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.

The peptidergic innervation of the rat parotid gland: effects of section of the auriculo-temporal nerve and/or of otic ganglionectomy.

The origin/pathways of peptidergic nerves contributing to the parotid gland contents of vasoactive intestinal peptide (VIP), substance P and calcitonin gene-related peptide (CGRP) were investigated by performing surgery on one side of the rat. Comparisons (based on total amount of peptide) were made between the gland on the operated side and the contralateral gland 7 days postoperatively. Otic ganglionectomy showed that almost all of the parotid gland contents of VIP (98%) and substance P (98%) were due to the otic connection, while this was true for only a minor portion (32%) of the CGRP-gland content. Section of the auriculo-temporal nerve showed that almost all of the VIP- and substance P-containing nerve fibres reached the parotid gland via this nerve, as judged by a reduction in the VIP-content by 88% and in the substance P-content by 93%, while the CGRP-content was only reduced by 37%. Section of the auriculo-temporal nerve combined with otic ganglionectomy did not reduce the gland contents of CGRP and substance P further than just otic ganglionectomy. Thus, the auriculo-temporal nerve is not likely to innervate the parotid gland with CGRP- and/or substance P-containing nerve fibres from the trigeminal ganglion.

Intraoral stimulation of salivary secretion with the cholinesterase inhibitor physostigmine as a mouth spray: a pilot study in healthy volunteers.

Dry mouth produces a deterioration in oral health and impairs quality of life. There is a need for a novel approach to the pharmacological treatment of dry mouth. With a view to enhancing the cholinergic drive on minor salivary glands, whilst at the same time minimising adverse systemic effects, the cholinesterase inhibitor physostigmine was therefore sprayed, in a fixed volume, onto the oral mucosa of seven healthy subjects. Three concentrations (0.5%, 1% and 2%) were tested. The mean salivary output over time (0-105 min) was higher than that of placebo (p<0.05), as the area under the curve increased by 61%, 91% and 66% at physostigmine 0.5%, 1% and 2%, respectively. Two subjects experienced nausea at the highest physostigmine concentration, thus reflecting systemic effects. Heart rate, blood pressure and respiration were unaffected by the physostigmine treatment.

Acetylcholine synthesis, muscarinic receptor subtypes, neuropeptides and secretion of ferret salivary glands with special reference to the zygomatic gland.

Studies on salivary secretion are usually focused on parotid and submandibular glands. However, the film of mucin, that protects the oral structures and is responsible for the feeling of oral comfort, is produced by the submucosal glands. The submucosal zygomatic and molar glands are particularly large in carnivores such as the ferret. Comparisons between the mucous sublingual, zygomatic and molar glands, serous parotid and sero-mucous submandibular glands showed the acetylcholine synthesis, in terms of concentration, to be three to four times higher in the mucous glands than in the parotid and submandibular glands. Bromoacetylcholine inhibited 95-99% of the synthesis of acetylcholine in the incubates of the five types of glands, showing the acetylcholine synthesis to depend on the activity of choline acetyltransferase. The high acetylcholine synthesis in the zygomatic gland was of nervous origin, since cutting the buccal nerve, aiming at parasympathetic denervation, and allowing time for nerve degeneration, reduced the acetylcholine synthesising capacity of the gland by 95%. A similar reduction (96%) in the parotid gland followed upon the avulsion of the parasympathetic auriculo-temporal nerve. Zygomatic saliva was very viscous. The salivary flow rate in response to electrical stimulation (20 Hz) of the buccal nerve (zygomatic gland), expressed per gland weight, was one-third of that to stimulation of the auriculo-temporal nerve (parotid gland) or the chorda-lingual nerve (submandibular gland). As previously shown for the parotid and submandibular gland, a certain fraction (25%) of the parasympathetic secretory response of the zygomatic gland depended on non-adrenergic, non-cholinergic transmission mechanisms, probably involving substance P and vasoactive intestinal peptide and possibly calcitonin gene-related peptide. Particularly, high concentrations of vasoactive intestinal peptide were found in the sublingual and molar glands, and of substance P in the submandibular, zygomatic and molar glands; notably, the concentration of calcitonin gene-related peptide of the sublingual gland was not detectable. All five muscarinic receptor subtypes were detected in the five glands. The receptor protein profile, as judged by immunoblotting and semi-quantitative estimations, was about the same in the glands: high level of M3, low level of M2 and levels roughly in the same range of M1, M4 and M5. Compared to the parotid and submandibular glands, the M5 receptor level was particularly low in the mucin-secreting glands. The present study points out both similarities and dissimilarities between the five types of glands investigated. The zygomatic gland, in particular, appears to be a suitable model for future studies aiming at causing relief of dry mouth by local pharmacological treatment.

Facial nerve section induces transient changes in sensitivity to methacholine and in acetylcholine synthesis in the rat parotid gland.

Nerves exert long-term influences on the salivary glands as e.g. revealed by increases in sensitivity to secretagogues following nerve degeneration. The objective was to study the effect of unilateral facial nerve section on the sensitivity of the parotid secretory cells 2-3 weeks postoperatively, i.e. at a time when the sensitisation is thought to be fully developed. Comparisons were made between pair of glands. However, no increase in the secretory response to increasing intravenous doses of methacholine of the duct-cannulated gland on the operated side was found; neither were any decrease in the acetylcholine synthesizing capacity of the gland found. In contrast, a slight supersensitivity had developed 1 week postoperatively supporting the idea of a functional influence of the facial nerve on the secretory cells under normal conditions. Furthermore, the results combined with the previous finding of ours of decreased acetylcholine synthesis in the parotid gland 1 week after facial nerve section, suggest a rapid restitution of the nervous influence on the secretory cells between 1 and 2-3 weeks postoperatively.

The otic ganglion in rats and its parotid connection: cholinergic pathways, reflex secretion and a secretory role for the facial nerve.

Otic ganglionectomy in rats was found to have affected the parotid gland more profoundly than section of the auriculotemporal nerve as assessed by reduction in gland weight (by 33 versus 20%) and total acetylcholine synthesizing capacity (by 88 versus 76%) 1 week postoperatively and, when assessed on the day of surgery under adrenoceptor blockade, by reflex secretion (by 99 versus 88%). The facial nerve contributed to the acetylcholine synthesizing capacity of the gland. Section of the nerve only, at the level of the stylomastoid foramen, reduced the acetylcholine synthesis by 15% and, combined with otic ganglionectomy, by 98% or, combined with section of the auriculotemporal nerve, by 82%. The facial nerve was secretory to the gland, and the response was of a cholinergic nature. The nerve conveyed reflex secretion of saliva and caused secretion of saliva upon stimulation. Most of the facial secretory nerve fibres originated from the otic ganglion, since after otic ganglionectomy (and allowing for nerve degeneration) the secretory response to facial nerve stimulation was markedly reduced (from 23 to 4 microl (5 min)(-1)). The persisting secretory response after otic ganglionectomy, exaggerated due to sensitization, and the residual acetylcholine synthesizing capacity (mainly depending on the facial nerve) showed that a minor proportion of pre- and postganglionic nerve fibres relay outside the otic ganglion. The great auricular nerve, which like the facial nerve penetrates the gland, caused no secretion of saliva upon stimulation. Avulsion of the auriculotemporal nerve was more effective than otic ganglionectomy in reducing the acetylcholine synthesizing capacity (by 94 versus 88%) and as effective as otic ganglionectomy in abolishing reflex secretion (by 99%). When aiming at parasympathetic denervation, avulsion may be the preferable choice, since it is technically easier to perform than otic ganglionectomy.