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
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ABSTRACT |
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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
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INTRODUCTION |
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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.
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METHODS |
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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.
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).
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RESULTS |
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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.
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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.
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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.
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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).
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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.
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DISCUSSION |
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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.
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ACKNOWLEDGEMENTS |
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We thank Rodney McNutt for assistance in preparing specimens for morphological evaluation.
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FOOTNOTES |
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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.
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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
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
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
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
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
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
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
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
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].