The effect of in vitro stimulation of rat parotid gland with the neuropeptides substance P, calcitonin gene-related peptide and galanin has been studied by microfilament fluorescence staining and in semithin sections, and compared to control incubations and in vitro stimulation with b&shy;adrenergic and muscarinic agonists. Clear-cut aspects of massive granule exocytosis and cytoplasm vacuolation, indicative of protein and fluid secretion respectively, were obvious only after substance P stimulation, whereas treatment with galanin and calcitonin gene-related peptide produced little to no morphological changes. The results being in agreement with the outcome of other methodological approaches, these procedures appear reliable, may be effectively applied to the study of the functional regulation of secretory mechanisms, and may be particularly useful in human tissue analyses

Del Fiacco, Marina

Lantini, M. Serenella

Quartu, Marina

Serra, M. Pina

Firenze University Press: E-Journals

© 2010 Firenze University Press 
ht tp://www.fupress .com/ijae
ITALIAN JOURNAL OF ANATOMY AND EMBRYOLOGY
IJAE 
Vo l .  115,  n .  1/2 :  59 - 64,  2010
Morphological changes induced by neuropeptide in 
vitro stimulation of the rat parotid gland
Marina Del Fiacco*, M. Serenella Lantini, M. Pina Serra, Marina Quartu
Dipartimento di Citomorfologia, Università di Cagliari, Cittadella universitaria di Monserrato, Monserrato (CA), 
Italy. *Corresponding author, Email: dfi acco@unica.it
Presented at a meeting in honour of Prof. G. Orlandini, Florence, February 15, 2010
Summary 
The effect of in vitro stimulation of rat parotid gland with the neuropeptides substance P, calci­
tonin gene­related peptide and galanin has been studied by microfi lament fl uorescence staining 
and in semithin sections, and compared to control incubations and in vitro stimulation with 
b­adrenergic and muscarinic agonists. Clear­cut aspects of massive granule exocytosis and cyto­
plasm vacuolation, indicative of protein and fl uid secretion respectively, were obvious only 
after substance P stimulation, whereas treatment with galanin and calcitonin gene­related pep­
tide produced little to no morphological changes. The results being in agreement with the out­
come of other methodological approaches, these procedures appear reliable, may be effectively 
applied to the study of the functional regulation of secretory mechanisms, and may be particu­
larly useful in human tissue analyses.
Key words 
Substance P, calcitonin gene­related peptide, galanin, immunohistochemistry, F­actin staining, 
semithin sections
Introduction
Saliva is a watery solution of electrolytes, small organic substances and a varie­
ty of proteins and glycoproteins with enzymatic, lubricating, defensive and protec­
tive functions. Salivary secretion is controlled by the synergistic activity of the two 
autonomic nervous system sections. The stimulation of parasympathetic muscarinic 
receptors causes a copious secretion of water and electrolytes, via Ca2+ mobilization 
from intracellular stores, whereas the stimulation of sympathetic b­adrenergic recep­
tors produces a small amount of secretion rich in exocytosed proteins, via c­AMP as 
second messenger (Schramm and Selinger, 1975). Morphological studies on rat sali­
vary glands show that a dramatic sequential reorganization of the luminal acinar cell 
membrane accompanies exocytosis (Amsterdam et al., 1969) and that stimulation of 
either b­adrenergic or muscarinic receptors induces two distinct exocytosis­coupled­
to­endocytosis mechanisms (Segawa and Riva, 1996). In fact, b­adrenergic agonists, 
together with extensive degranulation of acinar cells, cause the enlargement of the 
luminal space and the appearance, along the apical membrane, of many W­shaped 
contours enwrapped by microfi laments, whose size is similar to that of the secretory 
granules (Segawa et al., 2000). By contrast, muscarinic agonists elicit the growth of 
cytoplasmic vacuoles, whose membrane is not surrounded by microfi laments, con­
60 Marina Del Fiacco, M. Serenella Lantini, M. Pina Serra, et al.
nected to the luminal membrane and space (Segawa et al., 2000). On the other hand, 
besides the classical cholinergic and adrenergic neuromediators, numerous peptides, 
collectively designated as non­adrenergic non­cholinergic (NANC) agonists, are 
known to participate with different effectiveness in the secretory mechanisms and 
hence influence the composition of saliva (Gallacher, 1983; Ekström, 1989). Among 
them, substance P (SP) has a potent sialogogic activity in some species, including rat 
(Lembeck and Starke, 1968; Ekström, 1987), a property that has been exploited as a 
bioassay to obtain its purification (Leeman and Hammerschlag, 1967; Chang and Lee­
man, 1970), whereas calcitonin gene­related peptide (CGRP) may affect the activity of 
other sialogogic peptides, but causes no secretion on its own (Ekström, 1987; Ekström 
et al., 1988), and galanin (GAL) may be functionally relevant in some rat salivary 
glands (Konopka et al., 2004; Szőke et al., 2009).
In this study, the morphological changes consequent to the in vitro stimulation of 
the rat parotid gland with the neuropeptides SP, CGRP and GAL have been examined 
and compared to those induced by b­adrenergic and muscarinic agonists. The occur­
rence of those peptides in the gland was also checked by immunohistochemistry.
Materials and methods
Parotid glands of 10­15 week old male Wistar rats were used. 
Immunohistochemistry
Three rats were transcardially perfused with phosphate­buffered 4% paraformal­
dehyde, pH 7.3, and the glands excised, postfixed in the same fixative for 2 hours 
and rinsed overnight in 0.1 M phosphate buffer, pH 7.3. Indirect immunofluorescence 
was elicited in 10­14 mm thick cryostat sections placed on coated slides, using as pri­
mary antiserum either a rat monoclonal antibody against SP (gift of Dr A.C. Cuello, 
diluted 1:50), a rabbit antiserum against CGRP (Peninsula, diluted 1:300), or a rabbit 
antiserum against GAL (CRB, diluted 1:1200). After overnight incubation and rinsing, 
the sections were incubated for 1 hour at room temperature with FITC­conjugated 
anti­rat or anti­rabbit secondary antisera, as appropriate. All antisera were diluted in 
phosphate buffered saline (PBS) containing 0.2% Triton X­100. Slides were mounted 
in glycerol/PBS 3:1 (v/v).
In vitro stimulation
Glands were rapidly excised from 3 rats and cut in small pieces. Samples were 
incubated at 37°C, for 45 min in constant agitation, in Dulbecco’s modified Eagles 
minimum medium (Segawa et al., 1985) added with either 20 mM D,L­isoproterenol 
(IPR), 100 mM carbachol (CCh), 10­6 mM SP, 10­6 mM CGRP and 10­6 mM GAL, or with­
out chemicals as controls. For microfilament F­actin staining, samples were fixed with 
phosphate­buffered 4% paraformaldehyde, pH 7.3, for 1 hour, rinsed in 0.1 M phos­
phate buffer, pH 7.3, cryostat cut in sections of 10­14 mm and stained with TRITC­
conjugated phalloidin (Molecular Probes, OR). For bright­field analysis, samples 
were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer, for 1 hour at room 
61Neuropeptide in vitro stimulation of rat parotid
temperature, postfixed in 1% OsO4 at 4 °C, for 1 hour, dehydrated and embedded in 
epoxy resin. Semithin sections were stained with toluidine blue. 
Slides were observed with a Leitz Dialux 20 microscope equipped with epifluores­
cence optics.
Results
Nerve fibres immunoreactive to SP, CGRP and GAL were found mainly around 
blood vessels and ducts, but also scattered in close proximity to acinar cells, with 
higher frequency for SP than for CGRP and GAL (Fig. 1A­E).
Analysis of granule exocytosis by microfilament F­actin staining showed that, 
compared to control incubations, where plasmalemmal contours appeared smooth 
and intercellular canaliculi and acinar lumen narrow and regular (Fig. 1F), in vitro 
stimulation with IPR caused the appearance of many W­shaped profiles along the 
luminal cell membrane and dilation of the acinar lumen (Fig. 1G), whereas CCh stim­
ulation did not change the aspect detectable in controls (Fig. 1 H). Treatment with SP 
resulted in widespread presence of numerous W­shaped profiles crowded towards a 
dilated lumen in many acini (Fig. 1I), whereas aspects of modifications of the luminal 
membrane were rare after treatment with GAL (Fig. 1J) and absent after treatment 
with CGRP (Fig. 1K). 
In toluidine blue­stained semithin sections of control specimens the acinar cells 
contained numerous secretion granules distributed in a wide area of the apical cyto­
plasm and the acinar lumen and interacinar space were narrow (Fig. 1L). Specimens 
stimulated in vitro with IPR showed conspicuous granule exocytosis, apicalization 
of the remaining secretory granules and enlargement of the luminal and interacinar 
space (Fig. 1M), and CCh treated samples showed many vacuoles in the acinar cell 
cytoplasm (Fig. 1N). Stimulation with SP resulted in dramatic degranulation and api­
calization of the remaining secretory granules accompanied by marked luminal space 
swelling, as well as in cytoplasm vacuolization (Fig. 1O). GAL treatment produced 
only a moderate degree of granule apicalization with sporadic and modest enlarge­
ment of the luminal space (Fig. 1P). Incubation with CGRP had no marked effects on 
granule density and distribution (Fig. 1Q).
Discussion
Our results on the occurrence and distribution of SP­ and CGRP­positive innerva­
tion of the rat parotid gland agree with previous data (Sharkey and Templeton, 1984; 
Ekström, 1987; Ekström et al., 1988), whereas, to our knowledge, GAL­positive nerve 
fibres, though reported in other rat salivary glands (Konopka et al., 2004), have not 
been described in this organ previously. The source of SP innervation is largely par­
asympathetic, whereas a substantial amount of the CGRP­containing fibres also has 
a sensory origin (Sharkey and Templeton, 1984; Ekström et al., 1988). The morpho­
logical changes ensuing from SP in vitro stimulation appear in agreement with data 
showing that in the presence of autonomic blockade SP evokes amylase release and 
fluid secretion from rat parotid (Gallacher, 1983) and that parasympathetic stimula­
62 Marina Del Fiacco, M. Serenella Lantini, M. Pina Serra, et al.
tion not only evokes a copious watery secretion, but also causes protein release and 
granule exocytosis via NANC mediators (Ekström et al., 1998; Ekström, 2002). 
In a similar way, the outcome after CGRP stimulation is in agreement with the 
fact that, contrary to SP, this peptide does not evoke any flow of saliva after intrave­
nous injection and is little effective in evoking amylase release in vitro (Ekström et 
al., 1988). As for GAL, a major effect on the rat parotid has not been detected, though 
its functional relevance can not be excluded. 
In conclusion, this morphological approach appears as a simple and reliable way 
to contribute effectively to functional studies on the regulation of gland activity and 
secretory mechanisms. Considering the high interspecific variability, which advises 
against the extrapolation to man of data obtained in experimental animals, it may be 
particularly useful when studying human tissues, as it may overcome the hindrance 
to systemic in vivo treatments. 
Aknowledgements 
We thank Ms S. Bernardini and Mr A. Cadau for skilled technical help.
References
Amsterdam A., Ohad I., Schramm M. (1969) dynamic changes in the ultrastructure of 
the acinar cell of the rat parotid gland during the secretory cycle. J. Cell Biol. 41: 
753­773.
Chang M.M., Leeman S.E. (1970) Isolation of a sialogogic peptide from bovine 
hypothalamic tissue and its characterization as substance P. J. Biol. Chem. 245: 
4784­4790.
Ekström J. (1987) Neuropeptides and secretion. J Dent Res. 66: 524­530.
Ekström J. (1989) Autonomic control of salivary secretion. Proc. Finn. Dent. Soc. 85: 
323­331.
Ekström J. (2002) Muscarinic agonist­induced non­granular and granular secretion of 
amylase in the parotid gland of the anaesthetized rat. Exp. Physiol. 87.2: 147–152.
Ekström J., Ekman R., Håkanson R., Sjögren S., Sundler F. (1988) 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: 
933­949.
Ekström J., Asztély A., Tobin G. (1998) Parasympathetic non­adrenergic, non­cholin­
ergic mechanisms in salivary glands and their role in reflex secretion. Eur. J. Mor­
phol. 36 Suppl:208­212.
Gallacher D.V. (1983) Substance P is a functional neurotransmitter in the rat parotid 
gland. J. Physiol. (Lond.) 342: 483­498.
Konopka L. M., May V., Forehand C. J. (2004) Galanin­like innervation of rat sub­
mandibular and sublingual salivary glands: origin and effect on acinar cell mem­
branes. J. Comp. Neurol. 317: 271–282.
Leeman S.E., Hammerschlag R. (1967) Stimulation of salivary secretion by a factor 
extracted from hypothalamic tissue. Endocrinol. 81: 803­810.
63Neuropeptide in vitro stimulation of rat parotid
Lembeck F., Starke K. (1968) Substanz P und speichelsekretion. Naunyn­
Schmiedeberg’s Arch. Pharmak. Exp. Path. 258: 375­385.
Schramm M., Selinger Z. (1975) The functions of cyclic AMP and calcium as alterna­
tive second messengers in parotid gland and pancreas. Cyclic Nuc. Res. 1: 181­192.
Segawa A., Riva A. (1996) Dynamics of salivary secretion studied by confocal laser 
and scanning electron microscopy. Eur. J. Morphol. 34: 215­219.
Segawa A., Sahara N., Suzuki K., Yamashina S. (1985) Acinar structure and mem­
brane regionalization as a prerequisite for exocrine secretion in the rat submandib­
ular gland. J. Cell Sci. 78: 67­85.
Segawa A., Loffredo F., Puxeddu R., Yamashina S., Testa Riva F., Riva A. (2000) Cell 
biology of human salivary secretion. Eur. J. Morphol. 38: 237­241. 
Sharkey K.A., Templeton D. (1984) Substance P in the rat parotid gland: evidence for 
dual origin from the otic and trigeminal ganglia. Brain Res. 304:392­396.
Szőke E., Zelles T., Boros I., Fehér E. (2009) Effects of raw soy diet on the rat parotid 
gland. Acta Alimentaria 38: 237­248.
64 Marina Del Fiacco, M. Serenella Lantini, M. Pina Serra, et al.
Fig. 1 – A-E: Nerve fibres immunoreactive to SP (A,B), CGRP (C,D) and GAL (E) in perivascular, periductal and 
periacinar position; A and C are semiconsecutive sections: the two asterisks label the same blood vessels and 
the star labels the same duct. F-K: microfilament fluorescence staining of parotid acini in vitro incubated as 
control (F) and with IPR (G), CCh (H), SP (I), GAL (J), and CGRP (K); arrows in G and I point to obvious aspects 
of granule exocytosis. L-Q: toluidine-blue stained semithin sections of samples in vitro incubated as control 
(L) and with IPR (M), CCh (N), SP (O), GAL (P), and CGRP (Q); acinar degranulation is remarkable in M and O; 
arrows in M, O, P point to apical granules and luminal space swellings; arrowheads in N and O point to sites 
of vacuolization. Scale bars: A-D = 50 mm; E = 25 mm; F-Q = 20 mm.
Figures


Morphological changes induced by neuropeptide in vitro stimulation of the rat parotid gland

http://www.fupress.net/index.php/ijae/article/download/8700/8105

Morphological changes induced by neuropeptide in vitro stimulation of the rat parotid gland

Abstract

Similar works

Full text

Available Versions

Firenze University Press: E-Journals