Chem. Senses 27: 825-830,
2002
© Oxford University Press 2002
Immunohistochemical Investigation of the Nitrergic System in the Taste Organ of the Frog, Rana esculenta
Department of Animal Biology and Marine Ecology, Section of Cell and Evolutionary Biology, University of Messina, Italy 1 Department of Morphological and Biomedical Sciences, Section of Anatomy and Histology, University of Verona, Italy
Correspondence to be sent to: Prof. Giacomo Zaccone, Department of Animal Biology and Marine Ecology, Section of Cell and Evolutionary Biology, University of Messina, Salita Sperone 31—S. Agata, I-98166 Messina, Italy. e-mail: gzaccone{at}unime.it
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
We have studied by immunocytochemistry, the taste discs of the frog, Rana esculenta, with the aim of providing morphological and neurochemical data on the nitrergic system and of assessing the eventual presence of intrinsic neurons associated with the gustatory organs. In taste discs, antibodies against neuronal nitric oxide synthase (nNOS) revealed a positive immunoreaction in the taste receptor cell bodies and processes. The basal cells were also stained. All the fungiform papillae contained intragemmal nerve fibers showing nNOS immunoreactivity; these fiber were mainly located in the basal plexus. Immunoreactive nerve fibers were also visible at the periphery of the papilla-contacting ciliate cells, which form a ring around the taste disc. In conclusion, the findings obtained in this study suggest that the occurrence of nNOS-immunoreactivity in basal cells, taste cells and nerves might reflect a role for nitric oxide in taste mechanisms of Amphibia. The results may also sustain the physiological implication of NO as a molecule involved in the local target function of maintaining the taste bud mucosal integrity and in regulating the blood flow to the epithelium.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The free radical nitric oxide (NO) is a versatile signaling molecule that regulates a variety of cellular functions (Moncada and Higgs, 1993
).
This messenger molecule is produced by oxidation of L-arginine to NO and
L-citrulline, a reaction catalysed by nitric oxide synthase (NOS)
(Moncada et al.,
1991). Some studies have demonstrated that NO may contribute to
the regulation of certain autonomic functions by acting as a non-adrenergic,
non-cholinergic transmitter in the peripheral autonomic nervous system
(De Man et al., 1991;
Toda and Okamura, 1991). This
function has very recently also been argued for the visceral sensory system in
lower vertebrates (Green and Campbell,
1994; Bodegas et al.,
1995; Brüning et
al., 1996; Mauceri et
al., 1999; Zaccone et
al., 2002).
In taste organs, an important role for nitrergic mechanisms has recently
been suggested (Herness, 1996;
Kretz et al., 1998;
Rosenzweig et al.,
1999; Sbarbati et
al., 1999). In the rat vallate papilla, these studies
demonstrated that neurons of an intrinsic system are mainly nitrergic elements
(Sbarbati et al.,
2000). These data suggested that in taste organs nitrergic
ganglion cells are capable of sustaining and modulating local activities as
described in integrative circuits of other organs, such as heart, airways and
bladders (Burnstock et al.,
1987). However, to date, studies on nitrergic mechanisms and
intrinsic nervous systems associated with taste organs have only been
performed in mammals and comparative data on other species of vertebrates are
lacking.
Among the Amphibia, because of the peculiar characteristics of the taste
disc, the taste organs of the frog have been considered by several authors to
be a good model and used to obtain anatomical and physiological data. In this
species, the taste organs consist of large discs located at the top of the
fungiform papillae (Osculati and Sbarbati,
1995). In these structures four distinct cell types can be
recognized. Cell types I, II and III reach the surface and contact the
external environment. Type IV cells are found exclusively in the basal layer
of the taste disc. These cells share many morphological and
immunohistochemical characteristics with the Merkel cells of the skin
(Tachibana, 1995;
Zaccone et al., 2001)
and are different from the basal cells of mammalian taste buds. They are rich
in the biogenic monoamine serotonin (Delay
et al., 1993;
Zancanaro et al.,
1997), neuron-specific enolase
(Toyoshima, 1989) and
enkephalins (Zaccone et al.,
1995) and, moreover, they are considered as paraneurons.
The purpose of the present study is to provide morphological and neurochemical data on the nitrergic system and to assess the eventual presence of intrinsic neurons in the taste discs of the frog, Rana esculenta, using immunocyto-chemistry for the neuronal isoform of nitric oxide synthase (nNOS). These data, compared to those recently obtained in mammals, could be useful in understanding the phylogenesis of the taste system.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Specimens of R. esculenta were maintained in the laboratory at 10°C with access to tap water. Animals were killed by decapitation and their tongues removed. Lingual tissues were cut into 2-3 mm3 fragments. The specimens were fixed in 4% buffered formaldehyde for 5-6 h at 4°C, dehydrated by a series of graded ethanols and embedded in Paraplast. Immunostaining was performed with indirect immunoperoxidase visualization.
Serial sections (3-7 µm) were incubated in 10% normal goat serum for 2 h prior to incubation with primary antisera to reduce non-specific staining. Sections were next incubated in the polyclonal antibody against nNOS (Transduction Laboratories, Lexington) diluted 1:250 with 0.1 M phosphate buffered saline (PBS; pH 7.4). The tissues were subsequently washed in PBS and then incubated with secondary antiserum. The signals were developed by treatment with 0.05 M 3,3'-diaminobenzidine tetrahydrochloride.
After staining, sections were viewed and photographed in a Zeiss Axiophot microscope. In control experiments, the primary antibody was omitted or replaced with a non-immune serum. In addition, prior to the incubation of the sections, the antiserum was absorbed overnight at 4°C with 1 nmol of the respective antigen per millilitre of diluted antiserum.
Immunoblotting
The taste discs were cut free with small scissors under a dissection microscope and total number used was 25. The taste discs were then extracted by homogenization (Ultraturax) in 10-15 volumes of buffer A (2% SDS, 4 µM leupeptin, 4 µM aprotenin, 64 µM pepstatin A, 1 mM EGTA, 1 mM EDTA, 2 mM phenylmethylsulphonyl fluoride, 20 mM tetrahydrobiopterin). The homogenate was centrifuged at 106 000 g for 15 min at 4°C. The supernatant was removed and 5% ß-mercaptoethanol, glycerol and bromophenol blue was added. Proteins were separated by SDS—PAGE and electrotransferred to nitrocellulose membrane (Hybond ECL; Amersham). The membrane was blocked for 1 h at room temperature with 5% bovine serum albumin (BSA) followed by incubation with nNOS antibody at a dilution of 1:50 at 4°C overnight. The membrane was incubated with horseradish-peroxidase-conjugated goat anti-rabbit immunoglobulin antibody (1/5000; Sigma) for 2 h at room temperature. The binding of primary antibody was detected by enhanced chemiluminescence (ECL; Amersham).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Histology
Vertical sections of the taste organ show that it consisted of a taste disc located on a fungiform papilla. The papilla was surrounded by a ciliated epithelium (Figure 1c) and most of its surface was composed of glandular supporting cells. Apical processes of taste cells were seen between these cells (Figure 2b,c). Numerous basal cells were located in the peripheral basal portion of the papilla. These have been reported by earlier authors using immunohistochemistry and are reported here for the first time using nNOS antibodies (see below).
|
|
nNOS immunostaining
At low magnification, immunostaining was visible in fungiform papillae of the dorsal surface of the tongue. At higher magnification, in taste discs, antibodies against nNOS revealed a positive immunoreaction in the taste receptor cells bodies and processes (Figures 1c and 2b,c). In longitudinal sections, the immunoreactive taste cells have a spindle-shaped body with long, thin apical and basal processes lying between the glandular supporting cells (Figure 2b,c). The basal cells were also stained (Figures 1b and 3). All the fungiform papillae contained intragemmal nerve fibers showing nNOS immunoreactivity (IR). These fibers were mainly located in the basal plexus (Figures 1a,b and 2a). Immunoreactive nerve fibers were also visible at the periphery of the papilla contacting ciliate cells which form a ring around the taste disc (Figure 1c). Many nitrergic nerve fibers were seen in the connective tissues and were closely associated with the gland system. Some of the nitrergic nerves coursed among the secretory units in sections of the anterior, middle and posterior surface and the ventromedian region of the tongue (Figure 1d). Isolated nitrergic fibers or perivascular plexuses were detected along the walls of the lingual arteries. Numerous positive nerve fibers were demonstrated around the mucous glands. Nitrergic nerve fibers were also located in the connective tissue of the filiform papillae.
|
nNOS-IR cells bodies were not found in the connective tissue of fungiform papillae in the present study.
nNOS immunoblotting
The antibody directed against nNOS recognized a protein of molecular mass at least 160 kDa in extracts of frog taste discs (Figure 4).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The present study demonstrates a wide presence of nitrergic structures in the taste organ of an amphibian species. These results are in agreement with data previously obtained in the gustatory system of mammals (Sbarbati et al., 1999
,
2000). However, some
differences seem to exist between the distribution of nitrergic structures in
amphibians and mammals. In the frog, nNOS-IR has been detected in nerves and
epithelial cells, while in the rat it seems to be restricted to nerves. In
addition, the nervous intrinsic ganglia associated with taste organs in
mammals are not described in the frog. nNOS-IR in basal cells
In the present study we demonstrate the presence of nNOS-IR in basal cells
of the taste disc. The neurotransmitter function of these cells has been
postulated by several authors. They found, in immunohistochemical studies,
that these cells are clearly serotonin positive
(Zaccone, 1986;
Delay et al., 1993;
Tachibana, 1995;
Hamasaki et al.,
1998). Recently, immunoreactivity for leu-enkephalin has been
reported in the basal cells of taste buds of Ambystoma tigrinum
(Zaccone et al.,
1995). The significance of serotonin and of the bioactive
substance in the basal cells of taste buds is still unknown. According to
earlier workers (Delay et al.,
1993), these cells may be regarded as serotonergic neurons having
also a trophic role in the maintenance of the morphological integrity of frog
taste buds (Hamasaki et al.,
1998), but their function is still currently enigmatic.
nNOS-IR in taste cells
In the present study, nNOS-IR was also noticed in taste cells and their
processes. To our knowledge, this is reported for the first time in amphibian
taste buds. NO derived from nNOS is known to be an important signaling
molecule regulating several neuroendocrine and behavioral functions
(Gammie et al., 2000).
The occurrence of nNOS-IR in both basal and taste cells might reflect a role
for NO in transmission mechanisms; in particular, this molecule may be useful
for initiating the chemosensory stimulation of taste buds. It has been argued
that basal cells are stimulated to release serotonin during chemostimulation
of taste buds (Kim and Roper,
1995; Nagai et al.,
1996), although synaptic contacts between the taste cells and
basal cells have not been ascertained
(Osculati and Sbarbati, 1995).
NO is regulated to occur under special circumstances and in neuronal
processes, glutamate is the principal activator of NO release
(Gammie et al., 2000).
NO also diffuses radially to affect surrounding synapses, carrying information
in the opposite direction to neural transmission
(Beckmann, 1996). So, the
synthesis of NO by taste and basal cells may be indicative of the onset of
functional activity in these taste cells and the activation of diverse
afferent synaptic structures between the basal cells and/or the taste cells
and nerve fibers.
nNOS-IR in nerves
The present study demonstrates the presence of nervous intragemmal and
extragemmal nerve fibers immunostained with nNOS. Previous studies showed that
the presence of nNOS enzymes corresponds with that in both central and
peripheral nervous systems (Belai et
al., 1992; Hassal et
al., 1992). In absence of tracing and denervation
experiments, we cannot demonstrate the extrinsic innervation of most of the
immunohistochemically tested nerve fibers. Gustatory fibers usually arise from
the geniculate ganglion innervating taste buds and the somatic nerve fibers
distributing in the perigemmal epithelium, originate from the trigeminal
ganglion (Hu et al.,
1996). In the dog and rat, nNOS-IR nerve fibers are thought to be
of intrinsic origin, arising from the intralingual neurons
(Hu et al., 1996;
Sbarbati et al.,
1999). The tongue of the examined species receives a rich
innervation of nitrergic fibers, which presumably provides a vasodilator and
secretomotor action to the glands, but the origin of both intragemmal and
extragemmal NO-producing fibers remains to be clarified by further
investigations due to the failure to detect intralingual neurons. An
interesting finding obtained from the present experiments is the relationship
between ciliate cells and nitrergic nerves. The evidence seems to suggest that
the ring of cilia surrounding the receptorial surface is controlled by a
neural mechanism.
In conclusion, the findings obtained in previous research and in this study
suggest that the occurrence of nNOS-IR in basal cells, taste cells and nerves
might reflect a role for NO in the gustatory mechanisms of amphibians. The
results may also sustain the physiological implication of NO as a
cytoprotective molecule (Konturek and
Konturek, 1995) in the local target function of maintaining taste
bud mucosal integrity, in regulating the blood flow to the epithelium and in
modulation of ciliary activity. NO plays multiple physiological roles in the
regulation of the functions of numerous organs. Investigations continue to
expand rapidly and further studies are indeed needed to clarify the NO
function of the particular cell types of amphibian taste buds, responsible for
their multifunctional roles.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Beckmann, J.S. (1996) The physiological and pathological chemistry of nitric oxide. In Lancaster, J. Jr (ed.),Nitric Oxide—Principles and Actions . Academic Press, San Diego, CA, pp. 1-82.
Belai, A., Schmidt, H.H.H.W., Hoyle, C.H.Y., Hassal, C.J.S., Saffrey, M.J., Moss, J., Förstermann, U, Murad, F. and Burnstock, G. (1992) Colocalization of nitric oxide synthase and NADPH-diaphorase in the myenteric plexus of the rat gut. Neurosci. Lett., 143,60 -64.[Web of Science][Medline]
Bodegas, M.E., Villaro, A.C., Montuenga, L.M., Moncada, S., Riveros-Moreno, V. and Sesma, P. (1995) Neuronal nitric oxide synthase immunoreactivity in the respiratory tract of the frog, Rana temporaria. Histochem. J.,27 , 812-818.[Web of Science][Medline]
Brüning, G., Hattwig, K. and Mayer, B. (1996) Nitric oxide synthase in the peripheral nervous system of the goldfish, Carassius auratus. Cell Tissue Res.,284 , 87-98.[Web of Science][Medline]
Burnstock, G., Allen, T.G.J., Hassal, C.J.S. and Pittam, B.S. (1987) Properties of intramural neurones cultured from the heart and bladder. In Heym, C. (ed.), Experimental Brain Research Series, No. 16. Histochemistry and Cell Biology of Autonomic Neurons and Paraganglia, Springer, Heidelberg, pp.323 -328.
De Man, J.G., Pelckmans, P.A., Boeckxstaens, G.E., Bult, H., Oosterbosch, L., Herman, A.G. and Van Maerdke, Y.M. (1991) The role of nitric oxide in inhibitory non-adrenergic non-cholinergic neurotransmission in the canine lower oesophageal sphincter. Br. J. Pharmacol., 103,1092 -1096.[Web of Science][Medline]
Delay, R.J., Taylor, R. and Roper, S.D. (1993) Merkel-like basal cells in Necturus taste buds contain serotonin. J. Comp. Neurol.,335 , 606-613.[Web of Science][Medline]
Gammie, S.C., Dawson, V.L. and Nelson, R.J. (2000) Influence of nitric oxide on neuroendocrine function and behaviour. In Ignarro, L.J. (ed.), Nitric Oxide—Biology and Pathobiology. Academic Press, San Diego, CA, pp.429 -438.
Green, K. and Campbell, G. (1994) Nitric oxide formation is involved in vagal inhibition of the stomach of the trout (Salmo gairdneri). J. Auton. Nerv. Syst.,50 , 221-229.[Web of Science][Medline]
Hamasaki, K., Seta, Y., Yamada, K. and Toyoshima, K. (1998) Possible role of serotonin in Merkel-like basal cells of the taste buds of the frog Rana nigromaculata. J. Anat., 193,599 -610.[Medline]
Hassal, C.J.S., Saffrey, M.J., Belai, A., Hyle, C.H., Moules, E.W., Moss, J., Schmidt, H.H.H.W., Murad, F., Förstermann, U. and Burnstock, G. (1992) Nitric oxide synthase immunoreactivity and NADPH-diaphorase activity in a subpopulation of intrinsic neurons of the guinea pig heart. Neurosci. Lett.,143 , 65-68.[Web of Science][Medline]
Herness, S. (1996) Immunocytochemical localization of c-fos and nitric oxide synthase to von Ebner gland but not to posterior taste cells in rat tongue. Chem. Senses,116 , 614.
Hu, Z.L., Masuko, S. and Katsuki, T. (1996) Distribution and origins of nitric oxide producing nerve fibers in the dog tongue: correlated NADPH-diaphorase histochemistry and immunohistochemistry for calcitonin gene-related peptide using light and electron microscopy. Arch. Histol. Cytol.,59 , 491-503.[Web of Science][Medline]
Kim, D.J. and Roper, S.D. (1995) Localization of serotonin in taste buds: a comparative study in four vertebrates. J. Comp. Neurol., 353,364 -370.[Web of Science][Medline]
Konturek, S.K. and Konturek, P.C. (1995) Role of nitric oxide in the digestive system.Digestion , 56,1 -13.[Web of Science][Medline]
Kretz, O., Bock, R. and Lindemann, B. (1998) Occurrence of nitric oxide synthase in taste buds of the rat vallate papilla. Histochem. J.,30 , 293-299.
Mauceri, A., Fasulo, S., Ainis, L., Licata, A., Lauriano, E.R., Martinez, A., Mayer, B. and Zaccone, G. (1999) Neuronal nitric oxide synthase (nNOS) expression in the epithelial neuroendocrine cell system and nerve fibers in the gill of the catfish, Heteropneustes fossilis. Acta Histochem.,101 , 437-448.[Web of Science][Medline]
Moncada, S. and Higgs, A. (1993) The
L-arginine-nitric oxide pathway. N. Engl. J. Med.,329
, 2002-2012.
Moncada, S., Palmer, R.M.J. and Higgs, E.A. (1991) Nitric oxide: physiology, pathophysiology and pharmacology. Pharmacol. Rev., 43,109 -142.[Web of Science][Medline]
Nagai, T., Kim, D.-J., Delay, R.J. and Roper, S.D.
(1996) Neuromodulation of transduction and signal processing
in the end organs of taste. Chem. Senses,21
, 353-365.
Osculati, F. and Sbarbati, A. (1995) The frog taste disc: a prototype of the vertebrate gustatory organ.Progr. Neurobiol. , 46,351 -399.[Web of Science][Medline]
Rosenzweig, S., Yan, W., Dasso, M. and Spielman, A.I.
(1999) Possible novel mechanism for bitter taste mediated
through cGMP. J. Neurophysiol., 81,1661
-1665.
Sbarbati, A., Crescimanno, C., DeRossi, V., Bernardi, P. and Osculati, F. (1999) NADPH-diaphorase and NOS positive ganglion cells are found in the vallate papilla/von Ebner gland complex.Histochem. J. , 31,417 -424.[Web of Science][Medline]
Sbarbati, A., Crescimanno, C., Bernardi, P., Benati, D., Merigo, F. and Osculati, F. (2000) Postnatal development of the intrinsic nervous system in the circumvallate papilla-vonEbner gland complex. Histochem. J., 32,483 -488.[Web of Science][Medline]
Tachibana, T. (1995) The Merkel cells: recent findings and unresolved problems. Arch. Histol. Cytol., 58,379 -396.[Web of Science][Medline]
Toda, N. and Okamura, T. (1991) Role
of nitric oxide in neurally induced cerebroarterial relaxation. J.
Pharmacol. Exp. Ther., 258,1027
-1032.
Toyoshima, K. (1989) Fine structural and histochemical study of lingual taste organs of Rana catesbiana (Anura: Ranidae) transplanted to liver. J. Morphol.,200 , 29-36.
Zaccone, G. (1986) Neuron-specific enolase and serotonin in the Merkel cells of conger-eel (Conger conger) epidermis. Histochemistry, 5,29 -34.
Zaccone, G., Fasulo, S., Ainis, L., Mauceri, A., Licata, A. and Lauriano, E.R. (1995) Enkephalin immunoreactivity in the paraneurons of the tiger salamander (Ambystoma tigrinum) tongue. Neuropeptides, 28,257 -260.[Web of Science][Medline]
Zaccone, G. Kapoor, B.G., Fasulo, S. and Ainis, L. (2001) Structural, histochemical and functional aspects of the epidermis of fishes. Adv. Mar. Biol.,40 , 255-347.
Zaccone, G., Mauceri, A., Ainis, L., Licata, A. and Fasulo, S. (2002) Nitric oxide synthase in the gill and air sac of the Indian catfish Heteropneustes fossilis. In Kapoor, B.G., Arratia, G., Chardon, M. and Diogo, R.(eds), Catifishes. Oxford and IBH Publishing, New Delhi, Calcutta, in press.
Zancanaro, C., Sbarbati, A., Bolner, A., Accordini, C., Piemonte, G. and Osculati, F. (1997) Biogenic amines in the taste organ. Chem. Senses,20 , 329-335.[Medline]
Accepted August 30, 2002
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  T. Nakamura, Y. Murata, M. Mashiko, K. Okano, H. Satoh, M. Ozaki, and T. Amakawa The Nitric Oxide-Cyclic GMP Cascade in Sugar Receptor Cells of the Blowfly, Phormia regina Chem Senses, January 1, 2005; 30(suppl_1): i281 - i282. [Full Text] [PDF]
|
|
|
|
|
|
Y. Murata, M. Mashiko, M. Ozaki, T. Amakawa, and T. Nakamura Intrinsic Nitric Oxide Regulates the Taste Response of the Sugar Receptor Cell in the Blowfly, Phormia regina Chem Senses, February 1, 2004; 29(1): 75 - 81. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||