An analysis of parotid salivary gland function with desipramine and age in female NIA Fischer 344 rats.

Cyclic antidepressants are still a dominating group of psychotherapeutic drugs used in the treatment of depression. Dry mouth is one of their major side effects. In this study we analyzed the effects of the long-term administration of the tricyclic antidepressant desipramine and the reversibility of this treatment following a 15-day washout period on different parameters in parotid gland function in aging rats. We hypothesized that glandular function would be decreased, and recovery delayed with age. Drug treatment affected body weight, glandular weight, DNA synthesis, and the concentration of soluble and structural membrane proteins. Surprisingly, parotid flow rate was increased with desipramine in all ages. While the concentration of secreted proteins was generally decreased with treatment, total proteins secreted were quite stable. SDS/PAGE analysis revealed prominent changes with desipramine. Amylase activity was depressed with treatment, but only low residual cellular enzyme activity was detected in the glandular supernatant. Therefore, a secretory impairment with desipramine was excluded. The content of the antimicrobial proteins peroxidase and lysozyme was increased with desipramine in all age groups. Most parameters measured revealed delayed recovery with age. These data indicate that the tricyclic antidepressant desipramine has profound effects on parotid gland function, accented with age.

An analysis of submandibular salivary gland function with desipramine and age in female NIA Fischer 344 rats.

Dry 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.

Pathway for uptake and degradation of X-prolyl tripeptides in Streptococcus mutans VA-29R and Streptococcus sanguis ATCC 10556.

The growth of Streptococcus mutans and Streptococcus sanguis in the oral environment requires that these micro-organisms be able to degrade salivary proteins and to assimilate the resulting peptides as an amino nitrogen source. Our research is aimed at the definition of the proteolytic enzyme systems in these oral streptococci which allow them to utilize such substrates. In the present work, the nature of the hydrolytic activity expressed by S. mutans VA-29R and S. sanguis ATCC 10556 against X-Pro4-nitroanilide and X-Pro-Y tripeptide substrates was investigated. This activity was predominantly associated with a cytoplasmic dipeptidyl peptidase which preferentially catalyzes the release of an N-terminal dipeptide from substrates in which proline is the penultimate residue. These streptococci also possess a second cytoplasmic peptidase, pepD, which catalyzes the hydrolysis of X-Pro dipeptides. We found that Gly-Pro-Ala or Ala-Pro-Gly were transported into the bacterial cells only when an energy source such as glucose was present. Peptide uptake was time-dependent, and selective exodus of peptide-derived amino acids from the bacterial cells occurred during peptide uptake. Results from these studies provide evidence that S. mutans VA-29R and S. sanguis ATCC 10556 possess a pathway for the complete degradation of X-Pro tripeptides. Transport of the peptides into cells prior to hydrolysis provides an efficient way by which all amino acids of a peptide may be obtained at an energy expense equivalent to that associated with the transport of just one amino acid. In light of the abundance of proline in salivary polypeptides, this degradative pathway could be an important component in the proteolytic pathway for salivary polypeptide utilization in these oral streptococci.

Effects of beta-adrenergic antagonists on salivary secretory function in individuals of different ages.

The purpose of this study was to assess the effects of chronic beta-adrenergic antagonists on parotid and submandibular gland secretions in men and women of different ages. Unstimulated and stimulated saliva flow rates, total protein concentrations, and protein secretion rates were compared from medicated (various beta-antagonists, n = 25) and control (n = 60) subjects. Age-related decreases were found in unstimulated parotid saliva flow rate (p = .011) and protein secretion rate (p = .04), unstimulated submandibular salivary flow rate (p = .005) and protein secretion rate (p = .010), and in stimulated submandibular flow rate (p = .002) and protein secretion rate (p = .006). A drug-related effect was observed only in unstimulated parotid salivary flow (p = .033) and protein secretion rate (p = .04) from medicated subjects. Results from this study indicate that age and beta-adrenergic blockade alter salivary glandular function, but their effects differ with the type of salivary secretion examined.

Comparison of aminopeptidase activities in four strains of mutans group oral streptococci.

In this study, native cells of Streptococcus mutans VA-29R and Streptococcus rattus FA-1 displayed significantly higher aminopeptidase activity than did cells of Streptococcus cricetus AHT or Streptococcus sobrinus 6715 toward the nitroanilide derivatives of leucine, alanine, methionine, arginine, and lysine. These differences in cellular aminopeptidase activity led us to investigate the subcellular localization of the aminopeptidase in these mutans group streptococci. Following conversion of native cells to protoplasts by treatment with lysozyme, most of the aminopeptidase activity detected in the native-cell preparations remained associated with the intact protoplasts. After lysis of protoplasts and differential centrifugation, most of the total cellular aminopeptidase activity was recovered with the cytoplasmic fraction. Membrane-associated aminopeptidases represented only minor activities in these mutans group streptococci. Although the four strains showed no differences with respect to a predominant cytoplasmic localization for the aminopeptidase activities, the levels of activity in the cytoplasmic fractions from S. cricetus AHT and S. sobrinus 6715 were significantly lower than those measurable in the corresponding fractions from S. mutans VA-29R and S. rattus FA-1. These results support the conclusion that the differences in aminopeptidase activity expressed by these streptococci reflect quantitative differences rather than differences in enzyme subcellular localization.

Studies on the subcellular localization of protease and arylaminopeptidase activities in Streptococcus sanguis ATCC 10556.

Intact cells of Streptococcus sanguis ATCC 10556 possessed arylaminopeptidases exhibiting activity toward the nitroanilide (NA) derivatives of leucine, alanine, methionine, arginine, or lysine. Weak hydrolytic activity was observed in assays with the NA derivatives of valine, proline, glycine, or glutamic acid. Subcellular localization studies revealed that arylaminopeptidase activities were located in both the cell membrane and cytoplasm. Arylaminopeptidases exhibiting activity toward the leucine, alanine, or methionine NA substrates appeared to be more predominantly associated with the membrane, whereas enzymes exhibiting activity toward arginyl-NA or lysyl-NA were more prevalently located in the cytoplasm. Several results from this study suggest that the membrane-assocaited arginyl and lysyl arylaminopeptidases were located in such a way that their expression was restricted in the intact cell. The addition of 0.5 mol/L NaCl to protoplast preparations derived from mutanolysin-treated cells resulted in an almost complete solubilization of membrane-associated arylaminopeptidase activities. These observations support the conclusion that the association of arylaminopeptidases with the cell membrane may involve hydrophobic or electrostatic interactions, or both. S. sanguis ATCC 10556 also possessed at least one caseinolytic endopeptidase activity. This activity is most likely located near the membrane surface, as no association with the cell wall was evident. The location of membrane-associated endopeptidase and arylaminopeptidase activities, together with intracellular peptidases, is suggested to provide an efficient mechanism for the hydrolysis and subsequent utilization of polypeptide and oligopeptide substrates as sources of amino acids for growth by this microorganism.

Influence of hydrophobicity on oligopeptide utilization by oral streptococci.

The growth responses of Streptococcus mutans VA-29R, Streptococcus sanguis ATCC 10556, and Streptococcus mitior NIH to hydrophilic and hydrophobic peptides obtained following isopropanol fractionation of Trypticase were compared. Although the two fractions contained peptides of similar molecular size, differences were observed with respect to amino acid compositions. S. mutans VA-29R showed a pronounced difference in growth response to hydrophilic vs. hydrophobic peptides. While growth of this micro-organism on hydrophilic peptides was indistinguishable from that on unfractionated Trypticase, only very slow growth occurred on the hydrophobic peptides. S. sanguis ATCC 10556 and S. mitior NIH also displayed some selectivity, as evidenced by their faster relative growth rates on hydrophilic, as compared with hydrophobic, peptides. The results of this study support the conclusion that the properties of the substrate, as defined by its amino acid composition, may be more important than molecular size as a factor influencing recognition and subsequent utilization of oligopeptides as sources of amino acids for growth by these three oral streptococci.

Immunological cross-reactivity of anionic proteins from caries-free and caries-active salivas which differ in biological properties toward oral streptococci.

We examined whether the anionic antimicrobial proteins in the saliva of certain caries-free (CF) individuals are immunologically cross-reactive with proteins in caries-active (CA) saliva which display identical isoelectric points but which exhibit growth-supportive properties for the oral streptococci. In the immunoblotting experiments reported here, the proteins present in CF and CA whole salivas reacted similarly with an antiserum prepared against the anionic inhibitory proteins. This antiserum also showed identical reactivity with the purified protein fraction containing the anionic proteins of interest from whole saliva of either CF or CA subjects. Additionally, an antiserum to CA saliva was found to react with the CF anionic inhibitory proteins. The fact that the inhibitory and noninhibitory proteins react similarly with the antisera used provides further evidence for similarity between both classes of proteins.

Immunological relationship between anionic antimicrobial proteins from caries-free individuals and known salivary antimicrobial factors.

We examined whether the anionic inhibitory proteins identified in mixed saliva from certain caries-free individuals are fragments or degradation products of recognized salivary antimicrobial factors. In the experiments reported here, the anionic inhibitory proteins did not produce precipitin reactions with antisera to any of the established salivary antimicrobial factors examined. Additionally, native, heat-treated, or urea-denatured known salivary antimicrobial factors did not react with the antiserum to the anionic inhibitory proteins. However, the antiserum to the anionic inhibitory proteins was found to be reactive with a protein concentrate from mixed saliva or from separate submandibular and parotid secretions from a number of different donors, as well as with a purified protein fraction containing the homologous anionic inhibitory proteins. These findings suggest that the anionic inhibitory proteins represent intact and unique salivary proteins and not the degradation fragments of salivary antimicrobial protein factors within the oral environment.

Evidence for thiocyanate-sensitive peroxidase activity in human saliva.

A procedure was developed for determining the relative levels of lactoperoxidase, leukocyte myeloperoxidase, and thiocyanate-sensitive peroxidase in human saliva. With this procedure, most of the peroxidase activity in whole saliva from normal (those without cancer) subjects was found to be associated with lactoperoxidase and thiocyanate-sensitive peroxidase, with only a minor contribution from leukocyte myeloperoxidase. In contrast, thiocyanate-sensitive peroxidase and leukocyte myeloperoxidase were the major peroxidase activities present in the residual salivary secretion obtainable from two xerostomic patients examined, and these enzymes were present at concentrations much higher than those normally occurring in human saliva. The occurrence of thiocyanate-sensitive peroxidase in saliva has not been previously reported and may represent either an additional peroxidase activity of saliva or a form of lactoperoxidase which is particularly sensitive to inhibition by thiocyanate.

Comparative growth responses of oral streptococci on mixed saliva or the separate submandibular and parotid secretions from caries-active and caries-free individuals.

Growth of S. mutans on mixed or parotid saliva from CF individuals may be influenced by the availability of growth-supportive proteins or the inhibitory activity present in parotid saliva. A deficiency in growth-supportive proteins may explain the limited growth of S. sanguis on mixed or submandibular saliva from these individuals.

Cysteine toxicity for oral streptococci and effect of branched-chain amino acids.

Cysteine was bactericidal to strains of Streptococcus mutans and S. salivarius in concentrations that were nontoxic to S. sanguis, S. milleri, or S. mitior when these microorganisms were incubated in a saliva protein-based synthetic medium. Cysteine toxicity for S. mutans also occurred after incubation in synthetic base medium supplemented with amino acids as the nitrogen source for growth. The bactericidal effect of cysteine for S. mutans or S. salivarius in the saliva protein medium was influenced by the cysteine oxidative activity associated with the saliva protein fraction. Valine alone or in combination with leucine or isoleucine was effective in overcoming cysteine toxicity for susceptible strains of S. mutans or S. salivarius. Cysteine toxicity for these oral streptococci may be due to cysteine inhibition of an enzymatic step in the valine-leucine biosynthetic pathway.

Growth inhibition of oral streptococci in saliva by anionic proteins from two caries-free individuals.

Mixed saliva from two caries-free individuals possessed antimicrobial activity toward Streptococcus mutans and S. sanguis. This inhibitory activity was attributed to the presence of a group of four anionic proteins each of which strongly inhibited the growth of the oral streptococci in a saliva protein-based medium but not in a medium containing amino acids as a nitrogen source. These proteins, with isoelectric points of 4.70, 4.90, 4.98, and 5.05, respectively, neither reacted with antisera to immunoglobulin A, G, or M nor appeared to be functionally related to a number of salivary peroxidases, lactoferrin, or lysozyme. On this basis, they may represent a previously unreported group of growth-inhibitory antimicrobial factors occurring in the saliva of some individuals.

Utilization of hydroxyapatite adsorbable salivary proteins as growth substrates for plaque-forming oral streptococci.

Following treatment with hydroxyapatite, clarified mixed saliva from one donor source lost much of its growth-supportive activity for S. mutans VA-29R (type c). Growth of the organism in a basal medium containing proteins desorbed from HA with 0.067 M potassium phosphate buffer, pH 8.0, was accompanied by the disappearance of some of the proteins, including those with isoelectric points of 4.90, 5.72, and 6.00. These proteins are among those known to be specifically attacked by S. mutans. These findings suggest that some of the proteins selectively adsorbed from saliva with HA may serve as specific growth substrates for the plaque-forming oral streptococci.

Differential utilization of proteins in saliva from caries-active and caries-free subjects as growth substrates by plaque-forming streptococci.

Mixed or parotid saliva from caries-active individuals consistently supported better growth of Streptococcus mutans (type c) than that from caries-free individuals. Electrophoretic studies revealed that certain proteins in caries-active salivas were susceptible to microbial attack, but similar proteins in caries-free salivas were refractory.