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Distribution of calcitonin gene-related peptide immunoreactive nerve fibers in the human submandibular gland
Neuroscience Letters, 150 (1993) 137-140
137
© 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/93/5 06.00
NSL 09281
Distribution of calcitonin gene-related peptide immunoreactive nerve fibers in the human submandibular gland A. Salo a, J. Ylikoski b and H. Uusitalo a'c ~Department of Anatomy, University of Helsinki, Helsinki (Finland), bDepartment of Otorhinolaryngology, Laakso Hospital, Helsinki (Finland) and CDepartment of Clinical Sciences, University of Tampere, Tampere (Finland) (Received 25 May 1992; Revised version received l October 1992; Accepted 7 November 1992)
Key words: Immunohistochemistry; Innervation; Human; Submandibular gland; CGRP The indirect immunofluorescence technique was used ttr study the distribution of calcitonin gene-related peptide (CGRP) in human submandibular gland. A relatively low number of thin varicose fibers with intense immunofluorescence for CGRP was seen in samples from seven glands. These CGRP-immunoreactive (CGRP-IR) nerve fibers were mainly seen around or in close contact with intra- and interlobular blood vessels. Some CGRP-IR nerve fibers were also found in association with intra- and interlobular salivary ducts and a few around the submandibular acini. By visual estimation there was no difference in the density of CGRP-IR nerve fibers between specimens of recurrent duct obstruction and laryngeal carcinoma. The present results show that the distribution of CGRP-IR nerve fibers in the stroma and in the glandular secretory dements of the human submandibular gland is quite similar to that seen in the rat and the ferret, which have been reported earlier. Furthermore, the regional distribution of CGRP-IR fibers in the human submandibular gland suggests that CGRP has a physiological role in the regulation of salivary gland function in human salivary glands, e.g. blood flow and secretion.
Calcitonin gene-related peptide (CGRP) is a 37 amino acid peptide and its production is encoded by the calcitonin gene [1, 10]. CGRP is widely distributed in the central and peripheral nervous system. In the periphery it is associated with sensory and motor nerves [10]. It has been shown that CGRP often co-exist with substance P (SP) in a population of sensory neurons [8, 11]. Physiologically, CGRP has potent effects on various target tissues, e.g. the vascular system, where it is one of the most powerful vasodilators [3]. In the salivary glands of the rat CGRP is found mainly around blood vessels and interlobular ducts, and to a minor extent around acini and intralobular ducts [5, 7, 13]. Similarly, the major salivary glands of the other species, e.g. the ferret [14], are also known to contain CGRP. The CGRP-IR nerve fibers in the salivary glands are suggested to be mainly sensory. However, a proportion of these fibers seem to be parasympathetic, presumably originating in the otic and submandibular ganglia [7, 13]. Although the physiological role of CGRP in the salivary glands is still unsettled, there is evidence of its
Correspondence: A. Salo, Department of Anatomy, University of Helsinki, Siltavuorenpenger 20A, 00170 Helsinki, Finland.
effects both on blood flow and secretion in experimental animals [2, 4, 6, 7]. The detailed description of the distribution of CGRP in human salivary glands has not been reported previously. The present study was undertaken to examine by immunohistochemical means the presence and distribution of CGRP in the human submandibular gland. Specimens of human submandibular glands were taken from a total of 7 patients (mean age 56.6, range 34-68) suffering from recurrent submandibular duct obstruction (5) and laryngeal carcinoma (2). The samples were taken from the macroscopically normal area of the gland. The samples were immersion fixed in 4% paraformaldehyde in 0.1 M phosphate buffer, pH 6.0, for 1 h at 4°C, and then transferred to the same fixative pH 11.0. After fixation, the samples were transferred into 0.1 M phosphate buffer, pH 7.4, containing 30% sucrose for at least 18 h at 4 ° C. The tissue samples were frozen on solid CO2 ice. Cryostat sections (10/am) were cut and mounted on chromealum-gelatine-coated glass slides, air-dried for 60 min, and normal swine serum (5%) in phosphate buffered saline (PBS) containing 0.1% Triton X-100 was applied on the sections for 60 min at 4°C. The specimens were then incubated using the specific CGRP antiserum raised in a
138
Fig. I. CGRP-IR nerve fibers around blood vessels in the stroma of human submandibular gland. Bar = 50/lm. Fig. 2. CGRP-IR nerve fibers around an intralobular blood vessel in the human submandibular gland. Bar = 50/lm. Fig. 3. CGRP-IR nerve fibers in close contact with an interlobular salivary duct in the human submandibular gland. Bar -- 50 ,um. Figs. 4 and 5. C G R P - I R nerve fibers around the secretory acini in the human submandibular gland. Bar =- 50/lm. Fig. 6. CGRP-IR nerve fibers in the stroma of the human submandibular gland. Bar = 50/lm.
139 rabbit (RPN 1842, Amersham, UK) diluted 1:500-2000 in PBS-Triton in a humid atmosphere at 4°C for 20 h. After careful rinsing with PBS-Triton, the sections were then incubated with biotinylated anti-rabbit immunoglobulin (Amersham, UK), diluted 1:100 in PBS for 60 min at room temperature. The specimens were thoroughly rinsed with PBS for 60 min, and thereafter incubated with fluorescein-conjugated streptavidin (Amersham, UK) diluted 1:100 in PBS for 15 min at room temperature. After incubation, the specimens were thoroughly washed with PBS and cover glasses were mounted by using glycerin-PBS (3:1). The specimens were examined with a Leitz Aristoplan fluorescence microscope equipped with an epi-illuminator and a specific filter block for FITC. The photographs were taken on Kodak T-Max 400 film by using an automatic VarioOrthomat microscope camera. Thin varicose brightly fluorescent C G R P - I R nerve fibers were observed in all the seven submandibular glands studied. The C G R P - I R nerve fibers were mainly located around intra- and interlobular blood vessels (Figs. 1 and 2). Usually, the fibers closely surrounded blood vessels, but they were not seen to form plexuses. C G R P - I R nerve fibers were also detected in association with intra- and interlobular salivary ducts (Fig. 3). Some of these fibers were seen in close association with the basal lamina of the ductal cells. However, fibers penetrating the basal lamina were not seen. A few nerve fibers were observed around the acini (Figs. 4 and 5). Usually these fibers were running between two acini and were not seen to form 'basketZlike' structures. Occasionally, some varicose C G R P - I R nerve fibers were found in the stroma (Fig. 6). There was no obvious difference in the density or distribution of C G R P - I R nerve fibers in submandibular glands extirpated for laryngeal carcinoma and recurrent duct obstruction. The present findings clearly demonstrate that the human submandibular gland is innervated by C G R P - I R nerve fibers. Most of these fibers were found around intra- and interlobular blood vessels. However, C G R P IR nerve fibers were also seen around salivary ducts, and occasionally between the secretory acini as well. The distribution of C G R P - I R nerves in human submandibular gland seems to be quite similar to that of experimental animals [5, 7, 13]. However, the density of C G R P - I R nerve fibers in the human submandibular gland was significantly lower than in the rat. Whether this is based on true differences between species, on the age of the patients, or the less effective immersion fixation used instead of perfusion fixation in case of experimental animals, is not known. In this study, patients with recurrent submandibular duct obstruction were compared with patients suffering
from laryngeal carcinoma. N o clear differences in the density or distribution of the C G R P - I R nerve fibers were seen. This finding indicates that duct obstruction does not change C G R P innervation in these glands, although the content of C G R P in neuronal elements is very sensitive to various pathological conditions [9]. C G R P - I R nerve fibers in the rat submandibular gland seem to be mainly of sensory origin [13]. However, a proportion of the C G R P - I R nerve fibers has been shown to be resistant to sensory denervation. Furthermore, a proportion of the ganglion cells in the rat submandibular ganglion are immunoreactive for C G R P [12] indicating that nerves in the submandibular gland have a dual origin. In the case of human tissue, the origin of nerves innervating the salivary glands is not known. However the similar distribution of C G R P - I R nerve fibers in the human and rat submandibular glands suggests that there are no differences in this respect. Physiologically C G R P stimulates amylase secretion in the parotid gland of the rat [6]. C G R P in combination with substance P (SP) is known to enhance flow of saliva and it has been suggested that SP, substance K, vasoactive intestinal peptide and C G R P may play complementary roles in the regulation of exocrine function in salivary glands [6]. Both in the rat [4] and cat [2], i.v. infusion of C G R P has also been shown to increase regional blood flow in submandibular gland. Based on the similar distribution of C G R P - I R nerves in the rat and man it is possible that C G R P also acts as a neuroactive substance in the salivary glands of man by regulating salivary gland blood flow and secretion as in the experimental animals. The skillful technical assistance by Ms. Paula Hasenson and Mr. Reijo Karppinen is gratefully acknowledged. This study was supported by grants from T E K E S and the Finnish Dental Society.
1 Amara, S.G., Jonas, V. and Rosenfeld, M.G., Alternative RNA processing in calcitonin gene related expressiongenerates mRNAs encoding different polypeptide products, Nature, 298 (1982) 240244. 2 Andersson,S.E., Effectsof intravenouscalcitoningene-relatedpeptide (CGRP) on local blood flow in the cat, Acta Physiol. Scand., 137 (1989)259 270. 3 Brain, S.D., Williams, T.J., Tippins, J.R., Morris, H.R. and Maclntyre, I., Calcitonin gene-relatedpeptide is a potent vasodilator, Nature, 313 (1985) 54-56. 4 Br~ttveit,M., Haugan, A. and Helle, K.B., Calcitonin gene-related peptide (CGRP) effectson reginal blood flow in the anaesthetized rat, Acta Physiol. Scand., 132 (1988) 46A. 5 Ekman, R., Ekstrrm, J., H~tkanson,R., Sjrgren, S. and Sundler, F., Calcitonin gene related peptide in rat salivary glands, J. Physiol., 381 (1986) 36P. 6 Ekstrrm, J., Neuropeptides and secretion, J. Dent. Res., 66 (2) (1987) 524-530.
140 7 Ekstr6m, J., Ekman, R., H~kanson, R., Sj6gren, S. and Sundler, F., Calcitonin gene-related peptide in rat salivary glands: neuronal localization, depletion upon nerve stimulation, and effects on salivation in relation to substance P, Neuroscience, 26 (1988) 933-949. 8 Gibson, S.J., Polak, J.M., Bloom, S.R., Sabate, I.M., Mulderry, P.M., Ghatel, M.A., McGregor, G.P., Morrison, J.F.B., Kelly, J.S., Evans, R.M. and Rosenfeld, M.G., Calcitonin gene-related peptide immunoreactivity in the spinal cord of man and of eight other species, J. Neurosci., 4 (1984) 3101-3111. 9 Mapp, P.I., Kidd, B.L., Gibson, S.J., Terry, J.M., Revell, P.A., Ibrahim, N.B.M., Blake, D.R. and Polak, J.M,, Substance P-, calcitonin gene-related peptide and C-flanking peptide of neuropeptide Y-immunoreactive fibers are present in normal synovium but depleted in patients with rheumatoid arthritis, Neuroscience, 37 (1990) 143 153. 10 Rosenfeldt, M.G., Mermod, J.J., Amara, S.G., Swanson, L.W., Rivier, J., Wale, W.W. and Evans, R.M., Production of a novel
11
12
13
14
neuropeptide encoded by the calcitonin gene via tissue-specific RNA processing, Nature, 304 (1983) 129-35. Saria, A., Gamse, R., Lundberg, J.M., H6kfelt, T., TheodorssonNorheim, E., Peterman, J. and Fischer, J.A., Co-existence of tachykinins and calcitonin gene-related peptide in sensory nerves in relation to neurogenic inflammation. In R. H/tkanson and F. Sundler, (Eds.), Tachykinin Antagonists, Elsevier, Amsterdam 1985, pp. 149-157. Silverman, J. and Kruger, L., Calcitonin gene-related peptide immunoreactive innervation of the rat head with emphasis on specialized sensory structures, J. Comp. Neurol., 280 (1989) 303-330. Soinila, J., Salo, A., Uusitalo, H., Yanaihara, N. and H~ipp61~i,O., CGRP-immunoreactive sensory nerve fibers in the submandibular gland of the rat, Histochemistry, 91 (1989) 455~,60. Tobin, G., Luts, A., Sundler, F. and Ekstr6m, J., Peptidergic innervation of the major salivary glands of the ferret, Peptides, 11 (1990) 863 867.
137
© 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/93/5 06.00
NSL 09281
Distribution of calcitonin gene-related peptide immunoreactive nerve fibers in the human submandibular gland A. Salo a, J. Ylikoski b and H. Uusitalo a'c ~Department of Anatomy, University of Helsinki, Helsinki (Finland), bDepartment of Otorhinolaryngology, Laakso Hospital, Helsinki (Finland) and CDepartment of Clinical Sciences, University of Tampere, Tampere (Finland) (Received 25 May 1992; Revised version received l October 1992; Accepted 7 November 1992)
Key words: Immunohistochemistry; Innervation; Human; Submandibular gland; CGRP The indirect immunofluorescence technique was used ttr study the distribution of calcitonin gene-related peptide (CGRP) in human submandibular gland. A relatively low number of thin varicose fibers with intense immunofluorescence for CGRP was seen in samples from seven glands. These CGRP-immunoreactive (CGRP-IR) nerve fibers were mainly seen around or in close contact with intra- and interlobular blood vessels. Some CGRP-IR nerve fibers were also found in association with intra- and interlobular salivary ducts and a few around the submandibular acini. By visual estimation there was no difference in the density of CGRP-IR nerve fibers between specimens of recurrent duct obstruction and laryngeal carcinoma. The present results show that the distribution of CGRP-IR nerve fibers in the stroma and in the glandular secretory dements of the human submandibular gland is quite similar to that seen in the rat and the ferret, which have been reported earlier. Furthermore, the regional distribution of CGRP-IR fibers in the human submandibular gland suggests that CGRP has a physiological role in the regulation of salivary gland function in human salivary glands, e.g. blood flow and secretion.
Calcitonin gene-related peptide (CGRP) is a 37 amino acid peptide and its production is encoded by the calcitonin gene [1, 10]. CGRP is widely distributed in the central and peripheral nervous system. In the periphery it is associated with sensory and motor nerves [10]. It has been shown that CGRP often co-exist with substance P (SP) in a population of sensory neurons [8, 11]. Physiologically, CGRP has potent effects on various target tissues, e.g. the vascular system, where it is one of the most powerful vasodilators [3]. In the salivary glands of the rat CGRP is found mainly around blood vessels and interlobular ducts, and to a minor extent around acini and intralobular ducts [5, 7, 13]. Similarly, the major salivary glands of the other species, e.g. the ferret [14], are also known to contain CGRP. The CGRP-IR nerve fibers in the salivary glands are suggested to be mainly sensory. However, a proportion of these fibers seem to be parasympathetic, presumably originating in the otic and submandibular ganglia [7, 13]. Although the physiological role of CGRP in the salivary glands is still unsettled, there is evidence of its
Correspondence: A. Salo, Department of Anatomy, University of Helsinki, Siltavuorenpenger 20A, 00170 Helsinki, Finland.
effects both on blood flow and secretion in experimental animals [2, 4, 6, 7]. The detailed description of the distribution of CGRP in human salivary glands has not been reported previously. The present study was undertaken to examine by immunohistochemical means the presence and distribution of CGRP in the human submandibular gland. Specimens of human submandibular glands were taken from a total of 7 patients (mean age 56.6, range 34-68) suffering from recurrent submandibular duct obstruction (5) and laryngeal carcinoma (2). The samples were taken from the macroscopically normal area of the gland. The samples were immersion fixed in 4% paraformaldehyde in 0.1 M phosphate buffer, pH 6.0, for 1 h at 4°C, and then transferred to the same fixative pH 11.0. After fixation, the samples were transferred into 0.1 M phosphate buffer, pH 7.4, containing 30% sucrose for at least 18 h at 4 ° C. The tissue samples were frozen on solid CO2 ice. Cryostat sections (10/am) were cut and mounted on chromealum-gelatine-coated glass slides, air-dried for 60 min, and normal swine serum (5%) in phosphate buffered saline (PBS) containing 0.1% Triton X-100 was applied on the sections for 60 min at 4°C. The specimens were then incubated using the specific CGRP antiserum raised in a
138
Fig. I. CGRP-IR nerve fibers around blood vessels in the stroma of human submandibular gland. Bar = 50/lm. Fig. 2. CGRP-IR nerve fibers around an intralobular blood vessel in the human submandibular gland. Bar = 50/lm. Fig. 3. CGRP-IR nerve fibers in close contact with an interlobular salivary duct in the human submandibular gland. Bar -- 50 ,um. Figs. 4 and 5. C G R P - I R nerve fibers around the secretory acini in the human submandibular gland. Bar =- 50/lm. Fig. 6. CGRP-IR nerve fibers in the stroma of the human submandibular gland. Bar = 50/lm.
139 rabbit (RPN 1842, Amersham, UK) diluted 1:500-2000 in PBS-Triton in a humid atmosphere at 4°C for 20 h. After careful rinsing with PBS-Triton, the sections were then incubated with biotinylated anti-rabbit immunoglobulin (Amersham, UK), diluted 1:100 in PBS for 60 min at room temperature. The specimens were thoroughly rinsed with PBS for 60 min, and thereafter incubated with fluorescein-conjugated streptavidin (Amersham, UK) diluted 1:100 in PBS for 15 min at room temperature. After incubation, the specimens were thoroughly washed with PBS and cover glasses were mounted by using glycerin-PBS (3:1). The specimens were examined with a Leitz Aristoplan fluorescence microscope equipped with an epi-illuminator and a specific filter block for FITC. The photographs were taken on Kodak T-Max 400 film by using an automatic VarioOrthomat microscope camera. Thin varicose brightly fluorescent C G R P - I R nerve fibers were observed in all the seven submandibular glands studied. The C G R P - I R nerve fibers were mainly located around intra- and interlobular blood vessels (Figs. 1 and 2). Usually, the fibers closely surrounded blood vessels, but they were not seen to form plexuses. C G R P - I R nerve fibers were also detected in association with intra- and interlobular salivary ducts (Fig. 3). Some of these fibers were seen in close association with the basal lamina of the ductal cells. However, fibers penetrating the basal lamina were not seen. A few nerve fibers were observed around the acini (Figs. 4 and 5). Usually these fibers were running between two acini and were not seen to form 'basketZlike' structures. Occasionally, some varicose C G R P - I R nerve fibers were found in the stroma (Fig. 6). There was no obvious difference in the density or distribution of C G R P - I R nerve fibers in submandibular glands extirpated for laryngeal carcinoma and recurrent duct obstruction. The present findings clearly demonstrate that the human submandibular gland is innervated by C G R P - I R nerve fibers. Most of these fibers were found around intra- and interlobular blood vessels. However, C G R P IR nerve fibers were also seen around salivary ducts, and occasionally between the secretory acini as well. The distribution of C G R P - I R nerves in human submandibular gland seems to be quite similar to that of experimental animals [5, 7, 13]. However, the density of C G R P - I R nerve fibers in the human submandibular gland was significantly lower than in the rat. Whether this is based on true differences between species, on the age of the patients, or the less effective immersion fixation used instead of perfusion fixation in case of experimental animals, is not known. In this study, patients with recurrent submandibular duct obstruction were compared with patients suffering
from laryngeal carcinoma. N o clear differences in the density or distribution of the C G R P - I R nerve fibers were seen. This finding indicates that duct obstruction does not change C G R P innervation in these glands, although the content of C G R P in neuronal elements is very sensitive to various pathological conditions [9]. C G R P - I R nerve fibers in the rat submandibular gland seem to be mainly of sensory origin [13]. However, a proportion of the C G R P - I R nerve fibers has been shown to be resistant to sensory denervation. Furthermore, a proportion of the ganglion cells in the rat submandibular ganglion are immunoreactive for C G R P [12] indicating that nerves in the submandibular gland have a dual origin. In the case of human tissue, the origin of nerves innervating the salivary glands is not known. However the similar distribution of C G R P - I R nerve fibers in the human and rat submandibular glands suggests that there are no differences in this respect. Physiologically C G R P stimulates amylase secretion in the parotid gland of the rat [6]. C G R P in combination with substance P (SP) is known to enhance flow of saliva and it has been suggested that SP, substance K, vasoactive intestinal peptide and C G R P may play complementary roles in the regulation of exocrine function in salivary glands [6]. Both in the rat [4] and cat [2], i.v. infusion of C G R P has also been shown to increase regional blood flow in submandibular gland. Based on the similar distribution of C G R P - I R nerves in the rat and man it is possible that C G R P also acts as a neuroactive substance in the salivary glands of man by regulating salivary gland blood flow and secretion as in the experimental animals. The skillful technical assistance by Ms. Paula Hasenson and Mr. Reijo Karppinen is gratefully acknowledged. This study was supported by grants from T E K E S and the Finnish Dental Society.
1 Amara, S.G., Jonas, V. and Rosenfeld, M.G., Alternative RNA processing in calcitonin gene related expressiongenerates mRNAs encoding different polypeptide products, Nature, 298 (1982) 240244. 2 Andersson,S.E., Effectsof intravenouscalcitoningene-relatedpeptide (CGRP) on local blood flow in the cat, Acta Physiol. Scand., 137 (1989)259 270. 3 Brain, S.D., Williams, T.J., Tippins, J.R., Morris, H.R. and Maclntyre, I., Calcitonin gene-relatedpeptide is a potent vasodilator, Nature, 313 (1985) 54-56. 4 Br~ttveit,M., Haugan, A. and Helle, K.B., Calcitonin gene-related peptide (CGRP) effectson reginal blood flow in the anaesthetized rat, Acta Physiol. Scand., 132 (1988) 46A. 5 Ekman, R., Ekstrrm, J., H~tkanson,R., Sjrgren, S. and Sundler, F., Calcitonin gene related peptide in rat salivary glands, J. Physiol., 381 (1986) 36P. 6 Ekstrrm, J., Neuropeptides and secretion, J. Dent. Res., 66 (2) (1987) 524-530.
140 7 Ekstr6m, J., Ekman, R., H~kanson, R., Sj6gren, S. and Sundler, F., Calcitonin gene-related peptide in rat salivary glands: neuronal localization, depletion upon nerve stimulation, and effects on salivation in relation to substance P, Neuroscience, 26 (1988) 933-949. 8 Gibson, S.J., Polak, J.M., Bloom, S.R., Sabate, I.M., Mulderry, P.M., Ghatel, M.A., McGregor, G.P., Morrison, J.F.B., Kelly, J.S., Evans, R.M. and Rosenfeld, M.G., Calcitonin gene-related peptide immunoreactivity in the spinal cord of man and of eight other species, J. Neurosci., 4 (1984) 3101-3111. 9 Mapp, P.I., Kidd, B.L., Gibson, S.J., Terry, J.M., Revell, P.A., Ibrahim, N.B.M., Blake, D.R. and Polak, J.M,, Substance P-, calcitonin gene-related peptide and C-flanking peptide of neuropeptide Y-immunoreactive fibers are present in normal synovium but depleted in patients with rheumatoid arthritis, Neuroscience, 37 (1990) 143 153. 10 Rosenfeldt, M.G., Mermod, J.J., Amara, S.G., Swanson, L.W., Rivier, J., Wale, W.W. and Evans, R.M., Production of a novel
11
12
13
14
neuropeptide encoded by the calcitonin gene via tissue-specific RNA processing, Nature, 304 (1983) 129-35. Saria, A., Gamse, R., Lundberg, J.M., H6kfelt, T., TheodorssonNorheim, E., Peterman, J. and Fischer, J.A., Co-existence of tachykinins and calcitonin gene-related peptide in sensory nerves in relation to neurogenic inflammation. In R. H/tkanson and F. Sundler, (Eds.), Tachykinin Antagonists, Elsevier, Amsterdam 1985, pp. 149-157. Silverman, J. and Kruger, L., Calcitonin gene-related peptide immunoreactive innervation of the rat head with emphasis on specialized sensory structures, J. Comp. Neurol., 280 (1989) 303-330. Soinila, J., Salo, A., Uusitalo, H., Yanaihara, N. and H~ipp61~i,O., CGRP-immunoreactive sensory nerve fibers in the submandibular gland of the rat, Histochemistry, 91 (1989) 455~,60. Tobin, G., Luts, A., Sundler, F. and Ekstr6m, J., Peptidergic innervation of the major salivary glands of the ferret, Peptides, 11 (1990) 863 867.