Filiform papillae as a sensory apparatus in the tongue: An immunohistochemical study of nervous elements by use of neurofilamcnt protein (NFP) and S-100 protein antibodies

Summary Nervous elements supplying the filiform papillae of the tongue of cattle and rats were investigated using immunohistochemistry against neurofilament protein (NFP) and glia-specific S-100 protein. The rod-shaped bovine filiform papillae were heavily keratinized along their entire length and lacked the connective tissue core that occurs in other mammals. Instead, the core was located posterior to the filiform papilla. The base of the bovine filiform papillae was invaded vertically by laminar connective tissue papillae. The core contained a large number of NFP-positive nerve fibers, most of them terminating as free endings in its anterior margin. NFP-positive nerves gathered around the anterior ridge of the epithelium at the base of the core and occasionally penetrated into the epithelium. The laminar connective tissue papillae at the base of the filiform papilla also contained NFP-positive nerve fibers. The core contained S-100-immunoreactive lamellated corpuscles, which were identified as simple corpuscles in electron micrographs. The structure and innervation of the bovine filiform papilla suggest that they represent a specialized sensory apparatus. The pyramidal filiform papillae of the rat were smaller, each containing a simple connective tissue core. Few NFP-positive nerve fibers from the nerve plexus entered the core. Filiform papillae are thus less specialized in rats than in cattle.     

Distribution of nerve fibers immunoreactive to neurofilament protein in rat molars and periodontium

Summary The distribution of nerve fibers in molars, periodontal ligament and gingiva of the rat shows a complex pattern. Decalcified material including the alveolar bone was sectioned in three different planes and stained by means of immunohistochemistry for detection of the neurofilament protein (NFP); the immunoreactive neural elements were clearly visualized in three-dimensional analyses. NFP-positive nerve fibers formed a subodontoblastic plexus in the roof area of the dental pulp; some of them entered the predentin and dentin directly through the dentinal tubules. This penetration was found mainly in the pulp horn, and was limited to a distance of about 100 m from the pulpo-dentinal junction. In the periodontal ligament, NFP-positive nerve fibers were found densely distributed in the lower half of the alveolar socket. Two types of nerve terminals were recognized in the periodontal ligament: free nerve endings with tree-like ramifications, and expanded nerve terminals showing button- or glove-like shapes. The former tapered among the periodontal fibers, some even reaching the cementoblastic layer. The latter were located, frequently in groups, within the ligament restricted to the lower third of the alveolar socket. A well-developed plexus of NFP-positive nerves was revealed in the lamina propria of the free gingiva, the innervation being denser toward the epithelium of the gingival crevice. The characteristic distribution of NFP-immunoreactive nerve fibers revealed in this study is discussed in relation to region-specific sensations in the teeth and surrounding tissues.     

Innervation of periodontal ligament and dental pulp in the rat incisor: An immunohistochemical investigation of neurofilament protein and glia-specific S-100 protein

Summary Nervous elements in the periodontal ligament and dental pulp of rat incisors were investigated by means of immunohistochemistry for neurofilament protein (NFP) and glia-specific S-100 protein. The periodontal ligament in the incisors was densely innervated by NFP-immunoreactive nerve fibers; the distribution of the nerve fibers and their terminations differed markedly from those in molars. NFP-positive, thick nerve bundles entered the lingual periodontal ligament through slits located in the mid-region of the alveolar socket, and immediately formed numerous Ruffini-like corpuscles. In the labial periodontal ligament, all of the NFP-immunoreactive nerve fibers terminated in free endings. The restricted location of the stretch receptor, Ruffini-like corpuscle, in the lingual periodontal ligament appears to be an essential element, because this region is regularly extended during mastication. The nervous elements were restricted to the alveolar half of the periodontal ligament in every region; they avoided the dental half of the periodontal ligament, which presumably moves continuously with the tooth. Pulpal nerve fibers in incisors also showed a characteristic distribution different from those in molars; individual nerve fibers with beaded structures ran in the center of the pulp toward the incisai edge, and did not form the subodontoblastic nerve plexus of Raschkow.Immunostaining for S-100 protein revealed a distribution pattern of nervous elements similar to that for NFP, suggesting that the nerves supplying the periodontal ligament and dental pulp were mostly covered by a Schwann sheath.     

Neuron-specific enolase,neurofilament protein and S-100 protein in the olfactory mucosa of human fetuses

Summary Immunohistochemical examination for neuronspecific enolase (NSE), neurofilament protein (NFP), and S-100 protein was performed in the olfactory mucosa of human fetuses. NSE and NFP immunoreactivities were found in the olfactory receptor cells, while no S-100 immunoreactive cells were recognized within the olfactory epithelium. The anti-NSE serum stained various types of nerve bundles in the lamina propria mucosae; a population of the NSE-positive nerve bundles was also immunoreactive for NFP. The anti-S-100 serum clearly demonstrated Schwann cells associated with the nerve fibers in the lamina propria mucosae. These findings 1) suggest a possibility of NSE and NFP as new marker substances for olfactory cells and 2) indicate that immunohistochemistry is a useful tool to analyse the cellular components of the olfactory organs in normal and pathological conditions.     

Inhibition of aberrant and constitutive phosphorylation of the high-molecular-mass neurofilament subunit by CEP-1347 (KT7515), an inhibitor of the stress-activated protein kinase signaling pathway

Previous studies have implicated stress-activated protein kinases (SAPKs) in aberrant phosphorylation of the high-molecular-mass neurofilament subunit (NFH). We now present direct evidence for this involvement using CEP-1347, a specific inhibitor of SAPK activation. Inhibition by this drug of stress-induced phosphorylation of NFH and the middle-molecular-mass neurofilament subunit in the perikaryon of dorsal root ganglion (DRG) neurons paralleled the decrease in levels of activated SAPKs and was essentially complete at 1 microM: CEP-1347. In addition, a role for SAPKs in the constitutive phosphorylation of NFH was demonstrated. Longterm treatment of unstressed DRG neurons with CEP-1347 lowered the steady-state phosphorylation level of NFH in neurites. No such effect was seen in neurons treated with PD 098059, which blocks activation of extracellular signal-regulated kinase 1/2. DRG neurites were shown to contain high basal levels of activated SAPKs. These included a 55-kDa SAPK whose activation was completely abolished at 0.05 microM: CEP-1347 and a 45-kDa SAPK that was not affected by the drug. These results indicate that SAPKs are involved in both stress-induced and constitutive phosphorylation of NFH. The differing responses of SAPKs in neurites and cell bodies to CEP-1347 inhibition further suggest the presence of different signaling pathways in the two neuronal compartments.     

Developmental appearance of dentin matrix protein 1 during the early dentinogenesis in rat molars as identified by high-resolution immunocytochemistry

Dentin matrix protein 1 (DMP 1) is an acidic phosphoprotein that has been postulated to play an important role in mineralized tissue formation. We have examined rat molar tooth germs by applying a high-resolution immunocytochemical approach with the purpose to identify the temporal and spatial localization of DMP 1 at the onset of dentinogenesis. Upper molar tooth germs of 2- to 3-day-old Wistar rats were fixed in a cacodylate-buffered 0.1% glutaraldehyde + 4% formaldehyde fixative, left unosmicated and embedded in LR White resin. The sections were incubated with a polyclonal DMP 1 antibody for postembedding colloidal gold immunolabeling and examined in a Jeol 1010 transmission electron microscope. The earliest localization of DMP 1 was in the Golgi region as well as in the nucleus of differentiating odontoblasts. When mineralization spread from matrix vesicles to the surrounding matrix, DMP 1 was extracellularly detected around the mineralizing globules. In the regions of fully mineralized mantle dentin, it was present in the mineralized regions, mainly around the peritubular dentin. The appearance of DMP 1 during early dentinogenesis implies a direct role for this protein in both odontoblast differentiation and matrix mineralization.     

Major phosphorylation site (Ser55) of neurofilament L by cyclic AMP-dependent protein kinase in rat primary neuronal culture

Ser55 of neurofilament L (NF-L) is reported to be partly phosphorylated in neurons and to be phosphorylated by cyclic AMP-dependent protein kinase (PKA). Bovine NF-L was phosphorylated by PKA in a low concentration of MgCl2 (0.3 mM) and digested by trypsin. Trypsin-digested fragments were assigned by MALDI/ TOF (matrix-assisted laser desorption and ionization/ time-of-flight) mass spectrometry. Phosphorylation sites were found at Ser41, Ser55, and Ser62 in the head region, with Ser55 considered the preferred site. A site-specific phosphorylation-dependent antibody against Ser55 rendered NF-L phosphorylated at Ser55 detectable in primary cultured rat neurons. One-hour treatment with 20 nM okadaic acid increased the phosphorylation level of Ser55, and co-treatment with 10 microM forskolin enhanced it. However, forskolin alone did not elevate the phosphorylation level. As a consequence, NF-L may be phosphorylated at Ser55 by PKA or by a PKA-like kinase in vivo; however, the phosphorylation level of Ser55 may be modulated by certain phosphatases sensitive to okadaic acid.     

Immunohistochemical localization of a gap junction protein (connexin43) in the muscularis externa of murine,canine, and human intestine

Electron-microscopic studies have revealed a heterogeneous distribution of gap junctions in the muscularis externa of mammalian intestines. This heterogeneity is observed at four different levels: among species; between small and large intestines; between longitudinal and circular muscle layers; and between subdivisions of the circular muscle layer. We correlated results obtained with two immunomethods, using an antibody to the known gap-junctional protein (connexin43) with ultrastructural findings, and further evaluated the respective sensitivity of these two approaches. For comparative reasons we also included the vascular smooth muscle of coronary arteries into our study. Two versions of the immunotechnique (peroxidase-antiperoxidase and fluorescence methods) were applied to frozen sections of murine, canine, and human small and large intestines, as well as to pig coronary artery. In the small intestine of all three species a very strong reactivity marked the outer main division of the circular muscle layer, while the longitudinal muscle layer as well as the inner thin division of the circular muscle layer were negative. In murine and human colon both muscle layers were negative, while in canine colon the border layer between the circular muscle and the submucosa reacted strongly, and scattered activity was found in the portion of the circular muscle layer (one tenth of its thickness) closest to the submucosa. The remainder of the circular muscle layer and the entire longitudinal muscle layer were negative in the canine colon. In the coronary artery we could not confirm the positive, specific labeling reported by other investigators (l.c.). In conclusion, we found close correlations at all four above-mentioned levels in the distribution of gap junctions in the gut musculature, as determined by binding of anticonnexin43 in comparison to conventional ultrastructural studies. Since no significant immunostaining was found in (i) the outer border of the circular muscle layer of the canine colon and (ii) the border layer between the submucosa and the circular muscle layer of human colon, where rare gap junctions have been identified at the ultrastructural level, we conclude that the electron-microscopic analysis is the more sensitive of the two methods.     

Immunohistochemical and ultrastructural localisation of peptide-containing nerves and myocardial cells in the human atrial appendage

Summary The innervation and myocardial cells of the human atrial appendage were investigated by means of immunocytochemical and ultrastructural techniques using both tissue sections and whole mount preparations. A dense innervation of the myocardium, blood vessels and endocardium was revealed with antisera to general neuronal (protein gene product 9.5 and synaptophysin) and Schwann cell markers (S-100). The majority of nerve fibres possessed neuropeptide Y immunoreactivity and were found associated with myocardial cells, around small arteries and arterioles at the adventitial-medial border and forming a plexus in the endocardium. Subpopulations of nerve fibres displayed immunoreactivity for vasoactive intestinal polypeptide, somatostatin, substance P and calcitonin gene-related peptide. In whole-mount preparations of endocardium, substance P and calcitonin gene-related peptide immunoreactivities were found to coexist in the same varicose nerve terminals. Ultrastructural studies revealed the presence of numerous varicose terminals associated with myocardial, vascular smooth muscle and endothelial cells. Neuropeptide Y immunoreactivity was localised to large electron-dense secretory vesicles in nerve terminals which also contained numerous small vesicles. Atrial natriuretic peptide immunoreactivity occurred exclusively in myocardial cells where it was localised to large secretory vesicles. The human atrial appendage comprises a neuroendocrine complex of peptidecontaining nerves and myocardial cells producing ANP.     

Production of an antiserum against cyclic nucleotide phosphodiesterase and its use for the immunocytochemical demonstration of this enzyme in rat cerebellum

Summary A procedure for the separation of cyclic AMP phosphodiesterase from a commercially available preparation and for raising antibodies against this enzyme in rabbits is described. An antiserum thus obtained was used for the immunocytochemical detection of cyclic nucleotide phosphodiesterase in rat cerebellum. The molecular layer, the granular layer and the cerebellar white matter exhibited different degrees of immunoreactivity. Only a few cell bodies (possibly glial cells) were stained. Most of the antigenic sites were present in the neuropil of the molecular layer and around Purkinje cells. Cerebellar glomeruli, sites of synaptic interactions between mossy fibres, Golgi cells and granule cells, were also stained by this antiserum. Control reactions using preimmune serum were consistently negative.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthday.     

Production of an antiserum against cyclic nucleotide phosphodiesterase and its use for the immunocytochemical demonstration of this enzyme in rat cerebellum

A procedure for the separation of cyclic AMP phosphodiesterase from a commercially available preparation and for raising antibodies against this enzyme in rabbits is described. An antiserum thus obtained was used for the immunocytochemical detection of cyclic nucleotide phosphodiesterase in rat cerebellum. The molecular layer, the granular layer and the cerebellar white matter exhibited different degrees of immunoreactivity. Only a few cell bodies (possibly glial cells) were stained. Most of the antigenic sites were present in the neuropil of the molecular layer and around Purkinje cells. Cerebellar glomeruli, sites of synaptic interactions between mossy fibres, Golgi cells and granule cells, were also stained by this antiserum. Control reactions using preimmune serum were consistently negative.     

Central projections of fibers in the auditory and tensor nerves of cicadas (Homoptera: Cicadidae)

Summary The auditory and tensor nerves of cicadas are mixed nerves containing both afferent and efferent elements. In 17-year cicadas, and in Okanagana rimosa, the auditory nerve contains afferents from body hairs, from the detensor tympani-chordotonal organ, and some 1300–1500 afferents from the hearing organ. Within the fused metathoracic-abdominal ganglionic complex the receptors from both the auditory and tensor nerves form a neuropilar structure that reveals the metameric organization of this complex. A few fibers run anteriorly, projecting into the meso and prothoracic ganglia. Within the ganglionic complex a division of auditory nerve afferents into a dense intermediate and a more diffuse ventral neuropile is observed. In addition, a dorsal motor neuropile is outlined by arborizations of the timbal motor neuron. This neuron is one of several efferent cell types associated with the auditory nerve, and there is an indication that several efferent fibers innervate the timbal muscle. There is anatomical evidence for a possible neuronal coupling between the bilaterally symmetrical large timbal motor neurons. In general, central projections from the auditory and tensor nerves support evidence of a structural layering within the CNS of insects.     

Distribution of the vanilloid (capsaicin) receptor type 1 in the human stomach

Vanilloid receptor type 1 (TRPV1) is expressed in a capsaicin-sensitive and peptide-containing sub-population of primary sensory nerves that in the rat stomach seems involved in regulation of chlorhydropeptic secretion and gastroprotection. Our aim was to identify which cell types express TRPV1 in the human stomach in order to gain a better insight in the role of this receptor in the regulation of HCl secretion. Immunohistochemistry, by using three different commercially available anti-capsaicin antibodies, in situ hybridisation and Western blot analysis were performed on fragments surgically obtained from the gastric body on the large curvature. TRPV1 labelling was found in the parietal cells at the level of intra-cytoplasmatic granules matching mitochondrial features and distribution. Immunolabelled neurons and nerve fibres were also seen, the latter numerous in the submucosa and mucosa and often ending close to the parietal cells. TRPV1 presence was confirmed by Western blot analysis and in situ hybridisation. TRPV1 presence in nerve structures and parietal cells suggests the possibility of a combined effect of both neuronal and epithelial TRPV1 on chlorhydropeptic secretion. The presumed TRPV1 mitochondrial location inside parietal cells is in favour of the existence of a local pathway of auto-regulation of HCl secretion. Therefore, TRPV1 might modulate chlorhydropeptic secretion in the human stomach through more complex pathways than previously thought.     

Immunohistochemical localization of glial fibrillary acidic protein (GFAP) and vimentin in the subcommissural organ of the Mongolian gerbil (Meriones unguiculatus)

Summary The chemical composition of intermediate filaments (IF's) in the ependyma of the subcommissural organ (SCO) of the Mongolian gerbil (Meriones unguiculatus) was investigated immunohistochemically in paraffin-embedded tissue. Antibodies against glial fibrillary acidic protein (GFAP), vimentin, neurofilament proteins and cytokeratins were used. Only GFAP and vimentin were detected in the non-specialized diencephalic ependyma and in the ependymocytes of the SCO. Staining could be observed in apical and basal processes of the SCO-cells. The latter processes extended into the posterior commissure up to the subpial surface, thus establishing a well-developed leptomeningeal route of ependymal projections. In contrast to the homogeneous vimentin-labeling, the SCO was particularly immunoreactive for GFAP in its lateral aspects and in the supraand precommissural parts. The coexpression of GFAP and vimentin in a subclass of SCO-ependymocytes was demonstrated on differentially immunostained semithin sections. The present study confirms the glial nature of the SCO-ependyma, which has been a matter of debate recently. It appears from this investigation that the high degree of secretory activity in the SCO does not necessarily lead to the disappearance of glial IF proteins. Moreover, the SCO-cells belong to the expanding group of mature astroglia, which is characterized by coexpression of GFAP and vimentin. The morphological similarity between SCO-ependymocytes and tanycytes is underscored by their common immunoreactivity against these two IF proteins. In view of the absence of GFAP from the rat SCO, interspecific differences must be considered in the evaluation of the IF protein composition.     

Sequence of protein expression of bone sialoprotein and osteopontin at the developing interface between repair cementum and dentin in human deciduous teeth

Experimental periodontal regeneration studies have revealed the weak binding of repair cementum to the root surface, whereas attachment of cementum to dentin preconditioned by odontoclasts appears to be superior. The aim of this study has been, therefore, to analyze the structural and partial biochemical nature of the interface that develops between resorbed dentin and repair cementum by using human deciduous teeth as a model. Aldehyde-fixed and decalcified tooth samples were embedded in acrylic or epoxy resins and sectioned for light and transmission electron microscopy. Antibodies against bone sialoprotein (BSP) and osteopontin (OPN), two noncollagenous proteins accumulating at hard tissue interfaces in bone and teeth, were used for protein A-gold immunocytochemistry. Light microscopy revealed a gradually increasing staining intensity of the external dentin matrix starting after the withdrawal of the odontoclast. Labeling for both BSP and OPN was first detected among the exposed collagen fibrils and in the intratubular dentin matrix when odontoclasts had withdrawn but mesenchymal cells were present. Subsequently, collagen fibrils of the repair cementum were deposited concomitantly with the appearance of labeling for BSP and OPN over the intratubular, intertubular, and peritubular dentin matrix. Labeled mineralization foci indicated the advancing mineralization front, and the collagenous repair matrix became integrated in an electron-dense organic material that showed labeling for BSP and OPN. Thus, no distinct planar interfacial matrix layer lies between the resorbed dentin and the repair cementum. The results suggest that odontoclasts precondition the dentin matrix such that the repair cementum becomes firmly attached.This study was supported by the Clinical Research Foundation (CRF) for the Promotion of Oral Health, University of Berne, Berne, Switzerland.     

The thoracic muscular system and its innervation in third instar Calliphora vicina Larvae. II. Projection patterns of the nerves associated with the pro‐ and mesothorax and the pharyngeal complex

We describe the anatomy of the nerves that project from the central nervous system (CNS) to the pro‐ and mesothoracic segments and the cephalopharyngeal skeleton (CPS) for third instar Calliphora larvae. Due to the complex branching pattern we introduce a nomenclature that labels side branches of first and second order. Two fine nerves that were not yet described are briefly introduced. One paired nerve projects to the ventral arms (VAs) of the CPS. The second, an unpaired nerve, projects to the ventral surface of the cibarial part of the esophagus (ES). Both nerves were tentatively labeled after the structures they innervate. The antennal nerve (AN) innervates the olfactory dorsal organ (DO). It contains motor pathways that project through the frontal connectives (FC) to the frontal nerve (FN) and innervate the cibarial dilator muscles (CDM) which mediate food ingestion. The maxillary nerve (MN) innervates the sensory terminal organ (TO), ventral organ (VO), and labial organ (LO) and comprises the motor pathways to the mouth hook (MH) elevator, MH depressor, and the labial retractor (LR) which opens the mouth cavity. An anastomosis of unknown function exists between the AN and MN. The prothoracic accessory nerve (PaN) innervates a dorsal protractor muscle of the CPS and sends side branches to the aorta and the bolwig organ (BO) (stemmata). In its further course, this nerve merges with the prothoracic nerve (PN). The architecture of the PN is extremely complex. It innervates a set of accessory pharyngeal muscles attached to the CPS and the body wall musculature of the prothorax. Several anastomoses exist between side branches of this nerve which were shown to contain motor pathways. The mesothoracic nerve (MeN) innervates a MH accessor and the longitudinal and transversal body wall muscles of the second segment. J. Morphol. 271:969–979, 2010. © 2010 Wiley‐Liss, Inc.     

Immunohistochemical demonstration of cyclooxygenase-2 (COX-2) and prostaglandin receptors EP2 and FP expression in the bovine intercaruncular uterine wall around term

During parturition, uterine-derived prostaglandins (PG) play an outstanding role regarding the functional elimination of the corpus luteum and the promotion of uterine contraction. The rate-limiting enzyme cyclooxygenase-2 (COX-2), highly regulated in a cell-type and localization specific manner throughout pregnancy, is involved in uterine prostanoid production. Prostaglandins exert their effects via G-protein-coupled receptors. Distribution and cellular localization of these receptors are decisive factors for prostaglandin-mediated actions. Since both COX-2 and PG receptors have only been assessed during pregnancy in the cow, these parameters were localized immunohistochemically near term to evaluate their specific role at parturition. Thus, during two periods, segments of the intercaruncular uterine wall were collected from cows at slaughter being eight and nine months pregnant, from cattle during caesarean section, and after spontaneous calving.

Results reveal that COX-2 was mainly localized in the cytoplasm of surface epithelial cells with a high expression in animals with induced parturition. The enzyme could also be found in lower concentrations within the glandular epithelium without any effect of gestational time or labour. In contrast to relaxant prostaglandin E receptor type 2 (EP2), not showing any change in all tissue layers observed, contractile prostaglandin F₂ receptor (FP) was modulated during the peripartal period revealing a peak expression in animals with induced parturition. FP was localized in surface and glandular epithelial cells as well as in endometrial stroma and myometrial smooth muscle cells.

Our study indicates that labour and induction of parturition may have an effect on amounts of immunohistochemically detectable COX-2 and FP. EP2 remains rather unchanged during the peripartal period. COX-2 and FP thus contribute via changes in amount and distribution to mechanisms associated with parturition.     

Partial cloning and production of polyclonal antiserum against recombinant capsid protein of Hepatopancreatic Parvovirus (HPV) and its application for diagnostics in penaeid shrimp

Hepatopancreatic Parvovirus (HPV) causes infection in the early stages of shrimp leading to retarded growth, ultimaltely resulting in monetary loss to the shrimp farmers. To over come this situation screening of post-larvae (PL) by immunology-based diagnostics is required. Hence, the specific gene of capsid protein for HPV was cloned into pRSET B expression vector and rHCP overexpressed with 6-histidine tagged fusion protein in Escherichia coli BL21(DE3). Immunology-based methods like Western blot, dot blot and ELISA techniques were employed to detect HPV in infected samples using the antiserum raised in rabbits against recombinant HCP of HPV. The dot blot assay using anti-rHCP was found to be capable of detecting HPV in HPV infected post-larvae as early as at 24 h post infection. The antiserum could detect the HPV in the infected samples at 1 ng of total protein. HPV infection estimated by ELISA using anti-HCP and pure r-HCP as a standard was found to increase gradually during the course of infection from 24 h post infection. The sensitivity of antibody-based diagnostics employed in the present study was compared with that of PCR diagnostic method to screen the post-larvae for the detection of HPV.     

Vasoactive intestinal polypeptide (VIP) in the pig pancreas: role of VIPergic nerves in control of fluid and bicarbonate secretion

Vasoactive intestinal polypeptide (VIP) in the pig pancreas is localized to nerves, many of which travel along the pancreatic ducts. VIP stimulates pancreatic fluid and bicarbonate secretion like secretin. Electrical vagal stimulation in the pig causes an atropine-resistant profuse secretion of bicarbonate-rich pancreatic juice. In an isolated perfused preparation of the pig pancreas with intact vagal nerve supply, electrical vagal stimulation caused an atropine-resistant release of VIP, which accurately parallelled the exocrine secretion of juice and bicarbonate. Perfusion of the pancreas with a potent VIP-antiserum inhibited the effect of vagal stimulation on the exocrine secretion. It is concluded, that VIP is responsible for (at least part of) the neurally controlled fluid and bicarbonate secretion from the pig pancreas.