Reversible regulation of P2Y2 nucleotide receptor expression in the duct-ligated rat submandibular gland

Jae Suk Ahn1, Jean M. Camden1, Ann M. Schrader1, Robert S. Redman2, and John T. Turner1

1 Department of Pharmacology, School of Medicine, University of Missouri, Columbia, Missouri 65212; and 2 Oral Pathology Research Laboratory, Department of Veterans Affairs Medical Center, Washington, District of Columbia 20422


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Ligation of the main excretory duct of the rat submandibular gland (SMG) produces a pronounced atrophy that is reversed upon ligature removal. Based on previous studies by our group and others suggesting that P2Y2 nucleotide receptors are upregulated in response to tissue damage, we hypothesized that P2Y2 receptor activity and mRNA levels would increase after duct ligation and return to control levels after ligature removal. Our results support this hypothesis. Intracellular Ca2+ mobilization in response to the P2Y2 receptor agonist UTP in SMG cells was increased significantly after ligation periods of 1.5 to 7 days, whereas no significant response was observed in the contralateral, nonligated gland. P2Y2 receptor mRNA, as measured by semiquantitative RT-PCR, increased about 15-fold after 3 days of ligation. These increases reverted to control levels by 14 days after ligature removal. In situ hybridization revealed that the changes in P2Y2 receptor mRNA abundance occurred mostly in acinar cells, which also were more adversely affected by ligation, including an increase in the appearance of apoptotic bodies. These findings support the idea that P2Y2 receptor upregulation may be an important component of the response to injury in SMG and that recovery of normal physiological function may signal a decreased requirement for P2Y2 receptors.

salivary glands; tissue damage and regeneration; receptor regulation


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

EXTRACELLULAR NUCLEOTIDES exert a broad range of physiological responses, including modulation of vascular tone, platelet aggregation, and transepithelial chloride secretion, via activation of P2 nucleotide receptors (3, 18, 24, 25). Four P2 receptor subtypes, P2X4 and P2X7 ligand-gated ion channels and P2Y1 and P2Y2 G protein-coupled receptors, have been identified in mammalian salivary glands (39).

Data obtained in a previous study demonstrated that, when dispersed cell aggregates from rat submandibular gland (SMG) are placed in culture, the expression and activity of the P2Y2 receptor increase as a function of time in culture (41). P2Y2 receptor activity and mRNA levels were also found to increase in vivo after unilateral SMG duct ligation, whereas no change was observed in the contralateral, nonligated gland (41). These observations indicated that a component of the changes in gland organization and structure that occur upon organ disruption is an upregulation of P2Y2 receptors.

Studies of the responses of SMG to ligation of the main excretory duct have established that both acinar cells and cells of the granular ducts are markedly altered morphologically and functionally (9, 13, 26, 34). The altered morphology and function of ligated salivary glands recover toward the normal state after removal of the ligature (2, 4, 5, 19, 23, 36, 37). These observations suggested that the ligated SMG model might be a useful system for studying P2Y2 receptor changes associated with damage and repair processes. If P2Y2 receptors are specifically important in tissue responses to damage, the prediction would be that as the SMG recovers after ligature removal, P2Y2 receptor expression and activity would return to the low levels found in the normal gland.

Therefore, we studied the effects of excretory duct ligation and ligature removal in rat SMG to elucidate the relationship between duct ligation and removal of the ligature, on one hand, and P2Y2 receptor expression and activity on the other. Our results confirm the prediction that upregulation of functional P2Y2 receptors in SMG cells after duct ligation returns to the normal state after removal of the ligature.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

SMG duct ligation. Unilateral ligation of the SMG main excretory duct was performed as described by Turner et al. (41). Sprague-Dawley rats of either sex weighing between 150 and 200 g were anesthetized with pentobarbital, and the main excretory duct of the SMG on one side of the neck was dissected. Surrounding connective tissues were separated from the duct under a surgical microscope. The duct was tied securely with surgical sutures at a point ~7 mm distal to the gland hilum, with particular care taken to avoid ligation of the accompanying nerves and blood vessels. The incision was closed with surgical clamps. Groups of animals were killed by anesthetic overdose 1.5, 3, 5, or 7 days after ligation. In a second series of experiments, ligatures were removed after 3 days of ligation, and the glands were allowed to recover for 3, 7, 14, or 21 days. In both series of experiments, the SMG were removed, weighed, and used for the preparation of dispersed cell aggregates that were used in functional studies and as a source of mRNA for semiquantitative RT-PCR. Morphological assessments and in situ hybridization experiments were performed on a separate group of animals after these same surgical procedures. In each case, the contralateral, nonligated glands were removed and used as the control.

Morphological evaluation. SMG were prepared for microscopic evaluation as described previously (45). Briefly, after a representative portion of SMG was removed, minced pieces (ca. 1 × 1 × 2 cm) of the gland were placed for 3 h at room temperature in fixative [4% (vol/vol) glutaraldehyde, 2.5% (vol/vol) DMSO, and 0.1 mM CaCl2 in 0.1 M sodium phosphate buffer, pH 7.4], rinsed three times (5, 15, and 30 min) in 0.1 M sodium phosphate buffer (pH 7.4) containing 0.1 mM CaCl2 and 0.15 M sucrose, and postfixed in 1% (wt/vol) osmium tetroxide. After being embedded in epoxy resin, semithin (~1 µm) sections for high-resolution light microscopy were stained with methylene blue and azure II.

Preparation of dispersed cell aggregates from rat SMG. Dispersed cell aggregates from SMG were prepared as described by Turner and Camden (38). Rats were anesthetized by pentobarbital, and SMG were removed. The glands were finely minced with scissors in a dispersion medium consisting of DMEM-Ham's F-12 (1:1) containing 2.7 units of purified collagenase and 10 units of hyaluronidase/mg of tissue, and cells were incubated for 40 min at 37°C in the same medium with aeration of 95% O2-5% CO2. Cell aggregates were dispersed further by pipetting at 20, 30, and 40 min of incubation and then washed three times with enzyme-free assay buffer (in mM: 120 NaCl, 4 KCl, 1.2 KH2PO4, 1.2 MgSO4, 1 CaCl2, 10 glucose, and 15 HEPES, pH 7.4) containing 1% (wt/vol) BSA and filtered through nylon mesh. The cell preparation was then resuspended in assay buffer containing 0.1% BSA for use in functional assays and for isolation of total RNA.

Intracellular free Ca2+ concentration measurements. Intracellular free Ca2+ concentration ([Ca2+]i) was quantitated with the Ca2+-sensitive fluorescent dye, fura 2-AM, in a spectrofluorometer (Spex Industries, Edison, NJ) by the method of Grynkiewicz et al. (15), as modified for salivary gland aggregates (29). Dispersed cell aggregates in assay buffer were preloaded with 2.0 µM fura 2-AM for 30 min at 37°C, followed by incubation for 30 min in the absence of the dye. Cell aliquots (2 ml) were placed in cuvettes, and changes in the 340/380 nm fluorescence ratio (emission at 550 nm) in response to the P2Y2 receptor subtype-selective agonist, UTP (100 µM), were detected and converted to [Ca2+]i after minimum and maximum fluorescence values were obtained with digitonin and EGTA, respectively. Response to the muscarinic cholinoceptor agonist, carbamylcholine (carbachol), was used as a reference.

For measuring Ca2+ mobilization in individual cells, we used an InCyt Dual-Wavelength Fluorescence Imaging System (Intracellular Imaging, Cincinnati, OH). Dispersed cell aggregates from nonligated and 3-day ligated SMG were prepared and loaded with fura 2-AM using the procedure described above. The cells were adhered to coverslips treated with Cell-Tak (Becton Dickinson Labware, Bedford, MA) and incubated for 20 min at 37°C according to the manufacturer's instructions. Cells under the fluorescent microscope were stimulated with UTP (100 µM) or carbachol (500 µM). The minimum fluorescence ratio (Rmin) was determined by adding 8 mM Tris EGTA, and the maximum ratio (Rmax) was determined by adding 4 µM ionomycin and 13 mM CaCl2, according to the method of Melvin et al. (27).

Semiquantitative RT-PCR. Levels of specific SMG mRNAs were quantitated by methods described previously (41). Total RNA was isolated from SMG cells using a RNeasy kit (Qiagen, Chatsworth, CA) and treated with RNase-free DNase. The quantity and quality of total RNA were assessed by spectrophotometry and agarose gel electrophoresis before and after the DNase treatment. Total RNA (1 µg) was used as a template for oligo(dT)18-primed first strand cDNA synthesis using a cDNA synthesis kit (Clontech, Palo Alto, CA). Ten percent of the cDNA was used as template in the PCR, using primers specific for the P2Y2 receptor. The upstream (5'-CTTCAACGAGGACTTCAAGTACGTGC-3', representing nucleotides 78-103) and downstream (5'-CATGTTGATGGCGTTGA-GGGTGTGG-3', representing nucleotides 855-831) primers were designed from the coding region of the rat P2Y2 receptor cDNA (32). This primer set was used to construct a 607-bp exogenous DNA fragment using a PCR MIMIC construction kit (Clontech, Palo Alto, CA). This mimic fragment (0.02 amol) was then used as the internal standard in the PCR reaction. The PCR reaction was performed with 3 units of Vent(exo-) DNA polymerase, 0.25 units of Vent DNA polymerase, and 20 pmol of each primer in a 50-µl reaction volume. The PCR conditions were as follows: jump start for 1 min at 94°C, denaturation for 1 min at 94°C, annealing for 1 min at 60°C, and extension at 72°C for 47 s, for 30 cycles, followed by a 10-min extension at 72°C. The resulting amplified PCR products were resolved on a 2% (wt/vol) agarose ethidium bromide gel. Wild-type and mouse P2Y2 receptor mRNA-transfected 1321N1 astrocytoma cells (10) also were used as negative and positive controls, respectively. The amplified bands were visualized with ultraviolet light. Relative densities of P2Y2 receptor and mimic PCR products were quantified by Gel Doc 2000 gel documentation system (Bio-Rad, Hercules, CA).

In situ hybridization. In situ hybridization was performed using digoxigenin-labeled P2Y2 receptor riboprobes. The 778-bp fragment of P2Y2 was generated by RT-PCR with the same primers as described above and ligated into pCR II transcription vector according to manufacturer's guideline for TA cloning kit (Invitrogen, Carlsbad, CA). The plasmid pCR II-P2Y2 was used for synthesis of both antisense and sense P2Y2 riboprobes during in vitro transcription using a digoxigenin RNA labeling kit (Boehringer Mannheim, Indianapolis, IN). The P2Y2 riboprobe was identified on a 1% (wt/vol) agarose formaldehyde RNA gel and precipitated with 0.5 M LiCl and 75 µl of 100% ethanol at -80°C overnight, then resuspended in diethyl pyrocarbonate (DEPC) water.

Rat SMG were removed under anesthesia (pentobarbital) and immersed in 2-methylbutane in liquid nitrogen until frozen completely. Frozen SMG tissues were kept at -80°C before being mounted and sectioned with a cryostat at -20°C. The tissue sections (10 µm) were placed on positively-charged Superfrost/Plus microscope slides (Fisher Scientific) and stored at -80°C. SMG sections were defrosted on foil followed by fixation in 4% (wt/vol) paraformaldehyde in 1× PBS for 10 min at room temperature. All of the solutions for in situ hybridization (except posthybridization washes) were treated with 0.1% (vol/vol) DEPC and used at room temperature. After fixation, the sections were washed twice with 2× PBS for 5 min, treated with 0.25% (vol/vol) acetic anhydride in 1× tetraethylammonium for 10 min, and washed twice with 2× standard sodium citrate (SSC) for 5 min. For prehybridization, tissue sections were covered with hybridization buffer [4× SSC, 0.5× Denhardt's, 1 mM EDTA, 10 mM phosphate buffer, pH 7.0, 0.1 mg/ml salmon sperm DNA, 0.1 mg/ml yeast tRNA, 100 mg/ml dextran sulfate, and 50% (vol/vol) deionized formamide] and incubated at 45°C in a humidified chamber for at least 3 h. Riboprobes (100 ng/ml sense or antisense) in hybridization buffer were heated for 5 min at 65°C, quenched on ice, and then added to the tissue sections after removal of the buffer used in the prehybridization step. Tissue sections were covered with HybriSlip (RPI, Mount Prospect, IL) and hybridized overnight at 45°C in a humidified chamber. The following day, tissue sections were washed sequentially as follows: 2× SSC for 10 min at 45°C; standard Tris-EDTA (STE) buffer (20 mM Tris · HCl buffer, pH 7.5, containing 500 mM NaCl and 1 mM EDTA) for 5 min at room temperature; 10 µg/ml RNase A in STE buffer for 30 min at room temperature; STE buffer for 10 min at 50°C; twice with 50% formamide, 1× SSC for 20 min at 45°C; and 0.2× SSC for 20 min at 45°C. Immunological detection was preceded immediately by washing the sections for 1 min with washing buffer (DIG Wash and Block Buffer Set, Boehringer Mannheim). Sections were incubated with blocking buffer (DIG Wash and Block Buffer Set) for 30 min at 37°C, and then alkaline phosphatase-conjugated anti-digoxigenin antibody, diluted 1:250 in blocking buffer, was added for 1 h at room temperature. Sections were washed three times with washing buffer for 5 min and once for 10 min with detection buffer (DIG Wash and Block Buffer Set). The sections were incubated with the chromogenic substrate, nitro blue tetrazolium/5-bromo-4-chloro-3-indolylphosphate-p-toluidine salt (Boehringer Mannheim), for 16 h in the dark at room temperature. After the reaction was stopped with 10 mM Tris buffer (pH 8.0) containing 1 mM EDTA, sections were counterstained with nuclear fast red (Zymed, San Francisco, CA) and mounted with Histomount mounting solution (Zymed) following the manufacturer's guidelines.

Data analysis. The significance of differences between mean values was assessed by ANOVA with Bonferroni's post hoc test. Differences with P values <=  0.05 were considered significant.

Materials. The following materials were purchased from the indicated sources: chromatographically purified collagenase, Worthington Biochemical (Freehold, NJ); fura 2-AM, Molecular Probes (Eugene, OR); RNase-free DNase, Promega (Madison, WI); RNase inhibitor, Boehringer Mannheim (Indianapolis, IN); and Vent(exo-) DNA polymerase and Vent DNA polymerase, New England Biolabs (Beverly, MA). All other reagents were from Sigma Chemical (St. Louis, MO).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Changes in SMG weight associated with duct ligation. SMG wet weight was monitored during duct ligation and after recovery following ligature removal. Consistent with previous reports (8, 26, 34), the SMG exhibited an initial edematous swelling after duct ligation, as reflected by a 33% increase in gland wet weight after 1.5 days (Table 1). This increase was followed by a progressive decrease so that the ligated gland weight after 3 days (10% above control) had declined toward control values, and the longer ligation times of 5 and 7 days produced decreases in SMG wet weight of 38% and 44% below control, respectively.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Effect of duct ligation and ligature removal on the weight of rat submandibular gland

Morphological evaluation. The decrease in gland weight after duct ligation described above was accompanied by progressive atrophy of the gland (Fig. 1), similar to that reported previously by others (9, 13, 34, 36). After 3 days of duct ligation, light microscopy showed that acini and granular convoluted tubules (GCT) had atrophied and contained very few secretory granules (Fig. 1B), compared with the nonligated gland (Fig. 1A). The acini appeared to be fewer in number. Intracellular sequestration of cellular debris was common in the acini and intercalated ducts (Fig. 1B and Fig. 2), but much less frequent in the larger ducts and GCT. Scattered unequivocal apoptotic bodies were seen, mostly in the acini and intercalated ducts, beginning at 1 day and peaking at 3 days of ligation (Fig. 2), falling off rapidly after removal of ligature. However, most of the somewhat similar dense, darkly stained bodies in the GCT appeared to be coalesced secretory granules (Fig. 2). Mitoses were uncommon (ca. 1 per section) in the gland before ligation and increased only somewhat (ca. 2 to 3 per section) during ligation and for the first 3 days after removal of the ligature.


View larger version (166K):
[in this window]
[in a new window]
 
Fig. 1.   Morphological aspects of rat submandibular glands (SMG) during and after ligation. SMG were fixed, embedded in epoxy resin, and sectioned at ~1 µm as described in METHODS. A: nonligated control. B: after 3 days of duct ligation. C and D: 7 and 14 days after ligature removal, respectively. A, seromucous acini (lucent secretory granules); E, I, S, excretory, intercalated, and striated ducts, respectively; G, granular convoluted tubules (variously dark secretory granules). Methylene blue and azure II stains. Original magnification ×400.



View larger version (148K):
[in this window]
[in a new window]
 
Fig. 2.   Higher magnification photomicrograph of SMG after 3 days of duct ligation. Intracellular sequestration of cellular debris is common in acini (A) and intercalated ducts, but occurs less frequently in striated ducts and granular convoluted tubules (G). A mitotically dividing cell (m) is in the large duct (upper left), and immediately beneath this is a closely apposed pair of nuclei suggestive of a recent division. An adjacent intercalated duct contains a nucleus with the semilunar chromatin fragments typical of an apoptotic body (large arrow). The darkly stained, round objects (small arrow) in the phagosomes within the labeled granular convoluted tubule appear to be coalesced secretory granules rather than apoptotic bodies. Magnification ×830.

On removal of the ligature, the gland revealed morphological evidence of regeneration. One week after the ligature was removed, the acini were almost fully recovered, whereas the GCT still had very few secretory granules (Fig. 1C). Two weeks after removal of the ligature, all elements appeared to be fully recovered to the control condition (Fig. 1D).

Assessment of P2Y2 receptor activity during ligation and recovery. We reported previously that P2Y2 receptor activity and mRNA levels are increased after duct ligation, whereas little or no P2Y2 receptor activity or mRNA can be detected in the normal gland (41). To extend these results over a wider range of ligation times and to assess the effect of ligature removal on SMG P2Y2 receptor activity, changes in the intracellular free Ca2+ concentration were measured in dispersed SMG cells in response to the P2Y2 agonist, UTP, after various times of ligation and recovery. As shown in Fig. 3A, changes in [Ca2+]i in SMG cells in response to UTP (100 µM) were increased significantly at all time points after duct ligation, whereas no significant response to UTP was obtained in cells isolated from the contralateral, nonligated gland at any time point. These results thus reflect increased P2Y2 receptor activity in SMG cells after duct ligation.


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 3.   Effect of duct ligation on agonist-induced increases in intracellular free Ca2+ concentration ([Ca2+]i) in rat SMG cells. The main excretory ducts of rat SMG were unilaterally ligated, as described in METHODS. After 1.5, 3, 5, or 7 days of ligation, dispersed cell aggregates were prepared and assayed immediately for changes in [Ca2+]i in response to UTP (100 µM; A) or carbachol (500 µM; B), using approximately equal numbers of cells from each preparation. In each experimental animal, the contralateral, nonligated gland served as control. Values are means ± SE of 3 experiments except for 3 days of ligation where n = 7. * Indicates a significant difference from the contralateral, nonligated gland.

The well-established salivary gland [Ca2+]i increase in response to muscarinic cholinergic receptor activation (12, 22, 41) was used for comparison in these studies. Changes in [Ca2+]i in the presence of a maximally effective concentration (500 µM) of the muscarinic receptor agonist, carbachol, were unaffected by ligation periods that produced significant increases in P2Y2 receptor activity (Fig. 3B), although a trend toward reduced activity was seen after 7 days of ligation. The key observation from these studies is that duct ligation for up to 5 days increases P2Y2 receptor activity but does not profoundly alter overall gland responsiveness, as reflected by the maintenance of muscarinic receptor activity.

We next examined the effect of ligature removal on P2Y2 receptor activity. Ligatures were removed following a 3-day ligation, and the glands were allowed to recover for 3, 7, 14, or 21 days. During the recovery period, responses to UTP progressively declined and, after 14 days of recovery, returned to the low levels obtained in nonligated glands (Fig. 4A). There was no significant change in the carbachol-induced [Ca2+]i increase during the recovery period (Fig. 4B).


View larger version (22K):
[in this window]
[in a new window]
 
Fig. 4.   Effect of ligature removal on agonist-induced increases in [Ca2+]i in rat SMG cells. After 3 days of ligation, the ligatures were removed. At the indicated times, animals were killed, and dispersed SMG cell aggregates were prepared as described in METHODS. Changes in [Ca2+]i in response to UTP (100 µM; A) or carbachol (500 µM; B) were determined in approximately equal numbers of cells from each preparation. In each experimental animal, the contralateral, nonligated gland served as control. Values are means ± SE of 3 experiments except for the zero time point where n = 7. * Indicates a significant difference from the contralateral, nonligated gland.

Results from studies of Ca2+ mobilization in individual cells and small cell clusters confirmed that cells from nonligated SMG do not respond to UTP, whereas cells from 3-day ligated glands exhibited consistent Ca2+ responses to 100 µM UTP. Nearly all cells or cell clusters from 3-day ligated glands (96%, 23 out of 24) responded to UTP; the Ca2+ increase was 96 ± 10 nM (mean ± SE). The carbachol-induced [Ca2+]i increase with this methodology was 440 ± 90 nM (mean ± SE). Basal [Ca2+]i values were consistently in the 50 to 100 nM range.

Assessment of P2Y2 receptor mRNA levels during ligation and recovery. Semiquantitative RT-PCR was used to assess changes in the steady-state abundance of P2Y2 receptor mRNA in SMG cells during ligation and recovery from ligation. Based on our previous observations (41) and the functional results described above, the prediction was that P2Y2 receptor mRNA levels would increase after ligation and decrease as a function of time after ligature removal. The primers used were based on two conserved regions among the human, mouse, and rat P2Y2 receptor mRNA sequences and predicted to give a product of 778 bp in rat tissues. The mimic included in the PCR reactions was designed to generate a product of 607 bp to allow differentiation from the amplified P2Y2 receptor product after gel electrophoresis (41). As shown in Fig. 5, P2Y2 receptor mRNA, which is only faintly discernible in nonligated control glands, was increased about 15-fold after 3 days of duct ligation. During the recovery period, the P2Y2 receptor mRNA level declined as a function of time, with intermediate mRNA levels observed at 3 days and 7 days, and a return to control levels by 14 days. Thus there was a close correlation between changes in P2Y2 receptor activity (Fig. 4A) and mRNA levels (Fig. 5).


View larger version (34K):
[in this window]
[in a new window]
 
Fig. 5.   Effect of duct ligation and ligature removal on relative P2Y2 receptor (P2Y2-R) mRNA levels in rat SMG cells. Semiquantitative RT-PCR for P2Y2 receptor mRNA in control, ligated, and recovery SMG was performed as described in METHODS. Samples were electrophoresed on a 2% (wt/vol) agarose ethidium bromide gel, and bands were visualized under ultraviolet light (top). Relative densities of P2Y2 receptor and mimic PCR products were quantified by Gel Doc 2000 gel documentation system (Bio-Rad, Hercules, CA; bottom). Top right: wt-1321N1 and P2Y2-1321N1 represent vector only and mouse P2Y2 receptor mRNA-transfected 1321N1 astrocytoma cells, respectively. Top left: bp markers from RNA ladder.

Detection of P2Y2 receptor mRNA by in situ hybridization during duct ligation and recovery. To determine whether the increase in P2Y2 receptor mRNA levels was cell type specific or a generalized response, the abundance and cellular localization of P2Y2 receptor mRNA in frozen SMG sections were assessed by in situ hybridization using a 778-base riboprobe. As shown in Fig. 6, P2Y2 receptor mRNA in nonligated glands was only faintly discernible (Fig. 6A). After a 3-day ligation, P2Y2 receptor mRNA was substantially increased in acini and intercalated ducts and, to a lesser extent, in the larger ducts (Fig. 6C). This increase reverted back to control levels after 14 days of recovery (Fig. 6D). Figure 6B shows hybridization using the analogous sense riboprobe after 3 days of ligation.


View larger version (165K):
[in this window]
[in a new window]
 
Fig. 6.   In situ hybridization of P2Y2 receptor mRNA in rat SMG. Sections of rat SMG were prepared and incubated with digoxigenin-labeled P2Y2 receptor riboprobe as described in METHODS. A: nonligated control; B and C: 3 days after duct ligation; D: 14 days after ligature removal. In A, C, and D, the antisense probe was used, whereas in B, the sense probe was used. A, acini and intercalated ducts; D, large ducts, i.e., excretory and striated ducts and granular convoluted tubules. The punctate blue precipitate indicates the location and approximate relative abundance of P2Y2 receptor mRNA. The counterstain is nuclear fast red. Original magnification ×320.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The P2Y2 nucleotide receptor has been detected in a variety of permanent SMG and parotid gland cell lines of salivary gland origin (30, 31, 44). In addition, we have observed previously that, although only marginally detectable in freshly isolated dispersed salivary gland cells, P2Y2 receptor activity rises dramatically following short-term culture (41). This time-dependent increase in the response to UTP in SMG cells is contingent upon transcription and translation and is accompanied by increases in P2Y2 receptor mRNA over the same time scale. Furthermore, ligation of the main excretory duct of the rat SMG for 3 days was shown to also produce increases in P2Y2 receptor activity and mRNA level (41). These results suggested a role for P2Y2 receptors in the response to damage and disruption of normal salivary gland structure and function. Similarly, Seye et al. (33) observed increased P2Y2 mRNA levels in the cells of intimal thickenings of balloon catheterized rat aortas, compared with normal adult aorta. These findings are consistent with the idea that tissue damage and repair processes include increased P2Y2 receptor expression and activity. A reasonable extension of this argument is that P2Y2 receptor activity and expression should return to normal levels as the tissue recovers.

Our results from the studies examining Ca2+ responses in single cells or small clusters of cells indicated that the increased responsiveness to UTP is generalized, rather than restricted to only a few cells. The significance of this widespread, low-magnitude Ca2+ response to UTP after gland ligation remains to be determined. It is interesting to note that additional P2Y2 receptor-mediated effects also occur in the ligated glands, including an increase in UTP-stimulated mitogen-activated protein kinase (MAPK) phosphorylation (Ahn and Turner, unpublished observations).

There is a wealth of evidence indicating that the damage induced by rat SMG ligation is fully reversible after ligature removal (2, 19, 23, 35-37). We thus used this model system to determine whether or not the in vivo upregulation of P2Y2 receptors observed upon duct ligation was reversed as the gland recovered. Examination of the ligated glands by comparing wet weights of ligated vs. control glands and by microscopic morphological analysis confirmed the reversible nature of gland atrophy associated with the ligation and ligature removal protocol. A progressive decrease in gland weight was observed after duct ligation (Table 1). Morphological examination showed that 1) ligation of the SMG duct produced atrophic changes in the gland, with the acini being predominantly affected, and 2) after removal of the ligature, the glands revealed evidence of regeneration (Fig. 1). After 14 days of recovery, even though all elements in the SMG cells appeared to be fully recovered by light microscopic observation, gland weight was not restored to normal (Table 1). It has been suggested that apoptosis is implicated as the mechanism responsible for reduction in cell numbers during the involution of hyperplasia in liver (7) and pancreas (28). It also has been shown that apoptosis is involved in acinar atrophy after ligation of excretory duct of pancreas (42) and parotid gland (43) in rats. The occurrence of considerable intracellularly sequestered debris and occasional apoptotic bodies supports cell death as a factor in the overall gland atrophy observed in the present study. However, the extent to which these phenomena were the result of apoptosis and other types of cell death (11, 42, 43) or autophagocytosis of secretory granules and cellular structures in the process of cell atrophy and not cell death (16, 17) was not clear. The substantially smaller weight of the ligated glands, despite the rapid reconstitution of individual glandular elements after removal of ligature, indicates that there not only was a loss of cells but also a failure to recover cell number. This was supported by the surprisingly low prevalence of mitoses during and following ligation. Further studies are needed to determine the extent to which the reduced size of the SMG after 14 days of recovery from duct ligation is a consequence of the death, atrophy, and dedifferentiation of the several parenchymal cell types.

After removal of the duct ligature, the increases in P2Y2 receptor activity (Fig. 4) and mRNA expression (Fig. 5) in rat SMG cells due to ligation return to the low level found in nonligated glands (Fig. 6). Although no methods for accurately quantifying native P2Y2 receptor protein levels are currently available, the close relationship between the changes in receptor activity and mRNA levels supports the suggestion that the changes in responsiveness to UTP are due to altered P2Y2 receptor expression. These observations establish the ligated rat SMG as an attractive model for studying reversible P2Y2 receptor regulation in vivo.

To date, many genes encoding members of the P2 nucleotide receptor family have been cloned (1, 6, 21). Despite this progress, relatively little is known about the regulation of P2 receptor expression, including the P2Y2 receptor. Gorodeski et al. (14) found that retinoids increase P2Y2 receptor mRNA and activity in CaSki human uterine cervical cells. In addition, Hou and colleagues (20) have demonstrated that P2Y2 receptor upregulation in vascular smooth muscle cells involves MAPK pathways, a finding consistent with observations made in our laboratory with rat SMG cells in short-term culture (Ahn and Turner, unpublished data). A more complete elucidation of the regulatory processes involved in P2Y2 receptor gene expression in SMG and other tissues is an essential component in defining the role P2Y2 receptors have in the response to damage of salivary glands and other tissues.


    ACKNOWLEDGEMENTS

We thank Rodney McNutt for assistance in preparing specimens for morphological evaluation.


    FOOTNOTES

This work was supported by National Institute of Dental and Craniofacial Research Grant DE-07389 and the Department of Veterans Affairs.

Address for reprint requests and other correspondence: J. T. Turner, Dept. of Pharmacology, School of Medicine, Univ. of Missouri-Columbia, M561 Health Science Center, One Hospital Drive, Columbia, MO 65212 (E-mail: JTT{at}missouri.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. §1734 solely to indicate this fact.

Received 5 November 1999; accepted in final form 28 February 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Barnard, EA, Webb TE, Simon J, and Kunapuli SP. The diverse series of recombinant P2Y purinoceptors. Ciba Found Symp 198: 166-180, 1996[ISI][Medline].

2.   Bhaskar, SN, Lilly GE, and Bhussry B. Regeneration of the salivary glands in the rabbit. J Dent Res 45: 37-41, 1966[ISI][Medline].

3.   Boarder, MR, and Hourani SMO The regulation of vascular function by P2 receptors: multiple sites and multiple receptors. Trends Pharmacol Sci 19: 99-107, 1998[ISI][Medline].

4.   Burford-Mason, AP, Cummins MM, Brown DH, MacKay AJ, and Dardick I. Immunohistochemical analysis of the proliferative capacity of duct and acinar cells during ligation-induced atrophy and subsequent regeneration of rat parotid gland. J Oral Pathol Med 22: 440-446, 1993[ISI][Medline].

5.   Burgess, KL, and Dardick I. Cell population changes during atrophy and regeneration of rat parotid gland. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 85: 699-706, 1998[ISI][Medline].

6.   Burnstock, G. P2 purinoceptors: historical perspective and classification. Ciba Found Symp 198: 1-28, 1996[ISI][Medline].

7.   Columbano, A, Ledda-Columbano GM, Coni PP, Faa G, Liguori C, Santa Cruz G, and Pani P. Occurrence of cell death (apoptosis) during the involution of liver hyperplasia. Lab Invest 52: 670-675, 1985[ISI][Medline].

8.   Ekström, J, and Rosengren E. Changes in diamine and polyamine metabolism in the duct-ligated submaxillary and sublingual glands of the rat. Acta Physiol Scand 119: 287-292, 1983[ISI][Medline].

9.   Emmelin, N, Garrett JR, and Ohlin P. Secretory activity and the myoepithelial cells of salivary glands after duct ligation in cats. Arch Oral Biol 19: 275-283, 1974[ISI][Medline].

10.   Erb, L, Garrad R, Wang Y, Quinn T, Turner JT, and Weisman GA. Site-directed mutagenesis of P2U purinoceptors: positively charged amino acids in transmembrane helices 6 and 7 affect agonist potency and specificity. J Biol Chem 270: 4185-4188, 1995[Abstract/Free Full Text].

11.   Formigli, L, Papucci L, Tani A, Schiavone N, Tempestini A, Orlandini GE, Capaccioli S, and Orlandini SZ. Apoptosis: morphological and biochemical exploration of a syncretic process of cell death sharing apoptosis and necrosis. J Cell Physiol 182: 41-49, 2000[ISI][Medline].

12.   Foskett, JK, and Wong DCP [Ca2+]i inhibition of Ca2+ release-activated Ca2+ influx underlies agonist- and thapsigargin-induced [Ca2+]i oscillations in salivary acinar cells. J Biol Chem 269: 31525-31532, 1994[Abstract/Free Full Text].

13.   Garrett, JR, and Parsons PA. Changes in the main submandibular salivary duct of rabbits resulting from ductal ligation. Z Mikrosk Anat Forsch 93: 593-608, 1979[ISI][Medline].

14.   Gorodeski, GI, Burfeind P, Gan SU, Pal D, and Abdul-Karim FW. Regulation by retinoids of P2Y2 nucleotide receptor mRNA in human uterine cervical cells. Am J Physiol Cell Physiol 275: C758-C765, 1998[Abstract].

15.   Grynkiewicz, G, Poenie M, and Tsien RY. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260: 3440-3450, 1985[Abstract].

16.   Hand, AR. The effects of acute starvation on parotid acinar cells. Ultrastructural and cytochemical observations on ad libitum-fed and starved rats. Am J Anat 135: 71-92, 1972[ISI][Medline].

17.   Hand, AR, and Ho B. Liquid-diet-induced alterations of rat parotid acinar cells studied by electron microscopy and enzyme cytochemistry. Arch Oral Biol 26: 369-380, 1981[ISI][Medline].

18.   Harden, TK, Lazarowski ER, and Boucher RC. Release, metabolism and interconversion of adenine and uridine nucleotides: implications for G protein-coupled P2 receptor agonist selectivity. Trends Pharmacol Sci 18: 43-46, 1997[ISI][Medline].

19.   Hayashi, H. Experimental study on recovery process of duct-ligated submandibular gland. Shigaku 73: 1712-1727, 1986[Medline].

20.   Hou, M, Möller S, Edvinsson L, and Erlinge D. MAPKK-dependent growth factor-induced upregulation of P2Y2 receptors in vascular smooth muscle cells. Biochem Biophys Res Commun 258: 648-652, 1999[ISI][Medline].

21.   Humphrey, PPA, Buell G, Kennedy I, Khakh BS, Michel AD, Surprenant A, and Trezise DJ. New insights on P2X purinoceptors. Arch Pharmacol Res (Seoul) 352: 585-596, 1995.

22.   Izutsu, KT, Fatherazi S, Wellner RB, Herrington J, Belton CM, and Oda D. Characteristics and regulation of a muscarinically activated K current in HSG-PA cells. Am J Physiol Cell Physiol 266: C58-C66, 1994[Abstract/Free Full Text].

23.   Junqueira, LC, and Rabinovitch M. Reversibility of the phenomena induced by excretory duct ligature in the rat submaxillary gland. Tex Rep Biol Med 12: 94-97, 1954[ISI].

24.   King, BF, Townsend-Nicholson A, and Burnstock G. Metabotropic receptors for ATP and UTP: exploring the correspondence between native and recombinant nucleotide receptors. Trends Pharmacol Sci 19: 506-514, 1998[ISI][Medline].

25.   Kunapuli, SP, and Daniel JL. P2 receptor subtypes in the cardiovascular system. Biochem J 336: 513-523, 1998[ISI][Medline].

26.   Martinez, JR, Bylund DB, and Cassity N. Progressive secretory dysfunction in the rat submandibular gland after excretory duct ligation. Arch Oral Biol 27: 443-450, 1982[ISI][Medline].

27.   Melvin, JE, Koek L, and Zhang GH. A capacitative Ca2+ influx is required for sustained fluid secretion in sublingual mucous acini. Am J Physiol Gastrointest Liver Physiol 261: G1043-G1050, 1991[Abstract/Free Full Text].

28.   Oates, PS, Morgan RGH, and Light AM. Cell death (apoptosis) during pancreatic involution after raw soya flour feeding in the rat. Am J Physiol Gastrointest Liver Physiol 250: G9-G14, 1986[ISI][Medline].

29.   Park, MK, Garrad RC, Weisman GA, and Turner JT. Changes in P2Y1 nucleotide receptor activity during the development of rat salivary glands. Am J Physiol Cell Physiol 272: C1388-C1393, 1997[Abstract/Free Full Text].

30.   Quissell, DO, Barzen KA, Gruenert DC, Redman RS, Camden JM, and Turner JT. Development and characterization of SV40 immortalized rat submandibular acinar cell lines. In Vitro Cell Dev Biol 33: 164-173, 1997.

31.   Quissell, DO, Barzen KA, Redman RS, Camden JM, and Turner JT. Development and characterization of SV40 immortalized rat parotid acinar cell lines in vitro. Cell Dev Biol 34: 58-67, 1998.

32.   Rice, WR, Burton FM, and Fiedeldey DT. Cloning and expression of the alveolar type II cell P2u-purinergic receptor. Am J Respir Cell Mol Biol 12: 27-32, 1995[Abstract].

33.   Seye, CI, Gadeau AP, Daret D, Dupuch F, Alzieu P, Capron L, and Desgranges C. Overexpression of the P2Y2 purinoceptor in intimal lesions of the rat aorta. Arterioscler Thromb Vasc Biol 17: 3602-3610, 1997[Abstract/Free Full Text].

34.   Shiba, R, Hamada T, and Kawakatsu K. Histochemical and electron microscopical studies on the effect of duct ligation of rat salivary glands. Arch Oral Biol 17: 299-309, 1972[ISI][Medline].

35.   Sumitomo, S, Kunikata-Sumitomo M, Hashimoto J, Nambu M, Shrestha M, Kurenuma S, Jayasinghe N, and Mori M. Cell proliferation activities in duct-ligated submandibular gland. Acta Histochem 28: 1-9, 1995.

36.   Tamarin, A. Submaxillary gland recovery from obstruction. I. Overall changes and electron microscopic alterations of granular duct cells. J Ultrastruct Res 34: 276-287, 1971[ISI][Medline].

37.   Tamarin, A. Submaxillary gland recovery from obstruction. II. Electron microscopic alterations of acinar cells. J Ultrastruct Res 34: 288-302, 1971[ISI].

38.   Turner, JT, and Camden JM. The influence of vasoactive intestinal peptide receptors in dispersed acini from rat submandibular gland on cyclic AMP production and mucin release. Arch Oral Biol 35: 103-108, 1990[ISI][Medline].

39.   Turner, JT, Landon LA, Gibbons SJ, and Talamo BR. Salivary gland P2 nucleotide receptors. Crit Rev Oral Biol Med 10: 210-224, 1999[Abstract].

40.   Turner, JT, Redman RS, Camden JM, Landon LA, and Quissell DO. A rat parotid gland cell line, Par-C10, exhibits neurotransmitter-regulated transepithelial anion secretion. Am J Physiol Cell Physiol 275: C367-C374, 1998[Abstract/Free Full Text].

41.   Turner, JT, Weisman GA, and Camden JM. Upregulation of P2Y2 nucleotide receptors in rat salivary gland cells during short-term culture. Am J Physiol Cell Physiol 273: C1100-C1107, 1997[Abstract/Free Full Text].

42.   Walker, NI. Ultrastructure of the rat pancreas after experimental duct ligation. I. The role of apoptosis and intraepithelial macrophages in acinar cell deletion. Am J Pathol 126: 439-451, 1987[Abstract].

43.   Walker, NI, and Gobé GC. Cell death and cell proliferation during atrophy of the rat parotid gland induced by duct obstruction. J Pathol 153: 333-344, 1987[ISI][Medline].

44.   Yu, H, and Turner JT. Functional studies in the human submandibular duct cell line, HSG-PA, suggest a second salivary gland receptor subtype for nucleotides. J Pharmacol Exp Ther 259: 1344-1350, 1991[Abstract].

45.   Yu, JH, and Redman RS. Physostigmine-induced salivary secretion in the rat. Arch Oral Biol 35: 209-218, 1990[ISI][Medline].


Am J Physiol Cell Physiol 279(2):C286-C294
0363-6143/00 $5.00 Copyright © 2000 the American Physiological Society