Prostate-specific antigen: An unfamiliar protein in the human salivary glands.

OBJECTIVES: The presence of prostate-specific antigen (PSA) in saliva and salivary glands has been reported. Nevertheless, its release pathway in these glands remains to be elucidated. Here, we showed PSA subcellular distribution focusing on its plausible route in human salivary parenchyma. MATERIALS AND METHODS: Sections of parotid and submandibular glands were subjected to the immunohistochemical demonstration of PSA by the streptavidin-biotin method revealed by alkaline phosphatase. Moreover, ultrathin sections were collected on nickel grids and processed for immunocytochemical analysis, to visualize the intracellular distribution pattern of PSA through the observation by transmission electron microscopy. RESULTS: By immunohistochemistry, in both parotid and submandibular glands PSA expression was detected in serous secretory acini and striated ducts. By immunocytochemistry, immunoreactivity was retrieved in the cytoplasmic compartment of acinar and ductal cells, often associated with small cytoplasmic vesicles. PSA labeling appeared also on rough endoplasmic reticulum and in the acini's lumen. A negligible PSA labeling appeared in most of the secretory granules of both glands. CONCLUSIONS: Our findings clearly support that human parotid and submandibular glands are involved in PSA secretion. Moreover, based on the immunoreactivity pattern, its release in oral cavity would probably occur by minor regulated secretory or constitutive-like secretory pathways.

Tyrosine-hydroxylase, dopamine ß-hydroxylase and choline acetyltransferase-like immunoreactive fibres in the human major sublingual gland.

OBJECTIVE: To study the innervation of the major sublingual gland by means of immunohistochemistry. DESIGN: Bioptic and autoptic specimens of the major sublingual gland of humans were examined for the presence of immunoreactivity to tyrosine hydroxylase and dopamine-ß-hydroxylase, on one hand, and choline acetyltransferase, on the other, to indicate adrenergic and cholinergic nerves, respectively. RESULTS: Acini and ducts were supplied by both divisions of the autonomic nervous system. CONCLUSIONS: Mucous and seromucous cells of the human major sublingual glands may respond with secretion not only to parasympathetic activity but also to sympathetic activity. The major sublingual gland is therefore a potential contributor to the mucin secretion recently reported in the literature in response to high sympathetic activity during physical exercise.

Proteomics of the acid-soluble fraction of whole and major gland saliva in burning mouth syndrome patients.

OBJECTIVE: In the present study the salivary proteome of burning mouth syndrome patients and healthy subjects was characterized by a top-down proteomic approach and compared to highlight possible qualitative and quantitative differences that may give suggestions about the causes of this pathology which are still unknown. MATERIALS AND METHODS: Resting and stimulated whole saliva, stimulated parotid and submandibular/sublingual saliva samples were collected from burning mouth syndrome patients (n = 16) and age- and gender-matched healthy subjects (n = 14). An equal volume of 0.2% trifluoroacetic acid was added to each sample immediately after collection and the supernatants were analysed by liquid chromatography coupled to electrospray-ionisation mass spectrometry. Proteins and peptides were quantified using a label-free approach measuring the extracted ion current peak areas of the main salivary proteins and peptides. RESULTS: The quantitation of the main salivary proteins and peptides revealed a higher concentration of cystatin SN in resting saliva of burning mouth syndrome patients with respect to healthy controls and no other conspicuous changes. CONCLUSIONS: The reported data showed that the salivary protein profile was not affected, in composition and relative abundance, by the burning mouth syndrome, except for the cystatin SN, a protein up-regulated in several pathological conditions, that might be considered potentially indicative of the disease.

Melatonin release by exocytosis in the rat parotid gland.

Several beneficial effects on oral health are ascribed to melatonin. Due to its lipophilic nature, non-protein-bound circulating melatonin is usually thought to enter the saliva by passive diffusion through salivary acinar gland cells. Recently, however, using transmission electron microscopy (TEM), melatonin was found in acinar secretory granules of human salivary glands. To test the hypothesis that granular located melatonin is actively discharged into the saliva by exocytosis, i.e. contrary to the general belief, the ß-adrenergic receptor agonist isoprenaline, which causes the degranulation of acinar parotid serous cells, was administered to anaesthetised rats. Sixty minutes after an intravenous bolus injection of isoprenaline (5 mg kg-1 ), the right parotid gland was removed; pre-administration, the left control gland had been removed. Samples were processed to demonstrate melatonin reactivity using the immunogold staining method. Morphometric assessment was made using TEM. Gold particles labelling melatonin appeared to be preferentially associated with secretory granules, occurring in their matrix and at membrane level but, notably, it was also associated with vesicles, mitochondria and nuclei. Twenty-six per cent of the total granular population (per 100 µm2 per cell area) displayed melatonin labelling in the matrix; three-quarters of this fraction disappeared (P < 0.01) in response to isoprenaline, and melatonin reactivity appeared in dilated lumina. Thus, evidence is provided of an alternative route for melatonin to reach the gland lumen and the oral cavity by active release through exocytosis, a process which is under the influence of parasympathetic and sympathetic nervous activity and is the final event along the so-called regulated secretory pathway. During its stay in granules, anti-oxidant melatonin may protect their protein/peptide constituents from damage.

Electrostimulation of the lingual nerve by an intraoral device may lead to salivary gland regeneration: A case series study

BACKGROUND: Salivary gland function is controlled by the salivary reflex, whose efferent arm is composed by the parasympathetic and the sympathetic divisions of the autonomic nervous system. Parenchymal injury is the main salivary gland involvement of Sjögren's syndrome and head and neck radiotherapy, but neural damage has been reported as well. Recently an intraoral device for electrostimulation of the lingual nerve in vicinity to the lower third molar has been introduced. At this point this nerve carries efferent fibers for the innervation of the submandibular, sublingual and several minor salivary glands and afferent fibers of the salivary reflex. Therefore, excitation of these fibers potentially leads to increased secretion of all salivary glands. Thus, the study objective was to assess whether comprehensive neural activation by electrostimulation of the lingual nerve carries the potential to induce the regeneration of damaged salivary glands. MATERIAL AND METHODS: The device was tested on three patients with no collectable resting and stimulated secretion of saliva during a double blind, sham controlled period of two months and nine open-label months. RESULTS: All three subjects developed the capacity to spit saliva, not only in direct response to the electrostimulation but also after free intervals without electrostimulation. In addition, their symptoms of dry mouth severity and frequency improved. CONCLUSIONS: This recovery is probably due to the combined effect of increase in secretory functional gland mass and regain of nervous control of the secretory elements and blood vessels. Both are phenomena that would contribute to gland regeneration

Saliva on the oral mucosa and whole saliva in women diagnosed with burning mouth syndrome.

OBJECTIVE: The aim of the study was to examine mucosal saliva and unstimulated (UWS) and stimulated (SWS) whole saliva secretion rates and associated factors, in 56 female patients diagnosed with BMS and age-matched control women. MATERIAL AND METHODS: Mucosal saliva was assessed using the Periotron® method and blood flow using laser Doppler flowmetry. Diseases, drug usage and xerostomia were registered using questionnaires. RESULTS: The patients with BMS displayed less lingual and whole saliva, and more hyposalivation, xerostomia diseases/disorders and drug usage, compared to the controls. Only a low SWS and xerostomia differed after adjusting for drugs and systemic diseases. Regression analyses suggested an importance of saliva affecting drugs for saliva on the tongue and for SWS, and the total number of drugs used for UWS. Lingual saliva and UWS were also associated with systemic diseases in the patients. Xerostomia was significantly associated with drug use and whole saliva for all subjects but not in separate analyses of the groups. CONCLUSION: Less saliva in patients with BMS could be related to more systemic diseases and medication and not to the syndrome per se. Xerostomia in the patients was not related to any of these factors.

Marked Differences in the Submandibular Salivary Proteome between Sardinian Alcohol-Preferring and Sardinian Alcohol-Non Preferring Rats Revealed by an Integrated Top-Down-Bottom-Up Proteomic Platform.

Sardinian alcohol-preferring (sP) and Sardinian alcohol-non preferring (sNP) rats have been selectively bred for opposite alcohol preference and consumption. Aiming to verify possible differences at the proteomics level between sP and sNP rats, we investigated the salivary proteome by a a liquid chromatography-mass spectrometry top-down-bottom-up integrated approach. For this purpose, submandibular saliva was collected from alcohol-naive sP and sNP rats under isoprenaline stimulation. A total of 200 peptides and proteins were detected and quantified in the two rat lines, 149 of which were characterized in their naturally occurring structure. The data are available via ProteomeXchange with identifier PXD006997. Surprisingly, sP rats exhibited marked quantitative and qualitative differences with respect to sNP rats, namely higher levels of proteoforms originating from submandibular gland protein C, and from submandibular rat protein 2, as well as those of several unidentified peptides and proteins. sP rats expressed some proteins not detectable in sNP rats such as the glutamine and glutamic acid-rich protein (GRP)-CB. The isoform GRP-B, detectable in both rat lines, was more abundant in sNP rats. The submandibular saliva of sNP rats was also characterized by very high levels of GRP-B proteolytic peptides and rat salivary protein 1. Whether these differences could contribute to the opposite alcohol preference and consumption of sP and sNP rats is currently unknown and requires further investigation.

A Guide to Medications Inducing Salivary Gland Dysfunction, Xerostomia, and Subjective Sialorrhea: A Systematic Review Sponsored by the World Workshop on Oral Medicine VI.

BACKGROUND: Medication-induced salivary gland dysfunction (MISGD), xerostomia (sensation of oral dryness), and subjective sialorrhea cause significant morbidity and impair quality of life. However, no evidence-based lists of the medications that cause these disorders exist. OBJECTIVE: Our objective was to compile a list of medications affecting salivary gland function and inducing xerostomia or subjective sialorrhea. DATA SOURCES: Electronic databases were searched for relevant articles published until June 2013. Of 3867 screened records, 269 had an acceptable degree of relevance, quality of methodology, and strength of evidence. We found 56 chemical substances with a higher level of evidence and 50 with a moderate level of evidence of causing the above-mentioned disorders. At the first level of the Anatomical Therapeutic Chemical (ATC) classification system, 9 of 14 anatomical groups were represented, mainly the alimentary, cardiovascular, genitourinary, nervous, and respiratory systems. Management strategies include substitution or discontinuation of medications whenever possible, oral or systemic therapy with sialogogues, administration of saliva substitutes, and use of electro-stimulating devices. LIMITATIONS: While xerostomia was a commonly reported outcome, objectively measured salivary flow rate was rarely reported. Moreover, xerostomia was mostly assessed as an adverse effect rather than the primary outcome of medication use. This study may not include some medications that could cause xerostomia when administered in conjunction with others or for which xerostomia as an adverse reaction has not been reported in the literature or was not detected in our search. CONCLUSIONS: We compiled a comprehensive list of medications with documented effects on salivary gland function or symptoms that may assist practitioners in assessing patients who complain of dry mouth while taking medications. The list may also prove useful in helping practitioners anticipate adverse effects and consider alternative medications.

Salivary secretion and drug treatment in four 70-year-old Swedish cohorts during a period of 30 years.

OBJECTIVE: The aim was to examine the association between drug treatment and unstimulated and stimulated whole saliva in four 70-year-old Swedish cohorts, between 1971 and 2001. BACKGROUND: Both diseases and their medication can affect the salivary secretion rate. MATERIALS AND METHODS: The study was based on selected samples of four cohorts born in 1901/1902, 1906/1907, 1911/1912 and 1930/1931, respectively, a total of 1072 individuals. The response rate varied between 65% and 85%. RESULTS: The mean value for the stimulated salivary secretion rate was higher in men (1.3 ± 0.8 ml/min) than in women (1.0 ± 0.7 ml/min) (p < 0.001)). There was a significant association between the salivary secretion rate and the number of drugs among both women (p < 0.01) and men (p < 0.001). This influence was most pronounced in participants who were treated with cardiovascular drugs, mainly diuretics and non-selective ß-adrenoceptor blockers, but also with antipsychotics and antidepressants, even when adjusted for cohort, gender, number of teeth and other drugs. There was an increase in treatment with medicines during the three decades. CONCLUSION: In these four groups of 70-year-old participants, high drug consumption was associated with lower salivary flow. Unstimulated secretion was only affected in women and then, when taking four or more drugs. Pronounced hyposalivation was, however, uncommon. Cardiovascular drugs, antidepressants and antipsychotics were associated with low salivary secretion. In this age group, the frequently observed association between polypharmacy and a lower saliva secretion rate represents a risk of impaired dental health.

World Workshop on Oral Medicine VI: a systematic review of medication-induced salivary gland dysfunction: prevalence, diagnosis, and treatment.

OBJECTIVES: Medication-induced salivary gland dysfunction (MISGD) causes significant morbidity resulting in decreased quality of life. This systematic review assessed the literature on the prevalence, diagnosis, treatment, and prevention of MISGD. MATERIALS AND METHODS: Electronic databases were searched for articles related to MISGD through June 2013. Four independent reviewers extracted information regarding study design, study population, interventions, outcomes, and conclusions for each article. Only papers with acceptable degree of relevance, quality of methodology, and strength of evidence were retained for further analysis. RESULTS: There were limited data on the epidemiology of MISGD. Furthermore, various methods were used to assess salivary flow rate or xerostomia. Preventive and therapeutic strategies included substitution of medications, oral, or systemic therapy with sialogogues, use of saliva substitutes or of electro-stimulating devices. Although there are promising approaches to improve salivary gland function, most studies are characterized by small numbers and heterogeneous methods. CONCLUSIONS: Physicians and dentists should identify the medications associated with xerostomia and salivary gland dysfunction through a thorough medical history. Preferably, health care providers should measure the unstimulated and stimulated whole salivary flow rates of all their patients so that these values can be used as a baseline to rate the complaints of patients who subsequently claim to experience xerostomia or salivary gland dysfunction as well as the possibilities of effectively treating this condition. CLINICAL RELEVANCE: MISGD remains a major burden for the population. This systematic review provides a contemporary in-depth description of the diagnosis and treatment of MISGD.

World Workshop on Oral Medicine VI: clinical implications of medication-induced salivary gland dysfunction.

OBJECTIVE: This study aimed to systematically review the available literature on the clinical implications of medication-induced salivary gland dysfunction (MISGD). STUDY DESIGN: The systematic review was performed using PubMed, Embase, and Web of Science (through June 2013). Studies were assessed for degree of relevance and strength of evidence, based on whether clinical implications of MISGD were the primary study outcomes, as well as on the appropriateness of study design and sample size. RESULTS: For most purported xerogenic medications, xerostomia was the most frequent adverse effect. In the majority of the 129 reviewed papers, it was not documented whether xerostomia was accompanied by decreased salivary flow. Incidence and prevalence of medication-induced xerostomia varied widely and was often associated with number and dose of medications. Xerostomia was most frequently reported to be mild-to-moderate in severity. Its onset occurred usually in the first weeks of treatment. There was selected evidence that medication-induced xerostomia occurs more frequently in women and older adults and that MISGD may be associated with other clinical implications, such as caries or oral mucosal alterations. CONCLUSIONS: The systematic review showed that MISGD constitutes a significant burden in many patients and may be associated with important negative implications for oral health.

Top-down HPLC-ESI-MS characterization of rat gliadoralin A, a new member of the family of rat submandibular gland glutamine-rich proteins and potential substrate of transglutaminase.

During HPLC-ESI-MS/MS analysis of rat submandibular saliva secreted under isoprenaline stimulation, a protein with an experimental [M+H](1+) = 10,544.24 m/z was detected (17.5 ± 0.7 min). The MS/MS fragmentation pattern, manually investigated, allowed establishing an internal sequence in agreement with a DNA-derived sequence of an unknown rat protein coded D3Z9M3 (Swiss-Prot). To match the experimental MS/MS fragmentation pattern and protein mass with theoretical data, the removal from the N terminus of the signal peptide and from the C terminus of three amino acid (a.a.) residues (Arg-Ala-Val) and the cyclization of the N-terminal glutamine in pyroglutamic had to be supposed, resulting in a mature protein of 90 a.a. HPLC-ESI-MS/MS of the trypsin digest ensured 100% sequence coverage. For the high glutamine content (34/90 = 37.8%) we propose to name this protein rat gliadoralin A 1-90. Low amounts of five different isoforms were sporadically detected, which did not significantly change their relative amounts after stimulation. Gliadoralin A is substrate for transglutaminase-2, having Lys 60 and different Gln residues as major determinants for enzyme recognition. In silico investigation of superior structures evidenced that a small part of the protein adopts an α-helical fold, whereas large segments are unfolded, suggesting an unordered conformation.

Morphological evidence that pentagastrin regulates secretion in the human parotid gland.

Salivary secretion is principally regulated by autonomic nerves. However, recent evidence from in vivo animal experiments suggests that gastrointestinal peptide hormones can also influence saliva production. The aim of the present study was to define the secretagogue activity of the gastrin-analogue pentagastrin in human salivary glands. For this purpose, parotid tissues were exposed to pentagastrin in vitro. Morphological techniques were used to evaluate modifications to serous acinar cells associated with secretion. Using a variant of the osmium maceration method, high resolution scanning electron microscopy allowed assessment of the morphology of the cytoplasmic aspect of the plasmalemma to demonstrate secretory activity. To quantify responses to pentagastrin, we recorded morphometric data on microvilli, microbuds, and protrusions. Dose-dependent morphological changes were observed, whereas protein concentration increased in the incubate. The use of selective receptor antagonists showed pentagastrin to act principally via cholecystokinin-A receptors. The morphological responses observed following exposure to pentagastrin differed from those elicited following exposure to the pan-muscarinic agonist carbachol. This study provides the first demonstration of a direct secretory action of gastrointestinal peptides on salivary glands in humans.

Emetine treatment masks initial LTP without affecting long-term stability.

Applying emetine, a protein synthesis inhibitor, at 20-40µM for 90-120 min prior to LTP induction in hippocampal slices from young rats (2-3 weeks) and washing it out afterwards revealed a slowly developing potentiation that reached maximum after 20-30 min, distinct from the LTP observed under normal conditions. Nevertheless, the later phase of this potentiation was similar to standard LTP as judged by experiments lasting up to 8h after induction. Emetine preapplication for 3h without subsequent washout resulted in a substantial decay of evoked responses. By comparison between test and control pathways, LTP could still be assessed in these experiments for up to 4-6h after induction and was found not to differ from normal, except for the slow onset. The NMDA-R blocker AP5 fully blocked LTP; however, with emetine pretreatment there was an initial depression of responses with a gradual recovery during 20-30 min. This depression involved not only the field EPSP but also the presynaptic fiber volley. However, when using the protein synthesis inhibitors cycloheximide and anisomycin there was essentially no such depression. In conclusion, the present results support the idea that preexisting proteins are sufficient for inducing stable LTP. Moreover, emetine but not anisomycin or cycloheximide impairs presynaptic action potentials, leading to an apparent slow onset of LTP. The emetine-dependent effect could be due to a characteristic blocking spectrum of the drug, preferred targeting of presynaptic compartments or effects unrelated to protein synthesis.

Clozapine-induced salivation: interaction with N-desmethylclozapine and amisulpride in an experimental rat model.

Many drugs (e.g. amisulpride) have been used to treat troublesome clozapine-induced salivation; however, varying success has been achieved in this respect, probably because, until recently, the salivatory action of clozapine has been largely unexplained. In the rat, clozapine and its main metabolite, N-desmethylclozapine, were found to exert mixed secretory actions: excitatory, through muscarinic acetylcholine M1-receptors giving rise to a long-lasting, low-level flow of saliva; and inhibitory, through muscarinic M3-receptors and α(1) -adrenoceptors reducing the parasympathetically and sympathetically nerve-evoked flow of saliva. The aim of the present study was to define the interactions between clozapine and N-desmethylclozapine, and clozapine and amisulpride, with respect to the excitatory response. Submandibular glands, sensitized by chronic parasympathetic preganglionic denervation, were studied in pentobarbitone-anaesthetized rats. To prevent clozapine from being metabolized to N-desmethylclozapine by hepatic enzymes, the liver was, under terminal anaesthesia, excluded from the circulation. The weak receptor-stimulating clozapine prevented the strong receptor-stimulating N-desmethylclozapine, at specific ratios in humans and in rats, from exerting its full agonistic action. In conclusion, the contribution of N-desmethylclozapine to the clozapine-induced sialorrhoea was, at most, only partly additive. Furthermore, the present experimental set-up failed to demonstrate any anti-salivatory action of amisulpride on the clozapine-induced flow of saliva.

N-Desmethylclozapine exerts dual and opposite effects on salivary secretion in the rat.

N-Desmethylclozapine is a major metabolite of the atypical antipsychotic drug clozapine, used in the treatment of therapy-resistant schizophrenia. Patients under clozapine treatment report a troublesome sialorrhea. Recent experiments show clozapine to exert mixed agonist/antagonist actions on salivary secretion in rats. The present study was performed to define the secretory role of N-desmethylclozapine and to compare it with that of its parent compound. N-Desmethylclozapine evoked secretion by acting directly on the muscarinic acetylcholine M1-receptors of 'silent' duct-cannulated parotid and submandibular glands of the anaesthetized rat. In chronic surgically denervated glands, the secretory response was enlarged. The methacholine-evoked secretion, as well as the parasympathetic nerve-evoked secretion, were reduced by N-desmethylclozapine and involved blockade of M3-receptors, while the sympathetic nerve-evoked response was reduced, involving blockade of alpha(1)-adrenergic receptors. Synergistic interactions between N-desmethylclozapine and the beta-adrenergic receptor agonist, isoprenaline, occurred. Compared with clozapine, the excitatory efficacy of N-desmethylclozapine was higher and the inhibitory efficacy was lower (parasympathetic activity) or about the same (sympathetic activity). Theoretically, in humans treated with clozapine, an increase in the N-desmethylclozapine : clozapine ratio would contribute to salivation during the night and at rest, and, furthermore, the magnitude of the reduction in the reflexly elicited secretion is likely to diminish.

RP-HPLC-ESI-MS characterization of novel peptide fragments related to rat parotid secretory protein in parasympathetic induced saliva.

Two peptides (MW 1211.7 and 928.5 Da) were detected by RP-HPLC-ESI-MS analysis of parotid saliva secreted upon continuous parasympathetic stimulation. The peptide with the higher mass (PSPFr-A) corresponded to the N-terminal dodecapeptide (Fragment 1-12) of rat parotid secretory protein (PSP), while the peptide with the lower mass (PSPFr-B) corresponded to the 4-12 fragment of the same protein. During stimulation, the PSPFr-A secretion increased, while the PSPFr-B secretion decreased (HPLC-ESI-MS). In the presence of cycloheximide, PSPFr-A was not demonstrated, while the PSPFr-B secretion decreased. In the presence of aprotinin, the PSPFr-B secretion was almost abolished, while the PSPFr-A secretion increased to higher levels than those observed in the absence of the inhibitor. In vitro perfusion, with artificial solution, of stimulated rat parotid glands excluded that the fragments were derived from the circulation. Neither peptide occurred in enriched granule preparations from unstimulated glands. The results suggest that at least two pathways--granular and vesicular--are responsible for the generation of the two peptides. PSPFr-A is the first cleavage product in both pathways. PRPFr-B is probably generated from granular PSPFr-A only and, at the end of the granule mediated pathway, by the action of an enzyme of the serine protease class.

The cholinesterase inhibitor physostigmine for the local treatment of dry mouth: a randomized study.

Application of physostigmine to the oromucosal surface with the aim of stimulating underlying mucin-producing glands while reducing cholinergic systemic effects might be a strategy for treating dry mouth. Subjects suffering from dry mouth and with hyposalivation participated in a crossover, double-blind, randomized study. A gel containing physostigmine (0.9, 1.8, 3.6, and 7.2 mg) or placebo was applied to the inside of the lips and distributed with the tongue. The feeling of dryness was assessed using a visual analogue scale (VAS) (where a score of 100 = extremely dry) and systemic effects were registered. Based on assessments of efficacy and safety, the dose of 1.8 mg of physostigmine was selected for use in the second part of the study to make objective measurements of saliva volumes. Physostigmine (1.8 mg) produced long-lasting (120 min) relief (evident as a score reduction of 25 on the VAS) in the feeling of dryness. Judging from AUC values related to baseline over 180 min, the improvement for both mouth and lips in response to physostigmine was six times greater than that to placebo. At higher doses of physostigmine, gastrointestinal discomfort predominantly occurred. The volume of saliva collected in response to physostigmine was five times higher over 180 min than that collected in response to placebo. Physostigmine, applied locally, therefore appears to be a promising modality for dry-mouth treatment.

Persistent LTP without triggered protein synthesis.

Protein synthesis is believed to be involved in stabilizing synaptic plasticity. Effects lasting longer than about 2-3h are considered to require synthesis of new proteins, implying a functional separation between early (E) and late (L) components. However, the issue of constitutive vs. new protein synthesis is still unclear, especially in young animals. Here, we examined the effects of two protein synthesis inhibitors, anisomycin and emetine, on long-term-potentiation (LTP) in CA1 area of hippocampal slices from 12- to 20-day-old rats. Either drug was applied from -30 min to +30 min with respect to LTP induction, a time window previously reported to be critical. However, the LTP remained stable under the entire recording period of 4h (anisomycin), or 8h (emetine). Proper preparation of emetine solution was evidenced by the fact that, in separate experiments, prolonged treatment with emetine gradually blocked baseline responses. Although no corresponding effect was observed with anisomycin, the drug was judged to be potent by its ability to inhibit yeast growth. The ability of anisomycin to inhibit protein synthesis was further confirmed by radiolabeling experiments assessing the degree of leucine incorporation. Our data suggest that LTP up to at least 8h is not dependent on triggered protein synthesis but can be attained by utilizing proteins already available at induction time.

Melatonin-evoked in vivo secretion of protein and amylase from the parotid gland of the anaesthetised rat.

The intravenous infusion of melatonin (5 and 25 mg/kg over 10 min) evoked a dose-dependent output of protein and amylase but no overt fluid secretion from the parotid gland of the pentobarbitone-anaesthetised rat, as revealed by increased concentrations of protein and amylase activity in a subsequent wash-out flow of saliva in response to an intravenous bolus injection of methacholine (5 microg/kg) 10 min later. The secretory responses to melatonin occurred in the presence of alpha- and beta-adrenoceptor antagonists. They were not affected by the cholecystokinin A-receptor antagonist, lorglumide, and they were reproduced in eviscerated animals acutely subjected to postganglionic sympathetic and parasympathetic denervation of the gland. The responses to melatonin were partially dependent on nitric oxide generation, through the activity of nitric oxide synthase of the neuronal type. Immunoblotting showed both melatonin receptors of type 1 and type 2 to be expressed in parotid gland tissue. The relative specific melatonin 2-receptor antagonist luzindole prevented the expected secretory effects of melatonin. The results favour a direct action by melatonin on melatonin receptors of parotid secretory cells and suggest a potential physiological role for melatonin in the regulation of salivary glandular activities.