]>MAD2369S0047-6374(00)00176-710.1016/S0047-6374(00)00176-7Elsevier Science Ireland LtdFig. 1Box-plot of the incorporation of tritiated thymidine into submandibular glands in vivo with age and treatment. Results are expressed as counts per min per g gland of six animals. Effect of treatment: significant differences (5%) compared with WO and SAL are marked with (*), compared with SAL with (+) by one-way ANOVA; effect of age: significant differences (5%) compared with 3 m are marked with (†) by one-way ANOVA.Fig. 2Box-plots of submandibular flow rates with age and treatment. Results are expressed as μl/min per gland (a) and μl/min/g gland (b). Values represent six to eight animals in each group. Effect of treatment: significant differences (5%) compared with WO and SAL are marked with (*), compared with SAL with (+) by one-way ANOVA. Effect of age: significant differences (5%) compared with 3 m are marked with (†), compared with 12 m with (¥) by one-way ANOVA.Fig. 3Box-plots of (a) the protein concentration (μg/ml), and (b) of the total amount of protein secreted (μg) with age and treatment. Results represent the values of six to eight animals in each group. Effect of age: significant differences (5%) compared with 3 m are marked with (†) by one-way ANOVA.Fig. 4Submandibular saliva protein analyzed on 10% SDS polyacrylamide gels with a representative sample (15 μg of protein) of each of the three age and treatment groups (Coomassie blue staining). Pre-stained molecular standards (Std) were; phosphorylase B, 130 kDa; bovine serum albumin, (BSA) 80 kDa; ovalbumin, 49.5 kDa; carbonic anhydrase, 32.5 kDa; soybean trypsin inhibitor, 27.5 kDa; lysozyme, 18.5 kDa.Fig. 5Box-plots of (a) the salivary EGF-concentration (mg/ml), and of (b) the salivary EGF content per mg protein secreted (μg/mg). The results express the values of six animals in each group. Effect of treatment: significant differences (5%) compared with SAL are marked with (+), compared with WO with (≠) by one-way ANOVA; effect of age; significant differences (5%) compared with 3 and 12 m are marked with (#), compared with 3 m with (†), compared with 12 m with (¥) by one-way ANOVA.Fig. 6Box-plot of the salivary peroxidase (a) and lysozyme (b) content per mg protein secreted (ng/mg) with age and treatment. The results represent the values of six animals in each group. Effect of treatment: significant differences (5%) compared with WO and SAL are marked with (*), compared with SAL with (+) by one-way ANOVA; effect of age: significant differences (5%) compared with 3 m are marked with (†), compared with 12 m with (¥) by one-way ANOVA.Fig. 7Box-plot of the RNA content in submandibular glands with age and treatment. The results are expressed as μg per g gland of six to nine animals in each group. Effect of age; significant differences (5%) compared with 3 m are marked with (†) by one-way ANOVA.An analysis of submandibular salivary gland function with desipramine and age in female NIA Fischer 344 ratsMarkus MKollera*mmkoller@zzmk.unizh.chRichard ACowmanbMichael GHumphreys-BehercdPhilip JScarpacedeaDepartment of Oral and Maxillofacial Surgery, Claude Denson Pepper Center for Research on Oral Health in Aging, University of Florida, Gainesville, FL 32610, USAbDental Research Unit, Department of Veterans Affairs Medical Center, Miami, FL 33125, USAcDepartment of Oral Biology, University of Florida, Gainesville, FL 32610, USAdDepartment of Pharmacology and Therapeutics, University of Florida, Gainesville, FL 32610, USAeGeriatric Research, Education and Clinical Center, Department of Veterans Affairs Medical Center, Gainesville, FL 32608, USA*Present address: Center for Dental and Oral Medicine, Clinic for Geriatric and Special Care Dentistry, University of Zurich, P.O. Box 322, CH-8028 Zurich, Switzerland. Tel.: +41-1-6343341; fax: +41-1-6344319AbstractDry mouth is one of the major side effects of cyclic antidepressants, which are still a dominating group of psychotherapeutic drugs used in the treatment of depression. In this study we analyzed the effects of 28 day tricyclic antidepressant administration and the reversibility of this treatment following a 15 day washout period on different parameters in submandibular gland function in aging rats. We postulated that desipramine would decrease gland function, accented with age, and delay recovery in senescent animals. In contrast to body weight, desipramine had no effect on glandular wet weight. While glandular DNA synthesis was changed with age and treatment, the concentration of total membrane and soluble proteins was not affected. Flow rate was significantly changed with age, but desipramine increased salivary flow in the youngest animals only. Neither age nor treatment influenced salivary protein concentrations, but the total amount of proteins secreted, revealed perturbation with age. SDS- polyacrylamide gel analysis revealed changes in protein expression with treatment and age. Desipramine decreased epidermal growth factor (EGF) levels in all age groups, but increased the secretion of peroxidase and lysozyme. Analysis of total RNA showed a pronounced decrease with age. These data indicate that desipramine has profound effects on submandibular salivary gland function.KeywordsDesipramineEGFLysozymePeroxidaseSubmandibular glandSaliva1IntroductionThe primary function of salivary glands is to secrete saliva, a fluid composed of water, electrolytes, and various multifunctional proteins. The autonomic sympathetic and parasympathetic receptor systems regulate salivary gland secretion via a co-ordinated sequence of signal transduction and intracellular signalling events (Looms et al., 1998; Ambudkar, 2000). Saliva is an essential fluid for the health of human teeth and oral mucosal surfaces, maintains microbial balance, and supports other oral functions. The basic protective mechanism mediated by saliva is bacterial clearance. But saliva also contains many antibacterial, antifungal and antiviral substances. Salivary glands and saliva are a part of the mucosal immune system (Tomasi and Plaut, 1985). Besides the predominant immunoglobulin secretory IgA, the oral cavity is protected, as well by many salivary nonimmunoglobulin antimicrobial molecules such as lysozyme, lactoferrin, and peroxidases (Mandel and Ellison, 1985). Many of these antimicrobial factors interact with each other and depend on salivary flow rate (Mandel and Ellison, 1985).Pharmaceutical side effects, systemic diseases, and radiation therapy are reported to be the most common causes of acquired salivary gland dysfunction (Mandel, 1980), leading to many subjective complaints (Fox et al., 1987) and dysfunction related objective findings (Gråhn et al., 1988; Atkinson and Fox, 1992). With age, an increasing number of people have medical conditions and/or use therapeutics that may significantly affect salivary gland physiology and the oral ecology. Tricyclic antidepressants, the basic group of amine uptake inhibitors, have a wide pharmacological spectrum displaying α1-adrenoceptor antagonistic, antiserotonin, antimuscarinic, and antihistaminic properties. Unwanted side effects are common (Trindade et al., 1998), and become more common with age (Tümer et al., 1992). One of the most reported ailments of tricyclic antidepressant therapy is dry mouth (Fox et al., 1985; Remick, 1988). Decreased salivary flow rate (Wu and Ship, 1993), increased salivary proteins and electrolytes (Blackwell et al., 1980; von Knorring and Mörnstad, 1981; Mörnstad et al., 1986), and changes in oral microflora were found in humans (Parvinen et al., 1984). This may compromise oral and systemic health, and overall quality of life. Salivary gland derived health problems pose a significant disease burden for the host.As in humans, submandibular flow rate seems not to be reduced in aging rats (Koller et al., 1992), while a progressive age-related decline in the rates of secretory protein synthesis in submandibular glands has been observed (Baum et al., 1983). Epidermal growth factor (EGF) was found to decrease with age, as well (Koller et al., 1992). Catecholamine responsiveness declines with age and is primarily due to a decrease in β-adrenergic signal transduction (Scarpace et al., 1991). In peripheral tissue, for the most part, the density of β-adrenoceptors is maintained with age, whereas the ability to activate adenyl cyclase declines (Scarpace et al., 1991). Tricyclic antidepressants may indirectly (central nervous system), and/or directly (salivary glands) reduce salivary gland function as a result of their action on modulating neural transmission. It has been shown (Scarpace et al., 1992, 1993), that signal transduction in submandibular glands was perturbed after chronic administration of desipramine. This drug down-regulated β-adrenergic receptors at all ages, but receptor recovery after drug withdrawal was prolonged in the brain of aged animals (Greenberg et al., 1985). In addition, we recently reported that desipramine led to changes in protein secretion, what may have negatively influenced oral health in aging rats (Koller et al., 2000).In this study, we investigated physiological and biochemical changes in submandibular gland secretory function in vivo with age and the long-term administration of the tricyclic antidepressant desipramine. This drug is a relatively selective inhibitor of the reuptake of norepinephrine and has weak effects on serotonergic neuronal activity. Therefore we expected (a) that submandibular flow rate would decrease temporarily with treatment; (b) that total protein secretion, as well as the content of specific proteins (EGF, peroxidase, lysozyme) in submandibular saliva would be depressed transitorily, and (c) that age would modulate the treatment-related changes.2Materials and methods2.1AnimalsPathogen-free female, Fischer 344 NIA rats of 3, 12 and 24 months of age were purchased from Harlan Sprague-Dawley (Indianapolis, USA) under contract with the National Institute of Aging (NIA). Animals were housed individually in 7×10 in. stainless steel micro-isolated cages, in a room maintained at 25°C. No clinical evidence of infectious illness was detected. All animals were maintained on Purina rat chow and water ad libitum and exposed to 12-h light–12-h dark cycle starting at 07:00 h. Animals were acclimatized for a minimum of 10 day before starting the experiments. At the beginning of the experimental period the young animals weighed 176–200 g, the middle age group rats 216–261 g, and the oldest ones 310–352 g.2.2Experimental designAnimals received a daily intraperitoneal (i.p. between 07:00 and 09:00 h) injection of desipramine or saline (10 mg/kg body weight) for 28 days. One half of each group was sacrificed on the third day after the last dose (DMI and SAL groups), the other half after a washout (WO) period of 15 days (DMI/WO and SAL/WO groups). Body weight was measured weekly, the final weight was monitored at the day of the experiment.2.3Saliva collection and flow rateThe same person between 09:00 and 11:00 h, using two animals per day, always carried out saliva collections. The rats were deprived of food the day before the experiment at 16:00 h. Animals were anesthetized i.p. (sodium pentobarbital, 50 mg/kg) and tracheotomized. The submandibular duct of the animal's left side was canulated with calibrated (lumen and length) drawn PE-10 plastic tubes. To stimulate salivary flow, pilocarpine HCl (Sigma Chemical, USA; 5 mg/kg, i.p.) was administered; to induce discharge of stored proteins isoproterenol (Sigma Chemical, USA; 5 mg/kg, i.p.) was chosen. These agents were dissolved in 37°C isotonic saline. The initial drop of saliva was discarded and subsequent flow was collected for 30 min into pre-weighed plastic tubes kept on ice. The sample tubes were reweighed to obtain a gravimetric estimate of the volume of saliva, assuming that 1 μl of saliva equals 1 mg. Flow rate was calculated as μl/min per gland and corrected for glandular wet weight and/or body weight. Saliva was stored at −80°C until analyzed. Sodium ethylene diamine tetraacetate (EDTA 1%, pH 8.6) was added to submandibular saliva (100 μl/ml saliva) to prevent the formation of a calcium-protein precipitate (Abe et al., 1980).2.4Salivary proteinsSamples were aliquoted to avoid repetitive freezing and thawing, assayed at one time, and in duplicate, to minimize error. Proteins were analyzed at one time by the method of Bradford (1976), using bovine plasma gamma globulin as the standard.For EGF estimation the volume of total saliva was brought up to 1 ml with sterile water. The glacial acetic acid (58 μl) was added, the solution cooled on ice for 30 min and then centrifuged at 4°C for 30 min in an Eppendorf centrifuge. The supernatant was transferred to a new tube, frozen at −80°C and then lyophilized. The remaining proteins were resuspended in 200 μl sterile water. Then 100 μl were added to 100 μl of human placental microvilli membranes and [125I]-labeled human EGF. Following incubation at 37°C for 2 h the membrane was diluted in phosphate buffered saline (PBS), centrifuged for 20 min at 7000×g and the radiolabel associated with the membrane determined in a gamma counter. This method of membrane binding competition is independent of species origin for the EGF source (Booth et al., 1980).Salivary peroxidase was assayed by the ABTS [2,2′-azinobis (3-ethylbenzothiozaline-6-sulfonic acid)] method of Shindler et al. (1976). The μmol substrate oxidized per min at 20°C was calculated from the absorbance change at 412 nm using the extinction coefficient for the radical cation produced. The salivary activity values were compared with a plot of activity versus ng protein prepared using purified bovine milk lactoperoxidase (Sigma Chemical, USA) as the standard. Salivary peroxidase concentrations were expressed as ng peroxidase per μl saliva.Lysozyme was assayed by the lysoplate method of Greenwald and Moy (1976) using low sulfur content agarose, and Micrococcus lysodeikticus as the indicator bacterium. Plates were incubated in a humidity chamber at 35°C for 48 h. The diameter of zone clearing was measured to the nearest 0.1 mm in both a vertical and horizontal direction. The mean zonal diameter values were compared with a plot of zone clearing versus ng protein prepared using grade I hen egg-white lysozyme (Sigma Chemical, USA) as the standard. Lysozyme concentrations were expressed as ng lysozyme per μl saliva.2.5Membrane preparation, cellular proteins, RNA contentAt sacrifice, the circulatory system was perfused with 20 ml of cold saline. The two submandibular glands were excised and dissected free of lymph nodes, fat, and fascia, and the sublingual glands removed. The wet weight was determined on an analytical balance, and the tissues immersed in 0.9% NaCl at 4°C. All subsequent steps were carried out at 4°C. The glands were finely minced in 20 volumes of 0.25 M sucrose, 1 mM MgCl2, and 5 mM Tris–HCl (pH 7.4). To avoid protein degradation, protease inhibitors were added to the mixture (1 μM leupeptin, 100 μM benzamidine, and 100 μM phenylmethylsulfonylfluoride-PMSF final concentrations). Preparations were disrupted by a Tekmar Tissuemizer (Cincinnati, USA) for 20 s and subsequently homogenized with 10 strokes of a motor-driven, teflon-tipped pestle at moderate speed. The homogenate was passed through two layers of cheese cloth, and centrifuged at 48 000×g for 15 min. The resultant pellet was resuspended in 20 volumes of 8 mM MgCl2, 0.08 mM ascorbic acid, the above mentioned protease inhibitors, and 50 mM HEPES (N-hydroxyethyl piperazine-N′-2′ ethane sulfonic acid, pH 7.4). Protein assays for total membrane and supernatant were determined by the above mentioned method of Bradford (1976).Submandibular gland RNA was isolated as described by Chomczynski and Sacchi (1987). One gland was homogenized in 2 ml of denaturing solution consisting of 4 M guanidinium thiocyanate. 25 mM sodium citrate, pH 7, 0.5% sarcosyl, 0.1 M 2-mercaptoethanol were added followed by 0.35 ml of 3 M sodium acetate, pH 5.2, 5 ml of phenol (water saturated), 1 ml of chloroform-isoamyl alcohol mixture (49:1) to extract the proteins from nucleic acid. The suspension was mixed and cooled on ice for 15 min and centrifuged at 10 000×g for 20 min at 4°C. The aqueous phase containing the RNA was transferred to a new tube, mixed with 5 ml of isopropanol, and then placed at −20°C for 1 h to precipitate RNA. Sedimentation at 10 000×g for 20 min at 4°C was again performed. The resulting RNA pellet was dissolved in 0.3 ml of the denaturing solution and precipitated with 0.1 ml 3 M sodium acetate (pH 5.2) and 0.4 ml isopropanol at −20°C for 1 h. After centrifugation in an Eppendorf centrifuge for 10 min at 4°C the RNA pellet was resuspended in 1 ml of 99% ethanol, sedimented, vacuum dried (10 min), and dissolved in 100 ml DEP-treated water and 0.5% SDS. The RNA content of all samples was estimated at one time by absorption at a wavelength of 260 nm and expressed as μg/g gland.2.6DNA synthesisIn vivo DNA synthesis was determined in submandibular glands by the incorporation of [3H]-thymidine into DNA by the injection of radiolabel (50 μCi per 100 g) 1 h before pentobarbital anesthesia and 4 h prior to sacrificing. Samples (100 μl) for [3H]-thymidine incorporation were removed prior to membrane centrifugation, and analyzed by scintillation counting using 10 ml of Fisher premixed nonaqueous scintillation cocktail (Sci Vers 5X20-4).2.7Polyacrylamide gel electrophoresisSubmandibular saliva was subjected to electrophoresis on a 1.5 mm thick 10% sodium dodecyl-sulfate polyacrylamide gel (SDS/PAGE) using a modified Tris–glycine system of Laemmli, as described by Pugsley and Schnaitman (1979). Gels were fixed and stained by a modification of the method of Fairbanks et al. (1971) as described by Humphreys-Beher and Wells (1984). Samples for gels were made up at 1.0 mg/ml of sample buffer. 15 μg of protein per well was used for gel electrophoresis.2.8Statistical analysisThe null hypotheses, no change with age and desipramine treatment, were tested by two way analysis of variance (ANOVA). When the main effect was significant, subgroups were examined by one-way ANOVA. Body weight was analyzed by repeated measures ANOVA. The Bonferroni/Dunn multiple comparison procedure was carried out to determine significant differences among the means following a significant one- and two-way ANOVA. No significant differences between the two saline groups were detected for all data. Therefore, we did not differentiate between the two saline groups in the final analysis.3Results3.1Body weight, organ weight and DNA-synthesisLong-term desipramine administration significantly reduced body weight throughout the experimental period between control and drug treated animals in all age groups (P<0.0001), with the greatest loss occurring in senescent rats (around 80 g). The animals gradually regained weight after the cessation of treatment. By day 15 post-treatment, body weight was comparable to that of control animals in the youngest age group only. A significant interaction between the parameters age and treatment was observed (P<0.0001).Comparing the control groups, the wet gland weight significantly increased between 3 and 12 m (P<0.0001), while desipramine administration had no major effect on glandular weight as on cortex weight in any age group. The histosomatic index, the organ to body weight ratio, was significantly decreased with treatment in the oldest animals only (P<0.0001).With regard to the general state of the glandular cells we found, that the DNA synthesis, as measured by the incorporation of tritiated thymidine (counts per min/g gland), was significantly influenced by treatment (P<0.0001) and age (P<0.0001), with no significant interaction (Fig. 1). While DNA synthesis was significantly decreased with desipramine in the two younger age groups (P≤0.0010), the control animals showed a significant increase between 3 and 12 month of age (P=0.0002). Recovery from drug effects was significantly delayed in senescent rats (P=0.0076).3.2Flow rateSubmandibular flow rate (Fig. 2), expressed as μl/min per gland, was significantly affected by age (P=0.0013). Correction for differences in glandular wet weight (μl/min/g gland) and/or for body weight (μl/min/g gland per kg, μl/min per gland per kg) eliminated the age related effect (P=0.5099, 0.0435, 0.7598, respectively). Correction for body weight only revealed a treatment related effect (P=0.0004). With desipramine, significant changes in submandibular flow rate were observed in the youngest animals only. Comparing the control groups, submandibular flow rate significantly increased with age (P=0.0017) when expressed as volume per time, but significantly decreased with age when correcting for glandular wet weight and body weight (P=0.0118).3.3ProteinsNo significant age- and treatment-related changes in protein concentrations (μg/μl) were detected for total membrane (structural proteins) and supernatant (soluble proteins). For the ratio structural/soluble proteins, a significant age-related effect was noted between 3 and 12 month old animals (P=0.0099). Subgroup analysis revealed no significant differences.Salivary protein concentrations (μg/μl) were not significantly changed with age and treatment, but the three age groups reacted differently to the drug regimen (Fig. 3). While in young and adolescent animals a decrease with desipramine occurred (27 and 40%, respectively), senescent rats showed a treatment-related increase of 45%. Recovery during the 15 day washout period was quite complete in all age groups (108, 85 and 102%). Correction for the observed changes in flow rate (total proteins secreted in 30 min) demonstrated significant age-related changes (P=0.0007). Again, the three age groups showed different effects with treatment. While 3 and 12 month old animals showed depressed protein secretion with the administration of the antidepressant (30 and 27%), total proteins secreted were elevated with treatment in senescent rats (30%). Protein concentrations in control animals revealed a steady increase with age, being significant when comparing 3 and 24 month old animals (P=0.0073), while total proteins secreted showed a steady, but not significant increase with age.On SDS/PAGE gels, loaded with a representative sample of each of the three treatment and age groups (15 μg of salivary proteins), protein expression differed with treatment and age (Fig. 4). In the Coomassie blue-stained gel, changes were detected in the bands around 36 kDa. The synthesis of these proteins was most pronounced in the treatment groups. An unusual protein (around 49 kDa) was detected in the desipramine group of 24 month old animals.Estimating the concentration (mg/ml) of EGF (Fig. 5) tested the secretory capacity of the submandibular granular tubule cells. Both, age and treatment revealed significant changes in the salivary EGF concentration (P<0.0001), and the interaction age×treatment was significant, as well (P<0.0211). Comparing the control groups, submandibular saliva of 12 month old animals contained 40% more EGF per ml saliva than the saliva of the youngest animals (P=0.1282), 24 month old rats showed a decrease of 81% compared with the adolescent rats (P=0.0004). All age groups showed decreased EGF concentrations with desipramine, significant in the middle age group only (P<0.0001). Basing the content of EGF on 1 mg of total protein in the sample (μg/mg) revealed a quite similar pattern for age (P<0.0001) and treatment (P<0.0140), and the interaction was still significant (P=0.0367).The content of peroxidase in submandibular saliva (Fig. 6), expressed as ng/mg protein secreted, was significantly affected by age (P=0.0237) and treatment (P=0.0206), with a significant interaction between the two parameters (P=0.0439). With desipramine administration, an increase was noted in all age groups, significant in 12 month old animals (P=0.0007). Recovery was incomplete in this age group only (P=0.0036). Comparing the controls, saliva of adolescent rats showed a significant lower peroxidase content than in the youngest age groups tested (P≤0.0054).The immunoprotein lysozyme showed similar effects with age (P=0.0002) and treatment (P=0.0021) as peroxidase (Fig. 6). The increase with desipramine was significant in the oldest age group only (P=0.0021). The control animals showed no age-related effect.Total RNA (Fig. 7), expressed as μg/g gland, was significantly affected by age (P=0.0213), but not by treatment (P=0.3440). In control animals, RNA decreased from 3 to 12 month of age by 17% and by 16% from 12 to 24 month old animals.4DiscussionIn the brain, desipramine blocks the reuptake of norepinephrine, leading to desensitization of the postjunctional β-adrenergic receptors, involving both a rapid attenuation of the responsiveness of adenylate cyclase and a slower downregulation of receptor number (Hausdorff et al., 1990). In submandibular glands of young rats, we observed desensitization of isoproterenol-stimulated activity, unchanged post-receptor signal transduction, and complete recovery after a 15 day washout period (Scarpace et al., 1992). With age, desipramine led to down-regulation in receptor number and attenuated receptor stimulated adenylate cyclase activity in submandibular glands (Scarpace et al., 1993). In addition, drug attenuated G-protein linked post receptor adenylate cyclase activity persisted through the 15 day washout period. In 12 month old rats, submandibular receptor up-regulation and adenylate cyclase supersensitivity were observed. Therefore, the animals in the present study probably also experienced receptor downregulation and adenylate cyclase desensitization, and the revealed alterations in submandibular protein secretion are, at least partially, the consequences of these changes in the signal transduction cascade changes present in all ages.It is well known, that most exocrine protein secretion occurs subsequent to β-adrenoreceptor activation. In this study we confirmed, that long-term administration of aging rats to the tricyclic antidepressant desipramine results in changes in salivary protein synthesis. A stimulating effect on the biosynthesis and secretion of different specific salivary proteins (e.g. bands around 36 kDa, lysozyme, peroxidase) was observed in all age groups. To maintain stable levels of total proteins secreted, the excretion rate of other proteins must have been depressed with desipramine. This was the case for the secretion of EGF in all the age groups. In a parallel study with the same drug regimen we showed unaffected salivary lactoferrin contents with treatment (Koller et al., 2000). Thyroid hormone is part of the regulatory mechanisms in salivary gland function (Tumilasci et al., 1986; Johnson and Kalu, 1988; Rousseau et al., 1998), e.g. it regulates the synthesis of EGF (Rall et al., 1985; Gubits et al., 1986; Johnson et al., 1987). In addition, serum levels of tetra- and triiodothyronine are influenced by treatment with antidepressants (Dagogo-Jack, 1995). Therefore, the detected changes in EGF secretion with desipramine may have indirectly influenced submandibular gland physiology in this study. As growth factor and cytokine receptors, many steroid hormones and their receptors found in salivary cells play a major regulatory role in salivary gland proliferation and function (Baum and Wellner, 1999; Purushotham and Humphreys-Beher, 1995), the decline in serum levels of thyroid hormones may have influenced cellular DNA synthesis and specific protein synthesis in this study. In the brain, chronic antidepressant therapy has been shown to induce changes in the function of protein kinase C (PKC), cyclic AMP-dependent protein kinase, and calcium/calmodulin-dependent protein kinase (Popoli et al., 2000). Therefore, as protein phosphorylation is an obligate step for most signaling pathways, desipramine may directly affect salivary gland proliferation and inactivate certain enzymes through the inactivation of important second messenger signaling pathway components (Purushotham and Humphreys-Beher, 1995).Differences in the induction of specific proteins with desipramine may be due to either nonspecific interaction with β-adrenergic signal transduction or differences in the induction of protein synthesis in different glands (Bedi, 1993). Additionally, there may be a differential control of secretion of proteins synthesized by ductal and acinar cells. While the secretion of many ductal proteins, such as EGF, appears to be initiated by α-adrenergic receptors (Wallace and Partlow, 1976; Orstavik and Gautvik, 1977; Hirata and Orth, 1980), the synthesis and secretion of acinar cell products is regulated by β-adrenergic receptors (Johnson and Cortez, 1988). Therefore, in submandibular glands, desipramine may affect not only β-adrenergic signal transduction (Scarpace et al., 1992, 1993), but β-adrenergic and muscarinic signaling, as well. Hanft and Gross (1990) showed, that rat cortical α1a- and α1b-adrenoceptors are regulated in a subtype-selective manner by desipramine (Hanft and Gross, 1990). This mechanism may influence secretory functioning of peripheral organs, as well. Additionally, desipramine administration may alter other neurotransmitter receptors involved in salivary secretory events as vasoactive intestinal polypeptide (VIP), tachykinin and purine receptors.The appearance of structurally altered proteins (Danner and Holbrook, 1990) is a prominent age-related change observed in various organs and tissues. These changes affect cellular contents, as well as functional properties of glandular proteins. Inanaga et al. (1988) observed unusual proteins in parotid saliva of 8- and 15-month-old rats similar to those we detected in submandibular saliva in senescent rats treated with the antidepressant desipramine.Saliva, having antimicrobial and growth-stimulating factors simultaneously, and the oral microbiota are the main factors determining oral health. Salivary antibody and non-immunoglobulin antimicrobial factors (e.g. lysozyme, peroxidase) maintain ecological balance (Gråhn et al., 1988). Peroxidase and lysozyme are only minor constituents of submandibular saliva. The changes in their content with treatment may have only a minor influence on oral microbiota in rats, but may partially reflect the reported increased incidence of gingivitis with desipramine treatment (Koller et al., 2000).Contrary to our expectations, considering the antagonistic action of desipramine on α1-adrenergic and muscarinic-cholinergic receptors in the brain (Potter et al., 1991), significant increased submandibular flow rates were detected with desipramine administration in young animals. One can speculate, that the detected increase in flow rate was caused by a positive feedback mechanism through the muscarinic-cholinergic, α1-adrenergic and substance P signal transduction system due to the observed decreased protein concentration.In contrast to human studies (Fernstrom and Kupfer, 1988), long-term treatment of rats with tricyclic antidepressants caused decreased food intake and a related weight loss (Nobrega and Coscina, 1987; Orthen-Gambill and Salomon, 1990). Long-term desipramine administration altered food and water intake (Durcan et al., 1988). After an initial decrease, body weight progressively returned towards pretreatment levels. This was explained by a rapid suppressive effect (acute central action of desipramine), and an adaptive effect during treatment (changes in feeding associated brain regions). This confirms our results, as body weight, after an initial decrease (1–2 weeks), was quite stable during the drug regimen in the two younger age groups. The decrease in the oldest animals lasted until the end of treatment and recovery was incomplete. As mentioned before, food intake, mastication, and long-term desipramine administration influenced serum levels of tetra- and triiodothyronine. Therefore, the loss in body weight and related changes in food and water intake with treatment may have indirectly influenced submandibular gland function in this study.In conclusion, the complexity of psychotropic drug actions, and variations in the absorption and excretion of these compounds and their metabolites, make precise predictions of their effects on salivary gland function difficult. The correlation between receptor activation and intracellular responses is often not predictable. Various signaling pathways seem to intersect and crosstalk, to modify and influence the biological outcome of the extracellular signal. Therefore, interactions among various transmitters must be considered with desipramine (Hill, 1998; Looms et al., 1998).AcknowledgementsThis work was supported in part by PHS Grants DE 08845 (Claude Denson Pepper Center for Research on Oral Health in Aging, P.J.Scarpace and R.A.Cowman.), and DE 08778 (M.H.B.) from the National Institutes for Dental Research, USA, the Medical Research Service of the Department of Veterans Affairs (P.J.Scarpace. and R.A.Cowman.), USA, and the University of Zurich (M.M.K.), Switzerland.ReferencesAbe et al., 1980K.AbeK.YonedaR.FujitaY.YokotaC.DawesThe effects of epinephrine, norepinephrine, and pilocarpine on the types of proteins secreted by rat salivary glandsJ. Dent. Res.59198016271634Ambudkar, 2000I.S.AmbudkarRegulation of calcium in salivary gland secretionCrit. Rev. Oral. Biol. Med.112000425Atkinson and Fox, 1992J.C.AtkinsonP.C.FoxSalivary gland dysfunctionClin. Geriatr. Med.81992499511Baum and Wellner, 1999Baum, B.J., Wellner, R.B., 1999. 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