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Central Muscarinic Receptors Signal Pilocarpine-induced Salivation

Department of Physiology and Pathology, School of Dentistry, Paulista State University-UNESP, Rua Humaitá, 1680, 14801-903, Araraquara, SP, Brazil;

*corresponding author, menani{at}foar.unesp.br

	ABSTRACT

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Although cholinergic agonists such as pilocarpine injected peripherally can act directly on salivary glands to induce salivation, it is possible that their action in the brain may contribute to salivation. To investigate if the action in the brain is important to salivation, we injected pilocarpine intraperitoneally after blockade of central cholinergic receptors with atropine methyl bromide (atropine-mb). In male Holtzman rats with stainless steel cannulas implanted into the lateral ventricle and anesthetized with ketamine, atropine-mb (8 and 16 nmol) intracerebroventricularly reduced the salivation induced by pilocarpine (4 µmol/kg) intraperitoneally (133 + 42 and 108 + 22 mg/7 min, respectively, vs. saline, 463 + 26 mg/7 min), but did not modify peripheral cardiovascular responses to intravenous acetylcholine. Similar doses of atropine-mb intraperitoneally also reduced pilocarpine-induced salivation. Therefore, systemically injected pilocarpine also enters the brain and acts on central muscarinic receptors, activating autonomic efferent fibers to induce salivation.

KEY WORDS: muscarinic receptors • acetylcholine • atropine • salivary glands • parasympathetic

	INTRODUCTION

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Pilocarpine is a cholinergic agonist that is used therapeutically to reduce dryness of the oral mucosa in patients affected by salivary gland diseases (Ferguson, 1993; Wiseman and Faulds, 1995). It is well-accepted that pilocarpine stimulates salivary secretion by acting on cholinergic receptors in the glandular parenchyma. This idea is supported by the sialogogue effect of pilocarpine in isolated salivary glands (Compton et al., 1981). However, recent evidence suggests that systemically administered pilocarpine can also activate muscarinic receptors in the brain. The salivary secretion induced by pilocarpine is reduced after focal lesions in the forebrain (Renzi et al., 1993, 2002). The inhibition of pilocarpine-induced salivation may appear as early as six hours after damage of the pre-optic-periventricular tissue surrounding the anteroventral third ventricle (AV3V) or lateral hypothalamus, prior to morphological alterations in the salivary glands (Renzi et al., 1993, 2002). Pilocarpine injected into the lateral cerebral ventricles also induces salivation, an action that seems dependent on activation of sympathetic efferent fibers (Cecanho et al., 1999). Finally, pilocarpine-induced salivation is reduced by central, but not peripheral, injections of the anti-hypertensive agent moxonidine (Moreira et al., 2001). These findings are all consistent with the ability of pilocarpine to cross the blood-brain barrier (Freedman et al., 1989). Therefore, one may predict that systemic pilocarpine gains access to the brain, activates cholinergic receptors there, and thereby activates neural pathways subserving salivation.

To test if systemic pilocarpine induces salivation in rats through activation of central muscarinic receptors, we combined intraperitoneal (ip) injection of pilocarpine with intracerebroventricular (icv) injection of atropine methyl bromide, a muscarinic cholinergic antagonist that does not cross the blood-brain barrier (Ghelardini et al., 1990; Pertwee and Ross, 1991; Sanchez and Meier, 1993). In the present study, we achieved functional confirmation of the impermeability of the blood-brain barrier to atropine methyl bromide by testing the effect of icv injection of atropine methyl bromide on the changes in arterial pressure and heart rate (hypotension and bradycardia) induced by intravenous (iv) administration of acetylcholine. These responses to acetylcholine are known to be due exclusively to systemic action. Peripheral effects of the same doses of atropine methyl bromide on salivation and cardiovascular responses were also tested with atropine methyl bromide injected ip and iv, respectively.

	MATERIALS & METHODS

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Animals
Experiments were performed on male Holtzman rats weighing from 250 to 300 g each. The animals were housed individually in stainless steel cages, and kept in a room with controlled light (lights on from 7:00 AM to 7:00 PM), temperature (23 + 2°C), and humidity (55 + 10%). Standard Purina chow and tap water were available ad libitum. All experimental protocols were approved by the Institutional Ethical Committee.

Brain Surgery
The animals that received icv injections were anesthetized with ketamine (100 mg/kg of body weight, ip) and placed in a Kopf stereotaxic instrument with the bregma and lambda points in the same horizontal plane. A stainless steel cannula (10 x 0.7 mm o.d.) was implanted into the lateral cerebral ventricle (LV) 0.3 mm caudal to bregma, 1.5 mm lateral to midline, and 3.6 mm below the dura mater. The cannula was fixed to the cranium by means of dental acrylic resin and jeweler screws. A prophylactic dose of penicillin (50,000 IU) was given intramuscularly at the beginning of surgery. Salivation tests started four days after the cerebral surgery.

Cerebral Injections
Injections into the LV were made by means of a 10-µL Hamilton syringe connected by polyethylene tubing (PE-10) to an injection cannula that extended 2 mm beyond the tip of the guide cannula. The drugs were dissolved in isotonic saline and injected in a volume of 1 µL.

Drugs
Pilocarpine hydrochloride, atropine methyl bromide, and acetylcholine chloride were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Pilocarpine was injected ip at the dose of 4 µmol/kg of body weight. Atropine methyl bromide was injected icv (2, 4, 8, and 16 nmol/1 µL), ip (2, 4, 8, and 16 nmol/0.1 mL), or iv (4, 8, and 16 nmol/0.1 mL). Acetylcholine was injected iv at the dose of 8 nmol/0.1 mL.

Salivation
The rats were anesthetized with ketamine (100 mg/kg of body weight, ip). They received an injection of vehicle or atropine methyl bromide (2, 4, 8, or 16 nmol) either icv (in 1 µL saline) or ip (in 0.1 mL saline), followed 15 min later by an ip injection of pilocarpine (4 µmol/kg of body weight). Ten min after injection of pilocarpine, 4 cotton balls, pre-weighed to the nearest 0.0001 g, were inserted into the oral cavity of each animal: 2 underneath the tongue and 2 bilaterally medial to the teeth and oral mucosa. The cotton balls were removed 7 min later, and the amount of saliva produced was calculated as the change in the weight of the cotton balls. Each rat was tested twice, once in the presence of icv or ip atropine methyl bromide, and once in the presence of the vehicle followed by ip pilocarpine. Rats were tested in a counterbalanced order, with 3 days between experiments.

Arterial Pressure and Heart Rate Recordings
Three days after the last salivation test, the animals were anesthetized with ketamine (100 mg/kg of body weight ip). A PE-10 catheter was inserted into the femoral vein for administration of drugs, and a second catheter (PE-10 connected to PE-50) was implanted into the femoral artery for measurement of arterial pressure. Pulsatile arterial pressure, mean arterial pressure, and heart rate were recorded with a Statham Gould P23 Db pressure transducer (Madison, WI, USA) that was coupled to a pre-amplifier (model ETH-200 Bridge Bio Amplifier, CB Sciences, Dover, NH, USA) and a data acquisition system (model Powerlab 8SP, ADInstruments, Colorado Springs, CO, USA).

Ten min after the start of MAP and HR recording, saline was injected icv, and 15 min later, acetylcholine (8 nmol/0.1 mL/rat) was injected iv. Ten min after the first injection of acetylcholine, atropine methyl bromide (4, 8, and 16 nmol/1 µL) was injected icv, and 15 min later, the iv injection of acetylcholine was repeated. The effects of each dose of atropine methyl bromide on salivation and on cardiovascular responses were tested in the same rats.

To show that the same doses of atropine methyl bromide could affect cardiovascular responses to iv acetylcholine if the antagonist had reached the periphery, we injected iv atropine methyl bromide (4, 8, and 16 nmol/0.1 mL/rat) prior to iv acetylcholine in additional groups of rats, following the same protocol as used for icv injections.

Histology
At the end of the experiments, the animals that had received icv injections were deeply anesthetized with sodium thiopental (50 mg/kg of body weight) and perfused through the heart with saline, followed by 10% formalin. The brains were frozen, cut coronally into 50-µm sections, and stained with Giemsa stain. Only animals with injections into the LV were considered for statistical analysis.

Statistical Analysis
Data were expressed as mean + standard error of the means. For statistical comparisons, we used one-way analysis of variance followed by Student-Newman-Keuls’ post hoc test. Significance level was set at P < 0.05.

	RESULTS

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Effects of icv or ip Atropine Methyl Bromide on the Salivation Induced by ip Pilocarpine
The icv injection of atropine methyl bromide (8 and 16 nmol/1 µL) inhibited the salivation induced by ip pilocarpine (4 µmol/kg of body weight) by around 80%, [F(7, 33) = 27.52; p < 0.01] (Fig. 1A). Atropine methyl bromide (4 nmol/1 µL) reduced pilocarpine-induced salivation by 24% (Fig. 1A). Atropine methyl bromide (2 nmol/1 µL) did not alter pilocarpine-induced salivation (Fig. 1A).

View larger version (20K): [in this window] [in a new window]   Figure 1. Salivation (mg/7 min) induced by ip injection of pilocarpine (4 µmol/kg of body weight) in rats pre-treated with (A) icv atropine methyl bromide (ATR: 2, 4, 8, and 16 nmol/1 µL) (open bars) or saline (filled bars) and (B) ip atropine methyl bromide (ATR: 2, 4, 8, and 16 nmol/0.1 mL) (open bars) or saline (filled bars). The results are represented as means + SEM. n = number of rats. *Significantly different from saline + pilocarpine (Student-Newman-Keuls test, p < 0.05).

Effects of icv or iv Atropine Methyl Bromide on the Hypotension and Bradycardia Induced by iv Acetylcholine
The iv administration of acetylcholine (8 nmol/0.1 mL) caused a significant drop in heart rate and arterial pressure. These effects were transient, lasting less than 1 min. The icv injection of atropine methyl bromide (4, 8, and 16 nmol/1 µL) did not affect the hypotension [F(5, 41) = 1.54; p > 0.05] and bradycardia [F(5, 41) = 2.84; p > 0.05] induced by iv acetylcholine (Fig. 2).

View larger version (17K): [in this window] [in a new window]   Figure 2. Changes in mean arterial pressure (MAP) and heart rate (HR) produced by iv injection of acetylcholine (8 nmol/0.1 mL/rat) in rats pre-treated with icv atropine methyl bromide (ATR: 4, 8, and 16 nmol/1 µL) (open bars) or saline (filled bars). The results are represented as means + SEM. n = number of rats.

View larger version (16K): [in this window] [in a new window]   Figure 3. Changes in mean arterial pressure (MAP) and heart rate (HR) produced by iv injection of acetylcholine (8 nmol/0.1 mL/rat) in rats pre-treated with iv atropine methyl bromide (ATR: 4, 8, and 16 nmol/0.1 mL/rat) (open bars) or saline (filled bars). The results are represented as means + SEM. n = number of rats. *Significantly different from saline + acetylcholine (Student-Newman-Keuls test, p < 0.05).

	DISCUSSION

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Quaternary ammonium compounds, such as atropine methyl bromide, are known to diffuse only poorly through the blood-brain barrier (Ghelardini et al., 1990; Pertwee and Ross, 1991; Sanchez and Meier, 1993). Our finding that intracerebral injections of atropine methyl bromide did not affect the cardiovascular responses to acetylcholine administered systemically confirms this failure of the drug to cross the blood-brain barrier. The present results agree with those of previous studies showing that the effects of icv atropine methyl bromide are restricted to the brain (Sanchez and Meier, 1993). However, other studies have shown a partial inhibition of the cardiovascular effects of systemic acetylcholine by icv atropine methyl bromide (Brezenoff et al., 1988). It has been reported that insertion of probes into the brain can disrupt the blood-brain barrier (Johansson et al., 1996), but other studies suggest that a significant part of the blood-brain barrier function may remain intact after introduction of a cannula into the cerebral ventricle, or that function of the barrier may recover with time (Sanchez and Meier, 1993). In the present work, icv atropine methyl bromide efficiently discriminates central from peripheral effects of ip pilocarpine on salivation.

The present work provides unequivocal evidence for a centrally dependent mechanism of salivation induced by systemic pilocarpine. With atropine methyl bromide, it was possible to block the access of systemic pilocarpine to brain muscarinic receptors and inhibit salivation stimulated by pilocarpine. These results are consistent with disruption of peripheral pilocarpine-induced salivation by focal cerebral lesions or central ₂-adrenoceptor activation (Renzi et al., 1993; Moreira et al., 2001; Renzi et al., 2002), and with the peripheral and central effects of another cholinergic agonist, oxotremorine, on salivation (Sanchez and Meier, 1993). One difference from previous data on oxotremorine (Sanchez and Meier, 1993) is that, with 4 µmol/kg of pilocarpine, we did not detect tremors or other signs of seizures in the awake animal (unpublished results). On the contrary, at this dose, ip pilocarpine is dipsogenic (Gay et al., 1976), and the overall motor pattern of the behavior looks normal when the rat is drinking (unpublished results). In contrast to previous studies, in which pilocarpine-induced salivation was tested under urethane or tribromoethanol anesthesia (Renzi et al., 1993, 2002; Moreira et al., 2001), rats in the present study were anesthetized with ketamine. The baseline salivary secretion under ketamine anesthesia is very low (around 14 mg/7 min), and pilocarpine-induced salivation is not different from that under urethane or tribromoethanol anesthesia.

Cholinergic receptors are widely distributed in the forebrain (Vickroy et al., 1985; Brann et al., 1993). Focal lesion studies have suggested that the pre-optic-periventricular tissue surrounding the anteroventral third ventricle (AV3V) and the lateral hypothalamus are candidates for processing the information raised by central cholinergic activation after systemic administration of pilocarpine (Renzi et al., 1993, 2002). Viral tracing techniques have shown the existence of neural pathways from the AV3V region and the lateral hypothalamus to the submandibular and sublingual salivary glands (Hübschle et al., 1998, 2001). In these studies, neurons situated in the lamina terminalis or, more specifically, in the AV3V and the subfornical organ were retrogradely labeled after injection of pseudorabies virus into the submandibular or sublingual gland of rats (Hübschle et al., 2001). Viral tracing combined with glandular denervation suggests that structures from the lamina terminalis influence especially the parasympathetic control of the submandibular gland (Hübschle et al., 2001). Thus, it is possible that the signals produced by the action of pilocarpine on central muscarinic receptors converge on the AV3V and from the AV3V are relayed via the lateral hypothalamus to autonomic efferent fibers innervating the salivary glands.

Blockade of central or peripheral cholinergic receptors by atropine methyl bromide inhibits ip pilocarpine-induced salivation. Therefore, the salivation produced by pilocarpine acting on central muscarinic receptors seems to depend on parasympathetic activity. This suggestion is in accordance with results from viral tracing studies that showed mainly parasympathetic efferent fibers from the lamina terminalis to salivary glands (Hübschle et al., 2001). However, the salivation induced by icv administration of pilocarpine can be reduced by cervical ganglionectomy or antagonism of systemic ₁-adrenoceptors (Cecanho et al., 1999), which suggests that pilocarpine can also act on the brain to increase sympathetic output to the salivary glands. Since the reduction in salivation after cervical ganglionectomy is not total (Cecanho et al., 1999), it is possible that the activation of parasympathetic efferent fibers contributes to the effect of pilocarpine, which would be in agreement with the present results. Therefore, it is possible that the regulation of salivation by central cholinergic receptors depends on an interaction of sympathetic and parasympathetic mechanisms, perhaps an interaction in a synergistic manner (Emmelin, 1987; Garrett, 1987).

The results also show that icv atropine methyl bromide causes a large reduction in pilocarpine-induced salivation, but does not completely block it. This is in accordance with the well-known effect that pilocarpine has on cholinergic receptors of the salivary glands to induce salivation. Therefore, salivation induced by ip pilocarpine may depend on the activation of both central and peripheral muscarinic cholinergic receptors and probably involves sympathetic and parasympathetic efferent fibers. Further investigation is needed to define better the relative importance of autonomic efferent fibers and the particular salivary glands that respond to this activation.

	ACKNOWLEDGMENTS

 
We thank Dr. Gus Schoorlemmer for his help in the revision of the manuscript, Silas Pereira Barbosa, Reginaldo da Conceição Queiróz, and Silvia Fóglia for expert technical assistance, Silvana A.D. Malavolta for secretarial assistance, and Ana L.V. de Oliveira for animal care. This research was supported by public funding from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) and Conselho Nacional de Pesquisa (CNPq).

Received June 18, 2003; Last revision August 19, 2003; Accepted September 8, 2003

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