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Objective The goals of this study were to (i) establish a useful miniature pig (minipig) model for irradiation-induced oral mucositis and (ii) evaluate the effect of Tempol to prevent its development. Methods and Materials Minipigs were irradiated with 6 Gy for five consecutive days targeting the entire oral cavity. To prevent radiation damage, minipigs were treated with 30 mg kg−1 Tempol 10 min before irradiation (n = 4), while the radiation-alone group was similarly injected with saline (n = 4). Lesions were graded using an oral mucositis score and visual inspection every 3 days, and biopsy of multiple sites was performed at day 18. Weight and chest and abdominal circumferences were measured every 3 days. Results Lesions began about 12 days after the first irradiation fraction and healed about 30 days after irradiation. Epithelial thickness was calculated on the lingual and buccal mucosa on the 18th day after the first irradiation fraction. Tempol provided modest protection from ulceration after irradiation using this treatment strategy. Conclusions This study established a useful large animal model for irradiation-induced oral mucositis and showed modest beneficial effects of Tempol in limiting tissue damage. The latter finding may be potentially valuable in preventing oral mucositis in patients receiving irradiation for head and neck cancers.

Salivary glands are useful target organs for local and systemic gene therapeutics. For such applications, the regulation of transgene expression is important. Previous studies by us in murine submandibular glands showed that a rapamycin transcriptional regulation system in a single serotype 2, adeno-associated viral (AAV2) vector was effective for this purpose. This study evaluated if such a vector was similarly useful in rhesus macaque parotid glands.A recombinant AAV2 vector (AAV-TF-RhEpo-2.3w), encoding rhesus erythropoietin (RhEpo) and a rapamycin-inducible promoter, was constructed. The vector was administered to macaques at either of two doses [1.5 x 10(11) (low dose) or 1.5 x 10(12) (high dose) vector genomes] via cannulation of Stensen's duct. Animals were followed up for 12-14 weeks and treated at intervals with rapamycin (0.1 or 0.5 mg kg(-1)) to induce gene expression. Serum chemistry, hematology, and RhEpo levels were measured at interval.AAV-TF-RhEpo-2.3w administration led to low levels of rapamycin-inducible RhEpo expression in the serum of most macaques. In five animals, no significant changes were seen in serum chemistry and hematology values over the study. One macaque, however, developed pneumonia, became anemic and subsequently required euthanasia. After the onset of anemia, a single administration of rapamycin led to significant RhEpo production in this animal.Administration of AAV-TF-RhEpo-2.3w to macaque parotid glands was generally safe, but led only to low levels of serum RhEpo in healthy animals following rapamycin treatment.

It is possible that, during primary saliva formation, aquaporins (AQPs) facilitate transcellular water flow across acinar cells to the lumina of salivary glands. In the rat submandibular gland (rSMG) AQP5 is localized in the apical membranes of acinar cells. The presence of a basolateral AQP in the same cell type has not been reported. We have therefore used immunofluorescence confocal microscopy to determine the subcellular localization of a newly discovered aquaporin, AQP8, in rSMG epithelial cells. The antibodies we used were made against the amino- or carboxyl-terminus (anti-rAQP8NT and anti-rAQP8CT, respectively) of an AQP8 cloned from rat pancreas and liver (rAQP8). Two lines of evidence suggest that both antibodies are suitable for immunolocalization studies. First, results of immunofluorescence confocal microscopy studies show that both antibodies bind to the plasma membranes of 293 cells infected with an adenovirus encoding rAQP8. Second, results of immunoblots of membranes from infected cells suggest that both antibodies bind to glycosylated and non-glycosylated forms of rAQP8. When tested in frozen sections of rSMG, we could not detect the binding of anti-rAQP8NT to any membranes. In contrast, anti-rAQP8CT binds to the basolateral membranes of acinar (but not ductal) epithelia, suggesting that rAQP8 resides in the basolateral membranes of acinar cells. Lack of anti-rAQPNT binding to basolateral membranes suggests that this epitope is not available in the membranes. Our evidence for the basolateral localization of rAQP8 in acinar cells, coupled with previous findings that AQP5 is localized apically in the same cells, raises the possibility that water crosses the acinar epithelium through these channels during primary saliva formation.

Objectives: Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) is a multifunctional cytokine that has pro-apoptotic, pro-angiogenic and pro-inflammatory effects. In liver, TWEAK leads to proliferation of progenitor oval cells, but not of mature hepatocytes. This study evaluated the hypothesis that TWEAK overexpression in salivary glands would lead to the proliferation of a salivary progenitor cell. Methods: A recombinant, serotype 5 adenoviral vector encoding human TWEAK, AdhTWEAK, was constructed, initially tested in vitro, and then administered to male Balb/c mice via cannulation of Wharton’s duct. TWEAK expression in vivo was monitored as protein secreted into saliva and serum by enzyme-linked immunosorbent assays. Salivary cell proliferation was monitored by proliferating cell nuclear antigen staining and apoptosis was monitored using TUNEL staining. Results: AdhTWEAK administration led to a dose-dependent, transient TWEAK protein expression, detected primarily in saliva. Salivary epithelial cell proliferation was generalized, peaking on ∼days 2 and 3. TWEAK expression had no detectable effect on apoptosis of salivary epithelial cells. Conclusion: Transient overexpression of TWEAK in murine salivary glands leads to a general proliferation of epithelial cells vs a selective stimulation of a salivary progenitor cell.

OBJECTIVE: We examined the toxicity and biodistribution associated with a single administration of a first-generation, serotype 5, adenoviral vector encoding human growth hormone (hGH; AdCMVhGH) to a single rat submandibular gland in the presence of hydroxychloroquine (HCQ). Previously, we showed that hGH is primarily secreted into saliva (approximately ninefold serum level) when expressed as a transgene in salivary glands (e.g. Baumet al,1999),butadministrationofHCQsubstantially increases the hGH levels secreted into the bloodstream (Hoque et al, 2001). A potential application of this observation is for patients with adult hGH deficiency. METHODS: Six groups of male and female adult rats (n = 12 each) were studied, with zero to 1.5 · 10 11 particles of AdCMVhGH, ±HCQ, administered retroductally. Multiple clinical and pathological parameters, as well as vector tissue distribution, were assessed. RESULTS: All animals survived until the scheduled day of sacrifice, and essentially no untoward events were observed clinically or at gross necropsy. We observed no vector-related effects on clinical hematology evaluations and a single, transient significant change on clinical chemistry evaluations (increased serum globulin levels). Three days after AdCMVhGH administration, the vector distributed to all tissues analyzed with the exception of gonads and heart. By day 29, most organs, other than the targeted and contralateral submandibular glands, were negative for the presence of vector. On day 3, none of the animals tested positive for the presence of replication competent adenovirus in either their blood or saliva. CONCLUSION: Salivary gland delivery of AdCMVhGH ±HCQ appears associated with limited toxicity in rats. Oral Diseases (2006) 12, 137–144

We previously demonstrated that recombinant adeno-associated virus vectors based on serotype 2 (rAAV2) can direct transgene expression in salivary gland cells in vitro and in vivo. However, it is not known how other rAAV serotypes perform when infused into salivary glands. The capsids of serotypes 4 and 5 are distinct from rAAV2 and from each other, suggesting that they may direct binding and entry into different cell types. In the present study, we investigated the tropisms, transduction efficiencies, and antibody response to AAV vectors based on AAV serotypes 2, 4, and 5. Administration of rAAV2beta-galactosidase (betagal), rAAV4betagal, or rAAV5betagal to murine submandibular salivary glands by retrograde ductal instillation resulted in efficient transduction of salivary epithelial cells, with AAV4 and AAV5 producing 2.3 and 7.3 times more betagal activity compared with AAV2. Improved transduction with AAV5 was confirmed by QPCR of DNA extracted from glands and immunohistochemical staining for transgene expression. Like AAV2, AAV5 primarily transduced striated and intercalated ductal cells. AAV4 transduction was evident in striated, intercalated, and excretory ductal cells, as well as in convoluted granular tubules. In keeping with the encapsulated nature of the salivary gland, the majority of persistent viral genomes were found in the gland and not in other tissues. Neutralizing antibodies (NABs) found in the serum of virus-infused animals were serotype specific and there was no crossreactivity between serotypes. No NABs were detected in saliva but sialic acid conjugates present in saliva could neutralize AAV4 at low dilutions. Together our data suggest that because of differences in receptor binding and transduction pathways, other serotypes may have improved utility as gene transfer vectors in the salivary gland and these differences could be exploited in gene therapy applications.

The diagnosis and treatment of Sjögren's syndrome, which poses many severe complications, should be of critical interest to dentists, who are often the first practitioners to detect symptoms. Dentistry is an integral part of health care delivery for patients with this condition. Management of Sjögren's syndrome can be seen as a model for the expanded scope of dental care in the future.

This study compared the localization of the alpha(v)beta3/5 integrins in the different cell types of the rat submandibular gland with the susceptibility of these cells to infection by an intraductally administered replication-deficient recombinant type 5 adenovirus coding for rat aquaporin 5 (termed AdrAQP5). We used a panel of antibodies and immunohistochemical procedures to determine the luminal membrane distribution of the integrins. The alpha(v)beta3/5 integrin subunits were present in luminal membranes of all ductal cell types and acini; however, the alpha(v) subunit was found to a lesser extent in the acini. After AdrAQP5 infection, the expression of AQP5 exhibited a similar, though not identical, cellular localization to that seen for alpha(v)beta3/5 integrins. AdrAQP5 infected all cell types in these glands, except excretory ducts, after intraductal administration, directing the transient expression of AQP5. In addition, the localization of alpha(v)beta3/5 integrins was examined in rabbit, monkey, and human submandibular and parotid glands. Although there were some interspecies differences, glands generally displayed the presence of these integrin subunits on luminal membranes of ductal cells and most acinar cells. These findings show that the presence of alpha(v)beta3/5 integrins on the luminal membranes of rat salivary epithelial cells is associated with the susceptibility of these cells to act as targets for adenoviral-mediated gene transfer. However, the abundance of alpha(v)beta3/5 integrin expression was not necessarily predictive of the extent of transgene expression in a particular cell type. Furthermore, because the salivary glands of rabbits, monkeys, and humans show a similar luminal membrane distribution of these integrins, it is likely that recombinant type 5 adenoviruses may be able to mediate in vivo gene transfer to salivary glands in these species.

The mechanism of irradiation-induced hypofunction of the salivary glands is a process that is not fully understood. Here we examine the hypothesis that intracellular and redox-active ions of iron and copper, which are associated with the secretion granules, play a catalytic role in the irradiation-induced damage. Rats were subjected to head and neck irradiation (15 Gy X rays) and allowed to recover for 2 months. The function of the parotid and submandibular glands was then determined by pilocarpine-stimulated salivary secretion. A 45% decrease in the function of both glands was obtained when compared to nonirradiated controls. Treatment prior to irradiation (90 min) with cyclocytidine (200 mg/kg) led to a massive degranulation of the parotid gland and yielded nearly complete protection from radiation-induced damage. In contrast, pilocarpine stimulation prior to irradiation led to a marginal degranulation of the parotid gland and yielded only 13% protection. Neither agent caused degranulation of the submandibular gland mucous cells or yielded functional protection of this gland. Treatment with both agents yielded a marked increase in iron, copper and manganese levels in the parotid gland saliva. An analogous marked increase in the redox activity of iron and copper ions was recorded for the parotid saliva stimulated by pilocarpine and cyclocytidine. Pilocarpine-stimulated submandibular gland saliva contained metal levels similar to those of the parotid gland saliva. However, no redox activity and no increase in metal mobilization could be demonstrated in the submandibular gland saliva stimulated by both agents. The correlation between the patterns of gland degranulation, mobilization of redoxactive metals and the protection of gland function, for both parotid and submandibular glands, focuses attention on the catalytic roles played by transition metal ions in promoting free radical reactions, which likely participate in the process of injury to the tissue.

Recently we employed two enantiomers of the muscarinic antagonist quinuclidinyl iodobenzilate (IQNB), and pharmacokinetic analyses, to define and quantitate nonspecific and specific binding to rat parotid gland muscarinic acetylcholine receptors (mAChRs) in vivo (Hiramatsu et al., 1993). The present studies were designed to utilize this same approach for evaluating mAChRs in three other morphologically different rat exocrine glands: the submandibular, sublingual and lacrimal glands. The metabolism and tissue distribution of the intravenously injected IQNB enantiomers were determined, and the resulting data were assessed in terms of their goodness of fit to several multicompartmental models. All three exocrine glands showed substantial nonspecific ligand distribution as measured with the receptor-inert enantiomer (SS)-IQNB. Nonspecific distribution represented 45, 21 and 36% of total ligand distribution in submandibular, sublingual and lacrimal glands, respectively, as measured with the receptor-active enantiomer (RR)-IQNB. The rank order of the binding potential, kinetically equivalent to Bmax/Kd, for (RR)-IQNB and these mAChRs was lacrimalsublingualsubmandibular glands (674 +/- 235575 +/- 109345 +/- 29). These results demonstrate that specific mAChRs in the exocrine glands can be measured in vivo with the (RR)-IQNB enantiomer and that despite some small quantitative differences, the distribution of (RR)- and (SS)-IQNB is similar in the three exocrine glands but is substantially different from that in brain and heart.

The purpose of this study was to examine the effects of different doses of X irradiation on the major rat salivary glands. The flow rates of the salivary glands were measured simultaneously in both parotid and submandibular glands of mature male Wistar rats at 3 and 40 days after head and neck irradiation with single doses of 2.5, 5, 7.5, 10, or 15 Gy. The parotid and submandibular glands were weighed at the time of saliva collection and total body weight was obtained weekly. Significant reductions in parotid salivary flow at 3 days and parotid and submandibular flow at 40 days were found. Diminished saliva output was dose-dependent and significantly reduced at radiation exposures of 7.5 Gy and greater. Submandibular function deteriorated between 3 and 40 days and the extent of hypofunction was comparable to the parotid gland at the latter time. Parotid and submandibular gland weights were reduced by irradiation in a dose-dependent manner at both 3 and 40 days. The effects were similar for both glands at the latter time. Total body weight was also reduced by the head and neck irradiation in a dose-dependent manner. There was significant mortality in the group receiving 15 Gy irradiation between 7 and 14 days after irradiation. The results demonstrate that parotid and submandibular glands may be affected comparably by equal doses of head and neck irradiation when examined at later times. In the period immediately after irradiation, there are significant differences in the responses of the major salivary glands.

Radiation-induced damage to salivary glands has been recognized for over 80 years. However, the mechanisms responsible for the destruction of gland parenchyma are still not known. The purpose of this study was to describe alterations in salivary function in the rat within the first 24 h after irradiation. Saliva was collected from the parotid and submandibular glands during radiation treatment and at four times (0.5, 1, 4, and 24 h) after 15 Gy X irradiation delivered to the head and neck. Total body weight, submandibular gland weight, and food and water intake were monitored and the total protein, sodium, and potassium content of the salivas was analyzed. The effects of radiation on salivary glands of the rat could be demonstrated by 24 h. Parotid salivary flow was reduced and the sodium concentration was significantly less than that in control animals. Submandibular gland weights declined markedly by 24 h. These effects appear to be influenced significantly by the animals' limited intake of food and water during this period, as well as the anesthesia administered.

It is not clear if aging distinctions can be made at the level of an organ or organism. The purpose of this study was to determine if a general definition of systemic aging, primary aging (influence of the passage of time), versus secondary aging (influence of extrinsic factors), can be used to discriminate the functional status of an individual organ system, the oral cavity. Thirty healthy, nonmedicated subjects (that is, those who exhibit primary aging) and 42 persons being treated for medical problems and taking prescription medications (that is, those who exhibit secondary aging), aged 75 to 96 years, from the oral physiology component of the Baltimore Longitudinal Study of Aging were evaluated. A standardized examination assessed gingival, periodontal, dental, and oral mucosal tissues. There were few substantive differences in oral health and function between primary and secondary aging subjects. Thus use of broad definitions of aging in an organism did not lead to meaningful predictions of the health or function of an individual organ system. Furthermore, the similarity in the oral condition between both groups studied here suggests substantial resiliency of the oral cavity during aging.

There is little known about the regulation of gene expression in rat parotid glands after exposure to ionizing radiation. The present studies investigate the effects of in vivo ionizing radiation, with subsequent stimulation of beta-adrenergic receptors by isoproterenol, on parotid gland function and on the expression of the early response genes, c-fos, c-jun, and jun B. Ionizing radiation diminished parotid gland weight and saliva output. Treatment of irradiated rats with isoproterenol increased the gland weight to levels similar to those in nonirradiated rats. However, such treatment had no effect on saliva output as indicated by measurements of parotid salivary flow rate. Irradiation alone increased the expression of c-fos, c-jun, and jun B. The combination of irradiation and isoproterenol had an additional effect on the levels of c-fos and jun B mRNAs and proteins particularly at earlier experimental times (1 to 8 h). Isoproterenol alone induced high levels of c-fos and jun B mRNA but not of c-jun mRNA. However, c-jun mRNA was induced markedly by radiation and 8 h of isoproterenol treatment, indicating a combined effect on c-jun gene expression. These observations suggest that the expression of the proto-oncogenes c-fos, c-jun, and jun B is probably regulated through differential signal transduction pathways which may be activated by these external stimuli and may be associated with functional changes induced in the rat parotid gland by ionizing radiation and by ionizing radiation and isoproterenol.

An HIV-1/ATH8-cell cytopathic system was used to characterize the previously reported anti-HIV-1 activity of human saliva. Inhibitory activity was demonstrated by monitoring viable cell counts, HIV-1 p24 core antigen, and reverse transcriptase levels. Nonfiltered whole saliva, sterilized by irradiation, protected the ATH8 cells from HIV-1 infection. When HIV-1/saliva mixtures were filtered following incubation, the quantity of virus was significantly less (approximately 50%) than in HIV-1/media-filtered controls, suggesting that salivary aggregation and/or agglutination may be involved in the inhibitory activity. However, a sufficient number of apparently morphologically intact viral particles were still present in the HIV-1/saliva filtrates to lead to infection. When saliva was filtered prior to incubation with HIV-1, these filtrates showed substantial inhibitory activity, although reduced compared with that of non-prefiltered saliva. We conclude that saliva likely has several means by which to inhibit HIV-1 infectivity.

We studied the effects of pilocarpine hydrochloride, a para-sympathomimetic agent, on major salivary gland output and subjective responses in 31 patients with salivary hypofunction. Pilocarpine hydrochloride (5-mg capsules, three times daily) was given for 5 months and a placebo was randomly assigned for 1 month in a double-blind fashion. Objective measurements of major salivary gland output, subjective impressions of oral moisture, treatment-related side effects, and a number of physiologic measures were assessed monthly. Pilocarpine significantly increased salivary output in 21 of the 31 patients. Subjective improvement in the feeling of oral dryness, speaking, chewing, and swallowing were reported by 27 individuals. Side effects, while common, generally were mild and tolerable. There were no significant alterations in cardiovascular or other physiologic measures. We conclude that pilocarpine is an effective and safe treatment for salivary gland hypofunction and xerostomia in selected patients. The increase in major gland output provides beneficial natural secretions and relief of oral dryness.

Saliva is important for the preservation and maintenance of oral health. It is unclear, however, how much saliva is required to maintain normal oral function. Major salivary gland flow rates, objective measurements of oral health, and subjective complaints of oral problems were assessed in different-aged, healthy persons. Results suggest that the comparison of major salivary gland flow rates of an individual with population standards to identify patients susceptible to the effects of salivary dysfunction is unreliable. Changes in salivary function over time are a more meaningful gauge of the impact of saliva on oral health. The clinician should monitor salivary production to identify patients with declining salivary gland output.

We have utilized fura-2 and a Ca2+ surrogate, Mn2+, to assess the mechanism of Ca2+ entry involved in the refill of the internal agonist-sensitive Ca2+ pool in parotid acini. Both the muscarinic agonist, carbachol, and the alpha-adrenergic agonist, epinephrine, stimulate Mn2+ entry into dispersed parotid acini, which is detected as an augmentation in fura-2 fluorescence quench rate. The rate of Mn2+ entry into parotid acini, depleted of internal agonist-sensitive Ca2+ pools by prolonged carbachol stimulation in a nominally Ca2(+)-free medium, is not significantly changed by the addition of the muscarinic antagonist, atropine, but is significantly attenuated when these internal pools are allowed to either partially or totally reload with Ca2+. Also, we provide evidence which suggests that under conditions which promote refill, Mn2+ appears to directly enter the cytosol from the extracellular medium and is not accumulated into an internal Ca2+ pool either directly from the medium or via a cytosolic route. Thus, we suggest that during refill, Ca2+ enters into the cytosol prior to its recruitment into the internal agonist-sensitive Ca2+ pool and in turn, the magnitude of this entry is modulated by the refill status of this pool.

Thirty-seven HIV-1-positive patients contributed salivary samples from individual major salivary glands. Nineteen patients were unmedicated and asymptomatic, and 18 patients had developed signs of AIDS. Salivas from 15 healthy males served as controls. Levels of four salivary antimicrobial proteins (lactoferrin, lysozyme, secretory IgA, and histatins) were determined, as well as total fluid output of the major salivary glands. Concentrations of all four salivary antimicrobial proteins were found to be increased in the stimulated submandibular/sublingual saliva of all HIV-1-positive patients as well as the subset of unmediated HIV-1-positive patients. Those patients with evidence of oral candidiasis had the highest concentrations of lysozyme and histatins, potent antifungal proteins, in their saliva. Although the etiology of these protein increases is still unknown, these results further document salivary changes following HIV-1 infection.

Freshly collected parotid saliva collected from human donors were shown by polyacrylamide gel electrophoresis to continuously secrete a group of low-molecular-weight cationic polypeptides. Up to 14 bands could be identified by Coomassie blue staining, and all bands migrated more rapidly than purified human leukemic lysozyme in cationic polyacrylamide gel electrophoresis. These peptides could be isolated as a group relatively free of other salivary components and recovered in high yields from concentrated parotid saliva by Sephadex G-25 chromatography. In sodium dodecyl sulfate gel electrophoresis, the histidine-rich polypeptide bands appeared as just two bands migrating at the tracking dye and ahead of insulin chain B. Amino acid analysis of the mixture revealed an average content of at least 48% cationic residues, of which half were histidine. When stained bands were eluted from electrophoretic gels, hydrolyzed, and subjected to amino acid analyses, they were found to be enriched in histidine. There was also a correlation of the electrophoretic mobility with the content of basic amino acids. Sephadex G-25 chromatography is a convenient, simple method for preparing milligram quantities of the histidine-rich polypeptides for chemical and biochemical studies.

The effect of X irradiation on rat parotid acinar cell function was evaluated in vitro 1, 3, and 7 days following in vivo exposure to 2000 R. Several cellular functions were followed: protein secretion (amylase release), ion movement (K+ efflux and reuptake), amino acid transport (alpha-amino[14C]isobutyric acid), and an intermediary metabolic response ( [14C]glucose oxidation). In addition both the morphologic appearance and in vivo saliva secretory ability of parotid cells were assessed. Our results demonstrate that surviving rat parotid acinar cells, isolated and studied in vitro 1-7 days following 2000 R, remain functionally intact despite in vivo diminution of secretory function.

Parotid gland acinar cells, prepared from 12- and 24-month-old rats, show decreased physiological responsiveness to alpha-adrenergic stimulation in vitro compared to cells from 3-month-old rats. K+ efflux, an index of water and electrolyte secretion, was approximately 35% lower with 12- and 24-month-old parotid cells. No loss of alpha-adrenergic receptors, their binding affinity for specific alpha-adrenergic ligands, or their relative subtype distribution, accompanied the diminished exocrine function. Conversely, a significant reduction in alpha-adrenergic-mediated phospholipid turnover in, and 45Ca2+ efflux from, parotid cells of older rats was observed. These changes in phospholipid metabolism and Ca2+ flux were parallel to changes seen in K+ efflux as judged by dose-response studies. When the alpha-adrenergic receptor was by-passed by using the Ca2+-ionophore A-23187 to elicit K+ efflux, young and old parotid cells were equally responsive. In aggregate the findings suggest that parotid gland cells from older rats display an altered alpha-adrenergic signal transduction mechanism at a site between the receptor and phospholipid turnover/Ca2+ mobilization.

Parotid acinar cells, prepared from pharmacologically sympathectomized adult rats (reserpine, 0.1 mg/kg/day for 1 week), display decreased responsiveness to beta adrenergic stimulation in vitro compared to cells from control and surgically sympathectomized rats. Both methods of denervation increase amylase content (amylase activity per microgram of DNA). Percent release of amylase activity and percent release of CCl3COOH-precipitable [14C]leucine were used as indicators of protein secretion. Exposure of cells from pharmacologically sympathectomized rats to the beta adrenergic agonist, isoproterenol, resulted in a marked reduction in receptor-coupled secretion (67% and 75% relative to controls, respectively). 8-Bromo-cyclic AMP, like isoproterenol, was unable to surmount this reserpine-induced inhibition of stimulated secretion, suggesting that an alteration in receptor-adenylate cyclase coupling is not responsible for the observed secretion defect. Cells prepared from surgically sympathectomized rats displayed modest decreases in stimulated secretion when the same secretory markers were monitored (30% and 25% relative to controls, respectively). The number of beta adrenoreceptors [( 3H]dihydroalprenolol binding sites) increased (35%), with no change in binding affinity, in membrane preparations from reserpine-treated rats. Thus, the observed inhibition of beta adrenergic agonist-induced secretion is not likely the result of alterations in beta adrenergic receptor characteristics. Short-term (1 week) surgical denervation had no effect on the number of beta adrenergic receptor sites; however, an increase in ligand binding affinity was noted. The decrease in the apparent Kd (30%) was not the result of a shift in receptor subtype as determined by competition studies with specific beta-1 (atenolol) and beta-2 (ICI 118,551) receptor antagonists.(ABSTRACT TRUNCATED AT 250 WORDS)

In dispersed rat parotid gland acinar cells, the beta-adrenergic agonist (-)-isoproterenol, but not its stereoisomer (+)-isoproterenol, induced a transient 1.6-fold (at maximum stimulation, 2 x 10(-4) M) increase in cytosolic free calcium ([Ca2+]i) within 9 s, which returned to resting levels (approximately 190 nM) by 60 s. This [Ca2+]i response was not altered by chelating extracellular Ca2+ with [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) and could be completely blocked by the beta-adrenergic antagonists propranolol (beta 1 + beta 2) and ICI 118,551 (beta 2) but not by atenolol (beta 1). The muscarinic-cholinergic agonist carbachol (at maximum stimulation, 10(-5) M) induced a 3-4-fold elevation in [Ca2+]i within 6 s, which slowly returned to resting levels by 8-10 min. The peak carbachol [Ca2+]i response was not substantially altered by the addition of EGTA to the extracellular medium. However, if the cells were first stimulated with isoproterenol in the EGTA-containing medium, the peak carbachol response was decreased approximately 54%. When carbachol was added to cells in the presence of high extracellular calcium, at the isoproterenol-stimulated [Ca2+]i peak, the resulting [Ca2+]i level was equal to that achieved when carbachol was either added alone or added after propranolol and isoproterenol. 8-Bromo-cyclic AMP induced a [Ca2+]i response similar to that elicited by isoproterenol, which was not additive to that by carbachol. Carbachol induced a approximately 3.5-fold increase in inositol trisphosphate (IP3) production in parotid cells within 30 s. 8-Bromo-cAMP, N6,O2'-dioctanoyl-cAMP, and isoproterenol consistently induced a significant stimulation in IP3 production. The half-maximal concentration of isoproterenol required for [Ca2+]i mobilization and IP3 production was comparable (approximately 10(-5) M). Isoproterenol-induced IP3 formation was blocked by propranolol. The data show that in rat parotid acinar cells, beta-adrenergic stimulation results in IP3 formation and mobilization of a carbachol-sensitive intracellular Ca2+ pool by a mechanism involving cAMP. This demonstrates an interaction between the cAMP and phosphoinositide second messenger systems in these cells.

ATP-dependent Ca2+ transport was studied in basolateral membrane vesicles prepared from rat parotid gland slices incubated without or with agents which increase cyclic AMP. Isoproterenol (10(-5) M), forskolin (2 X 10(-6) M) and 8-bromocyclic AMP (2 X 10(-3) M) all increased ATP-dependent 45Ca2+ uptake 1.5- to 3-fold. The effect of isoproterenol was concentration-dependent and blocked by the beta-adrenergic antagonist propranolol. Enhanced uptake did not appear an artifact of vesicle preparation as apparent vesicle sidedness, 45Ca2+ efflux rates, specific activity of marker enzymes and equilibrium Ca2+ content were identical in vesicle preparations from control and 8-bromocyclic AMP-treated slices. Kinetic studies showed the ATP-dependent Ca2+ transport system in vesicles from 8-bromocyclic AMP-treated slices displayed a approximately 50% increase in Vmax and in Km Ca2+, compared to controls. The data suggest that physiological secretory stimuli to rat parotid acinar cells, which involve cyclic AMP, result in a readjustment of the basolateral membrane ATP-dependent Ca2+ pump.

To ascertain whether an age-related decrease in number of taste buds occurs in the tongue of aged rats, taste buds were counted in fungiform and circumvallate papillae of Wistar-derived rats aged 5-7 months and 23-24 months. There was no difference in number or size of taste buds in papillae in anterior and posterior areas of the tongue from the two age groups. However, both fungiform and circumvallate papillae were larger in old rats. These results complement a recent study demonstrating no difference in numbers of taste buds in human fungiform papillae from birth to old age (Arvidson, 1979). Both anatomical investigations and human taste threshold studies indicate that age-related differences in the gustatory system are not as substantial as investigators have suggested in the past.

Epinephrine stimulated glucose oxidation in isolated rat parotid cell aggregates through alpha- and beta-adrenergic mechanisms. The alpha-adrenergic component appeared to be of the alpha 1-receptor subtype as evidenced by inhibition studies with selective antagonists. Calcium mobilization in the presence of the ionophore A23187 was as capable as epinephrine in eliciting this response. Epinephrine stimulation of glucose oxidation was, however, only partially dependent on extracellular calcium. Only the upper 50% of the maximal (10(-5) M) epinephrine response required external calcium, since stimulation by 10(-6) M epinephrine, which elicits only about 40% of the maximal (10(-5) M epinephrine) response, was independent of external calcium. Furthermore, no external calcium dependence was observed for pure alpha- or beta-adrenergic stimulation alone, which comprised about 60 and 40%, respectively, of the maximal epinephrine response. Thus, adrenergic mediated parotid cell glucose oxidation may proceed by different mechanisms depending on the extent and nature of cellular stimulation.

Both unstimulated and stimulated parotid saliva samples were collected from 85 healthy, unmedicated individuals between the ages of 23 and 81 years. There were no significant differences in flow rate related to age with either unstimulated or stimulated secretion.

A physical examination procedure was used to evaluate oral motor functions in a population of 257 community-dwelling males and females ranging in age from 23-88 yr. An increased prevalence of altered motor performance with age was found for parameters measuring lip posture and masticatory muscle function. Also, males, but not females, showed a higher prevalence of alterations in tongue function and in swallowing.

The regulation of phosphatidylinositol turnover by alpha-adrenergic agonists in rat parotid acinar cell aggregates was examined with respect to kinetics and agonist-antagonist interactions. Phosphatidylinositol turnover was followed by the changes in the specific activities of [32P]phosphatidic acid and [32P]phosphatidylinositol. The specific activity of phosphatidic acid increased rapidly (within 1 min) after addition of epinephrine (10(-5) M), reached a maximal level within 12-16 min, and then decreased. Incorporation of 32P into phosphatidylinositol exhibited a lag phase of about 5 min and then increased continuously for an additional 40 min. The absolute amounts of phosphatidic acid and phosphatidylinositol did not change. The concentrations of epinephrine needed to stimulate 32P incorporation into phosphatidic acid and phosphatidylinositol, measured at 15 and 30 min, respectively, were similar; Ka values of 2.05 +/- 0.46 X 10(-6) M for phosphatidic acid and 2.98 +/- 0.30 X 10(-6) M for phosphatidylinositol were found. The effects of agonists on 32P labeling of phosphatidylinositol, in order of potency, were epinephrine greater than or equal to norepinephrine greater than phenylephrine much greater than normetanephrine. When various adrenergic antagonists were evaluated for their ability to inhibit 10(-5) M epinephrine-stimulated 32P incorporation into both phosphatidic acid and phosphatidylinositol, the order of antagonist potency was prazosin greater than or equal to phenoxybenzamine greater than phentolamine greater than or equal to yohimbine greater than much greater than propranolol. These findings indicate that phosphatidylinositol-phosphatidic acid turnover in the rat parotid gland is mediated by the alpha 1-adrenergic receptor system.

This report describes a variety of demographic, socioeconomic and dental characteristics of 254 (145 male, 109 female) participants in a longitudinal study of oral physiology and aging. All individuals are part of the U.S. National Institute on Aging's Baltimore Longitudinal Study of Aging. Subjects are generally in middle to upper middle socioeconomic strata, well educated and in good health (about 75% not taking prescription medication). Most participants practice regular oral hygiene habits and receive regular and comprehensive dental care. Less than 4% of all subjects are edentulous and the average number of natural teeth present for all participants was about 23. Data are presented on DMFT scores, cervical caries (active an restored) scores and gingival and periodontal disease indices for male by age group (20-39, 40-59, 60-88 years) and for females by menopause status (pre-, post-).

The possible role of cyclic AMP-mediated phosphorylation events in the regulation of exocrine secretion after beta-adrenergic stimulation was examined in vitro in dispersed acinar cell aggregates from rat parotid gland. l-Isoproterenol, a beta-adrenergic agonist, stimulated endogenous activity of cyclic AMP-dependent protein kinase, alterations in the 32P content of 3 parotid phosphoproteins (increased 32P in 2, Mr = 27,000 and 14,000; decreased 32P in the remaining, Mr = 13,600), and amylase secretion in a dose-dependent manner. All responses were half-maximal within a range of l-isoproterenol concentrations of approximately 4 X 10(-8) to 5 X 10(-7) M. Examination of the time course of these 3 processes revealed that by 30 s after addition of l-isoproterenol, significant elevations in cyclic AMP-dependent protein kinase activity and alterations in the 32P content of the 3 parotid proteins had occurred, whereas secretion of amylase from cells was first detected 1-2 1/2 min after hormonal stimulation. Dibutyryl cyclic AMP (2 mM) elicited the same changes in parotid protein 32P content as l-isoproterenol. Our results support the concept of a role for cyclic AMP-regulated protein phosphorylation in the sequence of cellular events leading to exocrine protein secretion from the rat parotid gland following beta-adrenergic stimulation.

The parotid gland of the aged rat provides an example of an altered alpha 1-adrenergic physiologic response (K+ efflux) resulting from a postreceptor perturbation in signal transduction mechanisms (Ito, H., Baum, B. J., Uchida, T., Hoopes, M. T., Bodner, L.Roth, G. S. (1982) J. Biol. Chem. 257, 9532-9538). This alteration in gland function can be completely circumvented by eliciting K+ efflux via the Ca2+-ionophore, A23187, at several Ca2+ concentrations (ibid.). Since Ca2+ is purported to mediate other secretory events in the rat parotid, we have probed neurotransmitter regulated Ca2+ mobilization and secretory mechanisms in this tissue by employing an aging paradigm. The responses studied were alpha-adrenergic- and muscarinic-cholinergic-mediated K+ efflux, 45Ca2+ release, and amylase secretion. No differences were detected between young (3 months) and old (24 months) cell preparations for any muscarinic-cholinergic agonist-induced response studied. Following alpha-adrenergic stimulation, K+ efflux and 45Ca2+ release from old cell preparations were reduced markedly, while no changes were found for the amylase secretion response. These results suggest that 1) alpha-adrenergic and cholinergic signal transduction mechanisms for K+ efflux and 45Ca2+ release are dissociated in cells of the rat parotid gland, and 2) following alpha 1-adrenergic stimulation, signal transduction likely proceeds by at least two pathways, one which is apparently involved in protein excytosis (intact in cells from old rats) and the other which is apparently involved in K+ efflux and 45Ca2+ release (perturbed in old cells).

The interaction of bromoacetylalprenololmenthane (BrAlpM), an irreversible beta adrenergic receptor antagonist, with rat parotid acinar cells was studied in vitro. In the presence of BrAlpM, the rate of (-)-isoproterenol-induced exocrine secretion from cells, measured as percentage of amylase release, was markedly reduced. The concentration of (-)-isoproterenol required to elicit half-maximal protein secretion was about 100 times greater (5 microM) in the presence of 1 microM BrAlpM than in control incubations (0.05 microM). BrAlpM and propranolol were similar in their ability to inhibit parotid protein release (IC50 approximately 10(-7) M). To demonstrate that BrAlpM functioned as an irreversible beta adrenergic antagonist, cells were preincubated with BrAlpM for varying amounts of time and then washed three to six times before adding (-)-isoproterenol. At least 10 min preincubation was required to show irreversibility. Alprenolol, under the same preincubation conditions, was unable to inhibit amylase release. BrAlpM inhibited the binding of [3H]dihydroalprenolol to parotid beta adrenoreceptors over a concentration range similar to that required for inhibition of protein secretion. Cells incubated in the absence or presence of BrAlpM displayed a comparable morphologic appearance when viewed by light and electron microscopy. The degree of inhibition of isoproterenol-induced exocytosis of secretory granules by BrAlpM appeared to vary from cell to cell. These findings suggest that BrAlpM should be a useful probe to study beta adrenoreceptor function and metabolism in rat parotid acinar cells.