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Pilocarpine-induced Salivation and Thirst in Conscious Rats
1 Departments of Biosciences and Correspondence: * corresponding author, ine{at}kyu-dent.ac.jp.
The muscarinic receptor agonist pilocarpine is widely used as a sialogogue. It has been well-established that it also induces water intake in animals. However, the mechanisms underlying the relationships between these events are unknown. To address this problem, we examined water intake and parotid salivary secretion in conscious rats. Intraperitoneally injected pilocarpine increased both water intake and salivary secretion. Intracerebroventricularly injected pilocarpine also induced water intake, but not salivary secretion. Intracerebroventricularly applied atropine, a muscarinic receptor antagonist, suppressed the water intake produced by pilocarpine applied intraperitoneally and intracerebroventricularly. However, it did not affect the salivary secretion induced by pilocarpine applied peripherally. We conclude that peripherally applied pilocarpine affects the parotid glands and the thirst center in the central nervous system, while it may induce salivary secretion mainly via peripheral responses, but water intake mainly via the central nervous system.
Key Words: pilocarpine • salivation • thirst • conscious rat
Elderly patients complain of dry mouth (xerostomia) more frequently with increasing age (Sreebny and Schwartz, 1997; Närhi et al., 1999). Xerostomia is also induced by several commonly used medications, as well as by Sjögren’s syndrome and radiation to the head and neck regions. Long-standing dry mouth leads to pain in the tongue and mucous membranes, and to bacterial infections and difficulty in swallowing. For relief of such conditions, muscarinic receptor agonists are used clinically as sialogogues, because activation of the muscarinic receptors in the salivary glands promotes salivary secretion. Pilocarpine, a representative sialogogue, has been reported to reduce depression of salivary secretion in humans (Gotrick et al., 2004) and in rodents (Omori et al., 2003). In general, it is considered that systemically administered pilocarpine affects the salivary glands. Additionally, it has been reported that intracerebroventricular injection of pilocarpine also induces salivary secretion in anesthetized rats (Moreira et al., 2002; Renzi et al., 2002), and that the salivation induced by intraperitoneally administered pilocarpine is inhibited by the pre-treatment with intracerebroventricular injection of atropine, a muscarinic antagonist (Takakura et al., 2003). Because of the absence of the blood-brain barrier in the circum-ventricular organs, peripherally administered drugs can affect neurons in these regions directly. It has thus been suggested that the salivary secretion elicited by systemically administered pilocarpine is mediated through the central nervous system, as well as through the salivary glands. In contrast, it is also well-known that when the thirst centers lying in the circumventricular organs are activated, animals start to drink. Hence, it has been reported that systemic administration of pilocarpine induces not only salivary secretion, but also water intake (Fregly, 1980; Fregly et al., 1981). This implies that while systemic administration of pilocarpine leads to moistening of the oral cavity by increasing the output of saliva, it simultaneously induces thirst in the mouth. This may raise serious problems when pilocarpine is used as a medication for xerostomia. Until now, there has been no comparative study of the salivary secretion and water intake induced by the peripheral administration of pilocarpine. In the previous studies showing pilocarpine-induced salivation, anesthetized animals were used, and rather high concentrations of pilocarpine and atropine, which may be toxic, were injected into the cerebral ventricle (Renzi et al., 1993, 2002; Moreira et al., 2002). Therefore, the present study was designed to investigate the effects of intraperitoneal and intracerebroventricular injections of low concentrations of pilocarpine and atropine on water intake and salivary secretion in conscious rats.
Animals The experiments were conducted on male Wistar rats (300–400 g). All were housed individually in plastic cages under regular light/dark conditions (lights on from 8:00 a.m. to 8:00 p.m.). The temperature was maintained at 23 ± 1°C, and the humidity was between 60 and 80%. The rats had access to water and laboratory pellets ad libitum, except during the experimental period. All experimental procedures were approved by the animal experiment committee of Kyushu Dental College.
Brain Surgery
Drug Application
Measurements of Water Intake and Salivary Secretion Cannulation into the parotid gland duct was conducted with the rats under sodium pentobarbital anesthesia (60 mg/kg, intraperitoneally injected). A Teflon tube (UT-02, Unique Medical, Tokyo, Japan), connected to polyethylene tubes (SP-10, Natsume, Tokyo, Japan, and No. 3, Hibiki, Tokyo, Japan), filled with saline, was inserted into the salivary duct. After recovery from the anesthesia, the end of the tube was connected to the polyethylene tube (No. 3), which was also filled with saline. Saliva was collected in a 1.5-mL sample tube every 5 min for 60 min, and measured to the nearest 1 mg. To ensure that the cannulation surgery into the duct was successful, we confirmed that saliva in excess of 100 µL was secreted when rats ate a 100-mg pellet (Ito et al., 2001).
Statistical Analysis
In conscious rats, the volumes of water intake and parotid salivary secretion for 60 min after intraperitoneal injections of pilocarpine were significantly increased by more than 4 µmol/kg and 1.2 µmol/kg, respectively, as compared with saline (Fig. 1  ). After intracerebroventricular injection of pilocarpine (0.1 nmol), the water intakes for 60 min were increased significantly, and the effects were dose-dependent (Fig. 2A). In spite of the increased water intake, salivary secretion was not induced by the intracerebroventricular injection of pilocarpine, even at 30 nmol (Fig. 2B).
To investigate the involvement of activation of muscarinic receptors in the brain, we injected atropine, a muscarinic antagonist, intracerebroventricularly 5 min before pilocarpine administration. We have previously reported, in an in vitro study (Xu et al., 2002), that high concentrations of atropine toxically excite some neurons in the circumventricular organ related to drinking behavior. Therefore, we tested whether intracerebroventricular injection of atropine at 1–10 nmol alone induced water intake. While the intracerebroventricular pre-injection of atropine at 10 nmol increased the water intake significantly (1.6 ± 0.7 mL for 60 min, P < 0.05), compared with saline, atropine at 1 nmol did not affect water intake (0.04 ± 0.02 mL for 60 min), and therefore 1 nmol of atropine was used in the antagonist experiments. After the intracerebroventricular injection of atropine, the increased water intakes by either intraperitoneal or intracerebroventricular injections of pilocarpine were suppressed significantly (Figs. 1A  , 2A). However, the increased salivary secretion produced by the intraperitoneal injection of pilocarpine was not changed by the intracerebroventricular pre-injection of atropine (Fig. 1B).
We compared the time courses of water intake produced by intraperitoneal injection at 12 µmol/kg with those following intracerebroventricular injection at 0.3 nmol (the water intakes for 60 min were 1.2 ± 0.3 mL for the intraperitoneal injection and 2.0 ± 0.5 mL for the intracerebroventricular injection). While the water intake elicited by the intracerebroventricular injection was transient, that induced by the intraperitoneal injection was long-lasting (Fig. 3A
Pilocarpine-induced Drinking Behavior In the present study, the increased water intakes produced by the intraperitoneal or intracerebroventricular injections of pilocarpine were obviously suppressed by the intracerebroventricular pre-injection of atropine. We have reported that muscarine and carbachol, both muscarinic receptor agonists, excite neurons in the subfornical organ (Xu et al., 2001; Honda et al., 2003; Inenaga et al., 2004). Because the subfornical organ is a circumventricular organ that lacks the blood-brain barrier, peripherally injected drugs can affect neurons in the region directly. Thus, the activation of muscarinic receptors in the subfornical organ may be related to pilocarpine-induced drinking behavior. Also, the intracerebroventricular injection of pilocarpine more rapidly and transiently induced water intake in comparison with the intraperitoneal injection. A previous study, which investigated the diffusion of Na+ after the intracerebroventricular injection of hypertonic NaCl solution (Nose et al., 1996), showed that the change of Na+ concentration in the brain regions containing the circumventricular organs was completed within 30 min. Although it is impossible to compare the diffusion of pilocarpine in the brain accurately with that of NaCl, it seems important that water intake by the intracerebroventricular injection of pilocarpine was almost finished in 30 min. In contrast, the water intake induced by intraperitoneal injection occurred slowly; thus, it is probable that the pilocarpine administered systemically was temporarily stored somewhere in the body and then released gradually to induce drinking behavior.
Pilocarpine-induced Parotid Salivary Secretion It has been reported that secretion of whole saliva induced by the intraperitoneal injection of pilocarpine in anesthetized rats is suppressed by the intracerebroventricular pre-injection of 2–16 nmol atropine (Takakura et al., 2003). We previously demonstrated, in an in vitro study (Xu et al., 2001), that a high concentration of atropine has toxic effects on neurons, and this action is not a consequence of its being a muscarinic antagonist. The present study showed that the intracerebroventricular injection of atropine at 10 nmol itself promoted water intake. Thus, we think that the high concentration of atropine used in the previous study might exert effects other than antagonistic influences on muscarinic responses. In this study, we observed only parotid saliva, not whole saliva. This may raise another possibility to explain the difference between our results and those of the previous study (Takakura et al., 2003). There are different physiological functions among major salivary glands. For example, submandibular, but not parotid, saliva is tightly related to thermoregulation in rodents. The center of thermoregulation lies in the circumventricular organs and the surrounding regions (Kanosue et al., 1990). A histological study has shown a neural connection from the circumventricular organs to the submandibular gland via the hypothalamus and the inferior salivary nucleus (Hubschle et al., 1998). Although there has been no report of a neural pathway from the circumventricular organs to the parotid gland, the inputs to the gland may be different from those of the submandibular and sublingual glands. Therefore, we cannot deny the possibility of different central effects of pilocarpine on salivation among the major salivary glands. We have reported that intracerebroventricular hypertonic stimulation decreased parotid salivary flow rate and induced thirst in conscious rats, implying that central thirst stimulation decreases salivary secretion through a specific neural pathway (Ito et al., 2002). Since intraperitoneal injection of pilocarpine induced thirst in the present study, the central action might suppress the increased salivary secretion by the peripheral action on the salivary gland. Therefore, we even expected that intraperitoneal pilocarpine-induced salivary secretion was increased by intracerebroventricular pre-injection of atropine, which blocked intraperitoneal pilocarpine-induced water intake. However, parotid salivary secretion was not changed by the treatment. Thus, it seems likely that salivation by the intraperitoneal injection of pilocarpine is not influenced by the central actions of pilocarpine. We conclude that systemically administered pilocarpine promotes parotid salivary secretion through a direct action on the glands, while it also centrally affects the thirst center and induces water intake. This result is important to the understanding of what happens when pilocarpine is used as a medication for dry mouth.
This work was supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan. Received for publication June 30, 2005. Revision received August 30, 2005. Accepted for publication September 22, 2005.
Journal of Dental Research, Vol. 85, No. 1,
64-68 (2006) This article has been cited by other articles:
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ABSTRACT
INTRODUCTION
) IP, 12 µmol/kg, n = 6) and intracerebroventricular injection of pilocarpine(•) ICV, 0.3 nmol, n = 6). There was a significant difference at 15 min between the intraperitoneal and intracerebroventricular injections (Bonferroni post-test followed by two-way ANOVA; *P < 0.05). (B) The time of the start of increased water intake induced by the intraperitoneal and intracerebroventricular injection of pilocarpine (open bar, 12 µmol/kg; filled bar, 0.3 nmol). **P < 0.01 by unpaired t test. (C) The time courses of parotid salivary secretion induced by the intraperitoneal injection of pilocarpine at 12 µmol/kg without (
), n = 5). The time courses were not significantly different by two-way ANOVA.
