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Alteration of salt taste sensitivity by the neonatal removal of sublingual glands in the rat
Camp. fliwhem. Phiof. Printed in Great B&in
0300-9629/%9 s3.00 + 0.00 s<: 1989 Pergamon Press plc
Vol. 94A, No. 1~ pp. 89-93, 1989
ALTERATION OF SALT TASTE SENSITIVITY BY THE NEONATAL REMOVAL OF SUBLINGUAL GLANDS IN THE RAT YUZO NINOMIYA, H~DEO KATSUKAWA and MASAYA FUNAKOSHI Department of Oral Physiology, Asahi University School of Dentistry, Hozumi, Motosu, Gifu 501-02, Japan. Telephone: 05832-6-6131 (Receioed 13 Febrtcury 1989) Abstract-l. Adult rats with the surgical removal of sublingual glands at their 10 days of age did not prefer NaCl solution of any concentration to water, whereas those with sham-operation or the removal of submandibular glands preferred 0.03 or 0.1 M NaCl to water. 2. Magnitudes of inhibition of chorda tympani responses to NaCl by the lingual treatment of 0.1 mM amiloride were greater in neonatally sublingual removed rats than in sham-operated or submandibular removed ones. 3. These results suggest that the removal of sublingual glands in neonatal period of the rat could increase the amount of the amiloride-sensitive Na receptor mechanism on the taste cell membrane in its adulthood.
INTRODUefION
MATERIALS AND
Several studies demonstrated that the surgical removal of the major salivary glands of adult rats increases intake of various taste solutions, such as NaCI, KCI, HCI and quinine, because of lowered overall taste sensitivities (Vance, 1965; Catalanotto and Sweeney, 1972, 1973; Nanda and Catalanotto, 1981). Interruption of flow from the salivary glands by drug or duct ligation also produced similar changes in preference-aversion behavior for taste solutions (Glaili et al., 1978, 1981). For the reason of such changes in taste sensitivities, a histological study of the rat dorsal tongue epithelium revealed increased keratosis after the desalivation (Nanda and Catalanotto, 1981). Cano et al. (1978) reported a significant increase in the rate of development of the intermediate type of rat taste bud cells after the removal of the submandibular-sublingual glands. It is known that taste sensitivities and salivary gland activities of rats develop after birth. Hall and Bryan (1981) reported that rat pups showed discrimination between water and sucrose in their mouthing behavior and general activity from 3 days of age, but they did not discriminate between water and quinine until 9 days of age. Hill and Bour (1985) showed that the amiIoride-sensitive Na receptor mechanism of the rat develops from at least 20 days of age. Also, maturation of rat submandibular and parotid glands takes a few weeks after birth, whereas sublingual glands of 1 day of age show similar function to those of adult rats (Redman, 1987). If the saliva could influence taste sensitivities in adult rats, as mentioned above, it is much more probable that the neonatal desalivation influences taste development. Present studies, therefore, examined possible effects of the removal of rat submandibular or sublingual glands of its neonatal period on the preferenceaversion behaviour for the four basic taste substances (sucrose, NaCl HCI and quinine HCl) in its adulthood and peripheral neural basis for the effects.
METHODS
The experimental animals were 28 male Wistar rats. Animals were divided into three groups, such as IO rats with sham-operation (Intact group), nine with the removal of submandibular glands (SMG group) and 9 with the removal of sublingual glands (SLG group). The surgery was done according to a similar method to that of Smith et al. (1979) for each rat at 10 days of age under the pentobarbital anesthesia (40 mg/kg, i.p.). After the operation, animals returned to their home cages and were maintained with their parents on ad libitum food and water until 4 weeks of age. At 4 weeks of age, each animal was individually housed in a cage. Cages were in a light-, temperature- and humiditycontrolled room. Preference-aversion behaviour was measured with a 48-hr two-bottle preference test which started when rats became 8 weeks of age. Rats were presented with a choice between water and each tasting solution for 48 hr; the bottles were switched each 24-hr period to control for any side preference. The total of each animal’s intake over 48 hr for each solution was measured and was used to calculate percent preference according to the following formula: % preference = volume of tasting solution (ml) x lOO/total volume of tasting solution and water (mf). Occurrence of preference or aversion for each tasting soiution was defined as when the mean preference percent for each solution was statistically signi~cantly greater or smaller (r-test, P <0.05) than that for water which was obtained from the control experiment exposing a waterwater pair with two bottles to-animals. Test sol&ions used were 0.003 M-1.0 M NaCI. 0.003-1.0 M sucrose. 0.1 mM10 mM HCl and 0.0001 mM-O.1 mM quinine Ha. After the finish of the preference test for all test solutions, responses of the chorda tympani nerve of each animal to test solutions applied to the tongue were recorded under pentobarbital anesthesia. Procedures for disection of the chorda tympani nerve and recording of the integrated chorda tympani responses are described elsewhere (Ninomiya and Funakoshi, 1989). Test solutions used for the electrophysiological study were 0.01-1.0 M NaCI, 0.5 M sucrose, 0.01 M HCI and 0.02 M quinine HCI. A much higher 89
Yuzo NINOMIYA et al.
90
concentration of quinine HCl was used for the electrophysiological study than that for the behavioural study to obtain measureable neural responses. The magnitude of the integrated chorda tympani response to each test solution was measured at 5 set after the stimulus onset, Since behavioural study demonstrated some differences in salt preference among the three groups, responses to NaCl were measured before and after the lingual treatment of 0.1 mM amiloride for 5 min (Ninomiya and Funakoshi, 1988) which is known as a specific inhibitor for salt taste responses in rats (Heck rf al., 1984). and were compared among the three groups. RESULTS
Behavioural taste responses (two-bottle preference test) Figure 1 shows mean preference percents of intact (open circles), SLG (filled circles) and SMG (open triangles) groups for several concentrations of NaCl, sucrose, HCI and quinine HCI. Preference percents for sucrose, HCI and quinine HCI were not significantly different among the three groups (t-test, P > 0.05). All three groups show high preference percents for sucrose of more than 0.01 M. and low preference percents for HCI of more than 0.1 mM and quinine HCI of more than 0.01 mM. However, there were apparent differences in preference percent for NaCl among three groups. SLG group did not prefer NaCl of any concentration to water, whereas the other two groups prefer 0.03 and 0.1 M NaCl to water. The difference in preference percent for 0.1 M NaCl between SLG (mean & SD: 41.1 _t 29.0%) and other two groups (intact, 75.9 + 12.1%; SMG, 73.6 k 16.5%) was statistically significant (ttest, P < 0.01). All three groups showed aversion for
100
1
1
NaCl of concentrations of 0.3 M or more. These results suggest that neonatal removal of sublingual glands in the rat inhibit salt preference in its adulthood, but the neonatal removal of submandibular glands was not effective. Neural taste responses (amiloride inhibition of NaCl response) Since behavioural study demonstrated the differences in salt preference between SLG and other two groups (intact and SMG groups), we examined chlorda tympani responses mainly to NaCl. Relative magnitudes of responses of the three groups to the four basic taste solutions, when the response to 0.01 M HCI was taken as a unity (1.0) were ranged, HCI : NaCl : sucrose : quinine
HCI
= 1.0: 1.2-1.4:0.&0.6:0.5JI.7. There was no significant difference among those of the three groups (t-test, p > 0.05). Figure 2 shows sample recordings of chorda tympani responses of each rat of the three groups to 0.1 M NaCl before and after the lingual treatment of 0.1 mM amiloride for 5 min. It is noted in this figure that the response to the SLG rat to NaCl before amiloride treatment was characterized by the longlasting, so-called, “off response” (ranging from 10 to 60 set). NaCl responses of all three groups were typically suppressed by the amiloride treatment. However, the magnitude of the amiloride inhibition of the NaCl response was greater in the SLG rat than other two groups of rats. Mean percent responses (NaCl response before amiloride = 100) to NaCl of all five concentrations after amiloride were smaller in
NaCl
1
HCI
0’ -4 Log
-3 Concentration
-2
o’
Q.HCI
-6
B
-4
(M)
Fig. 1. Preference percents of adult rats with sham-operation (Intact, open circles), the removal of sublingual glands (SLG, filled circles) or submandibular glands (SMG, open triangles) in their 10 days of age for NaCI, sucrose, HCl and quinine HCI (QHCl) solutions. **Preference percents for 0.1 M NaCl were significantly different between SLG and other two (Intact and SMG) groups (t-test, P < 0.01). Vertical bars indicate the standard errors (SE).
Sublingual desalivation abolished salt appetite
SLG group than in intact and SMG groups. Statistically significant differences were observed in the responses to 0.1 M NaCl between SLG and the other two groups (mean + SD, n = 5: SLG, 24.2 f 13.3%; Intact, 48.8 + 16.7%; SMG, 48.0+ 16.5%; f-test, P < 0.05). These results suggest that the neonatal removal of sublingual glands of the rat increases the amount of the amiloride-sensitive Na receptor mechanism on the taste cell membrane in its adulthood, and this increment of the amiloride-sensitive Na receptor mechanism possibly relates to the reduction of salt preference of the rat.
before
After
Ami.
after
1 Intact h
b-4
DISCUSSION
I
SLG
The present study demonstrated that adult rats with the removal of subligual glands at their 10 days of age did not show preference for NaCl at any concentration, although those with sham-operation or the removal of submandilar glands show typical preference for 0.03 and 0.1 M NaCl. Previous studies reported that the removal of all three major salivary glands or both submandibular and sublingual glands of adult rats also changes salt preference, although how the salt preference would change may be dependent on time intervals between surgery and preference testing. Catalanotto and Sweeney (1972) found marked enhancement of preference for NaCl solutions immediately after the surgery (1-15 days) but, in their subsequent experiment (Catalanotto and Sweeney, 1973), they found that at 6 months after the surgery rats showed rather lower preferences for NaCl than in the controls. In the present study, preference testing was performed about 7 weeks after the surgery, indicating that the interval is fairly long. Therefore, the disappearance of salt preference observed in neonatally sublingual removed rats in the present study does not contradict to the results of sialectomy of adult rats mentioned above. However, there is no report on the specific effect of the selective removal of sublingual glands of adult rats on salt preference. Catalanotto and Sweeney (1973) reported that rats with the removal of both submandibular and sublingual glands showed lower preferences for not
91
L
4%.
i
-
SMG
J-L
L-L
Fig. 2. Sample recordings of the integrated chorda tympani responses of adult rats with sham-operation (Intact), the removal of sublingual glands (SLG) or submandibular glands (SMG) at their 10 days of age to 0.1 M NaCl before and after the lingual treatment of 0.1 mM amiloride (Ami) for 5 min.
only NaCl but also sucrose and lower aversions for quinine and HCl than in the controls, suggesting the reduction of overall taste sensitivities. It is possible to think that the observed gland-selective effect is due to differences in maturation among the salivary glands in the rat at 10 days of age. Redman (1987) described that rat sublingual glands immediately after birth can function similarly to those of the adulthood, whereas, although submandibular glands have some secretary activity at 1 day of age, the functional maturation of the glands needs at least 2 weeks after birth. Neonatal development of parotid glands is slowest
. . Amllorlb
Intact
0.03 Concrntrrtion
_ 1oaac
0.1
0.3
SMG
l.OM
of NaCl
Fig. 3. Percent responses (response before amiloride = 100) of adult rats with sham-operation (Intact, open columns), the removal of sublingual glands (SLG, filled columns) and submandibular glands (SMG, striped columns) at their 10 days of age to NaCl of five different concentrations after the lingual treatment of 0.1 mM amiloride for 5 min. Number of animals in each group was five (*t-test, P c 0.05).
92
Yuzo NINOMIYA et a!.
among the three major glands. Significant secretion of the enzymes of parotid glands does not begin until the weaning process. Therefore, in the rat at 10 days of age, the removal of sublingual glands could much more severely affect other related organs than that of submandibular glands. However, the fact that the removal of sublingual gland changes salt preference without affecting preference-aversion behaviour for sucrose, HCl or quinine HCI suggests rather specific relationship between sublingual gland and salt taste sensitivities. Amiloride is known to be an inhibitor of passive sodium transport of various epithelial cells (Beno, 1982). Recent human psychophysical (Schiffman et al., 1983) and rat electrophysiological (Heck et al., 1984) studies demonstrated that this drug suppresses taste responses to NaCl. Hill and Bour (1985) found that amiloride did not suppress responses of the chorda tympani nerve to 0.5 M NaCl in the rat at 12-I 3 days of age, but the drug suppressed the NaCl responses by about 50% of the control in the rat at 29-3 1 days or more of age, suggesting rapid development of the amiloride-sensitive Na receptor mechanism between 2 and 4 weeks of age. The present study showed that, in submandibular removed and
sham-operated rats, amiloride suppressed chorda tympani responses to 0.1 M NaCl by about 50% of the control, similar to that shown by Hill and Bour (19X5), whereas in sublingual removed rats the drug inhibit the NaCl response by about 25% of the control, suggesting the increment of the amount of the amiloride-sensitive Na receptor mechanism by the neonatal removal of sublingual glands. Therefore, it is possible that some substance derived from sublingual glands of neonatal rats inhibits the development of the amiloride-sensitive Na receptor mechanism. Several studies (Richter, 1936; Fregly and Rowland, 1985) have suggested that reninangiotensin-aldosteron system plays important role for the induction of a salt appetite in the rat, because the lack of aldosterone by adrenolectomy produces enhancement of NaCl preference. Will et al. (1980) demonstrated, in isolated rat colon, that by raising aldosterone level, an amiloride-sensitjve component of short circuit current was induced, although normal rat colon does not display any amiloride sensitivity, If such induction of the amiloride-sensitive mechanism by aldosterone would be also the case in the rat taste cell membrane, a possible substance derived from neonatal sublingual glands would inhibit the activity of renin-angiotensin-aldosterone system. Concerned with this, it has shown that rat submandibular glands synthesize renin and angiotensin II (Gutman et al., 1973; Ito et al., 1979), although their physiological role and function are not known. The results of the present study in the rat suggest the possibility that the larger amount of the amiloride-sensitive Na receptor mechanism induces the lower salt preference. Correspondingly, our recent study in the mouse (Ninomiya et al., in preparation) demonstrated that the mouse inbred strain showing the higher magnitude of amiloride inhibition of the chorda tympani nerve to NaCl (C3H/He > C57BL/6 > BALB/c) exhibits the lower threshold of salt aversion (C3H/He < C57BL/6 < BALB/c) (they do not clearly prefer NaCl to water). Therefore, taste
information through the amiloride-sensitive Na receptor mechanism may relate to salt aversion in the rodents. There are many compounds synthesized in salivary glands of rats, such as NGF, EGF and so on, which are candidates for biological active substances, but their physiological role and function have not yet been clarified (Barka, 1980). Taking the observed results. together with the fact that the salivary system tightly connect with the taste system in taste-salivary reflex, it is probable that there exists some substance derived from salivary glands which affects development and differentiation of taste cells, as suggested in human (“Gustin” purified from parotid glands; Henkin ef al., 1975). To clarify this, however, further investigations
are needed.
Acknowledgements-This study was supported in part by Grants-in-Aid for Scientific Research (No. 61480394) from the Ministry of Education, Science and Culture of Japan. REFERENCES
Barka T. (1980) Biologically active polypeptides in submandibular glands. J. Histvchem. Cyfoehem. 28,836-859. Beno D. J, (1982) Amiloride: a molecular probe of sodium transport in tissue and ceils. Am. J. Physivl. 242, C131~-145. Cano J., Roza C. and Rodriguez-Echandia E. L. (1978) Effects of selective removal of the salivary glands on taste but cells in the vahate papilla of the rat. Experientia 34, 1290-1291. Catalanotto F. A. and Sweeney E. A. (1972) The effects of surgical desalivation of the rat upon taste acuity. Arch. Oral Biol. 17, 1455-1465. Catalanotto F. A. and Sweeney E. A. (1973) Long-term effects of selective desaiivation on taste acuity in the rat. Arch. Oral Biol. 18, 941-952. Fragly M. J. and Rowland N. E. (1985) Role of reninangiotensin-aldosteron system in NaCl appetite of rats. Am. J. Physivl. 248, RI-1 1. Galiii D., Maller 0. and Bri8htman V. (1978) Effects of drug-desalivation on feeding and taste preferences in the rat. Arch. Oral Bioi. 23, 459464. Galili D., Maller 0. and Brightman V. (1981) The effects of desalivation by duct ligation or salivary gland extirpation on taste preference in rats. Arch Oral Bid 26, 853-858. Gutman Y., Levy M. and Shorr J. (1973) Renin-like activity of the rat submaxillary gland: characterization and the effect of several drugs and stimuli. Br. J. Pharmacol. 47, 59-63. Hall W. G. and Bryan 7. E. ( 1981) The ontogeny of feeding in rats: IV taste development as measured by intake and behavioral responses to ora! infusions of sucrose and quinine. 1. camp. Physiol. Psychol. 95, 24G251. Heck G. L., Mierson S. and DeSimone J. A. (1984) Salt taste transduction occurs through an amiloride-sensitive sodium transport pathway. Science 223, 4031105. Henkin R. I., Lippoldt R. E., Bilstad J. and Edelhoch H. (1975) A zinc protein isolated human parotid saliva. Proc. natn. Acad. Sci. USA 72, 488-492. Hill 13. V. and Bour T. (1985) Addition of functionai amiloride-sensitive components to the receptor membrane: a possible mechanism for altered taste responses during development, Des! Brain Res. 20, 310-313. Ito S., Yamaguchi K., Kusumoto Y., Hama H. and Shibata A. (1979) Angiotensin-like immunoreactivity in the rat submaxillary gland. Arch. Hisrol. Jpn 42, 89-92. Nanda R. and Catalanotto F. A. (1981) Long-term effects of surgical desalivation upon taste acuity, fluid intake, and taste buds in the rat. J. Dent. Res. 60, 69-76.
Sublingual desalivation abolished salt appetite Ninomiya Y. and Funakoshi M. (1988) Amiloride inhibition of responses of rat single chorda tympani fibers to chemical and electrical tongue stimulations. Bruin Res. 451, 319-325. Ninomiya Y. and Funakoshi M. (1989) Selective procaine inhibition of rat chorda tympani responses to electric taste stimulation. Camp. Biocire~. Ph.&l. (in press). Schiffman S. S.. Lockhead E. and Maes F. W. (1983) Amiloride reduces the taste intensity to Na+ and Li+ salts and sweeteners. Proc. natn. Acad. Sci. USA 80, 136-6140.
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Smith S., Mazur A., Voyles N., Bathena S. and Reeant L. (1979) Is submaxillary gland immunoreactive glucagon import in carbohydrate homeostasis? Metabolism 28, 343-347.
Vance W. B. (1965) Observations on the role of the salivary secretions in the regulation of food and fluid intake in the white rat, PsycholMonogr. 79, l-22. Will P. C.. Lebowitz J. L. and Hoofer U. (1980) Induction of amiloride sensitive sodium transport in the rat colon by mineral-corticoids. Am. J. Physiol. 238, F261-268.
0300-9629/%9 s3.00 + 0.00 s<: 1989 Pergamon Press plc
Vol. 94A, No. 1~ pp. 89-93, 1989
ALTERATION OF SALT TASTE SENSITIVITY BY THE NEONATAL REMOVAL OF SUBLINGUAL GLANDS IN THE RAT YUZO NINOMIYA, H~DEO KATSUKAWA and MASAYA FUNAKOSHI Department of Oral Physiology, Asahi University School of Dentistry, Hozumi, Motosu, Gifu 501-02, Japan. Telephone: 05832-6-6131 (Receioed 13 Febrtcury 1989) Abstract-l. Adult rats with the surgical removal of sublingual glands at their 10 days of age did not prefer NaCl solution of any concentration to water, whereas those with sham-operation or the removal of submandibular glands preferred 0.03 or 0.1 M NaCl to water. 2. Magnitudes of inhibition of chorda tympani responses to NaCl by the lingual treatment of 0.1 mM amiloride were greater in neonatally sublingual removed rats than in sham-operated or submandibular removed ones. 3. These results suggest that the removal of sublingual glands in neonatal period of the rat could increase the amount of the amiloride-sensitive Na receptor mechanism on the taste cell membrane in its adulthood.
INTRODUefION
MATERIALS AND
Several studies demonstrated that the surgical removal of the major salivary glands of adult rats increases intake of various taste solutions, such as NaCI, KCI, HCI and quinine, because of lowered overall taste sensitivities (Vance, 1965; Catalanotto and Sweeney, 1972, 1973; Nanda and Catalanotto, 1981). Interruption of flow from the salivary glands by drug or duct ligation also produced similar changes in preference-aversion behavior for taste solutions (Glaili et al., 1978, 1981). For the reason of such changes in taste sensitivities, a histological study of the rat dorsal tongue epithelium revealed increased keratosis after the desalivation (Nanda and Catalanotto, 1981). Cano et al. (1978) reported a significant increase in the rate of development of the intermediate type of rat taste bud cells after the removal of the submandibular-sublingual glands. It is known that taste sensitivities and salivary gland activities of rats develop after birth. Hall and Bryan (1981) reported that rat pups showed discrimination between water and sucrose in their mouthing behavior and general activity from 3 days of age, but they did not discriminate between water and quinine until 9 days of age. Hill and Bour (1985) showed that the amiIoride-sensitive Na receptor mechanism of the rat develops from at least 20 days of age. Also, maturation of rat submandibular and parotid glands takes a few weeks after birth, whereas sublingual glands of 1 day of age show similar function to those of adult rats (Redman, 1987). If the saliva could influence taste sensitivities in adult rats, as mentioned above, it is much more probable that the neonatal desalivation influences taste development. Present studies, therefore, examined possible effects of the removal of rat submandibular or sublingual glands of its neonatal period on the preferenceaversion behaviour for the four basic taste substances (sucrose, NaCl HCI and quinine HCl) in its adulthood and peripheral neural basis for the effects.
METHODS
The experimental animals were 28 male Wistar rats. Animals were divided into three groups, such as IO rats with sham-operation (Intact group), nine with the removal of submandibular glands (SMG group) and 9 with the removal of sublingual glands (SLG group). The surgery was done according to a similar method to that of Smith et al. (1979) for each rat at 10 days of age under the pentobarbital anesthesia (40 mg/kg, i.p.). After the operation, animals returned to their home cages and were maintained with their parents on ad libitum food and water until 4 weeks of age. At 4 weeks of age, each animal was individually housed in a cage. Cages were in a light-, temperature- and humiditycontrolled room. Preference-aversion behaviour was measured with a 48-hr two-bottle preference test which started when rats became 8 weeks of age. Rats were presented with a choice between water and each tasting solution for 48 hr; the bottles were switched each 24-hr period to control for any side preference. The total of each animal’s intake over 48 hr for each solution was measured and was used to calculate percent preference according to the following formula: % preference = volume of tasting solution (ml) x lOO/total volume of tasting solution and water (mf). Occurrence of preference or aversion for each tasting soiution was defined as when the mean preference percent for each solution was statistically signi~cantly greater or smaller (r-test, P <0.05) than that for water which was obtained from the control experiment exposing a waterwater pair with two bottles to-animals. Test sol&ions used were 0.003 M-1.0 M NaCI. 0.003-1.0 M sucrose. 0.1 mM10 mM HCl and 0.0001 mM-O.1 mM quinine Ha. After the finish of the preference test for all test solutions, responses of the chorda tympani nerve of each animal to test solutions applied to the tongue were recorded under pentobarbital anesthesia. Procedures for disection of the chorda tympani nerve and recording of the integrated chorda tympani responses are described elsewhere (Ninomiya and Funakoshi, 1989). Test solutions used for the electrophysiological study were 0.01-1.0 M NaCI, 0.5 M sucrose, 0.01 M HCI and 0.02 M quinine HCI. A much higher 89
Yuzo NINOMIYA et al.
90
concentration of quinine HCl was used for the electrophysiological study than that for the behavioural study to obtain measureable neural responses. The magnitude of the integrated chorda tympani response to each test solution was measured at 5 set after the stimulus onset, Since behavioural study demonstrated some differences in salt preference among the three groups, responses to NaCl were measured before and after the lingual treatment of 0.1 mM amiloride for 5 min (Ninomiya and Funakoshi, 1988) which is known as a specific inhibitor for salt taste responses in rats (Heck rf al., 1984). and were compared among the three groups. RESULTS
Behavioural taste responses (two-bottle preference test) Figure 1 shows mean preference percents of intact (open circles), SLG (filled circles) and SMG (open triangles) groups for several concentrations of NaCl, sucrose, HCI and quinine HCI. Preference percents for sucrose, HCI and quinine HCI were not significantly different among the three groups (t-test, P > 0.05). All three groups show high preference percents for sucrose of more than 0.01 M. and low preference percents for HCI of more than 0.1 mM and quinine HCI of more than 0.01 mM. However, there were apparent differences in preference percent for NaCl among three groups. SLG group did not prefer NaCl of any concentration to water, whereas the other two groups prefer 0.03 and 0.1 M NaCl to water. The difference in preference percent for 0.1 M NaCl between SLG (mean & SD: 41.1 _t 29.0%) and other two groups (intact, 75.9 + 12.1%; SMG, 73.6 k 16.5%) was statistically significant (ttest, P < 0.01). All three groups showed aversion for
100
1
1
NaCl of concentrations of 0.3 M or more. These results suggest that neonatal removal of sublingual glands in the rat inhibit salt preference in its adulthood, but the neonatal removal of submandibular glands was not effective. Neural taste responses (amiloride inhibition of NaCl response) Since behavioural study demonstrated the differences in salt preference between SLG and other two groups (intact and SMG groups), we examined chlorda tympani responses mainly to NaCl. Relative magnitudes of responses of the three groups to the four basic taste solutions, when the response to 0.01 M HCI was taken as a unity (1.0) were ranged, HCI : NaCl : sucrose : quinine
HCI
= 1.0: 1.2-1.4:0.&0.6:0.5JI.7. There was no significant difference among those of the three groups (t-test, p > 0.05). Figure 2 shows sample recordings of chorda tympani responses of each rat of the three groups to 0.1 M NaCl before and after the lingual treatment of 0.1 mM amiloride for 5 min. It is noted in this figure that the response to the SLG rat to NaCl before amiloride treatment was characterized by the longlasting, so-called, “off response” (ranging from 10 to 60 set). NaCl responses of all three groups were typically suppressed by the amiloride treatment. However, the magnitude of the amiloride inhibition of the NaCl response was greater in the SLG rat than other two groups of rats. Mean percent responses (NaCl response before amiloride = 100) to NaCl of all five concentrations after amiloride were smaller in
NaCl
1
HCI
0’ -4 Log
-3 Concentration
-2
o’
Q.HCI
-6
B
-4
(M)
Fig. 1. Preference percents of adult rats with sham-operation (Intact, open circles), the removal of sublingual glands (SLG, filled circles) or submandibular glands (SMG, open triangles) in their 10 days of age for NaCI, sucrose, HCl and quinine HCI (QHCl) solutions. **Preference percents for 0.1 M NaCl were significantly different between SLG and other two (Intact and SMG) groups (t-test, P < 0.01). Vertical bars indicate the standard errors (SE).
Sublingual desalivation abolished salt appetite
SLG group than in intact and SMG groups. Statistically significant differences were observed in the responses to 0.1 M NaCl between SLG and the other two groups (mean + SD, n = 5: SLG, 24.2 f 13.3%; Intact, 48.8 + 16.7%; SMG, 48.0+ 16.5%; f-test, P < 0.05). These results suggest that the neonatal removal of sublingual glands of the rat increases the amount of the amiloride-sensitive Na receptor mechanism on the taste cell membrane in its adulthood, and this increment of the amiloride-sensitive Na receptor mechanism possibly relates to the reduction of salt preference of the rat.
before
After
Ami.
after
1 Intact h
b-4
DISCUSSION
I
SLG
The present study demonstrated that adult rats with the removal of subligual glands at their 10 days of age did not show preference for NaCl at any concentration, although those with sham-operation or the removal of submandilar glands show typical preference for 0.03 and 0.1 M NaCl. Previous studies reported that the removal of all three major salivary glands or both submandibular and sublingual glands of adult rats also changes salt preference, although how the salt preference would change may be dependent on time intervals between surgery and preference testing. Catalanotto and Sweeney (1972) found marked enhancement of preference for NaCl solutions immediately after the surgery (1-15 days) but, in their subsequent experiment (Catalanotto and Sweeney, 1973), they found that at 6 months after the surgery rats showed rather lower preferences for NaCl than in the controls. In the present study, preference testing was performed about 7 weeks after the surgery, indicating that the interval is fairly long. Therefore, the disappearance of salt preference observed in neonatally sublingual removed rats in the present study does not contradict to the results of sialectomy of adult rats mentioned above. However, there is no report on the specific effect of the selective removal of sublingual glands of adult rats on salt preference. Catalanotto and Sweeney (1973) reported that rats with the removal of both submandibular and sublingual glands showed lower preferences for not
91
L
4%.
i
-
SMG
J-L
L-L
Fig. 2. Sample recordings of the integrated chorda tympani responses of adult rats with sham-operation (Intact), the removal of sublingual glands (SLG) or submandibular glands (SMG) at their 10 days of age to 0.1 M NaCl before and after the lingual treatment of 0.1 mM amiloride (Ami) for 5 min.
only NaCl but also sucrose and lower aversions for quinine and HCl than in the controls, suggesting the reduction of overall taste sensitivities. It is possible to think that the observed gland-selective effect is due to differences in maturation among the salivary glands in the rat at 10 days of age. Redman (1987) described that rat sublingual glands immediately after birth can function similarly to those of the adulthood, whereas, although submandibular glands have some secretary activity at 1 day of age, the functional maturation of the glands needs at least 2 weeks after birth. Neonatal development of parotid glands is slowest
. . Amllorlb
Intact
0.03 Concrntrrtion
_ 1oaac
0.1
0.3
SMG
l.OM
of NaCl
Fig. 3. Percent responses (response before amiloride = 100) of adult rats with sham-operation (Intact, open columns), the removal of sublingual glands (SLG, filled columns) and submandibular glands (SMG, striped columns) at their 10 days of age to NaCl of five different concentrations after the lingual treatment of 0.1 mM amiloride for 5 min. Number of animals in each group was five (*t-test, P c 0.05).
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Yuzo NINOMIYA et a!.
among the three major glands. Significant secretion of the enzymes of parotid glands does not begin until the weaning process. Therefore, in the rat at 10 days of age, the removal of sublingual glands could much more severely affect other related organs than that of submandibular glands. However, the fact that the removal of sublingual gland changes salt preference without affecting preference-aversion behaviour for sucrose, HCl or quinine HCI suggests rather specific relationship between sublingual gland and salt taste sensitivities. Amiloride is known to be an inhibitor of passive sodium transport of various epithelial cells (Beno, 1982). Recent human psychophysical (Schiffman et al., 1983) and rat electrophysiological (Heck et al., 1984) studies demonstrated that this drug suppresses taste responses to NaCl. Hill and Bour (1985) found that amiloride did not suppress responses of the chorda tympani nerve to 0.5 M NaCl in the rat at 12-I 3 days of age, but the drug suppressed the NaCl responses by about 50% of the control in the rat at 29-3 1 days or more of age, suggesting rapid development of the amiloride-sensitive Na receptor mechanism between 2 and 4 weeks of age. The present study showed that, in submandibular removed and
sham-operated rats, amiloride suppressed chorda tympani responses to 0.1 M NaCl by about 50% of the control, similar to that shown by Hill and Bour (19X5), whereas in sublingual removed rats the drug inhibit the NaCl response by about 25% of the control, suggesting the increment of the amount of the amiloride-sensitive Na receptor mechanism by the neonatal removal of sublingual glands. Therefore, it is possible that some substance derived from sublingual glands of neonatal rats inhibits the development of the amiloride-sensitive Na receptor mechanism. Several studies (Richter, 1936; Fregly and Rowland, 1985) have suggested that reninangiotensin-aldosteron system plays important role for the induction of a salt appetite in the rat, because the lack of aldosterone by adrenolectomy produces enhancement of NaCl preference. Will et al. (1980) demonstrated, in isolated rat colon, that by raising aldosterone level, an amiloride-sensitjve component of short circuit current was induced, although normal rat colon does not display any amiloride sensitivity, If such induction of the amiloride-sensitive mechanism by aldosterone would be also the case in the rat taste cell membrane, a possible substance derived from neonatal sublingual glands would inhibit the activity of renin-angiotensin-aldosterone system. Concerned with this, it has shown that rat submandibular glands synthesize renin and angiotensin II (Gutman et al., 1973; Ito et al., 1979), although their physiological role and function are not known. The results of the present study in the rat suggest the possibility that the larger amount of the amiloride-sensitive Na receptor mechanism induces the lower salt preference. Correspondingly, our recent study in the mouse (Ninomiya et al., in preparation) demonstrated that the mouse inbred strain showing the higher magnitude of amiloride inhibition of the chorda tympani nerve to NaCl (C3H/He > C57BL/6 > BALB/c) exhibits the lower threshold of salt aversion (C3H/He < C57BL/6 < BALB/c) (they do not clearly prefer NaCl to water). Therefore, taste
information through the amiloride-sensitive Na receptor mechanism may relate to salt aversion in the rodents. There are many compounds synthesized in salivary glands of rats, such as NGF, EGF and so on, which are candidates for biological active substances, but their physiological role and function have not yet been clarified (Barka, 1980). Taking the observed results. together with the fact that the salivary system tightly connect with the taste system in taste-salivary reflex, it is probable that there exists some substance derived from salivary glands which affects development and differentiation of taste cells, as suggested in human (“Gustin” purified from parotid glands; Henkin ef al., 1975). To clarify this, however, further investigations
are needed.
Acknowledgements-This study was supported in part by Grants-in-Aid for Scientific Research (No. 61480394) from the Ministry of Education, Science and Culture of Japan. REFERENCES
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