synergism between the cholinergic and β-adrenergic systems has also been reported. 3  
Adrenergic innervation in the mouse LG, as reflected by markers such as TH, is sparse and far less extensive than is parasympathetic innervation. However, the fact that there is a high number of adrenergic receptors in the lacrimal gland and that systemic catecholamines circulating in the blood stream can enter the gland suggests that the adrenergic pathways is an important regulatory pathway. 41 42  
 
Figure 1.
 
Confocal images of α1- and β1-IR in the mouse LG. (A) Control for α1-receptor. (B) There was extensive α1-IR in the gland and it appeared that more α1-IR was associated with interstitial tissue, blood vessel, and ducts. This IR appeared as larger punctate labeling compared with that in contact with the acinar cells. (C) At higher magnification, α1-IR was observed on the surface and inside the acinar cells. (D) Control for the β1-receptor. (E) The lacrimal gland had extensive β1-IR. Little was found on the acinar cell surfaces, and most was inside the cells. Unlike the distribution of α1-IR, there was little β1-IR in the interstitium. (F) β1-IR at higher magnification. Note that β1-IR appeared to concentrate in the central areas within the acinar cells’ cytoplasm. Scale bars: (A, B, D, E) 25 μm; (C, F) 40 μm.
Figure 1.
 
Confocal images of α1- and β1-IR in the mouse LG. (A) Control for α1-receptor. (B) There was extensive α1-IR in the gland and it appeared that more α1-IR was associated with interstitial tissue, blood vessel, and ducts. This IR appeared as larger punctate labeling compared with that in contact with the acinar cells. (C) At higher magnification, α1-IR was observed on the surface and inside the acinar cells. (D) Control for the β1-receptor. (E) The lacrimal gland had extensive β1-IR. Little was found on the acinar cell surfaces, and most was inside the cells. Unlike the distribution of α1-IR, there was little β1-IR in the interstitium. (F) β1-IR at higher magnification. Note that β1-IR appeared to concentrate in the central areas within the acinar cells’ cytoplasm. Scale bars: (A, B, D, E) 25 μm; (C, F) 40 μm.
Figure 2.
 
Confocal images of double labeling of α1- and β1-receptor-IR and TH-IR in the mouse LG. (A) α1-IR (visualized with FITC) in the gland as punctate labeling. (B) A few TH-IR nerves (visualized with rhodamine red) were observed in the gland. Some of these nerves colocalized (arrow) or were in close association (arrowhead) with α1-IR. (C) Extensive β1-IR was found in the gland, and it appeared that none was in close contact or colocalized with TH-IR. A blood vessel (arrow) and a nerve (arrowhead) were stained with TH antibody in (D). Scale bar, 40 μm.
Figure 2.
 
Confocal images of double labeling of α1- and β1-receptor-IR and TH-IR in the mouse LG. (A) α1-IR (visualized with FITC) in the gland as punctate labeling. (B) A few TH-IR nerves (visualized with rhodamine red) were observed in the gland. Some of these nerves colocalized (arrow) or were in close association (arrowhead) with α1-IR. (C) Extensive β1-IR was found in the gland, and it appeared that none was in close contact or colocalized with TH-IR. A blood vessel (arrow) and a nerve (arrowhead) were stained with TH antibody in (D). Scale bar, 40 μm.
Figure 3.
 
Dose-response curves of protein secretion from mouse LG fragments in response to autonomic agonists. Each point represents the mean ± SEM of results in four to six experiments. *Significant difference from the corresponding basal secretion rate of each agonist.
Figure 3.
 
Dose-response curves of protein secretion from mouse LG fragments in response to autonomic agonists. Each point represents the mean ± SEM of results in four to six experiments. *Significant difference from the corresponding basal secretion rate of each agonist.
Figure 4.
 
The inhibitory effect of phentolamine (PL) on protein secretion induced by phenylephrine at EC50 (5 × 10−7 M). The ordinate is the percentage of inhibitory effect. Each point represents the mean ± SEM of results in four to six experiments.
Figure 4.
 
The inhibitory effect of phentolamine (PL) on protein secretion induced by phenylephrine at EC50 (5 × 10−7 M). The ordinate is the percentage of inhibitory effect. Each point represents the mean ± SEM of results in four to six experiments.
Figure 5.
 
Inhibitory effect of propranolol (PP) on protein secretory responses induced by isoproterenol at EC50 (3 × 10−5 M). The ordinate is the percentage of inhibitory effect. Each point represents the mean ± SEM of results in four to six experiments.
Figure 5.
 
Inhibitory effect of propranolol (PP) on protein secretory responses induced by isoproterenol at EC50 (3 × 10−5 M). The ordinate is the percentage of inhibitory effect. Each point represents the mean ± SEM of results in four to six experiments.
Figure 6.
 
Inhibitory effects of phentolamine (PL) and propranolol (PP) on norepinephrine (NE)-induced protein secretion. The protein secretory rate induced by NE was normalized to 100 and the ordinate is the percentage of secretion rate with the presence of antagonists compared with it: NE (10−6 M); PL and PP (10−5 M). NE and the antagonist were added simultaneously. Each point represents the mean ± SEM of results in four to six experiments.
Figure 6.
 
Inhibitory effects of phentolamine (PL) and propranolol (PP) on norepinephrine (NE)-induced protein secretion. The protein secretory rate induced by NE was normalized to 100 and the ordinate is the percentage of secretion rate with the presence of antagonists compared with it: NE (10−6 M); PL and PP (10−5 M). NE and the antagonist were added simultaneously. Each point represents the mean ± SEM of results in four to six experiments.
Figure 7.
 
Protein secretion in response to the simultaneous addition of phenylephrine (PE) and isoproterenol (Isop) is significantly lower (P < 0.05) than the theoretical addition of the secretion elicited by PE and Isop alone, indicating no synergism between these two agonists. Agonists were used at their half maximum concentrations (EC50): phenylephrine (5 × 10−7 M), isoproterenol (3 × 10−5 M). Ordinate is the secretion rate in micrograms per milliliter per gram tissue per minute. Each point represents the mean ± SEM of results in four to six experiments.
Figure 7.
 
Protein secretion in response to the simultaneous addition of phenylephrine (PE) and isoproterenol (Isop) is significantly lower (P < 0.05) than the theoretical addition of the secretion elicited by PE and Isop alone, indicating no synergism between these two agonists. Agonists were used at their half maximum concentrations (EC50): phenylephrine (5 × 10−7 M), isoproterenol (3 × 10−5 M). Ordinate is the secretion rate in micrograms per milliliter per gram tissue per minute. Each point represents the mean ± SEM of results in four to six experiments.
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Figure 1.
 
Confocal images of α1- and β1-IR in the mouse LG. (A) Control for α1-receptor. (B) There was extensive α1-IR in the gland and it appeared that more α1-IR was associated with interstitial tissue, blood vessel, and ducts. This IR appeared as larger punctate labeling compared with that in contact with the acinar cells. (C) At higher magnification, α1-IR was observed on the surface and inside the acinar cells. (D) Control for the β1-receptor. (E) The lacrimal gland had extensive β1-IR. Little was found on the acinar cell surfaces, and most was inside the cells. Unlike the distribution of α1-IR, there was little β1-IR in the interstitium. (F) β1-IR at higher magnification. Note that β1-IR appeared to concentrate in the central areas within the acinar cells’ cytoplasm. Scale bars: (A, B, D, E) 25 μm; (C, F) 40 μm.
Figure 1.
 
Confocal images of α1- and β1-IR in the mouse LG. (A) Control for α1-receptor. (B) There was extensive α1-IR in the gland and it appeared that more α1-IR was associated with interstitial tissue, blood vessel, and ducts. This IR appeared as larger punctate labeling compared with that in contact with the acinar cells. (C) At higher magnification, α1-IR was observed on the surface and inside the acinar cells. (D) Control for the β1-receptor. (E) The lacrimal gland had extensive β1-IR. Little was found on the acinar cell surfaces, and most was inside the cells. Unlike the distribution of α1-IR, there was little β1-IR in the interstitium. (F) β1-IR at higher magnification. Note that β1-IR appeared to concentrate in the central areas within the acinar cells’ cytoplasm. Scale bars: (A, B, D, E) 25 μm; (C, F) 40 μm.
Figure 2.
 
Confocal images of double labeling of α1- and β1-receptor-IR and TH-IR in the mouse LG. (A) α1-IR (visualized with FITC) in the gland as punctate labeling. (B) A few TH-IR nerves (visualized with rhodamine red) were observed in the gland. Some of these nerves colocalized (arrow) or were in close association (arrowhead) with α1-IR. (C) Extensive β1-IR was found in the gland, and it appeared that none was in close contact or colocalized with TH-IR. A blood vessel (arrow) and a nerve (arrowhead) were stained with TH antibody in (D). Scale bar, 40 μm.
Figure 2.
 
Confocal images of double labeling of α1- and β1-receptor-IR and TH-IR in the mouse LG. (A) α1-IR (visualized with FITC) in the gland as punctate labeling. (B) A few TH-IR nerves (visualized with rhodamine red) were observed in the gland. Some of these nerves colocalized (arrow) or were in close association (arrowhead) with α1-IR. (C) Extensive β1-IR was found in the gland, and it appeared that none was in close contact or colocalized with TH-IR. A blood vessel (arrow) and a nerve (arrowhead) were stained with TH antibody in (D). Scale bar, 40 μm.
Figure 3.
 
Dose-response curves of protein secretion from mouse LG fragments in response to autonomic agonists. Each point represents the mean ± SEM of results in four to six experiments. *Significant difference from the corresponding basal secretion rate of each agonist.
Figure 3.
 
Dose-response curves of protein secretion from mouse LG fragments in response to autonomic agonists. Each point represents the mean ± SEM of results in four to six experiments. *Significant difference from the corresponding basal secretion rate of each agonist.
Figure 4.
 
The inhibitory effect of phentolamine (PL) on protein secretion induced by phenylephrine at EC50 (5 × 10−7 M). The ordinate is the percentage of inhibitory effect. Each point represents the mean ± SEM of results in four to six experiments.
Figure 4.
 
The inhibitory effect of phentolamine (PL) on protein secretion induced by phenylephrine at EC50 (5 × 10−7 M). The ordinate is the percentage of inhibitory effect. Each point represents the mean ± SEM of results in four to six experiments.
Figure 5.
 
Inhibitory effect of propranolol (PP) on protein secretory responses induced by isoproterenol at EC50 (3 × 10−5 M). The ordinate is the percentage of inhibitory effect. Each point represents the mean ± SEM of results in four to six experiments.
Figure 5.
 
Inhibitory effect of propranolol (PP) on protein secretory responses induced by isoproterenol at EC50 (3 × 10−5 M). The ordinate is the percentage of inhibitory effect. Each point represents the mean ± SEM of results in four to six experiments.
Figure 6.
 
Inhibitory effects of phentolamine (PL) and propranolol (PP) on norepinephrine (NE)-induced protein secretion. The protein secretory rate induced by NE was normalized to 100 and the ordinate is the percentage of secretion rate with the presence of antagonists compared with it: NE (10−6 M); PL and PP (10−5 M). NE and the antagonist were added simultaneously. Each point represents the mean ± SEM of results in four to six experiments.
Figure 6.
 
Inhibitory effects of phentolamine (PL) and propranolol (PP) on norepinephrine (NE)-induced protein secretion. The protein secretory rate induced by NE was normalized to 100 and the ordinate is the percentage of secretion rate with the presence of antagonists compared with it: NE (10−6 M); PL and PP (10−5 M). NE and the antagonist were added simultaneously. Each point represents the mean ± SEM of results in four to six experiments.
Figure 7.
 
Protein secretion in response to the simultaneous addition of phenylephrine (PE) and isoproterenol (Isop) is significantly lower (P < 0.05) than the theoretical addition of the secretion elicited by PE and Isop alone, indicating no synergism between these two agonists. Agonists were used at their half maximum concentrations (EC50): phenylephrine (5 × 10−7 M), isoproterenol (3 × 10−5 M). Ordinate is the secretion rate in micrograms per milliliter per gram tissue per minute. Each point represents the mean ± SEM of results in four to six experiments.
Figure 7.
 
Protein secretion in response to the simultaneous addition of phenylephrine (PE) and isoproterenol (Isop) is significantly lower (P < 0.05) than the theoretical addition of the secretion elicited by PE and Isop alone, indicating no synergism between these two agonists. Agonists were used at their half maximum concentrations (EC50): phenylephrine (5 × 10−7 M), isoproterenol (3 × 10−5 M). Ordinate is the secretion rate in micrograms per milliliter per gram tissue per minute. Each point represents the mean ± SEM of results in four to six experiments.
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