ARTICLE |
Correspondence to: Mahmood S. Mozaffari, Dept. of Oral Biology & Maxillofacial Pathology, CB 3710, Medical College of Georgia School of Dentistry, Augusta, Georgia 30912-1128. E-mail: Mmozaffa@mail.mcg.edu
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Summary |
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Taurine exerts a number of actions in mammalian cells, including regulation of ion transport and osmoregulation. The production and secretion of saliva involve transepithelial ion transport, thereby making the plasma-like primary saliva hypotonic before secretion. Therefore, it is plausible to suggest modulation of salivary taurine by muscarinic agents that affect salivary gland function. One of the objectives of this study was to determine tissue content and localization of taurine in the submandibular gland of the rat. Further, we determined whether treatment with muscarinic drugs that either increase (e.g., pilocarpine) or decrease (e.g., propantheline) saliva secretion affects the submandibular gland taurine content. The results indicate that the submandibular gland contains an appreciable amount of taurine (8.9 ± 0.3 µmoles/g wet wt). Further, acute treatment of the rats with either of the muscarinic drugs did not significantly affect tissue taurine content compared to the control group. By contrast, chronic treatment with propantheline, but not pilocarpine, reduced the tissue content of taurine compared to the control rats (p<0.05). Utilizing light microscopic immunohistochemical techniques, intense immunoreactivity was found primarily in the striated ducts of the submandibular gland. Neither pilocarpine nor propantheline treatment led to differential distribution of immunoreactivity in this tissue. In conclusion, the submandibular gland contains an appreciable amount of taurine, primarily in the striated ducts, that can be decreased by chronic muscarinic receptor blockade.
(J Histochem Cytochem 50:527532, 2002)
Key Words: taurine, salivary gland, pilocarpine, propantheline, rat
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Introduction |
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Taurine (2-aminoethanesulfonic acid) is the most abundant free amino acid in mammalian cells. A number of physiological roles have been attributed to taurine, one of the most important being modulation of ion transport. This important action of taurine has been implicated in many of taurine's regulatory effects, including osmoregulation (see
The production and secretion of saliva involve transepithelial transport of ions, thereby regulating saliva tonicity (
The present investigation was prompted for several reasons. First, taurine is an important player in the adaptive responses of cells to osmotic stress (see
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Materials and Methods |
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Chronic Treatment with Muscarinic Drugs
Six-week-old male WistarKyoto rats were obtained from Harlan Laboratories (Indianapolis, Indiana). All rats were maintained at constant humidity (60 ± 5%), temperature (24 ± 1C), and light cycle (06001800 hr.). Two days after arrival the animals were randomly assigned to three groups: (a) the control group (n=5) was given tapwater, (b) the pilocarpine-treated group (n=5) received tapwater containing pilocarpine (5 mg/kg/day), and (c) the propantheline-treated group (n=5) was provided with tapwater containing propantheline bromide (20 mg/kg/day). To achieve these dosages, the concentration of each drug in the drinking fluid was adjusted on the basis of daily fluid intake and body weight before and after administration of each drug. Food and drinking fluid were available ad libitum throughout the study.
Six days after initiation of drug therapy the animals were anesthetized with sodium pentobarbital (60 mg/kg, IP). The left submandibular gland and the left kidney were removed from each rat, weighed, and homogenized in 2% chilled perchloric acid (1:10 w/v). The homogenates were centrifuged (4C, 10,000 rpm) and the supernatants used for the taurine assay (
Immunohistochemical Labeling
The right submandibular glands from the above animals were placed in formalin solution and used to establish tissue localization of taurine utilizing a light microscopic immunohistochemical technique (
Formalin-fixed submandibular glands used for the localization of taurine were dehydrated through a graded series of ethanols and xylene before embedding in paraffin. Five-µm paraffin-embedded tissue sections were cut and collected on glass slides for immunohistochemical labeling.
The polyclonal antibody AB137, used to localize taurine, was purchased from Chemicon International (Temecula, CA). This antibody was created using a taurineglutaraldehydepolylysine immunogen and shows minimal crossreactivity with other amino acids.
Sections of the submandibular glands were processed for immunohistochemical localization of taurine using the avidinbiotinperoxidase technique of
After immunohistochemical processing, the sections were counterstained with Mayer's hematoxylin (Sigma) to aid in morphological identification. Serial sections were also processed as negative controls without incubation in the primary antibody AB137.
Finally, as an index of the effectiveness of drug treatments, 5-µm paraffin-embedded tissue sections were cut and stained with periodic acidSchiff (PAS) reagent. The PAS stain was deemed appropriate because the submandibular gland contains both mucous and serous secretory cells and the PAS reagent labels both the mucous secretory component and other secretory granules of the serous component.
Acute Treatment with Muscarinic Drugs
Male WistarKyoto rats (170190 g) were anesthetized with sodium pentobarbital (60 mg/kg, IP) and implanted with a femoral venous catheter (PE 50 fused to PE 10;
Statistics
All quantitative comparisons between groups were analyzed by ANOVA and Duncan's test was employed for comparison of mean values (significance of criteria of p<0.05). Data are reported as means ± SEM.
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Results |
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Chronic Treatment with Muscarinic Drugs
Body weight was similar among the groups before initiation of drug therapy and was not affected by either pilocarpine or propantheline treatment [173 ± 5 g (control), 171 ± 3 g (pilocarpine-treated), and 171 ± 2 (propantheline-treated)]. Furthermore, no differences were noted among the groups in tissue weights of the left submandibular gland [197 ± 6 mg (control), 200 ± 11 mg (pilocarpine-treated), and 190 ± 9 mg (propantheline-treated)] and the left kidney [698 ± 14 mg (control), 704 ± 26 mg (pilocarpine-treated), and 695 ± 16 mg (propantheline-treated)].
Basal taurine content of the submandibular gland was slightly lower than that of the kidney (Fig 1A; p<0.05). Whereas treatment of the rats with pilocarpine did not affect the submandibular gland content of taurine, treatment with propantheline caused a significant decrease (15%) compared to the control group (Fig 1B).
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Light microscopic examination revealed significant immunoreactivity with taurine-specific antibody. In sections of the submandibular gland from the control group (Fig 2A and Fig 2B), localization was found mainly in the blood vessels and striated ducts, whereas the acini showed no immunoreactivity. Chronic treatment with either pilocarpine (Fig 2D and Fig 2E) or propantheline (Fig 2G and Fig 2H) did not alter the pattern of localization of immunoreactivity. Immunohistochemical processing of the tissue without the use of the primary anti-taurine antibody showed an absence of staining [ Fig 2C (control), 2F (pilocarpine-treated), and 2I (propantheline-treated)].
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To illustrate the effectiveness of the muscarinic drug regimens, tissue sections from the control and drug-treated rats were stained with PAS (Fig 2J2L). The staining intensity was reduced in the submandibular gland sections of the pilocarpine-treated rats compared to the control rats (Fig 2K vs 2J). By comparison, tissue sections from the propantheline-treated rats (Fig 2L) displayed intense staining compared to the tissue sections from either the control or the pilocarpine-treated rats (Fig 2J and Fig 2K, respectively). As a result, the difference in staining was most prominent between the propantheline-treated and the pilocarpine-treated rats (Fig 2L vs 2K). Muscarinic receptor stimulation (e.g., pilocarpine) increases but muscarinic receptor blockade (e.g., propantheline) reduces salivary gland secretion (
Acute Treatment with Muscarinic Drugs
Acute IV administration of either pilocarpine or propantheline to rats for either 5 (right gland) or 10 (left gland) min did not significantly affect tissue taurine content in the pilocarpine-treated [9.6 ± 0.4 (right gland) vs 9.7 ± 0.6 (left gland) µmoles/g wet wt] or propantheline-treated [9.0 ± 0.3 (right gland) vs 8.6 ± 0.1 (left gland) µmoles/g wet wt] rats compared to the control group [8.9 ± 0.4 (right) vs 8.8 ± 0.3 (left) µmoles/g wet wt]. Interestingly, however, the submandibular gland taurine content tended to be lower in the propantheline-treated rats compared to the pilocarpine-treated rats 10 min after initiation of drug administration (8.6 ± 0.1 vs 9.7 ± 0.6 µmoles/g wet wt).
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Discussion |
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The effects of taurine on cell function have received considerable attention in many tissues such as the kidney, heart, and brain (
The lack of a significant effect of acute exposure to either of the muscarinic drugs corroborates previous reports indicating that significant alterations in the tissue taurine pool occur after chronic but not acute experimental manipulations (
The reason for the chronic propantheline-induced reduction in submandibular gland taurine is not apparent from this study. One plausible scenario relates to chronic blockade of the muscarinic receptors and the attendant reduction in salivary flow rate, which would promote the formation of a more hypotonic saliva (see
The modification of the plasma-like primary saliva occurs via ductal reabsorption of Na+ and Cl-, essentially without water, thereby making the final saliva hypotonic. Several transport processes operate in concert to determine the ionic composition of saliva. These include the basolaterally located Na+-K+-ATPase, Cl- and K+ channels, and the Na+/H+ exchanger, together with luminally located Na+ and Cl- channels, K+/H+, Na+/H+, and the Cl-/HCO3- exchangers (see
In conclusion, the demonstration of an appreciable amount of taurine and its preferential localization in the striated ducts of the submandibular gland, as well as modulation of tissue taurine content by chronic muscarinic receptor blockade, suggest a role for taurine in salivary gland function. In light of these observations, it is plausible to postulate a dual role for taurine in salivary gland function, i.e., regulation of duct cell volume as well as determination of ionic composition and tonicity of saliva. In this context, the potential contributions of taurine efflux vs taurine influx to these processes may represent a fertile area for future investigation.
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Acknowledgments |
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Supported by a grant from Taisho Pharmaceutical Company of Japan.
We are grateful to Drs Worku Abebe and Rafik Abdelsayed (Department of Oral Biology and Maxillofacial Pathology, MCG School of Dentistry) for their critical review of the manuscript. We also thank Champa Patel and Anita Smith for technical assistance.
Received for publication May 14, 2001; accepted November 7, 2001.
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