Life Sciences, Vol. Printed in the USA
51, pp.
1493-1499
Pergamon
Press
EFFECTS OF CHRONIC CLONIDINE ADMINISTRATION ON PARASYMPATHETIC-EVOKED RAT SALIVA
Jia-Huey Yu Department of Physiology and Biophysics, Georgetown University Medical Center and Oral Pathology Research Laboratory, Department of Veterans Affairs Medical Center, Washington, DC
(Received
in final
form September
4, 1992)
Summary Effects of chronic administration of clonidine on parasympatheticevoked saliva from both parotid and s u b m a n d i b u l a r glands were investigated. Clonidine at 1 mg/kg/day for 5 or 7 days caused a significant reduction in the salivary secretion (flow rate and total volume) evoked by p a r a s y m p a t h e t i c nerve stimulation of parotid b u t not s u b m a n d i b u l a r glands. Ion concentrations (Na, K and Ca) of p a r a s y m p a t h e t i c a l l y nerve-evoked parotid saliva were not altered. However, the total protein concentration as well as output, amylase activity, and output of such saliva were markedly increased. Possible mechanisms for clonidine-induced increase in nerve-elicited salivary protein concentration include release of neuropeptides, and changes in adrenergic receptor binding which need further study. Clonidine, a centrally acting antihypertensive drug, produces a dry mouth in the majority of patients (1). From animal studies, it has been speculated that the dry mouth caused by clonidine in man is due to activation of peripheral presynaptic a 2adrenoceptors, resulting in inhibition of cholinergic transmission (2-4) in addition to an action of the drug on a-adrenoceptors in the brain stem involved in the central control of salivation (5). However, in a subsequent study (6), it was found that a high dose of clonidine at 10 mg/kg, i. p. was capable of inducing salivary secretion from both p a r o t i d and s u b m a n d i b u l a r glands by activating a l - r a t h e r than a2adrenoceptors. The a2-adrenoceptors appear to play a role in secretion similar to that of al-adrenoceptors only following reserpine treatment or sympathectomy (6). The effect of clonidine on parasympathetic stimulation of rat salivary gland is not clear. The present study was to investigate effects of chronic administration of the low doses of clonidine on parasympathetically evoked secretion from rat salivary glands.
Jia-Huey Yu, Oral Pathology Res. Lab. (151-I), Dept. of Veterans Affairs Medical Center, 50 Irving St.,N.W., Washington DC 20422 Copyright
0024-3205/92 $5.00 + .00 © 1992 Pergamon Press Ltd All rights reserved.
1494
C h r o n i c C l o n i d i n e on N e r v e - e v o k e d R a t S a l i v a
Vol.
51, No. 19, 1992
Materials and Methods Adult Long-Evans male rats (220-300g in body weight, Charles River Laboratories) were kept in temperature-controlled animal facilities with a daily cycle of 12 hrs of light and 12 hrs of darkness. The experimental and control rats were m a i n t a i n e d on commercial lab chow and water ad libitum until 18 hr before the experiments, when food but not w a t e r was removed. All animals were sacrificed between the hours of 9:00 and 11:00 a.m. The rats were anesthetized with pentobarbital sodium (50 mg/kg body weight, i.p.) and tracheostomy was performed with polyethylene tubing to provide a clear airway. I m o l a n t a t i o n of Alzet osmotic minipump; The stress of daily injections has the potential to disturb r a t feeding activity and adrenocortical hormones, both of which are involved in the regulation of salivary secretory proteins (7). Therefore, osmotic minipumps were used for delivering the test drug, or, in control animals, the drug vehicle. All surgical equipment was sterilized. Rats were anesthetized and the ventral skin of the lower abdomen was shaved, cleaned with isopropyl alcohol and swabbed with betadine solution. A small sagittal incision (3 cm in length) was made to one side of the midline, and fascia and muscle carefully separated. An Alzet osmotic minipump (model 2ML1 for 1 week obtained from Alza Co., Palo Alto, CA ) providing 1 mg/kg/day of clonidine (Sigma Chemical Co. Inc., St. Louis, MO, U.S.A.) was placed inside the abdomen. The muscle was sutured with 5.0 Dermalon (Davis & Geck, Cyanamid Canada Inc., Montreal, Canada) and the skin was closed with 4 to 5 wound clips. Rats were allowed to recover. S t i m u l a t i o n of P ~ r ~ y m ~ a t h e t i c Innervotion; P a r a s y m p a t h e t i c secretory activity of the parotid gland was produced by electrical s t i m u l a t i o n of the auriculotemporal nerve (AT). The AT, located between the pinna of the ear and the temporomandibular joint, was carefully exposed and bipolar electrodes were placed around it. Stimulation of the parasympathetic innervation to the submandibular gland was accomplished by placing bipolar electrodes around the main excretory duct at a point just below the level at which the lingual nerve (chorda tympani nerve) crosses the duct. The nerves were stimulated electrically by a Grass stimulator (model SD 9) which delivers square-wave shocks (5 msec in duration at a frequency of 16 Hz) and an intensity of 4 volts. At the end of 20 min nerve stimulation, both control and s t i m u l a t e d parotid as well as s u b m a n d i b u l a r gland were quickly removed, cleaned of extraneous tissue, blotted gently, weighed. For collection of submandibular saliva, a fine polyethylene cannula (ClayAdams, PE 10) was inserted to a distance of about 2-3 mm in the oral opening of one s u b m a n d i b u l a r duct. Saliva was collected from the end of the cannula using disposable micropipettes of 3 -50 pl. Parotid saliva was collected by micropipettes (1, 3, 5, or 20 pl) directly from Stensen's duct after it was severed at the midpoint of its path across the massester muscle. Flow rate was determined by measuring the time required for collection of a raven volume of saliva and relating this to weight of the gland and was expressed as pYmin/g of fresh tissue weight. Stimulation was continuous and collection of samples was continuous so that the total output of saliva and its components could be mesaured. Sample volumes of 3-20 ~l of saliva were used directly for determination of salivary Na, K and Ca concentration by atomic absorption spectrometry. Salivory Protein Concentration and AmTlase Activity: Salivary protein concentration was measured by the method of Lowry et al. (8) Salivary amylase activity was determined by the method of Myers et al. (9) and was expressed as mg of reducing substance (as glucose) formed in 15 rain digestion period at 37 °C, per ml of
Vol.
51, No.
19, 1992
C h r o n i c C l o n i d i n e on N e r v e - e v o k e d Rat S a l i v a
1495
saliva. The output of amylase was calculated on the basis of the specific activity of the pure enzyme of 2500 units/mg protein (10). Statistical Analysis: Differences in the above parameters between animals receiving clonidine and vehicle were tested by analysis of variance, with P < 0.05 being regarded as significant. Results
Parotid
Saliva
160
---0---
Control
.... O---
Clonidine-5
days
- -"%- -
Clonidine-7
days
140 o9
120 100 80 60
rj"j ~ 40 20 0
I
I
I
I
2.5
5.0
7.5
10.0
I
Submandibular
12.5
15.0
I
17.5
20.0
Saliva Control
280
. . . . El---
Clonidine-5
7 d- "a y s - " - '
days
-
240 200 160 120 r.D ~
80 40 0
i
i
i
i
i
2.5
5.0
7.5
10.0
12.5
Time
15.0
i
i
17.5
20.0
(min)
Fig. 1. Change in flow rate of r a t saliva evoked by p a r a s y m p a t h e t i c nerve stimulation following chronic administration of clonidine (1 mg/kg/day for 5 or 7 days). Values are means + SEM of 4-8 rats.
1496
Chronic
Clonidine
on Nerve-evoked
Rat
Saliva
Vol.
51,
No.
19,
1992
The data in Figure 1 show that clonidine at 1 mg/kg/day for 5 or 7 days caused a significant reduction in the salivary flow evoked by parasympathetic nerve stimulation of parotid but not submandibular glands. Salivary flow rates from the parotid gland of the clonidine-treated rat were significantly lower than those of the vehicle-treated rat at all time intervals studied (p
51, pp.
1493-1499
Pergamon
Press
EFFECTS OF CHRONIC CLONIDINE ADMINISTRATION ON PARASYMPATHETIC-EVOKED RAT SALIVA
Jia-Huey Yu Department of Physiology and Biophysics, Georgetown University Medical Center and Oral Pathology Research Laboratory, Department of Veterans Affairs Medical Center, Washington, DC
(Received
in final
form September
4, 1992)
Summary Effects of chronic administration of clonidine on parasympatheticevoked saliva from both parotid and s u b m a n d i b u l a r glands were investigated. Clonidine at 1 mg/kg/day for 5 or 7 days caused a significant reduction in the salivary secretion (flow rate and total volume) evoked by p a r a s y m p a t h e t i c nerve stimulation of parotid b u t not s u b m a n d i b u l a r glands. Ion concentrations (Na, K and Ca) of p a r a s y m p a t h e t i c a l l y nerve-evoked parotid saliva were not altered. However, the total protein concentration as well as output, amylase activity, and output of such saliva were markedly increased. Possible mechanisms for clonidine-induced increase in nerve-elicited salivary protein concentration include release of neuropeptides, and changes in adrenergic receptor binding which need further study. Clonidine, a centrally acting antihypertensive drug, produces a dry mouth in the majority of patients (1). From animal studies, it has been speculated that the dry mouth caused by clonidine in man is due to activation of peripheral presynaptic a 2adrenoceptors, resulting in inhibition of cholinergic transmission (2-4) in addition to an action of the drug on a-adrenoceptors in the brain stem involved in the central control of salivation (5). However, in a subsequent study (6), it was found that a high dose of clonidine at 10 mg/kg, i. p. was capable of inducing salivary secretion from both p a r o t i d and s u b m a n d i b u l a r glands by activating a l - r a t h e r than a2adrenoceptors. The a2-adrenoceptors appear to play a role in secretion similar to that of al-adrenoceptors only following reserpine treatment or sympathectomy (6). The effect of clonidine on parasympathetic stimulation of rat salivary gland is not clear. The present study was to investigate effects of chronic administration of the low doses of clonidine on parasympathetically evoked secretion from rat salivary glands.
Jia-Huey Yu, Oral Pathology Res. Lab. (151-I), Dept. of Veterans Affairs Medical Center, 50 Irving St.,N.W., Washington DC 20422 Copyright
0024-3205/92 $5.00 + .00 © 1992 Pergamon Press Ltd All rights reserved.
1494
C h r o n i c C l o n i d i n e on N e r v e - e v o k e d R a t S a l i v a
Vol.
51, No. 19, 1992
Materials and Methods Adult Long-Evans male rats (220-300g in body weight, Charles River Laboratories) were kept in temperature-controlled animal facilities with a daily cycle of 12 hrs of light and 12 hrs of darkness. The experimental and control rats were m a i n t a i n e d on commercial lab chow and water ad libitum until 18 hr before the experiments, when food but not w a t e r was removed. All animals were sacrificed between the hours of 9:00 and 11:00 a.m. The rats were anesthetized with pentobarbital sodium (50 mg/kg body weight, i.p.) and tracheostomy was performed with polyethylene tubing to provide a clear airway. I m o l a n t a t i o n of Alzet osmotic minipump; The stress of daily injections has the potential to disturb r a t feeding activity and adrenocortical hormones, both of which are involved in the regulation of salivary secretory proteins (7). Therefore, osmotic minipumps were used for delivering the test drug, or, in control animals, the drug vehicle. All surgical equipment was sterilized. Rats were anesthetized and the ventral skin of the lower abdomen was shaved, cleaned with isopropyl alcohol and swabbed with betadine solution. A small sagittal incision (3 cm in length) was made to one side of the midline, and fascia and muscle carefully separated. An Alzet osmotic minipump (model 2ML1 for 1 week obtained from Alza Co., Palo Alto, CA ) providing 1 mg/kg/day of clonidine (Sigma Chemical Co. Inc., St. Louis, MO, U.S.A.) was placed inside the abdomen. The muscle was sutured with 5.0 Dermalon (Davis & Geck, Cyanamid Canada Inc., Montreal, Canada) and the skin was closed with 4 to 5 wound clips. Rats were allowed to recover. S t i m u l a t i o n of P ~ r ~ y m ~ a t h e t i c Innervotion; P a r a s y m p a t h e t i c secretory activity of the parotid gland was produced by electrical s t i m u l a t i o n of the auriculotemporal nerve (AT). The AT, located between the pinna of the ear and the temporomandibular joint, was carefully exposed and bipolar electrodes were placed around it. Stimulation of the parasympathetic innervation to the submandibular gland was accomplished by placing bipolar electrodes around the main excretory duct at a point just below the level at which the lingual nerve (chorda tympani nerve) crosses the duct. The nerves were stimulated electrically by a Grass stimulator (model SD 9) which delivers square-wave shocks (5 msec in duration at a frequency of 16 Hz) and an intensity of 4 volts. At the end of 20 min nerve stimulation, both control and s t i m u l a t e d parotid as well as s u b m a n d i b u l a r gland were quickly removed, cleaned of extraneous tissue, blotted gently, weighed. For collection of submandibular saliva, a fine polyethylene cannula (ClayAdams, PE 10) was inserted to a distance of about 2-3 mm in the oral opening of one s u b m a n d i b u l a r duct. Saliva was collected from the end of the cannula using disposable micropipettes of 3 -50 pl. Parotid saliva was collected by micropipettes (1, 3, 5, or 20 pl) directly from Stensen's duct after it was severed at the midpoint of its path across the massester muscle. Flow rate was determined by measuring the time required for collection of a raven volume of saliva and relating this to weight of the gland and was expressed as pYmin/g of fresh tissue weight. Stimulation was continuous and collection of samples was continuous so that the total output of saliva and its components could be mesaured. Sample volumes of 3-20 ~l of saliva were used directly for determination of salivary Na, K and Ca concentration by atomic absorption spectrometry. Salivory Protein Concentration and AmTlase Activity: Salivary protein concentration was measured by the method of Lowry et al. (8) Salivary amylase activity was determined by the method of Myers et al. (9) and was expressed as mg of reducing substance (as glucose) formed in 15 rain digestion period at 37 °C, per ml of
Vol.
51, No.
19, 1992
C h r o n i c C l o n i d i n e on N e r v e - e v o k e d Rat S a l i v a
1495
saliva. The output of amylase was calculated on the basis of the specific activity of the pure enzyme of 2500 units/mg protein (10). Statistical Analysis: Differences in the above parameters between animals receiving clonidine and vehicle were tested by analysis of variance, with P < 0.05 being regarded as significant. Results
Parotid
Saliva
160
---0---
Control
.... O---
Clonidine-5
days
- -"%- -
Clonidine-7
days
140 o9
120 100 80 60
rj"j ~ 40 20 0
I
I
I
I
2.5
5.0
7.5
10.0
I
Submandibular
12.5
15.0
I
17.5
20.0
Saliva Control
280
. . . . El---
Clonidine-5
7 d- "a y s - " - '
days
-
240 200 160 120 r.D ~
80 40 0
i
i
i
i
i
2.5
5.0
7.5
10.0
12.5
Time
15.0
i
i
17.5
20.0
(min)
Fig. 1. Change in flow rate of r a t saliva evoked by p a r a s y m p a t h e t i c nerve stimulation following chronic administration of clonidine (1 mg/kg/day for 5 or 7 days). Values are means + SEM of 4-8 rats.
1496
Chronic
Clonidine
on Nerve-evoked
Rat
Saliva
Vol.
51,
No.
19,
1992
The data in Figure 1 show that clonidine at 1 mg/kg/day for 5 or 7 days caused a significant reduction in the salivary flow evoked by parasympathetic nerve stimulation of parotid but not submandibular glands. Salivary flow rates from the parotid gland of the clonidine-treated rat were significantly lower than those of the vehicle-treated rat at all time intervals studied (p
