1Centre for Visual Sciences, Research School of Biological Sciences, Australian National University, Canberra, Australia; and 2Department of Physiology and Biophysics, Health Sciences Center, State University of New York, Stony Brook, New York
Submitted 28 October 2004 ; accepted in final form 21 May 2005
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ABSTRACT |
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To maintain significant ion and water secretion, the intracellular concentrations of K+ and Cl must remain sufficiently high to drive the passive transport through the apical membrane. In other tissues that transport water using similar mechanisms (e.g., cornea, salivary gland), there are several cotransporter systems that serve to replenish the cells with Cl and K+. One system is the Cl/HCO3 exchanger acting in concert with the Na+/H+ exchanger (24) and another is the Na+-K+-2Cl cotransporter (NKCC1) (10, 14, 15, 20). Although the NKCC1 has been identified cornea and salivary gland, where its functional significance is well established, there is only fragmentary evidence for its presence and role in fluid secretion in lacrimal gland. The mRNA for the transporter has been reported to be present in rabbit lacrimal glands (19) and application of furosemide, an inhibitor of NKCC1, reduced Cl ion flow (35) as well as fluid flow measured on the ocular surface in situ (2).There are no published data on mouse lacrimal glands, which are often used as model systems for dry-eye disease. Therefore, we have examined mouse exorbital lacrimal glands to determine if NKCC1 is present and to evaluate its role in fluid secretion. We show using immunocytochemical methods that antisera against NKCC1 bind to the plasma membranes of acinar and duct cells. Furthermore, we developed a new method for continuous topical drug application to lacrimal glands in situ that was used to demonstrate that furosemide treatment reduced fluid flow measured in the lacrimal duct. Finally, we demonstrate that furosemide blunts stimulus-induced shrinkage in lacrimal acinar cells in primary culture. Together, these data strongly suggest a major role for the NKCC1 transporter in fluid secretion by the mouse exorbital lacrimal gland.
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METHODS |
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Immunocytochemistry. Glands were removed and fixed for 4 h at 4°C in 4% paraformaldehyde in 0.1 M phosphate buffer. The tissue was then placed in 30% sucrose in buffer at 4°C for at least 12 h and up to 5 days. The tissue was frozen in frozen tissue medium and then cut on a cryostat at 14 µm. Sections were collected on slides, dried and then stored at 20°C until staining. Staining was accomplished by rehydrating the slides in buffer (0.1 M phosphate buffer, pH 7.4, with 0.25% Triton X-100) for 30 min. The tissue was then treated with nonspecific goat serum (Cappel Labs) for 1 h at room temperature. The slides were then drained and exposed to either of three primary antibodies to NKCC1. One was supplied by Dr. Turner (30) and was used at 1:1,000 dilution. Another antiserum was provided by Dr. Delpire (21) and was used at 1:200 dilution. Both antisera were made in rabbits. The third primary antiserum used was from Santa Cruz Biotechnology (Santa Cruz, CA), and was made from goat serum. It was used at a dilution of 1:100. After an overnight incubation at room temperature in a humidified chamber, the slides were drained and washed for 1 h in the buffer with Triton X-100. As controls, some slides were exposed to normal goat serum instead of the primary antisera. All slides were then exposed to secondary goat anti-rabbit antisera coupled to FITC (Cappel) diluted 1:75 for 1 h at room temperature. The slides were then washed in buffer plus Triton X-100 for 1 to 2 h and were cover-slipped using Vectashield medium (Vector Laboratories). The sections were examined using a Zeiss epifluorescence microscope and photographed using a Sony DKC 5000 digital camera.
In vivo measurement of lacrimal secretion.
Mice were anesthetized with Inactin/ketamine (110130 mg/kg Inactin, intraperitoneal; 100120 mg/kg ketamine, intramuscular) and placed on a heated surgical table. Rectal temperature was maintained at 37°C. A tracheal cannula was inserted to avoid aspiration of saliva. Catheters were inserted into the left jugular vein (for isotonic saline infusion of 0.5 ml·h1·100 g body wt1 and additional anesthetic as needed), and into the right femoral artery for arterial pressure monitoring and blood sampling. Heparin was added to the fluid in the arterial line to prevent clotting.
The experimental procedure is illustrated in Fig. 1. The animal is placed on its side and the head is immobilized with nonpenetrating steel pins. The right lacrimal gland is exposed with a small incision along an axis defined by the outer junction of the eyelids and the ear. The duct and gland were then freed via blunt dissection under a stereo microscope. Care was taken not to injure nearby nerves and blood vessels. The thin connective tissue capsule enclosing the gland was then carefully opened and removed from the upper surface to maximize penetration of topically applied drugs and chemicals.
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The lacrimal duct was then sectioned as far distally as possible (7080% of the duct remains), and the severed end of the duct was placed into a constant bore glass microcapillary tube (Microcaps; Drummond Scientific) that had been fire polished briefly. By using microcaps of different volumes (1, 2, and 5 µl), both basal and stimulated flows could be measured. The inner diameter of the collection capillary must be sufficiently large to contain the duct without impeding flow; the total time of collection was limited by the volume of the collection tube. Fluid collection into the capillaries occurred spontaneously, and we measured the rate of fluid flow by monitoring the movement of the fluid meniscus with a charge-coupled device camera mounted on a stereomicroscope. Recordings of the images were obtained using a video printer or on a S-VHS videocassette recorder. Minute flows were measured on the recordings by determining the increase in length of the fluid column per minute and multiplying by the volume per unit length for the capillary tube.
After a 15-min recovery period with continuous superfusion, the duct was placed into the collection tube, and one of two protocols was used. In the first protocol, the superfusate was switched to a KBR solution containing 10 µM carbachol. This resulted in rapid increase in lacrimal secretion that declined to a slowly waning plateau (see Fig. 3). After 10 min, the perfusate was switched to KBR solution with 100 µM furosemide without carbachol. After an additional 10 min, the superfusate was switched to a KBR solution containing furosemide and 10 µM carbachol. The flow response was then monitored for 10 min. After the flow response was measured, the superfusate was switched to KBR solution for 20 min, after which a repeat measure of the flow response to carbachol was obtained. The magnitude of the early peak in flow was highly variable and was attenuated with repeated stimulation in an individual gland, whereas the magnitude of the plateau phase was more consistent (see Fig. 3). Hence, we computed the flow-time average during the last 3 min of the plateau for each response and compared the value with furosemide with the average of the pre- and postfurosemide responses.
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Ion concentration in lacrimal fluid.
Some tear fluid samples were sealed in the collection tubes with silicone clay (Critoseal) for determination of Na+ and K+ ion concentrations with ion-selective microelectrodes obtained from commercial sources (Diamond General). Aliquots (500 nl) were obtained from the tear samples using an oil-filled micropipette and transferred into the lumen of a 2-µl microcapillary tube. The aliquots were placed within a column of mineral oil to prevent evaporation. The ion-selective electrode and an appropriate reference were then inserted into the lumen of the capillary tube into the sample. The resulting potential was measured with a Keithley electrometer. The measurement of sample potential was performed in duplicate, and each determination was bracketed by potential measurements in similar aliquots of standard solutions. Corrections for cross-selectivity between K+ and Na+ ions were computed using selectivity coefficients provided by the vendor. Cl ion concentrations were measured in nanoliter aliquots by performing electrochemical titration in a bath of water-equilibrated mineral oil (39).
Preparation of cultured acinar cells. Cells are isolated using a method modified from that of Hann et al. (16) The glands are removed, placed into a soybean trypsin inhibitor (STI), and cut into small pieces using two sharp, sterile scalpels. The pieces were then washed with Hanks' balanced salt solution (11885-084; GIBCO, Grand Island, NY) and incubated at 37°C for 15 min. After being washed again with STI, the cells were incubated in a 37°C agitated mixture of collagenase, DNase, and hyaluronidase in Dulbecco's modified Eagle's medium 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 into small sterile dishes that had small coverslips on the bottom that were coated with Matrigel. The dishes were kept in a tissue culture incubator overnight and used the next day.
Measurement of carbachol-induced acinar cell shrinkage.
Stimulation of secretory epithelial cells resulted in cell shrinkage, owing to a net steady-state loss of intracellular solute during active fluid secretion (7). Harvested acinar cells were plated on Matrigel-coated glass coverslips during overnight incubation at 37°C. After being washed with mammalian Tyrode solution, coverslips were placed into a chamber on an inverted microscope and perfused with a warmed (37°C) isotonic (320 mosmol/l) solution (in mM: 125 NaCl, 20 NaHCO3, 4 KCl, 1 MgCl2, 1 CaCl2, 10 glucose, and 5 HEPES, adjusted to pH 7.4 after equilibration with 95% O2-CO2). After 30-min rest, control images of the cell were obtained during the course of 3 min via a videomicroscopic system through a x40 lens objective. The perfusate was then switched to one of five test solutions (control Tyrode solution, Tyrode solution plus 10 µM carbachol, Tyrode solution plus carbachol + 100 µM furosemide, Tyrode solution plus 80 mM mannitol, and Tyrode solution plus 80 mM mannitol plus 100 µM furosemide). Images of the cell were obtained every 30 s after application of the test solution for 1020 min. Cell volumes (V) were estimated from the videotape images by outlining the cells and computing the enclosed area (A) using NIH Image software. Relative volume was then computed as follows:
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Statistical analysis. Comparisons were performed with paired or two-sample t-tests as indicated. Comparisons of cell volume responses were performed using two-way ANOVA, followed by the Student-Newman-Keuls multiple-comparison test using SigmaStat software (Systat).
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RESULTS |
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Figure 5 shows the effect of furosemide on carbachol-stimulated lacrimal gland fluid secretion. The data show that 10-min preexposure to furosemide reduced fluid secretion (Fig. 5, inverted triangle) compared with the average of the bracketing preexposure and recovery responses (Fig. 5, closed circle) obtained in the same glands. The difference in flows (Fig. 5, open circle) was significantly different from 0 at times 2 min (P < 0.05, paired t-test; n = 7). The relative reduction was
30%.
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The effects of 20-min preexposure to furosemide are shown in Fig. 6C. These studies were undertaken because the interval of furosemide pretreatment corresponded to the measurement of in vivo lacrimal flow as presented in Fig. 5. After a latent period of several minutes, cell volume began a slow decline of 20% in volume for 20 min. Exposure to carbachol at that point elicited a further, more rapid decrease in cell volume. The slow loss of solute before carbachol exposure likely represented the leak conductance of unstimulated the acinar cells. The leak pathway required Cl conductance, because the cell shrinkage was completely blocked by 0.1 mM flufenamic acid, an inhibitor of Cl ion channels (Fig. 6C).
Previous patch-clamp measurements of membrane currents induced by carbachol in isolated cells showed that there were two dominant currents: an inward Cl current and an outward K+ current (3, 11). Application of furosemide with carbachol did not change the magnitude of either of these currents (data not presented). Flufenamic acid exposure reduced the Cl current but did not greatly affect the K+ current (11).
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DISCUSSION |
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In salivary glands, the duct cells are known to play an active role in the exchange of Na+ for K+ in the salivary fluid (49). However, the localization of the NKCC1 on the basolateral membranes of lacrimal duct cells, together with the measurements of the composition of fluid collected from the distal lacrimal duct, suggests that lacrimal duct cells may be fundamentally different from salivary gland duct cells. Unlike salivary secretions that are hypotonic, high in Na+ and Cl, and low in K+ (44), the lacrimal gland fluid was hypertonic and high in both Cl and K+. The localization of NKCC1 on the basolateral membrane rather than on the apical membrane of the duct cells suggests K+ and Cl secretion rather than absorption (14).
The high density of staining observed on the duct cells certainly suggests active ion transport. However, the question of the mechanisms of ductal cell transport and whether this is associated with substantial water transport is unclear. The basis for this assertion is data provided in the study by Alexander et al. (1) showing that the composition and the degree of hypertonicity of lacrimal fluid collected from the terminal duct is remarkably independent of flow rate. At very low flows, the concentration of K+ and Na+ was higher, but the degree of hypertonicity did not appear to change. If the ductal epithelium were sufficiently water permeable to permit osmotic water transport secondary to ductal cell ion transport, one would clearly expect that the hypertonicity of the lacrimal fluid would be dissipated at low flow rates. Because salivary and pancreatic ducts and the distal renal tubule all show a strong dependency of ion composition on fluid flow, the transport process in the ductal system of the lacrimal gland appears to be unique and worthy of further study. In this regard, the delivery of hypertonic fluid to the ocular surface may be of physiological importance because it would promote osmotic water transport into the tear film from the cornea and conjunctiva.
We evaluated the physiological role of the NKCC1 transporter with two complementary techniques: a new method for the measurement of lacrimal gland secretion in vivo and evaluation of secretagogue-induced changes in isolated acinar cell volume. The new method provides continuous topical exposure to test substances without intravenous administration; in our experiments, the use of intravenous carbachol often resulted in severe hypotension in anesthetized mice. The method also permits measurement of the temporal pattern of secretion and the collection of samples of the fluid secreted by the lacrimal gland. The primary disadvantages of the method are that the extent of penetration of test substances is difficult to quantify and that the access of hydrophobic transport inhibitors to the apical membrane may be limited in an intact gland. These limitations are obviated in studies of isolated acinar cells, in which consistent drug exposure to both basolateral and apical membranes can be achieved. However, isolated cells are subjected to some degree of trauma, and cell volume changes are only an indirect indicator of ion efflux across the apical membrane.
The data obtained from intact, in situ lacrimal glands reveal an interesting temporal pattern in fluid secretion after stimulation. In the majority of glands, the initial stimulation was associated with a prominent early peak flow of highly variable magnitude that quickly subsided to a lower, more stable plateau. Flow in the plateau phase of the response was relatively stable for up to 20 min in most glands; in some glands, however, the flow waned after 10 min of stimulation. The mechanism responsible for the flow peak is unclear. The fact that the peak was attenuated on subsequent stimulations to a greater degree than the magnitude of the flow in the plateau phase suggests a minor role for receptor desensitization. It is unlikely that the reduced flow as a result of the second and subsequent stimuli were due to cell death, because we have been able to collect fluid for several hours and the volume of fluid collected per unit of time from the second stimulus onward was relatively consistent in many cases. It is possible that the initial peak in flow was associated with a receptor-simulated event that was only slowly reversible, such as protein secretion and the fusion of secretory vesicles with the apical membrane. With the ability of our new method to collect pure samples lacrimal gland fluid, it should be possible to elucidate the temporal pattern of lacrimal protein secretion to determine whether it shows a similar early peak.
Our analysis of the ionic composition of lacrimal fluid is in good agreement with the earlier measurements published by Alexander et al. (1) in the rat and by Botelho and Martinez (2) in the rabbit. In the rat, Alexander et al. found values for K+, Na+, and Cl of 46 ± 3, 135 ± 5, 123 ± 1 mM, while in the rabbit, Botelho and Martinez found that K+, Na+, and Cl values were 42 ± 4, 107 ± 4, 126 ± 5 mM. The high K+ values suggest substantial secretion of K+ by the acinar cells into the lumen of the acini. This suggests that there are apically located K+ channels, and immunocytochemistry using a rabbit antibody to the -subunit of the maxi-K+ channels supports that conclusion (48). The high Cl concentration also suggests apical Cl channels. There are a number of patch-clamp studies showing that acinar cells have many Cl channels (8, 39, 44), but there is little direct evidence for them to be located predominantly on the apical membrane (41). In the salivary gland, a similar location of Cl channels was suggested (29). The concentrations of both K+ and Cl ions in the fluid in the lacrimal ducts suggest that there is movement of these ions across the apical membranes.
Furosemide reduced fluid secretion from stimulated intact lacrimal glands and blunted carbachol-induced cell shrinkage in isolated acinar cells. The inhibition was partial in both cases. The most plausible explanation for continuing secretion in the presence of furosemide is that acinar cells have other Cl and K+ transport systems that can move these ions into the cells. For example, Cl can enter the cells via basolateral Cl/HCO3 exchangers (23). In many transporting epithelia, the Cl/HCO3 exchanger works in parallel with the Na+/H+ exchanger, which extrudes H+ and prevents marked cellular acidification (32, 34, 36, 42). The net result is the uptake of Cl and Na+, and most of the transported Na+ is exchanged for K+, because Na+ is extruded from the cytoplasm by the Na+ pump. This process depends on cytosolic carbonic anhydrase, which has been identified in rabbit and rat (4) and rat and mouse lacrimal glands (18, 34).
In the isolated cells, exposure to furosemide alone during a 20-min period caused the cells to shrink slowly (Fig. 6C), an effect inhibited by a Cl channel blocker (flufenamic acid). This could be due either to furosemide affecting the uptake of Cl ions by the cells or by furosemide blocking the exit of Cl, or both. Given that furosemide had little effect on the membrane currents induced by carbachol, it seems unlikely that the effect of furosemide is on the Cl channels. This finding suggests that in these isolated cells, there is a leakage of Cl from the cells that is replaced by an inward movement of Cl due to a constitutively active NKCC1. If NKCC1 is blocked, the cytosolic pool of Cl decreases and the cells shrink as a result of the decrease in total ion concentration within the cell. This would also suggest that the pool of Cl ions available to move across the apical membrane is relatively small and is rapidly depleted on stimulation.
That furosemide produced significant inhibition, however, certainly supports the argument that NKCC1 is important for fluid production by the mouse lacrimal gland. It is an efficient transport system that moves one K+ and two Cl ions for each Na+ ion that enters. In an in vivo perfusion system of rabbit lacrimal glands, it also has been shown that systemic furosemide significantly reduces carbachol-induced fluid delivery to the ocular surface (5), findings that suggest a similar role for the NKCC1 transporter in other species. In other cell types, stimulation results in activation of NKCC1 and/or upregulation of its expression (11, 13, 25). In some tissues, the decrease in intracellular Cl ions that occurs upon cell activation stimulates NKCC1 (40). In the salivary gland, -adrenergic activation of acinar cells results in upregulation of NKCC1 as a result of phosphorylation, although other agents that cause increased activity do not involve phosphorylation (45). In a human tracheal epithelial cell line, activation of NKCC1 is mediated via a protein kinase C (25, 26). Nitric oxide (NO) has been shown to inhibit NKCC1 activity in renal epithelial cells (17). Some nerves and acinar cells in mouse lacrimal glands have neural nitric oxide synthase (nNOS), which could produce NO and could result in NKCC1 inhibition and reduced fluid production (6). Increased nNOS activity or the failure of the normal control pathways to increase NKCC1 activity or expression would effectively reduce fluid production by the lacrimal gland and therefore could result in a dry-eye condition.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
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2. Botelho SY and Martinez EV. Electrolytes in lacrimal gland fluid and in tears at various flow rates in the rabbit. Am J Physiol 225: 606609, 1973.
3. Brink PR, Peterson E, Banach K, and Walcott B. Electrophysiological evidence for reduced water flow from lacrimal gland acinar epithelium of NZB/NZW F1 mice. In: Lacrimal Gland, Tear Film, and Dry Eye Syndromes 2: Basic Science and Clinical Relevance, edited by Sullivan DA, Dartt DA, and Meneray AM. New York: Plenum, 1998, p. 209219.
4. Bromberg BB, Welch MH, Beuerman RW, Chew SJ, Thompson HW, Ramage D, and Githens S. Histochemical distribution of carbonic anhydrase in rat and rabbit lacrimal gland. Invest Ophthalmol Vis Sci 34: 339348, 1993.[Abstract]
5. Dartt DA, Moller M, and Poulsen JH. Lacrimal gland electrolyte and water secretion in the rabbit: localization and role of (Na+ and K+)-activated ATPase. J Physiol 321: 557569, 1981.[Abstract]
6. Ding C, Walcott B, and Keyser KT. Neuronal nitric oxide synthase is expressed in the mouse lacrimal gland and neurons of pterygopalatine ganglion. Adv Exp Med Biol 506: 9195, 2002.[ISI][Medline]
7. Douglas IJ and Brown PD. Regulatory volume increase in rat lacrimal gland acinar cells. J Membr Biol 150: 209217, 1996.[CrossRef][ISI][Medline]
8. Edelman JL, Loo DD, and Sachs G. Characterization of potassium and chloride channels in the basolateral membrane of bovine nonpigmented ciliary epithelial cells. Invest Ophthalmol Vis Sci 36: 27062716, 1995.[Abstract]
9. Evans MG and Marty A. Calcium-dependent chloride currents in isolated cells from rat lacrimal glands. J Physiol 378: 437460, 1986.[Abstract]
10. Evans RL, Park K, Turner RJ, Watson GE, Nguyen HV, Dennett MR, Hand AR, Flagella M, Shull GE, and Melvin JE. Severe impairment of salivation in Na+/K+/2Cl cotransporter (NKCC1)-deficient mice. J Biol Chem: 2672026726, 2000.
11. Farokhzad OC, Sagar GD, Mun EC, Sicklick JK, Lotz M, Smith JA, Song JC, O'Brien TC, Sharma CP, Kinane TB, Hodin RA, and Matthews JB. Protein kinase C activation down regulates the expression and function of the basolateral Na+/K+/2Cl cotransporter. J Cell Physiol 181: 489498, 1999.[CrossRef][ISI][Medline]
12. Findlay I. A patch-clamp study of potassium channels and whole-cell currents in acinar cells of the mouse lacrimal gland. J Physiol 350: 179195, 1984.[Abstract]
13. Garay RP, Alvarez-Guerra M, Alda JO, Nazaret C, Soler A, and Vargas F. Regulation of renal Na-K-Cl cotransporter NKCC2 by humoral natriuretic factors: relevance in hypertension. Clin Exp Hypertens 20: 675682, 1998.[ISI][Medline]
14. Haas M and Forbush B III. The Na-K-Cl cotransporter of secretory epithelia. Annu Rev Physiol 62: 515534, 2000.[CrossRef][ISI][Medline]
15. Haas M and Forbush B III. The Na-K-Cl cotransporters. J Bioenerg Biomembr 30: 161172, 1998.[CrossRef][ISI][Medline]
16. Hann LE, Tatro JB, and Sullivan DA. Morphology and function of lacrimal gland acinar cells in primary culture. Invest Ophthalmol Vis Sci 30: 145158, 1989.[Abstract]
17. He H, Podymow T, Zimpelmann J, and Burns KD. NO inhibits Na+-K+-2Cl cotransport via a cytochrome P-450-dependent pathway in renal epithelial cells (MMDD1). Am J Physiol Renal Physiol 284: F1235F1244, 2003.
18. Hennigar RA, Schulte BA, and Spicer SS. Immunolocalization of carbonic anhydrase isozymes in rat and mouse salivary and exorbital lacrimal glands. Anat Rec 207: 605614, 1983.[CrossRef][ISI][Medline]
19. Iserovich P, Bildin VN, Kuang K, and Fischbarg J. Evidence for the presence of mRNA for Na+-K+-2Cl cotransporter and MDR in rabbit lacrimal glands (ARVO Abstract 3697). Invest Ophthalmol Vis Sci 39, Suppl: S798, 1998.
20. Jelamskii S, Sun XC, Herse P, and Bonanno JA. Basolateral Na+-K+-2Cl cotransport in cultured and fresh bovine corneal endothelium. Invest Ophthalmol Vis Sci 41: 488495, 2000.
21. Kaplan MR, Plotkin MD, Brown D, Hebert SC, and Delpire E. Expression of the mouse Na-K-2Cl cotransporter, mBSC2, in the terminal inner medullary collecting duct, the glomerular and extraglomerular mesangium, and the glomerular afferent arteriole. J Clin Invest 98: 723730, 1996.
22. Kotera T and Brown PD. Calcium-dependent chloride current activated by hyposmotic stress in rat lacrimal acinar cells. J Membr Biol 134: 6774, 1993.[ISI][Medline]
23. Lambert RW, Bradley ME, and Mircheff AK. Cl-HCO3 antiport in rat lacrimal gland. Am J Physiol Gastrointest Liver Physiol 255: G367G373, 1988.
24. Lambert RW, Bradley ME, and Mircheff AK. pH-sensitive anion exchanger in rat lacrimal acinar cells. Am J Physiol Gastrointest Liver Physiol 260: G517G523, 1991.
25. Liedtke CM and Cole TS. PKC signalling in CF/T43 cell line: regulation of NKCC1 by PKC- isotype. Biochim Biophys Acta 1495: 2433, 2000.[CrossRef][ISI][Medline]
26. Liedtke CM, Papay R, and Cole TS. Modulation of Na-K-2Cl cotransport by intracellular Cl and protein kinase C- in Calu-3 cells. Am J Physiol Lung Cell Mol Physiol 282: L1151L1159, 2002.
27. Lytle C, Xu JC, Biemesderfer D, and Forbush B III. Distribution and diversity of the Na-K-Cl cotransport proteins: a study with monoclonal antibodies. Am J Physiol Cell Physiol 269: C1496C1505, 1995.
28. Marty A, Tan YP, and Trautmann A. Three types of calcium-dependent channel in rat lacrimal glands. J Physiol 357: 293325, 1984.[Abstract]
29. Melvin JE. Chloride channels and salivary gland function. Crit Rev Oral Biol Med 10: 199209, 1999.
30. Moore-Hoon ML and Turner RJ. Molecular and topological characterization of the rat parotid Na+-K+-2Cl cotransporter 1. Biochim Biophys Acta 1373: 261269, 1998.[ISI][Medline]
31. Nelson MT and Quayle JM. Physiological roles and properties of potassium channels in arterial smooth muscle. Am J Physiol Cell Physiol 268: C799C822, 1995.
32. Nguyen HV, Stuart-Tilley A, Alper SL, and Melvin JE. Cl/HCO3 exchange is acetazolamide sensitive and activated by a muscarinic receptor-induced [Ca2+]i increase in salivary acinar cells. Am J Physiol Gastrointest Liver Physiol 286: G312G320, 2004.
33. Nilius B and Droogmans G. Amazing chloride channels: an overview. Acta Physiol Scand 177: 119147, 2003.[CrossRef][ISI][Medline]
34. Ogawa Y, Matsumoto K, Maeda T, Tamai R, Suzuki T, Sasano H, and Fernley RT. Characterization of lacrimal gland carbonic anhydrase VI. J Histochem Cytochem 50: 821827, 2002.
35. Ozawa T, Saito Y, and Nishiyama A. Mechanism of uphill chloride transport of the mouse lacrimal acinar cells: studies with Cl-sensitive microelectrode. Pflügers Arch 412: 509515, 1988.[CrossRef][ISI][Medline]
36. Park K, Evans RL, Watson GE, Nehrke K, Richardson L, Bell SM, Schultheis PJ, Hand AR, Shull GE, and Melvin JE. Defective fluid secretion and NaCl absorption in the parotid glands of Na+/H+ exchanger-deficient mice. J Biol Chem 276: 2704227050, 2001.
37. Park KP, Beck JS, Douglas IJ, and Brown PD. Ca2+-activated K+ channels are involved in regulatory volume decrease in acinar cells isolated from the rat lacrimal gland. J Membr Biol 141: 193201, 1994.[ISI][Medline]
38. Petersen OH. Calcium-activated potassium channels and fluid secretion by exocrine glands. Am J Physiol Gastrointest Liver Physiol 251: G1G13, 1986.
39. Ramsay J, Brown RHT, and Croghaw P. Electrometric titration of Cl in small volumes. J Exp Biol 32: 822829, 1955.[ISI]
40. Russell JM. Sodium-potassium-chloride cotransport. Physiol Rev 80: 211276, 2000.
41. Saito Y, Ozawa T, Hayashi H, and Nishiyama A. The effect of acetylcholine on chloride transport across the mouse lacrimal gland acinar cell membranes. Pflügers Arch 409: 280288, 1987.[ISI][Medline]
42. Saito Y, Ozawa T, and Nishiyama A. Effects of intra- and extracellular H+ and Na+ concentrations on Na+-H+ antiport activity in the lacrimal gland acinar cells. Pflügers Arch 417: 382390, 1990.[CrossRef][ISI][Medline]
43. Sundermeier T, Matthews G, Brink PR, and Walcott B. Calcium dependence of exocytosis in lacrimal gland acinar cells. Am J Physiol Cell Physiol 282: C360C365, 2002.
44. Tandler B, Gresik EW, Nagato T, and Phillips CJ. Secretion by striated ducts of mammalian major salivary glands: review from an ultrastructural, functional and evolutionary perspective. Anat Rec 264: 121145, 2001.[CrossRef][ISI][Medline]
45. Tanimura A, Kurihara K, Reshkin SJ, and Turner RJ. Involvement of direct phosphorylation in the regulation of the rat parotid Na+-K+-2Cl cotransporter. J Biol Chem 270: 2525225258, 1995.
46. Trautmann A and Marty A. Activation of Ca-dependent channels in rat lacrimal glands. J Physiol 357: 293325, 1984.[Abstract]
47. Walcott B. The lacrimal gland and its veil of tears. News Physiol Sci 13: 97103, 1998.[ISI][Medline]
48. Walcott B, Birzgalis A, Claros N, and Brink PR. A model of fluid secretion by the acinar cells of the mouse lacrimal gland. Adv Exp Med Biol 506: 191197, 2002.[ISI][Medline]
49. Zhao H, Xu X, Diaz J, and Muallem S. Na+, K+, and H+/HCO3 transport in submandibular salivary ducts: membrane localization of transporters. J Biol Chem 270: 1959919605, 1995.
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