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Localization of vasoactive intestinal polypeptide-receptor-immunoreactivity in human salivary glands
Neuropeptides (1992) 22,89-92 0 Longman GroupUK Ltd 1992
Localization of Vasoactive Intestinal PolypeptideReceptor-lmmunoreactivity in Human Salivary Glands W. M. HERBST*, W. KUMMERt and CH. HEYMt *Department of Dermatology, University of Erlangen-Niirnberg, Hartmannstr. 14, W-8520 Erlangen and tlnstitute for Anatomy and Cell Biology I, University of Heidelberg, Im Neuenheimer Fe/d 307, W-6900 Heidelberg
Abstract -A monoclonal VIP-receptor antibody derived from a human adenocarcinoma cell line (HT 29) was used in combination with immunogold silver staining for the immunohistochemical demonstration of VIP-receptor (ret) immunoreactivity (IR) in paraffinembedded human salivary glands (parotid, palatal, labial glands). VIP-ret-IR was localized to mucous endpieces of labial and-to a lesser extent - palatal glands, intercalated ducts of the parotid gland, and excretory ducts of all glands investigated. The findings correlate well with known effects of VIP on mucous release and electrolyte transport in salivary glands. Lack of VIP-ret-IR at serous acini may point to immunologically different receptor subtypes in these glands.
Introduction
and trachea (5, 11). Its effects are mediated by specific VIP-receptors coupled to adenylate cyclase and increasing intracellular CAMP content (12- 15). The distribution of VIP binding sites in secretory organs of different species has been studied by means of radiolabelled (I?) VIP and autoradiography without permitting a detailed analysis of the cellular distribution of the VIP-receptor. Therefore, a monoclonal VIP-receptor antibody derived from the human adenocarcinoma cell line HT 29 (16) was used in combination with immunogold silver staining for immunohistochemical demonstration of VIP-receptor immunoreactivity (VIP-ret IR) in paraffin-embedded human salivary glands. This antibody reduces VIP-induced CAMP production in HT 29 cells by 70% and competes with VIP in bind-
Vasoactive intestinal poly-peptide (VIP) - a regulatory peptide of 28 amino acids - was first isolated from porcine intestine by Said and Mutt in 1970 (1). It is frequently present in parasympathetic cholinergic nerves of salivary glands surrounding the acini and, to a smaller extent, the ducts and blood vessels (2). In salivary glands VIP causes an increase in blood flow and potentiates the secretomotor effect of acetylcholine (3- 10). Furthermore, VIP is known to be a powerful stimulator of epithelial chloride transport in several organs such as colon, pancreas
Date received 2 March 1992 Date accepted 2 March 1992
89
90
NEUROPEPTIDES
ing assays, thus it probably recognizes an epitope which may be closely related to but not fully occupy the binding region of the receptor (14, 16).
Material and Methods Samples of parotid, labial and palatal glands were supplied from the files of the Department of Dermatology, University of Erlangen-Niimberg. The specimens were fixed in 10% formalin, routinely dehydrated and embedded in paraffin. Sections were cut at 7 pm, deparaffiized and rinsed in phosphate buffered saline (PBS) containing 2% normal swine serum. Monoclonal rat anti-VIPreceptor (Dianova, FRG; clone 109, for ref. see Pichon et al. 1983; used at a concentration of 2.5-5 pg/ml) served as primary antibody. Incubation was carried out overnight at room temperature (r.t.). The rat anti-VIP-receptor was visualized by anti-rat IgG labelled with colloidal gold (Janssen, Olen, Belgium; 1:40; 1 h at r.t.) followed by silver enhancement (IntenSE MR; Janssen). As the preabsorption of the primary antibody was impossible (the epitope to which the rat anti-VIPreceptor has not been characterised), sections were incubated with normal rat serum instead of the monoclonal antibody or only with the detection system itself. Fig 1.
Results Parotid gland. Homogenous staining for VIP-recIR was observed at the intercalated ducts (Fig. 1a). The large excretory ducts also contained VIP-recIR cells, but they were irregularly mixed with nonreactive cells (Fig. lb). Striated ducts and serous acini were devoid of immunolabelling except a few single VIP-ret-IR cells located in the acini (Fig. lb). The exact identity of these cells could not be determined. They either constitute a particular subgroup of acinar cells or may represent the initial portion of the intercalated ducts. Pulutulgkznds. VIP-ret-IR in palatal glands showed considerable variability in the acinar regions: The mucous endpieces of some glands showed moderate VIP-ret-IR predominantly at their basal aspect (Fig. lc), whereas acini of other glands entirely
lacked VIP-ret-IR. Yet, it is not clear whether these changes are due to regional differences in the palatal glands or to technical difficulties which unavoidably occur when dealing with human material obtained at biopsy. As a consistent finding, however, VIPret-IR was observed in excretory ducts. Labial glands. The mucous acinar cells showed intense immunolabeling of the membranes, particularly at the luminal side, but also at the basolateral circumference. Granular immunoreaction products were also obtained in the cytoplasm (Fig. Id). Serous endpieces remained unlabeled in most instances and displayed only occasionally faint staining. The majority of columnar cells of the excretory ducts were strongly immunoreactive at their luminal membrane and contained a single VIP-ret-IR spot in the cytoplasm just apical to the nucleus (Fig. le).
LOCALJZATION
OF VASOACTIVE
INTESTINAL
POLYPEPTIDE-RECEPTOR-LMhKJNOREACTIVITY
Staining of lateral membranes was seen in various intensities, whereas the basal membranes lacked immunolabeling in most cases. Myoepithelial cells, blood cells or other structural elements of the glands and surrounding tissues did not react with the VIP-ret antibody.
91
tor in the regulation of the secretory process of major and minor salivary glands. These observations may be of pathophysiological importance in syndromes associated with xerostomia or in diseases like cystic fibrosis (25,26).
Acknowledgements Discussion The staining pattern obtained with the VIP-ret antibody correlates in several aspects with pharmacological observations: the demonstration of VIP-ret-IR in mucous endpieces of labial and- to a lesser extent - palatal glands agrees well with a recent report on VIP-stimulated mucin release from rat submandibular gland (17). Moreover, VIP modifies the composition of the primary saliva by reabsorption (18). This may be reflected by the demonstration of VIP-ret-IR at different sites of the ductal system. Reabsorption depends on the direct activation of apical membrane chloride transport, while sodium is recycled across the basolateral membrane (9, 19). The intense VIP-ret-IR at the luminal side as also observed in human sweat glands (20) is in accordance with the reported luminal localization of adenylate cyclase (19) to which the VIPreceptor is coupled (21). However, not all effects evoked by VIP in salivary glands can be explained by the distribution of VIP-ret-IR revealed by the monoclonal antibody 109. For example, VIP-induced stimulation of amylase secretion strongly points to the existence of VIP-receptors on serous acinar cells (22), which were not positive in our immunohistochemical study. Also, vasodilatation in response to application of VIP (1) is not reflected by VIP-ret-IR distribution on blood vessels (18, 22, 23). A similar mismatch of known VIP actions and VIP-rec-distribution was also observed in the human colonic muscle (24). The presence of immunologically different types of VIP-ret molecules may account for this difference. In fact, abnormalities in the VIP-ret coupled signal transduction pathway involved in mucin release have recently been pointed out (17), so that evidence for diversity of VIP-receptors in salivary glands is also provided by biochemical findings. Both physiological and immunohistochemical data speak for a significant role of VIP and its recep-
This study was supported by the FSP 23 of the Ministry for Science and Art, Baden-Wiirttemberg, and by the DFG, Grant He 91916-3. Thanks are due to Ms. U. Preissler and U. Sonnek for skilful technical assistance.
References 1. Said, S. 1. and Mutt, V. (1970). Polypeptide with broad biological activity: Isolation from small intestine. Science 169: 1217-1218. 2. Wharton, J., Polak, J., Bryant, M. G., Van Noorden, S., Bloom, S. R. and Pearce, A. G. E. (1979) .Vasoactive intestinal polypeptide (VIP)-like immunoreactivity in salivary glands. Life Sci. 25: 273-280. 3. Ekstriim, J., Mansson, B. and Tobin, G. (1983). Vasoactive intestinal peptide evoked secretion of fluid and protein from rat salivary glands and the development of supersensitivity. Acta Physiol. Stand. 119: 169-175. 4. Larsson, O., Dun&-Engstrom, M., Lundberg, J. M., Fredhohn, B. B. and iinggard, A. (1986). Effects of VIP, PHM and substance P on blood vessels and secretory elements of the human submandibular gland. Regulat Peptides 13: 319-326. 5. Lundberg, J. M., Anggard, A. and Fahrenkrug, J. (1981). Complementary role of vasoactive intestinal polypetide (VIP) and acetylcholine for cat submandibular gland blood flow and secretion. II: Effects of cholinergic antagonists and VIP antiserum. Acta Physiol. Stand. 113: 329-336. 6. Lundberg, J. M., Anggard, A., Fahrenkrug, J., Hiikfelt, T. and Mutt, V. (1980). Vasoactive intestinal polypeptide in cholinergic neurons of exocrine glands, functional significance of coexisting transmitters for vasodilatation and secretion. Proc. Natl. Acad. Sci. 77: 1651-1655. 7. Lundberg, J. M. (198 1). Evidence for coexistence of vasoactive intestinal polypeptide (VIP) and acetylcholine in neurons of cat exocrine glands. Acta Physiol. Stand. 112 Suppl 496: 1-57. 8. Lundberg, J. M., Anggard, A., Fahrenkrug, J., Lundgren, G. and Holmstedt. B (1982). Corelease of VIP and acetvlcholine in relation to blood flow and salivary secretion in cat submandibular salivary gland. Acta Physiol. Stand. 115: 525528. 9. Putney, J. W. (1986). Identification of cellular activation mechanisms associated with salivary secretion. Amut. Rev. Physiol. 48: 75-88. 10. Reid, A. M. and Heywood, L. H. ( 1986). The effects of exogenous vasoactive intestinal polypeptide on secretion from the parotid salivary gland of the pig. Proc. Aust. Physiol. Pharmacol. Sot. 17: 83P. 11. Lundberg, J. M., Martling, C. R. and Hiikfelt, T. (1988). Airway, oral cavity and salivary glands: classical transmit-
92
12.
13.
14.
15.
16.
17.
18.
NEUROPEPTIDES
ters and peptides in sensory and autonomic motor neurons. In: Handbook of Chemical Neuroanatomy, Vol. 6: The peripheral nervous system. Editors: Bjiirklund, A., Hiikfelt, T. and Owman, C. Elsevier Scientific Publishers pp. 391-444. Fredhohn, B. and Lundberg, J. M. (1982). VIP-induced cyclic AMP formation in the cat submandibular gland. Acta Physiol. Stand. 114: 157-159. Luis, J., Martin, J. M., El Battari, A., Marvaldi, J. and Pichon, J. (1988). The vasoactive intestinal peptide (VIP) receptor: recent data and hypothesis. Biochimie 70: 131 l-1322. Marvaldi, J., Luis, J., Muller, J. M., El Battari, A., Fantini, J., Martin, J. M., Abadie, B., Tirard, A. andpichon, J. (1986). Characterization of the vasoactive intestinal peptide (VIP) binding site: a biochemical and immunological approach. Peptides 7, Suppl 1: 137-146. Cripps, M. J. and Bemtet, D. J. (1990). Peptidergic stimulation and inhibition of lacrimal gland adenylate cyclase. Gphthalmol. Vis. Sci. 31: 2145-2150. Pichon, J., Him, M., Muller, J. M., Mangeat, P. and Marvaldi, J. (1983). Anticell surface monoclonal antibodies which antagonize the action of VIP in a human adenocarcinoma cell line (HT29). EMBO J 2: 1017-1022. Turner, J. T. and Camden, J. M. (1990). The influence of vasoactive intestinal peptide receptors in dispersed acini from rat submandibular gland on cyclic AMP production and mucin release. Archs. oral Biol. 35: 103-108. Ekstrom, J., Brodin, E., Ekman, R., Hakansson, R., Mansson, B. and Tobin, G. (1985). Depletion of neuropeptides in rat parotid glands and declining atropine-resistant salivary
19.
20.
21.
22.
23.
24.
25.
26.
secretion upon continuous parasympathetic nerve stimulation. Regul. Peptides 11: 353-359. Durham, J. P., Galanti, M. and Revis, N. W. (1975). The purification and characterization of plasma membranes and subcellular distribution of adenylate cyclase in mouse parotid glands. Biocbim. Biophys. Acta 394: 388-405. Kummer, W., Herbst, W. M. and Heym, Ch. (1990). VIPreceptor like immunoreactivity in human sweat glands. Neurosci. Lett. 110: 239-243. Robberecht, P., Chatelain, P., de Neer, P., Camus, J. C., Waelbroeck,M.andChristophe, J. (198l).Presenceofvasoactive intestinal peptide receptors coupled to adenylate cyclase in rat lung membranes. Biochii. Biophys. Acta 678: 76-82. Reid, A. M. and Tichen, D. A. (1985). Effects ofvasoactive intestinal polypeptide on ovine parotid and submandibular saliva. Proc. Aust. Physiol. Sot. 16: 27P. Bloom, S. R. and Edwards, A. V. (1980). Vasoactive intestinal polypeptide in relation to atropine resistant vasodilatation in the submaxillary gland of the cat. J. Appl. Physiol. 300: 41-53. Kummer,W. (1990). Simultaneousdemonstrationofvasoactive intestinal polypeptide (VIP)- and its receptor in the human colon. Histochem. J. 22: 249-256. Heinz-Erian, P., Dey, R. D., Flux, M. and Said, S. I. (1985). Deficient vasoactive intestinal peptide innervation in the sweat gland of cystic fibrosis patients. Science 229: 14071408. Quinton, P. M. (1983). Chloride impermeability in cystic fibrosis. Nature 301: 421-422.
Localization of Vasoactive Intestinal PolypeptideReceptor-lmmunoreactivity in Human Salivary Glands W. M. HERBST*, W. KUMMERt and CH. HEYMt *Department of Dermatology, University of Erlangen-Niirnberg, Hartmannstr. 14, W-8520 Erlangen and tlnstitute for Anatomy and Cell Biology I, University of Heidelberg, Im Neuenheimer Fe/d 307, W-6900 Heidelberg
Abstract -A monoclonal VIP-receptor antibody derived from a human adenocarcinoma cell line (HT 29) was used in combination with immunogold silver staining for the immunohistochemical demonstration of VIP-receptor (ret) immunoreactivity (IR) in paraffinembedded human salivary glands (parotid, palatal, labial glands). VIP-ret-IR was localized to mucous endpieces of labial and-to a lesser extent - palatal glands, intercalated ducts of the parotid gland, and excretory ducts of all glands investigated. The findings correlate well with known effects of VIP on mucous release and electrolyte transport in salivary glands. Lack of VIP-ret-IR at serous acini may point to immunologically different receptor subtypes in these glands.
Introduction
and trachea (5, 11). Its effects are mediated by specific VIP-receptors coupled to adenylate cyclase and increasing intracellular CAMP content (12- 15). The distribution of VIP binding sites in secretory organs of different species has been studied by means of radiolabelled (I?) VIP and autoradiography without permitting a detailed analysis of the cellular distribution of the VIP-receptor. Therefore, a monoclonal VIP-receptor antibody derived from the human adenocarcinoma cell line HT 29 (16) was used in combination with immunogold silver staining for immunohistochemical demonstration of VIP-receptor immunoreactivity (VIP-ret IR) in paraffin-embedded human salivary glands. This antibody reduces VIP-induced CAMP production in HT 29 cells by 70% and competes with VIP in bind-
Vasoactive intestinal poly-peptide (VIP) - a regulatory peptide of 28 amino acids - was first isolated from porcine intestine by Said and Mutt in 1970 (1). It is frequently present in parasympathetic cholinergic nerves of salivary glands surrounding the acini and, to a smaller extent, the ducts and blood vessels (2). In salivary glands VIP causes an increase in blood flow and potentiates the secretomotor effect of acetylcholine (3- 10). Furthermore, VIP is known to be a powerful stimulator of epithelial chloride transport in several organs such as colon, pancreas
Date received 2 March 1992 Date accepted 2 March 1992
89
90
NEUROPEPTIDES
ing assays, thus it probably recognizes an epitope which may be closely related to but not fully occupy the binding region of the receptor (14, 16).
Material and Methods Samples of parotid, labial and palatal glands were supplied from the files of the Department of Dermatology, University of Erlangen-Niimberg. The specimens were fixed in 10% formalin, routinely dehydrated and embedded in paraffin. Sections were cut at 7 pm, deparaffiized and rinsed in phosphate buffered saline (PBS) containing 2% normal swine serum. Monoclonal rat anti-VIPreceptor (Dianova, FRG; clone 109, for ref. see Pichon et al. 1983; used at a concentration of 2.5-5 pg/ml) served as primary antibody. Incubation was carried out overnight at room temperature (r.t.). The rat anti-VIP-receptor was visualized by anti-rat IgG labelled with colloidal gold (Janssen, Olen, Belgium; 1:40; 1 h at r.t.) followed by silver enhancement (IntenSE MR; Janssen). As the preabsorption of the primary antibody was impossible (the epitope to which the rat anti-VIPreceptor has not been characterised), sections were incubated with normal rat serum instead of the monoclonal antibody or only with the detection system itself. Fig 1.
Results Parotid gland. Homogenous staining for VIP-recIR was observed at the intercalated ducts (Fig. 1a). The large excretory ducts also contained VIP-recIR cells, but they were irregularly mixed with nonreactive cells (Fig. lb). Striated ducts and serous acini were devoid of immunolabelling except a few single VIP-ret-IR cells located in the acini (Fig. lb). The exact identity of these cells could not be determined. They either constitute a particular subgroup of acinar cells or may represent the initial portion of the intercalated ducts. Pulutulgkznds. VIP-ret-IR in palatal glands showed considerable variability in the acinar regions: The mucous endpieces of some glands showed moderate VIP-ret-IR predominantly at their basal aspect (Fig. lc), whereas acini of other glands entirely
lacked VIP-ret-IR. Yet, it is not clear whether these changes are due to regional differences in the palatal glands or to technical difficulties which unavoidably occur when dealing with human material obtained at biopsy. As a consistent finding, however, VIPret-IR was observed in excretory ducts. Labial glands. The mucous acinar cells showed intense immunolabeling of the membranes, particularly at the luminal side, but also at the basolateral circumference. Granular immunoreaction products were also obtained in the cytoplasm (Fig. Id). Serous endpieces remained unlabeled in most instances and displayed only occasionally faint staining. The majority of columnar cells of the excretory ducts were strongly immunoreactive at their luminal membrane and contained a single VIP-ret-IR spot in the cytoplasm just apical to the nucleus (Fig. le).
LOCALJZATION
OF VASOACTIVE
INTESTINAL
POLYPEPTIDE-RECEPTOR-LMhKJNOREACTIVITY
Staining of lateral membranes was seen in various intensities, whereas the basal membranes lacked immunolabeling in most cases. Myoepithelial cells, blood cells or other structural elements of the glands and surrounding tissues did not react with the VIP-ret antibody.
91
tor in the regulation of the secretory process of major and minor salivary glands. These observations may be of pathophysiological importance in syndromes associated with xerostomia or in diseases like cystic fibrosis (25,26).
Acknowledgements Discussion The staining pattern obtained with the VIP-ret antibody correlates in several aspects with pharmacological observations: the demonstration of VIP-ret-IR in mucous endpieces of labial and- to a lesser extent - palatal glands agrees well with a recent report on VIP-stimulated mucin release from rat submandibular gland (17). Moreover, VIP modifies the composition of the primary saliva by reabsorption (18). This may be reflected by the demonstration of VIP-ret-IR at different sites of the ductal system. Reabsorption depends on the direct activation of apical membrane chloride transport, while sodium is recycled across the basolateral membrane (9, 19). The intense VIP-ret-IR at the luminal side as also observed in human sweat glands (20) is in accordance with the reported luminal localization of adenylate cyclase (19) to which the VIPreceptor is coupled (21). However, not all effects evoked by VIP in salivary glands can be explained by the distribution of VIP-ret-IR revealed by the monoclonal antibody 109. For example, VIP-induced stimulation of amylase secretion strongly points to the existence of VIP-receptors on serous acinar cells (22), which were not positive in our immunohistochemical study. Also, vasodilatation in response to application of VIP (1) is not reflected by VIP-ret-IR distribution on blood vessels (18, 22, 23). A similar mismatch of known VIP actions and VIP-rec-distribution was also observed in the human colonic muscle (24). The presence of immunologically different types of VIP-ret molecules may account for this difference. In fact, abnormalities in the VIP-ret coupled signal transduction pathway involved in mucin release have recently been pointed out (17), so that evidence for diversity of VIP-receptors in salivary glands is also provided by biochemical findings. Both physiological and immunohistochemical data speak for a significant role of VIP and its recep-
This study was supported by the FSP 23 of the Ministry for Science and Art, Baden-Wiirttemberg, and by the DFG, Grant He 91916-3. Thanks are due to Ms. U. Preissler and U. Sonnek for skilful technical assistance.
References 1. Said, S. 1. and Mutt, V. (1970). Polypeptide with broad biological activity: Isolation from small intestine. Science 169: 1217-1218. 2. Wharton, J., Polak, J., Bryant, M. G., Van Noorden, S., Bloom, S. R. and Pearce, A. G. E. (1979) .Vasoactive intestinal polypeptide (VIP)-like immunoreactivity in salivary glands. Life Sci. 25: 273-280. 3. Ekstriim, J., Mansson, B. and Tobin, G. (1983). Vasoactive intestinal peptide evoked secretion of fluid and protein from rat salivary glands and the development of supersensitivity. Acta Physiol. Stand. 119: 169-175. 4. Larsson, O., Dun&-Engstrom, M., Lundberg, J. M., Fredhohn, B. B. and iinggard, A. (1986). Effects of VIP, PHM and substance P on blood vessels and secretory elements of the human submandibular gland. Regulat Peptides 13: 319-326. 5. Lundberg, J. M., Anggard, A. and Fahrenkrug, J. (1981). Complementary role of vasoactive intestinal polypetide (VIP) and acetylcholine for cat submandibular gland blood flow and secretion. II: Effects of cholinergic antagonists and VIP antiserum. Acta Physiol. Stand. 113: 329-336. 6. Lundberg, J. M., Anggard, A., Fahrenkrug, J., Hiikfelt, T. and Mutt, V. (1980). Vasoactive intestinal polypeptide in cholinergic neurons of exocrine glands, functional significance of coexisting transmitters for vasodilatation and secretion. Proc. Natl. Acad. Sci. 77: 1651-1655. 7. Lundberg, J. M. (198 1). Evidence for coexistence of vasoactive intestinal polypeptide (VIP) and acetylcholine in neurons of cat exocrine glands. Acta Physiol. Stand. 112 Suppl 496: 1-57. 8. Lundberg, J. M., Anggard, A., Fahrenkrug, J., Lundgren, G. and Holmstedt. B (1982). Corelease of VIP and acetvlcholine in relation to blood flow and salivary secretion in cat submandibular salivary gland. Acta Physiol. Stand. 115: 525528. 9. Putney, J. W. (1986). Identification of cellular activation mechanisms associated with salivary secretion. Amut. Rev. Physiol. 48: 75-88. 10. Reid, A. M. and Heywood, L. H. ( 1986). The effects of exogenous vasoactive intestinal polypeptide on secretion from the parotid salivary gland of the pig. Proc. Aust. Physiol. Pharmacol. Sot. 17: 83P. 11. Lundberg, J. M., Martling, C. R. and Hiikfelt, T. (1988). Airway, oral cavity and salivary glands: classical transmit-
92
12.
13.
14.
15.
16.
17.
18.
NEUROPEPTIDES
ters and peptides in sensory and autonomic motor neurons. In: Handbook of Chemical Neuroanatomy, Vol. 6: The peripheral nervous system. Editors: Bjiirklund, A., Hiikfelt, T. and Owman, C. Elsevier Scientific Publishers pp. 391-444. Fredhohn, B. and Lundberg, J. M. (1982). VIP-induced cyclic AMP formation in the cat submandibular gland. Acta Physiol. Stand. 114: 157-159. Luis, J., Martin, J. M., El Battari, A., Marvaldi, J. and Pichon, J. (1988). The vasoactive intestinal peptide (VIP) receptor: recent data and hypothesis. Biochimie 70: 131 l-1322. Marvaldi, J., Luis, J., Muller, J. M., El Battari, A., Fantini, J., Martin, J. M., Abadie, B., Tirard, A. andpichon, J. (1986). Characterization of the vasoactive intestinal peptide (VIP) binding site: a biochemical and immunological approach. Peptides 7, Suppl 1: 137-146. Cripps, M. J. and Bemtet, D. J. (1990). Peptidergic stimulation and inhibition of lacrimal gland adenylate cyclase. Gphthalmol. Vis. Sci. 31: 2145-2150. Pichon, J., Him, M., Muller, J. M., Mangeat, P. and Marvaldi, J. (1983). Anticell surface monoclonal antibodies which antagonize the action of VIP in a human adenocarcinoma cell line (HT29). EMBO J 2: 1017-1022. Turner, J. T. and Camden, J. M. (1990). The influence of vasoactive intestinal peptide receptors in dispersed acini from rat submandibular gland on cyclic AMP production and mucin release. Archs. oral Biol. 35: 103-108. Ekstrom, J., Brodin, E., Ekman, R., Hakansson, R., Mansson, B. and Tobin, G. (1985). Depletion of neuropeptides in rat parotid glands and declining atropine-resistant salivary
19.
20.
21.
22.
23.
24.
25.
26.
secretion upon continuous parasympathetic nerve stimulation. Regul. Peptides 11: 353-359. Durham, J. P., Galanti, M. and Revis, N. W. (1975). The purification and characterization of plasma membranes and subcellular distribution of adenylate cyclase in mouse parotid glands. Biocbim. Biophys. Acta 394: 388-405. Kummer, W., Herbst, W. M. and Heym, Ch. (1990). VIPreceptor like immunoreactivity in human sweat glands. Neurosci. Lett. 110: 239-243. Robberecht, P., Chatelain, P., de Neer, P., Camus, J. C., Waelbroeck,M.andChristophe, J. (198l).Presenceofvasoactive intestinal peptide receptors coupled to adenylate cyclase in rat lung membranes. Biochii. Biophys. Acta 678: 76-82. Reid, A. M. and Tichen, D. A. (1985). Effects ofvasoactive intestinal polypeptide on ovine parotid and submandibular saliva. Proc. Aust. Physiol. Sot. 16: 27P. Bloom, S. R. and Edwards, A. V. (1980). Vasoactive intestinal polypeptide in relation to atropine resistant vasodilatation in the submaxillary gland of the cat. J. Appl. Physiol. 300: 41-53. Kummer,W. (1990). Simultaneousdemonstrationofvasoactive intestinal polypeptide (VIP)- and its receptor in the human colon. Histochem. J. 22: 249-256. Heinz-Erian, P., Dey, R. D., Flux, M. and Said, S. I. (1985). Deficient vasoactive intestinal peptide innervation in the sweat gland of cystic fibrosis patients. Science 229: 14071408. Quinton, P. M. (1983). Chloride impermeability in cystic fibrosis. Nature 301: 421-422.