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Articles

Ascorbate Deficiency Impairs the Muscarinic-Cholinergic and ß-Adrenergic Receptor Signaling Systems in the Guinea Pig Submandibular Salivary Gland^{1 ,2}

Departments of Biochemistry and Oral and Craniofacial Biological Sciences, Schools of Medicine and Dentistry, University of Maryland, Baltimore, MD

³To whom correspondence should be addressed.

	ABSTRACT

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Ascorbic acid is preferentially concentrated in the hypothalamus, pituitary and adrenal glands. Its level in the acini of salivary glands is relatively high. We therefore hypothesized that ascorbate may have a role in salivary gland function. Ascorbate-deficient guinea pigs had lower stimulated whole salivary flow rates than well-fed, age-matched controls (P < 0.005). Total salivary protein concentration was also markedly (P < 0.005) reduced in the deficient guinea pigs. SDS-PAGE and densitometric quantification of protein bands confirmed significant reduction in specific salivary proteins (e.g., amylase, proline-rich proteins) in the saliva samples of malnourished guinea pigs. Some protein bands not seen in control saliva were detected in the saliva of malnourished guinea pigs. Ascorbate deficiency also produced a significant (P < 0.005) reduction in the ß-adrenergic receptor density (subtype 1; 95 ± 19 fmol/mg protein compared with 179 ± 27 fmol/mg protein for the controls). No significant difference was observed between the two groups with respect to the ß-adrenergic receptor subtype 2. Additionally, ascorbate-deficient guinea pigs had significantly lower muscarinic-cholinergic receptor densities (50 ± 5 vs. 74 ± 8 fmol/mg protein for controls). Our data support the conclusion that diminished membrane receptors might impair the capacity of the transmembrane signaling system, resulting in salivary gland hypofunction in ascorbate-deficient guinea pigs. Without implying extrapolation of our findings in experimental animals to humans, it is perhaps relevant that many conditions often associated with salivary gland hypofunction in humans (e.g., smoking or drug ingestion) deplete cellular ascorbate.

KEY WORDS: • ascorbate deficiency • salivary glands • signal transduction systems • membrane receptors • guinea pigs

	INTRODUCTION

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Present in eukaryotic salivary glands are cell-surface receptors that elicit secretion of saliva on stimulation by the autonomic nervous system (Baum 1987a , Quissell et al. 1992 ). The stimulated receptors couple through G proteins to promote intracellular signaling events leading to generation of specific messenger molecules. Noradrenaline (sympathetic) and acetylcholine (parasympathetic) stimulate the adrenergic ( and ß) and muscarinic-cholinergic (mAChR)⁴ receptors, respectively, and are the key neurotransmitters involved in secretory activity of the major salivary glands (Baum 1987a and 1987b , Baum et al. 1993 ). Stimulation of the ß-adrenergic receptors activates adenylate cyclase and promotes cAMP accumulation, resulting mainly in protein secretion with relatively little increase in saliva volume (Baum 1987 ). Stimulation of the mAChR activates phospholipase C, resulting in hydrolysis of phosphatidylinositol-4,5-bisphosphate into inositol 1,4,5-trisphosphate (IP₃) sn-1,2-diacylglycerol (Baum 1987a , Gallacher 1988 , Quissell et al. 1992 ). The increased generation of IP₃ promotes release of Ca²⁺ from intracellular stores, leading ultimately to increased saliva volume (Baum 1987 ). It must be emphasized, however, that the ß-adrenergic cAMP-coupled process and the phosphoinositide-coupled fluid transport process do not function as two entirely independent systems in view of documented evidence of interactions between the two pathways (Baum et al. 1993 ).

Disease states, including malnutrition, influence secretory processes (Johnson 1987 ) and, by inference, receptor numbers and/or signal transduction systems. High concentrations of intracellular ascorbic acid (AA) are found in neurohormonal secretory cells of the hypothalamus, pituitary and adrenal glands (Levine 1986 ) as well as in exocrine acini of the salivary glands (von Zastrow et al. 1984 ). In an earlier report (Sawiris et al. 1995 ), we presented evidence for a significantly impaired mAChR transmembrane signaling cascade system, particularly the depressed generation of IP₃ and Ca²⁺ in stimulated submandibular glands of AA-deficient guinea pigs compared with controls. In view of reports (Houslay 1985 , Wiseman 1996 ) that dietary nutrients, including the vitamins, influence membrane fluidity, structure and function, this study was designed primarily to examine the effect of marginal ascorbate deficiency on the density (B_max) of membrane receptors in the guinea pig submandibular gland. Also evaluated was the effect of the vitamin deficiency state on the profiles of stimulated salivary proteins, including amylase, peroxidase and the proline-rich proteins.

	MATERIALS AND METHODS

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Induction of ascorbate deficiency.

The detailed protocol for this study was approved by the Institutional Animal Care and Use Committee of the University of Maryland, Baltimore, MD. On arrival in our laboratory, the male Hartley guinea pigs (Charles River Breeding Laboratory, Wilmington, MA) aged 21 d and weighing 300–350 g were housed individually in plastic cages. After a brief quarantine period of 3–5 d, the guinea pigs were weighed; those that failed to show a reasonable gain in body weight were eliminated from the study. The remaining guinea pigs were assigned randomly to two groups, an ascorbate-deficient group and a control group. The commercial diets used in the study (Table 1 ) were prepared in pelleted form by Purina Test Diets, Richmond, IN. Ascorbic acid levels in the diets were verified by analysis in our laboratory. Control guinea pigs were fed a Reid-Briggs diet containing 0.2 g/100 g AA. The ascorbate-deficient group initially consumed ad libitum for the first 2 wk a Reid-Briggs diet totally devoid of ascorbic acid, followed by a Reid-Briggs diet containing 50 µg/g ascorbate. By first feeding the guinea pigs the ascorbate-devoid diet, tissue levels of the vitamin were rapidly reduced but not completely depleted. Feeding the deficient guinea pigs a diet containing 50 µg/g ascorbic acid thereafter permitted a fairly normal food intake and thus prevented the complications of severe anorexia and marked body weight loss usually encountered in guinea pigs fed a diet totally devoid of AA for >2 wk (Ginter 1989 ). This approach was necessary to isolate the specific effects of ascorbate status from other nutrient deficiencies, particularly those caused by energy deficit. The ascorbate-deficient guinea pigs consumed the marginal ascorbic acid (50 µg/g) diet for at least 2–4 wk before they were killed for analyses. During the same period, control guinea pigs consumed ad libitum the diet containing adequate ascorbic acid.

Food was withdrawn from the guinea pigs overnight before autopsy. Dissections and procurement of tissues for analyses were performed between the hours of 0900 and 1100 h, i.e., no longer than 3–5 h after the start of the light cycle to minimize complications resulting from the effects of circadian rhythm on variables such as plasma glucocorticoid levels.

For collection of submandibular glands, the guinea pigs were anesthetized with a cocktail containing 6.7–10.0 mg/100 g body ketamine-HCl, 1.0 mg/100 g body xylazine-HCl and 0.15 mg/100 g body butorphanol tartrate. Unless indicated otherwise, all chemicals used were from Sigma Chemical, St. Louis, MO. The guinea pigs were placed on a heating pad maintained at 37 °C. For collection of whole saliva, the trachea was exposed and a small piece of polyethylene tubing (1 mm i.d.; 1.5 mm o.d.) inserted to maintain an open airway. To stimulate saliva secretion, an intraperitoneal injection containing (per 100 g body) 5 mg isoproterenol-HCl and 4 mg pilocarpine-HCl was administered. The volume of the whole saliva collected over a 20-min period served as an index of flow rate. Saliva samples were centrifuged at 1500 x g for 10 min (Sorvall RC-5B, Global Medical Instrumentation, St. Paul, MN) to remove debris, promptly divided into 500-µL aliquots and stored at -80°C. The submandibular glands were exposed, dissected free of fat and connective tissues, and excised. After weighing, the glands were frozen at -80°C.

Blood was taken by cardiac puncture into evacuated, heparinized and EDTA-treated polypropylene tubes kept in the cold, and plasma separated within 0.5 h of blood collection by centrifugation in a refrigerated centrifuge for 5 min at 2000 x g.

Electrophoresis of proteins in saliva.

Separation of salivary proteins was by SDS-PAGE as described by Laemmli (1970) . Densitometric spectra of the electrophoretograms were analyzed using the NIH Image Program (software version 1.49).

Membrane preparation for saturation binding assays.

For the ß-adrenergic saturation binding assays, one pair of submandibular glands was placed in a 50-mL polypropylene tube (Falcon Plasticware, Oxnard, CA) containing 8 volumes of ice-cold 50 mmol/L phosphate buffer, 10 mmol/L EDTA, pH 7.4. The glands were minced with scissors and homogenized for 20 s at 4°C using a Polytron Homogenizer (Brinkmann Instruments, Westbury, NY) adjusted to a speed setting of 6. The homogenate was centrifuged at 500 x g in a refrigerated Sorvall RC-5B centrifuge for 10 min. The supernatant was transferred to a clean 50-mL polypropylene test tube and centrifuged at 31,000 x g for 10 min. The supernatant was decanted and the pellet was resuspended in 1 mL of 50 mmol/L phosphate buffer containing 10 mmol/L MgCl₂, pH 7.4. The protein concentration of this suspension was determined by the method of Arneberg (1970) and then diluted to 10 g protein/L. In all assays, the total reaction volume was 200 µL and the protein concentration was 250 µg. For the mAChR receptor density assay, the submandibular glands were processed as for the ß-adrenergic saturation binding assays.

Assay conditions for receptor densities.

To measure ß-adrenergic saturation binding, the reaction mixture consisted of 10 concentrations (ranging from 0.3 to 25 nmol/L) of the ß-adrenergic specific receptor antagonist 1-[4,6, propyl 3H]-dihydroalprenolol (DHA; specific activity 95 Bq/mmol, Amersham, Arlington Heights, IL) and 250 µg of membrane protein in 50 mmol/L phosphate buffer, 10 mmol/L MgCl₂, pH 7.4. Each sample was assayed in triplicate. To measure nonspecific binding, the reaction mixture consisted of the same DHA and protein concentrations and also 40 µmol/L of the ß-adrenergic antagonist propranolol (ICN Biochemicals, Cleveland, OH). Total radioactivity was determined by adding only the DHA to 4 mL of scintillation fluid and counting the sample in a Beckman LS 5801 liquid scintillation counter (Beckman Instruments, Fullerton, CA). The reaction was stopped by addition of 5 mL of ice-cold 50 mmol/L phosphate buffer, pH 7.4. The membrane fraction was collected on GF/C filters (Brandel, Gaithersburg, MD) with a cell harvester. The tube was washed 3 times with ice-cold 50 mmol/L phosphate buffer, pH 7.4. The filter paper was then placed in a scintillation vial, to which was added 4 mL Opti-Fluor scintillation fluid (Packard Instrument Company, Downers Grove, IL), and allowed to dark-adapt overnight. Samples were counted in a Beckman LS 5801 liquid scintillation counter.

To measure specific binding, the reaction mixture consisted of 10 concentrations (ranging from 0.8 to 13 nmol/L) of the mAChR receptor antagonist L-[benzilic-4–4'-³H]-quinunclidinyl benzilate (QNB; specific activity 43 Bq/mmol, NEN, Wilmington, DE) and 250 µg of membrane protein in 50 mmol/L phosphate buffer containing 2 mmol/L MgCl₂, pH 7.4. For the nonspecific binding assay, the reaction mixture contained QNB at the same concentrations listed previously and also included 20 µmol/L atropine, a mAChR receptor antagonist. Total radioactivity used was determined by adding only the radioactive QNB to 4 mL of scintillation fluid and counting radioactivity in a Beckman scintillation counter. All assays were done after incubation for 90 min at 37°C. Saturation isotherms of QNB binding were plotted, and the B_max (expressed as fmol/mg protein) of mAChR receptors were determined by Scatchard analysis using the program KaleidaGraph (Version 2.1, 1990, Abelbeck Software,Reading,PA).

ß₁,ß₂-Adrenergic competitive binding assay.

Competitive binding assays were performed to characterize the ß-adrenergic subtypes present in submandibular gland tissue. Submandibular glands were prepared as for ß-adrenergic saturation binding assays except that the glands were homogenized in 8 volumes of ice-cold 50 mmol/L Tris-HCl, 10 mmol EDTA, pH 7.4, and the postmitochondrial pellet was resuspended in 1 mL of 50 mmol/L Tris-HCl containing 10 mmol/L MgCl₂, pH 7.4. The assay conditions were identical to those described for the ß-adrenergic saturation binding assay except for inclusion of the ß₁ adrenergic blocker metoprolol at 12 concentrations ranging from 10 nmol/L to 10 mmol/L. All assays were carried out in triplicate. After determining the percentage displaced (tritiated DHA displaced by the metoprolol), the values were used to generate a Hofstee plot in which the percentage of DHA displaced by metoprolol was plotted on the y-axis and the ratio of the percentage of DHA displaced by metoprolol to metoprolol concentration was plotted on the x-axis. Initial estimates of the B_max were obtained in this manner.

Final estimates for these values were obtained using the nonlinear curve fitting program Ligand (Munson and Rodbard 1980 ). This program is part of a collection of radioligand binding analysis programs (Kinetic, EDBA, Ligand, Lowry) called KELL supplied by Biosoft (Ferguson, MO).

Amylase and peroxidase measurements in submandibular glands.

Submandibular gland acinar cell preparations were obtained by enzymatic digestion. Details on the procedure and subsequent processing have been described in Baum et al. (1990) . Initially, it was desirable for us to determine the carbachol concentration at which maximal stimulation of the submandibular gland would occur. This was found to be 20 µmol/L as determined by measuring amylase and peroxidase activities in submandibular gland acinar cell preparations in the presence of 5, 10, 20 and 40 µmol/L carbachol. Carbachol was added to acinar cell preparations to a final concentration of 20 µmol/L after which the tissue was gassed with 95% O₂/5% CO₂ for 10 s, and incubated in a metabolic shaker at 37°C, 110 rpm. At 20-min intervals, the tissue was regassed and triturated with a 10-mL pipet to aid in the dispersion. After 60 min, the resulting acinar cell suspension was centrifuged at 4°C for 10 s (Sorvall RC-5B Superspeed Centrifuge) at 400 x g. A 1-mL aliquot of the supernatant was saved for extracellular protein and amylase measurements. The tissue was then resuspended in incubation medium (80 g/L). After resuspension, the tissue was homogenized for 30 s at a setting of 6 (Brinkmann Instruments). Another 1-mL aliquot of the homogenate was taken for intracellular protein and amylase measurements.

Amylase assay.

Amylase was measured using an assay kit (Sigma) according to Sigma protocol number 577. The absorbance was recorded at 405 nm using water as a reference. This was the baseline absorbance. Incubation was continued for another 2 min and the absorbance recorded again at exactly 1 and 2 min after the baseline absorbance reading. The reading recorded at 2 min was the final absorbance reading.

Peroxidase assay.

Peroxidase activity was assayed according to the method of Carlsoo et al. (1974) . Saliva samples (100 µL) were added to 16.7 mmol/L pyrogallol in 200 mmol/L sodium phosphate buffer, pH 6.0, in total volume of 3 mL. One milliliter was added to a cuvette, placed in a Beckman spectrophotometer, and 19 µL of 88 mmol/L H₂O₂ added to a final concentration of 1.67 mmol/L. The baseline absorbance was recorded at 400 nm. The incubation was continued for 2 min and the absorbance recorded at exactly 2 min after the baseline absorbance reading. The reading at 2 min was the final absorbance reading. Peroxidase activity was then calculated as for the amylase assay and expressed as A/(min · g gland).

Total proteins in saliva.

These were measured according to Arneberg (1970) and Lowry et al. (1952) . The former method measures the absorbance of all peptide bonds in a protein and gives higher readings than the latter in which the dye does not detect aromatic amino acid residues. The protein levels in the samples were calculated from a standard curve generated using five dilutions of bovine serum albumin.

Measurement of noradrenaline levels in the submandibular gland.

Noradrenaline levels in the submandibular gland from control and ascorbate-deficient guinea pigs were measured using HPLC (Devalia et al. 1985 ). Immediately after dissection, the glands were homogenized in 4 volumes of ice-cold 0.4 mol/L perchloric acid using a Brinkmann polytron homogenizer with a speed setting of 6 (Brinkmann Instruments). The homogenate was treated with sodium chloride (0.7 kg/L), vortexed for 15 min and centrifuged at 1500 x g for 15 min (Sorvall RC-5B centrifuge). The supernatant was diluted 1:10 with 0.5 mol/L sodium acetate, pH 6.5. The latter was passed through an ion exchange column of 1.0 mL Amberlite CG-50 contained in a 10-mL polypropylene Econo column (Bio-Rad, Melville, NY).

Noradrenaline levels were measured using a Perkin Elmer Series 400 HPLC (Broxburn, UK). Chromatography was done on a Nucleosil 250 x 3.2 mm column (Phenomenex, Torrance, CA) with a particle size of 5 µm and a pore volume of 0.7 mL/g. The mobile phase was a mixture of 0.2 mol/L sodium acetate and tetrahydrofuran (75:25 v/v) adjusted to pH 5.1 with 12 mol/L HCl and pumped at a constant flow rate of 1.0 mL/min. A Gilson model 121 fluorometer (Gilson, Middleton, WI) was used to detect noradrenaline after derivatization with O-phthaladehyde reagent. The fluorometer was set at an emission wavelength of 305–395 nm and an excitation wavelength of 430–470 nm. Chromatographic peaks were recorded with a Shimadzu Chromatopac C-R3A data processor (Shimadzu, Japan).

Statistical analysis.

The statistical analyses used in this study included Student’s t test, the Mann-Whitney test and ANOVA. Student’s t test was used in cases in which two equal group means were compared. When data were not normally distributed, the Mann-Whitney U-test (two tailed; 5% significance level) was used. In cases in which more than two group means were compared, ANOVA was used. Data were reported as group means ± SEM, and a difference was considered significant when a P-value 0.05 (95% confidence interval) was obtained.

	RESULTS

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Clinical and biochemical features.

The ascorbate-deficient guinea pigs had a normal food intake and there were no differences in the final body weights and the paired salivary gland weights between the control and experimental groups (Table 2 ). Edema of the subcutaneous tissues between the forelimbs and in the orofacial area was common in the ascorbate-deficient guinea pigs. In addition, ascorbate-deficient guinea pigs exhibited some hair loss as well as decreased mobility, likely as a result of bleeding into the joints. Plasma levels of AA were 22.9 ± 2.7 µmol/L for the control group and 3.0 ± 0.4 µmol/L for the ascorbate-deficient group, confirming that the latter were deficient in the vitamin. Ascorbate-deficient guinea pigs exhibited significantly (P < 0.005) reduced saliva flow rates. There were also markedly lower glandular noradrenaline concentrations in ascorbate-deficient guinea pigs compared with controls (Table 2) .

The gel shown in Figure 1 (obtained by destaining in the presence of organic solvents) compares whole saliva samples collected from control guinea pigs (lanes C and D) with saliva samples from ascorbate-deficient guinea pigs (lanes E and F). Protein bands were numbered consecutively starting from those with the highest molecular weights to those with the lowest. Lanes A and B refer to high- and low-molecular-weight markers. Visual inspection of the gels revealed at least 9 protein bands for each of the saliva samples analyzed. Band 1 protein had an electrophoretic mobility close to that of myosin (MW 200 kDa) and although present in the saliva of control guinea pigs, it was not evident in the saliva of ascorbate-deficient guinea pigs. Band 2 protein migrated at a rate between that of bovine serum albumin (MW 66 kDa) and ovalbumin (MW 45 kDa) and was identified as -amylase. This protein appeared to be in smaller quantity in the malnourished guinea pigs than in controls. Bands 3, 4 and 5, whose identities are yet to be clarified, had electrophoretic mobilities between those of ovalbumin (MW 45 kDa) and carbonic anhydrase (MW 31 kDa) and had higher concentrations in the ascorbate-deficient guinea pigs than in controls. Band 6 migrated at a rate almost equal to that of carbonic anhydrase (MW 31 kDa) and appeared slightly larger in the ascorbate-deficient guinea pigs. Band 7 protein, with an electrophoretic mobility between that of carbonic anhydrase (MW 31 kDa) and soybean trypsin inhibitor (MW 21 kDa), was present in the saliva of control guinea pigs, but was not readily apparent in the saliva of ascorbate-deficient guinea pigs. Band 8 protein had an electrophoretic mobility almost similar to that of soybean trypsin inhibitor (MW 21 kDa) and appeared to be present in equal quantities in the saliva samples of both control and ascorbate-deficient guinea pigs. Band 9 protein, with electrophoretic mobility between that of soybean trypsin inhibitor (MW 21 kDa) and lysozyme (MW 14 kDa), was present in the saliva of controls but not in that of malnourished guinea pigs. When the gel was stained with Coomassie Brilliant Blue R-250 and destained in the absence of organic solvents (data not shown), more protein bands were noted than in Figure 1 . There was good evidence of more phosphorylated proline-rich proteins in saliva samples of controls than in those from ascorbate-deficient guinea pigs.

View larger version (82K): [in this window] [in a new window]   Figure 1. SDS-PAGE of guinea pig whole saliva. The gel was stained with Coomassie Blue. All samples applied to the gel were of equal volume and contained 50 µg of protein. Lanes A and B are high- and low-molecular-weight standards, respectively. Lanes C and D, control guinea pigs. Lanes E and F, ascorbate-deficient guinea pigs.

Total peroxidase activity was significantly lower (P < 0.01) in ascorbate-deficient (Table 3 ) than in control guinea pigs. Treatment with carbachol released significantly (P < 0.001) less peroxidase from glands of ascorbate-deficient guinea pigs than from controls.

ß-Adrenergic and mAChR receptor densities in acinar cells from the submandibular gland.

Saturation isotherms were used to compare total, specific and nonspecific binding. Nonspecific binding was typically <20% of specific binding. In the ß-adrenergic binding assays (using equivalent amounts of membrane), total and specifically bound DHA for control guinea pigs were approximately twice those for ascorbate-deficient guinea pigs at any given DHA concentration. In the mAChR binding assays, total and specifically bound QNB for control guinea pigs were 3–5 times the levels noted for the ascorbate-deficient guinea pigs at any given QNB concentration (data not shown).

ß-Adrenergic receptor densities were obtained through representative reverse Scatchard analysis (Hofstee plots). Analysis of the plots suggested the presence of two different populations of ß-adrenergic receptor subtypes. Measurements of mAChR receptor densities were obtained from Scatchard plots. Summarized in Table 4 are the effects of ascorbate deficiency on the maximal ß-adrenergic and mAChR receptor densities (B_max) in acinar cells from the guinea pig submandibular glands. Vitamin-deficient guinea pigs showed a marked (P < 0.005) reduction in submandibular gland receptor concentrations of ß-adrenergic receptors (subtype 1) and mAChR receptors.

	DISCUSSION

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This study demonstrated significantly reduced (P < 0.005) mAChR receptor density in the submandibular salivary glands of vitamin C–deficient guinea pigs (Table 4) , an observation consistent with their reduced saliva flow rate compared with findings in control guinea pigs (Table 2) . In a previous study (Sawiris et al. 1995 ), we reported significantly impaired generation of IP₃ as well as markedly reduced intracellular calcium ion level in dispersed submandibular gland acinar cells stimulated with carbachol compared with controls. The significantly impaired mAChR transmembrane signaling cascade system in ascorbate-deficient guinea pigs could explain the relative hyposalivation in these malnourished guinea pigs compared with the well-fed controls because stimulation of the mAChR and the -adrenergic receptors results primarily in fluid secretion (Baum 1987a and 1987b ).

Our findings confirmed published reports that ß-adrenergic receptors in salivary glands are mainly of the ß1 type (Schneyer 1986 ). This study also demonstrated that ascorbate deficiency promoted significant reduction (P < 0.005) in the ß-adrenergic receptor density (Type 1) but had no effect on the Type 2 receptor (Table 4) . Previous published data indicate that despite increasing evidence of interactions among the various signaling systems in salivary glands (Baum et al. 1993 , Horn et al. 1988 , Mills et al. 1993 ), stimulation of the ß-adrenergic receptors results primarily in protein secretion (Baum 1987 ). The observed changes in ß-adrenergic receptor density in the ascorbate-deficient guinea pigs could account in part for the significantly reduced stimulated total protein level (Table 2) as well as carbachol-stimulated releasable peroxidase and amylase activities (Table 3) in whole saliva from these guinea pigs compared with well-fed guinea pigs. It should be underscored that in addition to the diminished ß-adrenergic receptor density observed in this study (Table 4) , studies of hypothalamic neurons in culture suggest that this vitamin enhances Forskolin-induced cAMP production and pro-atrial natriuretic factor mRNA expression (Huang et al. 1993 ). For the various cell types studied, high intracellular ascorbate is reported to increase cAMP partly through reversible inhibition of cAMP phosphodiesterase (EC 3.1.4.17) (Buck and Zadunaisky 1975 ), stimulation of active transport of Cl^- (Buck and Zadunaisky 1975 ), enhancement of levels of cGMP (Atkinson et al. 1979 , Pickett et al. 1979 ) and modulation of many cellular events involving participation of the calcium ion (LeVine et al. 1983 ).

Electrophoretic analyses of stimulated saliva from ascorbate-deficient and control guinea pigs revealed a greater number of protein bands in the region between ovalbumin (MW 45 kDa) and carbonic anhydrase (MW 31 kDa) in the former group (Fig. 1) . These proteins, which we have not yet identified, were more evident in saliva from ascorbate-deficient guinea pigs (Fig. 1) than in saliva from controls. The importance of this finding is not immediately clear. Published studies (Ikeda et al. 1998 ) suggest that AA deficiency changes hepatic gene expression of acute phase proteins in scurvy-prone ODS rats (genotype od/od with a hereditary defect in ascorbate biosynthesis), and that serum concentration of interleukin-6, an inflammatory cytokine that stimulates gene expression of some acute phase proteins, is significantly higher in rats subjected to total ascorbate deficiency for 14 d than in controls. The relevance of the latter to our observation in saliva of ascorbate-deficient guinea pigs (Fig. 1) is not clear. Although hypercortisolemia occurs in guinea pigs fed diets totally devoid of ascorbate for 2 wk (Enwonwu 1990 ), as well as in those fed marginal AA for a prolonged period (Sawiris et al. 1995 ), the observed changes in saliva protein profiles could not be readily attributed to increased circulating cortisol in these guinea pigs because changes in hepatic mRNA levels of some acute phase proteins in ascorbate-deficient scurvy-prone ODS rats are not affected by adrenalectomy (Ikeda et al. 1998 ).

Ascorbate has been reported to be a constituent of secretory granules of salivary glands and is suggested to have an important role in the secretory process (von Zastrow et al. 1984 ). It is also the major factor in brain extracts responsible for increasing the density of acetylcholine receptor sites in a muscle cell culture line, an effect mediated through transcriptional regulation (Knaack and Podleski 1985 ). In this study, the model of vitamin C deficiency employed permitted fairly normal food intake by the guinea pigs (Enwonwu et al. 1995 , Ginter 1989 ), thus circumventing the complications of reduced ingestion of energy, proteins and other essential nutrients. This approach was necessary because various forms of malnutrition have been reported to affect salivary gland function (Johnson 1987 ). Protein-energy malnutrition (PEM), for example, significantly reduces stimulated whole saliva flow rates in humans (Govindam et al. 1985 ), an observation confirmed in experimental animal models of PEM, which also exhibit a marked reduction in ß-adrenergic receptor densities in both parotid and submandibular glands, with no change in dissociation constants (Johansson and Ryberg 1991 , Johnson 1987 ). Protein deficiency also impairs incorporation of (NaH₂³²)PO₄ into phosphorylated derivatives of inositol in rodent exocrine pancreas (Butani et al. 1986 ), a secretory organ with close similarities to the major salivary glands. Additionally, dietary fatty acid types influence membrane phospholipids and transmembrane signaling in the rat submandibular salivary gland (Ahmad et al. 1990 ). In a relatively recent review, Wiseman (1996) examined dietary influences on membrane function and their importance in protection against oxidative damage. Membrane lipid peroxidation promotes loss of polyunsaturated fatty acids, decreases membrane fluidity and causes severe structural alterations, resulting in loss of enzyme and receptor activities involved in the second messenger system and cell signaling (Wiseman 1996 ). One of the most important cellular antioxidants is AA (Frei et al. 1989 ), whose level in tissues is a valuable biomarker of oxidative stress. Ascorbic acid status affects lipid metabolism (Ginter 1989 ), particularly the integrity of cell membranes (Halliwell 1994 ). Studies in guinea pigs have shown that chronic marginal AA deficiency, under conditions similar to those in our present work, produces lipid peroxidation in all of the organs evaluated including liver, kidney, lung, adrenal gland and testes (Chakraborty et al. 1994 ).

Features clearly identified in ascorbate deficiency in humans include loss of salivary and lacrimal secretion, with swelling of the major salivary glands (Hodges 1971 ). It is perhaps relevant that many seemingly unrelated human conditions that promote cellular depletion of and/or increased requirement for ascorbate, such as stress, smoking, exposure to ionizing radiation, chronic drug ingestion, uncontrolled diabetes, aging and hypercholesterolemia (Gaby and Singh 1991 , Murata 1991 ), are all implicated in the genesis of salivary gland hypofunction and the associated feeling of dry mouth (xerostomia) (Sreebny and Valdini 1987 ). Future studies will address the potential role of cellular ascorbate deficiency in metabolically induced feeling of dry mouth in humans. It will also be rewarding to examine the relationships among ascorbate deficiency, tissue levels of proinflammatory cytokines and alterations of gene expressions in salivary glands.

	FOOTNOTES

 
¹ Data reported in this manuscript were presented in partial fulfillment for the degree of Ph.D. by P.G.S., University of Maryland, Baltimore, MD.

² Supported by Research Grant DEO9653 from the United States Public Health Service.

⁴ Abbreviations used: AA, ascorbic acid; DHA, 1-[4,6,propyl ³H]-dihydroalprenolol; IP₃, inositol 1,4,5-trisphosphate; mAChR, muscarinic-cholinergic; PEM, protein-energy malnutrition; QNB, L-[benzilic-4,4'-³H]-quinuclidinyl benzilate.

Manuscript received March 14, 2000. Initial review completed May 4, 2000. Revision accepted August 22, 2000.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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