Non-inflammatory salivary gland hypertrophy (sialadenosis) is caused by a variety of metabolic or secretory disturbances. The repeated reduction of lower and/or upper incisors in rodents induces sialadenosis. The increase in gland size is due to both hypertrophy and hyperplasia of the acinar cells and requires the presence of both sympathetic and parasympathetic nerve fibres, being less pronounced in parasympathetically or sympathetically denervated glands. Duct cells seem to be unaffected.

Sialadenosis can also be induced experimentally by the chronic administration of isoproterenol, by anabolic androgenic steroids, by intraoral papain or by a bulk diet consisting of 50% inert cellulose.

Little is known about the ability of the hypertrophied glands to secrete saliva. Ohlin and Perec (1968) found that secretory responses to epinephrine (adrenaline) increased as glandular size increased but that the responses to isoproterenol were unchanged. Only a single dose of agonist was used in these experiments. Abe et al. (1979), showed that there were changes in the types and concentrations of proteins secreted by hypertrophied submandibular glands in response to a single dose of the ₁-adrenergic agonist methoxamine or the ?-adrenergic agonist isoproterenol. These stimuli did not produce significant changes in the volume of saliva. Our aims now were to evaluate, in an experimental model of sialadenosis, the noradrenergic neuroeffector control of the induced salivary secretion and its relation to the noradrenergic receptors that mediate these physiological responses.

All experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals (NIH 85-23, revised in 1985).

One hundred and sixty-two adult female Wistar rats (250–280 g) were used. The animals were divided into three experimental groups: (i) incisor-reduced, (ii) sham-operated (animals subjected only to repeated light ether anaesthesia) and (iii) untreated controls. Incisors were reduced every 2 days under light ether anaesthesia for 3 weeks by grinding the tooth to a height of 2 or 3 mm above the gingival margin with a miniature electrical drill. All groups were maintained on a soft diet of crushed pellets mixed with milk.

Secretory responses were determined on the day after the last incisor reduction. The rats were anaesthetized with chloralose (100 mg/kg) given intravenously after ether induction. The trachea was intubated, and the submandibular ducts on both sides exposed and cannulated with fine-glass cannulae. No resting (unstimulated) flow of saliva was observed. The secretion induced by the different agents was collected in preweighed vessels and weighed. Dose–response curves for the two agonists were obtained in two different sets of animals (a total of 36 rats per set, 12 in each group) by the sequential injection, into the femoral vein, of increasing doses of either phenylephrine (10, 30, 60 and 100 ?g/kg) or isoproterenol (0.3, 1.0, 3.0 and 10 ?g/kg). The secretory response to the lower doses of the two agonists subsided in less than 3 min. Two more minutes were then allowed until the next dose was injected. After the injection of 10 ?g/kg of isoproterenol and 60 and 100 ?g/kg of phenylephrine a longer secretory response was obtained. In these cases, the next dose was injected 2 min after the response to the previous dose had subsided. Clonidine (10 ?g/kg) was injected into all the animals intravenously 15 min before the determination of a second dose–response curve to the same agonist. Rats were killed with an overdose of ether, the submandibular glands carefully dissected, separated from the sublingual glands, and weighed and dried at 110°C for 24 hr. The gland weight for each animal was determined as the mean value from both glands.

In other sets of rats, following the last incisor reduction, the animals were anaesthetized with ether and both submandibular glands were dissected out and weighed. Membranes for radioligand binding were prepared by homogenizing the tissue in 20 vol of ice-cold buffer (50 mM Tris–HCl, pH 7.4, for [³H]clonidine and [³H]prazosin and pH 7.8 for [³H]dihydroalprenolol) with a Sorvall homogenizer and centrifuged in a Sorvall RC 2C centrifuge at 250 g for 10 min. The supernatant was kept at 0°C, and the pellet was rehomogenized in fresh ice-cold buffer and centrifuged at 250 g for 10 min. Both supernatants were pooled and recentrifuged at 35,000 g for 45 min. The pellet was suspended and recentrifuged once more at 35,000 g for 45 min. The final pellet was stored at ?70°C or immediately used for binding assays. The pellet was suspended in 30 vol of Tris buffer, corresponding to 35–50 mg original gland weight/ml and containing 700–1000 ?g of protein.

For binding assays, triplicate samples of 1 ml (0.2 ml for [³H]dihydroalprenolol) of the suspensions were incubated for 30 min at 25°C (10 min at 37°C for [³H]dihydroalprenolol) with different concentrations of the [³H]ligands. The final volume used for incubation was 4 ml for [³H]prazosin, 0.4 ml for [³H]dihydroalprenolol and 2 ml for [³H]clonidine. The final concentrations of labelled drugs were: 0.02–1.28 nM for [³H]prazosin, 0.1–2.4 nM for [³H]dihydroalprenolol and 0.1–6.4 nM for [³H]clonidine. The samples were then diluted with 5 ml of ice-cold 50 mM Tris–HCl buffer and rapidly filtered under reduced pressure through CF/B glass-fibre Whatman filters. Assay tubes and filters were washed with four 5-ml portions of Tris–HCl buffer. Filters were dried overnight at room temperature and radioactivity determined by scintillation spectrophotometry (efficiency 40%).

The density (maximum number of binding sites; B_max) and affinity (apparent affinity constant; K_d) of receptors were determined from saturation experiments using graded concentrations of the radioligands. Binding inhibited in the presence of 10 ?M phentolamine (for [³H]prazosin), 10 ?M propranolol (for [³H]dihydroalprenolol) and 10 ?M clonidine (for [³H]clonidine) was defined as specific binding. The resulting saturation-binding data (presented as Scatchard plots) are from typical experiments with each point determined in triplicate. Three experiments were performed for each ligand. A total of 90 animals was used for these experiments: 15 for [³H]clonidine (five by group), 6 for [³H]dihydroalprenolol (two by group) and 9 for [³H]prazosin (three by group).

The data on glandular weight and on secretory responses were compared by analysis of variance in the incisor-reduced, control and sham-operated groups. Individual comparisons were made with Tukey’s test.

Comparison of B_max and K_d values between incisor-reduced, control and sham groups in the binding experiments was made with student’s t-test, in which the variance of these values was calculated using Fieller’s theorem; p<0.05 was considered statistically significant.

[³H]clonidine (59.9 Ci/mmol), [³H]prazosin (82.0 Ci/mmol) and [³H]dihydroalprenolol (111.5 Ci/mmol) were obtained from New England Nuclear (Boston, MA). Isoproterenol hydrochloride, phenylephrine hydrochloride, propranolol hydrochloride, clonidine hydrochloride, phentolamine hydrochloride and Trizma base were purchased from Sigma Chemical Co. (St. Louis, MO).

Table 1 shows that, during the 3-week observation period, the increase in body weight was significantly smaller in the incisor-reduced animals than in the control or sham-operated animals (p<0.01), while the increase in weight of the submandibular gland was greater in the incisor-reduced groups than in the control or sham-operated groups. Because of these opposite changes, the comparison of the secretory responses between the three groups is presented by normalizing the data by glandular weight (?g saliva/mg of wet tissue) and as secretion of total gland (mg of saliva/gland). For the same reason, the B_max values in the radioligand binding assays are also presented in two ways, as fmol/mg of protein and as fmol/gland. The dry weight as percentage of total weight was the same in all groups.