Departments of 1 Neurobiology and Behavior and 2 Physiology and Biophysics, State University of New York, Stony Brook, New York 11794 and 3 Institute of Genetics, University of Bonn, 53117 Bonn, Germany
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ABSTRACT |
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In glands such as the liver and pancreas, gap junctions containing connexin 26 and 32 (Cx26 and Cx32, respectively) couple the secretory cells. Uncoupling these junctions compromises the secretory function of these glands. Lacrimal glands also contain extensive arrays of gap junctions consisting of Cx26 and Cx32. We wanted to determine the role of these junctions in fluid secretion. In Cx32-deficient mice, immunocytochemistry showed that, in the male lacrimal gland, the remaining Cx26 was found evenly distributed in the membrane whereas there was little in the membranes of female glands. Western blot analysis of Cx26 showed that female Cx32-deficient mice expressed Cx26. Patch-clamp analyses of acinar cell coupling showed that the cell pairs from male glands were coupled whereas those from female glands were not. Stimulated fluid production by the glands from Cx32-deficient mice was abnormally low in female glands compared with controls at low topical doses of carbachol. The protein secretory response to different doses of carbachol was the same in all animals. These data suggest that gap junctions are essential for optimal fluid secretion in lacrimal glands.
connexin 26; connexin 32; mouse; tears
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INTRODUCTION |
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IN EXOCRINE
GLANDS, THE ACINAR cells that produce the secretory product are
coupled by gap junctions (10). Gap junction channels,
which link the cytoplasms of adjacent cells, are made up of
membrane-spanning proteins, the connexins. In most glands, the major
connexins are 32 and 26 (Cx32 and Cx26, respectively). The integrity of
these junctions is apparently necessary for normal glandular secretory
function. In the liver, for example, Cx32-deficient mice showed a 78%
reduction in glucose mobilization on neuronal stimulation
(12). Blocking liver gap junctions with
18-glycyrrhetinic acid reduced hormonal control of bile secretion
(11). In Cx32-deficient mice, circulating levels of
amylase were higher, suggesting that coupling in the pancreas is
involved in regulation of its secretion (3).
Lacrimal glands also contain gap junctions (14) with extensive arrays of Cx32 and Cx26 patches (7). The vertebrate lacrimal gland has three major functions: to synthesize and secrete certain tear-specific proteins, to transport secretory IgA across the epithelium, and, finally, to transport water to form the tear fluid (19). There are few data on the role of gap junctions in any of these major functions of the lacrimal gland. Patch-clamp studies on isolated cell pairs of acinar cells from mouse lacrimal glands showed that the cells are highly coupled and that junctional conductance is affected by neurotransmitters and second messengers (5, 16). Loss of gap junctions or alterations in their regulation, therefore, could affect the secretory ability of the gland and thus lead to some form of dry eye disorder.
Here, we investigated the role of gap junctions in lacrimal gland function using a strain of Cx32-deficient mice. We have found that isolated cell pairs from male Cx32-deficient glands are electrically coupled whereas those from female Cx32-deficient animals are not. We have determined the ability of lacrimal glands from both Cx32-deficient and wild-type mice to secrete both protein and fluid. Our data show that application of carbachol, an acetylcholine agonist, to gland fragments from Cx32-deficient mice induced normal protein secretion. In contrast, topical application of carbachol to the gland in situ showed that fluid production from female Cx32-deficient glands was significantly less sensitive to the neurotransmitter agonist than that in glands from male Cx32-deficient mice, suggesting that the normal organization of gap junctions is important in the regulation of fluid secretion by the gland.
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METHODS |
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Cx32-deficient mice were generated by homologous recombination as has been described elsewhere (12) and were raised in an approved animal facility. Control C57B/6 mice were obtained from Jackson Laboratories (Bar Harbor, ME).
Immunocytochemistry. Animals were killed with halothane, and the glands were removed. The tissue was immersion fixed for 3 h in 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, at 4°C. The tissue was rinsed in 30% sucrose in 0.1 M phosphate buffer and then stored overnight in fresh sucrose buffer at 4°C. Cryostat sections were cut at 14 µm, mounted on slides, and stored at 4°C until they were stained. Commercial rabbit polyclonal antibodies to Cx32, Cx26, and synaptophysin, a protein found at neurotransmitter release sites, were obtained from Zymed Laboratories (San Francisco, CA). Sections were hydrated in 0.1 M phosphate buffer and then exposed to normal goat serum diluted 1:75 for 1 h. The goat serum was drained off, and the primary antisera, diluted 1:200 with the phosphate buffer, were applied to the sections overnight at room temperature in a humidified chamber. Control slides were always prepared in parallel and consisted of sections exposed to the normal goat serum overnight instead of the primary antibody. Slides were then washed three times in phosphate buffer and then exposed to FITC-coupled goat anti-rabbit secondary antisera for 1 h in the dark at room temperature. The slides were then washed in phosphate buffer for 1 h in the dark and then cover slipped with use of Vectashield mounting medium. Sections were examined by using a Zeiss epifluorescence microscope equipped with a Sony DKC 5000 camera attached to a personal computer. Images were captured by using Photoshop software and were printed on a digital Sony printer (UPD 8800).
Western blot.
Fresh gland tissue was blotted dry, weighed, and then homogenized in
cold sample buffer (0.1 M Tris · HCl, pH 6.8) with 2% SDS and
2 -mercaptoethanol to a constant gland weight to buffer volume of
0.1 g/ml. The protein concentration of each sample was determined by
using the method of Peterson (15). Sufficient volumes of
sample were added to each well so that 90 µg of each sample were
loaded into the wells of a 2-20% polyacrylamide gradient gel
(Bio-Rad, Hercules, CA) with prestained molecular weight standards (SeeBlue Plus2, Novex, CA) being placed in the two outer wells. The gel
was transferred to polyvinylidene difluoride protein transfer membrane
(Schleicher and Schuell, Keene, NH) and blocked with 1% milk powder in
buffer. The membrane was exposed to a 1:1,000 dilution of a mouse
monoclonal antibody to Cx26 (Zymed Laboratories) overnight. The
membrane was then washed and exposed to a goat anti-mouse antibody
coupled to alkaline phosphatase (Bio-Rad). Finally the membrane was
treated with a chemiluminescence system (Immuno-Star chemiluminescent
protein detection system, Bio-Rad) and exposed to X-ray film for
3 min.
Protein secretion.
Glands were removed as described and were weighed before being cut into
0.5-mm slices with a scalpel blade. The slices from a gland were placed
in 5 ml of an artificial saline solution (in mM: 116 NaCl, 5.4 KCl, 1.8 CaCl2, 0.81 MgCl2 1.01 NaH2PO4, 26.2 NaHCO3, 5.6 dextrose,
and 1.0 -hydroxybutyric acid, pH 7.4) that was maintained at 37°C
and was vigorously aerated with 95% O2 and 5%
CO2 for 10 min. The solution was changed three times and discarded. The gland fragments were then placed in 5 ml of medium, and,
after 10 min, 1 ml of medium was removed and replaced with fresh
medium. This was repeated three times, and the 1 ml of the collected
medium after each of the 10-min incubations was saved. In the last
exchange, the medium that was added to replace that removed contained
sufficient carbachol to make the final concentration in the bath either
0.1, 1, or 10 µM. After 10 min, 1 ml was removed. All four 1-ml
samples were analyzed for their total protein content by using a
Coomassie colorimetric system (Pierce, Rockford, IL). BSA was used as a
standard protein, and standards were run with each batch of
experiments. Unknown protein concentrations were determined from the
standard curves measured with each experiment.
Patch-clamp studies. For patch-clamp analysis, acinar cells were isolated from freshly dissected glands by using a method developed for rat lacrimal glands (6). The mice were anesthetized with halothane and then decapitated. The glands were removed, placed in soybean trypsin inhibitor (0.1 mg/ml, Sigma Chemical) and cut into small pieces by using two sharp sterile scalpel blades. The pieces were then washed with Hanks' balanced salt solution and incubated at 37°C for 15 min. After being washed again with soybean trypsin inhibitor, the cells were incubated at 37°C in an agitated mixture of collagenase (225 U/ml, GIBCO), DNase (10 U/ml, Sigma Chemical), and hyaluronidase (600-700 U/ml, Sigma Chemical) in DMEM for 25 min. The resulting mixture was centrifuged at 1,200 rpm for 5 min, and the pellet was resuspended in medium. The suspension was filtered through sterile mesh to remove the large fragments that remained, and the resulting cell suspension was centrifuged again. The pellet was resuspended in medium, and 0.5-ml aliquots were plated on small sterile dishes that had been coated with Matrigel. The plates were incubated overnight to allow the cells to settle and were used the next day.
For patch clamping, the dishes were drained of medium, and the cells were covered with saline (in mM: 110 NaCl, 1 MgCl2, 1 CaCl2, and 10 HEPES, pH 7.2). Electrodes were produced on a P-57 Sutter puller, and an Axopatch 200 series patch amplifier was employed for recording. The pipette solution contained (in mM) 110 KCl, 0.9 EGTA, 0.1 CaCl2, 0.1 MgCl2, and 10 HEPES at pH 7.05. Cell pairs were patched in a whole cell configuration, and coupling was measured by use of conventional methods.Fluid secretion.
To measure the fluid production of the exorbital lacrimal gland, we
anesthetized mice with 120-130 mg/kg Inactin (RBI, Natick, MA) and
120-130 mg/kg ketamine. The animal was placed on a heated surgical
table; rectal temperature was maintained at ~37°C. A tracheal
cannula was inserted to avoid aspiration of saliva, the flow of which
can rise during carbachol (acetylcholine agonist) stimulation. A
catheter was inserted into the left jugular vein for infusion of 0.5 ml · h1 · 100 g body wt
1
isotonic saline during surgery and measurements. Another catheter was
placed in the right femoral artery for arterial pressure monitoring.
Statistics. Differences between group means were tested with ANOVA, either one- or two-way, as appropriate, and Tukey's multiple comparison test. Differences between proportions were tested with the z-test. A significance level of 0.05 was assumed.
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RESULTS |
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Immunocytochemistry.
We examined the Cx32 and Cx26 immunoreactivity in exorbital lacrimal
gland tissue from control animals. The pattern of staining seen with
both antisera was similar (Fig. 1) in
males and females, consisting of numbers of small patches of
immunoreactivity associated with the secretory acinar cells. Many of
the patches were linearly arranged as if associated with the plasma
membranes of adjacent cells. In addition, we found immunoreactivity
above control levels that appeared to be localized within the cells.
The duct cells, distinguished by their obvious lumen and more cuboidal
shape (see Fig. 1A), showed no positive staining for either
of the connexins.
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Western blot.
Figure 3 shows a Western blot stained
with a monoclonal antibody to Cx26. This blot compares the glands from
a male and a female C57BL/6 control mouse and from a male and a female
Cx32-deficient animal. In all cases, there is a major band at ~30,000
Da that shows positive immunoreactivity. Additional blots on glands
pooled from four different animals of each type and sex showed similar results, with Cx26 being present in both male and female glands of
Cx32-deficient animals as well as control animals.
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Protein secretion.
Figure 4 shows the amount of protein
released by gland fragments in response to three different
concentrations of the acetylcholine agonist carbachol. The data show
that there is dose-dependent protein secretion in response to carbachol
in glands from both male and female Cx32-deficient animals as well as
control strain (Swiss Webster) female glands. At the lowest dose of
carbachol (0.1 µM), the Cx32-deficient female gland fragments
secreted significantly more protein than fragments from the other
glands (t-test, P < 0.05). At 1 µM carbachol, there was no significant difference, whereas at 10 µM
again the female Cx32-deficient gland fragments secreted more protein
than the others (P < 0.05). The protein secretion from
the female Cx32-deficient gland fragments was dose dependent and
robust.
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Cell coupling.
Patch-clamp analysis of cell pairs from control glands showed that the
majority of cells were coupled (Fig. 5),
as has been previously reported (14). The majority of cell
pairs from Cx32-deficient female glands, however, were not coupled, and
those that were coupled had a much lower average conductance (1.8 ± 1.2 vs. 10.0 ± 2.5 ps) than cell pairs from control animals.
Cell pairs from Cx32-deficient males were coupled and showed a
conductance similar to that of control animals.
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Fluid secretion.
As described in METHODS, the secretory responses with no
stimulation and then with topical carbachol at low (2-6 µl at
0.12 mM) and high (2-4 µl at 6.0 mM) doses were measured. In all
animals, our methods were not able to measure significant amounts of
basal, nonstimulated flow. In control female glands, the peak flow rate induced by the lower dose of carbachol was significant but not as high
as that induced by the higher dose (Fig.
6). The carbachol diffused at least some
distance into the gland because there was a relatively rapid (within 1 min) drop in mean arterial pressure after the application of the dose
of carbachol. In each group (n = 6 in all groups), the
dose response to carbachol was significant (P < 0.05 ANOVA). In both the control and Cx32-deficient groups, the response to
carbachol was significantly higher in males than in females
(P < 0.001, two-way ANOVA). This is likely a
reflection of the larger gland size in male mice. However, deficiency
of Cx32 compromised flow responses only in female mice and, in
particular, only at the low topical dose of carbachol
(P < 0.05, two-way ANOVA, Tukey's test). Application
of the high dose of carbachol induced a flow rate in Cx32-deficient
female glands indistinguishable from that of the control female glands.
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DISCUSSION |
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The data presented here show that the mouse exorbital lacrimal gland acinar cells are highly coupled by gap junctions and that these junctions consist of Cx32 and Cx26. Such junctions have been shown to exist in the rat exorbital lacrimal gland (10), where they develop in number and size as the gland increases its secretory ability with growth and development (7). Also in the rat lacrimal gland, several patch-clamp studies have shown that the acinar cells are highly coupled and that the conductance of these junctions is modulated by acetylcholine independently of the internal calcium levels (13). This regulation of junctional conductance seems to require activation of protein kinase C (16). The rat salivary glands also have Cx32 and Cx26 junctions between acinar cells, and the conductance of these junctions is also affected by acetylcholine via a protein kinase C system (8, 9). Although it is clear that gap junctions are present and can be regulated, there are no data on either mouse or rat lacrimal or salivary gland systems to show the role of gap junctions in the secretory process itself. We have found that the Cx32-deficient female mice showed a reduced fluid secretion in response to low doses of topically applied carbachol. Only one of seven animals (see Fig. 6) had any significant fluid flow, whereas the others had little to no response. This is in contrast to control animals as well as Cx32-deficient males, in which there was always fluid secretion in response to topically applied low doses of carbachol. These results correlate with the coupling data, which showed that female Cx32-deficient lacrimal gland acinar cell pairs were electrically uncoupled, whereas those from male Cx32-deficient cell pairs were coupled, as were cell pairs from control animals. The observation that topical applications of high doses of carbachol induced normal fluid flow in all animals suggests that, in the Cx32-deficient female glands, the acetylcholine receptor second message system is not significantly different from that in control glands. The integrity of the muscarinic acetylcholine-mediated second messenger system is further supported by the observation that protein secretion, which is also induced by acetylcholine, was similar in gland fragments from all glands. A simple interpretation of these data would be that gap junctions are necessary for the spread of excitation from the surface cells to deeper cells within the gland. When low doses of topical carbachol are used, the carbachol does not diffuse at a high enough concentration to cause excitation of many of the deeper cells within the gland tissue. In glands where the cells are coupled, excitation can be spread through the tissue via the gap junctions. In Cx32-deficient females, the junctions are defective, so perhaps there is minimal excitation of deep cells.
In other secretory tissues such as liver and pancreas, loss of Cx32 or blockage of the gap junction channels has a profound effect on the regulation of the secretory ability of the glands (10, 12). The livers of Cx32-deficient mice, for example, secreted less glucose in response to glucagon or norepinephrine stimulation when half-maximum doses were used (17). However, secretion of glucose in response to saturating doses was the same between Cx32-deficient and normal animals. This behavior is similar to what we observed on the lacrimal gland fluid secretion. The liver is also controlled by sympathetic neurons. In Cx32-deficient animals, stimulation of the sympathetic innervation to the liver resulted in a significantly reduced (78%) production of glucose (12). Thus reduction of coupling between cells in some glands affects the ability of those glands to respond to either endocrine or neural stimulation.
The immunocytochemical data (Figs. 1 and 2) showed that there was a difference in the morphology of the Cx26 immunoreactivity in the lacrimal glands between the control and Cx32-deficient mice. In control mice, the pattern of staining was predominantly patchlike, often in rows along the cell margins, although there was some diffuse staining in the cytoplasm. In the Cx32-deficient animals, Cx26 was expressed, but its distribution was different between males and female animals. In Cx32-deficient males, the staining pattern observed was of a uniform staining on the plasma membranes as well as diffuse cytoplasmic staining. In the female Cx32-deficient glands, there was little membrane-associated Cx26 immunoreactivity, but there was diffuse cytoplasmic staining. The Western blot data (Fig. 3) showed that there was little difference in the expression of Cx26 between male and female control mouse glands and that the female Cx32-deficient glands contained Cx26 as well even though most cell pairs isolated from these glands showed no coupling. The male Cx32-deficient glands, whose cells were still electrically coupled, also showed an expression of Cx26. These data can be explained by the following hypotheses: either the deletion of the Cx32 gene could affect Cx26 expression and thus its ability to normally insert in the membrane, or expression of both Cx32 and Cx26 may be needed for the normal insertion or retention of Cx26 in the membrane. Alternatively, the difference in the placement of the Cx26 in male and female Cx32-deficient animals may be due to androgens that could play a role in the normal expression and insertion of these connexins into the membrane.
Androgens affect the lacrimal glands in several ways. In many species, including mice and humans, the lacrimal gland acini of males are larger in area than those from females (4). Studies of the activity of certain enzymes in the lacrimal glands of rabbits show that significant sex-related changes occurred as the animals matured, which indicated that the glands were influenced by the androgens and estrogens of the sexually maturing animals (1). Lacrimal gland acinar cells have androgen-binding sites in their nuclei (18). Glandular fluid secretion is dependent on the presence of androgens (2), as ovariectomy of female rabbits, for example, reduced blood androgen levels and resulted in a reduced ability of the glands from those animals to secrete fluid (20). These studies show that androgens have a significant role in the maintenance and function of lacrimal glands. It is therefore likely that the difference in Cx26 placement between the male and female Cx32-deficient animals could be due to the different androgen levels in these animals. The mechanism by which the androgens exert their influence on the gap junction channel proteins remains to be determined.
Our results on Cx32-deficient mice suggest that gap junctions play an important role in the excitation secretion process of lacrimal glands. The determination of whether older Cx32-deficient females have drier eyes than males, however, is not a simple task. The ocular fluid is derived from a number of sources such as the accessory glands, Harderian glands, cornea, and conjunctiva, all secreting different types of fluids (19). To grossly measure the total fluid on the surface by means of Schirmer's strips, for example, would not necessarily show any difference. However, there could well be differences in the amounts of lacrimal gland-specific proteins, such as peroxidase or lactoferrin, that reach the ocular surface because it is likely that fluid flow is necessary to move these proteins down the lumen of the acini and the ducts. Uncoupling of acinar cells due to either reduced expression, failure to insert the connexin channels in the membrane, and/or reduced permeability due to regulatory effects could result in reduced fluid flow from the lacrimal glands and hence dry eye and/or could affect the composition of the tear fluid.
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ACKNOWLEDGEMENTS |
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We are grateful to Chris Picken for doing PCR on tail clips of the knockout mice.
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FOOTNOTES |
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This research was supported by National Eye Institute Grant EY-0940607.
Address for reprint requests and other correspondence: B. Walcott, Dept. of Neurobiology and Behavior, State Univ. of New York, Stony Brook, New York 11794-5230 (E-mail: bwalcott{at}ms.cc.sunysb.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajpcell.00004.2001
Received 20 August 2001; accepted in final form 30 October 2001.
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