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Suppression of reflex saliva from rat parotid gland following intracerebroventricular injection of hypertonic NaCl and sucrose
Archives of Oral Biology 47 (2002) 93–97
Short communication
Suppression of reflex saliva from rat parotid gland following intracerebroventricular injection of hypertonic NaCl and sucrose Kayoko Ito a,b , Masao Morikawa b , Kiyotoshi Inenaga a,∗ b
a Department of Physiology, Kyushu Dental College, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu 803-8580, Japan Department of Removable Prosthodontics, Kyushu Dental College, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu 803-8580, Japan
Accepted 24 July 2001
Abstract These effects were studied in conscious rats. Salivary volume and flow rate induced by eating solid diet were decreased by both the hypertonic solutions, compared with the effects of normal saline. This finding suggests that central osmotic perception affects parotid salivary secretion in rats. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Saliva; Parotid gland; Central osmotic stimulation
The volume of urine and sweat is regulated by a system of body fluid homeostasis. Because saliva is produced from blood, the volume of saliva, like that of urine and sweat, changes with the osmotic pressure and volume of body fluid. It has been reported that osmotic stimulation induced by intraperitoneal (Ito et al., 2001) or intravenous injection (Miyoshi et al., 1969) of hypertonic solutions in in vivo experiments modifies salivary secretion. An in vitro study on the isolated, perfused rat submandibular gland also demonstrates that hyperosmotic stimulation reduces the salivary flow rate (Nakahari et al., 1997). These observations indicate a direct action of hyperosmosis on the salivary gland. In addition, some reports have indicated that central osmotic stimulation can also influence salivary secretion. Olsson (1976) reports that the intracerebroventricular injection of hypertonic NaCl solution reduced the parotid salivary flow rate in conscious goats. In contrast, the injection of hypertonic glucose or glycerol solutions affected salivary secretion in the opposite way. On the other hand, Yoshimura et al. (1968) report that the volume of salivary secretion induced by pilocarpine was increased by the intracerebroventricular injection of hypertonic NaCl solution in dogs. Thus, there is no
∗
Corresponding author. Tel.: +81-93-582-1131/ext. 6632; fax: +81-93-582-8288. E-mail address: [email protected] (K. Inenaga).
consensus about the central regulation of salivary secretion by hyperosmotic stimulation. There is considerable evidence that the intracerebroventricular injection of hypertonic NaCl or other hyperosmotic solutions evokes both vasopressin release and drinking behaviour (Bourque et al., 1994). It is believed that intracerebroventricularly (i.c.v.) injected hyperosmotic solutions exert their effects directly upon osmosensitive neurones in the circumventricular and hypothalamic regions. Bourque et al. (1994) describe the existence of stretch-inactivated cationic channels in neurones in these areas in rats. These non-selective channels are opened when cell volume shrinks during hyperosmotic stimulation. If a similar cellular mechanism is involved in salivary secretion, then any hypertonic stimulants, after central administration, may influence salivary secretion in the same direction. The present study was designed to investigate the effects of the intracerebroventricular injection of hypertonic solutions on reflex parotid salivary secretion. For this purpose, we used rats as experimental animals because their central osmoregulation is well understood. All experimental procedures were approved by the Animal Experiment Committee, Kyushu Dental College. Experiments were conducted on male Wistar rats weighing 250–300 g. All rats were individually housed in plastic cages in a temperature- and humidity-controlled room under light/dark conditions (lights on: 08.00–20.00 h). They
0003-9969/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 0 3 - 9 9 6 9 ( 0 1 ) 0 0 0 8 4 - X
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K. Ito et al. / Archives of Oral Biology 47 (2002) 93–97
were allowed access to water and laboratory pellets ad libitum, except during the experimental period. Before the experiments, the rats were trained in a test box for 1 week to eat and to drink water immediately after being served. A series of experiments was carried out at a fixed time (10.00–15.00 h) for each rat, considering its circadian rhythm. Well-trained rats were used for the experiments. To install a stainless-steel guide cannula for the intracerebroventricular injection of hypertonic solutions, the rat was anaesthetised with sodium pentobarbital (50 mg/kg, intraperitoneal) and was placed in a stereotaxic apparatus (Takahashi Co. Ltd., Tokyo, Japan). A 24G stainless-steel guide cannula was inserted into the brain (0.8 mm posterior to the bregma, 1.4 mm to the right lateral side and 2 mm below from the surface). After a recovery period of at least 1 week, the rats underwent another operation for the measurement of salivary secretion, as described by Ito et al. (2001). In brief, the rats were anaesthetised with sodium pentobarbital (50 mg/kg, intraperitoneal), and a polyethylene cannula (PE–10; Becton Dickinson, MD, USA) was introduced into the duct of the parotid gland. The other end of the tube was connected to a second polyethylene tube (0.8 mm i.d., 1.1 mm o.d.). After recovering from anaesthesia, the rat was transferred to a test box, and the second polyethylene tube was connected to a pressure transducer (Nihon Kohden, Tokyo,
Japan). To test the effect of hyperosmosis on salivary secretion, 1 M NaCl and 2 M sucrose dissolved in isotonic saline were used. Solutions were injected by a microinfusion pump (Microprocessor-Controlled Syringe Pump; Stoelting Co., IL, USA) at a rate of 4 l/min for 5 min. The volume of secreted saliva was evaluated as an increase of pressure in the transducer. The output from the pressure transducer was relayed to an AD converter (MacLab; AD Instruments, NSW, Australia) via a bridge amplifier (Bridge Amp; AD Instruments, NSW, Australia). To avoid too great a pressure on the salivary glands, a solenoid valve was used to leak the same amount of liquid as the secreted saliva. The open period was 2 s and the closed period was 18 s. The pressure value was calibrated as saliva volume by the microinfusion pump. Saliva volume was evaluated as the total volume of saliva secreted from the parotid gland in response to a given diet (200 mg). Salivary flow rate (l/min) was evaluated as the volume of saliva secreted during 60 s when the secretion was at a maximum. For statistical analysis, the Friedman test was used. Reflex salivary secretion from the parotid gland, stimulated with 200 mg pellets, was repeatable (Fig. 1). The consecutive traces in Fig. 1 show the change in salivary secretion induced by the injection of isotonic saline, 1 M NaCl and 2 M sucrose. Fig. 2 shows the averaged changes in parotid salivary secretion. After the injection of either hypertonic
Fig. 1. The effects of i.c.v. injected NaCl and sucrose on reflex parotid saliva in the conscious rat. Consecutive traces show the reflex secretion. At the arrows, isotonic saline in (A) 1 M NaCl in (B) and 2 M sucrose in (C) were injected i.c.v. (4 l/min, 5 min). The bars show the duration of handling 200 mg solid diet.
K. Ito et al. / Archives of Oral Biology 47 (2002) 93–97
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Fig. 2. The effect of i.c.v. injected NaCl and sucrose on the volume and flow rate of reflex parotid saliva: (A) and (B) show the average changes of saliva volume per 200 mg solid diet (a) and the salivary flow rate (b) from the parotid gland in 11 rats for NaCl injection and 10 rats for sucrose injection, respectively. The i.c.v. injection of osmotic solutions started at 0 min (4 l/min, 5 min, i.c.v.). After the hypertonic NaCl and sucrose injections (䊉), both the saliva volume and the salivary flow rate were reduced, compared to the isotonic saline injection (䊊). Each plot shows mean ± S.E.M. (n = 11 for NaCl injection; n = 10 for sucrose injection). (∗ ) P < 0.05 (Friedman test).
NaCl or sucrose, the saliva volume was less than after the isotonic saline injection (Fig. 2A(a) and B(a)). However, the time-course of the responses to hypertonic NaCl and sucrose was different. In response to hypertonic NaCl, the saliva volume and the salivary flow rate rapidly decreased, and recovered to control values within 40 min (Fig. 2A). By contrast, in response to the hypertonic sucrose injection, the saliva volume and the salivary flow rate decreased gradually. The maximum change occurred 30 min after the injection, and recovered towards the control value over 60 min (Fig. 2B). It is believed that the intracerebroventricular injection of hyperosmotic solutions stimulates circumventricular structures such as the subfornical organ and the organum vasculosum lamina terminalis. These regions, in which osmoreceptors exist (Bourque et al., 1994), are involved in the integrative control of body fluid and electrolyte homeostasis (Honda et al., 1990; McKinley et al., 1992, 1996). Body fluid balance is closely related to salivary secretion; indeed, dehydration in human beings decreases parotid salivary flow rates (Ship and Fischer, 1997). There are efferent projections from the circumventricular organs to the salivary glands (H¨ubschle et al., 1998) and lesions in these hypothalamic structures induce morphological changes in the salivary glands (Renzi et al., 1990). Thus, neuronal efferents originating from the
integrative osmoregulatory hypothalamic structures could possibly modulate saliva production and secretion under the condition of hypertonicity. This is, to the best of our belief, the first report that the reflex secretion of parotid saliva is influenced through osmoperception in the central nervous system in conscious rats. The central mechanisms of osmoreception have been clarified by a number of experiments, particularly in the rat brain. Bourque et al. (1994) report the existence of non-selective cation channels in osmoreceptive neurones located in the hypothalamus and in circumventricular organs of the forebrain. These channels, which are opened by hyperosmosis, permeate cations including sodium ions. Many years ago, Olsson (1976) reported that the salivary flow rate was decreased by intracerebroventricular injection of hypertonic NaCl solution in goats, but increased by d-glucose. Those findings, as for the hypertonic NaCl injection, were consistent with ours whereas those for the hypertonic glucose injection seem to be contradictory. However, as d-glucose is highly membrane-permeant, it may not be an effective substance for stimulating the osmoreceptors in the brain (Greger, 1996). Furthermore, Olsson (1976) used distilled water to dissolve d-glucose and then injected the solution into the ventricle. This procedure may produce
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a reduction in the concentration of sodium in the cerebrospinal and extracellular fluids. Thus, the net effect of the infusion of hypertonic glucose may in fact be a reduction in the effective osmotic stimulus to central osmoreceptors. The time-course of salivary secretion was different after the intracerebroventricular injections of hypertonic NaCl and sucrose solutions. NaCl decreased the saliva volume and salivary flow rate 10 min after the injection; the secretion gradually recovered to the control value by 40 min. Sucrose also decreased significantly the saliva volume and the salivary flow rate, but unlike the effects of hypertonic NaCl, those of sucrose were long-lasting, and were maintained until 60 min after the injection. Thus, while both hypertonic solutions inhibited the reflex parotid salivary secretion, the pattern of the responses was different. The diffusion coefficients of sucrose in aqueous solution (Robinson and Stokes, 1970) and in the dog brain (Patlak and Fenstermacher, 1975) were approx. two times smaller than those of NaCl (7 × 10−6 cm2 /s versus 1.5 × 10−5 cm2 /s for aqueous solution and 3.1 × 10−6 cm2 /s versus 5.6 × 10−6 cm2 /s for the dog brain). These findings suggest that sucrose diffuses more slowly and is cleared more slowly than NaCl in the cerebrospinal fluid (CSF) of the ventricle and brain tissue. Therefore, it seems likely that sucrose affects the osmoreceptors in the circumventricular organs for much longer than NaCl. Further, re-uptake of NaCl by the cells in the ventricular walls might be faster than that of sucrose. Alternatively, it has been suggested that, in addition to osmoreception, the brain also detects increased sodium concentration in initiating natriuretic mechanisms (McKinley et al., 1994). If sodium-sensing receptors exist in the circumventricular organs, and their related mechanisms are different from those of the osmoreceptors, these may be responsible for the time-course difference between NaCl and sucrose. We studied the reflex secretion of saliva in response to eating pellets, and such secretion is also influenced by the central mechanisms that control feeding behaviour. Electrophysiological (Ishizuka and Murakami, 1995) and histochemical (Hosoya and Matsushita, 1981; Jansen et al., 1992; H¨ubschle et al., 1998; Matsuo, 1999) studies have suggested that neuronal activity in the salivary nucleus in the brainstem is modulated by the feeding centre, the lateral hypothalamic area. Further, there appear to be osmosensitive neurones in the lateral hypothalamic area as well as the circumventricular organs (Leng, 1982). If the osmosensitive neurones in the lateral hypothalamic area are stimulated by the i.c.v. injected and diffused NaCl and sucrose, these responses might contribute to the time-course difference between NaCl and sucrose, due to their different diffusion coefficients. However, the lateral hypothalamic area is obviously much further from the ventricle, than are the circumventricular organs. Therefore, even if the injected NaCl and sucrose did diffuse in the brain tissues and directly affected neurones in the lateral hypothalamic area, the influence of this might be not so large.
We recently reported the effects of intraperitoneal injection of hypertonic NaCl and 1 day’s water deprivation on reflex salivary secretion (Ito et al., 2001). The saliva volume secreted during eating a pellet was increased, whereas the salivary flow rate was decreased. In the case of these stimuli, both the central and peripheral influence of hyperosmosis should be considered. However, it is likely that the action in the present study was confined to the central nervous system. Even if all of the hypertonic NaCl that was injected into the ventricle leaked into blood, the increased osmolarity would amount to only a few mosmol/kg, a change much smaller than that induced by the intraperitoneal injection of hypertonic NaCl. At present, we have no clear explanation of the difference between our previous and present results. However, it is feasible that systemic hyperosmotic stimulation changes the sensitivity of oral sensation and increases the threshold for swallowing, and induces the increment in saliva volume during the eating of a specific amount of food. An intracerebroventricular injection of hyperosmotic solutions affected the central mechanism of thermoregulation and increased the temperature of the preoptic area in the hypothalamus of rabbits (Turlejska and Baker, 1986). Local warming of these regions in the hypothalamus increased secretion from salivary glands in rats (Kanosue et al., 1990). The intracerebroventricular hyperosmotic stimulation in our study, on the other hand, suppressed the reflex saliva from the rat parotid gland, possibly accompanied by a similar increase of hypothalamic temperatures. If increased hypothalamic temperature induced the secretion from the parotid, the temperature effects on the secretion might mask the suppression of salivary secretion by intracerebroventricular hyperosmotic stimulation. Beside their role in digestion and swallowing, the salivary glands of rodents function as thermoregulatory effector organs, with heat dissipation by evaporative water loss, which could be related to body fluid homeostasis (Stricker and Hainsworth, 1970). However, as we discussed earlier (Ito et al., 2001), there are functional differences between the parotid and submandibular salivary glands: the submandibular plays an import part in thermoregulation, but the parotid does little. Osmotic stimulation of the circumventricular organs in the forebrain increases vasopressin in the plasma (Bourque et al., 1994). Increased plasma vasopressin might have suppressed salivary secretion in our study by acting on the parotid gland ducts (Ferguson, 1994). However, this possibility is contradicted by the findings of a study on the intranasal administration of desmopressin, a vasopressin analogue (Ferguson, 1994), which did not positively support the notion of suppressive effects of vasopressin on salivary secretion. In conclusion, the intracerebroventricular injection of hypertonic solutions suppresses reflex salivary secretion from the rat parotid gland through central osmoreceptive mechanisms. Further experiments should be done to elucidate the neural mechanisms underlying the control system.
K. Ito et al. / Archives of Oral Biology 47 (2002) 93–97
Acknowledgements This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Japan.
References Bourque, C.W., Oliet, S.H.R., Richard, D., 1994. Osmoreceptors, osmoreception, and osmoregulation. Frontiers Neuroendocrinol. 15, 231–274. Ferguson, D.B., 1994. The effect of an analogue of antidiuretic hormone on salivary composition. J. Dental Res. 73, 815. Greger, R., 1996. Central control of water and salt metabolism. In: Greger, R., Windhorst, U. (Eds.), Comprehensive Human Physiology. Springer, Berlin, pp. 1625–1648. Honda, K., Negoro, H., Dyball, R.E., Higuchi, T., Takano, S., 1990. The osmoreceptor complex in the rat: evidence for interactions between the supraoptic and other diencephalic nuclei. J. Physiol. 431, 225–241. Hosoya, Y., Matsushita, M., 1981. Brainstem projections from the lateral hypothalamic area in the rat, as studied with autoradiography. Neurosci. Lett. 24, 111–116. H¨ubschle, T., McKinley, M.J., Oldfield, B.J., 1998. Efferent connections of the lamina terminalis, the preoptic area and the insular cortex to submandibular and sublingual gland of the rat traced with pseudorabies virus. Brain Res. 806, 219–231. Ishizuka, K., Murakami, T., 1995. Response of the superior salivatory nucleus to stimulation of the lateral hypothalamic area in the cat. Jpn. J. Oral Biol. 37, 481–483. Ito, K., Morikawa, M., Inenaga, K., 2001. The effect of food consistency and dehydration on stimulated parotid and submandibular salivary secretion in conscious rats. Arch. Oral Biol. 46, 353–363. Jansen, A.S.P., Ter Horst, G.J., Mettenleiter, T.C., Loewy, A.D., 1992. CNS cell groups projecting to the submandibular parasympathetic preganglionic in the rat: a retrograde transneuronal viral cell body labeling study. Brain Res. 572, 253–260. Kanosue, K., Nakayama, T., Tanaka, H., Yanase, M., Yasuda, H., 1990. Modes of action of local hypothalamic and skin thermal stimulation on salivary secretion in rats. J. Physiol. 424, 459– 471. Leng, G., 1982. Lateral hypothalamic neurones: osmosensitivity and the influence of activating magnocellular neurosecretory neurones. J. Physiol. 326, 35–48.
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Matsuo, R., 1999. Interrelation of taste and saliva. In: Garrett, J.R., Ekstr¨om, J., Anderson, L.C. (Eds.), Neural Mechanisms of Salivary Gland Secretion. Karger, Basel, pp. 185–195. McKinley, M.J., Bicknell, R.J., Hards, D., McAllen, R.M., Vivas, L., Weisinger, R.S., Oldfield, B.J., 1992. Efferent neural pathways of the lamina terminalis subserving osmoregulation. Progress Brain Res. 91, 395–402. McKinley, M.J., Harvey, R.B., Vivas, L., 1994. Reducing brain sodium concentration prevents post-prandial and dehydration-induced natriuresis. Acta Physiol. Scand. 151, 467– 476. McKinley, M.J., Pennington, G.L., Oldfield, B.J., 1996. Anteroventral wall of the third ventricle and dorsal lamina terminalis: headquarters for control of body fluid homeostasis. Clin. Exp. Pharmacol. Physiol. 23, 271–281. Miyoshi, M., Okamoto, T., Sueki, M., Yoshimura, H., 1969. Influence of blood osmolality on the concentration of protein and hexosamine in canine saliva. Jpn. J. Physiol. 19, 841–850. Nakahari, T., Steward, M.C., Yoshida, H., Imai, Y., 1997. Osmotic flow transients during acetylcholine stimulation in the perfused rat submandibular gland. Exp. Physiol. 82, 55–70. Olsson, K., 1976. Influence of altered CSF solute composition on parotid salivary secretion in goats. Acta Physiol. Scand. 97, 196–201. Patlak, C.S., Fenstermacher, J.D., 1975. Measurements of dog blood-brain transfer constants by ventriculocisternal perfusion. Am. J. Physiol. 229, 877–884. Renzi, A., Lopes, R.A., Sala, M.A., Camargo, L.A., Menani, J.V., Saad, W.A., Campos, G.M., 1990. Morphological, morphometric and stereological study of submandibular glands in rats with lesion of the anteroventral region of the third ventricle (AV3V). Exp. Pathol. 38, 177–187. Robinson, R.A., Stokes, R.H., 1970. Electrolyte Solutions, 2nd edition. Butterworths, London. Ship, J.A., Fischer, D.J., 1997. The relationship between dehydration and parotid salivary gland function in young and older healthy adults. J. Gerontol. Ser. A: Biol. Sci. Med. Sci. 52, M310–319. Stricker, E.M., Hainsworth, F.R., 1970. Evaporative cooling in the rat: effects of dehydration. Can. J. Physiol. Pharmacol. 48 (1), 18–27. Turlejska, E., Baker, M.A., 1986. Elevated CSF osmolality inhibits thermoregulatory heat loss responses. Am. J. Physiol. 251, R749–754. Yoshimura, H., Inoue, T., Hirai, K., 1968. Effect of changes in osmotic pressure in the third ventricular fluid upon the salt secretion into saliva. Med. Biol. 76, 227–231.
Short communication
Suppression of reflex saliva from rat parotid gland following intracerebroventricular injection of hypertonic NaCl and sucrose Kayoko Ito a,b , Masao Morikawa b , Kiyotoshi Inenaga a,∗ b
a Department of Physiology, Kyushu Dental College, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu 803-8580, Japan Department of Removable Prosthodontics, Kyushu Dental College, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu 803-8580, Japan
Accepted 24 July 2001
Abstract These effects were studied in conscious rats. Salivary volume and flow rate induced by eating solid diet were decreased by both the hypertonic solutions, compared with the effects of normal saline. This finding suggests that central osmotic perception affects parotid salivary secretion in rats. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Saliva; Parotid gland; Central osmotic stimulation
The volume of urine and sweat is regulated by a system of body fluid homeostasis. Because saliva is produced from blood, the volume of saliva, like that of urine and sweat, changes with the osmotic pressure and volume of body fluid. It has been reported that osmotic stimulation induced by intraperitoneal (Ito et al., 2001) or intravenous injection (Miyoshi et al., 1969) of hypertonic solutions in in vivo experiments modifies salivary secretion. An in vitro study on the isolated, perfused rat submandibular gland also demonstrates that hyperosmotic stimulation reduces the salivary flow rate (Nakahari et al., 1997). These observations indicate a direct action of hyperosmosis on the salivary gland. In addition, some reports have indicated that central osmotic stimulation can also influence salivary secretion. Olsson (1976) reports that the intracerebroventricular injection of hypertonic NaCl solution reduced the parotid salivary flow rate in conscious goats. In contrast, the injection of hypertonic glucose or glycerol solutions affected salivary secretion in the opposite way. On the other hand, Yoshimura et al. (1968) report that the volume of salivary secretion induced by pilocarpine was increased by the intracerebroventricular injection of hypertonic NaCl solution in dogs. Thus, there is no
∗
Corresponding author. Tel.: +81-93-582-1131/ext. 6632; fax: +81-93-582-8288. E-mail address: [email protected] (K. Inenaga).
consensus about the central regulation of salivary secretion by hyperosmotic stimulation. There is considerable evidence that the intracerebroventricular injection of hypertonic NaCl or other hyperosmotic solutions evokes both vasopressin release and drinking behaviour (Bourque et al., 1994). It is believed that intracerebroventricularly (i.c.v.) injected hyperosmotic solutions exert their effects directly upon osmosensitive neurones in the circumventricular and hypothalamic regions. Bourque et al. (1994) describe the existence of stretch-inactivated cationic channels in neurones in these areas in rats. These non-selective channels are opened when cell volume shrinks during hyperosmotic stimulation. If a similar cellular mechanism is involved in salivary secretion, then any hypertonic stimulants, after central administration, may influence salivary secretion in the same direction. The present study was designed to investigate the effects of the intracerebroventricular injection of hypertonic solutions on reflex parotid salivary secretion. For this purpose, we used rats as experimental animals because their central osmoregulation is well understood. All experimental procedures were approved by the Animal Experiment Committee, Kyushu Dental College. Experiments were conducted on male Wistar rats weighing 250–300 g. All rats were individually housed in plastic cages in a temperature- and humidity-controlled room under light/dark conditions (lights on: 08.00–20.00 h). They
0003-9969/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 0 3 - 9 9 6 9 ( 0 1 ) 0 0 0 8 4 - X
94
K. Ito et al. / Archives of Oral Biology 47 (2002) 93–97
were allowed access to water and laboratory pellets ad libitum, except during the experimental period. Before the experiments, the rats were trained in a test box for 1 week to eat and to drink water immediately after being served. A series of experiments was carried out at a fixed time (10.00–15.00 h) for each rat, considering its circadian rhythm. Well-trained rats were used for the experiments. To install a stainless-steel guide cannula for the intracerebroventricular injection of hypertonic solutions, the rat was anaesthetised with sodium pentobarbital (50 mg/kg, intraperitoneal) and was placed in a stereotaxic apparatus (Takahashi Co. Ltd., Tokyo, Japan). A 24G stainless-steel guide cannula was inserted into the brain (0.8 mm posterior to the bregma, 1.4 mm to the right lateral side and 2 mm below from the surface). After a recovery period of at least 1 week, the rats underwent another operation for the measurement of salivary secretion, as described by Ito et al. (2001). In brief, the rats were anaesthetised with sodium pentobarbital (50 mg/kg, intraperitoneal), and a polyethylene cannula (PE–10; Becton Dickinson, MD, USA) was introduced into the duct of the parotid gland. The other end of the tube was connected to a second polyethylene tube (0.8 mm i.d., 1.1 mm o.d.). After recovering from anaesthesia, the rat was transferred to a test box, and the second polyethylene tube was connected to a pressure transducer (Nihon Kohden, Tokyo,
Japan). To test the effect of hyperosmosis on salivary secretion, 1 M NaCl and 2 M sucrose dissolved in isotonic saline were used. Solutions were injected by a microinfusion pump (Microprocessor-Controlled Syringe Pump; Stoelting Co., IL, USA) at a rate of 4 l/min for 5 min. The volume of secreted saliva was evaluated as an increase of pressure in the transducer. The output from the pressure transducer was relayed to an AD converter (MacLab; AD Instruments, NSW, Australia) via a bridge amplifier (Bridge Amp; AD Instruments, NSW, Australia). To avoid too great a pressure on the salivary glands, a solenoid valve was used to leak the same amount of liquid as the secreted saliva. The open period was 2 s and the closed period was 18 s. The pressure value was calibrated as saliva volume by the microinfusion pump. Saliva volume was evaluated as the total volume of saliva secreted from the parotid gland in response to a given diet (200 mg). Salivary flow rate (l/min) was evaluated as the volume of saliva secreted during 60 s when the secretion was at a maximum. For statistical analysis, the Friedman test was used. Reflex salivary secretion from the parotid gland, stimulated with 200 mg pellets, was repeatable (Fig. 1). The consecutive traces in Fig. 1 show the change in salivary secretion induced by the injection of isotonic saline, 1 M NaCl and 2 M sucrose. Fig. 2 shows the averaged changes in parotid salivary secretion. After the injection of either hypertonic
Fig. 1. The effects of i.c.v. injected NaCl and sucrose on reflex parotid saliva in the conscious rat. Consecutive traces show the reflex secretion. At the arrows, isotonic saline in (A) 1 M NaCl in (B) and 2 M sucrose in (C) were injected i.c.v. (4 l/min, 5 min). The bars show the duration of handling 200 mg solid diet.
K. Ito et al. / Archives of Oral Biology 47 (2002) 93–97
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Fig. 2. The effect of i.c.v. injected NaCl and sucrose on the volume and flow rate of reflex parotid saliva: (A) and (B) show the average changes of saliva volume per 200 mg solid diet (a) and the salivary flow rate (b) from the parotid gland in 11 rats for NaCl injection and 10 rats for sucrose injection, respectively. The i.c.v. injection of osmotic solutions started at 0 min (4 l/min, 5 min, i.c.v.). After the hypertonic NaCl and sucrose injections (䊉), both the saliva volume and the salivary flow rate were reduced, compared to the isotonic saline injection (䊊). Each plot shows mean ± S.E.M. (n = 11 for NaCl injection; n = 10 for sucrose injection). (∗ ) P < 0.05 (Friedman test).
NaCl or sucrose, the saliva volume was less than after the isotonic saline injection (Fig. 2A(a) and B(a)). However, the time-course of the responses to hypertonic NaCl and sucrose was different. In response to hypertonic NaCl, the saliva volume and the salivary flow rate rapidly decreased, and recovered to control values within 40 min (Fig. 2A). By contrast, in response to the hypertonic sucrose injection, the saliva volume and the salivary flow rate decreased gradually. The maximum change occurred 30 min after the injection, and recovered towards the control value over 60 min (Fig. 2B). It is believed that the intracerebroventricular injection of hyperosmotic solutions stimulates circumventricular structures such as the subfornical organ and the organum vasculosum lamina terminalis. These regions, in which osmoreceptors exist (Bourque et al., 1994), are involved in the integrative control of body fluid and electrolyte homeostasis (Honda et al., 1990; McKinley et al., 1992, 1996). Body fluid balance is closely related to salivary secretion; indeed, dehydration in human beings decreases parotid salivary flow rates (Ship and Fischer, 1997). There are efferent projections from the circumventricular organs to the salivary glands (H¨ubschle et al., 1998) and lesions in these hypothalamic structures induce morphological changes in the salivary glands (Renzi et al., 1990). Thus, neuronal efferents originating from the
integrative osmoregulatory hypothalamic structures could possibly modulate saliva production and secretion under the condition of hypertonicity. This is, to the best of our belief, the first report that the reflex secretion of parotid saliva is influenced through osmoperception in the central nervous system in conscious rats. The central mechanisms of osmoreception have been clarified by a number of experiments, particularly in the rat brain. Bourque et al. (1994) report the existence of non-selective cation channels in osmoreceptive neurones located in the hypothalamus and in circumventricular organs of the forebrain. These channels, which are opened by hyperosmosis, permeate cations including sodium ions. Many years ago, Olsson (1976) reported that the salivary flow rate was decreased by intracerebroventricular injection of hypertonic NaCl solution in goats, but increased by d-glucose. Those findings, as for the hypertonic NaCl injection, were consistent with ours whereas those for the hypertonic glucose injection seem to be contradictory. However, as d-glucose is highly membrane-permeant, it may not be an effective substance for stimulating the osmoreceptors in the brain (Greger, 1996). Furthermore, Olsson (1976) used distilled water to dissolve d-glucose and then injected the solution into the ventricle. This procedure may produce
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a reduction in the concentration of sodium in the cerebrospinal and extracellular fluids. Thus, the net effect of the infusion of hypertonic glucose may in fact be a reduction in the effective osmotic stimulus to central osmoreceptors. The time-course of salivary secretion was different after the intracerebroventricular injections of hypertonic NaCl and sucrose solutions. NaCl decreased the saliva volume and salivary flow rate 10 min after the injection; the secretion gradually recovered to the control value by 40 min. Sucrose also decreased significantly the saliva volume and the salivary flow rate, but unlike the effects of hypertonic NaCl, those of sucrose were long-lasting, and were maintained until 60 min after the injection. Thus, while both hypertonic solutions inhibited the reflex parotid salivary secretion, the pattern of the responses was different. The diffusion coefficients of sucrose in aqueous solution (Robinson and Stokes, 1970) and in the dog brain (Patlak and Fenstermacher, 1975) were approx. two times smaller than those of NaCl (7 × 10−6 cm2 /s versus 1.5 × 10−5 cm2 /s for aqueous solution and 3.1 × 10−6 cm2 /s versus 5.6 × 10−6 cm2 /s for the dog brain). These findings suggest that sucrose diffuses more slowly and is cleared more slowly than NaCl in the cerebrospinal fluid (CSF) of the ventricle and brain tissue. Therefore, it seems likely that sucrose affects the osmoreceptors in the circumventricular organs for much longer than NaCl. Further, re-uptake of NaCl by the cells in the ventricular walls might be faster than that of sucrose. Alternatively, it has been suggested that, in addition to osmoreception, the brain also detects increased sodium concentration in initiating natriuretic mechanisms (McKinley et al., 1994). If sodium-sensing receptors exist in the circumventricular organs, and their related mechanisms are different from those of the osmoreceptors, these may be responsible for the time-course difference between NaCl and sucrose. We studied the reflex secretion of saliva in response to eating pellets, and such secretion is also influenced by the central mechanisms that control feeding behaviour. Electrophysiological (Ishizuka and Murakami, 1995) and histochemical (Hosoya and Matsushita, 1981; Jansen et al., 1992; H¨ubschle et al., 1998; Matsuo, 1999) studies have suggested that neuronal activity in the salivary nucleus in the brainstem is modulated by the feeding centre, the lateral hypothalamic area. Further, there appear to be osmosensitive neurones in the lateral hypothalamic area as well as the circumventricular organs (Leng, 1982). If the osmosensitive neurones in the lateral hypothalamic area are stimulated by the i.c.v. injected and diffused NaCl and sucrose, these responses might contribute to the time-course difference between NaCl and sucrose, due to their different diffusion coefficients. However, the lateral hypothalamic area is obviously much further from the ventricle, than are the circumventricular organs. Therefore, even if the injected NaCl and sucrose did diffuse in the brain tissues and directly affected neurones in the lateral hypothalamic area, the influence of this might be not so large.
We recently reported the effects of intraperitoneal injection of hypertonic NaCl and 1 day’s water deprivation on reflex salivary secretion (Ito et al., 2001). The saliva volume secreted during eating a pellet was increased, whereas the salivary flow rate was decreased. In the case of these stimuli, both the central and peripheral influence of hyperosmosis should be considered. However, it is likely that the action in the present study was confined to the central nervous system. Even if all of the hypertonic NaCl that was injected into the ventricle leaked into blood, the increased osmolarity would amount to only a few mosmol/kg, a change much smaller than that induced by the intraperitoneal injection of hypertonic NaCl. At present, we have no clear explanation of the difference between our previous and present results. However, it is feasible that systemic hyperosmotic stimulation changes the sensitivity of oral sensation and increases the threshold for swallowing, and induces the increment in saliva volume during the eating of a specific amount of food. An intracerebroventricular injection of hyperosmotic solutions affected the central mechanism of thermoregulation and increased the temperature of the preoptic area in the hypothalamus of rabbits (Turlejska and Baker, 1986). Local warming of these regions in the hypothalamus increased secretion from salivary glands in rats (Kanosue et al., 1990). The intracerebroventricular hyperosmotic stimulation in our study, on the other hand, suppressed the reflex saliva from the rat parotid gland, possibly accompanied by a similar increase of hypothalamic temperatures. If increased hypothalamic temperature induced the secretion from the parotid, the temperature effects on the secretion might mask the suppression of salivary secretion by intracerebroventricular hyperosmotic stimulation. Beside their role in digestion and swallowing, the salivary glands of rodents function as thermoregulatory effector organs, with heat dissipation by evaporative water loss, which could be related to body fluid homeostasis (Stricker and Hainsworth, 1970). However, as we discussed earlier (Ito et al., 2001), there are functional differences between the parotid and submandibular salivary glands: the submandibular plays an import part in thermoregulation, but the parotid does little. Osmotic stimulation of the circumventricular organs in the forebrain increases vasopressin in the plasma (Bourque et al., 1994). Increased plasma vasopressin might have suppressed salivary secretion in our study by acting on the parotid gland ducts (Ferguson, 1994). However, this possibility is contradicted by the findings of a study on the intranasal administration of desmopressin, a vasopressin analogue (Ferguson, 1994), which did not positively support the notion of suppressive effects of vasopressin on salivary secretion. In conclusion, the intracerebroventricular injection of hypertonic solutions suppresses reflex salivary secretion from the rat parotid gland through central osmoreceptive mechanisms. Further experiments should be done to elucidate the neural mechanisms underlying the control system.
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Acknowledgements This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Japan.
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