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Experimental Physiology 89.2 pp 219-227
DOI: 10.1113/expphysiol.2003.002648
© The Physiological Society 2004
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Nitric oxide-dependent protein synthesis in parotid and submandibular glands of anaesthetized rats upon sympathetic stimulation or isoprenaline administration

Shariel Sayardoust and J. Ekström

Department of Pharmacology, Sahlgrenska Academy at Göteborg University, Medicinaregatan 15 D, Göteborg 413 90, Sweden


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
In anaesthetized female rats, the ß-adrenoceptor agonist isoprenaline was intravenously infused (20 µg kg–1 min–1) for 30 min or the ascending cervical sympathetic nerve trunk was intermittently stimulated (50 Hz, 1 s every tenth second) on one side for 30 min. The incorporation of [3H]leucine into trichloroacetic acid (TCA)-insoluble material was used as an index of protein synthesis. In response to isoprenaline, the [3H]leucine incorporation increased by 79% in the parotid glands and by 82% in the submandibular glands. The neuronal type NO-synthase inhibitor N-PLA, reduced (P < 0.001) this response to 26% and 20%, respectively. Sympathetic stimulation under {alpha}-adrenoceptor blockade increased the [3H]leucine incorporation by 192% in the parotid glands and by 35% in the submandibular glands. N-PLA reduced the corresponding percentage figures to 86% (P < 0.01) and 8% (P < 0.05). When tested in the parotid glands, the non-selective NO-synthase inhibitor L-NAME reduced (P < 0.01) the nerve-evoked response to 91%. The increase in [3H]leucine incorporation in response to sympathetic stimulation under ß-adrenoceptor blockade was not affected by N-PLA in the parotid (139%versus 144%) and submandibular glands (39%versus 34%). In non-stimulated glands, the [3H]leucine incorporation was not influenced by the NO-synthase inhibitors. In conclusion, ß-adrenoceptor mediated salivary gland protein synthesis is largely dependent on NO generation by neuronal type NO-synthase, most likely of parenchymal origin.

(Received 29 August 2003; accepted after revision 8 January 2003)
Corresponding author J. Ekström: Department of Pharmacology, Sahlgrenska Academy at Göteborg University, Medicinaregatan 15 D, Göteborg 413 90, Sweden. E-mail: jorgen.ekstrom{at}pharm.gu.se


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Salivary ß-adrenoceptor-stimulated protein secretion is usually associated with an elevation of intracellular cyclic AMP (Baum & Wellner, 1999). Recently, the ß-adrenoceptor agonist isoprenaline was found to evoke secretion of amylase from rat parotid lobules in vitro that was partly dependent on the mobilization of a nitric oxide (NO)/cyclic GMP intracellular pathway and which involved the activity of neuronal type NO-synthase, most likely of parenchymal origin (Sayardoust & Ekström, 2003). Isoprenaline induces synthesis of secretory proteins in parotid and submandibular gland tissues and the effect is thought to be mediated through increases in levels of intracellular cyclic AMP (Proctor, 1998). In the light of a role for NO in the secretion of amylase, in response to the administration of isoprenaline, we wished to consider the possibility that ß-adrenoceptor activation also involves the stimulation of a NO-dependent synthesis of salivary gland proteins.

Thus, in the present study the incorporation of tritium-labelled leucine into trichloroacetic acid-insoluble material of parotid and submandibular glands of anaesthetized female rats was used as an index of protein synthesis. Protein synthesis was measured in response to electrical stimulation of the ascending cervical sympathetic nerve trunk, in the presence of {alpha}- or ß-adrenoceptor blockade, or to intravenous infusion of isoprenaline with or without inhibition of NO generation.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
A total of 59 female rats, aged 9–12 weeks, of a Sprauge-Dawley strain (B & K Universal AB, Sollentuna, Sweden), were fed a pelleted standard diet (B & K Universal) and fasted over-night with free access to water. The animals were anaesthetized with pentobarbitone (50 mg kg–1, I.P.) and cannulated with a femoral venous polyethylene catheter and a tracheal cannula. The body-temperature of the anaesthetized animal, measured with a rectal probe, was maintained at 38°C using a thermostatically controlled blanket. In the animals subjected to unilateral sympathetic nerve stimulation, the parotid duct was exposed externally on the stimulation side and the duct was cannulated with a fine polyethylene tube. Moreover, in these animals the arterial blood pressure was measured continuously by means of a pressure transducer connected to a catheter inserted into the femoral artery. The study was approved by the Ethics Committee for Animal Experiments, Göteborg.

Sympathetic nerve stimulation

The cervical sympathetic trunk was exposed in the neck on one side, cut and the ascending nerve was placed in a bipolar electrode and stimulated supramaximally (6 V, 2 ms; Grass S48 stimulator and isolation unit (SIU), Grass SIU 5 A; Grass, Quincy, MA, USA) for 30 min using an intermittent mode of stimulation (50 Hz for 1 s every tenth second) as recommended by Anderson et al. (1988) to avoid prolonged restriction of the blood flow to the gland (Anderson & Garrett, 1998). To exclude any possiblity of irradiation of the stimulation to the vagal nerve, this nerve was cut and a piece of the nerve was removed. To check the efficiency of the nerve stimulation, the parotid saliva secreted in response to the stimulation was, in most cases, collected over 10 min periods in ice-chilled preweighed tubes, which were then re-weighed to measure the amount of saliva produced. In the few experiments where the parotid saliva was not monitored, filter papers had been placed under the tongue to roughly monitor the submandibular response. When appropriate, the non-selective {alpha}-adrenoceptor blocker phentolamine (2 mg kg–1) and/or the non-selective ß-adrenoceptor blocker propranolol (2 mg kg–1) were injected intravenously 20 min before the nerve stimulation began. Two types of NO-synthase inhibitors were tested, 30 mg kg–1 of each, injected intravenously 10 min before the start of the stimulation: the non-selective inhibitor N{omega}-nitro-L-arginine methyl ester (L-NAME Moncada, 1992); and the highly selective inhibitor of neuronal type NO synthase (nNOS) N{omega}-propyl-L-arginine (N-PLA; Zhang et al. 1997). The following groups were studied: 10 rats (mean body-weight and S.E.M., 269 ± 9 g) given phentolamine; 5 rats (247 ± 5 g) given phentolamine and L-NAME; 5 rats (279 ± 9 g) given phentolamine and N-PLA; 5 rats (269 ± 11 g) given propranolol; 5 rats (267 ± 8 g) given propranolol and N-PLA; 3 rats (282 ± 2 g) without any of the autonomic blockers and, finally, 3 rats (294 ± 6 g) with both blockers.

Isoprenaline infusion

Isoprenaline (20 µg kg–1 min–1) was continuously infused intravenously for 30 min in a volume of 0.3 ml (CMA/microinjection pump; Carneige Medicin, Stockholm, Sweden). When appropriate, N-LPA (30 mg kg–1) was injected 10 min before the start of the infusion. The secretory response was roughly monitored by placing filter papers in the mouth of the animal. Control rats received saline infusion (0.3 ml) for 30 min. After the end of the infusion period, propranolol (2 mg kg–1, I.P.), was injected to interrupt the stimulation of the ß-adrenoceptors; the control rats were also given propranolol. To study the effect of isoprenaline administration the groups were as follows: 8 rats (300 ± 12 g) received saline infusion; 8 rats (291 ± 4 g) received isoprenaline; and 7 rats (293 ± 5 g) received isoprenaline and N-PLA.

Leucine incorporation

[3H]Leucine (500 µCi kg–1 in 0.5 ml saline) was injected intravenously as a bolus dose 30 min after the end of the 30 min long period of nerve stimulation or drug infusion. Fifteen minutes after the injection of the bolus dose of labelled leucine and still under anaesthesia, the abdomen was opened, the aorta cut and the animal exsanguinated. The parotid and submandibular glands on both sides were rapidly removed, washed in saline, pressed gently between gauze pads, placed on filter paper to remove adherent tissue (if any), weighed, frozen (–20°C) and stored (–70°C) until further processed.

Processing of tissues

Each gland was placed in 1 ml of cold 5 mM NaOH, homogenized (Ultra-Turrax; Janke & Kunkel KG, Breisgan, Germany) and then diluted 4 times. To 100 µl of this solution, 5% trichloroacetic acid (TCA) was added to precipitate the gland homogenate and the mixture was centrifuged at 3000 g for 5 min. This procedure was repeated twice; at the last centrifugation, the supernatant was virtually devoid of radioactivity. To the final precipitate, 500 µl Soluene 100 (Packard, PerkinElmer, Boston, MA, USA) was added and left overnight; the blank consisted of 500 µl of Soluene. Then 8 ml Optiphase HiSafe 2 (Fisons Chemicals, PerkinElmer) was added and the mixture was analysed in a scintillation counter (LKB Wallac, PerkinElmer). The amount of [3H]leucine incorporated was expressed as disintegration per minute (d.p.m.) per gland (or per mean of left and right glands of rats exposed to infusions).

Substances

Isoprenaline hydrochloride and propranolol hydrochloride were from Sigma Chemical Co (St. Louis, MO, USA)while phentolamine mesylate was from Novartis Pharma AG (Basel, Switzerland). L-NAME was from Sigma and N-LPA from Tocris Cookson Ltd. (Bristol, UK). Radiolabelled [3H]leucine was from Amersham Biosciences Europe GMBH (Uppsala, Sweden).

Statistics

Statistical significance was calculated either by Student's t test for unpaired or paired values (comparisons between nerve stimulated gland and its contralateral unstimulated gland) or by one-way analysis of variance (ANOVA) followed by Fisher's protected least significant difference. Probabilities of less than 5% were considered significant. When the glands of the right and left side were subjected to the same treatment, a mean value was calculated for the two sides and used for the statistical analysis. To present percentage figures for radiolabelled leucine incorporation, the mean of each control group was set to 100%. The individual values of control and experimental glands of each study group were related to this reference point. Values presented are mean ±S.E.M.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Isoprenaline infusion

Parotid glands  Expressed per gland, the [3H]leucine incorporation increased by 79 ± 8% (P < 0.001, ANOVA based on raw data) into the TCA-insoluble material of the glands (mean gland weight 127 ± 6 mg) of eight isoprenaline-stimulated rats (500 x 103± 23 x 103 d.p.m.) and by 26 ± 4% (P < 0.05, ANOVA, based on raw data) into that of the glands (145 ± 7 mg) of seven rats subjected to both N-PLA and isoprenaline (351 x 103± 12 x 103 d.p.m) above the level of the glands (137 ± 8 mg) of the eight control rats (280 x 103± 25 x 103 d.p.m), Fig. 1A. The increase was significantly lower in the presence of N-PLA than in its absence (P < 0.001, ANOVA based on raw data). The stimulated glands with or without N-PLA showed no oedema, and there were no statistically significant differences in gland weights between the various groups (ANOVA).



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  		Figure 1.  [3H]Leucine incorporation into the trichloroacetic acid-insoluble material of A, parotid glands and B, submandibular glands in response to the intravenous infusion of isoprenaline (20 µg kg–1 min–1) for 30 min without or with the neuronal type NO-synthase inhibitor N-PLA (30 mg kg–1, I.V.), and then followed by propranolol (2 mg kg–1, I.V.)
Animals serving as controls received saline infusion followed by propranolol, as above. Columns represent means and vertical bars S.E.M. Significant differences, based on raw data and using the ANOVA, were found between glands of control rats and those treated with isoprenaline (***P < 0.001), and between those treated with isoprenaline and those treated with N-PLA in addition (***P < 0.001). # Denotes comparison between glands of control rats and glands of rats subjected to both isoprenaline and N-PLA, *P < 0.05, **P < 0.01. Number of observations (and rats) in brackets.

 
Submandibular glands  Isoprenaline increased the [3H]leucine incorporation by 82%± 8% (P < 0.001; ANOVA based on raw data) into the TCA-insoluble material of the glands (173 ± 6 mg) of eight rats (400 x 103± 17 x 103 d.p.m) compared with that of the glands (169 ± 7 mg) of the eight control rats (220 x 103± 11 x 103 d.p.m). Isoprenaline combined with N-PLA resulted in a 20 ± 4% increase (P < 0.05, ANOVA based on raw data) in the glands (165 ± 6 mg) of seven rats (264 x 103± 8 x 103 d.p.m) compared with the control glands (Fig. 1B). The increase was significantly lower in the presence of N-PLA than in its absence (P < 0.001, ANOVA based on raw data). The stimulated glands with or without N-PLA did not appear oedematous, and there were no statistically significant differences in gland weights between the various groups (ANOVA).

Sympathetic nerve stimulation {alpha}-adrenoceptor blockade with or without L-NAME or N-PLA

Parotid glands  The incorporation of [3H]leucine into the TCA-insoluble material of the stimulated glands (146 ± 11 mg) of 10 rats increased by 192 ± 31% above the level of the contralateral glands (142 ± 6 mg) in the absence of either NO-synthase inhibitor (554 x 103± 59 x 103 d.p.m. versus 190 x 103± 18 x 103 d.p.m.; P < 0.001; paired t test), Fig. 2A. In the presence of L-NAME (30 mg kg–1I.V.), the incorporation of [3H]leucine into the TCA-insoluble material of the stimulated glands (135 ± 7 mg) of five rats increased by 91 ± 14% above the level of the contralateral glands (133 ± 4 mg; 353 x 103± 27 x 103 d.p.m. versus 185 x 103± 18 x 103 d.p.m.; P= 0.001; paired t test). The increase in [3H]leucine incorporation into TCA-insoluble material of the stimulated glands (154 ± 8 mg) of five rats in the presence of N-PLA (30 mg kg–1I.V.) was about the same as after L-NAME. It was 86 ± 8% compared with the contralateral glands (149 ± 10 mg; 463 x 103± 20 x 103 d.p.m. versus 249 x 103± 36 x 103 d.p.m.; P < 0.01; paired t test). The percentage increases in [3H]leucine incorporation were less in the presence of L-NAME or N-PLA than in their absence (P < 0.01; ANOVA). The administration of L-NAME or N-LPA did not affect the [3H]leucine incorporation into the TCA-insoluble material of the contralateral gland on the non-stimulated side in a statistically significant way (unpaired t test). There were no signs of glandular oedema and statistically significant differences in the weights of stimulated and contralateral non-stimulated glands in the various groups (paired t test).



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  		Figure 2.  [3H]Leucine incorporation into the trichloroacetic acid-insoluble material of parotid glands in response to intermittent stimulation of the sympathetic innervation 50 Hz (1 s every tenth s) for 30 min on one side
A, stimulation under {alpha}-adrenoceptor blockade (phentolamine 2 mg kg–1, I.V.) without or with either the non-selective NO-synthase inhibitor L-NAME (30 mg kg–1, I.V.) or the neuronal type NO-synthase inhibitor N-PLA (30 mg kg–1, I.V.). B, stimulation under ß-adrenoceptor blockade (propranolol 2 mg kg–1, I.V.) without or with N-PLA. Columns represent means and vertical bars S.E.M. Comparisons were made between stimulated gland and contralateral gland (#, paired t test based on raw data) or between stimulated glands of the various groups (A, ANOVA or B, unpaired t test based on percentage figures). Number of observations (and rats) in brackets. **P < 0.01, ***P<= 0.001.

 
Submandibular glands  The incorporation of [3H]leucine into the TCA-insoluble material of the stimulated glands (171 ± 7 mg) of eight rats increased by 35 ± 16% above the level of the contralateral glands (167 ± 7 mg) in the absence of NO-synthase inhibition (259 x 103± 31 x 103 d.p.m. versus 192 x 103± 21 x 103 d.p.m.; P < 0.01; paired t test), Fig. 3A. In the five animals subjected to N-PLA, there was no statistically significant increase (8 ± 7%) in [3H]leucine incorporation into the TCA-insoluble material of the stimulated glands (170 ± 8 mg) compared with the contralateral glands (176 ± 6 mg; 197 x 103± 18 x 103 d.p.m. versus 185 x 103± 20 x 103 d.p.m.). The percentage figures found in the N-PLA treated animals were significantly lower (P < 0.05; unpaired t test) than those found in the absence of the inhibitor. The incorporation of [3H]leucine into the TCA-insoluble material of the contralateral glands on the non-stimulated side was not affected by N-PLA (unpaired t test). The stimulated glands were not oedematous, and there were no statistically significant differences in the weights of stimulated and contralateral non-stimulated glands in the various groups (paired t test).



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  		Figure 3.  [3H]Leucine incorporation into the trichloroacetic acid-insoluble material of submandibular glands in response to intermittent stimulation of the sympathetic innervation 50 Hz (1 s every 0.1 s) for 30 min on one side
A, stimulation under {alpha}-adrenoceptor blockade (phentolamine 2 mg kg–1, I.V.) without or with the neuronal type NO-synthase inhibitor N-PLA (30 mg kg–1, I.V.). B, stimulation under ß-adrenoceptor blockade (propranolol 2 mg kg–1, I.V.) without or with N-PLA. Columns represent means and vertical bars S.E.M. Comparisons were made between stimulated gland and contralateral gland (#, paired t test based on raw data) or between stimulated glands of the various groups (unpaired t test based on percentage figures). Number of observations (and rats) in brackets. *P < 0.05, **P < 0.01.

 
ß-adrenoceptor blockade with or without N-PLA

Parotid glands  Sympathetic stimulation caused the incorporation of [3H]leucine to increase into the TCA-insoluble material of the stimulated glands (144 ± 8 mg) of five rats by 139 ± 20% over that of the contralateral glands (131 ± 11 mg; 494 x 103± 42 x 103 d.p.m. versus 206 x 103± 22 x 103 d.p.m.; P < 0.01, paired t test), Fig. 2B. The presence of N-PLA did not affect the increase in [3H]leucine incorporation as shown in another group of five rats. In the stimulated glands (154 ± 8 mg) the incorporation was 144 ± 14% above the level in the contralateral glands (149 ± 10 mg; 532 x 103± 31 x 103 d.p.m. versus 218 x 103± 22 x 103 d.p.m.; P= 0.001; paired t test). The incorporation of [3H]leucine into the TCA-insoluble material of the contralateral glands on the non-stimulated side was not affected in a statistically significant way (unpaired t test) by the administration of L-NAME. The stimulated glands did not appear oedematous. There were no statistically significant differences in the weights of stimulated and contralateral non-stimulated glands in the various groups (paired t test).

Submandibular glands  In the stimulated glands (167 ± 4 mg), of five rats the [3H]leucine incorporation was increased by 39 ± 14% compared with the contralateral glands (170 ± 6 mg; 258 x 103± 25 x 103 d.p.m. versus 186 x 103± 32 x 103 d.p.m.; P < 0.05; paired t test), Fig. 3B. In the presence of N-PLA, the increase in the stimulated glands (179 ± 6 mg), of five rats of 34 ± 16% above that in the contralateral glands (176 ± 7 mg) was not statistically different (unpaired t test) from the increase in the absence of the NO-synthase inhibitor (323 x 103± 38 x 103 d.p.m. versus 241 x 103± 32 x 103 d.p.m.; P < 0.05; paired t test). The incorporation of [3H]leucine into the TCA-insoluble material of the contralateral glands on the non-stimulated side was not affected by N-PLA (unpaired t test). Also here, signs of oedema were lacking, and there were no statistically significant differences in the weights of stimulated and contralateral non-stimulated glands in the various groups (paired t test).

The effect of combined {alpha}- and ß-adrenoceptor blockade

The incorporation of [3H[leucine in the stimulated parotid and submandibular glands in response to sympathetic stimulation was 252 ± 90% and 94 ± 30%, respectively, over that of the contralateral glands in a group of three rats, whereas in another group of three rats, and in the presence of phentolamine and propranolol, the corresponding figures were 8 ± 27% and 5 ± 2%.

Parotid secretion of saliva

The total volume of saliva secreted from the parotid gland in response to the 30 min long period of stimulation was 33 ± 5 mg, n= 6, in the presence of just the {alpha}-adrenoceptor blocker, 44 ± 9 mg, n= 4, in the presence of the {alpha}-adrenoceptor blocker combined with N-PLA and 15 ± 3 mg, n= 4, in the presence of the {alpha}-adrenoceptor blocker combined with L-NAME. The response in the presence of L-NAME was significantly lower than that in the presence of N-PLA (P < 0.01, ANOVA) and also to that in the absence of any NO-synthase blocker (P < 0.05, ANOVA), while the response in the presence of N-PLA was not significantly different from that in the absence of {alpha}-blockade. The total volume secreted in the presence of the ß-adrenoceptor blocker and N-PLA, 76 ± 11 mg, n= 4, was not statistically significant different from that in the presence of just ß-blockade, 61 ± 9 mg, n= 4 (unpaired t test). In the absence of any blocker, the response in the three animals examined was 88 ± 17 mg saliva, while in the presence of both blockers there was no secretion at all in another group of three animals in response to the sympathetic stimulation. There were no significant differences in the gland weights on the stimulated side of the various groups (ANOVA).

Blood pressure recordings

The administration of N-PLA was, in contrast to the administration of L-NAME, not followed by any restoration of the blood pressure from the lowered level caused by the administration of either of the two types of adrenoceptor blockers (Fig. 4). Under {alpha}-adrenoceptor blockade, the mean blood pressure increased from 68 ± 2 mmHg to 130 ± 5 mmHg in response to L-NAME (n= 5, P < 0.001, t test for paired data), whereas the corresponding figures were 69 ± 3 mmHg before and 66 ± 3 mmHg after administration of N-PLA (n= 5).



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  		Figure 4.  Effects of L-NAME (30 mg kg–1, I.V.) and N-PLA (30 mg kg–1, I.V.) on mean blood pressure recorded over 5-min periods under A, {alpha}-adrenoceptor blockade by phentolamine (2 mg kg–1, I.V.) or B, ß-adrenoceptor blockade by propranolol (2 mg kg–1, I.V.)
Columns represent means and vertical bars S.E.M. Open and narrow diagonal columns before and after the administration of either adrenoceptor blocker; filled columns represent the initial and final periods of the 30-min long period of sympathetic stimulation; and wide diagonal columns represent the end of the 30-min long resting period, preceding the administration of the radiolabelled amino acid. When appropriate, either NO-synthase inhibitor was administered 10 min before the start of the stimulation as indicated by the arrow. Number of observations (and rats) in brackets.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
The results of the present study make it likely that NO generation in response to activation of ß-adrenoceptors by sympathetic neuronal activity is of importance for the synthesis of salivary gland proteins in the rat. Of the isoprenaline-induced 80% increase in the incorporation of [3H]leucine into the TCA-insoluble material of both the parotid and submandibular glands, 55% and 60%, respectively, depended on NO-generation. Stimulation of the sympathetic innervation in the presence of the ß-adrenoceptor blocker propranolol or the {alpha}-adrenoceptor blocker phentolamine showed the NO-generation in response to the sympathetic stimulation to depend on ß-adrenoceptor activation and not on {alpha}-adrenoceptor activation. The NO generation contributed to more than half of the sympathetically nerve-evoked ß-adrenoceptor mediated 190%-increase in the [3H]leucine incorporation into the TCA-insoluble material of the parotid gland and to almost the whole 35%-increase into that of the submandibular gland. A combination of propranolol and phentolamine prevented completely the increase in the [3H]leucine incorporation in response to the sympathetic stimulation. In the absence of receptor blockade, the NO generation may, theoretically, contribute to about 40% of the increase in the [3H]leucine incorporation in the two types of glands in response to the sympathetic stimulation.

The rapid incorporation of radiolabelled leucine associated with the glandular stimulation reflects mainly the synthesis of secretory products such as amylase in the parotid gland (Sreebny et al. 1971) and glycoproteins in the submandibular gland (Bogart, 1976; Baum & Kuyatt, 1981). Individual glandular proteins were not examined in the present study. The experimental protocols used do not induce any hyperplastic effect in the parotid and submandibular glands of the rat as judged by the absence of any increase in radiolabelled thymidine 18 h after the end of the stimulation (Ekström J, Çevic H and Sayardoust S, unpublished observation). It may be noted, that in the present study [3H]leucine was injected into the blood stream as a bolus dose 30 min after the end of the stimulation of the glands, and 15 min later the animals were killed. Since the two types of glands in the anaesthetized rat do not secrete saliva in the absence of stimulation, loss of radioactivity by way of salivation did not occur under the present experimental set-up. Labelled proteins in blood or serum are unlikely to have contaminated the gland homogenates to any degree of importance for the outcome of the present study. Upon inspection the stimulated glands did not appear watery, and there were no statistically significant weight differences between stimulated and control glands. The rats were exsanguinated, and the glands were washed. Sreebny et al. (1971) found in the rat that 15 min after an intravenous injection of [3H]leucine, all of the radioactivity of the serum was confined to the trichloroacetate-soluble fraction. The [3H]leucine incorporation technique is a convenient and well recognized technique for studies on protein synthesis. Although not affecting the interpretation of the present results, it is, however, less accurate than the ‘flooding dose’ technique for calculations of the rate of protein synthesis (Proctor et al. 1993).

The inhibitor L-NAME is non-selective for endothelial, neuronal and inducible types of NO-synthase (Moncada, 1992). N-LPA, the selective inhibitor of the neuronal type NO-synthase (Zhang et al. 1997), was found to cause an inhibition of the [3H]leucine incorporation of the same magnitude as L-NAME in the parotid gland in response to the sympathetic stimulation under {alpha}-blockade. These results suggest that the ß-mediated NO-dependent [3H]leucine incorporation was entirely due to the activity of the neuronal type of NO-synthase, at least in the parotid gland, where both types of inhibitors were tested. As expected, due to its inhibitory effect on NO-synthase of endothelial type, administration of L-NAME raised the systemic blood pressure from a low level in the {alpha}-adrenoceptor blocked animals. The vasoconstriction induced by L-NAME (still present at the end of the 30-min long resting period following the stimulation period as reflected by the maintained high blood pressure) most likely influenced the presentation rate of [3H]leucine to the stimulated gland and its contralateral gland on the non-stimulated side but this appeared to have little, if any effect, since the uptake of the radiolabelled amino acid into the contralateral glands was not different from that in the absence of L-NAME.

The fact that neither L-NAME nor N-PLA reduced the [3H]leucine incorporation in the non-stimulated glands of the groups of rats subjected to sympathetic stimulation suggests that in those glands on-going NO production, if present, was of little importance for the [3H]leucine incorporation.

Although the isoform of NO-synthase involved in the [3H]leucine incorporation evoked by sympathetic neuronal activity or isoprenaline infusion was of the neuronal type, the enzyme is localized to the parenchymal cells rather than to the nerves. The sympathetic innervation of the rat salivary glands lacks NO-synthase (Alm et al. 1995, 1997; Takai et al. 1999). Isoprenaline causes parotid tissue to secrete amylase depending on the activity of neuronal type NO-synthase regardless of whether the parotid tissue is from an innervated or from a chronically denervated parotid gland (Sayardoust & Ekström, 2003). Moreover, rat parotid acinar cells, loaded with a fluorescent indicator of NO, increase their fluorescence intensity strongly upon exposure to isoprenaline (Looms et al. 2000).

Intracellularly, NO activates soluble guanyl cyclase to catalyse the formation of cyclic GMP, and by preventing NO from activating this enzyme, the isoprenaline evoked in vitro secretion of amylase from rat parotid gland tissue is markedly reduced (Sayardoust & Ekström, 2003). Since in the rat parotid gland, cyclic GMP may reduce the rate of enzymatic degradation of cyclic AMP by inhibiting the activity of phosphodiesterases (Imai et al. 1995), there is the possiblity that the NO/cyclic GMP pathway contributes to elevated levels of cyclic AMP in the glands. Interestingly, sympathetic nerve stimulation as well as isoprenaline administration increase not only the level of cyclic AMP but also the level of cyclic GMP as shown in rat and mouse parotid glands (Durham et al. 1974; Templeton et al. 1977; Schneyer et al. 1986).

The inhibition of the neuronal type NO-synthase by the use of N-PLA did not reduce the volumes of parotid saliva produced by the activation of either {alpha}-or ß-adrenoceptors in response to the stimulation of the sympathetic innervation. The lack of an effect by N-PLA on the nerve-evoked ß-mediated volume response combined with the previously reported reduction in the isoprenaline-evoked ß-mediated in vitro secretion of amylase upon administration of N-PLA (Sayardoust & Ekström, 2003), suggests that the two phenomena are non-uniformly influenced by the inhibition of NO generation. The lowered fluid response to the stimulation of the sympathetic innervation upon administration of L-NAME in the presence of {alpha}-adrenoceptor blockade was most likely due to impaired blood flow through the gland (Anderson & Garrett, 1998). That reduced blood flow through the glands may hamper the secretory volume response was originally suggested by Langley (1891), and more recently paid attention to by Edwards and coworkers (Harrison et al. 2002).

The present finding of an increase in protein synthesis after nerve-evoked activation of the {alpha}-adrenoceptors of both types of glands, deserves a comment since in previous in vitro studies on rat parotid and submandibular gland tissues, {alpha}-adrenoceptor agonists have been reported to cause the opposite response (McPherson & Hales, 1978; Takuma et al. 1984; Anderson, 1988). The intermittent mode of nerve stimulation in the present study compared with prolonged exposure to a high concentration of an {alpha}-agonist in vitro may perhaps be one explanation for this difference. Interestingly, a low concentration of a cholinergic agonist increased the amino-acid incorporation into proteins in dispersed submandibular acinar cells, while a high concentration of the agonist decreased the incorporation (Anderson, 1988).


   References
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
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   Acknowledgements
 
This study was supported by grants from the Swedish Science Council (05927), Willhelm and Martina Lundgren's Foundation, and the Dental Society of Göteborg. It is a particular pleasure to acknowledge the competent technical assistance provided by Mrs Ann-Christine Reinhold.




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