Histochemistry (1991) 96:153 156 0301556491000808 Histochemistry

© Springer-Verlag 1991

Neuromodulators of the lingual von Ebner gland: an immunocytochemical study I.M. Roberts 1, S.E. Solomon 1, O.A. Brusco 1, W. Goldberg 2, and J.J. Bernstein 2

1 Division of Gastroenterology, Departments of Medicine and 2 Physiology, George Washington University Medical Center, Room 5-401, 2150 Pennsylvania Ave., N.W., Washington, DC 20037, USA

Received July 31, 1990 / Accepted March 7, 1991

Summary. The serous lingual glands of von Ebner secrete lingual lipase, an enzyme that begins fat digestion in the stomach. The objective of this study was to charac- terize the neuromodulators in the rat tongue and von Ebner glands using immunocytochemical techniques. Rat lingual tissues were fixed in formalin, embedded in paraffin and sectioned at 4 gm for light microscopic studies. Immunocytochemical localization of neuromo- dulators was performed with monospecific anti-rat neu- romodulator IgG or control (preimmune) IgG as the primary antibody, using the peroxidase-antiperoxidase (PAP) technique. No staining was seen with control anti- rat IgG. Immunospecific staining for vasoactive intesti- nal peptide (VIP), tyrosine hydroxylase and choline ace- tyltransferase (CHAT) was observed in nerves in the tongue, and cells containing immunospecific staining for serotonin (5-hydroxytryptamine) were seen in the stroma between the lingual glands. Selected cells in the serous glands stained positively for the presence of substance P and somatostatin. Adrenergic, VIP-containing and cholinergic nerves appear to innervate the tongue and serous glands. Substance P and somatostatin were iden- tified in cells of the lingual serous glands and may be additional local modulators regulating lingual lipase re- lease.

Introduction

Lingual lipase, secreted by the serous von Ebner glands, is an important enzyme in intragastric fat digestion (Ro- berts et al. 1984; Roberts 1985). We have previously pur- ified rat lingual lipase to homogeneity and have studied the molecular properties of the enzyme (Roberts et al. 1984; Roberts 1985; Roberts and Jaffe 1986). The ultra- structure of the von Ebner gland resembles the pancreas and salivary glands (maxillary, submandibular and paro- tid). The yon Ebner gland contains acini which empty

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into ductules similar to those found in other serous exoc- rine glands (Hand •970). The pancreas and salivary glands have been shown to be innervated by both para- sympathetic (cholinergic) and sympathetic (adrenergic) nerves (Holst 1986; Williams •984). Parasympathetic tone is felt to be the most important neuromodulator that regulates pancreatic secretion, while the salivary glands contain major sympathetic inputs in addition to parasympathetic nerves (Holst 1986; Williams 1984). Al- though studies of the innervation of the von Ebner gland are sparse, Hand was able to identify nerve processes within the parenchyma of the gland (Hand 1970). Other investigators have suggested that lingual lipase may re- leased in response to cholinergic and adrenergic agents (Hamosh •978; Roberts and Jaffe •986; Ruellan et al. 1988). In order to further delineate the specific innerva- tion of the lingual von Ebner gland, we used the peroxi- dase-antiperoxidase 1Lechnique to determine which neu- romodulators are present in the rat tongue and von Ebner gland.

Materials and methods

Animals

Male Sprague-Dawley rats (240-260 g) were obtained from the Hilltop Laboratories, Scottdale, Pa., USA. Rats were fed a stan- dard laboratory diet and water ad libitum. Animals were fasted overnight before all experiments (see below). Rats were sacrificed using CO2 asphyxiation prior to the removal of tissue.

Chemicals

Antibodies to the following proteins were obtained from these ven- dors: Neurofilament 70 kDa protein (Chemicon International, E1 Segundo, Calif., USA), synaptophysin and tyrosine hydroxylase (Boehringer-Mannheim Biochemicals, Indianapolis, Ind., USA) CHAT (choline acetyl transferase), substance P, VIP (vasoactive intestinal peptide) and somatostatin (all from Incstar Corporation, Stillwater, Minn., USA). VIP (vasoactive intestinal peptide), sub- stance P and somatostatin were products of Sigma Chemical, St.

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Louis, Mo., USA). All chemicals used to make buffers were ob- tained from Sigma and were ACS-grade. All other reagents were of the highest purity available.

Irnmunocytochemical localization of lingual nerves

Light microscopy. Lingual tissues were prepared for light microsco- py as previously described (Roberts et al. 1986; Roberts et al. 1987). The specimens were processed on an Autotechnicon and embedded in Tissue Prep (paraffin and synthetic polymer). Sections were cut at 4~5 ~tm, hydrated through xylene and 95% alcohol to water. Endogenous tissue peroxidases were depleted with 1% hydrogen peroxide in methanol for 30 min. The PAP method was used detect the presence of neurons (using monoclonal antibody to neurofilament 70-kDa protein), synaptic vesicles (using antibody to synaptophysin), specific peptide neuromodulators (using anti- bodies to somatostatin, VIP, or substance P) or adrenergic and cholinergic nerves (using antibodies to CHAT and tyrosine hydrox- ylase, respectively). The "first" antibody consisted of one of the specific antibodies above. The "second" antibody was a goat or swine anti-rat IgG. Finally, a rat peroxidase-antiperoxidase was applied followed by the peroxidase substrate (diaminobenzidine). Immunospecific staining was not observed when sections were stained with either control preimmune serum or "second" anti- body without primary ("first") antibody. Kodak ASA 32 Pana- tomic X film was used in the camera and a 4~ 5 green filter was placed on the light source to enhance contrast.

Antisera

Antisera for substance P, VIP, and somatostatin were polyclonal and raised in rabbits. Pretreatment of a 1:100 dilution of anti- substance P with 100 gg of substance P completely eliminated stain- ing. There was no cross-reactivity of a 1:1000 dilution of VIP antisera tested against 100 pmole amounts of secretin, motilin, pan- creatic polypeptide, gastric inhibitory polypeptide, insulin, gluca- gon, somatostatin, gastrin, CCK-8, growth hormone releasing fac- tor, ACTH, serotonin, or substance P by the method of Larsson (Larsson 1981). Pretreatment of a 1:500 dilution of anti-VIP with 200 gg of VIP eliminated tissue staining. Anti-somatostatin stain- ing was completely eliminated by pretreatment of a 1 : 500 dilution of the antibody with 200 gg of somatostatin.

Antibodies to CHAT, tyrosine hydroxylase, neurofilament 70 kDa, and synaptophysin were all monoclonal. Anti-neurofila- merit 70 was an IgG1 raised in a mouse hybridoma ascites as de- scribed by Shaw and Chau (1988). Anti-CHAT was an IgG~ de- rived from a rat hybridoma that produced the antibody in response to bovine CHAT (Incstar). Anti-synaptophysin was an IgG1 pro- duced from a mouse hybridoma (Wiedenmann 1985). Anti-tyrosine hydroxylase was an IgG2a prepared from a mouse hybridoma (Rohrer et al. 1985). No staining was observed when control mouse IgG1 or IgGza were used as "first" antibodies.

Results

Nerve fibers were identified in the tongue and in the areas in and near the serous von Ebner glands (see Figs. 1-3). Application of " f i r s t " antibodies to neurofi- lament 70-kDa protein and synaptophysin, followed by " s e c o n d " ant ibody- immunoperoxidase complex, local- ized nerves successfully (Fig. 4); 70-kDa neurofi lament protein is a nerve-specific antigen (Lazarides 1980; Liem et al. 1978) and synaptophysin is a protein found only in neurosecretory granule membranes (DeCamilli et al.

1988; Wiedenmann and Frank 1985; Thomas etal . 1988). We used these markers to help definitively identify a cell as a neuron. Nerves that contained tyrosine hyd- roxylase, choline acetyl transferase (CHAT) and vasoac- rive intestinal peptide were then indentified in the rat tongue (Figs. 1-3). In addition, selected cells in the ser- ous von Ebner gland of the tongue stained with anti- bodies directed again substance P, somatostat in (Figs. 5 and 6) and serotonin (not shown).

Discussion

There is some evidence that lingual lipase secretion is stimulated by cholinergic and possibly adrenergic neu- romodula tors (Field and Hand 1987; Hamosh 1978; Ruellan et al. 1988). Field and Hand (1987) demon- strated evidence of endocytosis in the rat von Ebner gland after t reatment with carbachol. Hamosh (1978) found that total lipase content of the von Ebner gland diminished after administrat ion of isoprenaline, an ad- renergic agent. Ruellan et al. (1988) found that carba- chol, epinephrine and isoproternol all led to secretion of lingual lipase in vitro.

Salivary gland secretion is modulated by both choli- nergic and adrenergic agents (Ludford and Tulamo 1980). Cholinergic tone is a more important regulator of pancreatic secretion than adrenergic tone (Williams 1984; Holst 1986). We have demonstrated that adrener- gic nerves (containing the enzyme tyrosine hydroxylase), cholinergeric nerves (containing the enzyme C H A T ) and vasoactive intestinal peptide (VIP)-containing nerves are present in the rat tongue. Although cholinergic and ad- renergic nerves have been found to innervate both the pancreas and salivary glands, the presence of VIP-con- taining nerves in the tongue is of particular interest. VIP exerts a st imulatory influence on salivary, Brunner gland and pancreatic secretion in concert with cholinergic nerves (Bosshard et al. 1989). VIP may increase the af- finity of muscarinic receptors and may be co-localized in the same neurons as acetylcholine (Lundberg et al. 1982). Furthermore, acinar cells that contain somatosta- tin, substance P, and serotonin were also found in the von Ebner glands. These hormones might function as paracrine regulators of lingual lipase and amylase secre- tion. The endocrine cells of the Brunner 's glands of the duodenum have been also found to contain somatosta- tin, gastrin, and peptide-YY (Bosshard et al. 1989). Most investigators believe that somatostat in is an inhibitory regulator of glandular secretion (Walsh 1981).

In conclusion, the rat von Ebner gland resembles both the pancreas and the major salivary glands with respect to the presence of specific neuromodula tors and nerves that regulate secretory processes in the gastrointestinal tract. How somatostat in, VIP, substance P and serotonin may control von Ebner gland secretion is of particular interest. The degree to which cholinergic, adrenergic and gastrointestinal hormonal modulators influence lingual von Ebner gland secretion will require further explora- tion.

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Fig. 1. Cholinergic neurons (arrows) in the serous glands stained with anti-choline acetyltransferase (CHAT) (peroxidase-diamino- benzidine; x 450). This nerve is synapsing in the von Ebner gland tissue

Fig. 2. Cateeholamine-containing nerve (arrow) in the serous acini stained with anti-tyrosine hydroxylase (peroxidase-antiperoxidase; x 450)

Fig. 3. Vasoactive intestinal peptide (VIP)-containing nerve (ar- rows) in the rat tongue in a subepithelial location (peroxidase- diaminobenzidine; x 450)

Fig. 4. Synaptophysin staining neural vesicles (arrow) in the von Ebner gland (peroxidase-diaminobenzidine; x450). This stain identifies nerve-containing gland sections. Specific antineuromodu- lator staining can be performed subsequently

Fig. 5. Somatostatin-containing cells (arrows) in the rat serous von Ebner gland (peroxidase-diaminobenzidine; x 200)

Fig. 6. Substance P-containing cell (arrow) in the serous acini (per- oxidase-diaminobenzidine; x 450)

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Acknowledgements. Grant support: This work was supported by Cystic Fibrosis Foundation Research Grant ~ G191 and NIDKK FIRST Award q~ R29-DK38729 (to IMR).

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