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Expression of muscarinic and nicotinic receptor mRNA in the salivary gland of rats: a study by in situ hybridization histochemistry
Molecular Brain Research, 17 (1993) 335-339 © 1993 Elsevier Science Publishers B.V. All rights reserved 0169-328x/93/$06.00
335
BRESM 80157
Short Communications
Expression of muscarinic and nicotinic receptor mRNA in the salivary gland of rats: a study by in situ hybridization histochemistry T o r u S h i d a a, A t s u s h i T o k u n a g a a, Eiji K o n d o a, Y u t a k a U e d a a, Koji O h n o T a k a n o r i Saika b, H i r o s h i K i y a m a c a n d M a s a y a T o h y a m a c
b,
a Department of Anesthesiology, Osaka Dental University, Osaka (Japan) b Department of Otolaryngology, and c Department of Anatomy II and Neuroscience, Osaka University Medical School, Osaka (Japan) (Accepted 10 November 1992)
Key words: Acetylcholine; Muscarinic receptor; Nicotinic receptor; mRNA; Salivary gland; In situ hybridization; Rat
Expression of muscarinic receptor mRNA subtypes (ml-5) and nicotinic receptor subunits (a2-4, and /32) was examined in the rat submandibular gland by in situ hybridization histochemistry, using oligonucleotide probes for the muscarinic receptor and RNA probes for the nicotinic receptors, m2, a3, and/32 mRNA were strongly expressed in the submandibular ganglion, and m3, a2, a3, a4,and/32 were expressed in the striated and interlobular duct cells. Both muscarinic and nicotinic receptors were coexpressed in the same ganglion neurons, while none of these mRNA were detected in the terminal secretory units.
The salivary glands are composed of parenchymal elements which are derived from the oral epithelium and consist of terminal secretory units leading into ducts. The terminal secretory units are composed of serous, mucous, and myoepithelial cells arranged into acini or secretory tubules. Within a lobule, the smallest ducts are the intercalated ducts which connect the terminal secretory units to the striated ducts. The secretions of terminal secretory units are collected by the intercalated ducts. Cholinergic innervation originating from the parasympathetic ganglion in the salivary gland is well established. Some acinar cells exhibit changes in their membrane potential following parasympathetic stimulation; this causes the marked secretion of watery saliva. Some duct cells also respond to the parasympathetic stimulation ~,4,~4. These findings suggest that acinar and duct cells in the salivary glands are target cells for acetylcholine. Cholinergic receptors are divided into two major classes, muscarinic and nicotinic. Muscarinic receptors, which have been classified into three subtypes, M1-3 ~t, on the basis of the~,r aiiinity for different antagonists,
play a major role in cholinergic transmission in the rat submandibular gland 1'14. The presence of MI and M3 receptors in the salivary gland has been revealed by binding analysis n'm~6, while recent advances in molecular biology have shown more complicated heterogeneity of muscarinic receptors. To date, five subtypes of muscarinic receptors m l - 5 have been cloned, and the content of ml-5 mRNA in various tissues has been measured ~5'~7. In the submaxillary gland, the ml and m3 subtypes are the most abundant ~°'n'm6. However, as salivary glands consist of heterogenous cellular subpopulations, i.e., parasympathetic ganglion cells, acinar cells, and duct cells, it has not yet been possible to identify the precise localization of muscarinic receptor subtypes at the cellular level. Nicotinic receptors, however, which mediate cholinergic transmission, were recently shown to consist of two subunits (a and /3) in the nervous system. Some subtypes of the a and /~ subunits~ a2-7 and/32-57"9, have also been identified. In this study, using i~ :?', hybridization histochemistry, we examined both muscarinlc and nicotinic receptor subsets in the rat submandibular gland.
Correspondence: T. Shida, Department of Anesthesiology, Osaka Dental University, 1-5-31 Ohtemae Chuo-ku, Osaka 540, Japan.
336 Nine male Wistar rats weighing 100-150 g were used. The animals were anesthetized with pentobarbital (50 mg/kg, i.p.), and the glands were quickly extirpated from each animal. The submandibular gland was then frozen quickly with powdered dry ice, and cut on a cryostat into 15-/zm-thick sections. We used synthesized antisense oligonucleotide probes for visualizing the mRNA of the m l - 5 subtypes of muscarinic receptors, and RNA probes for the a 2 - 4 and f12 subunits of the nicotinic receptors. The oligonucleotide probes (each 48 met) were synthesized using an Applied Biosystems DNA synthesizer and were then purified by high pressure liquid chromatography (ODS column chromatography). The probes consisting of antisense nucleotides were derived from rat DNA sequences complementary to m l - 5 muscarinic receptors; complementary to bases 4-51 for the ml probe, to bases 4-51 for the m2 probe, to bases 4-51 for the m3 probe, to bases 4-48 for the m4 probe, and to bases 4-51 for the m5 probe. The RNA probes of the a 2 - 4 and f12 subunits of the nicotinic receptors were kind gifts from Dr. K. Wada (NIH, USA). The isolat.~on and characterization of these probes have been described nT. The hybridization procedure for the muscarinic receptor probes used has been described previously 2. Briefly, sections were fixed in 4% paraformaldehyde in 0.1 M PBS, and then rinsed with 0.1 M PBS, rinsed in 4 x sodium chloride-sodium citrate buffer (SSC), immersed in 4 x SSC containing 1 x Denhardt's solution, dehydrated through a gradient of ethanol solu-
tion, delipidated in chloroform, and then washed in 100% ethanol. Oligonucleotide probes were labeled with a-[35S]dATP using the terminal deoxynucleotidyl transferase, giving a specific activity of 1.5-2.0 × 10 9 dpm//~g. Hybridization was performed by incubating the sections with a buffer (50% deionized formamide, 4 × SSC, 0.12 M PB, 20% sarcosyl, 100 × Denhardt's solution, 2.5% tRNA, 10% dextran sulfate) containing labeled probes for 24 h at 41°c. Hybridization specificity was comfirmed by the competition test and ribonuclease pretreatment. As control experiments, an excess of non-labelled m l - 5 muscarinic receptor probes was used to compete with the the labelled muscarinic receptors probes. Both the labelled and non-labelled probes were added to the hybridization buffer at the same time. Sections hybridized with an excess of nonlabelled probe did not show any specific hybridization signals. In addition, ribonuclease A (RNase, 20/zg/ml) pretreatment (30 min at room temperature) was carried out just before hybridization, and the sections were fixed with 4% paraformaldehyde. RNase pre~ treatment also abolished any hybridization signals, indicating that these muscarinic receptor probes did not bind non-specifically to the tissue section. To identify the neurons expressing the mRNA of the m l - 5 muscarinic receptors, and those expressing the mRNA of the a 2 - 4 and /32 subunits of the nicotinic receptors, the grain densities of the cells and the background densities of the sections were determined for our probes, as reported elsewhere t3. The signal to noise
m
.~41, Q
°O
m. B
b
0 s,.
p,
,! Q
p
~o
o
m
Q
,
Fig. 1. Dark- and brigm-nempnotomtcrograpnsshowingthe expression of mRNAof the m2- (a, b), and m3- (c, d) muscarinicreceptor subtypes. The m2-muscafinicreceptor subtype is expressedstronglyin the submandibularganglion(a, b; arrows)and the m3-muscarinicreceptor subtype is expressed in the interlobularducts (c, d; arrowheads). Bar -- 100 ~tm.
337
ratio (N/S) for the probes was calculated. We considered cells that had a grain density at least three times higher than the background to be positive. Fig. la and b show the localization of the m2 muscarinic receptor mRNA subtype in the submandibular ganglion. Most of the ganglion cells were labeled strongly by the m2 probe, but the neurons and satellite cells were below the detection level or background level with other probes for muscarinic receptor sub-
types. Fig. lc and d show the expression of the m3 mRNA subtype in the parenchymal cells. The labeling of interiobular duct cells by this probe was very strong. However, other probes of the muscarinic receptor subtype did not label the interlobular duct cells. Labeling in the intercalated region by all the muscarinic receptor subtypes was below the detection level. In addition, cells in the terminal secretory unit were negligibly labeled by all receptor subtypes. The grain density of
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Fig. 2. Dark- and bright-field photomicrographs showing the expression of mRNA of the a2- (a, b), a3- (c, d), a4- (e, f), and fi2- (g, h) nicotinic receptor subunits. The a2-nicotinic receptor subunit is expressed in the striated and interiobular duct (a, b; arrowheads), the a3-nicotinic receptor subunit is expressed in the submandibular ganglion (c, d; arrows), the a4-nicotinic receptor subunit is expressed in the striated ~nd interlobular duct (e, f; arrowheads), and the fi2-nicotinic receptor subunit is expressed in the submandibular ganglion (g, h; arrow) and in !We striated and interlobular duct (arrowheads). Bar = 100 izm.
33g TABLE I
Detection of mRNA of mte;carinic receptor subtypes and nicotinic receptor subunits in the rat submandibular gland + +: moderately labeled ( S / N ratio > 5); +: weakly labeled ( S / N ratio 3-5); - : unlabeled ( S / N ratio < 3).
ml Ganglion Intercalated duct
.
Striated
.
duct
lnterlobular duct Acinar cells
.
m2
m3
+ +
.
.
.
.
.
. .
.
.
m4
.
. .
m5 . .
a2 .
.
+ + .
. +
.
the cells in the terminal secretory unit was always less than three-fold the level of the background density. As shown in Figures 2c and g, most of the ganglion cells were strongly labeled by the probes for a3 (Fig. 2c) and /32 (Fig. 2g). However, grain density in ganglion neurons for the a2 and a4 subunits was below the detection level. The a2, a4, and/32 subunits of the nicotinic receptors were detected in the interlobular and striated ducts (Fig. 2a,e,g). Intercalated ducts, satellite cells, and terminal secretory units were not labeled by any of the probes for any of the nicotinic receptor subunits. Table 1 summarizes these findings. Parasympathetic ganglion neurons have been pharmacologically shown to express nicotinic receptors s. The present study confirmed this finding and further clarified that nicotinic receptors expressed in the parasympathetic submandibular ganglion are composed of a3 and/32 subunits. Pharmacological studies have suggested that submandibular ganglion neurons do not express the m2muscarinic receptor subtype n,15. However, the present study demonstrated the localization of the m2 mRNA subtype. These findings suggest that the mRNA exists but that the protein is not translated or that the translated protein somehow does not act as a receptor of the m2 subtype. Furthermore, the differences between our findings and those of the studies above suggests that some inhibitory mechanism for the binding of antagonists or agonists specific to m2 subtypes exists in the submandibular ganglion, or that the antagonists or agonists that have been employed so far do not bind to the m2 subtype in the submandibular ganglion neurons in particular. Alternatively, the failure to detect the m2 receptor in pharmacological binding assay may have been due to the small amount of m2 receptor protein. The m2-specific ligand binding in the ganglion neurons may be reduced to a negligible level with the excess amount of protein from acinar cells, which cells are independent of the m2 receptor. The present study revealed that most of the ganglion cells were labeled by the m2 probe, and by the a3 and the/32 subunit probes, suggesting that both mus-
.
+ .
a3
a4
~2
+ + .
--
+ +
-
+
+
+
+
.
.
carinic and nicotinic receptors are expressed in the same neurons. Whether or not two types of receptors are expressed on the same postsynaptic membrane is important, particularly in the analysis of the neural circuit. If two of these receptors are coexpressed on the same postsy* naptic membrane, differences in the affinity of acetylcholine to the receptors or differences in the acetylcholine content in the synaptic cleft may drive two different receptor systems separately by a common ligand, resulting in different functions. If two types of receptors are expressed on different postsynaptic membranes, the neural circuit of the preganglionic neurons should be analyzed. Binding studies have shown that the salivary gland is rich in muscarinic receptors of the m3 subtype 12. The present study supported this finding, and further showed that duct cells express the m3 subtype very strongly. Since duct cells are related to the secretion and reabsorption of electrolytes and secretion of proteins, acetylcholine released from the parasympathetic postganglionic nerves could regulate these functions via m3 subtype muscarinic receptors. On the other hand, since parasympathetic stimulation causes a marked secretion of saliva and since the terminal secretory unit was negative for m l - 5 probes, this suggests that acetylcholine does not influence the production of saliva directly by binding to these receptor subunits, but by binding to other, as yet unknown, subunits of the muscarinic receptors. We thank Dr. Keiji Wada (NIH, USA) for kindly providing plasmid DNA-encoded nicotinic acetylcholine receptor ¢2, a3, a4, a n d / 3 2 subunits. This study was supported, in part, by Grants-in-Aid from the Ministry of Education, Science and Culture of Japan and the Science and Technology Agency of Japan. I Bhaskar, S.N., Oral Histology and Embriology, Mosby Year Book, New York, 1989, pp. 337-369. 2 Bloch, E., Papovici, T., Le Guellee, D., Normand, E., Chouham, S., Guitteny, A.F. and Bohlen, P., In situ hybridization histochemistry for analysis of gene expression in the endocrine and nervous system tissue: a three years experience, Y. Neurosci. Res., 16 (1986) 183-200. 3 Bundgaard, M., Moiler, M. and Poulsen, J.H., Localization of
339 sodium pump sites in cat salivary glands, ./. Physiol., 273 (1977) 339-348. 4 Bonner, T.I., Buckley, M.J., Young, A.C. and Brann, M.R., Identification of a family of muscarinic acetyicholine receptor genes, Science, 237 (1987) 527-532. 5 Bonner, T.I., Young, A.C., Brann, M.R. and Buckley, N.J., Cloning and expression of the human and rat m5 muscarinic acetylcholine receptor genes, Neuron, 1 (1988) 403-410. 6 Bonner, T.H., The molecular basis of muscarinic receptor diversity, Trends Neurosci., 12 (1¢89) 148-151. 7 Boulter, J., Goldman, D., Martin, D., Treco, D., Heinemann, S. and Patrick, J., Isolation of a cDNA clone coding for possible neuronal nicotinic ~cetylcholine receptor a-subunit, Nature, 319 (1986) 368-374. 8 Boyd, T. R., Jacob, M.H., Couturier, S., Ballivet, M. and Berg, D.K., Expression and regulation of neuronal acetylcholine receptor mRNA in chick ciliary ganglia, Neuron, 1 (1988) 495-502. 9 Deneris, E.S., Connoly, J., Boulter, J., Wada, E., Wada, K., Swanson, W., Patrick, J. and Swanson, L.W., Primary structure and expression of /32: a novel subunit of neuronal nicotinic acetylcholine receptors, Neuron, 1 (1989)45-54. 10 Hammer, R., Giraldo, E., Schiavi, G.B., Monferini, E. and Ladinsky, H., Binding profile of a novel cardioselective muscarinic receptor antagonist, AF-DX 116, to membranes of peripheral tissues and brain in the rat, Life Sci., 38 (1986) 1653-1662.
11 Hummer, R., Berrie, C.P., Birdsall, N.J.M., Burgen, A.S.V. and Hulme, E.C., Pirenzepin distingushies between different subclasses of muscarinic receptors, Nature, 283 (1980) 90-92. 12 Martos, F., Monferini, E., Giraldo, E., Paoli, A.M. and Hammer, R., Characterization of muscarinic recel~iors in salivary and lacrimal glands of the rat, Eur. J. Pharmacol., 143 (1987) 189-194. 13 Noguchi, K., Morita, Y., Kiyama, H., Ono, IL and Tohyama, M., A noxious stimulus induces the preprotachykinin-A gene expression in the rat dosal root ganglion: a quantitative study using in situ hybridization histochemistry, Mol. Brain Res., 4 (1988) 31-35. 14 Ten Cate, A.R., Oral histology: Salivary Gland, Mosby, St. Louis, 1989, pp. 312-340. 15 Vanderheyden, P., Gies, J.P., Ebinger, G., Keyser, J.D., Landry, Y. and Vauquelin, G., Human M1-, M2-, and M3-muscarinic cholinergic receptors: Binding characteristics of agonists and antagonists, J. Neurol. Sci., 97 (1990) 67-80. 16 Vilar6, M.T., Palacios, J.M. and Mengod, G., Localization of m5 muscarinic mRNA in rat brain examined by in situ hybridization histochemistry, Neurosci. Left., 114 (1990) 154-159. 17 Wada, E., Wada, K., Boulter, J., Deneris, E., Heinemann, S., Patrick, J. and Swanson, L.W., Distribution of alpha2, alpha3, alpha4, and beta2 neuronal nicotinic receptor subunit mRNAs in the central nervous system: A hybridization histochemicai study in the rat, .l. Comp. Neurol., 284 (1989) 314-335.
335
BRESM 80157
Short Communications
Expression of muscarinic and nicotinic receptor mRNA in the salivary gland of rats: a study by in situ hybridization histochemistry T o r u S h i d a a, A t s u s h i T o k u n a g a a, Eiji K o n d o a, Y u t a k a U e d a a, Koji O h n o T a k a n o r i Saika b, H i r o s h i K i y a m a c a n d M a s a y a T o h y a m a c
b,
a Department of Anesthesiology, Osaka Dental University, Osaka (Japan) b Department of Otolaryngology, and c Department of Anatomy II and Neuroscience, Osaka University Medical School, Osaka (Japan) (Accepted 10 November 1992)
Key words: Acetylcholine; Muscarinic receptor; Nicotinic receptor; mRNA; Salivary gland; In situ hybridization; Rat
Expression of muscarinic receptor mRNA subtypes (ml-5) and nicotinic receptor subunits (a2-4, and /32) was examined in the rat submandibular gland by in situ hybridization histochemistry, using oligonucleotide probes for the muscarinic receptor and RNA probes for the nicotinic receptors, m2, a3, and/32 mRNA were strongly expressed in the submandibular ganglion, and m3, a2, a3, a4,and/32 were expressed in the striated and interlobular duct cells. Both muscarinic and nicotinic receptors were coexpressed in the same ganglion neurons, while none of these mRNA were detected in the terminal secretory units.
The salivary glands are composed of parenchymal elements which are derived from the oral epithelium and consist of terminal secretory units leading into ducts. The terminal secretory units are composed of serous, mucous, and myoepithelial cells arranged into acini or secretory tubules. Within a lobule, the smallest ducts are the intercalated ducts which connect the terminal secretory units to the striated ducts. The secretions of terminal secretory units are collected by the intercalated ducts. Cholinergic innervation originating from the parasympathetic ganglion in the salivary gland is well established. Some acinar cells exhibit changes in their membrane potential following parasympathetic stimulation; this causes the marked secretion of watery saliva. Some duct cells also respond to the parasympathetic stimulation ~,4,~4. These findings suggest that acinar and duct cells in the salivary glands are target cells for acetylcholine. Cholinergic receptors are divided into two major classes, muscarinic and nicotinic. Muscarinic receptors, which have been classified into three subtypes, M1-3 ~t, on the basis of the~,r aiiinity for different antagonists,
play a major role in cholinergic transmission in the rat submandibular gland 1'14. The presence of MI and M3 receptors in the salivary gland has been revealed by binding analysis n'm~6, while recent advances in molecular biology have shown more complicated heterogeneity of muscarinic receptors. To date, five subtypes of muscarinic receptors m l - 5 have been cloned, and the content of ml-5 mRNA in various tissues has been measured ~5'~7. In the submaxillary gland, the ml and m3 subtypes are the most abundant ~°'n'm6. However, as salivary glands consist of heterogenous cellular subpopulations, i.e., parasympathetic ganglion cells, acinar cells, and duct cells, it has not yet been possible to identify the precise localization of muscarinic receptor subtypes at the cellular level. Nicotinic receptors, however, which mediate cholinergic transmission, were recently shown to consist of two subunits (a and /3) in the nervous system. Some subtypes of the a and /~ subunits~ a2-7 and/32-57"9, have also been identified. In this study, using i~ :?', hybridization histochemistry, we examined both muscarinlc and nicotinic receptor subsets in the rat submandibular gland.
Correspondence: T. Shida, Department of Anesthesiology, Osaka Dental University, 1-5-31 Ohtemae Chuo-ku, Osaka 540, Japan.
336 Nine male Wistar rats weighing 100-150 g were used. The animals were anesthetized with pentobarbital (50 mg/kg, i.p.), and the glands were quickly extirpated from each animal. The submandibular gland was then frozen quickly with powdered dry ice, and cut on a cryostat into 15-/zm-thick sections. We used synthesized antisense oligonucleotide probes for visualizing the mRNA of the m l - 5 subtypes of muscarinic receptors, and RNA probes for the a 2 - 4 and f12 subunits of the nicotinic receptors. The oligonucleotide probes (each 48 met) were synthesized using an Applied Biosystems DNA synthesizer and were then purified by high pressure liquid chromatography (ODS column chromatography). The probes consisting of antisense nucleotides were derived from rat DNA sequences complementary to m l - 5 muscarinic receptors; complementary to bases 4-51 for the ml probe, to bases 4-51 for the m2 probe, to bases 4-51 for the m3 probe, to bases 4-48 for the m4 probe, and to bases 4-51 for the m5 probe. The RNA probes of the a 2 - 4 and f12 subunits of the nicotinic receptors were kind gifts from Dr. K. Wada (NIH, USA). The isolat.~on and characterization of these probes have been described nT. The hybridization procedure for the muscarinic receptor probes used has been described previously 2. Briefly, sections were fixed in 4% paraformaldehyde in 0.1 M PBS, and then rinsed with 0.1 M PBS, rinsed in 4 x sodium chloride-sodium citrate buffer (SSC), immersed in 4 x SSC containing 1 x Denhardt's solution, dehydrated through a gradient of ethanol solu-
tion, delipidated in chloroform, and then washed in 100% ethanol. Oligonucleotide probes were labeled with a-[35S]dATP using the terminal deoxynucleotidyl transferase, giving a specific activity of 1.5-2.0 × 10 9 dpm//~g. Hybridization was performed by incubating the sections with a buffer (50% deionized formamide, 4 × SSC, 0.12 M PB, 20% sarcosyl, 100 × Denhardt's solution, 2.5% tRNA, 10% dextran sulfate) containing labeled probes for 24 h at 41°c. Hybridization specificity was comfirmed by the competition test and ribonuclease pretreatment. As control experiments, an excess of non-labelled m l - 5 muscarinic receptor probes was used to compete with the the labelled muscarinic receptors probes. Both the labelled and non-labelled probes were added to the hybridization buffer at the same time. Sections hybridized with an excess of nonlabelled probe did not show any specific hybridization signals. In addition, ribonuclease A (RNase, 20/zg/ml) pretreatment (30 min at room temperature) was carried out just before hybridization, and the sections were fixed with 4% paraformaldehyde. RNase pre~ treatment also abolished any hybridization signals, indicating that these muscarinic receptor probes did not bind non-specifically to the tissue section. To identify the neurons expressing the mRNA of the m l - 5 muscarinic receptors, and those expressing the mRNA of the a 2 - 4 and /32 subunits of the nicotinic receptors, the grain densities of the cells and the background densities of the sections were determined for our probes, as reported elsewhere t3. The signal to noise
m
.~41, Q
°O
m. B
b
0 s,.
p,
,! Q
p
~o
o
m
Q
,
Fig. 1. Dark- and brigm-nempnotomtcrograpnsshowingthe expression of mRNAof the m2- (a, b), and m3- (c, d) muscarinicreceptor subtypes. The m2-muscafinicreceptor subtype is expressedstronglyin the submandibularganglion(a, b; arrows)and the m3-muscarinicreceptor subtype is expressed in the interlobularducts (c, d; arrowheads). Bar -- 100 ~tm.
337
ratio (N/S) for the probes was calculated. We considered cells that had a grain density at least three times higher than the background to be positive. Fig. la and b show the localization of the m2 muscarinic receptor mRNA subtype in the submandibular ganglion. Most of the ganglion cells were labeled strongly by the m2 probe, but the neurons and satellite cells were below the detection level or background level with other probes for muscarinic receptor sub-
types. Fig. lc and d show the expression of the m3 mRNA subtype in the parenchymal cells. The labeling of interiobular duct cells by this probe was very strong. However, other probes of the muscarinic receptor subtype did not label the interlobular duct cells. Labeling in the intercalated region by all the muscarinic receptor subtypes was below the detection level. In addition, cells in the terminal secretory unit were negligibly labeled by all receptor subtypes. The grain density of
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'
4~
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.
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.-
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;
, : •
.
.
,.0
.
: r
,.."
~"
-,
.
. |
'
d
o.
°°
iii~!~i!:;! ¸
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Fig. 2. Dark- and bright-field photomicrographs showing the expression of mRNA of the a2- (a, b), a3- (c, d), a4- (e, f), and fi2- (g, h) nicotinic receptor subunits. The a2-nicotinic receptor subunit is expressed in the striated and interiobular duct (a, b; arrowheads), the a3-nicotinic receptor subunit is expressed in the submandibular ganglion (c, d; arrows), the a4-nicotinic receptor subunit is expressed in the striated ~nd interlobular duct (e, f; arrowheads), and the fi2-nicotinic receptor subunit is expressed in the submandibular ganglion (g, h; arrow) and in !We striated and interlobular duct (arrowheads). Bar = 100 izm.
33g TABLE I
Detection of mRNA of mte;carinic receptor subtypes and nicotinic receptor subunits in the rat submandibular gland + +: moderately labeled ( S / N ratio > 5); +: weakly labeled ( S / N ratio 3-5); - : unlabeled ( S / N ratio < 3).
ml Ganglion Intercalated duct
.
Striated
.
duct
lnterlobular duct Acinar cells
.
m2
m3
+ +
.
.
.
.
.
. .
.
.
m4
.
. .
m5 . .
a2 .
.
+ + .
. +
.
the cells in the terminal secretory unit was always less than three-fold the level of the background density. As shown in Figures 2c and g, most of the ganglion cells were strongly labeled by the probes for a3 (Fig. 2c) and /32 (Fig. 2g). However, grain density in ganglion neurons for the a2 and a4 subunits was below the detection level. The a2, a4, and/32 subunits of the nicotinic receptors were detected in the interlobular and striated ducts (Fig. 2a,e,g). Intercalated ducts, satellite cells, and terminal secretory units were not labeled by any of the probes for any of the nicotinic receptor subunits. Table 1 summarizes these findings. Parasympathetic ganglion neurons have been pharmacologically shown to express nicotinic receptors s. The present study confirmed this finding and further clarified that nicotinic receptors expressed in the parasympathetic submandibular ganglion are composed of a3 and/32 subunits. Pharmacological studies have suggested that submandibular ganglion neurons do not express the m2muscarinic receptor subtype n,15. However, the present study demonstrated the localization of the m2 mRNA subtype. These findings suggest that the mRNA exists but that the protein is not translated or that the translated protein somehow does not act as a receptor of the m2 subtype. Furthermore, the differences between our findings and those of the studies above suggests that some inhibitory mechanism for the binding of antagonists or agonists specific to m2 subtypes exists in the submandibular ganglion, or that the antagonists or agonists that have been employed so far do not bind to the m2 subtype in the submandibular ganglion neurons in particular. Alternatively, the failure to detect the m2 receptor in pharmacological binding assay may have been due to the small amount of m2 receptor protein. The m2-specific ligand binding in the ganglion neurons may be reduced to a negligible level with the excess amount of protein from acinar cells, which cells are independent of the m2 receptor. The present study revealed that most of the ganglion cells were labeled by the m2 probe, and by the a3 and the/32 subunit probes, suggesting that both mus-
.
+ .
a3
a4
~2
+ + .
--
+ +
-
+
+
+
+
.
.
carinic and nicotinic receptors are expressed in the same neurons. Whether or not two types of receptors are expressed on the same postsynaptic membrane is important, particularly in the analysis of the neural circuit. If two of these receptors are coexpressed on the same postsy* naptic membrane, differences in the affinity of acetylcholine to the receptors or differences in the acetylcholine content in the synaptic cleft may drive two different receptor systems separately by a common ligand, resulting in different functions. If two types of receptors are expressed on different postsynaptic membranes, the neural circuit of the preganglionic neurons should be analyzed. Binding studies have shown that the salivary gland is rich in muscarinic receptors of the m3 subtype 12. The present study supported this finding, and further showed that duct cells express the m3 subtype very strongly. Since duct cells are related to the secretion and reabsorption of electrolytes and secretion of proteins, acetylcholine released from the parasympathetic postganglionic nerves could regulate these functions via m3 subtype muscarinic receptors. On the other hand, since parasympathetic stimulation causes a marked secretion of saliva and since the terminal secretory unit was negative for m l - 5 probes, this suggests that acetylcholine does not influence the production of saliva directly by binding to these receptor subunits, but by binding to other, as yet unknown, subunits of the muscarinic receptors. We thank Dr. Keiji Wada (NIH, USA) for kindly providing plasmid DNA-encoded nicotinic acetylcholine receptor ¢2, a3, a4, a n d / 3 2 subunits. This study was supported, in part, by Grants-in-Aid from the Ministry of Education, Science and Culture of Japan and the Science and Technology Agency of Japan. I Bhaskar, S.N., Oral Histology and Embriology, Mosby Year Book, New York, 1989, pp. 337-369. 2 Bloch, E., Papovici, T., Le Guellee, D., Normand, E., Chouham, S., Guitteny, A.F. and Bohlen, P., In situ hybridization histochemistry for analysis of gene expression in the endocrine and nervous system tissue: a three years experience, Y. Neurosci. Res., 16 (1986) 183-200. 3 Bundgaard, M., Moiler, M. and Poulsen, J.H., Localization of
339 sodium pump sites in cat salivary glands, ./. Physiol., 273 (1977) 339-348. 4 Bonner, T.I., Buckley, M.J., Young, A.C. and Brann, M.R., Identification of a family of muscarinic acetyicholine receptor genes, Science, 237 (1987) 527-532. 5 Bonner, T.I., Young, A.C., Brann, M.R. and Buckley, N.J., Cloning and expression of the human and rat m5 muscarinic acetylcholine receptor genes, Neuron, 1 (1988) 403-410. 6 Bonner, T.H., The molecular basis of muscarinic receptor diversity, Trends Neurosci., 12 (1¢89) 148-151. 7 Boulter, J., Goldman, D., Martin, D., Treco, D., Heinemann, S. and Patrick, J., Isolation of a cDNA clone coding for possible neuronal nicotinic ~cetylcholine receptor a-subunit, Nature, 319 (1986) 368-374. 8 Boyd, T. R., Jacob, M.H., Couturier, S., Ballivet, M. and Berg, D.K., Expression and regulation of neuronal acetylcholine receptor mRNA in chick ciliary ganglia, Neuron, 1 (1988) 495-502. 9 Deneris, E.S., Connoly, J., Boulter, J., Wada, E., Wada, K., Swanson, W., Patrick, J. and Swanson, L.W., Primary structure and expression of /32: a novel subunit of neuronal nicotinic acetylcholine receptors, Neuron, 1 (1989)45-54. 10 Hammer, R., Giraldo, E., Schiavi, G.B., Monferini, E. and Ladinsky, H., Binding profile of a novel cardioselective muscarinic receptor antagonist, AF-DX 116, to membranes of peripheral tissues and brain in the rat, Life Sci., 38 (1986) 1653-1662.
11 Hummer, R., Berrie, C.P., Birdsall, N.J.M., Burgen, A.S.V. and Hulme, E.C., Pirenzepin distingushies between different subclasses of muscarinic receptors, Nature, 283 (1980) 90-92. 12 Martos, F., Monferini, E., Giraldo, E., Paoli, A.M. and Hammer, R., Characterization of muscarinic recel~iors in salivary and lacrimal glands of the rat, Eur. J. Pharmacol., 143 (1987) 189-194. 13 Noguchi, K., Morita, Y., Kiyama, H., Ono, IL and Tohyama, M., A noxious stimulus induces the preprotachykinin-A gene expression in the rat dosal root ganglion: a quantitative study using in situ hybridization histochemistry, Mol. Brain Res., 4 (1988) 31-35. 14 Ten Cate, A.R., Oral histology: Salivary Gland, Mosby, St. Louis, 1989, pp. 312-340. 15 Vanderheyden, P., Gies, J.P., Ebinger, G., Keyser, J.D., Landry, Y. and Vauquelin, G., Human M1-, M2-, and M3-muscarinic cholinergic receptors: Binding characteristics of agonists and antagonists, J. Neurol. Sci., 97 (1990) 67-80. 16 Vilar6, M.T., Palacios, J.M. and Mengod, G., Localization of m5 muscarinic mRNA in rat brain examined by in situ hybridization histochemistry, Neurosci. Left., 114 (1990) 154-159. 17 Wada, E., Wada, K., Boulter, J., Deneris, E., Heinemann, S., Patrick, J. and Swanson, L.W., Distribution of alpha2, alpha3, alpha4, and beta2 neuronal nicotinic receptor subunit mRNAs in the central nervous system: A hybridization histochemicai study in the rat, .l. Comp. Neurol., 284 (1989) 314-335.